HK1088242B - Ceramic based nanoparticles for entrapping therapeutic agents for photodynamic therapy and method of using same - Google Patents

Description

Ceramic nanoparticles for encapsulating therapeutic agents for photodynamic therapy and methods of use thereof

This application claims priority from U.S. provisional patent application No. 60/442,237, filed 24/1/2003, the contents of which are incorporated herein by reference.

Technical Field

The present invention relates generally to the field of photodynamic therapy, and more particularly to a novel drug carrier system for photodynamic therapy.

Background

Photodynamic therapy (PDT) is a new therapeutic approach for the treatment of various neoplastic, cardiovascular, dermatological and ophthalmological diseases [1, 2]. Photodynamic therapy of cancer relies on the concept that photosensitizing substances or agents (PS) can be preferentially localized to tumor tissue by systemic administration [2, 3]. When visible at a suitable wavelengthWhen light or near infrared light (NIR) is irradiated on the photosensitizer, the activated molecules conduct their energy to the surrounding oxygen molecules, resulting in the production of Reactive Oxygen Species (ROS), such as singlet oxygen ((R))¹O₂) Or the generation of free radicals. The active oxygen species can oxidize various cell membranes including plasma membrane, mitochondrial membrane, lysosomal membrane, nuclear membrane, etc., resulting in irreversible damage to cells [2-6]. Under appropriate conditions, photodynamic therapy has the advantage of efficiently and selectively destroying diseased tissue without damaging surrounding healthy tissue [3]。

However, most photosensitive drugs (PS) are hydrophobic, e.g. poorly water soluble, thus preventing their formulation as parenteral injectable pharmaceutical formulations [2-7 ]. To overcome this difficulty, various strategies have been developed, usually with the aid of delivery vehicles, to facilitate stable dispersion of these drugs in aqueous systems. When administered systemically, the drug-incorporated vehicle is taken up more preferentially by tumor tissue due to the 'Enhanced Permeability and Retention (EPR)' [2, 8-10 ]. Vehicles include oily dispersions (micelles), liposomes, polymeric micelles, hydrophilic drug-polymer compositions, and the like. Oily substance formulations (micellar systems) using non-ionic polyoxyethylated castor oils (e.g., tween-80, Cremophor-EL, CRM, etc.) show increased drug-carrying capacity and tumor uptake, presumably due to interaction with cytoplasmic lipoproteins in the blood [11-13 ]. However, such emulsifiers have also been reported to cause acute hypersensitivity reactions in vivo [14, 15 ]. Liposomes are concentric phospholipid bilayers encapsulating aqueous compartments and can contain both hydrophilic and lipophilic drugs [2 ]. Although uptake of drugs by tumors is superior to simple aqueous dispersions after encapsulation by liposomes, the drug loading of liposomes is low and self-polymerization of drugs in encapsulated state is increased [2, 16-18 ]. Liposomes are also easily opsonized and then captured by the body's major defense system (reticuloendothelial system, RES) [18 ]. Recently, drugs incorporated inside pH-sensitive polymeric micelles in vitro have shown enhanced tumor phototoxicity compared to CRM formulations, however in vivo studies have shown that tumor regression is less and accumulation in normal tissues is increased [7, 19, 20 ].

Hydrated ceramic-based nanoparticles incorporating photosensitizing drugs provide bright prospects for solving problems associated with free or polymer-packaged drugs. Such ceramic particles have many advantages over organic polymer particles. First, the preparation process is very similar to the well-known sol preparation process, requiring only simple and room temperature conditions [21, 22 ]. These microparticles can be made to the desired size, shape and porosity and are extremely stable [22 ]. Their extremely small size (below 50 nm) can help them escape the capture of the reticuloendothelial system (RES). And, they do not swell or change their porosity as the pH changes; at the same time, these particles are less susceptible to microbial attack [23 ]. These microparticles also effectively protect the molecules (enzymes, drugs, etc.) incorporated therein against denaturation caused by extremes of pH and temperature [24 ]. These particles are also known as: silica, alumina, titania, etc. are compatible with biological systems [24, 25, 26 ]. Furthermore, their surfaces are easily functionalized with different groups [26, 27], so they can be attached to various monoclonal antibodies and other ligands to enable them to be targeted to desired sites in the body.

Although the synthesis of ceramic nanoparticles, mostly but not exclusively based on silica, has been widely reported in the literature [28-30], their use in drug delivery has not yet been fully exploited. Therefore, there is still a need to develop new drug delivery systems for photodynamic therapy.

Summary of The Invention

The present invention provides compositions and methods for photodynamic therapy. The compositions of the present invention comprise ceramic nanoparticles encapsulated (enterrapped) with one or more therapeutic agents.

The invention also provides methods of synthesizing ceramic nanoparticles coated with one or more therapeutic agents. These microparticles, when synthesized by the process of the present invention, were found to be spherical and to have a highly uniform dispersion.

In one embodiment, the present invention provides a method for synthesizing photosensitizer dye/drug doped silica nanoparticles (30 nm diameter) by controlled alkaline hydrolysis of ceramic materials (e.g., triethoxyvinylsilane (VTES) in micellar media.

In one example, the photosensitive dye/drug 2-devinyl-2- (1-hexyloxyethyl) pyropheophorbide (HPPH), a potent photosensitizer, has been introduced in phase I/II clinical trials at Roswell park cancer institute (Buffalo, N.Y.; U.S.; 32, 33). Although the microparticle matrix does not interfere with the visible absorption spectrum of the encapsulated photosensitizing drug, the quenching of fluorescence of the drug is largely suppressed in aqueous media. Singlet oxygen detection experiments revealed that the encapsulated photosensitizer is able to interact with surrounding molecular oxygen and that the generated singlet oxygen is able to pass out of the silica matrix. Such drug-incorporated microparticles can be actively taken up by tumor cells and then irradiated with light of a suitable wavelength to cause irreversible destruction of those cells which take up such microparticles. Therefore, the ceramic particles of the present invention can be used as a carrier for photodynamic therapy drugs.

Brief description of the drawings

FIG. 1 is a transmission electron micrograph of HPPH doped silica nanoparticles showing highly uniformly dispersed particles with an average diameter of 30 nm.

FIG. 2 is a schematic representation of the synthesis and purification of HPPH doped silica nanoparticles in micellar medium.

FIG. 3 is a UV-VIS absorption spectrum of (a) HPPH dissolved in AOT/BuOH/Water micelles, (b) HPPH doped in silica nanoparticles, and (c) empty silica nanoparticles.

FIG. 4 is a graph of the emission spectra of (a) HPPH dissolved in AOT/BuOH/Water micelles before dialysis, (b) HPPH doped in silica nanoparticles before dialysis, (c) HPPH dissolved in AOT/BuOH/Water micelles after dialysis, and (d) HPPH doped in silica nanoparticles after dialysis, with excitation wavelength 414 nm.

FIG. 5 shows the results of (a) dissolution in AOT/BuOH/D under 1270nm wavelength irradiation₂HPPH in O micelles (solid line), (b) dispersed in D₂HPPH doped in silica nanoparticles in O and (c) dispersed in D₂Singlet oxygen phosphorescence emission spectra produced by the hollow silica nanoparticles in O.

FIG. 6 shows the results of (a) dissolution in AOT/BuOH/D upon irradiation with light₂HPPH in O micelles, (b) Dispersion in D₂HPPH doped in silica nanoparticles in O and (c) dispersed in D₂The time-dependent bleaching of the singlet oxygen generated by the hollow silica nanoparticles in O removes the graphic representation of the dye ADPA (maximum wavelength 400 nm).

FIG. 7 is a two-photon confocal fluorescence image of single tumor cells (KB) treated with HPPH-doped nanoparticles, and the inset is a cytoplasmic local fluorescence spectrum of the treated cells.

FIG. 8 is a graph showing the survival rate of UCI-107 cells measured by MTT method

Detailed Description

The present invention provides ceramic nanoparticles coated with one or more therapeutic agents. The invention also provides methods of synthesizing and using ceramic microparticles loaded with one or more therapeutic agents. The term "nanoparticle" is used herein to refer to particles having a diameter of 100nm or less.

The ceramic nanoparticles of the present invention are useful for photodynamic therapy to deliver therapeutic agents, such as light sensitive drugs or dyes, to cells. For example, these drugs may be hydrophobic and hydrophilic.

In one embodiment, the silica particles used in the present invention are organically modified silicate (ORMOSIL) nanoparticles loaded with a hydrophobic photosensitive (also referred to herein as photosensitizer) drug. To prepare the nanoparticles, the photosensitizer is coated onto the nonpolar core of sodium bis (2-ethylhexyl) sulfosuccinate (AOT)/1-butanol/water micelles dissolved in an alkoxy-organosilicon (e.g., triethoxyvinylsilane), and then precipitated by basic hydrolysis (e.g., stirring at room temperature for 24 hours) under mild conditions (e.g., with ammonia, ammonium compounds, or amine-containing compounds). Dialysis of the precipitated material to remove the surfactant can extend the dialysis time (e.g., 50 hours), indicating a stable dispersion of the encapsulated material over an extended period of time.

The main advantages of ORMOSIL nanoparticles over other nanoparticles are: under appropriate conditions, the hydrophobic and hydrophilic groups present in the precursor alkoxy-silicones help them self-assemble into forward and reverse micelles. The resulting micellar (and reverse micellar) cores can be used to encapsulate biomolecules such as: drugs, proteins, etc. Such a system has many advantages, including: (a) they can be loaded with hydrophobic and hydrophilic dyes, (b) they can be precipitated in oil-in-water microemulsions, thus avoiding the use of aggressive solvents such as cyclohexane, and complicated purification steps such as solvent evaporation, ultracentrifugation, etc. (c) Their organic groups may be further modified to allow attachment to a target molecule. (d) They can be biodegraded by biochemical decomposition of silicon-carbon bonds. The presence of organic groups also reduces the overall rigidity and density of the particles, which is expected to increase the stability of the particles in aqueous systems without settling.

The spherical, uniformly dispersed silica gel microparticles can be conveniently prepared by hydrolysis of tetraalkylsilane. This method, commonly referred to as the "sol-gel" method, can be further extended to the synthesis of organically modified silica (ORMOSIL) microparticles, in which the precursor alkoxysilane molecule also contains one or two organic groups. The incorporation of organic groups modifies the final structure of the silica network, for example: resulting in the formation of a porous matrix characterized by the formation of an ordered and uniform porous network structure. These porous matrices can accommodate a variety of optically and biologically active molecules, such as fluorescent dyes, proteins, anti-cancer drugs, imaging contrast agents, and the like.

With respect to the use of the ceramic microparticles of the present invention, the drug-loaded microparticles may be administered locally or systemically. Local administration can be effected, for example, by injecting a mixture containing drug/dye loaded microparticles near the target tissue or within the tumor. In the topical treatment of superficial tumors, the nanoparticles may be incorporated into standard topical compositions, including lotions, suspensions or ointments for topical application. Systemic administration can be by intravenous, subcutaneous, intramuscular, intraperitoneal or rectal routes. Dosage forms for these methods of administration are well known in the art; exemplary dosage forms are described, for example, in Ramington's pharmaceutical sciences (Easton, Pa.; Mack publishing Co.).

The drug/dye encapsulated nanoparticles may be administered in a composition of suitable formulation suitable for the intended use; such as: oral, parenteral or topical administration. Suitable dosage forms for oral administration are suspensions, dispersions, emulsions in the form of tablets, dispersible powders, granules, capsules, syrups and elixirs. Inert diluents and carriers for tablets include, for example: calcium carbonate, sodium carbonate, lactose and talc. Tablets may also contain granulating and disintegrating agents, such as starch and alginic acid; binding agents, such as starch, gelatin and acacia, and lubricating agents, such as magnesium stearate, stearic acid and talc. Tablets may also be uncoated or coated by known techniques to delay disintegration and absorption. Inert diluents and carriers that can be used in the capsule include, for example, calcium carbonate, calcium phosphate and china clay. Suspensions, syrups and elixirs may contain conventional excipients, for example methylcellulose, tragacanth, sodium alginate; wetting agents, such as lecithin and polyoxyethylene stearate; and preservatives, such as p-ethyl-hydroxy benzoate. Formulations suitable for parenteral administration include ceramic nanoparticle suspensions, dispersions, emulsions, and the like.

Typically, ceramic microparticles are highly stable and do not release any encapsulated biomolecules even under extreme pH and temperature conditions [24 ]. Conventional drug delivery methods require a carrier to release the encapsulated drug to produce the appropriate biological response [3 ]. However, this is not required when using macromolecular carriers for delivery of light sensitive drugs in photodynamic therapy [3, 31 ]. In the present invention, we have developed ceramic nanoparticles as carriers for photosensitive drugs used in photodynamic therapy. The porous matrix of the nanoparticles is permeable to oxygen molecules and reactive oxygen species (singlet oxygen and free radicals), so the nanoparticles do not have to release any significant amount of the encapsulated drug into the aqueous environment. Thus, the desired photo-destructive effect can be maintained even in the wrapped form.

By adopting the method, the silicon dioxide nanoparticles coated with the water-insoluble photosensitive anticancer drug, such as HPPH, can be synthesized in the micelle medium. Particles of 30nm monomodal size distribution coated with drug can be made stable aqueous dispersions. Although the drug is encapsulated in the particulate matrix, it can be activated by irradiation with light of a suitable wavelength, and the singlet oxygen produced can diffuse out of the particulate matrix. The drug-encapsulated nanoparticles are actively taken up by tumor cells, and irradiation of the taken-up cells with light can result in significant cell death. These observations demonstrate that various ceramic matrices can be used as drug carriers for photodynamic therapy. The microparticles of the present invention can be used as an injectable formulation for safe and effective delivery to tumor tissue in vivo.

In one embodiment, a ligand that specifically targets a tumor can be attached to the surface of the ceramic nanoparticle. Recently, our group has synthesized silica nanoparticles that encapsulate a magnetic core [39 ]. These microparticles are functionalized with a polypeptide hormone targeting agent, luteinizing hormone releasing hormone (LH-RH). The results show that the generated nanoclinic can selectively target tumor cells containing LH-RH receptors. The tumor cells are then exposed to a DC magnetic field that selectively causes magnetic cytolysis of only the receptor-positive cells (U.S. Pat. No. 6,514,481, incorporated herein by reference). Thus, the ceramic surface of the nanoparticles of the invention can be functionalized with different ligands to enable the targeting of the particles to tumor cells containing receptors specific for these ligands.

The invention is further described by the examples set forth below, which are intended to be illustrative of the invention and are not intended to be limiting in any way.

Example 1

This example describes the preparation of drug-loaded silica nanoparticles. To illustrate this example, silica particles loaded with HPPH were synthesized. Surfactant aerosol OT (AOT, 98%), co-surfactant n-butanol complex (99.8%) and triethoxyvinylsilane (VTES, 97%) were purchased from Aldrich. MTT and isopropanol are products from Sigma. Deuterium oxide (99.9 atomic percent) was obtained from Isotec corporation, usa. The dye/drug HPPH 2-devinyl-2- (1-hexyloxyethyl) pyropheophorbide-acid [38] was gifted by the Tom Dougherty Bausch, Roswell park cancer institute, Buffalo, N.Y.. Dye disodium dipropylanthracene acid salt (ADPA) was purchased from Molecular Probes, USA. Cell culture products, such as MEM-alpha medium, 5% Fetal Bovine Serum (FBS), Phosphate Buffered Saline (PBS), etc., were purchased from GIBCO, USA. All of the above chemicals were used without further purification.

Both empty and drug-loaded nanoparticles were synthesized in the non-polar center of the AOT/n-butanol/water micelle, and the synthesis scheme is shown in fig. 2. 0.44 g of AOT and 800. mu.l (0.56 g) of n-butanol were dissolved in 20 ml of double distilled water and stirred vigorously and magnetically into micelles. To the resulting clear solution, 40. mu.l of DMF (15mM) with HPPH dissolved and 10. mu.l of pure DMF were added, dissolved by magnetic stirring (when empty microparticles were prepared, 50. mu.l of DMF was added without HPPH added), and then 200. mu.l of pure triethoxyvinylsilane (VTES) was added to the micelle system, and mixed and stirred for about one hour, or stirred until the solution was clear. Then, 10. mu.L of an aqueous ammonia (10M) solution was added to the sub-system, and stirred for about 20 hours. The whole reaction process is carried out at room temperature. At the end of this step, the production of a blue-white transparent liquid was observed, indicating that nanoparticles had formed. After the formation of the drug-incorporated nanoparticles, the surfactant AOT and co-surfactant n-butanol were completely removed by dialysis with water for 40 hours through a cellulose membrane with a cut-off of 12-14kD (available from Spectrum Laboratories, Inc., USA). The dialysis solution was then filtered through a 0.2 micron filter (Nalgene, usa) for subsequent experiments.

The morphology and size of the resulting aqueous dispersion of nanoparticles was determined by Transmission Electron Microscopy (TEM) using a JEOL JEM 2020 Electron microscope operating at an acceleration voltage of 200 kV. The results are shown in FIG. 1 as a Transmission Electron Micrograph (TEM) of drug-loaded nanoparticles. The particles were spherical, with a monomodal size distribution and an average size of 30nm (product prepared using the above protocol).

Example 2

This example demonstrates that the emission spectrum of the encapsulated drug is characterized by the same emission spectrum as the non-encapsulated drug. To illustrate this example, the UV-visible absorption spectra of both drug-coated silica particles and free drug were recorded using a Shimadzu UV-3101 computer spectrophotometer using a 1 cm thick quartz glass; fluorescence spectra were recorded using a Shimadzu RF 5000U spectrofluorometer using a 1 cm thick quartz glass. The uv-vis absorption spectrum of HPPH in AOT/BuOH/water micelles as encapsulated in nanoparticles showed the same peak position (fig. 3). This demonstrates that encapsulation of HPPH within nanoparticles does not cause a change in peak position. Control experiments with empty nanoparticles showed no substantial absorption in the visible and near infrared wavelength regions, which are the regions of interest (treatment regions) in photodynamic therapy because of the high tissue transmittance of light. Thus, such microparticles are useful for photodynamic therapy because they do not interfere with the therapeutic light used to excite the light sensitive drug.

FIG. 4 represents the fluorescence emission spectra of HPPH in AOT/BuOH/Water micelles and encapsulated in nanoparticles. Spectra were recorded at the excitation wavelength of 414nm for both samples before and after double distilled water dialysis (effective removal of surfactant and co-surfactant molecules used). Although the emission spectra of HPPH in micelles or in nanoparticles are almost identical before dialysis, a substantial difference between the two is observed after dialysis. Although the emission intensity of the nanoparticles loaded with HPPH was still significant after dialysis (approximately 75% of the intensity before dialysis), in practice, no emission of HPPH/micelles could be detected after dialysis. This can be explained by the fact that HPPH, as a non-polar molecule, can undergo aggregation in polar solvents, thus causing its fluorescence to self-quench. As long as the HPPH molecules are in the non-polar core of the micelle, they can remain separated from each other, but as the surfactant is removed, the molecules gradually come into contact with the surrounding water, thus starting to aggregate. However, in the case of nanoparticles incorporating HPPH, it can be seen that the drug molecules are dispersedly embedded in the particle matrix (see FIG. 1), and therefore do not cause aggregation and significant loss of emitted light intensity when contacting aqueous systems. This property of encapsulating drugs/dyes against self-quenching in aqueous media can be exploited to make microprojections for imaging biological systems.

Example 3

This example demonstrates that the encapsulated drug can generate singlet oxygen by phosphorescence spectroscopy. Singlet oxygen has been widely reported¹O₂) Through which the phosphorescence emission spectrum at 1270nm [34, 35 ]]And (4) detecting. Because of the fact that¹O₂The life cycle in water is very short (2-5 microseconds) and detection by the above method is very difficult. We employ deuterium oxide (D)₂O) because of¹O₂Has a longer life cycle (50-60 microseconds) [36 ] in this solvent]. In a typical experiment, 3 ml of 22.5. mu.M HPPH was coated in dispersion D₂Nanoparticles in O. Respectively adopting the solution in AOT/BuOH/D₂HPPH in O micelles and dispersion in D₂Empty nanoparticles in O served as positive and negative controls. The singlet oxygen phosphorescence emission spectrum was recorded with a SPEX 270M spectrophotometer (Jobin Yvon) equipped with an In-Ga-As Optical probe (Electro-Optical systems, USA). A solid-state crystal diode-driven laser (Verdi, Coherent) was used as the excitation light source (532 nm). A1 cm thick square quartz cup containing the sample solution was placed directly in front of the incident light slit of the spectrophotometer and the emission signal from the side of the quartz cup at 90 degrees to the excitation laser beam was collected. In the light ofAnother NIR long wave edge filter (Andover, usa) was also placed before the probe.

The results demonstrate that because ceramic matrices are generally porous, photosensitizing drugs encapsulated therein can interact with oxygen molecules diffusing through these pores. Any Reactive Oxygen Species (ROS) generated by the interaction of oxygen molecules with the excited photosensitizer will diffuse out of the porous matrix and into the surrounding environment where we have studied the generation of singlet oxygen, which is the ROS reactive oxygen species generated by HPPH, by the generated 1270nm phosphorescence spectrum. FIG. 5 shows the spectra of HPPH solubilized in micelles and entrapped in nanoparticles. Both spectra show a peak at 1270nm, indicating that singlet oxygen is produced in both cases. The control spectrum obtained with empty nanoparticles did not show this phosphorescence peak. This demonstrates that photosensitized HPPH in the encapsulated state does produce singlet oxygen that can diffuse out through the pores of the ceramic matrix, interacting with the surrounding environment.

Example 4

This example demonstrates the detection of singlet oxygen generated by the encapsulated drug by chemical means using disodium dipropylanthracene acid (ADPA). In addition to phosphorescence spectroscopy, singlet oxygen production can also be detected chemically using the disodium salt of ADPA (9, 10-dipropylanthracene acid) as the singlet oxygen sensor [36 ]]. The disodium salt of ADPA (a water-soluble anthracene derivative) is bleached to its corresponding endoperoxide upon reaction with singlet oxygen, and the optical density drop at 400nm (λ max of ADPA) is then recorded spectrophotometrically. In a typical experiment, 150. mu.l of a D2O solution of disodium salt of ADPA (5.5mM) was mixed with 3 ml of 15. mu.M HPPH (dissolved in AOT/D)₂O micelle, and D dispersed in the nano particle₂O) and filled in a 1 cm thick quartz glass cup. ADPA disodium salt for control experiment and empty nanoparticles are mixed and dispersed in D₂In O. The resulting solutions were irradiated with a 650 nm laser source (solid state crystal diode driven laser) and their optical densities were recorded by a spectrophotometer every 10 minutes.

Figure 6 shows the time-dependent bleaching of ADPA, which was obtained by observing the decrease in Optical Density (OD) at 400nm (maximum absorption of ADPA) after incubation with different samples and light irradiation. The curves for HPPH dissolved in micelles and incorporated in nanoparticles show a rapid decrease in OD with increasing illumination time, indicating that a large amount of singlet oxygen is generated in both cases. The curve for the empty nanoparticles incubated with ADPA did not show any decrease in OD with time, indicating that the bleaching of ADPA was due to singlet oxygen production rather than light.

Example 5

This example demonstrates that silica nanoparticles coated with photosensitizer drug/dye can be taken up by cells. To study the uptake of nanoparticles, the KB cell line (human oral epithelial cancer cells) was used. Cells were cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS) at 37 deg.C (containing 5% CO)₂Moist environment). In a 60X 15mm tissue culture plate, 3 ml of culture medium cultured monolayer cells were incubated for 1 hour with 100. mu.l of an aqueous dispersion of HPPH (15. mu.M) doped nanoparticles. After washing the cells with sterile Phosphate Buffered Saline (PBS), they were directly observed under a confocal laser scanning microscope.

Having established that HPPH-doped nanoparticles generate cytotoxic singlet oxygen molecules, we examined tumor cell uptake of such drug-doped nanoparticles by fluorescence imaging techniques. Two-photon fluorescence images (fig. 7A) of tumor cells (KB) showed significant staining in the cytoplasm and membranes, indicating that the nanoparticles were concentrated in these regions. FIG. 7B is a cytoplasmic localization spectrum showing the characteristic emission peak (665 nm) of HPPH, which effectively separates HPPH fluorescence from autofluorescence of these cells. The viability of the treated cells was verified by their morphology, indicating that the cells were viable even after 10 hours of staining.

Example 6

This example demonstrates that the silica nanoparticles of the present invention can be used for lightDynamic therapy. By way of illustration, cell viability was studied after cells that had taken up drug-loaded silica nanoparticles, with or without light irradiation. UCI-107 (university of California, Europe, USA) tumor cell line was used to study cell viability. Cells were incubated in MEM alpha Medium containing 5% Fetal Bovine Serum (FBS) at 37 deg.C (containing 5% CO)₂Moist environment). Prior to the experiment, cells were seeded in 24-well plates (7.5X 10)⁵Cells/well) and cultured overnight. After removal of the medium, the cells were washed 3 times with sterile PBS. After careful rinsing, 2 ml of fresh medium was added to each well. The drug concentration was previously determined by densitometry, for example: mu.M HPPH was dissolved in 120. mu.L of 0.25% Tween-80/water micelles, (b) 20. mu.M HPPH was coated in 120. mu.L of the aqueous dispersion of nanoparticles, (c) 120. mu.L of 0.25% Tween-80/water micelles and (d) 120. mu.L of the aqueous dispersion of empty nanoparticles, which were added to the calibrated wells, and the plates were returned to the incubator for 2 hours. The culture wells were washed 3 times with sterile PBS and replaced with 2 ml/well volume of fresh medium. Immediately after addition of fresh medium, the wells were placed under a 650 nm light source (laser driven by solid state crystal diode) for 10 minutes per well. After the light source irradiation is completed, the plate is returned to the incubator to be cultured overnight. Cell viability was determined by the colorimetric MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) assay [ 37%]. The MTT method only detects viable living cells and found that the absorbance at 570 nm is directly proportional to the number of cells. Briefly, MTT was dissolved in sterile PBS at a concentration of 5 mg/ml. To each well was added 200. mu.l of this solution. Plates were then incubated at 37 ℃ with 5% CO₂Incubate for 4 hours in ambient. After incubation, the wells were carefully aspirated of medium containing MTT. 2 ml of a 0.1N solution of hydrochloric acid in anhydrous isopropanol was added to each well to dissolve the purple MTT formazan crystals formed. Using Bausch&The absorbance value at 570 nm of the resulting purple solution was measured by a Lomb Spectronic 601 spectrophotometer. The average absorbance value of control cells incubated with serum-containing medium alone represents 100% cell survival. Making four-hole for each medicine and light doseThe experiment was repeated three times.

The results in fig. 8 show the cell viability of UCI-107 tumor cells treated with different agents (pure medium as control) following subsequent photoactivation. Significant cell death was observed after HPPH in tween-80 micelles, approximately 7% cell viability for the former and approximately 11% for the latter, and both when HPPH incorporated into nanoparticles was treated. In addition, substantial cytotoxicity was observed with empty tween-80 micelles, and tween-80 was generally considered to be a practically non-toxic surfactant; while the empty nanoparticles are almost non-toxic. Overall, the following conclusions can be drawn: as a drug/vehicle system, HPPH was incorporated into nanoparticles or dissolved in tween-80 micelles with almost the same efficiency for killing tumor cells.

Example 7

This example describes the use of the nanoparticles of the invention in animals. Intraperitoneal injection of HPPH-loaded nanoparticles (10 per gram of body weight) into female SCID mice loaded with human tumors¹⁴Microparticles). After injection, individual animals were incubated for 24 hours in absolute darkness. Mice were anesthetized with 5.5 micrograms of fentanyl and 5.5 milligrams of medetomidine hydrochloride (dissolved in 0.9% NaCl) (Sulzbach JanssenCilag, germany) per 100 grams of body weight and the mouse hairs were cut before light irradiation. The tumor was exposed to a laser at 532 nm and 50 mW for 2 minutes to reach 30J/cm²Total irradiation dose (ambient temperature 25 ℃). The light irradiation may also be conducted via a fiber optic transmission system. Animal tumors that were not irradiated served as controls. The assessment of tumor response can be determined by changes in tumor size as well as changes in histological morphology. According to the description provided herein, the nanoparticles may be used in photodynamic therapy (PDT) of other individuals, including humans.

While the invention has been demonstrated by the examples provided herein, those of skill in the art will recognize that modifications can be made in the various embodiments and that such modifications are intended to be included within the scope of the invention as described in the specification and claims.

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Claims (18)

1. A method of making ceramic nanoparticles carrying one or more photosensitive drugs, the method comprising the steps of:

a) preparing micelles coated with photosensitive drugs;

b) adding an alkoxyorganosilane to the micelle to form a complex of silica and micelle;

c) subjecting the complex of silica and micelles to alkaline hydrolysis to precipitate silica nanoparticles coated with photosensitive drug molecules; and

d) the precipitated nanoparticles were isolated by dialysis.

2. The method of claim 1, wherein the alkoxyorganosilane is triethoxyvinylsilane.

3. The method of claim 1, wherein the micelle comprises AOT and 1-butanol.

4. The process of claim 1, wherein the alkaline hydrolysis is carried out with aqueous ammonia.

5. The method of claim 1, wherein the alkaline hydrolysis is performed with an ammonium compound.

6. The method of claim 3, wherein the photosensitive drug is 2-devinyl-2- (1-hexyloxyethyl) pyropheophorbide.

7. A composition comprising ceramic nanoparticles, wherein one or more photosensitive drugs are encapsulated in said composition by:

a) preparing micelles coated with photosensitive drugs;

b) adding an alkoxyorganosilane to the micelle to form a complex of silica and micelle;

c) subjecting the complex of silica and micelles to alkaline hydrolysis to precipitate silica nanoparticles coated with photosensitive drug molecules; and

d) the precipitated nanoparticles were isolated by dialysis.

8. The composition of claim 7, wherein the drug is 2-devinyl-2- (1-hexyloxyethyl) pyropheophorbide.

9. The composition of claim 7, further comprising a pharmaceutically acceptable carrier.

10. The composition of claim 7, wherein the alkaline hydrolysis is performed with ammonia or an ammonium compound.

11. The composition of claim 7, wherein the ceramic nanoparticles have a size exhibiting a unimodal distribution with an average size of 30nm in diameter.

12. A method of inhibiting cell growth in vitro, comprising the steps of:

a) delivering a composition comprising ceramic nanoparticles coated with one or more photosensitive drugs prepared by the method of claim 1 to a cell, allowing the nanoparticles to be taken up by the cell; and

b) exposing the cell to light radiation, said exposure to light radiation resulting in the production of reactive oxygen species, said reactive oxygen species inhibiting the growth of the cell.

13. The method of claim 12, wherein the reactive oxygen species comprises singlet oxygen.

14. The method of claim 13, wherein the photosensitive drug is 2-devinyl-2- (1-hexyloxyethyl) pyropheophorbide.

15. Use of ceramic nanoparticles coated with one or more photosensitive drugs prepared by the method of claim 1 in the manufacture of a medicament for reducing tumor growth in a subject, wherein a composition comprising the ceramic nanoparticles is administered to a subject in need of treatment; allowing the ceramic nanoparticles to be taken up by tumor cells; and irradiating the area of the tumor with radiation causes the photosensitive drug to produce reactive oxygen species, wherein the reactive oxygen species produced will result in a reduction in tumor growth.

16. The use of claim 15, wherein the photosensitive drug is 2-devinyl-2- (1-hexyloxyethyl) pyropheophorbide.

17. The use of claim 15, wherein said composition comprises a pharmaceutically acceptable carrier.

18. The use of claim 15, wherein the reactive oxygen species comprises singlet oxygen.