HK1097449B - Methods for in vivo delivery of biologics and compositions useful therefor - Google Patents

Description

Method for in vivo delivery of biologicals and compositions for use in the method

The present application is a divisional application, and the original application has application number 94191236.1(PCT/US94/01985), application date 2/22 of 1994, entitled "method for delivering biological products in vivo and composition for the method".

The present invention relates to in vivo delivery of biologicals. In one aspect, the biologic is combined with a polymeric shell made of a biocompatible material. The biological product may be held together by the polymer shell itself and/or the biological product (or the biological product suspended/dispersed in the biocompatible dispersant) may be contained within the polymer shell. In another aspect, the biologic associated with the polymeric shell is administered to a subject, or is dispersed in a suitable biocompatible liquid.

Microsomes and exosomes present in blood are generally cleared from the blood circulation by the "blood filtration organs", i.e., spleen, lung and lung. The particulate matter contained in normal whole blood includes red blood cells (typically 8 microns in diameter), white blood cells (typically 6-8 microns in diameter) and platelets (typically 1-3 microns in diameter). Microcirculation in most organs and tissues allows these blood cells to pass freely. When there is a small thrombus (clot) in the blood circulation, with a diameter greater than 10-15 microns, there is a risk of capillary occlusion or obstruction, resulting in ischemia or oxygen deficiency and possible tissue necrosis. Thus, injection of particles with a diameter of more than 10-15 microns into the blood circulation must be avoided. Despite this, particle suspensions of less than 7-8 microns are relatively safe and have been used to deliver pharmaceutically active ingredients, nutritional agents, and imaging contrast agents in the form of liposomes and emulsions.

The size of the particles and the manner in which they are transported determine their biological function. Strand et al [ inMicros phere-Biomedical Applications，ed.A.Rembaum，PP193-227，CRC Press(1988)]It has been reported that the transport of particles depends on their size. After interstitial injection, particles between a few nanometers and 100 nanometers in size enter the lymphatic capillaries and phagocytosis occurs in the lymph nodes. Following intravenous/arterial injection, particles smaller than about 2 microns are rapidly cleared by the reticuloendothelial system (RES), also known as the Mononuclear Phagocyte System (MPS), in the blood. Particles larger than about 7 microns are trapped by pulmonary capillaries following intravenous injectionAnd (4) capturing. After arterial injection, the particles are trapped in the first capillary bed reached. The vesicular macrophages entrap the inhaled particles.

For drugs that are insoluble or poorly soluble in water and sensitive to the gastric acid environment, they cannot be administered in the conventional manner (e.g., intravenous or oral administration). Parenteral administration of oil-stable drugs can be accomplished by emulsifying the drugs with an aqueous solution (e.g., physiological saline) in the presence of a surfactant or emulsion stabilizer to form a stable microemulsion. Emulsifiers can be injected intravenously, as long as their composition is inert in pharmacology. For example, U.S. Pat. No.4,073,943 describes administration of a water-insoluble pharmacologically active ingredient dissolved in oil emulsified with water in the presence of a surfactant such as lecithin, Pluronic (a copolymer of polypropylene glycol and polyethylene glycol), polyglycerol oleate, etc. PCT International patent publication No. NO, WO85/00011 describes tiny drops of anesthetic drugs encapsulated in a phospholipid, such as dimyristoyl lecithin, having a size range suitable for intradermal or intravenous injection.

There are reports in the literature of using protein microparticles as carriers for pharmacological or diagnostic agents. Albumin microspheres have been prepared by heat denaturation or chemical crosslinking. Heat-denatured microspheres are produced from an emulsified mixture (e.g., albumin, the agent to be added, and a suitable oil) at a temperature between 100 ℃ and 150 ℃. The microspheres are then washed with a suitable solvent and stored. Leucuta et al (International-al Journal of pharmaceuticals vol 41: 213-217(1988)) describe the preparation of heat-denatured microspheres.

The process for preparing chemically crosslinked microspheres involves treating the emulsion with glutaraldehyde to crosslink the proteins, followed by washing and storage. Lee et al [ Science Vol.213: 233-235(1981) and U.S. Pat. No.4,671,954 report such a preparation method.

The above-described method for preparing protein microspheres as carriers for pharmacologically active ingredients, while suitable for delivery of water-soluble agents, is not capable of carrying water-insoluble agents. This limitation is inherent to formulation techniques based on cross-linking or heat-denaturing of protein components in the aqueous phase of water-in-oil emulsions. Any water soluble agent dissolved in the aqueous phase containing the protein may be entrapped by the resulting cross-linked or heat denatured protein matrix, but agents that are poorly soluble in water or oil cannot be incorporated into the protein matrix formed by these methods.

Thus, many poorly water soluble biologics present problems for administration to humans. Indeed, if oral administration is ineffective in delivering a drug, delivery of such pharmacologically active ingredients that are otherwise insoluble or poorly soluble in aqueous media can be severely impaired. Thus, the current formulations for delivery of pharmacologically active ingredients that are otherwise insoluble or poorly soluble in aqueous media require the addition of agents to stabilize the pharmacologically active ingredient. Nevertheless, agents used to stabilize pharmacologically active ingredients (e.g., emulsifiers) often cause severe allergic reactions. Thus, the general method of administration is to treat the patient with antihistamines and steroids prior to injection of the pharmacologically active ingredient to reduce the allergic effects of the agent added for drug delivery.

In an effort to improve the solubility of drugs that are otherwise insoluble or poorly soluble in aqueous media, some researchers have routinely modified the structure of drugs with functional groups that impart strong water solubility. The chemical modifications described in the prior art are the preparation of sulfonated derivatives [ Kingston et al, U.S. Pat. No. 5,059,699(1991) ], and amino acid esters [ Mathew et al, j.med.chem.vol.35: 145-151(1992) ], which exhibit significant biological activity. The water-soluble derivatives produced by this modification facilitate intravenous delivery of drugs that are otherwise insoluble or poorly soluble in water in an aqueous medium (dissolved in a non-hazardous carrier such as physiological saline). Nevertheless, such modifications add cost to the manufacture of the drug, and may also cause undesirable side effects and/or allergic reactions, and/or reduce the effectiveness of the drug.

A biological product that is often difficult to transport is oxygen. Indeed, the requirement for clinically safe and effective oxygen carrying media for use as red blood cell substitutes ("blood substitutes" or "artificial blood") cannot be overemphasized. Some possible uses for these mediators include (a) general infusion applications, including replacement of acute blood loss in both routine and emergency situations, (B) support of organs outside of organ transplant precursors or in vivo surgery, (C) enhanced oxygen delivery to ischemic tissues and organs in vivo, (D) enhanced oxygen delivery to tumors with insufficient vascular supply to increase the effectiveness of radiotherapy or chemotherapy, (E) support of organs or animals in experimental studies, and (F) increased oxygen delivery to living cells in culture.

Blood transfusions may be used to supplement the hemodynamic system of patients suffering from a variety of diseases, including a reduction in blood volume (e.g., due to blood loss), a reduction in the number of red blood cells (e.g., due to bone marrow destruction), or damage or destruction of blood cells (e.g., due to hemolytic anemia). Blood transfusions not only increase intravascular volume, but also provide red blood cells carrying dissolved oxygen to assist in providing oxygen to the tissue.

During blood transfusions to patients who have had excessive blood loss, the blood types of the donor and recipient are carefully matched to be compatible. This often subjects the patient to an adverse period of hypoxia. Moreover, even with autologous red blood cells that have been provided by the patient themselves by prior blood collection and storage, the oxygen carrying capacity and safety of these autologous cells are reduced due to the storage time. Thus, the patient may be in a non-optimal oxygen delivery state for a period of 24 hours after infusion. Finally, there is always a risk of viral and/or bacterial infection of the patient during the transfusion of all whole blood or red blood cells derived therefrom.

Accordingly, there is a recognized need for an alternative substance for oxygen transport and delivery under normal environmental conditions that has the following characteristics. Ideally, the substance used for oxygen transport and delivery should be capable of carrying and delivering oxygen to devices, organs and tissues so as to maintain normal oxygen pressures for these environments. Ideally such a substance should be safe and non-toxic, free of bacterial and/or viral contamination, and non-antigenic and non-pyrogenic (i.e. less than 0.25 EU/ml). In addition, the substance used for oxygen transport and delivery should have viscosity, colloidal and osmotic properties that match those of blood. It is also desirable that such substances remain in the patient's blood system for extended periods of time, thereby allowing the patient's own red blood cells to regenerate and mature. Furthermore, it is desirable that the substance used does not affect or hinder the production of red blood cells.

Currently, there are several intravenous fluids available for the treatment of acute hypovolemia, including crystalloid, such as lactated ringer's solution or normal saline, and colloidal solutions, such as normal human serum albumin. Crystalloid and colloidal solutions can temporarily correct the insufficiency of blood volume, but cannot directly supplement oxygen delivery to the tissue. Although blood transfusion is the preferred treatment modality, the ability to have a sufficient amount of a safe source of blood is a permanent issue.

Other biologicals that are often insoluble or poorly soluble in aqueous media, and which are intended to be administered dissolved in a non-toxic carrier such as physiological saline, and which are useful for minimizing undesirable side effects and/or allergic reactions, are diagnostic agents, such as contrast agents.

Contrast agents are desirable in radiological imaging procedures because they can facilitate the visualization of organs (i.e., their location, size, and morphology) and other cellular structures from the surrounding tissue. For example, even though soft tissues have significantly different biological functions (e.g., liver and spleen), they have a similar cellular composition (i.e., they consist essentially of water).

Magnetic Resonance Imaging (MRI) or magnetic resonance imaging techniques are applied to examine specific nuclei under applied magnetic field strength using radio frequency radiation. It is similar in some respects to X-ray Computed Tomography (CT), which can provide (in some cases) tomographic imaging of organs, and possibly with significant soft tissue resolution. In current applications, its image is a scatter plot of protons in organs and tissues. However, unlike X-ray computed tomography, MRI does not use ionizing radiation. Therefore, MRI is a safe and non-hazardous medical imaging technique.

Although the NMR phenomenon was discovered as early as 1954, it was only a matter of recent years to use it as a tool to describe internal structure for medical diagnostics. The technology is disclosed by Lauterbur [ Nature 242: 190-.

It is well known that nuclei with suitable nuclear spin are aligned in the direction of the applied magnetic field. The nuclear rotation can be arranged in a straight line in two ways: in the same or opposite direction to the external magnetic field. The arrangement along the magnetic field direction is more stable; while in an unstable state the alignment (i.e. the anti-magnetoacoustic direction) must absorb energy. For protons, at 1 tesla (1 tesla ═ 10)⁴Gaussian) magnetic field at which radiated radio frequency pulses (RF) excite nuclei and change their direction of rotation to align with the direction of the opposing magnetic field. After the RF pulse, the excited nuclei "relax" or return to an equilibrium state or align along the direction of the magnetic field. Relaxation signal attenuation may be described in two relaxation terms. T is₁The spin-lattice relaxation time, or longitudinal relaxation time, is the time required for the nucleus to return to equilibrium along the direction of the externally applied magnetic field. Second, T₂Or spin-spin relaxation time, is related to the initial consecutive precession time interval of a single proton spin. Relaxation times of various body fluids, organs and tissues in different species of mammals are well documented.

One of the advantages of MRI is that different scan plane and slice thicknesses can be selected without loss of resolution. This enables high quality transverse, coronal and sagittal images to be obtained directly. The absence of any moving parts in the MRI device ensures a high degree of reliability. MRI is generally considered to have greater potential than X-ray Computed Tomography (CT) for selectively examining tissue. In CT, the X-ray attenuation coefficient alone determines the image contrast, and there are at least three separate variables (T)₁，T₂And nuclear spin density) determines the magnetic resonance image.

Due to subtle physio-chemical differences in organs and tissues, MRI is able to distinguish tissue types and detect diseases that X-ray or CT is not possible to detect. In contrast, CT and X-ray are only sensitive to differences in electron density in tissues and organs. The images that can be obtained by MRI techniques, due to the better stereo resolution, also enable physicians to examine smaller structures than can be examined by CT. In addition, any image scan plane, including transverse, coronal, and sagittal, can be readily obtained using MRI techniques.

Currently, MRI is widely used for the aided diagnosis of many medical conditions. For example, joint injury, bone marrow disease, soft tissue tumors, mediastinal infiltrates, lymphadenopathy, cavernous hemangiomas, hemophilia, cirrhosis, renal cell carcinoma, uterine leiomyoma, endometriosis, breast cancer, stenotic lesions, coronary artery disease, segmental aneurysm, lipoma hypertrophy, atrial septal disease, constrictive pericarditis, and the like. [ see, e.g., Edelman & Warac, Medical Progress 328: 708-716 (1993); edelman & Warac, New England J.of Medicine 328: 785-791(1993)].

The magnetic resonance imaging conventionally used is currently based on proton signals from intracellular water molecules. Therefore, it is often difficult to interpret images and to distinguish individual organs and cellular structures. There are two possible ways to better distinguish the proton signals. The first is the use of contrast agents to alter the T of water molecules within a region₁Or T₂For comparison with other regions. For example, gadolinium ethylene triamine pentaacetate (Gd-DTPA) may shorten the proton T of the water molecule in its vicinity₁Relaxation time, to enhance the image obtained.

As mentioned above, paramagnetic cations, such as Gd, Mn, and Fe, are good MRI contrast agents. They are able to shorten the protons T of the surrounding water₁The relaxation time is used to enhance the MRI images obtained that would otherwise be unreadable.

A second method of distinguishing between individual organs and cellular structures is to introduce another nucleus (i.e., an imaging agent) that is capable of being imaged. With the second method, imaging can only be performed where the contrast agent arrives. The advantage of this method is that the image is obtained by eliminating the interference of the surrounding water. Suitable contrast agents must be biocompatible (i.e., non-toxic, chemically stable, non-reactive with tissue) and capable of being eliminated from the body for a limited period of time.

Although hydrogen has been chosen as the typical basis for MRI scanning (because of the large amount of hydrogen in the body), it produces very poor image areas due to the lack of contrast. Thus, the use of other active MRI nuclei (e.g. fluorine) is advantageous. Matery has described in the paper [ see SPIE, 626, XIV/PACS IV, 18-23(1986) ] the use of certain perfluorocarbons in various diagnostic imaging techniques such as ultrasound, magnetic resonance, radiography and computed tomography. The use of fluorine is beneficial because fluorine is not naturally present in the body.

The fluorine-containing compounds mentioned in the prior art that can be used for magnetic resonance for medical diagnostic purposes are limited to a selection from a group of fluorine-containing molecules that are water-soluble or capable of forming an emulsion. Thus, the prior art fluorocarbon emulsions using water-soluble fluorocarbons have several disadvantages, such as 1) the use of unstable emulsions, 2) the lack of organ specificity and targeting, 3) the potential for inducing allergic reactions due to the use of emulsifiers and surfactants (e.g., lecithin and egg yolk lecithin), 4) limited delivery capacity, and 5) the rapid dilution of such water-soluble fluorocarbons by blood following silent electrophoretic injection.

According to the present invention there is provided a composition in the form of microparticles suitable for parenteral administration as an aqueous suspension for the in vivo delivery of biologicals. The compositions of the present invention comprise a biologic (solid, liquid or gaseous) in combination with a polymeric shell. The polymeric shell is a biocompatible material that is cross-linked in the presence of disulfide bonds. The polymeric shell associated with the biologic may be suspended in a biocompatible matrix for administration. Delivery of biologicals using the compositions of the present invention eliminates the need to administer biologicals in the form of an emulsion containing, for example, ethanol and polyethoxylated castor oil, diluted with saline (see, for example, Norton et al, InAbstract of the 2nd National Cancer Institute workshop Taxol & Taxus, September 23-24, 1992). A disadvantage of these known compositions is that they may give rise to allergic side effects.

In accordance with another aspect of the present invention, it has been surprisingly and unexpectedly discovered that insoluble structures of hemoglobin (Hb) prepared in accordance with the present invention can reversibly bind oxygen. The insoluble hemoglobin structure (IHC) of the present invention binds oxygen with an oxygen affinity similar to that obtained with soluble hemoglobin molecules in red blood cells or soluble modified hemoglobin molecules as described in the prior art for use as potential blood substitutes.

According to another aspect of the invention, a method of encapsulating a biologic in a polymeric shell is also provided. In another aspect of the invention, methods of obtaining local oxygen and temperature data and obtaining magnetic resonance fluorine images of organs and tissues in vivo are also provided.

Delivery of biologics in the form of suspensions of microparticles to target organs such as the liver, lung, spleen, lymphatic circulation, etc. can be achieved to some extent by allowing particles of different sizes and by administration by different routes. The delivery method of the present invention also allows administration of biological products, such as substantially water-insoluble pharmacologically active ingredients, in smaller amounts of liquid and with greatly reduced administration times than the administration volumes and times required by prior art delivery systems (i.e., delivery of a typical human dose of 200-400mg of paclitaxel requires intravenous infusion of about 1 to 2 liters of liquid over a 24 hour period).

For example, a cocktail of polymer shells of the invention can be administered intravenously, enabling the generation of images of vascularized organs (e.g., liver, spleen, lymph and lung) and bone marrow. Organ target specificity is obtained because the reticuloendothelial system (RES), also known as the mononuclear phagocyte (MNP) system, is able to take up micron-sized polymer shells containing organofluorine. Organs such as the liver and spleen play an important role in the removal of foreign material (e.g., particulate matter) from the blood, and are therefore often referred to as "blood filtration organs".

These organs constitute the majority of the reticuloendothelial system. In addition, the lymph in the lymphatic circulation is bound by cells of the reticuloendothelial system. Therefore, imaging of the lymphatic system is possible using the micron-sized organofluoropolymer-containing shells of the present invention. Orally administered or in the form of a suppository, and also for imaging the stomach and gastrointestinal tract. The suspension can also be injected into non-vascular sites, such as the cerebral spinal cavity, to image these sites.

As a further embodiment of the invention paramagnetic cations such as gd.mn, Fe etc. can be linked to polyanions such as alginate and serve as an effective MRI contrast agent.

The present invention overcomes the deficiencies of the prior art by providing 1) injectable suspensions of polymer shells containing biologicals, 2) biologicals in a form with enhanced stability compared to simple emulsions, 3) specificity for the target organ (e.g., liver, spleen, lung, etc.) due to uptake of the polymer shells of the present invention by the RES or MNP system, 4) an emulsifier-free system, thereby avoiding agents that could potentially trigger allergic reactions, and 5) the ability to inject relatively small doses of biologicals and still obtain a better response, due to the ability of the polymer shells containing biologicals of the present invention to target a particular organ.

FIG. 1 is a schematic representation of a polymeric shell prepared in accordance with the present invention. In the figure, A refers to the insoluble disulfide crosslinked polymer shell, B refers to the interior of the polymer shell, which contains oxygen or other gas, contains oxygen-dissolved hydrofluorocarbons, a biocompatible oil in which biologicals are dissolved, a water-in-oil emulsion in which biologicals are dissolved in an aqueous matrix, a suspension of solid particles dispersed in a liquid, and the like, C refers to the thickness of the polymer shell, generally about 5-50 nm, and D refers to the diameter of the polymer shell, generally in the range of about 0.1 to 20 μm.

FIG. 2 shows the oxygen binding curve of a stroma-free hemoglobin solution (dashed curve) and a solution containing an insoluble hemoglobin structure of the invention (solid curve) (i.e., curves expressed as a function of Hill's coefficient and oxygen partial pressure). The actual numerical points of the insoluble hemoglobin structure of the present invention are shown as filled boxes.

FIG. 3 shows the oxygen binding curve (dashed curve) for a stroma-free hemoglobin solution and for a solution containing an insoluble hemoglobin structure of the invention treated with 1.7mM allosteric effector, 2, 3-bisphosphate glycerol (2, 3-BPG) (solid curve). The actual numerical points of the insoluble hemoglobin structure of the present invention are shown as filled boxes.

According to the present invention there is provided a composition for in vivo delivery of a biological product, wherein said biological product is selected from the group consisting of:

a solid substantially completely contained within a polymeric shell, which may be dispersed in a biocompatible dispersing agent,

a liquid substantially completely contained within a polymeric shell, which may be dispersed in a biocompatible dispersing agent,

a gas substantially completely contained within a polymer shell, which may be dispersed in a biocompatible dispersing agent,

a gas, or a mixture of any two or more thereof,

wherein the shell has a maximum cross-sectional diameter of no more than about 10 microns, wherein the polymeric shell comprises a biologically acceptable material substantially cross-linked by disulfide bonds, and

wherein the exterior of the polymer shell is optionally modified with a suitable agent, wherein the agent is covalently attached to the polymer shell.

The term "in vivo delivery" as used herein means delivery of a biological product by oral, intravenous, subcutaneous, intraperitoneal, intrathecal, intramuscular, intracranial, inhalation, topical, transdermal, suppository (rectal), pessary (occlusion) and the like routes of administration.

The term "biological product" as used herein refers to pharmaceutically active agents (e.g., analgesics, anesthetics, antiasthmatics, antibiotics, antidepressants, antidiabetics, antifungals, antihypertensives, anti-inflammatory agents, antineoplastic agents, anxiolytic agents (anxiolytic a-genes), enzymatic agents, nucleic acid structures, immune enhancers, immunosuppressive agents, physiologically active gases, vaccines, etc.), diagnostic agents (e.g., ultrasound contrast agents, radiocontrasts, or magnetic contrast agents), nutritionally valuable agents, and the like.

The term "micrometer" as used herein refers to a unit of measurement of one thousandth of a millimeter.

Many biocompatible materials can be used in the practice of the present invention to form a polymeric shell. The term "biocompatible" as used herein refers to a substance that does not significantly alter or affect the biological system into which it is introduced in any harmful manner. Essentially any material containing sulfhydryl or disulfide linkages in natural or synthetic structures can be used to prepare disulfide-crosslinked shells. The sulfhydryl or disulfide bond may be pre-existing in the structure of the biocompatible substance or they may be introduced by a suitable chemical modification. For example, naturally occurring biocompatible materials such as proteins, polypeptides, polynucleotides, polysaccharides (e.g., alginate esters or salts, high mannuronic acid content alginate esters or salts, polymannuronate, hyaluronic acid, hyaluronate, chitosan, chitin, cellulose, starch, glycogen, guar gum, locust bean gum, dextran, levan, inulin, cyclodextran, agarose, xanthan gum, karaagan, heparin, pectin, gellan gum, scleroglucan, or any combination of two or more thereof), lipids, and the like are selected for such modification. Other linkages such as ester, amide, ether linkages and the like can also be formed during the ultrasonic irradiation step (provided that the necessary functional groups are contained in the starting materials).

As suitable examples of biocompatible materials, naturally occurring or synthetic proteins may be used so long as they have sufficient sulfhydryl or disulfide linkages to form crosslinks (e.g., via disulfide bond formation due to oxidation in ultrasound irradiation). Suitable examples of proteins include albumin (containing 35 cysteine residues), insulin (containing 6 cysteine residues), hemoglobin (each alpha is₂β₂Units containing 6 cysteine residues), lysozyme (containing 8 cysteine residues), immunoglobulins, alpha-2-macroglobulin, fibronectin (fibronectin), vitronectin (vitronectin), fibrinogen, and the like, as well as combinations of any two or more thereof.

The presently preferred protein for forming the polymeric shell is albumin. Other preferred proteins for forming the polymeric shell are hemoglobin. Still other preferred proteins for forming the polymeric shell are mixtures of albumin and hemoglobin. Alternatively, a protein such as alpha-2-macroglobulin, a known opsonin, can be used to facilitate the uptake of the coat of the encapsulated biologic by macrophage-like cells or the uptake of the coat by the liver, spleen. Other functional proteins, such as antibodies or enzymes, which facilitate delivery of the biological product to the desired target site may also be used in forming the polymeric shell.

Similarly, synthetic polypeptides containing sulfhydryl or disulfide linkages are good candidates for forming particles having a polymeric shell. In addition, polyglycols (e.g., linear or branched), polyvinyl alcohols, polyhydroxyethylmethacrylate, polyacrylic acids, polyethyloxazoline, polyacrylamides, polyvinylpyrrolidone, and the like are good candidates for chemical modification (introduction of thiol and/or disulfide bonds and shell formation (by causing their crosslinking).

One can optionally use a dispersant to suspend or solubilize biologicals in the preparation of the compositions of the present invention. Dispersants that can be used in the practice of the present invention include any liquid that can suspend or dissolve the biological product but does not chemically react with the polymer used to form the housing or the biological product itself. For example, water, vegetable oils (e.g., soybean oil, mineral oil, corn oil, rapeseed oil, coconut oil, olive oil, safflower oil, cottonseed oil, etc.), aliphatic, cycloaliphatic, or aromatic hydrocarbons having 4 to 30 carbon atoms (e.g., n-dodecane, n-decane, n-hexane, cyclohexane, toluene, benzene, etc.), aliphatic or aromatic alcohols having 1 to 30 carbon atoms (e.g., octanol, etc.), aliphatic or aromatic esters having 2 to 30 carbon atoms (e.g., ethyl octanoate, etc.), alkyl, aryl, cyclic ethers (e.g., diethyl ether, tetrahydrofuran, etc.), alkyl halides or aryl halides having 1 to 30 carbon atoms (and may be one or more halogen substituents, e.g., CH, rapeseed oil, coconut oil, olive oil, safflower oil, cottonseed oil, etc.), aliphatic or aromatic alcohols having 1 to 30 carbon atoms (e.g., octanol, etc.), aliphatic or aromatic₃Cl，CH₂Cl₂，CH₂-Cl-CH₂Cl, etc.), ketones having 3 to 30 carbon atoms (e.g., acetone, butanone, etc.), polyglycols (e.g., polyethylene glycol, etc.), or mixtures of any two or more thereof.

Particularly preferred dispersant compositions include volatile liquids such as methylene chloride, ethyl acetate, benzene, and the like (i.e., solvents having a high degree of solubility for the pharmaceutically active agent and being soluble in the other dispersant used) and a less volatile dispersant. These volatile additives, when added to other dispersing agents, help to promote dissolution of the pharmaceutically active ingredient in the dispersing agent. This mixture is most desirable since this step is often time consuming. After dissolution is complete, these volatile components can be removed by evaporation (optionally under vacuum).

The particles of the biological product prepared according to the invention, substantially completely contained in or associated with the polymeric shell, may be administered in the form of particles alone or as a suspension in a biocompatible matrix. Such a matrix may be selected from water, a buffered aqueous matrix, physiological saline, buffered physiological saline, an optionally buffered amino acid solution, an optionally buffered protein solution, an optionally buffered sugar solution, an optionally buffered carbohydrate solution, an optionally buffered vitamin solution, an optionally buffered synthetic polymer solution, a lipid containing emulsion, or a combination of any two or more thereof.

According to yet another embodiment of the present invention, there is provided a method of preparing a biologic preparation for in vivo delivery, the method comprising subjecting a substrate comprising a biocompatible material capable of being cross-linked by disulfide bonds and a biologic to high intensity ultrasound conditions for a time sufficient to promote cross-linking of the biocompatible material by disulfide bonds; wherein the biologic is substantially completely contained within the polymeric shell, and wherein the shell has a maximum cross-sectional diameter of no greater than 10 microns.

Thus, in accordance with the present invention, the bioproduct contained in the polymeric shell is synthesized using high intensity ultrasound. Two non-linear acoustic processing steps (i.e., sonification and cavitation) are involved in forming a stable polymer shell first, sonification disperses the bioproduct in an aqueous protein solution. The resulting dispersion is then chemically cross-linked and stabilized by the formation of disulfide bonds. The residues of cysteine (the polymer here is a protein such as albumin) are oxidized by peroxide generated by acoustic cavitation to form disulfide bonds.

The resulting suspension may optionally be filtered with a centrifugal filter (100kDa cut-off) and the filtered structures or microcapsules suspended in physiological saline or a suitable buffer. A schematic of such a structure is shown in figure 1. The average diameter of these structures is around 2 microns. The particle size range determined with an Elzone particle counter is quite narrow (a gaussian distribution with an average diameter around 3 microns is generally observed). The particle size range obtained with this technique is between 0.1 and 20 microns. The preferred particle size range is between 0.5 and 10 microns, with the most preferred range being 1 to 5 microns. This particle size is particularly suitable for medical applications, since intravenous or intra-arterial injections can be carried out without the risk of small vessel occlusion and secondary tissue necrosis (ischemia due to oxygen deficiency). As a control, normal red blood cells are approximately 8 microns in diameter.

The non-obvious feature of the above procedure is the choice of dispersant, which relates to the polarity choice of the dispersant. The formation of the shell of the relevant bioproduct particle involves reorienting the biocompatible material at the interface between the aqueous phase and the non-aqueous phase such that the hydrophilic regions of the biocompatible material are exposed to the aqueous phase and the hydrophobic regions of the biocompatible material are oriented to the non-aqueous phase. In the case where the biocompatible material is a protein, energy must be supplied to the polymer in order to effect unfolding or change of its conformation. The interfacial free energy (interfacial tension) between the two liquid phases, i.e., aqueous and non-aqueous, causes a change in protein conformation at the interface. Thermal energy is also the energy reservoir required for conformational expansion and/or alteration of proteins.

The heat energy input is a function of the following variables, namely: the sound power used in the high intensity ultrasonic wave radiation process, the time of the high intensity ultrasonic wave radiation, the properties of the material subjected to the high intensity ultrasonic wave radiation, and the like. The sound power during the irradiation of high intensity ultrasound waves varies widely, typically from 1 to 1000 tex/cm²Within the range of (1); 50 to 200 watts/cm²Is the preferred range of sound power. Similarly, the time of exposure to high intensity ultrasound radiation also varies widely, typically in the range of a few seconds to about 5 minutes. The exposure time in the high intensity ultrasound radiation is preferably in the range of about 15 to 60 seconds. It is well known to those skilled in the art that the higher the acoustic power used, the shorter the exposure time required to irradiate with high intensity ultrasound and vice versa.

The interfacial free energy is proportional to the difference in polarity between the two liquids. The minimum free energy at the interface between the two liquids at a given operating temperature is the essential energy to form the desired polymer shell. Thus, if a homologous series of dispersants is used to gradually change its polarity, e.g., alkyl acid ethyl esters, the greater the homology the less polar, i.e., the interfacial tension between these dispersants and water increases with the number of carbon atoms in the ester. Therefore, we have found that while ethyl acetate is water immiscible (i.e. an ester of a two carbon acid), this dispersant alone does not produce a large yield of polymer shell-encased particles at room temperature (-20 ℃). In contrast, a higher ester such as ethyl octanoate (an ester of an 8 carbon acid) produced higher yields of polymer shell-coated particles. In fact, ethyl heptanoate (an ester of an acid of 7 carbon atoms) gives moderate yields of particles while lower esters (esters of an acid of 3, 4,5 or 6 carbon atoms) give lower yields. Thus, one can set a condition of minimum aqueous dispersant interfacial tension required to form high yield polymer shell-encased particles at a given temperature.

Temperature is another variable that can be controlled to affect the yield of polymer shell-encased particles. In general, the surface tension of a liquid decreases with increasing temperature. The rate of change of temperature versus surface tension often varies from liquid to liquid. So, for example, the interfacial tension (Δ γ) between two liquids may be T₁Delta gamma at temperature₁And T₂Delta gamma of temperature₂. If T is₁Lower [ Delta ] gamma₁Close to the minimum required to form the polymer shell of the present invention, and, if Δ γ₂(T₂At temperature) greater than Δ γ₁Then, from T₁To T₂Will increase the yield of the polymer shell. In fact, this variation was observed with ethyl heptanoate, which gave moderate yields at 20 ℃ and higher yields at 10 ℃.

Temperature also affects the vapor pressure of the liquid used. The lower the temperature, the lower the total vapor pressure. The lower the total vapor pressure, the more effective the collapse of the cavitation bubbles. Increased collapse Rate of ultrasound radiated cavitation bubbles and peroxide (HO)₂ ^-) The increase in the rate of formation correlates. Increased peroxide generation can result in increased polymer shell yields at lower temperatures. However, in contrast to this, however, the reactivity of oxidizing mercapto groups (i.e., forming disulfide bonds) with peroxide ionsIs increased with increasing temperature. Therefore, there is a relatively narrow optimum temperature operating range for a given liquid subjected to ultrasonic irradiation conditions that can achieve a high yield of polymer shells.

The combination of the two effects, i.e. the change in surface tension with temperature (which directly affects the unfolding and/or conformational changes of the polymer) and the change in reaction yield with temperature (which refers to the cross-linking of the polymer by the formation of disulfide bonds) thus represents the overall change and yield of the polymer shell-coated particles. Suitable temperatures for preparing the polymer shells of the present invention are in the range of about 0-80 ℃.

The ultrasonic irradiation step described above may be operated to produce polymer shell-encased particles containing bioproducts having a range of sizes. The preferred particle radius here is in the range of about 0.1 to 5 microns. A narrow size distribution in this range is very suitable for intravenous administration of biologicals. Preferably, the polymer-coated particles are suspended in a biocompatible matrix (as described herein) and administered in an appropriate manner.

Furthermore, the polymer shell may be modified with a suitable agent, wherein the agent is bound to the polymer shell by an arbitrary covalent bond. Covalent bonds that can be used for such bonding are ester bonds, ether bonds, urethane bonds, diester bonds, amide bonds, secondary or tertiary amine bonds, phosphate bonds, sulfate bonds, and other similar bonds. Suitable agents for such optionally modified polymeric shells are synthetic polymers (polyglycols (e.g. linear or branched polyethylene glycols)), polyvinyl alcohols, polyhydroxyethylmethacrylate, polyacrylic acids, polyethyloxazoline, polyacrylamide, polyvinylpyrrolidone, etc.), phospholipids (e.g. Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI), sphingomyelin, etc.), proteins (e.g. enzymes, antibodies, etc.), polysaccharides (e.g. starch, cellulose, dextran, alginates, chitosan, pectin, hyaluronic acid, etc.), chemical modifiers (e.g. pyridoxal 5' -phosphate, derivatives of pyridoxal, dialdehydes, succinylsalicylate, etc.), or mixtures of any two or more thereof.

The general condition of a monday polymeric shell-encased dissolved biological product can vary. A suspension of the biologic particles in a biocompatible dispersant (instead of a biocompatible dispersant containing dissolved biologic) can be used to produce a polymeric shell containing dispersant-suspended biologic particles. In other words, the polymeric shell can contain a saturated solution of the biologic in the dispersant. Another variation is a polymer shell containing a solid core of a biologic, which is prepared by first dissolving the biologic in a volatile organic solvent (e.g., benzene) to form a polymer shell, and then evaporating the volatile solvent or lyophilizing the entire suspension under vacuum, e.g., in a rotary evaporator. This results in a structure that surrounds a core of a biological product community with a polymer coating. The latter method is particularly beneficial for delivering relatively small volume, high dose quantities of biological products. In some cases, the biocompatible material forming the core shell may itself be a therapeutic or diagnostic agent, e.g., for insulin, which may be delivered as part of a polymeric shell formed during the ultrasound irradiation step described above. In other cases, the polymer forming the outer shell can also participate in the delivery of the biologic, for example, when the biologic is insulin, it can be delivered as part of the polymeric shell formed during the ultrasonic irradiation step described above, thereby providing a blood substitute with a high degree of oxygen bonding.

Variations of the polymer shell are also possible. For example, a small amount of PEG containing a thiol group can be included in the polymer. Upon exposure to ultrasonic radiation, PEG crosslinks with and forms part of the polymer. Alternatively, PEG may be attached to the polymeric shell after the shell is prepared (rather than as part of the matrix from which the shell is made).

PEG is well known for its non-adhesive properties and has been attached toProteins and enzymes to increase their circulation time in vivo (Abu-chowski et al, J.biol.chem.Vol.252: 578 (1977)). PEG is also attached to phospholipids forming lipid bilayers in liposomes to reduce their uptake in vivo and to prolong their residence time in vivo (Klibanov et al, FEBS Letters vol.268:235(1990)). Thus, incorporation of PEG into the wall of the crosslinked protein coat can alter their blood circulation time. This property can be used to maintain a higher blood concentration of the biologic and can extend the release time of the biologic.

Suitable for modifying the polymer shell are electrophilic PEG derivatives, including PEG-imidazole, succinimidyl succinate, nitrophenyl carbonate, tres-ates, and the like; nucleophilic PEG derivatives, including PEG-amines, amino acid esters, hydrazides, thiols, and the like. The PEG-modified polymer shell is able to last longer in the cycle than the unmodified shell. The modification of the polymer shell with PEG can be performed before or after the shell is formed. The presently preferred technique is to modify the polymer shell after it is formed. Other polymers including dextran, alginates, hydroxyethyl starch, and the like may be used for modification of the polymer shell.

Those skilled in the art will envision many variations that are within the scope and spirit of the invention. For example, the dispersant in the polymeric shell can be varied during the formation of the polymeric shell wall, a number of different biologics can be used, and a wide range of proteins and other natural and synthetic polymers can be used. Its application range is also quite wide. In addition to biomedical applications such as the delivery of drugs, diagnostic agents (for use in imaging), artificial blood (sonochemically crosslinked hemoglobin), and parenteral nutrition, the polymeric shell structure of the present invention can also be incorporated into cosmetic applications such as use in skin creams or hair care products, perfumes, use in pressure sensitive inks, use in pesticides.

According to one embodiment of the present invention, the polymeric shell made as described above may be used for in vivo delivery of biological products, such as drugsBioactive agents, diagnostic agents or nutritionally valuable agents. Examples of pharmaceutically active agents that may be used in the practice of the present invention are analgesics (e.g., minophen acetate (acetominophen), aspirin, ibuprofen, morphine and derivatives thereof, etc.), anesthetic gases (e.g., cyclopropane, enflurane, halothane, isoflurane, methoxyflurane, nitrous oxide, etc.), antiasthmatics (e.g.,benfoperazine, ketotifen, traxanox, etc.), antibiotics (e.g., neomycin, streptomycin, chloramphenicol, cephalosporin, ampicillin, penicillin, tetracycline, etc.), antidepressants (e.g., tolyloxacine, oxyphenpazole, imipramine, chloropiperidone (trazadone), etc.), antidiabetic agents (e.g., biguanides, hormones, sulfonylurea derivatives, etc.), antifungal agents (e.g., amphotericin B, nystatin, candicidin, etc.), antihypertensive agents (e.g., propranolol, propiophenol, propranolol, cardamine, reserpine, etc.), steroidal antiinflammatory agents (e.g., cortisone, hydrocortisone, dexamethasone, prednisone, fluorometholone, etc.), non-steroidal antiinflammatory agents (e.g., indomethacin, ibuprofen, lamivune (ramifenizone), piroxicam, etc.), antitumor agents (e.g., doxorubicin, cyclophosphamide, actinomycin, bleomycin, clarithromycin (Duanorubicin), doxorubicin, epothilones (epirubicin), doxycycline, methotrexate, fluorouracil, Carboplatin (Carboplatin), carmustine (BCNU), cisplatin, etoposide, interferons, benzene mustard cholesterol, paclitaxel (the term "paclitaxel" as used herein is intended to include paclitaxel analogs and prodrugs thereof, taxanes and other drugs similar to paclitaxel, such as taxotere and the like), camptothecin and derivatives thereof (which compounds have a more reliable therapeutic effect in treating colon cancer), vinblastine, vincristine, and hormonal antineoplastics, such as estrogens, progestogens, tamoxifen and the like), anxiolytics (such as nifedipine, analgin), enzymatic agents (such as ribonucleases, ribozymes (ribozymes), etc.), nucleic acid structures (such as IGF-1, factor VIII coding sequences, factor IX coding sequences, antisense nucleotide sequences, etc.)Examples of such pharmaceutically active agents include, but are not limited to, pestilence stimulants (e.g., whitecresins, interferons, vaccines, etc.), immunosuppressants (e.g., cyclosporin (C, a), azathioprine, brevicine (mizorobi-ne, FK506, prednisone, etc.), physiologically active gases (e.g., air, oxygen, argon, nitrogen, carbon monoxide, carbon dioxide, helium, xenon, nitrous oxide, nitric oxide, nitrogen dioxide, etc., and mixtures of any two or more thereof), and other pharmaceutically active agents such as cimetidine, o-chlorobenzenedichloride, visadine (visadine), halogenated nitrosoureas (halonitrosteroureas), anthracyclines (hrantacyline), ellipticine, benzocaine, barbiturates, etc.

Examples of diagnostic agents that can be used in the practice of the present invention are ultrasound contrast agents, radiological contrast agents (e.g., iodooctanones, halocarbons, renal contrast agents, etc.), magnetic contrast agents

(e.g., fluorocarbons, fat-soluble paramagnetic compounds, GdDTPA, aqueous paramagnetic compounds, etc.), and other agents (e.g., gases, argon, nitrogen, carbon monoxide, carbon dioxide, helium, xenon, nitrous oxide, nitric oxide, nitrogen dioxide, and the like, and mixtures of any two or more thereof).

Examples of nutritionally valuable agents which may be used in the practice of the invention include amino acids, sugars, proteins, nucleic acids, carbohydrates, fat-soluble vitamins (e.g. vitamins a, D, E, K, etc.) or fats, or mixtures of any two or more thereof.

The main differences between the biologics-containing polymeric shells of the present invention and the prior art protein microspheres are the nature of the construction and the final state of the protein after formation of the polymeric shell, as well as the ability to transport poorly or substantially water-insoluble agents. According to the present invention, the polymer (e.g., protein) is formed by the interaction of amino acids found in the natural structure of many proteins. For example, cysteine forms disulfide bonds for selective chemical crosslinking. Ultrasonic irradiation is used to disperse a dispersion containing dissolved or suspended biological products into an aqueous solution of a biocompatible material having a sulfhydryl or disulfide group (e.g., albumin), thereby forming a crosslinked polymer shell around the droplets of water-insoluble matrix. The ultrasonic irradiation step creates cavitation in the liquid, causing a large local generation of heat, resulting in the formation of peroxide ions that crosslink the polymer by oxidizing sulfhydryl residues (and/or breaking existing disulfide bonds) to form new crosslinked disulfide bonds.

In contrast to the present procedure, the glutaraldehyde cross-linking methods of the prior art are non-specific and react essentially with any nucleophilic groups (e.g., amine, thiol, and hydroxyl) present in the protein structure. Heat denaturation, as described in the prior art, can significantly and irreversibly alter the structure of a protein. In contrast, the disulfides formed in the present invention are very specific and do not substantially denature proteins. In addition, because the polymer shell produced by the method of the present invention is relatively thinner than the diameter of the encapsulated particles, the bioproduct particles or droplets contained in the polymer shell are different from the crosslinked or heat denatured protein microspheres of the prior art. The "shell thickness" of the polymer coating has been measured (by transmission electron microscopy) to be about 25 microns, while the diameter of the coated particles is 1 micron (1000 nanometers). In comparison, prior art microspheres have no protein shell, but rather have protein dispersed throughout the volume of the microsphere.

The polymeric shell containing the solid, liquid or gas bioproduct core can deliver large doses of bioproducts with relatively small volumes. This will reduce patient discomfort when receiving large volumes of fluid and will reduce hospital stays. In addition, the walls of the polymeric shell are generally fully degradable in vivo by proteolytic enzymes (e.g., when the polymer is a protein) so that the delivery system has no side effects, as is often the case with current formulations.

According to this embodiment of the invention, the droplets or particles of the biological product are contained within a shell having a cross-sectional diameter of no more than about 10 microns. Cross-sectional diameters of less than 5 microns are more preferred, while cross-sectional diameters of around 2 microns are most preferred for intravenous administration.

In accordance with another embodiment of the present invention, it has been found that the polymeric shells described herein, when prepared from hemoglobin, have surprisingly high oxygen binding capacity and can therefore be used as blood substitutes. Hemoglobin (Lehninger, in Biochemistry, Worth Publishers, Inc. New York, PP.145-149, 1975) is a 64,500MW protein composed of tetramers (two alpha and two beta chains). Each of the α and β chains is non-covalently linked to a heme residue. The alpha and beta bonds are also held together by non-covalent bonds formed by hydrogen bonding and van der waals forces. These four heme groups, one in each subunit, are capable of binding four molecules of oxygen. These flat heme groups contain an iron ion in the square plane of coordination. In an intact molecule, these four heme groups are located relatively far from one another.

In tetrameric hemoglobin molecules, the oxygen-binding capacity of each heme unit is greatly increased by the interaction or synergy between heme units in binding oxygen. In general, a single heme unit should be capable of binding a single molecule of oxygen. However, the synergistic effect of adjacent heme units in a hemoglobin molecule increases the oxygen binding per heme unit. This synergy is described by the term "hill coefficient", the value of which reflects the number of oxygen binding sites that interact. For native hemoglobin, the hill coefficient is about 2.8.

Soluble hemoglobin accounts for about 90% of the total protein in erythrocytes. 100ml of whole blood can absorb about 21ml of oxygen due to the binding force of hemoglobin. Equally important to binding oxygen, hemoglobin is also effective in releasing the bound oxygen to the tissue. This ability of hemoglobin to bind and release oxygen is often quantified as P₅₀(or). For example, P of whole blood₅₀I.e. the partial pressure of oxygen that results in 50% saturation of hemoglobin, is about 28 mmHg.

The relationship between oxygen partial pressure and percent hemoglobin saturation can be represented by a sigmoidal curve whose position is affected by pH (Bohr effect). The higher the pH of the hemoglobin solution at a defined partial pressure of oxygen, the greater the percentage of oxygen saturation, and P₅₀The lower; the oxygen saturation curve moves to the left on the abscissa. Conversely, the lower the pH of the hemoglobin solution, the lower the percentage of oxygen saturation, and P₅₀The higher; the oxygen saturation curve is shifted to the right on the abscissa. Thus, when hemoglobin moves from the relatively basic pH lung to the relatively acidic pH hypoxic tissue (lactic acid produced by hyperbaric respiration), the hemoglobin will have a tendency to release its carried oxygen. Therefore, the affinity of hemoglobin for oxygen is P for hemoglobin₅₀Varying in the opposite direction.

Changes in the hemoglobin molecule or its conformation are associated with changes in oxygen binding affinity. For example, binding to 2, 3-diphosphoglycerol (2, 3-DPG, present in erythrocytes) relaxes the binding of oxygen to hemoglobin, thereby facilitating the release of oxygen to the tissue; for example, at high altitude and during pregnancy, these physiological conditions require increased oxygen delivery, the 2, 3-DPG content in plasma rises. The oxidation of the iron ion from fe (ii) to fe (iii) in the heme substituent results in the formation of methemoglobin (met-Hb), which binds so tightly with water as to impede oxygen transport. This oxidation, or "autoxidation," is a process that occurs in vivo and is constantly monitored by the redox enzyme system in the red blood cells.

Hemoglobin, a protein that transports and transfers oxygen, can be separated from the membrane of the red blood cell wall or from the matrix (which contains the specific antigens that determine the blood group) and from other cellular and plasma components. If such separation and isolation is effective, the resulting media-free hemoglobin is free of antigenic material; therefore, blood matching according to blood group is not necessary.

Stroma-free hemoglobin (SFH) extracted from the erythrocyte microenvironment has been found to have particularly tight oxygen binding properties (low P)₅₀) And also has a short circulating half-life after transfusion. Lower P₅₀Is a response to a leftward shift in the hemoglobin oxygen binding curve, due in part to the results of exposure of the mediator-free hemoglobin to a plasma pH (7.4) that is higher than the pH in the red blood cells (7.2); moreover, when hemoglobin is removed from red blood cells, the natural relationship between hemoglobin and 2, 3-diphosphoglyceride is disrupted, thereby further reducing P₅₀. With respect to clearance from circulation, we observed that mediator-free hemoglobin is rapidly cleared by the kidneys, transfusion half-life: () The Hill coefficient of SFH was in the range of 2.3 to 2.8 for only about 100 minutes.

We have determined that chemically modified hemoglobin overcomes some of the disadvantages of mediator-less hemoglobin. The improved methods described in the prior art include various intramolecular cross-linking methods of mediator-free hemoglobin; a method of intramolecular and intermolecular crosslinking of mediator-free hemoglobin with a low molecular weight reagent; and methods of mediator-less hemoglobin complexation with other polymers.

Methods for mediator-free intramolecular cross-linking of hemoglobin are known in the art. See, for example, U.S. Pat. Nos.4,584,130, 4,598,064, and 4,600,531. The method is to modify the mediator-free hemoglobin by covalently linking a lysine 99 residue to the alpha chain of the protein through a fumarate bridge. As a result of this intramolecular cross-linking, the bis-aspirin cross-linked hemoglobin has an oxygen affinity equivalent to that of blood. Moreover, the bisaspiralin-crosslinked hemoglobin (molecular weight 64,500) was not able to re-decompose to the dimer (molecular weight 32,250). Therefore, the retention time of the bis-aspirin α - α cross-linked hemoglobin is 4 to 8 hours (which is two to four times the retention time of the mediator-free hemoglobin). Nevertheless, when a patient loses a large amount of blood, it is necessary to have a blood transfusion system that can be carried in several daysOxygen carriers for oxygen delivery also do not provide adequate maintenance for the treatment of acute bleeding. P of bis-aspirin crosslinked hemoglobin₅₀This is also true in the biological range (24-28mmHg) Hill coefficient (2.5-2.8).

The use of low molecular weight cross-linking agents also allows the hemoglobin molecules to undergo intramolecular cross-linking. For example, it is described in us patent 4,336,248 that preferably following the addition of pyridoxal phosphate, dialdehyde is used to complex the hemoglobin molecules with each other and/or with plasma proteins and gelatin derivatives. Crosslinking with difunctional or polyfunctional low molecular weight crosslinkers is described in U.S. Pat. Nos.4,001,401, 4,001,200, 4,05,590 and 4,061,736. The intermolecular hemoglobin crosslink products are often not single soluble tetramers, but soluble oligomers formed by covalent bonding of multiple hemoglobin tetramers. Typically, such products of intermolecular cross-linking often have oxygen carrying and transport properties (comparable to P for whole blood) that are not equivalent to those of blood₅₀Value 28 comparison of glutaraldehyde-polymerized hemoglobin P₅₀18-23) and its hill coefficient is in the range of 1.8-2.8. Furthermore, it is known in the art that the products of intermolecular cross-linking by glutaraldehyde are antigenic [ see D H Marks et al, Militry Med.152：473(1987)]。

Generally, intramolecular and intermolecular hemoglobin crosslinking reduces some of the renal toxicity problems that result from dissociation of unmodified hemoglobin into α β -dimers. However, the Colloid Osmotic Pressure (COP) generated by soluble hemoglobin is not significantly reduced by intramolecular cross-linking. This therefore limits the dosage of the soluble hemoglobin blood substitute suitable for administration. Generally, an increase in COP will cause a decrease in hydrostatic pressure and a corresponding decrease in glomerular filtration rate, resulting in oliguria and, in severe cases, anuria. Administration of soluble hemoglobin as described in the prior art has resulted in bradycardia, increased blood pressure and decreased creatinine clearance. Vasoconstriction and vascular obstruction have been found to be the cause of renal effects, both of which are associated with the use of soluble hemoglobin blood substitutes. These problems can be alleviated by using a highly polymerized form of hemoglobin prepared as described herein as a blood substitute.

Highly fluorinated compounds, particularly perfluorocarbons, have also been considered as red blood cell substitutes due to their high solubility for oxygen, and suitable highly fluorinated compounds for this purpose are perfluorocarbons such as perfluorodecalin, perfluoroindane, perfluoromethyladamantane, perfluorotripropylamine, perfluorotributylamine, perfluorooctylbromide, and the like. In intravenous use, these perfluorocarbon types, which are immiscible with water, must be dispersed in an injectable emulsion. Commonly used emulsifiers in these applications are egg yolk lecithin and lecithin, both of which have the ability to precipitate the allergic reaction. See, for example, PCT 92/06517, which describes an emulsion containing fluoride and phospholipids, such as lysophosphatidylcholine and lysophosphatidylethanolamine, as surfactants, or PCT 93/11868, which describes an emulsion using egg yolk lecithin as an emulsifier, which contains a highly fluorinated, chlorine-substituted, acyclic organic compound as an oxygen carrier.

Fluosol-DA (a therapeutic agent), an emulsion of perfluorodecalin and perfluorotripropylamine, is the only FDA approved drug for the prevention of transient ischemia in balloon coronary angioplasty. Another fluorocarbon product, oxygenates (alliances Pharmaceuticals) or Perfluorosina, has also been approved as an oral formulation. For a review of the use of perfluorinated compounds as blood substitutes see Riess et al, Angew chem.17.621-634(1978).

The blood substitutes described in the prior art use only soluble hemoglobin as an oxygen carrier. Indeed, it is generally accepted conventionally that an insoluble hemoglobin molecule (e.g., one that is excessively polymerized or cross-linked with other hemoglobin molecules to the extent that it is insoluble, or one that is rendered insoluble by excessive denaturation, etc.) is not suitable for reversibly binding oxygen due to the high probability that the oxygen binding site in the molecule. Furthermore, the hill coefficient of the soluble hemoglobin of the prior art is not greater than that of unmodified native hemoglobin.

In comparison, the polymer shell made from hemoglobin described herein is a "giant" macroscopic molecule (since it heavily polymerizes or crosslinks many tetrameric hemoglobin molecules) that is insoluble in aqueous media because of its large volume. Polymerization occurs during the ultrasound irradiation step due to cross-linking of thiol groups on cysteine residues of the protein. The polymeric shells prepared according to the invention generally contain at least 10⁴A cross-linked polymer molecule, and has a molecular weight of 10¹²The individual hemoglobin tetramers are cross-linked into a macroscopic "macropolymer" (megamer) of hemoglobin. It has been unexpectedly found that oxygen can reversibly bind to these insoluble structures with affinity that is in the useful range for a Red Blood Cell (RBC) substitute, i.e., P₅₀Between about 10mmHg and about 50 mmHg.

Another surprising and unexpected finding concerning the insoluble hemoglobin structure (IHC) of the present invention is that it has an extremely high Hill coefficient (n). The hill coefficient is an index for determining the degree of synergy between oxygen binding sites (heme units) in hemoglobin tetrameric molecules. The maximum hill coefficient of native hemoglobin is about 2.8, whereas prior art modified hemoglobins generally report a hill coefficient of less than 2.8. The measured Hill factor for the insoluble hemoglobin structures of the present invention is particularly large, generally in the range of about 5 to about 25. Without wishing to be bound by any theory of action, these extremely high values result from the interaction or interconnection between oxygen binding sites of adjacent cross-linked tetrameric hemoglobin units. Basically, it is believed that the hill coefficient increases, indicating that multiple tetramers in an insoluble structure act synergistically in converting from a deoxy-T (strained) state to an oxy-R (relaxed) state upon incorporation of oxygen.

The unexpectedly large Hill factor observed in the hemoglobin structure of the present invention has the advantage that the amount of oxygen carried per tetrameric hemoglobin unit is far in excess of that achievable with native hemoglobin or prior art modified hemoglobin. This increased oxygen carrying capacity greatly benefits the present invention for use as an RBC substitute.

The hemoglobin structure of the present invention has a maximum Hill factor at an oxygen partial pressure in the range of about 4 to 100 mmHg. In other words, maximum synergy can be achieved within this oxygen partial pressure range. Because of the typical alveolar pO₂Within this range, the hemoglobin structure can provide maximum oxygen uptake in the lung when the structure of the invention is used as a blood substitute.

On the other hand, the structure of the invention releases oxygen to the tissue very similarly to physiological hemoglobin, i.e., in typical tissue pO₂(< 40mmHg), most of the oxygen bound to the insoluble hemoglobin structure is released to oxygenate the tissue. Therefore, the cross-linked insoluble hemoglobin of the present invention has a higher unusual oxygen binding capacity (due to the larger Hill factor) than prior art hemoglobin at typical loading pressures (e.g., in the lung) and retains an effective oxygen release capacity at typical pressures encountered in tissues.

Due to the cross-linking nature of the insoluble hemoglobin structure of the invention and its volume, it may have a considerably longer in vivo circulation time than prior art Red Blood Cell (RBC) substitutes. Moreover, due to their large molecular (macroscopic) volume, they are unlikely to induce nephrotoxicity problems, which are the problems with hemoglobin in the general tetramer or oligomer soluble form described in the prior art.

The empty ("bubble" or microbubble) insoluble hemoglobin structures of the present invention can be loaded with a suitable gas within the hemoglobin envelope or membrane. Thus, when hemoglobin "microbubbles" are equilibrated with oxygen, for example in an external device or in the lungs, the central core of the structure or bubble is saturated with unbound or free oxygen, which diffuses into the core through the molecule. This structure therefore carries unbound molecular oxygen in the small space of its hollow core, in addition to the oxygen bound to the hemoglobin forming the microcapsule shell or membrane. The ability of such systems to transport unbound (but encapsulated) oxygen greatly increases the oxygen carrying capacity of the nuclear system, beyond the amount of oxygen carried by hemoglobin alone. This ability to bind oxygen to hemoglobin while also carrying unbound oxygen through small vacuoles is not described in the prior art.

The insoluble hemoglobin structure can also be pre-loaded or saturated with oxygen prior to intravascular administration. This allows for maximum oxygen delivery in a short-term use like coronary angioplasty or tumor therapy.

The "cell" dispersing properties of the insoluble hemoglobin structures of the present invention allow them to transport oxygen in a physiological manner, without distinction from erythrocytes in the body. Due to the "large polymer" nature of the insoluble hemoglobin structures of the present invention, they produce a colloid osmotic or oncotic pressure that is negligible compared to any equivalent (in terms of oxygen loading) soluble hemoglobin of the prior art. This allows intravenous infusion of high concentrations of the hemoglobin structure of the invention, whereas prior art soluble hemoglobins can only be infused at maximum concentrations of 6-8g/dl due to concerns about severe dehydration of perivascular tissue caused by osmotic gradients.

The invention also extends to the use of other oxygen binding proteins as RBC substitutes. As an example, myoglobin has a single oxygen-binding heme group (but no crosslinkable cysteine residue) and should function in the same manner as the present invention. A genetically engineered myoglobin having at least two cross-linkable cysteine residues can be used to generate an insoluble myoglobin structure. Compositions of oxygen binding proteins and proteins having no affinity for oxygen may be used to form the insoluble structures of the invention, e.g., hemoglobin and myoglobin may be used.

The compositions of the present invention have significant advantages over prior art microencapsulated hemoglobin compositions. The prior art liposome formulations of hemoglobin contain soluble hemoglobin in the liposome shell. The prior art liposome-encapsulated hemoglobin compositions have several disadvantages that have been overcome by the present invention. Soluble hemoglobin cleaved from the liposome composition may cause renal toxicity. The insoluble structure of the present invention will not lyse soluble hemoglobin due to its extensively cross-linked nature. Aggregation of liposomes is known to activate complement protein C3 α. If it is an insoluble structure, such aggregation would not be possible because its volume is much larger than the volume range of the liposomes.

The insoluble cross-linked hemoglobin composition of the present invention avoids the toxicity associated with prior art soluble hemoglobin compositions. Renal toxicity or nephrotoxicity of hemoglobin is primarily associated with clearance of soluble dimer, tetramer, or oligomer hemoglobin from the circulation. The hemoglobin of the invention, which is already extensively cross-linked or "macropolymer", is not cleared by the kidney and is therefore not potentially nephrotoxic. The insoluble structures of the present invention are not cleared by the kidney, thus solving this problem. Another advantage of the extensively cross-linked hemoglobin structure of the present invention over the prior art is the increased retention characteristics within the blood vessel due to its insoluble form.

The morphology of Insoluble Hemoglobin (IHC) was measured by Transmission Electron Microscopy (TEM). To obtain TEM micrographs of cross sections of bovine IHC, the IHC was fixed with glutaraldehyde, stained with osmium tetroxide and potassium ferrocyanide (to provide a control for high protein concentration zones), embedded with a low viscosity resin and microtomed (slice thickness 75 nm). Since there may be some shrinkage of the overall diameter and some distortion of the shape of the IHC during these steps, the true diameter of the IHC is best represented by the distribution of solution particle sizes (3 microns; std. dev.1) rather than by direct measurement of TEM micrographs. Careful observation of the TEM micrograph reveals three distinct regions: a clear central region; a thin layer of dark particles; and a loosely adherent, discrete, speckled gray zone associated with the outer surface of the particle. The thin black layer is the IHC shell. It contains a high density protein, and the color development is most pronounced during the staining step. Loosely attached gray appears to be a native protein that sticks to the IHC shell during the fixation step of the sample preparation. For this photo and many othersPreliminary measurements of the micrographs indicated that the IHC of bovine hemoglobin has a shell thickness of about 25-35 nm. Hemoglobin is a coarse globular protein with a diameter of 5.5mn (l.stryer,Biochemistryfree-man, New York, 1988). Therefore, the protein coat of IHC is about 4 to 20 times thicker than the hemoglobin molecule (tetramer). Thus, a bubble of 3.0 μm diameter should contain about 10⁴To 10¹²A hemoglobin molecule.

Examination of the insoluble hemoglobin structures (IHC) (microbubbles or microspheres) of the present invention using circular dichroism revealed that the content of alpha-helices and beta-sheets in IHC is not significantly different from purified mediator-free hemoglobin (SFH). This finding is important because it suggests that the crosslinking step and formation of insoluble hemoglobin does not result in protein denaturation (i.e., changes in tertiary and quaternary structure). This finding is of course also confirmed by functional data representing that the synergy between reversible oxygen-binding and oxygen-binding heme units is retained after the synthetic step.

The oxygen binding properties of IHC have been measured. Since hemoglobin in the form of met-fe (iii) cannot bind oxygen, the reduction system of Hyashi et al (a.hyashi, t.suzuki, m.shin) can be used.Biochim.Biophys.Acta310, 309, 1973) to reduce fe (iii) to fe (ii). The reduction system consists of glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP, ferredoxin reductase and catalase at different concentrations. The reduction system was added to IHC and held at 4 deg.C for 24-36 hours prior to each oxygen binding experiment.

Bovine and human hemoglobin IHC can be synthesized as described in example 14. As will be appreciated by those skilled in the art, the hemoglobin employed may be derived from any of the gene manipulation products of a vertebrate, non-vertebrate or eukaryotic source, or a vertebrate, non-spinal pusher or eukaryotic cell. Table 1 provides a summary of the results herein.

TABLE 1

Sonicated Hb microbubbles and non-sonicated BHb at different concentrationsOf phosphate ester of (A) and P₅₀Summary of the invention

Note: the hill coefficient (n) of BHb microbubbles is calculated according to the formula:wherein Y is an oxygenating moiety and P₀₂Is the oxygen pressure, and for microbubbles, each Delalog (Y/1-Y) is the average of five consecutive points

All binding experiments were performed in tris buffer (pH 7.4) at 25 ℃. As confirmed by IHC UV-Vis spectroscopy, IHC retains its reversible oxygen binding properties, indicating the presence of met-Fe (III), oxygen-Fe (II), and deoxy-Fe (II) forms. IHC is able to cycle between deoxygenated and aerobic states, and is not substantially degraded over ten cycles. This is important because it indicates that the environment surrounding the active heme site is not significantly altered in the process of preparing the IHC red blood cell substitute.

These oxygen binding data indicate that IHC is substantially free of denatured hemoglobin. If it is denatured, no (or little) physiological reactivity is observed.

The oxygen binding curves for both reduced hemoglobin IHC and native mediator-free hemoglobin in the absence of phosphate were sigmoidal in shape, indicating the effect of oxygen binding. In both curves P₅₀Values (half of the pressure required for oxygen to bind to the oxygen binding sites present on hemoglobin) were similar (21 torr versus 22 torr). This result indicates that IHC binds and releases oxygen at similar oxygen pressures as native hemoglobin. Order toSurprisingly, the maximum Hill coefficient, nmax (representing the degree of synergy between oxygen binding sites) of IHC was significantly higher than that of the mediator-free hemoglobin solution (9.5 vs. 2.6; see FIG. 2). The hill coefficient (n) is calculated using the following formula:

wherein:

y is an oxygenated moiety, and

PO₂partial pressure of oxygen

Each (. DELTA.) log (Y/1-Y) value was obtained using the average of 5 consecutive points and then plotted against some smoothing curve.

Allosteric effectors of native hemoglobin, such as Inositol Hexaphosphate (IHP) and 2, 3-bisphosphoglyceride (2, 3-BPG), have been shown to increase P₅₀(i.e., lower oxygen affinity) and improved synergy. The same effect can also be seen in IHC. I.e. using P₅₀The same increase in value also allows for a more dramatic effect on the synergy of IHC. The nmip increased more significantly than native hemoglobin in the presence of 1.7mm ihp (17.6 vs. 2.8) and 2, 3-BPG (14 vs. 2.8) (see fig. 3 and table 1).

The unexpectedly large enhancement in synergy is evident due to the covalent linkage between the hemoglobin tetramers in the IHC shell. The hill coefficient cannot be larger than the number of interaction sites. A value of about 2.8 in native hemoglobin reflects the cooperativity of one tetramer. Nevertheless, in the IHC shell, there is a link (through the formation of disulfide bonds) between several cross-linked tetramers upon binding of oxygen. The interaction between the nearest tetramers may be strongest; however, weaker interactions also exist between tetramers that are further apart. n maximum essentially representing bound oxygenThe synergistic effect of multiple tetramers during the transition from deoxy-T to oxy-R state in the IHC shell. TEM micrographs of hemoglobin IHC also reflect the shell thickness of approximately 6 hemoglobin polymers. Bubbles of 20 μm diameter should contain about 10⁴To 10¹²A hemoglobin molecule.

The number of particles at different times after preparation was used to determine the IHC storage stability. IHC can be stored in normal saline at 4 deg.C for up to 6 months. At month 3, the concentration of IHC had dropped by approximately 10%, while at month 6, the concentration had dropped by approximately 25-30%.

The rate of IHC autooxidation (conversion of Fe (II) to met-Fe (III)) was measured at 37 deg.C, 25 deg.C and 4 deg.C for greater than 60 hours, 96 hours and 25 days, respectively. These results are obtained without special precautions, kept in an inert atmosphere. The prior art clearly demonstrates that preservation in an inert gas such as nitrogen is beneficial in reducing the autoxidation rate of hemoglobin. Storage under such conditions will result in a dramatic increase in the fraction of fe (ii) hemoglobin that is stored over a longer period of time.

In addition, storing the IHC suspension with the Hyashi et al reduction system as described above prevents autooxidation.

We have investigated pasteurization as a method of final stage sterilization of IHC suspensions. Several different pasteurization conditions were used. The effect of temperature on IHC was measured by the number of particles sterilized under each condition.

Condition 1: the temperature of the IHC suspension was raised from 25 ℃ to 62.8 ℃ in 8 minutes and maintained at this temperature for 30 minutes. The number of particles indicates a degradation of less than 20%.

Condition 2: the temperature of the IHC suspension was raised from 25 ℃ to 71.7 ℃ in 10 minutes and held at this temperature for 15 seconds. The number of particles indicates less than 20% degradation.

Condition 3: the temperature of the IHC suspension was raised from 25 ℃ to 89.5 ℃ in 12 minutes and held at this temperature for 2 seconds. The number of particles indicates a severe degradation of more than 70%.

Therefore, conditions 1 and 2 were found to be suitable as pasteurization methods. Gamma irradiation is also suitable as a final stage sterilization process.

Chemical modification of hemoglobin with known allosteric effectors can alter the oxygen affinity (or P) of IHC₅₀). Modification of hemoglobin generally limits the transition between the two conformations, aerobic and deoxygenated, so the oxygenation function is almost always altered in some way. For example, modification of hemoglobin in the aerobic form is generally beneficial for high oxygen affinity, whereas modification under deoxygenated conditions is counterproductive. Pyridoxal derivatives can be used as modifiers since they behave very much like the natural allosteric effector 2, 3-glyceropyridoxal Diphosphate (DPG). They can bind to the terminal amino group of hemoglobin. For example, increasing P by reacting pyridoxal 5/-phosphate (PLP) with hemoglobin, which is similar to the natural interaction of 2, 3-DPG₅₀. Also useful as modifiers are other pyridoxal derivatives such as 2-nor-2-formyl PLP (a bifunctional reagent capable of linking to the beta chain of hemoglobin) or pyridoxal tetraphosphate. Other crosslinking agents such as sodium acyl trimethyl phosphate) may also be used to crosslink the beta chains.

Aldehyde modifiers may also be used. For example, polymerization of hemoglobin with glutaraldehyde and attachment of PLP with valeraldehyde.

Bis-aspelins, such as 3, 5-bis (bis-salicyl), fumaric acid and the corresponding mono-aspirin, may be used as allosteric modifiers. Aspirin is bound to an internal lysine between the a-chain of hemoglobin and the monofunctional agent. Both of which increase the P of hemoglobin₅₀。

Thus, a "low affinity" or "high affinity" structure can be prepared for use in situations other than trauma and acute blood loss, such as where local delivery of oxygen is required and beneficial.

A "low affinity" structure produced by the above techniqueI.e. with a higher P₅₀(> 28mmHg) has been used as an adjuvant when oxygen is used in the radiation or chemical treatment of tumors. This structure is loaded with the maximum volume of oxygen in vitro and then administered into the tumor circulation. It releases a large amount of oxygen at the tumor site. Reactive oxygen species generated in the presence of radiation or chemotherapy produce greater cytotoxic activity at the tumor site.

"high affinity" structures (P)₅₀< 28mmHg) is suitable for "ischemic oxygen delivery". Ischemia or tissue hypoxia occurs in a number of pathological conditions, such as stroke, myocardial infarction, and the like. Preferential release of oxygen at these sites will help minimize sustained tissue damage. Oxygen carriers or RBC substitutes that have similar oxygen affinity as whole blood do not preferentially release oxygen at such sites. However, it has a high oxygen affinity (i.e., P compared to whole blood)₅₀Lower) and thus retain most of its oxygen under normally encountered oxygen gradient conditions, alternately releasing oxygen preferentially at such ischemic sites due to the greater oxygen gradient between blood and tissue. The affinity of the insoluble hemoglobin structure of the present invention can be readily manipulated by changing the nature of the cross-linking to obtain a value (P) suitable for such use, using a suitable native hemoglobin having the desired affinity, or a hemoglobin having a suitable affinity obtained by genetic engineering methods₅₀)。

The insoluble hemoglobin structure of the present invention can be encapsulated and thus used as an effective carrier for oxygen carriers (e.g., fluorocarbons), pharmaceuticals, diagnostic agents, and the like in pharmaceuticals. Encapsulated Fluorocarbons (FC) are effective oxygen carriers that deliver dissolved oxygen in a linear relationship with oxygen partial pressure, while the hemoglobin shell of IHC delivers and releases bound oxygen in a sigmoidal relationship with oxygen pressure. This particular combination of hemoglobin and fluorocarbon in the same formulation allows for maximum oxygen transport and release in the body.

None of the prior art discloses a technique for simultaneously transporting hemoglobin (Hb) and Fluorinated Carbon (FC). Encapsulated fluorocarbon energy in the core of hemoglobin coatAs a reservoir for oxygen. This binding allows the transport of carrier-bound oxygen to be sigmoidal (i.e., for hemoglobin) as well as linear (i.e., for fluorocarbon) with respect to pressure. This association also allows for its application to tissue pO₂Oxygen release from the "background" of the linear (from the fluorocarbon) and the "bolus" (bolus) of the S-form (from the hemoglobin). In particular, it allows for more efficient oxygen delivery when large amounts of oxygen are needed in a short time, such as during tissue ischemia or tumor therapy.

Hb/FC shows its advantages in combination with the ability to externally monitor the location of the delivered dose within the vessel. Since it is easy to make by MRI¹⁹F-nuclear imaging, and therefore the accumulation of the transport suspension in vessels and tissues is likely to be perceived. This has great advantage in tumor therapy, since oxygen is used as an adjuvant to radiation or chemotherapeutic agents during this process, it allows accurate monitoring of the delivery of the oxyhemoglobin/FC suspension to the intended site.

In the practice of the present invention, there are a number of Fluorinated Carbons (FC)_s) Suitable for use, as described in detail below.

Furthermore, an oxygen-free, binding but cross-linkable protein of cysteine residues or sulfhydryl groups (naturally or artificially introduced) can be coated with biocompatible carbon fluorides suitable for oxygen affinity to be used as a blood substitute. As an example thereof, hemoglobin-coated perfluorodecalin or perfluorotripropylamine is used as a blood substitute.

There are some drugs that are suitable for encapsulation in the hemoglobin microcapsules of the present invention. Some chemotherapeutic agents require the presence of oxygen to achieve maximum tumor cytotoxicity. Delivery of such drugs in the structure of an oxygen carrier such as hemoglobin can effectively incorporate the basic components of the cytotoxin into a single package. Many suitable cytotoxic drugs are oil soluble. These drugs are soluble in a fluorocarbon or other biocompatible oil such as soybean oil, safflower oil, coconut oil, olive oil, cottonseed oil, and the like. The oil/drug solution and a hemoglobin solution are irradiated together with ultrasound to produce oil/drug microspheres encased in a cross-linked insoluble hemoglobin shell. The suspension was saturated with oxygen prior to intravascular administration. Oil-soluble cytotoxic drugs include cyclophosphamide, GCNU, levo-sarcolysin, doxycycline, paclitaxel and its derivatives, taxotere and its derivatives, camptothecin, doxorubicin, etoposide, tamoxifen, vinblastine, vincristine, etc.; non-steroidal anti-inflammatory agents such as: ibuprofen, aspirin, piroxicam, cimetidine, and the like; steroids such as estrogen, prednisolone, cortisone, hydrocortisone, difloron, etc., drugs such as cholesteryl chlorambucil, o-chlorobenzene-p-chlorobenzene dichloroethane, Visadine (Visadine), halogenated nitrosoureas (heliotropoureas), anthracyclines (anthracyclines), ellipticine, diazepam, etc.; immunosuppressants such as cyclosporin, azathioprine, FK506 and the like.

The water soluble drug may also be encapsulated in the IHC shell in the form of a capsule using the double emulsion method. First, an aqueous drug solution is emulsified with a biocompatible oil to obtain a water-in-oil (w/o) emulsion. The w/o emulsion is used as an oil phase and subjected to ultrasonic irradiation together with the above-mentioned aqueous hemoglobin solution to produce a micro-suspension of the desired water-soluble drug containing IHC in the shell, and the emulsifying agent suitable for this embodiment of the present invention includes Pluronics (block copolymer of polyethylene oxide and polypropylene oxide), phospholipids derived from egg yolk (e.g., lecithin, egg yolk lecithin, etc.); fatty acid esters (e.g., glycerol mono-and distearate, glycerol mono-and dipalmitate, etc.). Water-soluble drugs useful in this embodiment of the invention include antineoplastic agents such as actinomycin, bleomycin, cyclophosphamide, daunorubicin (duanorubicin), doxorubicin, epothilones (epirubicin), fluorouracil, carboplatin, cisplatin, interferons, white mustard, methotrexate, doxycycline, tamoxifen, estrogens, progestins, and the like.

The double emulsion method is also suitable for delivery of other water soluble therapeutic agents, diagnostic agents or nutritionally valuable substances. For example, encapsulation of hemoglobin microemulsions in IHC can increase the hemoglobin content of IHC.

To make IHC more red blood cell-like, a phospholipid bilayer may be formed around the cross-linked hemoglobin microbubbles. Such bilayers form a true "red cell analog" and charged phospholipids or lipids that can be formed into such bilayer structures in a two-step process include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, ditetradecylphosphate, dihexadecylphosphate, sarcosinate (sarcosinamide), betaine, monomeric and dimeric alkyds, and the like. Nonionic lipids may also be used in the present invention and include polyethylene fatty acid esters, polyethylene fatty acid ethers, diethanolamides, long chain acyl hexose amides, long chain acyl amino acid amides, long chain amino acid amines, polyoxyethylene sorbitol esters, polyoxy mono-and diglycerides, mono-and distearyl glycerides, mono-and diglycerides, and mono-and diglycerides palmitate glycerides

Another variation on this technique is to use photopolymerizable lipids or lipids that are readily cross-linked via chemical reaction to produce a more stable lipid "film" coating. Photopolymerizable lipids that can be used in the present invention are acrylate or methacrylate substituted lipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, ditetradecylphosphonic acid, dihexadecylphosphonic acid, etc.), naturally polymerizable unsaturated lipids (e.g., unsaturated phosphatidylcholine with malondialdehyde or linkable dienyl groups, etc.), and the like. Lipids that are readily cross-linked via thio-disulfide exchange are also suitable for forming stable lipid coatings for IHC. Examples of such lipids are derivatives of lipoic acid esterified phosphatidyl choline, and the like.

The IHC's synthesized by ultrasound irradiation may be administered as a suspension in a biocompatible medium as described above, or may be administered together with other nutrients.

Preferred routes of administration in vivo are intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, buccal, inhalation, topical, transdermal, suppository, pessary and the like.

In summary, the insoluble hemoglobin structure of the present invention has many advantages over prior art soluble hemoglobins, encapsulated soluble hemoglobins, fluorocarbon blood substitutes or oxygen carriers. These advantages include:

-a higher oxygen capacity;

-variable oxygen affinity;

insoluble "macropolymer" hemoglobin, which remains in the circulation for a longer time than the soluble hemoglobin of the tetramer or oligomer of the prior art;

renal cytotoxicity with lower probability due to the larger molecular size;

the potential for hemoglobin leakage is less than liposome-encapsulated hemoglobin;

because of its larger volume than liposomes, it is not possible to have aggregates that provoke complement proteins;

due to its independent "cellular" nature, it acts more like an RBC than soluble haemoglobins of the prior art;

-a reservoir capable of transporting unbound oxygen while carrying oxygen bound to hemoglobin;

-a Fluorinated Carbon (FC) carrier capable of acting as a latent allergy and toxic free emulsifier;

cross-linked hemoglobin in the Hb/FC structure provides greater stability than the emulsifying systems of the prior art using lecithin and/or other synthetic surfactants;

the oxygen release profile from Hb/FC is relative to tissue pO₂A combined sigmoidal and linear curve;

is available to¹⁹FMRI assay and monitoring Hb/FC structure in vivo;

in addition to transporting oxygen, the hemoglobin or Hb/FC structure can also be used as a drug carrier;

lipid bilayer membranes can be used for hemoglobin structure to make it more physiological;

modification of the hemoglobin structure with polymers such as PEG further increases the intravascular retention time.

In another aspect of the invention, such difficult to administer organofluorine-containing compounds, which are generally hydrophobic and immiscible with water, can be encapsulated in a polymeric shell (as described above) to allow for easy delivery. Organic fluorine containing compounds coated with a polymeric shell are easy to use and are biocompatible. The polymer shell particle size produced according to the present invention has an average diameter of about 2 microns, and is most desirable for medical applications because it can be used for intravenous or intra-arterial injection without the risk of small vessel occlusion and subsequent tissue damage (e.g., oxygen loss due to local blood loss), as compared to red blood cells having a diameter of about 8 microns (so the injectable biomaterial should have a diameter of less than 8-10 microns to prevent vessel occlusion).

Naturally occurring fluorine atom (¹⁹F) Can give a clear nuclear magnetic resonance signal, and can be used as contrast agent or "probe" in MRI¹⁹Particular advantages of F include: 1) relatively low natural concentrations in vivo (fluorine is generally not present in vivo), 2) high sensitivity to nuclear magnetic resonance, 3) and¹h is close to the magnetic rotation ratio, so that the magnetic rotation ratio can be used by only slightly changing the existing MRI device¹⁹F magnetic resonance imaging, and 4) most organic fluorine-containing compounds have low toxicity.

Fluorocarbons are generally non-toxic and biocompatible. Since the carbon-fluorine bond of the fluorinated carbon is strong (about 130 kcal/mole), it is stable and not reactive and therefore not likely to be metabolized. In contrast, the C-H bond (about 100 kcal/mole) is weak and highly reactive, and two fluorinated carbons, perfluorotripropylamine and perfluorodecalin, have been approved by the FDA for medical use under the trade name Fluosol DA as a blood substitute.

Many different fluorinated carbons can be used in the practice of the present invention. For example, compounds corresponding to the following general formula may be incorporated into the polymer shells used in the methods of the invention described herein:

(a)CxF_2x+y-zA_zwherein

x is 1 to 30, preferably 5 to 15,

y is 2; or when X is more than or equal to 2, y is 0 or-2; or when x is more than or equal to 4, y is-4,

z is any integer from 0 to (2x + y-1); and

a is selected from the group consisting of H, halogen other than F, -CN, -OR, wherein R is H, alkyl, fluoroalkyl, alkenyl, fluoroalkenyl, alkynyl, fluoroalkynyl, aryl, fluoroaryl, alkanoyl, fluoroalkanoyl, alkenoyl, fluoroalkenoyl, alkynoyl, fluoroalkynyl, (b) [ C ]_xF_2x+y-zA_z]_aJR_b-aWherein

X, Z, A and R are as defined above,

y' ═ 1; when X is not less than 2, Y' is-1 or-3; when X is more than or equal to 4, Y' is-5,

j ═ O, S, n.p, Al or Si,

a is 1,2, 3 or 4, and

b is 2 for divalent J, or

For a trivalent J value of 3, the average molecular weight,

for a tetravalent J which is 4,

(c)A′-[(CF₂)_x-O]_c-a ", wherein:

x is as defined above, and X is as defined above,

a' is selected from the group consisting of H, a halogen atom, -CN, -OR, wherein R is H, alkyl, fluoroalkyl, alkenyl, fluoroalkenyl, alkynyl, fluoroalkynyl, aryl, fluoroaryl, alkanoyl, fluoroalkanoyl, alkenoyl, fluoroalkenoyl, alkynoyl, fluoroalkynyl,

a' is selected from H or R, wherein R is as defined above,

c is 1-300, preferably 2-50, or

Wherein:

x is as defined above, and

c ═ 2 to 20, preferably 2 to 8, and mixtures of any two or more thereof.

Included in the above formula are compounds having the formula:

C_xF_2xfor example, perfluoro-1-hexene (C)₆F₁₂) Perfluoro-n-hexene (C)₆F₁₂) Perfluoro-3-hexene (C)₆F₁₂) Etc.;

Cyclic-C_xF_2xFor example, perfluorocyclohexane (C)₆F₁₂) Perfluoro cyclooctane (C)₈F₁₆) And the like,

C_xF_2x-2for example, perfluoro-1-hexyne (C)₆F₁₀) Perfluoro-2-hexyne (C)₆F₁₀) Perfluoro-3-hexyne (C)₆F₁₀) And the like,

bicyclic ring-C_xF_2x-2For example perfluorodecalin (C)₁₀F₁₈) And the like,

C_xF_2x+2for example, perfluorohexane (C)₆F₁₄) Perfluorooctane (C)₈F₁₈) Perfluoro nonane (C)₉F₂₀) Perfluoro decane (C)₁₀F₂₂) Perfluorododecane (C)₁₂F₂₆) And the like,

C_xF_2x-4such as perfluoro-2, 4-hexadiene and the like,

C_xF_2x+1a, for example, perfluorotripropylamine [ (C)₄F₉)₃N]Perfluorotributylamine, perfluorotri-tert-butylamine, and the like,

C_xF_2x-2A₂e.g. C₁₀F₁₈H₂And the like, as well as such highly fluorinated compounds as perfluoro-1, 2-indane, perfluoromethyladamantane, perfluorooctylbromide, perfluorodimethylcyclooctane, perfluorocyclooctylbromide, perfluorocrown ethers, and the like.

In addition to the linear, branched, and cyclic fluorocompounds described above, fluorinated crown ethers (e.g., perfluoro-12-crown-4, perfluoro 15-crown-5, perfluoro 18-crown-6, etc.) may also be used in the practice of the present invention.

In order to obtain good magnetic resonance images with a high signal-to-noise ratio, it is advantageous to use a large amount of equivalent fluorine, which as used herein refers to fluorine substituents of those fluorine-containing compounds that are present in substantially similar microenvironments (i.e., substantially similar magnetic environments), which will produce an imaging signal. A large number of equivalent fluorines will produce a strong signal that is not diluted by the competing signal of the "non-equivalent" fluorine.

As used herein, the term "non-equivalent fluorine" refers to those fluorine substituents of a fluorine-containing compound that are present in a substantially dissimilar microenvironment (i.e., a substantially dissimilar magnetic environment) as compared to other fluorine substituents on the same fluorine-containing compound. Thus, non-equivalent fluorine generates multiple signals due to its different chemical shifts compared to equivalent fluorine. Therefore, while compounds with large amounts of non-equivalent fluorine may be satisfactory for MRI applications, these compounds are not ideal for obtaining optimal images.

Of particular interest for the application of vascular imaging is a fluorocarbon-containing polymer shell with extended circulation time. Currently used angiography techniquesThis is a damaging approach using X-ray contrast agents. Have recently demonstrated¹H-MRI can be used for angiography [ Edelman and Warach, New Eng-land J328：785-791(1993)]. In the same way as above, the first and second,¹⁹F-MRI is suitable for angiography and has many advantages, such as the ability to obtain a relatively strong contrast to the reference of the surrounding tissue (which does not contain any native fluorine). Examples of methods that may be used include diagnosis and identification of intracranial aneurysms, arteriovenous malformations, superior vena cava occlusion, inferior vena cava occlusion, portal vein occlusion, pelvic vein occlusion, renal mesenteric artery occlusion, peripheral mesenteric artery occlusion, and the like.

The fluorine-containing compound encapsulated with a polymer shell according to the present invention can be used for various purposes, such as obtaining magnetic resonance images of various organs and/or tissues, obtaining fluorine distribution of organs and/or tissues, and measuring local temperature. The contrast agents of the present invention are not limited to MRI use, but can also be used in ultrasonography and radiology. Other isotopes of fluorine¹⁸F can also be used as a contrast agent in Positron Emission Tomography (PET). Therefore, using one fluorine-containing contrast agent, both PET and MRI diagnoses can be performed. Other imaging agents such as technetium and thallium compounds that are useful in radiocontrast agents can also be captured. Two examples of such contrast agents are nerve relaxants (neurolytes) and heart relaxants (cardiolytes).

The determination of oxygen using the compositions of the invention is based on the presence of paramagnetic substances such as oxygen¹⁹Significant change in NMR relaxation rate of F. Since oxygen is paramagnetic, it will interact with the fluorine nucleus, increasing¹⁹Relaxation rate of F from excited state to normal state. By monitoring this change in relaxation rate, the local region oxygen concentration can be determined (calibrating the MRI signal to a known oxygen concentration).

For example, the novelty of this system is 1) the information that oxygen can be obtained using MRI; 2) paramagnetic effect using oxygen¹⁹FMRI (NMR) signal; 3) the use of a polymeric shell provides a constant protective environment that is also permeable to oxygen, and the like.

By using solid fluorine-containing compounds (e.g., high molecular weight compounds, or mixtures of fluorine-containing compounds) that undergo a phase transition in the physiological temperature range, MRI can also be used to measure local temperature. The relaxation time of the solid is longer than that of the liquid. The relaxation time will be significantly reduced when the phase transition (i.e. from solid to liquid) is reached. A significant change in the NMR spectrum is observed in the solid to liquid phase transition. The shape of the MRI signal for a given fluorochemical can be corrected to a known temperature. The contents of the polymer shell are selected to provide a desired temperature range (typically in the range of about 22-55 ℃) for phase transition to occur, either by using a high molecular weight fluorochemical (i.e., a fluorochemical having a melting point of 15 ℃ or greater) within the polymer shell, or by using a mixture of a fluorochemical and a non-fluorochemical within the polymer shell. The carbon fluoride within the shell will undergo a phase change from solid to liquid within a specified temperature range, with a consequent change in the observed relaxation rate, and the temperature in the body can be determined. Local temperature data would be particularly useful, for example, in monitoring patients in hyperthermia treatment of cancer or in determining cancer cells (which are colder than normal cells).

The fluorochemical composition used will determine the temperature range of the phase transition. Thus, this technique can be used over a wide temperature range simply by changing the components of the fluorochemical composition. For example, pure perfluorododecane (C) encapsulated in a polymer shell₁₂F₂₆) The solid to liquid phase transition will occur at the melting point of the fluorocarbon (75 c). However, this transition is sharp and only a small amount of temperature data is available. To obtain extensive data, the melting point of the fluorochemical composition can be extended over a wide range, for example, by simply adding other components to the neat fluorochemical composition. It is well known in the art that mixtures have a lower and broader melting point range than the corresponding pure components. Thus, for example, perfluorododecane formulated with a lower molecular weight fluorocarbon will broaden the melting point range of encapsulated compositions. Similarly, with an alkane (e.g. pentane) and an alkane containingMixtures of fluorine compounds (e.g., perfluorododecane) will broaden the melting range of the coating composition.

In addition, chemically modified long chain fatty acids (e.g., n-heptadecanoic acid [ C ]) to which fluorine is chemically added can also be used in the practice of the present invention₁₇H₃₄O₂]Nonadecanoic acid [ C ]₁₉H₃₈O₂]Etc.), alcohols (e.g. nonadecanol [ C ]₁₉H₄₀O]Docosanol [ C ]₂₂H₄₆O]Etc.). For example, perfluoro-tert-butanol (t- [ C)₄F₉-OH；PCR CHEMI-CALS)]The dehydration coupling reaction with any of the reactive oxygenates described above will produce one molecule undergoing a solid-to-liquid phase transition and one molecule having 9 equivalent fluorines. Also, for example, a fluorinated fatty acid widens the melting point range and thus can be used to perform local temperature measurements.

The novelty of such thermometry systems is, for example, that 1) temperature data for spatial decomposition can be obtained using MRI, 2) the use of temperature dependence on MRI (nmr) signals, 3) the use of a fluorocarbon-containing composition that undergoes a solid to liquid phase transition in a specified temperature range, 4) the use of a polymeric shell to provide a constant and protective environment for the medium, and 4) the temperature data can be obtained at the same time as the morphological data.

In accordance with the present invention, particles of the fluorochemical composition are encapsulated in a shell having a cross-sectional diameter of no more than about 10 microns (the term "microns" as used herein refers to thousandths of a measurement in millimeters). More preferably, the cross-sectional diameter is less than 5 microns, and for intravenous administration, cross-sectional diameters of less than 1 micron are presently most preferred.

The contrast agents of the present invention may be introduced to the in vivo site in a variety of ways depending on the need for imaging. For example, aqueous suspension solutions can be introduced into the gastrointestinal tract by oral ingestion or in the form of suppositories (e.g., to obtain an image of the stomach and gastrointestinal tract), injected into non-vascular sites such as the cerebral spinal cavity, or injected into the vascular system or vessels of specific organs such as the coronary arteries. In addition, the inventive contrast agents can also be injected into other body parts, such as the anterior and posterior ocular cavities, the ear, the bladder (e.g., through the urethra), the peritoneal cavity, the ureter, the renal pelvis of the urethra, the bone joint cavity, the lymphatic vessel, the subarachnoid cavity, the ventricular cavity, the asphyxia cavity, and the like.

The polymeric shell containing the solid or liquid core of the fluorochemical composition enables the direct delivery of high doses of the fluorochemical composition agent in relatively small volumes. This reduces discomfort to the patient when receiving large amounts of liquid.

According to other embodiments of the present invention, a solution to the problem of administering substantially water-insoluble drugs, such as paclitaxel, is provided that is not described in the prior art. We have therefore found that the delivery of such drugs can be achieved using aqueous suspensions of micron-sized particles, or of drugs containing such drug particles or dissolved in a biocompatible non-aqueous liquid. This approach facilitates the delivery of high concentrations of such drugs and thereby avoids the use of emulsifiers and their associated toxic side effects.

According to yet another embodiment of the present invention, the above-described mode of administration may be improved by a novel drug-containing composition wherein a substantially water-insoluble drug, such as paclitaxel, is suspended in a biocompatible liquid and the resulting suspension contains particles of such drug, such as paclitaxel, having a cross-sectional size of no greater than about 10 microns, and the desired size of the particles, such as less than 10 microns, can be achieved by various means, such as milling, spray drying, precipitation, ultrasonic irradiation, and the like.

Crystals of a water-insoluble drug such as paclitaxel obtained by conventional methodsGreater than 20 microns, solid particles of such drugs (e.g., paclitaxel) have not been delivered in suspension in carriers such as saline. However, the present invention discloses the delivery of a substantially water-insoluble drug (e.g., paclitaxel) by grinding into particles having a size of less than about 10 microns, preferably less than about 5 microns, and most preferably less than about 1 micron, and administering the suspension of the particles, wherein the particles are largeSmall enough to be delivered intravenously in suspension without the risk of blockage of the organ and tissue microcirculation.

Due to the particulate nature of the delivered drug, organs with a reticuloendothelial system such as the spleen, liver, and lung are able to clear most of the drug from the circulation. This allows the physiologically active agent in the form of particles to reach these sites in the body.

The biocompatible liquids used in this embodiment are the same as those described above. In addition, parenteral nutrition agents such as Intralipid (trade name for commercially available fat emulsions for use as parenteral nutrition; commercially available from Kabi vitrrum, Inc-, Clayton, Nchrh (Carolina)), Nutralipid (trade name for commercially available fat emulsions for use as parenteral nutrition; available from McGaw, Irvine, alifornia), Liposon III (trade name for commercially available fat emulsions for use as parenteral nutrition (containing 20% soybean oil, 1.2% lecithin and 2.5% glycerol), available from Abbott laboratories, North Chicago, Illinois), etc. may be used as a carrier for the drug particles. Such as those described above.

In accordance with another embodiment of the present invention, there is also provided a composition for in vivo delivery of paclitaxel, wherein the paclitaxel is dissolved in a parenteral nutrition agent.

According to one embodiment of the present invention, there is provided a composition for in vivo delivery of a biological product, wherein the biological product is selected from the group consisting of:

a solid, optionally dispersed in a biocompatible dispersing agent, which is substantially completely encapsulated in a polymeric shell,

a liquid, optionally dispersed in a biocompatible dispersing agent, which is substantially completely encapsulated in a polymeric shell,

a gas, optionally dispersed in a biocompatible dispersant, which is substantially completely encapsulated in a polymeric shell,

a gas associated with the polymer shell, or a combination of any two or more thereof,

wherein said shell has a maximum cross-sectional diameter of no more than 10 microns, wherein said polymeric shell is made of a biocompatible material that is substantially cross-linked by disulfide bonds, and wherein said polymeric shell is optionally modified with an appropriate agent that is covalently bonded to said polymeric shell by an optional covalent bond.

Further wherein said suitable agent is selected from the group consisting of synthetic polymers, phospholipids, proteins, polysaccharides, surfactants, chemical modifiers, or combinations thereof, wherein said agent is bound to said polymer shell by any covalent bond.

According to yet another embodiment of the present invention, there is provided a composition for in vivo delivery of a biological product, wherein the biological product is selected from the group consisting of:

a solid, optionally dispersed in a biocompatible dispersing agent, which is substantially completely encapsulated in a polymeric shell,

a liquid, optionally dispersed in a biocompatible dispersing agent, which is substantially completely encapsulated in a polymeric shell,

a gas, optionally dispersed in a biocompatible dispersant, which is substantially completely encapsulated in a polymeric shell,

a gas associated with the polymer shell, or a combination of any two or more thereof,

wherein said shell has a maximum cross-sectional diameter of no more than 10 microns, wherein said polymeric shell is made of a biocompatible material that is substantially cross-linked by disulfide bonds, and wherein said polymeric shell is optionally modified with an appropriate agent that is covalently bonded to said polymeric shell by an optional covalent bond.

Further, the biological product is selected from a pharmaceutically active agent, a diagnostic agent or a nutritional agent. Further, the diagnostic agent is selected from an ultrasound contrast agent, a radiation contrast agent, or a magnetic contrast agent. Further, the magnetic contrast agent is a fluorine-containing magnetic resonance imaging agent.

According to yet another embodiment of the present invention, there is provided a composition for in vivo delivery of a biological product, wherein the biological product is selected from the group consisting of:

a solid, optionally dispersed in a biocompatible dispersing agent, which is substantially completely encapsulated in a polymeric shell,

a liquid, optionally dispersed in a biocompatible dispersing agent, which is substantially completely encapsulated in a polymeric shell,

a gas, optionally dispersed in a biocompatible dispersant, which is substantially completely encapsulated in a polymeric shell,

a gas associated with the polymer shell, or a combination of any two or more thereof,

wherein said shell has a maximum cross-sectional diameter of no more than 10 microns, wherein said polymeric shell is made of a biocompatible material that is substantially cross-linked by disulfide bonds, and wherein said polymeric shell is optionally modified with an appropriate agent that is covalently bonded to said polymeric shell by an optional covalent bond.

Further wherein said crosslinked polymer is a naturally occurring polymer, a synthetic polymer, or combinations thereof,

wherein said polymer, prior to crosslinking, has covalently attached sulfhydryl groups or disulfide bonds.

Further, the naturally occurring polymer is selected from the group consisting of a thiol and/or disulfide group-containing protein, a thiol and/or disulfide group-containing polypeptide, a thiol and/or disulfide group-containing lipid, a thiol and/or disulfide group-containing polynucleic acid, and a thiol and/or disulfide group-containing polysaccharide.

Further wherein the protein is selected from the group consisting of hemoglobin, myoglobin, albumin, insulin, lysozyme, immunoglobulin, alpha-2-macroglobulin, fibronectin, vitronectin, fibrinogen, or a combination of any two or more thereof.

The invention will be illustrated in more detail by the following non-limiting examples.

Example 1

Preparation of oil-containing protein hulls

3ml of USP (United states Pharmacopeia) 5% human serum albumin (Alpha therapeutics) solution was placed in a cylindrical tube containing a sonicator probe (Heat System XL 2020). 6.5ml of USP grade soybean oil (soybean oil) was overlaid on the albumin solution. The tip of the sonic probe was placed at the interface of the two solutions and the combined set up was kept in a cold bath at 20 ℃. After the system was allowed to equilibrate, the acoustic wave device was turned on for 30 seconds. Vigorous mixing gave a white milky suspension. The suspension was diluted 1: 5 with physiological saline. The size distribution and concentration of the oleosin shell was determined using a particle counter (particle data system, Elzone, model 280 PC). The protein shell after assay was determined to have a maximum transverse diameter of about 1.35. + -. 0.73. mu.m, at a concentration of-10 in the original suspension⁹Pieces/ml shell.

As a control, the composition is free of protein and does not form a stable microemulsion when subjected to ultrasonic radiation. This result indicates that proteins are essential for microsphere formation. This will be confirmed by electron micrograph observation and transmission electron microscopy studies as described below.

Example 2

Parameters influencing the formation of the outer shell of a polymer

Several variables, such as protein concentration, temperature, sonication time, concentration of management actives and sonic intensity were tested in order to obtain the best way of preparing the polymer shell. These parameters were determined by cross-linked bovine serum albumin shell containing toluene.

The size and number of polymer shells prepared from solutions with protein concentrations of 1%, 2.5%, 5% and 10% were measured using a particle counter and found to not vary greatly with protein concentration, but the number of polymer shells per ml prepared from a "milky suspension increased with increasing protein concentration below 5%. No significant change in the number of polymer shells was found above this protein concentration.

For optimal preparation of the polymer shell, it was found that the initial temperature of the test tube is important. Typically, the initial tube temperature is maintained between 0-45C. The water-oil interfacial tension of the oil used for polymer shell formation is an important parameter, which also varies as a function of temperature. The pharmacologically active agent has no significant effect on the production of the protein coat. Whether the pharmacologically active agent is added in solution or suspended in the dispersion medium, it is relatively unimportant.

Sonication time is an important factor in determining the number of polymer shells produced per ml. Sonication times of greater than 3 minutes were found to reduce the total amount of polymer shells, indicating that excessive sonication may result in destruction of the polymer shells, and sonication times of less than 3 minutes resulted in a sufficient number of polymer shells.

According to the nomogram provided by the sounder manufacturer, the sounder used in addition has large ultrasonic powerAbout 150w/cm². Three progressively higher power settings were applied and the highest power setting was found to yield the greatest number of polymer shells.

Example 3

Preparation of polymer shells containing dissolved paclitaxel

Paclitaxel was dissolved in USP grade soybean oil at a concentration of 2 mg/ml. 3ml USP 5% human serum albumin solution was placed in a cylindrical tube with a sonicator attached. The albumin solution was overlaid with 6.5ml of soybean oil/paclitaxel solution and the sonotrode tip was placed at the interface of the two solutions. The combination was allowed to equilibrate and the sounder was turned on for 30 seconds. Mixing vigorously to obtain a stable white milky suspension containing a protein wall polymer shell coated with a solution of paclitaxel.

To achieve a higher loading of the crosslinked protein shell, a mutual solvent of oil and drug, in which the drug has considerable solubility, is mixed with the oil. As long as the mutual solvent is relatively non-toxic (e.g., ethyl acetate), it can be injected with the original carrier and, on the other hand, can be removed by evaporation of the liquid under vacuum after the polymer shell is prepared.

Example 4

Stability of Polymer Shell

The stability of polymer shell suspensions of known concentrations was analyzed at 3 different temperatures (i.e., 4 ℃,25 ℃, and 38 ℃). Stability was determined by the change in particle number over a specified time. A cross-linked protein (albumin) shell containing soybean oil was prepared as described above (see example 1), diluted in saline to a final oil concentration of 20%, and stored at the above temperature. The number of particles (Elzone) per sample obtained as a function of time is summarized in table 2.

TABLE 2

The above data illustrate that: the concentration of the counted particles (i.e. the polymer shell) remained fairly constant over the experimental time. Quite constant in the range of 7-9X 10¹⁰Between/ml. It shows that the polymer shell has good stability over almost four weeks at different temperatures.

Example 5

In vivo biodistribution-Cross-Linked protein Shell containing fluorophores

To determine the uptake and biodistribution of the fluid encapsulated within the outer shell of the protein polymer following intravenous injection, a fluorescent dye (rubrene, available from Aldrich) was encapsulated within the outer shell of the Human Serum Albumin (HSA) polymer and used as a marker. In this way, rubrene was dissolved in toluene and a toluene/rubrene containing cross-linked albumin shell was prepared by ultrasound irradiation as described above. The resulting milky suspension was diluted 5-fold in physiological saline. Then 2ml of the diluted suspension was injected into the tail vein of the mouse at a rate of 10 minutes. One animal was sacrificed 1 hour after injection and the other 24 hours after injection. Frozen sections of 100 microns of lung, liver, stomach, spleen, bone marrow were examined under a fluorescence microscope to determine the presence of a polymer shell encapsulating or releasing the fluorescent dye. At 1 hour, most of the polymer shell was intact (i.e., exhibited bright fluorescent particles, about 1 micron in diameter) and located in the lung and liver. For 24 hour sections, the dye was observed in lung, liver, spleen, and bone marrow. Observations of ordinary tissue staining showed that the shell wall of the polymer shell had been hydrolyzed (digested) and the dye was released therefrom. This is consistent with the expected results, indicating the potential use of the compositions of the present invention to delay and control the release of a pharmaceutically active agent (e.g., paclitaxel) from the contents.

Example 6

Toxicity of Polymer Shell containing Soybean oil (SBO)

A polymer shell containing soybean oil was prepared as described in example 1 and diluted in physiological saline to two different solutions, one containing 20% soybean oil and the other containing 30% soybean oil.

Lactone, a commercially available TPN agent, contains 20% soybean oil (SBO). When injecting lactone at a rate of 1 ml/min, half the lethal dose of mice is 120ml/kg, or about 4ml for 1 mouse of 30 g.

Two groups of mice (3 of 1 group each weighing about 30g) were treated with the composition of the invention containing soybean oil as follows. Each mouse was injected with 4ml of the prepared suspension containing soybean oil polymer shells. Each mouse in one group received a suspension containing 20% SBO and each mouse in the other group received a suspension containing 30% SBO.

After such treatment, all 3 mice in the group receiving the suspension containing 20% SBO survived, and all tissues or organs showed no macroscopic toxicity observed one week after SBO treatment. Only 1 of a group of three mice receiving a suspension containing 30% SBO died after injection. This result clearly shows that the oil contained in the polymer shell according to the invention is non-toxic at half its lethal level compared to the commercially available SBO formulation (lactone). This effect may be a result of the slow release (i.e., controlled bioavailability) of the oil within the polymer housing. Such slow release prevents the onset of lethal levels of oil, as compared to commercially available high oil emulsions.

Example 7

In vivo bioavailability of soybean oil released from polymeric shells

After injection of the polymer shell suspension into the rat bloodstream, an experiment was performed to determine the slow or sustained release of the substance encapsulated by the polymer shell. A polymer shell containing cross-linked protein (albumin) walls of soybean oil (SBO) was prepared by sonication as described above. The resulting oily polymer shell suspension was diluted with physiological saline to a final 20% oil suspension. 5ml of this suspension was injected over 10 minutes into the cannulated external jugular vein of mice. At several time points after injection, blood was taken from these mice and the triglyceride content in the blood (soybean oil is mainly triglycerides) was determined by routine analysis.

5ml of a commercially available fat emulsion (lactone, an aqueous parenteral nutrition-containing 20% soybean oil, 1.2% egg yolk lecithin and 2.25% glycerol) was used as a control. This control utilizes lecithin as an emulsifier to stabilize the emulsion. A comparison of the serum levels of triglycerides in both cases will give a direct comparison of the bioavailability of the oil as a function of time. In addition to the polymer shell suspension containing 20% oil, a sample of polymer shell suspension diluted in 5ml of physiological saline to a final concentration of 30% oil was injected. Of the three groups, 2 mice were used per group. The triglyceride levels in the blood are listed in Table 3 in each case in mg/dl.

TABLE 3

The column labeled "before injection" indicates the level of triglycerides in the blood before injection. It is clear that for the lactone control, the triglyceride levels are seen to be high after injection, taking approximately 24 hours for the triglyceride to start to drop to the pre-injection levels. It can be seen that this oil can be used immediately after injection in metabolism.

Suspensions of the oily polymer shells containing the same total amount of oil (20%) as the lactone showed a very different utilisation of the triglycerides detectable in the serum. Its content rises to around 2 times its normal value and is maintained at this level for several hours. This indicates that triglycerides are slowly or continuously released into the blood at a level quite close to normal. The polymer shell group receiving 30% oil showed higher triglyceride levels (with higher dosing) and dropped to normal within 48 hours. This was repeated once, and the blood triglyceride level was not greatly increased in this group compared to the control group receiving lactone. This again indicates a slow and sustained release of the oil in the composition of the invention, which has the advantage that dangerously high blood concentrations of the substance in the polymeric shell are avoided and are utilized at acceptable levels over a longer period of time.

It is clear that the same advantages will be achieved with the drug delivered within the polymeric shell of the present invention.

The soybean oil-containing polymer shell system can be suspended in an aqueous solution consisting of amino acids, basic electrolytes, vitamins and sugars to form a Total Parenteral Nutrition (TPN). Such TPN cannot be made with the fat emulsions (e.g., lactones) currently used because the emulsions in the presence of electrolytes are unstable.

Example 8

Preparation of a crosslinked protein wall polymeric shell containing a solid core of a pharmaceutically active agent an alternative method of delivering a poorly water soluble drug, such as paclitaxel, within a polymeric shell is to prepare a polymeric shell that surrounds a solid drug core. Such "protein coated" drug particles can be obtained as follows. The procedure described in example 3 was repeated using an organic solvent to solubilize paclitaxel to a higher concentration. Commonly used solvents are organic solvents such as benzene, toluene, hexane, diethyl ether and the like. The polymer shell was prepared as in example 3.5ml of the milky suspension of polymer shell containing dissolved paclitaxel was diluted to 10ml with physiological saline. The suspension was placed in a rotary evaporator at room temperature and the volatile organics were removed by vacuum. After 2 hours in the rotary evaporator, the polymer shells exposed a hazy core when viewed under a microscope, indicating that essentially all of the organic solvent had indeed been removed and that solid paclitaxel was present within the protein shell.

The polymeric shell with the drug core dissolved in the organic solvent can also be lyophilized to obtain a non-dry powder which can be resuspended in saline (or other suitable liquid) at the time of use. In the case of drugs that do not remain solid at room temperature, a liquid core polymer shell is obtained. This method can be used to prepare cross-linked protein wall shells containing non-dilute drugs. Particle size analysis showed that these polymer shells were smaller than the oil-containing polymer shells. Although albumin is currently the preferred protein for preparing the polymer shell, other proteins such as alpha-2-macroglobulin, a known opsonin, may also be used to increase the uptake of the polymer shell by macrophages. Also, PEG-thiol may be added to the polymer shell during its formation to create a polymer shell that extends circulation time in vivo.

Example 9

Circulation and release kinetics of polymer shells in vivo

A solid core polymer shell containing paclitaxel was prepared as described above (see, example 3) and suspended in physiological saline. The concentration of paclitaxel in the suspension was determined by HPLC as described below. First, paclitaxel within the polymer shell is liberated by the addition of 0.1M mercaptoethanol (causing the exchange of protein disulfide crosslinks and breaking the crosslinks of the polymer shell); the free paclitaxel is then extracted from the suspension with acetonitrile. The resulting mixture was centrifuged and the supernatant was lyophilized. The lyophilisate was dissolved in methanol and injected on HPLC to determine the concentration of paclitaxel in the suspension, approximately 1.6 mg/ml.

Mice were injected with 2ml of this suspension via jugular vein catheter, the animals were sacrificed at 2 hours and the amount of paclitaxel present in the liver was determined by HPLC. This required homogenisation of the liver, followed by extraction with acetonitrile and freeze-drying of the supernatant after centrifugation. The lyophilisate was dissolved in methanol and injected on HPLC. About 15% of the administered amount of paclitaxel was recovered from the liver for 2 hours. Showing an effective dose to the liver. This result is consistent with the known function of the reticuloendothelial system of the liver to clear small particles from the blood.

Example 10

Preparation of Cross-Linked PEG-wall Polymer shells

Thiol-group-containing polyethylene glycols are prepared which are substitutes for thiol-group-containing proteins for the preparation of the polymer shells of the invention or are additives for the preparation of the polymer shells of the invention. PEG is known to be non-toxic to cells, non-inflammatory, non-adhesive, and generally non-biologically active. It has been attached to proteins to reduce its antigenicity and to liposomes that constitute lipids to prolong their circulation time in vivo. Thus, conjugation of PEG to the basic protein shell can be expected to extend the cycle time and improve the stability of the polymer shell. By varying the concentration of the thioglycol added to the 5% albumin solution, it is possible to obtain polymer shells with different in vivo stability. Mercaptopolyethylene glycols can be prepared by techniques available in the literature (e.g., Harris and Herati, published in Polymer preprints Vol.32: 154-155 (1991)).

Mercaptopolyethylene glycol having a molecular weight of 2000g/mol was dissolved in a 5% albumin solution at a concentration of 1% (0.1g added to 10 ml). This protein/PEG solution was coated with oil as described in example 1 and after sonication produced an oil-containing polymer shell with a shell wall composed of cross-linked protein and PEG. The stability of these polymer shells was tested as described in example 4.

Other water-soluble synthetic polymers that can be modified with sulfhydryl groups and used in place of PEG include: polyvinyl alcohol, polyhydroxyethyl methacrylate, polyacrylic acid, polyvinyl oxazoline, polyacrylamide, polyvinyl pyrrolidone, polysaccharide, etc. (e.g., chitosan, alginate, hyaluronic acid, dextran, starch, pectin, etc.).

For example, fluorocarbon containing protein shells, which extend circulation time in vivo, are particularly beneficial for imaging the vascular system. These shells remain in circulation for an extended period of time compared to shells without PEG in the shell wall. For example, it can visualize the cardiac cycle and provide a non-invasive method of assessing coronary circulation instead of traditional invasive techniques such as angiography.

Example 11

Delivery of immunosuppressants to transplanted organs by intravenous administration of immunosuppressant-containing polymeric shells

Immunosuppressive agents are widely used in organ transplantation to prevent rejection as follows. In particular, cyclosporin (Cyclosporine), a potent immunosuppressant, prolongs allograft survival, including: transplantation of skin, heart, kidney, pancreas, bone marrow, small intestine and lung. Cyclosporin has been shown to inhibit certain humoral immunity and to a greater extent cell-mediated responses such as species graft rejection, delayed-type hypersensitivity, experimental allergic encephalomyelitis, branheimer-adjuvant arthritis and graft-versus-host transplantation disease of different organs in many animal species. Renal, hepatic and cardiac allografts have been successfully performed in humans using cyclosporine.

Cyclosporin is currently administered orally and may be administered in the form of capsules containing cyclosporin solutions, both in ethanol and oil solutions (e.g., corn oil, polyoxyethylated glycerides, etc.), as well as solutions of olive oil, polyoxyethylated glycerides, etc. of cyclosporin. It can also be administered by intravenous injection, in which case it is dissolved in ethanol (about 30%) and Cremaphor (polyoxyethylated castor oil) solution, which must be diluted 1: 20 to 1: 100 in physiological saline or 5% glucose before injection. The absolute bioavailability of oral solutions compared to intravenous infusion is approximately 3% (Sandoz pharmaceutical Corporation, public SDI-Z10(A4), 1990). Generally, intravenous administration of cyclosporin has similar problems to that of paclitaxel currently used, namely, allergy and allergy which are considered to be caused by the intravenous administration carrier Cremaphor. In addition, intravenous administration of encapsulated drugs (e.g., cyclosporine) as described herein avoids dangerous blood spikes immediately following administration. For example, comparison of the currently used cyclosporin formulations with the above-described cyclosporin formulations in encapsulated form shows a 5-fold decrease in peak blood levels immediately after injection.

To avoid the problems associated with Cremaphor, cyclosporin encapsulated in a polymeric shell as described above can be administered by intravenous injection. It may be dissolved in a biocompatible oil or many other solvents. It is then dispersed into the polymer shell by sonication as described above. In addition, an important advantage of polymeric shell-encapsulated cyclosporin (or other immunosuppressive agents) administration is the localized character of the intrahepatic reticuloendothelial system due to ingestion of the injected material. To a certain extent, systemic toxic effects can be avoided and effective doses reduced due to local localization features. Following intravenous administration, paclitaxel encapsulated in a polymer shell can be delivered efficiently to the liver as described in example 9. Similar results would be obtained for delivery of cyclosporine (or other possible immunosuppressive agents) according to the present invention.

Example 12

Antibody targeting of polymeric shells

The polymer shell of the present invention is characterized in that monoclonal or polyclonal antibodies can be attached to the polymer shell or antibodies can be incorporated into the polymer shell. The antibody may be incorporated into the polymeric shell when the polymeric microcapsule shell is prepared, or may be attached to the polymeric microcapsule shell after preparation. Standard protein immobilization techniques can be used to achieve this. For example, protein microcapsules made from proteins such as albumin, a large number of amino groups on lysine residues of albumin can be used to attach a suitably modified antibody. For example, when preparing the polymeric shell, an anti-tumor antibody is incorporated into the polymeric shell; or attaching an anti-tumor antibody to the polymeric microcapsule shell after preparation, thereby delivering the anti-tumor agent to the tumor site. For another example, antibodies directed against receptors on target cells are incorporated into the polymer shell during the preparation of the polymer shell, and antibodies directed against receptors on target cells are attached to the polymer microcapsule shell after preparation. Allowing the gene product to be delivered to specific cells (e.g., hepatocytes or certain stem cells in the bone marrow); alternatively, monoclonal antibodies directed against the core receptor may be used to target the encapsulated product to the nucleus of a particular cell type.

Example 13

Polymer shells as vectors for polynucleotide constructs, enzymes and vaccines

Gene therapy is becoming more and more widely accepted as a viable alternative therapy (currently, more than 40 human gene transfer protocols have been approved by the NIH and/or FDA committee). However, one of the obstacles to the practice of this therapy is the reluctance to use viral vectors to introduce genetic material into the human cell genome. The virus itself is toxic and therefore the risk of using viral vectors in gene therapy, particularly for the treatment of non-lethal, non-genetic diseases, is unacceptable. Unfortunately, plasmids that have not been transferred using viral vectors are not usually introduced into the genome of the target cell. In addition, as with conventional drugs, the half-life of such plasmids in vivo is limited. Thus, the general limitations of using gene therapy (as well as antisense therapy, which is an inverse form of gene therapy in which nucleic acids or oligonucleotides are introduced to inhibit gene expression) do not allow for efficient delivery of nucleic acids or oligonucleotides into the body because they are too large to penetrate the cell membrane.

The coating of DNA, RNA, plasmid, oligonucleotide, enzyme, etc. in the protein microcapsule shell can promote the target transmission of the protein microcapsule shell to liver, lung, spleen, lymph and bone marrow. Thus, according to the present invention, these biologics can be delivered to the inside of cells without accompanying risks associated with the use of viruses (vectors). This type of formulation may promote non-specific uptake of the polymer shell or the granulocyte uptake by RES cells directly from the blood stream. Either by intramuscular injection into muscle cells or by direct injection into tumors. Alternatively, monoclonal antibodies directed against the core receptor may be used to deliver the encapsulated product target to the nucleus of a cell of a certain cell type.

The diseases that can be treated by the structure include diabetes, hepatitis, hemophilia, bladder fibrosis, multiple sclerosis, tumor, influenza, AIDS and the like. For example, the insulin-like growth factor gene (IGF-1) may be encapsulated in protein microcapsules for administration to treat diabetic peripheral neuropathy and cachexia. Genes encoding factor IX and factor VIII (for treatment of hemophilia) may be encapsulated in the protein microcapsule shell of the invention for specific delivery to the liver. Similarly, Low Density Lipoprotein (LDL) receptor genes can be encapsulated in the protein microcapsule shells of the invention and delivered specifically to the liver for treatment of atherosclerosis.

Other genes suitable for use in the practice of the present invention are those that reactivate the immune response of the body to cancer cells. For example, an antigen encoded by the DNA contained in the plasmid, such as HLA-B7, can be incorporated into the protein microcapsule shell of the present invention and injected directly into a cancer focus (e.g., skin cancer). Once inside the tumor, the antigen will excite cancer specific cells, causing elevated levels of cytokines (e.g., IL-2), which can make the tumor a target for immune system attack.

As another example, a plasmid containing a portion of the adeno-associated viral genome can be encapsulated in the protein microcapsule shell of the present invention. In addition, the protein microencapsulate shells of the present invention can be used to deliver therapeutic genes to CD8+ T cells as an acceptable immunotherapy for various cancers and infectious diseases.

The protein microencapsule shells of the present invention can also be used as a delivery system against infectious diseases to target delivery of, for example, anti-hepatitis B virus antisense nucleotides. An example of such an antisense oligonucleotide is a 21-mer phosphorothioated nucleotide (phosphothioate) that is resistant to the poly A signal of hepatitis B virus.

The protein microcapsule shell of the present invention may also be used to deliver Cystic Fibrosis Transmembrane Regulator (CFTR). Persons lacking this anchorage may develop cystic fibrosis and may be treated by spraying the protein microencapsulate shells of the present invention containing the CFTR gene and inhaling directly into the lungs.

Enzymes can also be delivered using the microcapsule shells of the present invention. For example, an enzyme (DNAse) can be encapsulated and its target delivered to a virally encapsulated protein or a virally infected cell by attaching a suitable antibody to the outside of the polymer shell. Vaccines can also be encapsulated in the polymeric microcapsules of the present invention and used for subcutaneous, intramuscular or intravenous administration.

Example 14

Preparation of insoluble hemoglobin structure (HIC) for use as a substitute for red blood cells

A20 ml glass reaction vessel, titanium generator and gasket, and all instruments used were rinsed with ethanol and sterile saline prior to synthesis. In a typical reaction, 3.5ml of 5% W/v hemoglobin (human or bovine) is added to a reaction dish and an ultrasound generator (Heat Systems XL2020, 20KHz, 400W maximum power) is attached. Then the sounder and the reaction vessel are immersed in a temperature control tank at 55 ℃, although the product can be synthesized in a wider temperature range (0-80 ℃), the optimal temperature is 55 ℃, and the pH value is 6.8. Temperature control is critical for high yields of material, and the optimum temperature depends on the particular experimental design. The ultrasonic source is switched on at the power 7 position. The output power was estimated to be about 150W/cm using the alignment chart of the manufacturer². The reaction was completed in 30 seconds. The yield is lower for longer or shorter reaction times. For bovine hemoglobin, the 2.5% w/v solution was passed through a Sephadex G-25 gel permeation column to remove all anions, such as phosphate. In the synthesis of a typical human hemoglobin IHC, an sonicator is placed at the interface of the gas and water. The resulting homogeneous suspension contains proteinaceous red blood cells. The aqueous suspension was stored in a sterile container at 4 ℃.

Typical reaction yields contain about 3X 10 per ml⁸The IHC shell solution had a shell mean diameter of 3 microns with a standard error of 1 micron. This synthesis process produces high concentrations of micron-sized biological material with a narrow size distribution.

After synthesis, IHC remained as a suspension in the native protein solution. To separate IHC from unreacted protein, several methods are available: filtration, centrifugation and dialysis. The 1 st method involves filtering the mixture through an Anotop syringe filter with a pore size of 0.2 μm (Whatman, Inc.) and rinsing the filter with several volumes of water until the filtrate contains little or no protein (as determined by UV-vis spectroscopy). The IHC was flushed back (backwashed) out of the filter and resuspended in the same volume of saline. The second purification process involved the use of centripetal or centrifugal filters with a cut-off molecular weight of 100 kD. Centrifugal filtration is a centrifuge tube separated therefrom by a filter membrane. Most of the unreacted hemoglobin (64.5kD) was allowed to pass through the filtration membrane by centrifuging the IHC solution at 1000G for 5 minutes. Finally, the IHC was purified by dialysis against a large molecular weight (300kD) filtration membrane. However, this method requires dialysis for about 2 days. A preferred method of purifying IHC is to use centripetal centrifugation.

Example 15

Preparation of insoluble blood 32 protein/albumin structures (IHAC) for use as red blood cell substitutes

Before synthesis, a 20ml glass reaction dish, titanium sounder and gasket, and all instruments used were washed with ethanol and sterile saline. In a typical reaction, 3.5ml of 5% m/v hemoglobin and albumin (human or bovine, with a hemoglobin/albumin ratio of between 0.5 and 2) are added to a reaction vessel, an ultrasonic generator (Heat Systems, XL2020, 20kHz, maximum power 400W) is connected, and the acoustic generator and the reaction vessel are immersed in a temperature-controlled bath at 55 ℃. Although the product can be synthesized in a wide temperature range (0-80 deg.C), the reaction is best carried out at 55 deg.C. The pH was 6.8. Control of the temperature is critical for high yields of product, and the optimum temperature depends on the particular experimental design. The ultrasonic source was turned on at power 7 and the output power was estimated to be about 150W/cm using the alignment chart from the manufacturer². The reaction was completed in 30 seconds. The yield is lower for longer or shorter reaction times. The resulting homogeneous suspension containing proteinaceous red blood cellsAnd (4) substitution. The aqueous suspension was filtered, washed, resuspended in sterile buffered saline and stored in a sterile container at 4 ℃.

As noted above, a typical reaction yields a solution containing about 10 per ml⁸The mean diameter of the shells of the shell solution was 3 microns with a standard error of 1 micron. This synthetic process produces high concentrations of micron-sized biological material with a narrow size distribution.

An in-line system may also be used to allow the IHC formation reaction to proceed continuously. This system consists of a peristaltic pump that continuously pumps a stream of hemoglobin and either biosoluble oil or fluorocarbon into a reaction cuvette with a sound-generating acoustic probe. The IHC is recovered by overflowing the reaction vessel into a recovery tank while remaining in the reaction vessel for a suitable period of time. The unreacted hemoglobin solution is recirculated into the reaction vessel.

Example 16

Preparation of insoluble hemoglobin structure containing encapsulated fluorocarbons

A20 ml glass reaction cuvette, titanium generator and gasket, and all instruments used were rinsed with ethanol and sterile saline prior to synthesis. In a typical reaction, 3.5ml of 5% m/v hemoglobin (human or bovine) is added to a reaction dish and an ultrasound generator (Heat Systems XL2020, 20KHz, 400W maximum power) is connected. A fluorocarbon, perfluorodecalin 3.5ml, was added to the reaction vessel. The ultrasonic generator and the reaction vessel were then immersed in a temperature controlled bath at 20 ℃. The pH of this aqueous phase was 6.8. The ultrasonic source is switched on at the power 7 position. The nomogram of the manufacturer is used to estimate that the output power is about 150W/cm². The reaction was completed in 30 seconds. The resulting homogeneous suspension contains microcapsules or microspheres of a cross-linked insoluble hemoglobin shell having perfluorodecalin encapsulated therein. This milky suspension was filtered, washed, resuspended in sterile buffered saline as described above, and stored in a sterile container at 4 ℃.

As described above, it is typicalThe reaction produced a reaction product containing about 10 per ml⁸The mean diameter of the shells of the shell solution was 3 microns with a standard error of 1 micron. This synthesis process produces high concentrations of micron-sized biological material with narrow size distribution.

Example 17

Preparation of insoluble albumin structures containing encapsulated fluorocarbons

Before synthesis, a 20ml glass reaction dish, titanium sounder and gasket, and all instruments were rinsed with ethanol and sterile saline. In a typical reaction, 3.5ml of 5% W/v albumin (human or bovine) is added to a reaction dish and an ultrasound generator (Heat Systems XL2020, 20KHz, 400W maximum power) is connected. 3.5ml of perfluorodecalin (or perfluorotripropylamine), a fluorocarbon, was added to the reaction vessel. The ultrasonic acoustic generator and the reaction vessel were then immersed in a temperature controlled bath at 20 ℃. The pH of this aqueous phase was 6.8. The ultrasonic source is switched on at the power 7 position. The nomogram of the manufacturer is used to estimate that the output power is about 150W/cm². The reaction was completed in 30 seconds. The resulting homogeneous suspension contains microcapsules or microspheres having a shell of crosslinked insoluble albumin with perfluorodecalin (or perfluorotripropylamine) encapsulated inside. This milky suspension was filtered, washed, resuspended in sterile buffered saline as described above, and stored in a sterile container at 4 ℃.

As noted above, a typical reaction yields a solution containing about 10 per ml⁸The mean diameter of the shells of the shell solution was 3 microns with a standard error of 1 micron. This synthesis process produces high concentrations of micron-sized biological material with narrow size distribution.

Example 18

Insoluble hemoglobin structure further modified with allosteric modifiers such as pyridoxal 5' -phosphate (PLP)

To obtain a mixture with different affinities for oxygen (i.e. different P)₅₀Further reaction with IHC and PLP, a known allosteric modulator. Mixing trihydroxymethyl aminomethaneThe suspension of IHC (obtained as in example 14) in buffer was deoxygenated under nitrogen at 10 ℃. 10ml of deoxygenated IHC suspension was placed in 6 separate reaction dishes. The PLP/Hb molar ratio charged in each reaction dish was different and was: 0.1/3.0,0.75/3.0,1.5/3.0,3.0/3.0,4.2/3.0,6.0/3.0. After 30 minutes, more than 10 times the amount of sodium borohydride was added for 30 minutes to reduce the schiff base. The suspension was then filtered by centrifugation, back-flushed 3 times with buffered saline, resuspended in buffered saline, and stored at 4 ℃. This modification acts on the amino terminal gene on the 6-globin chain in deoxyhemoglobin. In this sense, this modification is very similar to the effect of 2, 3-DPG in its attachment to lysine EF6(82) b in stabilizing the deoxy conformation.

The results of these six different degrees of modification are: p of IHC₅₀Increase with increasing PLP substitutions (P)₅₀I.e., a decrease in oxygen affinity).

Example 19

Insoluble structure with hemoglobin and polyethylene glycol cross-linked shell

Polyethylene glycol (PEG) is known to be non-toxic to cells, non-inflammatory, non-adhesive, and generally non-biologically active. Proteins attached to PEG are rarely found to be antigenic. It was found that the circulation of liposomes was increased by attachment or conjugation of solid PEG. Thus, this incorporation of PEG into RBCs would be expected to prolong circulation time. By varying the concentration of sulfhydryl PEG added to a protein (e.g., hemoglobin), PEG hemoglobin RBCs with varying stabilities can be prepared. Sulfhydryl PEGs may be prepared by conventional techniques (e.g., Harris and Heart Polymer Preprints 32: 154 (1991)).

MercaptPEG with a molecular weight of 2000g/mol was dissolved in a 5% hemoglobin solution at a concentration of 1% (i.e.0.1 g in 10 ml). This protein-Peg solution was sonicated to generate a protein red blood cell substitute as described in example 14.

Example 20

Insoluble hemoglobin structure with polyethylene glycol covalently attached to the exterior of the shell

IHC was prepared as described in example 14. Polyethylene glycol (PEG 10K) having a molecular weight of 10,000 was reacted with 1, 1' monocarbonyldiimidazole (CDI) using a technique known in the literature (Beaucamp et al analytical Biochemistry 131: 25-33, 1983). IHC was suspended in 50mM pH 8.0 boric acid buffer, and PEG-CDI (2 times molar excess over the total amount of hemoglobin lysine) was added, the reaction mixture was stirred at room temperature for 6 hours, and the resulting PEG-IHC was isolated by filtration, washed with saline, and resuspended in sterile buffered saline.

Example 21

Parameters affecting the formation of insoluble hemoglobin structures

Several variables, such as protein concentration, temperature, sonication time, sound intensity, pH, were examined to obtain the best mode of production of IHC.

These substances are prepared from 1%, 2.5%, 5% and 10% hemoglobin solutions, and also from mixed protein solutions, such as hemoglobin and human, bovine albumin, with concentrations varying from 1-10%. The size and concentration were determined with a particle counter. It was found that the size does not change significantly with the initial concentration of protein. Between the initial concentration of protein and 5%, the amount obtained increases with increasing concentration. Above this concentration, no significant change in the amount was observed.

The initial tube temperature was found to be important for optimal preparation of IHC. The typical initial temperature is maintained at 0-80 deg.C, and the optimal initial temperature is about 70 deg.C.

Sonication time is also an important factor in determining the amount of IHC produced per ml. The ultrasonic treatment is about 30 seconds, which is beneficial to synthesizing IHC with high concentration. Longer or shorter sonication times produce sufficient but less IHC than before.

The ultrasound generation for this experiment was performed according to a nomogram supplied by the ultrasound generator manufacturerThe sound power of the device is about 150W/cm². Other settings of power may also produce a large number of IHCs.

Example 22

Insoluble hemoglobin structure as carrier for oil soluble drugs

The cytotoxic effects of several antineoplastic drugs are greatly enhanced in the presence of oxygen. Thus, it is desirable to increase the oxygen concentration at that site while delivering the drug to the tumor site. The hemoglobin microspheres of the present invention have this effect. Example 16 above describes the encapsulation of a fluorocarbon liquid by an insoluble hemoglobin shell. Cytotoxic drugs, such as cyclophosphamide, BCNU, levophenylalanine mustard, paclitaxel, camptothecin, doxorubicin, etoposide, and the like, can be dissolved in fluorocarbon or other suitable oils, such as soybean oil, and encapsulated within the hemoglobin structure.

Paclitaxel was dissolved in soybean oil (SBO) at a concentration of 5mg/ml, 3.5ml of 5% hemoglobin solution was added to the reaction, and then 3.5ml of SBO/paclitaxel was added. The biphasic mixture was sonicated as in example 16 to give a crosslinked insoluble hemoglobin shell containing SBO/paclitaxel.

Example 23

Polymeric shells as carriers for water-soluble drugs

Several water-soluble drugs were selected for encapsulation in a polymeric shell. For example, the carbamoylic acid is dissolved in water at a concentration of 5 mg/ml. Each ml of this aqueous solution was emulsified with 4ml of soybean oil using pluronic-65 (a block copolymer of polyethylene oxide and polypropylene oxide) to form a stable water-in-oil (w/o) microemulsion. 3.5ml of this w/o microemulsion was overlaid on 3.5ml of 5% hemoglobin solution. And performing sound treatment for 30 seconds to obtain an insoluble hemoglobin structure containing the encapsulated aminomethylfolic acid microemulsion.

Example 24

Polymeric shell as protein carrier

Several proteins are selected to be encapsulated in a polymer shell, such as hemoglobin, albumin, and the like. For example, as a method for increasing the hemoglobin load of IHC, hemoglobin was encapsulated in IHC instead of the water-soluble drug of example 23. Hemoglobin was dissolved in water at a concentration of 10%. 1ml of this aqueous solution was emulsified with 4ml of soybean oil using Pluronic (Pluronic) -65 (a block copolymer of polyethylene oxide and polypropylene oxide) to form a stable water-in-oil (w/o) microemulsion. 3.5ml of the water-in-oil microemulsion containing hemoglobin is covered on 3.5ml of 5% hemoglobin solution. The biphasic mixture was sonicated for 30 seconds to give an insoluble hemoglobin structure containing encapsulated microemulsion which itself also contained hemoglobin. This approach increases the total hemoglobin content of each IHC microsphere, thereby increasing the oxygen carrying capacity for binding oxygen.

Example 25

In vivo administration of albumin/fluorocarbon structures-magnetic resonance imaging (19F-MRI) to detect biodistribution

Perfluorononane-containing albumin structures were prepared as in example 17. The final suspension consisted of sterile saline containing 20% by volume of carbon fluoride. 2ml of this suspension was injected via the tail vein into Sprague Dawley mice anesthetized with ketamine. By using a Bruker500MHz NMR instrument to measure¹⁹F-MRT detects the distribution of the carbon fluoride in the living body. Mice were placed in a 10cm rack¹⁹Using T in F coil₁The weighted sequence acquires images with TR 1 sec, TE 20 ms and a 256 × 128 data matrix.

Most FC accumulation in liver, lung, spleen was found 1 hour after administration. Some FC was also detected in bone marrow. The hemoglobin structure has exactly the same effect as expected in terms of tissue localization and accumulation. These observations have important implications for the treatment of liver, lung tumors, and for the treatment of bone marrow tumor cells with localized administration of high-dose oxygen-complexed cytotoxic drugs or as an adjunct to radiation therapy.

Example 26

In vivo administration of structures with drugs

Insoluble hemoglobin structures containing encapsulated paclitaxel (in SBO) were prepared as in example 22, and the resulting suspension consisted of sterile saline containing 20% SBO by volume. 2ml of this suspension was injected via the tail vein into Sprague Dawley mice anesthetized with ketamine.

After 2 hours of injection, the mice were sacrificed and the liver was removed. The liver was homogenized with a small amount of saline and extracted with ethyl acetate. The extract was lyophilized, dissolved in methanol and injected onto an HPLC column. Approximately 15% of the starting unmetabolized dose of paclitaxel was recovered from the liver. This suggests that it is feasible to deliver antineoplastic drugs specifically to the liver while delivering oxygen to these sites.

Example 27

Stable blood replacement model as insoluble hemoglobin blood substitute

Anesthetized Sprague-Dawley mice were intubated via the external jugular vein. About 70% of the blood volume was removed in 10 minutes. The mice remained in this state for an additional 10 minutes. Then re-implanting a P₅₀An oxygenated isotonic suspension of IHC at 28 mmHg. Mice were continuously monitored for mean arterial pressure, heart rate and respiratory rate. These mice survived over time.

Example 28

Effect of insoluble hemoglobin on reversal of tissue ischemia

The function of poorly soluble hemoglobin (IHC) to selectively deliver oxygen to ischemic sites was studied. Having "high affinity", i.e. P₅₀IHCs of < 28mmHg are suitable for this purpose because they normally encounter a site of high oxygen gradient in the circulatory system, i.e. so-called oxygen release at the site of ischemia. Will have P₅₀IHC at 20mmHg forThis is done for this purpose.

The rat bilateral carotid artery occlusion model was used as the "stroke" or cerebral ischemia model. Both arteries of the Sprague-Dawley mice anesthetized with ketamine by temporary ligation were occluded and in the control group, the ligation was removed after 15 minutes to restore normal blood flow. In laboratory rats, after the IHC suspension was oxygenated externally with an oxygenating instrument, 1ml of high affinity IHC saline suspension was injected directly into the carotid artery on each side. After 24 hours of treatment, mice were sacrificed and their brains removed, fixed, sectioned and stained with nitroblue tetrazolium (NBT) or trypan blue to determine the extent of cell death. The determination by trypan blue staining revealed a lower degree of cell death in experimental mice receiving IHC of the invention.

Example 29

Estimation of half-life of insoluble hemoglobin structure in living circulation

Anesthetized Sprague-Dawley mice (350-. An isotonic suspension injection of IHC equivalent to 20% of the animal's blood volume was administered through a catheter. Blood is collected over a sampling time range of between 0.25 and 92 hours. The blood samples were centrifuged and the plasma was observed for signs of hemolysis or the appearance of hemolyzed hemoglobin. Since the "microbubbles" in IHC have gaseous spaces (and therefore lower density than water), they rise to the surface of the plasma after centrifugation. These microbubbles were filtered off, resuspended in saline, and counted with a particle counter. The IHC half-life in the circulation was determined. The IHC was found to exhibit an increased circulating half-life compared to prior art artificial plasma substitutes derived from hemoglobin.

Example 30

Organ preservation by IHC-preservation of rat Heart

Hearts of anesthetized Sprague-Dawley rats were surgically removed and artificially breathed using room air. Immersing the heart in a medium having the same composition as the IHC (or IHC/FC, or albumin/FC) preservation mediumAnd a crystal matrix without a hemoglobin component ("cardioplegic matrix" — CM). The heart was soaked with CM for a few minutes and cooled to 10 ℃. The heart was then stored at 12 ℃ for 12 hours using 140ml of the IHC preservation medium. IHC matrices were passed continuously through the heart at low pressure (18mmHg) and at 95% O₂/5％CO₂Allowing it to equilibrate continuously. After 12 hours of storage, the heart was examined for contraction, relaxation and functional activity by using an isolated working rat heart test device

Example 31

Cardioplegia in cardiac surgery using IHC matrices

Cardiopulmonary bypass was performed and after appropriate aortic cross-closure and opening, 500 to 100ml of the bolus was delivered to the aortic root by bolus injection at 4 ℃ using an isotonic suspension containing IHC (or IHC/FC, or albumin/FC) as an oxygen carrier. Additional doses of cold substrate are delivered to the left and right coronary ostia, and in the case of surgical bypass, media is also delivered to the ends of the graft prior to the final anastomosis. The matrix is delivered in an amount sufficient to maintain a lower myocardial temperature every 15 to 20 minutes. After the entire procedure is completed, the aortic clamp is removed and the heart begins to return to warmth.

Example 32

Use of IHC matrices in angioplasty or artery angioplasty (Atherectomy)

IHC (or IHC/F, or albumin/FC) media is administered during an interventional procedure to restore perfusion to an occluded or hypoperfused area of an organ. Examples of such procedures are angioplasty or arteriotomy. Administration of oxygenated IHC matrix at a rate of about 60 ml/min through the central lumen of an inflation catheter during inflation in a percutaneous transluminal angioplasty procedure can alleviate ischemic conditions. The matrix is administered at body temperature and contains, for example, physiologically compatible Ringer's electrolyte and substrate. A dose of oxygen-balanced IHC matrix is injected during each inflation cycle. A similar approach may also be used during the inflation user phase of an arterial pull-through procedure, which may be used to physically clear a blockage in a blood vessel in the form of a knife or laser. Direct injection of the matrix into the occluded blood vessel during enzymatic hemolysis can provide oxygen to the occlusion as it dissolves. Currently, Fluosol-DA is used during some angioplasty procedures, and the IHC (or IHC/FC, or albumin/FC) matrix of the present invention can replace Fluosol-DA.

Example 33

Synthesis of Dodecafluorononane (C) encased by a polymeric casing₉F₂₀)

A 20ml glass reaction cell, titanium sounder, cannula and all equipment used had to be washed clean with ethanol and sterile saline before the synthesis reaction proceeded. In this typical reaction, 3.5ml of sterile 5% W/v USP (United states Pharmacopeia standard) human serum albumin (supplied by Alpha healthcare) was added to a reaction chamber connected to an ultrasonic sound generator (Heat Systems, XL2020, 20KHz, 400W max). The sounder was then immersed with the tank in a water bath at a constant temperature of 22 ℃. The reaction is most suitably carried out at 22 ℃ and the product can be synthesized over a broader temperature range (0 ℃ to about 40 ℃). Temperature control is critical for high throughput, and the optimum temperature conditions depend on the particular experimental conditions.

6ml of dodecafluorononane (C) are then added₉F₂₀) And the ultrasonic knob is screwed to the seventh gear power position. The amount of fluorocarbon added can vary from less than 1ml to about 13ml, where the yield of protein polymer shell is good. The reaction was complete in about 30 seconds. Both too short a reaction time and too long a production amount are reduced. The homogeneous suspension produced contained dodecafluorononane, surrounded by a protein polymer shell, with approximately 60% by volume perfluorononane. This aqueous suspension was stored in a sterile container at 4 ℃.

Typical reaction-produced solutions contain approximately 1X 10 per ml⁹A housing. The average shell diameter was 2 microns with a standard deviation of 1 micron. Observe thatThis synthesis produces high concentrations of micron-sized biologics with narrow size distribution.

Example 34

Synthesis of Perfluorotributylamine (C) encased by a Polymer Shell₁₂F₂₇N) or perfluorotripropylamine (C)₉F₂₁N)

Human serum albumin (3.5ml) and a fluoroamine compound (6ml) at 5% w/vUSP were added to a glass reaction vessel and irradiated with high intensity ultrasound under conditions such that the power was set at 7 th, the water bath temperature was 22 ℃ and the reaction time was approximately 30 seconds. Once again, a high concentration of perfluorotripropylamine [ (C) encapsulated by a protein polymer shell is synthesized₃F₇)₃N]And perfluorotributylamine [ (C)₄F₉)₃N](1×10⁹Individual shells/ml) having an average shell diameter of 2 microns.

Example 35

Synthesis of perfluorodecalin (C) encased by a polymeric casing₁₀F₁₈)

Human serum albumin (3.5ml) at 5% w/vUSP and perfluorodecalin (C)₁₀F₁₈(ii) a 6ml) was added to a glass reaction tank and irradiated with high-intensity ultrasonic waves. The reaction conditions were that the power was set at the seventh stage, the water bath temperature was 22 ℃ and the reaction time was approximately 30 seconds. Synthesize perfluorodecalin with high concentration and narrow size distribution range wrapped by protein polymer shell. In addition, as perfluorodecalin and perfluorotripropylamine are both FDA approved fluorocarbons, the major components of Fluosol DA, the use of these compounds for medical imaging is readily recognized by regulatory agencies.

Example 36

Synthesis of perfluoro 15-crown-5 (C) encapsulated by a Polymer sheath₁₀F₂₀O₅)

Human serum albumin (3.5ml) and fluoro crown ether (C) at 5% w/vUSP₁₀F₂₀O₅(ii) a 6ml) was added to a glass reaction tank and irradiated with high intensity ultrasonic waves. The reaction conditions were set at the seventh stage of power, the bath temperature was 22 ℃ and the reaction time was approximately 30 seconds. As before, high concentrations of the fluorcrown ether encapsulated by the protein polymer shell were synthesized with a narrow particle size distribution. This experimental procedure for the synthesis of fluorocarbons encased in a polymeric shell is typical of all fluorocarbon research methods.

Example 37

Synthesis of perfluoro-tert-butylbutene (C) encased by a polymeric Shell₁₀F₁₈H₂)

Human serum albumin (3.5ml) and C at 5% w/vUSP₁₀F₁₈H₂(6ml) was added to a glass reaction vessel and irradiated with high intensity ultrasound, with power set at seventh level, water bath temperature 22 ℃ and reaction time approximately 30 seconds. By this process, a high concentration of perfluoro-tert-butylbutene encapsulated by a protein polymer can be synthesized.

Example 38

Toxicity of fluorocarbon encapsulated by polymeric shell

Five rats were injected over 10 minutes with 5ml of 2% v/v fluorocarbon suspension (perfluorononane coated with a polymer shell of HSA protein) via an extrapolated intravenous catheter. Typical fluorocarbons are not toxic due to their strong fluorine-carbon bonds; indeed, fluorocarbons have been successfully used as FDA approved artificial plasma substitutes (Fluosol DA). Rats were collected at specific times and dissected. In addition to observing the general health of the rats, the liver, spleen, lung and kidney were carefully examined. Rats were healthy with no inflamed tissue or organ at 0.5, 2, 8 and 24 hour examinations. The fifth rat survived 90 days later and was still healthy. As a control, the amount of soybean oil used in this FDA approved rat was half the lethal amount, further confirming that the fluorocarbon compound is non-toxic and safe.

Example 39

Of pure fluorocarbons and fluorocarbons surrounded by a polymeric shell¹⁹F nuclear magnetic resonance spectroscopy

NMR spectra of fluorocarbon encapsulated by protein polymer shell and pure fluorocarbon were obtained on a Bruker500mhz NMR spectrometer. The instrument is adjusted to¹⁹F, its resonant frequency is 470.56 NHz. Deuterium solvent was used as a blocker and all spectra were reported at 0ppm Freon (CCl)₃F) Is an objective reference standard. Reacting perfluorononane with CDCl₃Put into a 5mm NMR tube. The spectra of pure perfluorononane gave two sets of peaks, one at-87 ppm, the second at-127, -128 and-133 ppm.

Perfluorononane suspension encapsulated by a HSA protein Polymer Shell in D₂Resuspend in O and obtain a similar N MR spectrum. Strong signals are obtained from 20% v/v fluorocarbon suspensions with peaks or resonant frequencies at-81, -121, -122 and-126 ppm. The fluorocarbon encapsulated by the polymer shell did not cause a change in the perfluorononane chemistry or structure during ultrasonic irradiation. For example, for C₉F₂₀Two sets of resonance frequencies were observed separately: one set corresponds to about-80 ppm, the second set resonance frequency is about-125 ppm, and CF₂The radicals correspond.

Example 40

Using fluorocarbons¹⁹F nuclear magnetic resonance spectrometry for determining local temperature

In Bruker500MH_zThe variable temperature NMR spectrum of the fluorocarbon was measured on an NMR spectrometer. The instrument is adjusted to¹⁹F, its resonance frequency is 470.50 Hz. With deuterium solvent (d)₆-dimethyl sulfoxide [ d₆-DMSO)]) As a blocking agent, and all spectra were at 0ppm Freon (CCl)₃F) Is an objective reference standard. Perfluorododecane has a melting point of 77 ℃ and is reacted with d₆Together with MDSO, the mixture was placed in a 5mm NMR tube at room temperature. Light with fluorine detected at different temperaturesSpectra were taken and their line widths were measured, and the line width at-81 ppm was expressed as a function of temperature as follows:

line widths of 81ppm (Hz) Temperature (. degree.C.)

51.1 102

57.0 82

64.6 560

As the temperature rises, the broad spectrum at low temperatures begins to sharpen, since perfluorododecane is in the phase transition from solid to liquid. This change changes sharply and rapidly with changes in temperature, as measured for pure substances.

To lower the melting point temperature and widen the melting point temperature range, pentane (about 2% v/v) was added to perfluorododecane. As shown above, in the phase transition stage of perfluorododecane from solid to liquid, the broad peak at low temperature becomes sharp. The line widths of the perfluorododecane/pentane mixtures as a function of temperature are indicated below:

spectral line width (Hz)

-82ppm -123.3ppm Temperature (. degree.C.)

21.26 87.17 77

165.89 280.50 67

216.6 341.2 57

290.77 436.15 47

578.27 451.33 37

577.62 525.11 27

Perfluorododecane/pentane mixtures have the required low melting point in a wide range. With this system, temperature measurements can be made in the range of 27 ℃ to 77 ℃. Thus, the temperature at that time can be determined given only one linewidth value.

For example, using this technique to measure the local temperature of a living body involves injecting a protein shell containing a fluorocarbon mixture (as described above) whose broad melting phase transition has a temperature-line width correlation (which can be obtained experimentally). Such preparations may be used in addition to¹⁹In addition to FMRI as a contrast agent, it acts on the liver and spleen, and therefore can be used to measure locally varying temperatures within organs (to elucidate apparently abnormal pathological conditions within tissues).

EXAMPLE 41

¹⁹F nuclear magnetic resonance model image

Two types of polymer shell-wrapped fluorocarbons were used for this simulated imaging study. Perfluorononane and perfluorotributylamine encapsulated by HSA protein polymer shells were synthesized as described in examples 33 and 34. The resulting suspension containing 60% fluorocarbon per volume was diluted with saline and 2ml was placed in a polystyrene test tube. These polystyrene tubes were placed into a commercially available Siemens 2T MRI instrument (10 cm) operating at 1.5 Tesla¹⁹F coil). Tubes were acquired over a 5 minute period with 10 milliseconds echo Time (TE) and 300 seconds repetition Time (TR) (256X 256 matrix)¹⁹F nuclear magnetic resonance image.

Perfluorononanes coated with a polymeric shell

Dilution of [ concentration of]，M Definition of imaging

11.8 is excellent

1/20.9 is excellent

1/40.45 is preferable

1/100.18 is preferred

1/500.09 is preferred

1/1000.02 is marginal

Good MR simulation imaging was observed even at low concentrations of perfluorononane encapsulated by a polymeric shell. Very close values can also be observed in perfluorotributylamine encapsulated by a polymer shell. Imaging quality and resolution were poor only at high dilutions (1/100; 0.02M).

Example 42

In vitro of the liver and spleen¹⁹F nuclear magnetic resonance imaging

300g rats were injected with 2ml of a 20% v/v perfluorononane suspension surrounded by a polymer coat of HSA protein. Rats were sacrificed 2 hours later and 5 days later, respectively, and the liver, spleen, kidney, and lung were removed. For example, a4 Tesla MRI machine is used to place the entire liver immediately into a 10cm piece of 10¹⁹The F coil is operated. Using T₁Weighted sequence and TR 1 sec, TE 20 ms, data matrix 256 × 128 (i.e., 128 encoding steps, 16 signal averages) obtained for liver, spleen and kidney¹⁹F magnetic resonance images.

Of the liver¹⁹The F MRI images exhibit locally variable intensity related to the variability of the uptake of the polymer sheath by the liver. For example, dark areas corresponding to portal veins can be observed where no polymer-encapsulated perfluorononane is visible, since most of the capsid is concentrated in the RES cells of the liver.

The average imaging intensity of the liver scanned two hours after injection was approximately 20-30% higher than the imaging intensity recorded by the scan five days after injection. This indicates that some perfluorononane may be dispersed by the rupture of the polymer shell. In summary, a high quality imaging showing the morphology of the liver is obtained. Indicating the feasibility of this technique in diagnosing and localizing abnormal pathological changes within the liver.

Example 43

Internal liver and spleen¹⁹F nuclear magnetic resonance imaging

150g rats were injected over 10 minutes with 2ml of 20% v/v perfluorononane (c) encapsulated by a HSA polymer shell₉F₂₀). The rats were then placed in a4 Tesla MRI machine using 10cmThe coil is operated. Rats were anesthetized with ketamine before images were collected. By T₁The weighted sequence sum TR 1 sec, TE 20 ms, and the data matrix 256 × 128 (i.e. 128 encoding steps, 16 signal averages) to obtain the sum for the entire rat and for individual organs such as liver, spleen and kidney¹⁹F magnetic resonance images.

Rats were imaged 15 minutes, 2 hours and 24 hours after injection of perfluorononane encapsulated by a polymer coat of HSA protein, respectively. In summary, high quality imaging showing liver and spleen morphology was obtained, demonstrating the feasibility of this technique in diagnosing and localizing abnormal pathological changes in organs containing the liver RES.

Example 44

By using in vivo¹⁹Local temperature determination by F-NMR imaging

300g of rats were injected over 10 minutes with 5ml of 20% v/v perfluorononane/2% pentane (or perfluorononadecanoic acid and 1% cholesterol) encapsulated by a HSA polymer shell. The rat was then placed in a 15cm coil (Siemens 1.5 Tesla MRI magnetic resonance machine). Imaging was collected with a TE of 10 milliseconds and a TR of 300 seconds (256X 256 matrix). Rats were anesthetized with ketamine prior to data acquisition. The liver and spleen were imaged in 5mm slices over a 15 minute period. The mild rats were wrapped with a heating pad and data was collected at room temperature and about 37 ℃ respectively.

Example 45

By using¹⁹In vivo oxygen determination by F NMR imaging

300g of rats were injected over 10 minutes with 5ml of 20% v/v perfluorononane encapsulated by an HSA polymer shell. The rats were then placed in a 16cm coil (Sienens 1.5 tesla MRI magnetic resonance machine). Imaging was acquired with a TE of 70 milliseconds and a TR of 3 seconds (256X 256 matrix). Rats were placed in a controlled device prior to data acquisition. Rats were first placed in an oxygen chamber to increase oxygen metabolism, and then line widths were collected and imaged. The rats were next injected with ketamine to reduce oxygen consumption and again line widths and images were collected and observed to vary in line width and image intensity, consistent with the dissolved oxygen content of the rats. The maximum line width was measured at higher oxygen concentrations. The liver and spleen were imaged after 15 minutes using a 5mm thick patch. The anesthetized rats were wrapped with a heating pad and two sets of data were collected at room temperature and 37 ℃ respectively.

Example 46

Preparation of paclitaxel particles

Paclitaxel crystals (Sigma Chemical) were milled with a ball mill until solid particles of paclitaxel with a particle size below 10 microns were obtained. The particles were suspended in isotonic saline and their Particle size was determined by counting with the aid of a Particle counter (Elzone, Particle Data). Milling was continued until 100% of the particles had a particle size below 5 microns. The preferred particle size for intravenous infusion is less than 5 microns, most desirably less than 1 micron.

Paclitaxel particles can also be obtained by sonicating a suspension of paclitaxel in water until all particles are below 10 microns in size.

Paclitaxel is precipitated from its ethanol solution by adding water to the paclitaxel ethanol solution until a turbid solution is obtained, and paclitaxel particles below 10 μm can also be obtained, or the paclitaxel solution can be sonicated during the addition of water until a turbid solution is obtained. The final mixture is filtered and dried to obtain pure paclitaxel particles of desired particle size.

High quality paclitaxel particles can be prepared by spray drying a solution of paclitaxel in a volatile organic solvent such as ethanol. The solution was passed through an ultrasonic nozzle to form droplets of paclitaxel containing ethanol. With the evaporation of ethanol in the spray dryer, high quality paclitaxel particles were obtained. The particle size can be varied by varying the concentration of the paclitaxel ethanol solution, adjusting the flow rate of the liquid through the nozzle, and the intensity of the sonication.

Example 47

Synthesis of paramagnetic cation attached to polyanion

For example, by dispersing alginate into gadolinium dichloride (GdCl)₃) In solution, synthesis of gadolinium alginate can be achieved, for example, by reacting gadolinium-containing ions (e.g., GdCl)₃) The solution is irradiated with ultrasonic waves and a small amount of sodium alginate solution is added to synthesize small spherical gadolinium alginate suitable for intracellular injection. Dispersing alginate into gadolinium ion solution, and performing ultrasonic irradiation and crosslinking of multivalent gadolinium ions. Producing micron sized gadolinium alginate granules. In addition to the use of ultrasonic radiation, low or high speed mixing methods can also be applied.

Alternatively, the sodium alginate solution is covered on or covered with an immiscible organic solvent or oil (e.g., soybean oil, sunflower oil, toluene, methylene chloride, chloroform, and the like), the liquid is subjected to ultrasonic irradiation, and thereby the alginate-containing aqueous phase is dispersed into the organic phase, and then a multivalent ion solution (e.g., GdCl) is added₃，Mn-Cl₃，FeCl₃And the like). The sodium alginate is therefore cross-linked, producing extremely small gadolinium alginate spherical particles suitable for use as MRI contrast agents after intravascular injection. Any basic synthetic technique can be used to make spheres, fibers, flat sheets, etc. from alginate and multivalent cations.

The invention has been described with particular reference to certain preferred embodiments thereof, and it is pointed out that any modifications and variations are within the scope of the description and the claims.

Claims (223)

1. A composition suitable for in vivo delivery of a substantially water-insoluble biological product for therapeutic, diagnostic or nutritional purposes, comprising a substantially water-insoluble biological product,

wherein the substantially water-insoluble biological product is a solid or liquid, substantially completely contained within the polymeric shell,

wherein the maximum cross-sectional diameter of the housing is no greater than about 10 microns,

wherein said polymeric shell comprises a biocompatible polymer substantially cross-linked by disulfide bonds.

2. The composition of claim 1, wherein the maximum cross-sectional diameter of the shell is no greater than about 2 microns.

3. The composition according to claim 1, wherein the polymeric shell is modified.

4. The composition according to claim 3, wherein the polymeric shell is modified with an agent selected from the group consisting of synthetic polymers, phospholipids, proteins, polysaccharides, surfactants, chemical modifiers, and combinations thereof.

5. The composition of claim 4, wherein the agent is attached to the polymeric shell.

6. The composition of claim 5, wherein the agent is attached to the polymeric shell by a covalent bond.

7. The composition according to claim 1, wherein said substantially water-insoluble biological product is contained solely within said shell.

8. The composition according to claim 1, wherein the substantially water-insoluble biological product is dispersed in a biocompatible dispersant.

9. The composition according to claim 8, wherein said biocompatible dispersing agent is selected from the group consisting of soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil,

aliphatic, alicyclic or aromatic hydrocarbons having 4 to 30 carbon atoms,

aliphatic or aromatic alcohols having 2 to 30 carbon atoms,

aliphatic or aromatic esters having 2 to 30 carbon atoms,

alkyl, aryl or cyclic ethers having 2 to 30 carbon atoms,

alkyl or aryl halides having 1 to 30 carbon atoms, optionally having more than one halogen substituent,

ketones having 3 to 30 carbon atoms, polyalkylene glycols, and combinations of any two or more thereof.

10. The composition of claim 1, wherein said biocompatible polymer is a naturally occurring polymer, a synthetic polymer, or a combination thereof.

11. The composition of claim 10, wherein said biocompatible polymer is a naturally occurring polymer selected from the group consisting of a protein, a polypeptide, a lipid, a polynucleic acid or a polysaccharide, wherein said naturally occurring polymer contains a sulfhydryl group, a disulfide group or a combination of both groups.

12. The composition according to claim 11, wherein the biocompatible polymer is a polysaccharide selected from the group consisting of alginate, high M-content alginate, polymannuronate, hyaluronic acid, hyaluronate, heparin, dextran, chitosan, chitin, cellulose, starch, glycogen, guar gum, locust bean gum, levan, inulin, cyclodextran, agarose, xanthan gum, carageenan, pectin, gellan gum, scleroglucan and combinations of any two or more thereof.

13. The composition of claim 10, wherein said biocompatible polymer is a synthetic polymer selected from the group consisting of synthetic polyamino acids, synthetic polypeptides, polyvinyl alcohol, polyhydroxyethylmethacrylate, polyacrylic acid, polyvinyloxazoline, polyacrylamide, polyvinylpyrrolidone, and polyalkylene glycol, wherein said synthetic polymer contains a sulfhydryl group, a disulfide group, or a combination of both groups.

14. The composition of claim 10, wherein said substantially water-insoluble biological product is contained solely within said shell.

15. The composition according to claim 10, wherein the substantially water-insoluble biological product is dispersed in a biocompatible dispersant.

16. The composition of claim 1, wherein said biocompatible polymer has a sulfhydryl group or disulfide bond covalently attached thereto.

17. The composition of claim 1, wherein said biocompatible polymer is a protein or oligopeptide.

18. The composition of claim 17, wherein said substantially water-insoluble biological product is contained solely within said shell.

19. The composition according to claim 17, wherein the substantially water-insoluble biological product is dispersed in a biocompatible dispersant.

20. The composition of claim 17, wherein said biocompatible polymer is a protein.

21. The composition of claim 20, wherein said substantially water-insoluble biological product is contained solely within said shell.

22. The composition according to claim 20, wherein the substantially water-insoluble biological product is dispersed in a biocompatible dispersant.

23. The composition of claim 20, wherein the protein is selected from the group consisting of hemoglobin, myoglobin, albumin, insulin, lysozyme, immunoglobulin, alpha-2-macroglobulin, fibronectin, vitronectin, fibrinogen, and combinations of any two or more thereof.

24. The composition of claim 23 wherein said protein is hemoglobin.

25. The composition of claim 23 wherein said protein is a combination of albumin and hemoglobin.

26. The composition of claim 23, wherein said protein is albumin.

27. The composition of claim 26, wherein said substantially water-insoluble biological product is contained solely within said shell.

28. The composition according to claim 26, wherein the substantially water-insoluble biological product is dispersed in a biocompatible dispersant.

29. The composition of claim 26, wherein said protein is human serum albumin.

30. The composition of claim 29, wherein said substantially water-insoluble biological product is contained solely within said shell.

31. The composition according to claim 29, wherein the substantially water-insoluble biological product is dispersed in a biocompatible dispersant.

32. The composition of any one of claims 1 to 31, wherein the polymeric shell containing the substantially water-insoluble biological product is suspended in a biocompatible medium.

33. The composition according to claim 32, wherein the biocompatible medium is selected from the group consisting of water, buffered aqueous media, saline, buffered saline, amino acid solutions, protein solutions, sugar solutions, vitamin solutions, carbohydrate solutions, synthetic polymer solutions, emulsions containing lipids, and combinations of any two or more thereof.

34. The composition according to claim 32, wherein the substantially water-insoluble biological product is a solid.

35. The composition according to claim 32, wherein the substantially water-insoluble biological product is a liquid.

36. The composition of any one of claims 1 to 31, wherein the substantially water-insoluble biologic is a solid.

37. The composition of any one of claims 1 to 31, wherein the substantially water-insoluble biological product is a liquid.

38. The composition of any one of claims 1 to 31, wherein the substantially water-insoluble biologic is a pharmaceutically active agent.

39. The composition of claim 38, wherein the pharmaceutically active agent is a solid.

40. The composition of claim 38, wherein the pharmaceutically active agent is a liquid.

41. The composition according to claim 38, wherein said polymeric shell containing the pharmaceutically active agent is suspended in a biocompatible medium.

42. The composition of claim 38, wherein the pharmaceutically active agent is selected from the group consisting of analgesics, anesthetics, antiasthmatics, antibiotics, antidepressants, antidiabetics, antifungals, antihypertensives, anti-inflammatory agents, antineoplastic agents, anxiolytic agents, enzymatic active agents, nucleic acid constructs, immune enhancers, immunosuppressive agents or vaccines.

43. The composition according to claim 42, wherein said pharmaceutically active agent is a solid.

44. The composition of claim 42, wherein the pharmaceutically active agent is a liquid.

45. The composition of claim 42, wherein the pharmaceutically active agent is an antineoplastic agent.

46. The composition of claim 45, wherein said antineoplastic agent is a solid.

47. The composition of claim 45, wherein said antineoplastic agent is a liquid.

48. The composition of claim 45, wherein the pharmaceutically active agent is an immunosuppressive agent.

49. The composition of claim 48, wherein said immunosuppressive agent is a solid.

50. The composition of claim 48, wherein said immunosuppressive agent is a liquid.

51. The composition of claim 42, wherein the pharmaceutically active agent is an anti-inflammatory agent.

52. The composition according to claim 51, wherein said anti-inflammatory agent is a solid.

53. The composition of claim 51, wherein said anti-inflammatory agent is a liquid.

54. The composition of claim 42, wherein the pharmaceutically active agent is an antibiotic.

55. The composition of claim 54, wherein said antibiotic is a solid.

56. The composition of claim 54, wherein said antibiotic is a liquid.

57. The composition of claim 42, wherein the pharmaceutically active agent is a nucleic acid construct.

58. The composition of claim 38, wherein the pharmaceutically active agent is a taxane.

59. The composition of claim 58 wherein said taxane is paclitaxel or a derivative thereof.

60. The composition of claim 59, wherein said taxane is paclitaxel.

61. The composition according to claim 58, wherein said polymeric shell containing the taxane is suspended in a biocompatible medium.

62. A composition according to claim 60, wherein said composition does not include polyethoxylated castor oil.

63. The composition of claim 58, wherein said taxane is paclitaxel.

64. The composition of any one of claims 1 to 31, wherein the substantially water-insoluble biological is a diagnostic agent.

65. The composition of claim 64, wherein said diagnostic agent is a solid.

66. The composition of claim 64, wherein said diagnostic agent is a liquid.

67. The composition of claim 64, wherein said diagnostic agent is selected from the group consisting of an ultrasound contrast agent, a radiological contrast agent, and a magnetic contrast agent.

68. The composition of claim 67, wherein said diagnostic agent is a fluorine-containing magnetic resonance imaging agent.

69. The composition according to claim 68, wherein the fluorine-containing magnetic resonance imaging agent is selected from the group consisting of:

(a)C_XF_2X+Y-ZA_Zwherein

X＝1-30，

Y is 2; or Y is 0 or-2 when X is not less than 2; or Y is-4 when X is more than or equal to 4,

z is any integer from 0 to (2X + Y-1), and

a is selected from the group consisting of hydrogen, halogen other than fluorine, -CN, -OR, wherein R is hydrogen, alkyl, fluoroalkyl, alkenyl, fluoroalkenyl, alkynyl, fluoroalkynyl, aryl, fluoroaryl, alkanoyl, fluoroalkanoyl, alkenoyl, fluoroalkynyl, alkynoyl, fluoroalkynyl,

(b)[C_XF_2X+Y′-ZA_Z]_aJR_b-awherein:

x, Z, A and R are as defined above,

y' ═ 1; or Y' is-1 or-3 when X is not less than 2; or Y' is-5 when X is greater than or equal to 4,

J-O, S, N, P, Al or Si,

a is 1,2, 3 or 4 and

b is 2 for 2-valent J, or

To J having a valence of 3 is 3, or

For a valence of 4, J is 4,

(c)A′-[(CF₂)_X-O]_c-a ", wherein:

x is as defined above, and X is as defined above,

a' is selected from the group consisting of hydrogen, halogen, -CN, -OR, wherein R is H, alkyl, fluoroalkyl, alkenyl, fluoroalkenyl, alkynyl, fluoroalkynyl, aryl, fluoroaryl, alkanoyl, fluoroalkanoyl, alkenoyl, fluoroalkenoyl, alkynoyl, fluoroalkynyl,

a' is selected from H or R, wherein R is as defined above,

c is 1-300, or

(d)[(CF₂)_X-O]c′

Wherein:

x is as defined above, and

c′＝2-20，

and mixtures of any two or more thereof.

70. The composition of claim 68, wherein said diagnostic agent undergoes a change in relaxation rate as a result of a change in local oxygen concentration.

71. The composition of claim 68, wherein said diagnostic agent undergoes a solid to liquid phase transition at a temperature in the range of about 22 ℃ to 55 ℃.

72. The composition of any one of claims 1 to 31, wherein the substantially water-insoluble biological product is an agent of nutritional value.

73. The composition according to claim 72, wherein the agent of nutritional value is a solid.

74. A composition according to claim 72, wherein the agent of nutritional value is a liquid.

75. The composition according to claim 72, wherein the agent of nutritional value is selected from the group consisting of amino acids, proteins, nucleic acids, sugars, carbohydrates, fat-soluble vitamins, fats, and combinations of any two or more thereof.

76. The composition of any one of claims 1 to 31, wherein the composition is suitable for in vivo delivery by a route selected from intravenous, subcutaneous, intraperitoneal, intrathecal, intramuscular, and intracranial.

77. The composition of any one of claims 1 to 31, wherein the composition is suitable for in vivo delivery by the intravenous route.

78. The composition of any one of claims 1 to 31, wherein the composition is suitable for in vivo delivery by intraperitoneal route.

79. Use of a composition according to any one of claims 1 to 31 in the manufacture of a medicament for the treatment of a disease, wherein the substantially water-insoluble biological agent is a pharmaceutically active agent.

80. Use of a composition according to any one of claims 1 to 31 in the manufacture of a medicament for delivering a substantially water-insoluble biological product to a subject.

81. The use according to claim 80, wherein said polymeric shell containing a substantially water-insoluble biological product is suspended in a biocompatible medium.

82. The use according to claim 81, wherein the biocompatible medium is selected from the group consisting of water, buffered aqueous media, saline, buffered saline, amino acid solutions, protein solutions, sugar solutions, vitamin solutions, carbohydrate solutions, synthetic polymer solutions, emulsions containing lipids and combinations of any two or more thereof.

83. The use according to claim 80, wherein the substantially water-insoluble biological product is a solid.

84. The use according to claim 80, wherein the substantially water-insoluble biological product is a liquid.

85. The use according to claim 80, wherein the substantially water-insoluble biological product is a pharmaceutically active agent.

86. The use according to claim 85, wherein said pharmaceutically active agent is a solid.

87. The use according to claim 85, wherein said pharmaceutically active agent is a liquid.

88. The use according to claim 85, wherein the polymeric shell containing the pharmaceutically active agent is suspended in a biocompatible medium.

89. The use according to claim 85, wherein the pharmaceutically active agent is selected from the group consisting of analgesics, anesthetics, antiasthmatics, antibiotics, antidepressants, antidiabetics, antifungals, antihypertensives, anti-inflammatory agents, antineoplastic agents, anxiolytic agents, enzymatic active agents, nucleic acid constructs, immune enhancers, immunosuppressive agents or vaccines.

90. The use according to claim 89, wherein said pharmaceutically active agent is a solid.

91. The use according to claim 89, wherein said pharmaceutically active agent is a liquid.

92. The use according to claim 89, wherein said pharmaceutically active agent is an antineoplastic agent.

93. The use according to claim 92, wherein the anti-neoplastic agent is a solid.

94. The use according to claim 92, wherein the anti-neoplastic agent is a liquid.

95. The use according to claim 89, wherein said pharmaceutically active agent is an immunosuppressant.

96. The use according to claim 95, wherein said immunosuppressive agent is a solid.

97. The use according to claim 95, wherein said immunosuppressive agent is a liquid.

98. The use according to claim 89, wherein said pharmaceutically active agent is an anti-inflammatory agent.

99. The use according to claim 98, wherein said anti-inflammatory agent is a solid.

100. The use according to claim 98, wherein said anti-inflammatory agent is a liquid.

101. The use according to claim 89, wherein said pharmaceutically active agent is an antibiotic.

102. The use according to claim 101, wherein said antibiotic is a solid.

103. The use according to claim 101, wherein said antibiotic is a liquid.

104. The use according to claim 89, wherein said pharmaceutically active agent is a nucleic acid construct.

105. The use according to claim 85, wherein said pharmaceutically active agent is a taxane.

106. The use according to claim 105, wherein said taxane is paclitaxel or a derivative thereof.

107. The use according to claim 106, wherein said taxane is paclitaxel.

108. The use according to claim 105, wherein said polymeric shell containing a taxane is suspended in a biocompatible medium.

109. The use according to claim 107, wherein said composition does not comprise polyethoxylated castor oil.

110. The use according to claim 105, wherein said taxane is taxol.

111. The use according to claim 80, wherein the substantially water-insoluble biological is a diagnostic agent.

112. The use according to claim 111, wherein said diagnostic agent is a solid.

113. The use according to claim 111, wherein said diagnostic agent is a liquid.

114. The use according to claim 111, wherein said diagnostic agent is selected from the group consisting of ultrasound contrast agents, radiological contrast agents, and magnetic contrast agents.

115. The use according to claim 114, wherein said diagnostic agent is a fluorine-containing magnetic resonance imaging agent.

116. The use according to claim 115, wherein the fluorine-containing magnetic resonance imaging agent is selected from the group consisting of:

(a)C_XF_2X+Y-ZA_Zwherein

X＝1-30，

Y is 2; or Y is 0 or-2 when X is not less than 2; or Y is-4 when X is more than or equal to 4,

z is any integer from 0 to (2X + Y-1), and

a is selected from the group consisting of hydrogen, halogen other than fluorine, -CN, -OR, wherein R is hydrogen, alkyl, fluoroalkyl, alkenyl, fluoroalkenyl, alkynyl, fluoroalkynyl, aryl, fluoroaryl, alkanoyl, fluoroalkanoyl, alkenoyl, fluoroalkynyl, alkynoyl, fluoroalkynyl,

(b)[C_XF_2X+Y′-ZA_Z]_aJR_b-awherein:

x, Z, A and R are as defined above,

y' ═ 1; or Y' is-1 or-3 when X is not less than 2; or Y' is-5 when X is greater than or equal to 4,

J-O, S, N, P, Al or Si,

a is 1,2, 3 or 4 and

b is 2 for 2-valent J, or

To J having a valence of 3 is 3, or

For a valence of 4, J is 4,

(c)A′-[(CF₂)_X-O]_c-a ", wherein:

x is as defined above, and X is as defined above,

a' is selected from the group consisting of hydrogen, halogen, -CN, -OR, wherein R is H, alkyl, fluoroalkyl, alkenyl, fluoroalkenyl, alkynyl, fluoroalkynyl, aryl, fluoroaryl, alkanoyl, fluoroalkanoyl, alkenoyl, fluoroalkenoyl, alkynoyl, fluoroalkynyl,

a' is selected from H or R, wherein R is as defined above,

c is 1-300, or

(d)[(CF₂)_X-O]c′

Wherein:

x is as defined above, and

c′＝2-20，

and mixtures of any two or more thereof.

117. The use according to claim 115, wherein said diagnostic agent undergoes a change in relaxation rate as a result of a change in local oxygen concentration.

118. The use according to claim 115, wherein said diagnostic agent undergoes a solid to liquid phase transition at a temperature in the range of about 22 ℃ to 55 ℃.

119. Use according to claim 80, wherein the substantially water-insoluble biological product is an agent of nutritional value.

120. The use according to claim 119, wherein the agent of nutritional value is a solid.

121. The use according to claim 119, wherein the agent of nutritional value is a liquid.

122. The use according to claim 119, wherein the agent of nutritional value is selected from the group consisting of amino acids, proteins, nucleic acids, sugars, carbohydrates, fat-soluble vitamins, fats, and combinations of any two or more thereof.

123. The use according to claim 80, wherein said medicament is for delivery by a route selected from the group consisting of intravenous, subcutaneous, intraperitoneal, intrathecal, intramuscular, and intracranial.

124. The use according to claim 123, wherein said medicament is for delivery by an intravenous route.

125. The use according to claim 123, wherein said medicament is for delivery by the intraperitoneal route.

126. Use of a composition according to any one of claims 1 to 31 in the manufacture of a medicament for the treatment of cancer, wherein the substantially water-insoluble biological is an anti-tumour agent.

127. The use according to claim 126, wherein said polymeric shell containing substantially water-insoluble biological products is suspended in a biocompatible medium.

128. The use according to claim 127, wherein said biocompatible medium is selected from the group consisting of water, buffered aqueous media, saline, buffered saline, amino acid solutions, protein solutions, sugar solutions, vitamin solutions, carbohydrate solutions, synthetic polymer solutions, lipid-containing emulsions, and combinations of any two or more thereof.

129. The use according to claim 126, wherein the substantially water-insoluble biological product is a solid.

130. The use according to claim 126, wherein the substantially water-insoluble biological product is a liquid.

131. The use according to claim 79, wherein said pharmaceutically active agent is a solid.

132. The use according to claim 79, wherein said pharmaceutically active agent is a liquid.

133. The use according to claim 79, wherein said pharmaceutically active agent is an antineoplastic agent.

134. The use according to claim 133, wherein said anti-neoplastic agent is a solid.

135. The use according to claim 133, wherein said anti-neoplastic agent is a liquid.

136. The use according to claim 79, wherein said pharmaceutically active agent is an immunosuppressant.

137. The use according to claim 136, wherein said immunosuppressive agent is a solid.

138. The use according to claim 136, wherein said immunosuppressive agent is a liquid.

139. The use according to claim 79, wherein said pharmaceutically active agent is an anti-inflammatory agent.

140. The use according to claim 139, wherein said anti-inflammatory agent is a solid.

141. The use according to claim 139, wherein said anti-inflammatory agent is a liquid.

142. The use according to claim 79, wherein said pharmaceutically active agent is an antibiotic.

143. The use according to claim 142, wherein said antibiotic is a solid.

144. The use according to claim 142, wherein said antibiotic is a liquid.

145. The use according to claim 79, wherein said pharmaceutically active agent is a nucleic acid construct.

146. The use according to claim 126, wherein said pharmaceutically active agent is a taxane.

147. The use according to claim 146, wherein said taxane is paclitaxel or a derivative thereof.

148. The use according to claim 147, wherein said taxane is paclitaxel.

149. The use according to claim 146, wherein said polymeric shell containing the taxane is suspended in a biocompatible medium.

150. The use according to claim 148, wherein said composition does not comprise polyethoxylated castor oil.

151. The use according to claim 146, wherein said taxane is taxol.

152. The use according to claim 126, wherein said medicament is adapted for in vivo delivery by a route selected from the group consisting of intravenous, subcutaneous, intraperitoneal, intrathecal, intramuscular, and intracranial.

153. The use according to claim 152, wherein said medicament is adapted for in vivo delivery by the intravenous route.

154. The use according to claim 152, wherein said medicament is adapted for in vivo delivery by the intraperitoneal route.

155. A method of preparing a biological product suitable for in vivo delivery of a substantially water-insoluble biological product for therapeutic, diagnostic or nutritional purposes, the method comprising subjecting a mixture of an aqueous medium comprising a biocompatible polymer capable of being cross-linked by disulphide bonds and the substantially water-insoluble biological product in a dispersing agent to high intensity ultrasound conditions at a temperature of from about 0 ℃ to about 80 ℃,

the high intensity ultrasound conditions cause the biocompatible polymer to be cross-linked by disulfide bonds,

wherein the substantially water-insoluble biological product is a solid or liquid, substantially completely contained within the polymeric shell,

and wherein the maximum cross-sectional diameter of the housing is no more than about 10 microns.

156. A method as in claim 155, wherein the maximum cross-sectional diameter of the shell is no greater than about 2 microns.

157. The method of claim 155, wherein the biocompatible polymer is a naturally occurring polymer, a synthetic polymer, or a combination thereof.

158. The method of claim 157, wherein said biocompatible polymer is a naturally occurring polymer selected from the group consisting of a protein, a polypeptide, a lipid, a polynucleic acid or a polysaccharide, wherein said naturally occurring polymer contains a sulfhydryl group, a disulfide group or a combination of both groups.

159. The method of claim 157, wherein the biocompatible polymer is a polysaccharide selected from the group consisting of alginate, high M-content alginate, polymannuronic acid, polymannuronate, hyaluronic acid, hyaluronate, heparin, dextran, chitosan, chitin, cellulose, starch, glycogen, guar gum, locust bean gum, levan, inulin, cyclodextran, agarose, xanthan gum, carageenan, pectin, gellan gum, scleroglucan and a combination of any two or more thereof.

160. The method of claim 157, wherein said biocompatible polymer is a synthetic polymer selected from the group consisting of synthetic polyamino acids, synthetic polypeptides, polyvinyl alcohol, polyhydroxyethylmethacrylate, polyacrylic acid, polyvinyloxazoline, polyacrylamide, polyvinylpyrrolidone, polyalkylene glycol, wherein said synthetic polymer contains a sulfhydryl group, a disulfide group, or a combination of both groups.

161. The method of claim 155, wherein the biocompatible polymer is a protein or oligopeptide.

162. The method of claim 161, wherein the biocompatible polymer is a protein.

163. The method of claim 162, wherein said protein is selected from the group consisting of hemoglobin, myoglobin, albumin, insulin, lysozyme, immunoglobulin, alpha-2-macroglobulin, fibronectin, vitronectin, fibrinogen and combinations of any two or more thereof.

164. The method of claim 163, wherein the protein is hemoglobin.

165. The method of claim 163, wherein the protein is a combination of albumin and hemoglobin.

166. The method of claim 163, wherein the protein is albumin.

167. The method of claim 166, wherein said protein is human serum albumin.

168. The method of any one of claims 155 to 167 wherein the substantially water-insoluble biological product is a solid.

169. The method of any one of claims 155 to 167 wherein the substantially water-insoluble biological product is a liquid.

170. The method of any one of claims 155 to 167, wherein the substantially water-insoluble biological product is a pharmaceutically active agent.

171. A method in accordance with claim 170, wherein the pharmaceutically active agent is a solid.

172. The method of claim 170, wherein the pharmaceutically active agent is a liquid.

173. The method of claim 170, wherein the pharmaceutically active agent is selected from the group consisting of an analgesic, an anesthetic, an antiasthmatic, an antibiotic, an antidepressant, an antidiabetic, an antifungal, an antihypertensive, an anti-inflammatory, an antineoplastic, an anxiolytic, an enzymatic active agent, a nucleic acid construct, an immune enhancer, an immunosuppressive agent, and a vaccine.

174. The method of claim 173, wherein said pharmaceutically active agent is a solid.

175. The method of claim 173, wherein the pharmaceutically active agent is a liquid.

176. The method of claim 173, wherein the pharmaceutically active agent is an antineoplastic agent.

177. The method of claim 176, wherein said anti-neoplastic agent is a solid.

178. The method of claim 176, wherein said antineoplastic agent is a liquid.

179. The method of claim 173, wherein the pharmaceutically active agent is an immunosuppressive agent.

180. The method of claim 179, wherein the immunosuppressive agent is a solid.

181. The method of claim 179, wherein the immunosuppressive agent is a liquid.

182. The method of claim 173, wherein said pharmaceutically active agent is an anti-inflammatory agent.

183. The method of claim 182, wherein said anti-inflammatory agent is a solid.

184. The method of claim 182, wherein said anti-inflammatory agent is a liquid.

185. The method of claim 173, wherein the pharmaceutically active agent is an antibiotic.

186. The method of claim 185, wherein the antibiotic is a solid.

187. The method of claim 185, wherein the antibiotic is a liquid.

188. The method of claim 173, wherein said pharmaceutically active agent is a nucleic acid construct.

189. The method of claim 170, wherein the pharmaceutically active agent is a taxane.

190. The method of claim 189, wherein the taxane is paclitaxel or a derivative thereof.

191. The method of claim 190, wherein the taxane is paclitaxel.

192. The method of claim 189, wherein the taxane is paclitaxel.

193. The method of any one of claims 155 to 167, wherein the substantially water-insoluble biological is a diagnostic reagent.

194. The method of claim 193, wherein said diagnostic agent is a solid.

195. The method of claim 193, wherein said diagnostic agent is a liquid.

196. The method of claim 193, wherein said diagnostic agent is selected from the group consisting of an ultrasound contrast agent, a radiation contrast agent, and a magnetic contrast agent.

197. The method of claim 193, wherein said diagnostic agent is a fluorine-containing magnetic resonance imaging agent.

198. The method of claim 197, wherein the fluorine-containing magnetic resonance imaging agent is selected from the group consisting of:

(a)C_XF_2X+Y-ZA_Zwherein

X＝1-30，

Y is 2; or Y is 0 or-2 when X is not less than 2; or Y is-4 when X is more than or equal to 4,

z is any integer from 0 to (2X + Y-1), and

a is selected from the group consisting of hydrogen, halogen other than fluorine, -CN, -OR, wherein R is hydrogen, alkyl, fluoroalkyl, alkenyl, fluoroalkenyl, alkynyl, fluoroalkynyl, aryl, fluoroaryl, alkanoyl, fluoroalkanoyl, alkenoyl, fluoroalkynyl, alkynoyl, fluoroalkynyl,

(b)[C_XF_2X+Y′-ZA_Z]_aJR_b-awherein:

x, Z, A and R are as defined above,

y' ═ 1; or Y' is-1 or-3 when X is not less than 2; or Y' is-5 when X is greater than or equal to 4,

J-O, S, N, P, Al or Si,

a is 1,2, 3 or 4 and

b is 2 for 2-valent J, or

To J having a valence of 3 is 3, or

For a valence of 4, J is 4,

(c)A′-[(CF₂)_X-O]_c-a ", wherein:

x is as defined above, and X is as defined above,

a' is selected from the group consisting of hydrogen, halogen, -CN, -OR, wherein R is H, alkyl, fluoroalkyl, alkenyl, fluoroalkenyl, alkynyl, fluoroalkynyl, aryl, fluoroaryl, alkanoyl, fluoroalkanoyl, alkenoyl, fluoroalkenoyl, alkynoyl, fluoroalkynyl,

a' is selected from H or R, wherein R is as defined above,

c is 1-300, or

(d)[(CF₂)_X-O]c′

Wherein:

x is as defined above, and

c′＝2-20，

and mixtures of any two or more thereof.

199. The method of any one of claims 155 to 167 wherein the substantially water-insoluble biological product is an agent of nutritional value.

200. The method of claim 199, wherein the nutritionally valuable agent is a solid.

201. The method of claim 199, wherein the nutritionally valuable agent is a liquid.

202. The method of claim 199, wherein the agent of nutritional value is selected from the group consisting of amino acids, proteins, nucleic acids, sugars, carbohydrates, fat soluble vitamins, fats, and combinations of any two or more thereof.

203. A composition comprising a substantially water-insoluble bioproduct, wherein the substantially water-insoluble bioproduct is a solid or a liquid that is substantially completely contained within a polymeric shell, wherein the substantially water-insoluble bioproduct within the shell is dispersed in a dispersant comprising an alkyl halide or an aryl halide having 1-30 carbon atoms, wherein the shell has a maximum cross-sectional diameter of no more than about 10 microns, wherein the polymeric shell comprises a biocompatible polymer that is substantially cross-linked by means of disulfide bonds.

204. The composition of claim 203, wherein said biocompatible polymer is a protein or oligopeptide.

205. The composition of claim 204, wherein said biocompatible polymer is a protein.

206. The composition of claim 205, wherein said protein is albumin.

207. The composition of claim 206, wherein said protein is human serum albumin.

208. A composition according to claim 203, wherein said dispersing agent comprises CH₃Cl。

209. The composition of claim 208, wherein said biocompatible polymer is a protein or oligopeptide.

210. A composition according to claim 209, wherein said biocompatible polymer is a protein.

211. The composition of claim 210, wherein said protein is albumin.

212. The composition of claim 211, wherein said protein is human serum albumin.

213. The composition of any one of claims 203 to 212, wherein the substantially water-insoluble biologic is a pharmaceutically active agent.

214. The composition of claim 213, wherein the pharmaceutically active agent is an anti-neoplastic agent.

215. The composition of claim 213, wherein the pharmaceutically active agent is an immunosuppressive agent.

216. The composition of claim 213, wherein the pharmaceutically active agent is an antibiotic.

217. The composition of claim 213, wherein the pharmaceutically active agent is a taxane.

218. The composition of claim 217, wherein the taxane is paclitaxel or a derivative thereof.

219. The composition of claim 218, wherein said taxane is paclitaxel.

220. The composition of claim 217, wherein said taxane is paclitaxel.

221. The composition of any one of claims 203 to 212, wherein the substantially water-insoluble biological is a diagnostic agent.

222. A composition according to claim 221, wherein said diagnostic agent is a fluorine-containing magnetic resonance imaging agent.

223. The composition of any one of claims 203 to 212, wherein the substantially water-insoluble biological product is an agent of nutritional value.