WO2014145242A1 - Peptide-coated polymer carriers - Google Patents

Abstract

The invention provides a gelatin particles comprising a gelatin core, one or more drugs or diagnostic agents impregnated into the gelatin core, and a layer of peptides conjugated to the surface of the gelatin core. The peptides can be targeting peptides that include about 4 to about 100 amino acid residues including a sequence of amino acids cleavable by an enzyme overexpressed in cancer cells. The targeting peptides can also include a fluorophore at one terminus of the peptide, a quencher molecule at a separate portion or terminus of the peptide. The layer of targeting peptides can inhibit or prevent the release of the drugs or diagnostic agents from the gelatin core. In the presence of enzymes overexpressed in cancer cells, the conjugated peptides can be cleaved and the drugs or diagnostic agents are thereby released from the gelatin particles.

Description

PEPTIDE-COATED POLYMER CARRIERS

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 61/788,911, filed March 15, 2013, which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. W81XWH-10-1-0859 awarded by the U.S. Army Medical Research Acquisition. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The most important aspect of cancer chemotherapy is delivering high doses of drug molecules to tumor sites for maximum treatment efficacy while minimizing systemic delivery to normal organs. The small size, customized surface, improved solubility and multi-functionality of nanoparticles provide a versatile vehicle for in vivo targeted cancer chemotherapy drug delivery. Despite the emerging and promising research in the field of in vivo targeted cancer delivery using nanoparticles, there are still many difficulties to overcome to create an ideal nanocarrier for targeted delivery.

Important challenges for current in vivo drug delivery research using nanoparticles include difficulties with the conjugation of developed nanoparticles with the host molecules, cargo loading, biodegradability and toxicity. Additionally, 40% of anticancer drug candidates suffer from poor solubility due to formation of the crystal phase in the nanocarrier resulting in a lower solubility of drug. Larger drug carriers such as porous silicon films and microparticles have been proposed and have shown high loading and releasing capacity. However, nanoparticles with diameters between 20-100 nm are believed to be more ideal for cancer therapy. Such particles are small enough to penetrate tumor vessel pores, large enough to avoid renal filtration, and are not too large to induce transient poration of the cell membrane and cytotoxicity.

Many of the existing nanoparticle drug delivery techniques still impose mild toxicity due to the material used for nanoparticle fabrication. Additionally, off-target drug release from such particle drug carriers still occurs due to the fast nonspecific drug release from nanoparticles during circulation. Moreover, the in vivo fate of nanomaterials strongly determines their biomedical efficacy and imaging has become an indispensable tool in cancer research, for example, to monitor nanomaterials in vivo. Therefore, new types of nanocarriers that can overcome the limitations of existing tools are needed. Also needed are nanocarriers that can be easily detected and imaged with high-resolution imaging systems, that are biodegradable and inert with respect to the treatment of a subject, and that can easily embed drug molecules without adversely interacting with them. SUMMARY

The invention provides novel drug delivery particles or nanocapsules activated by cancer-specific biomarker enzymes for high-precision cancer chemotherapy. Off-target cancer drug uptake by benign tissues often causes serious side-effect and compromised treatment efficiency in cancer chemotherapy. The invention provides a novel nano-bio hybrid drug capsule from which cancer drug release can be triggered and tuned by the biomarker oncoproteins in cancer cells and the extracellular matrix. The cancer drug therefore is only released where cancer tissues are present and the release dosage is inherently proportional to localized cancer status.

The drug delivery particles or nanocapsules can be gelatin nanocarriers that are biodegradable and easily prepared in desired diameters in a cost-effective manner. These nanocarriers overcome the problem of early dissolution and off-target drug release by the conjugation of peptides to the surface of the particles to provide a protective coating. Additionally, the nanocarriers described herein are suitable for high resolution ultrasound and fluorescence imaging for monitoring drug release in real time to demonstrate the targeted release localized near tumors.

Accordingly, the invention provides a gelatin particle comprising a gelatin core, one or more drugs or diagnostic agents impregnated into the gelatin core, and a layer of peptides conjugated to the surface of the gelatin core. The peptides can be targeting peptides each independently having about 4 to about 100 amino acid residues including a sequence of amino acids cleavable by an enzyme overexpressed in cancer cells. The targeting peptides can also include a fluorophore at one terminus of the peptide, and a quencher molecule near another terminus of the peptide, or at a different location of the peptide separated from the fluorophore by a protease cleavage site. The layer of targeting peptides can inhibit or prevent release of the drugs or diagnostic agents from the gelatin core in the absence of the enzyme overexpressed in cancer cells. The diameter of the particle can be, for example, about 20 nm to about 20 μιη, about 50 nm to about 5 μηι, or about 200 nm to about 800 nm.

In some embodiments, the gelatin polymers of the gelatin core are crosslinked. The crosslinking can be derived from the condensation of crosslinking agents, such as dialdehyde compounds, with free amine groups of the gelatin particle matrix.

In some embodiments, the targeting peptide is cleavable between the fluorophore and the quencher molecule by an enzyme, such as a protease. In some embodiments, the targeting peptides can include about 5- 50 amino acids, about 5-25 amino acids, about 5-20 amino acids, or about 5-15 amino acids. These amino acids can include protease-recognition sequences that can be targeted by proteases that are overexpressed in cancer cells, including some sequences that are found in all cancers. Specific examples of several cancer types that overexpress these proteases include breast cancer, colon cancer, colorectal cancer, epithelial cancer, esophageal cancer, head and neck cancer, lung cancer, occult cancer, ovarian cancer, pancreatic cancer, prostate cancer, and stomach cancer.

In some embodiments, the amino acids of the targeting peptide are cleavable between the fluorophore and the quencher molecule by a serine protease, a cysteine protease, an aspartyl protease, or a metalloprotease. The protease can be factor Xa, trypsin, chymotrypsin, thrombin, protein specific antigen (PSA), peanut mottle, polyvirus Nla protease, papaine, bromelaine, cathepsin B, cathepsin L, HIV protease, S. cerevisiae yapsin 2, cathepsin D, thermolysin, peptidyl-Lys metalloendopeptidase, peptidyl-Asp metalloendopeptidase, coccolysin, autolysin, gelatinase A (MMP-2), human neutrophil collagenase (MMP-8), or a combination thereof.

The targeting peptide can include at least one protease recognition site sequence selected from Ile- Gly-Gly-Arg*; Lys*; Arg*; Tyr*; Phe*; Leu*; He*; Val*; Trp*; and His* at high pH; Arg*; Glu-Xaa-Xaa- Tyr-Gln*(Ser/Gly); Arg*; Lys*; Phe*; Lys*; Ala*; Tyr*; Gly*; Arg*Arg; Phe*Arg; Phe*Arg; Phe*Pro; Lys*; Arg*; Phe*Phe; Phe*Lys; Leu*Phe; Leu*Tyr; *Tyr; *Phe; *Leu; *Ile; *Val; *Τ ; and *His; Xaa*Lys;

Xaa*Asp; Xaa*Glu; Xaa*Cys; *Leu; *Phe; *Tyr; *Ala; Leu-Trp-Met*Arg-Phe-Ala; Pro-Gln-Gly*Ile-Ala- Gly-Gln; and Gly-Leu-Ser-Ser-Asn-Pro*Ile-Gln-Pro; wherein the asterisk represents the site of cleavage. In some embodiments, the targeting peptide comprises at least one protease recognition site sequence selected from Phe-Phe, Phe-Lys, Leu-Phe, and Leu-Tyr, for example, when the protease is cathepsin D.

In some embodiments, the targeting peptide cleavable by Cathepsin D contains a Phe-Phe-Arg-Asp sequence or a Phe-Phe-Arg-Leu sequence.

In some embodiments, the fluorophore is a blue fluorophore, such as methoxycoumarin (MCA). In various embodiments, the quencher molecule is 2,4-dinitrophenyl (DNP). Other fluorophores and quencher molecules can be used and suitable equivalents are well known to those of skill in the art.

In some embodiments, the diameter of the particle is about 10 nm to about 100 μιη, about 50 nm to about 75 μιη, about 100 nm to about 50 μιη, about 200 nm to about 20 μιη, about 1 μιη to about 10 μιη, about 10 μιη to about 50 μιη, about 10 nm to about 200 nm, about 20 nm to about 100 nm, about 100 nm to about 900 nm, about 1 μιη to about 2 μιη, about 1 μιη to about 20 μιη, about 5 μιη to about 10 μιη, 50 nm to about 5 μιη, about 100 nm to about 2 μιη, about 100 nm to about 1 μιη, about 200 nm to about 1 μιη, about 200 nm to about 900 nm, about 200 nm to about 800 nm, or a range from one to another of any two of the preceding integers.

The invention also provides a pharmaceutical composition comprising a plurality of particles described herein and a pharmaceutically acceptable diluent or carrier. The particles can include fluorophores and quencher moieties, or the particles can be absent of fluorophores and quencher moieties.

The invention further provides a method of delivering a drug to a subject having a cancer tumor comprising administering an effective amount of a plurality of particles described herein to a subject, wherein the location of the cancer tumor has elevated protease levels compared to normal tissue and the particles accumulate at a cancer tumor, the proteases at the cancer tumor cleave the targeting peptides conjugated to the surface of the particles, thereby releasing the drug from the particles and delivering the drug to the cancer tumor, and treating the cancer tumor, or killing or inhibiting the growth of cancer cells in the tumor. Enzymes such as collagenase 1A can then disintegrate the deprotected gelatin particle and release further amounts of the cancer drugs. The method can further include monitoring the cleavage of the targeting peptides by fluorescence microscopy, and/or monitoring the movement of the particles in the body using high resolution ultrasound imaging.

The invention also provides a method comprising in-vivo imaging biomarker activated chemotherapy drug delivery by administering a plurality of particles described herein to a subject having a cancer tumor and monitoring the cleavage of the targeting peptides by fluorescence microscopy. In some embodiments, the invention provides in-vivo imaging of biomarker activated chemotherapy drug delivery with nanoparticle capsules described herein, for example, for high resolution ultrasound imaging.

The invention additionally provides a method of monitoring the progress of a therapeutic method comprising administering to a subject having a cancer tumor a plurality of particles described herein and monitoring the area of the tumor for fluorescence, wherein the particles arrive at the tumor site, an enzyme at the tumor site cleaves the targeting peptide on the surface of the particles, thereby releasing the drug, diagnostic agent, or combination thereof, allowing for the fluorophore of the targeting peptide to fluoresce, which fluorophores are thereby detected by the monitoring the area of the subject having the tumor.

The invention also provides a method of treating breast cancer comprising administering to a subject having breast cancer an effective amount of a plurality of particles described herein, wherein the location of the breast cancer has elevated protease levels compared to normal (e.g., non-cancerous) tissue and the particles accumulate at location of the breast cancer, the proteases cleave the targeting peptides conjugated to the surface of the particles, thereby releasing the drug from the particles to the breast cancer and treating the breast cancer, or inhibiting the growth of cancer cells in the tumor.

The invention further provides a method of killing or inhibiting the growth of cancer cells comprising contacting cancer cells with a plurality of particles described herein, wherein proteases associated with the cancer cells cleave the targeting peptides conjugated to the surface of the particles, thereby releasing the drug from the particles to the cancer cells and killing or inhibiting the growth of cancer cells. The particles can be gelatin particles described herein. The cancer cells can be, for example, cells of breast cancer, colon cancer, colorectal cancer, epithelial cancer, esophageal cancer, head and neck cancer, lung cancer, occult cancer, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer. In one specific embodiment, the cancer cells are breast cancer cells. Accordingly, the invention also provides in vitro Cathepsin D activated drug release in breast cancer cell secretions.

The invention yet further provides a method of delivering a drug or diagnostic agent to a cell comprising preparing a gelatin particle described herein and contacting the cell with the gelatin particle under conditions sufficient to permit release of the drug by the gelatin particle. The invention also provides a method of inducing apoptosis in a tumor cell, comprising contacting the tumor cell with a gelatin particle described herein.

The methods provided herein provide an amount of drug released from the gelatin particles that is self-regulated by the local concentration of the biomarker enzymes, which is related to cancer progression, thus leading to effective drug delivery with minimized side effects and little or no systemic release of the drug. The invention further provides for the use of the compositions described herein for use in medical therapy. The medical therapy can be treating cancer, for example, breast cancer, lung cancer, pancreatic cancer, prostate cancer, colon cancer, or another cancer described herein. The invention also provides for the use of a composition as described herein for the manufacture of a medicament to treat a disease in a mammal, for example, cancer in a human. The medicament can include a pharmaceutically acceptable diluent, excipient, or carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

Figure 1. Illustration of a gelatin micro/nano particle drug carrier protected by a proteolytic peptide substrate. After a proteolytic reaction, drug molecules are released from nanopores of the particle to the tumor site.

Figure 2. Scanning electron microscopy (SEM) image of gelatin nanoparticles with diameter sizes of 200-800 nm, according to an embodiment.

Figure 3. Double Passive Cavitation Detection of gelatin microspheres (GMS) was performed using a manually constructed ultrasound imaging system in control ultrasound imaging experiments. Three confocally aligned transducers were held in place during imaging.

Figure 4. Gelatin microparticle drug carriers. (A) Scanning electron microscopy (SEM), (B) optical microscopy, and (C) confocal laser scanning microscopy images of Type B gelatin microspheres after cross- linking and impregnation of DOX drugs. The scale bars are 20 μιη, 50 μιη, and 50 μιη, respectively. (D) Zeta potentials of gelatin microparticles, and (E) loading efficiencies of toluidine blue O (TBO) in GMS cross- linked with 0.625% w/v glutaraldehyde (GA) as a function of pH. (F) Loading efficiencies of TBO in cross- linked GMS, (G) Zeta potentials of gelatin measured at pH 11, and (H) Swelling ratio of cross-linked GMS (C), as a function of GA concentration.

Figure 5. Peptide hydrolysis and doxorubicin (DXR) drug release from gelatin microparticles. (A)

Schematic illustration of peptide-conjugated GMS with a representative peptide sequence shown. The peptide can be hydrolyzed at the Phe-Phe bond by Capthesin D enzymes and then show fluorescence. (B) Peptide fluorescence intensity under different incubated conditions. (C) Fluorescence intensity of released DOX drug under various conditions. The tested samples include pure buffer solution, purified Cathepsin D solution, purified Collagenase 1A solution, and 2 -day-incubation culture media of MCF7 human breast cancer cells, 3T3 mouse fibroblast cells and HeLa human cervical cancer cells. Figure 6. In vitro cancer cell chemotherapy using peptide-conjugated gelatin microspheres as a doxorubicin (DOX) drug carrier. Optical microscopy images of microspheres cultured with (A) MCF7 human breast cancer cells; (B) 3T3 mouse fibroblast cells; and (C) HeLa human cervical cancer cells. The darker microspheres are gelatin microparticles loaded with DOX and smaller cells are next to them in the same field of view. The darker microparticle drug carrier disintegrates only in the MCF7 cell culture (A) but not in either 3T3 (B) or HeLa (C) cell cultures. (D) Cell count for the above three cases at every two hours after the chemotherapy starts during a 10-hour period of time.

Figure 7. In vitro cancer cell chemotherapy experiments using DOX loaded gelatin nanoparticles in MCF-7 breast cancer cell, 3T3 mouse fibroblast, and 4T1 breast cancer cell cultures, (i) Cell concentration counting at various times after incubating gelatin nanocapsules with the three cell cultures, (ii) Cell concentration counting for 3T3 mouse fibroblast cultures with and without incubating with DOX loaded gelatin nanoparticles. (iii) Cell concentration counting for 4T1 breast cancer cultures with and without incubating with DOX loaded gelatin nanoparticles. The initial viability experiment results obtained via hemocytometer for 3T3 and 4T1 cells.

Figure 8. Cell viability of (A) 4T1 and (B) MCF7 cells was reduced for cells incubated with DOX nanoparticles, while control cells (untreated with nanoparticles) continued their growth. (A) Left column, GMS treated cells; right column, control cells.

Figure 9. Unique signal types observed caused by the detected gelatin particles.

Figure 10. Setup of gelatin nanoparticle ultrasound imaging using the VisualSonics imaging system (top), and the obtained ultrasound images of imaged samples of water, without (left) and with (right) addition of nanoparticles (1-2 μιη in diameter).

Figure 11. High-frequency ultrasound images of the blood vessel in the heart of a nude mouse during the injection of gelatin nanocapsules. Ultrasound imaging of vena cava vessel (A) before and (B) several seconds after the injection of particles via the tail vein into the mouse body. The nanocapsules in flow can be clearly identified in (B), inside the vena cava.

Figure 12. Fluorescence imaging of chicken breast tissue with injected samples showing fluorescing DOX. Shown are (A) ten wells with a thin chicken breast layer on top, and (B) a piece of chicken breast that is injected with DOX sample.

Figure 13. Fluorescence images of mice experiments with the tail injection of DOX loaded gelatin nanoparticles (0.1 mL of gelatin nanoparticles in saline solution), or DOX nanocapsules. (A): control

(normal) mouse without tumor growth and without injection. (B): control (normal) mouse without tumor growth and with injection. (C): cancerous mouse with xenografted 4T1 breast tumor growth and with DOX nanoeapsule injection. The fluorescence intensity is approximately proportional to the localized nanocapsules and release of DOX molecular concentration. DETAILED DESCRIPTION

The invention provides gelatin nanocapsules having drug molecules loaded into the gelatin matrix of the particles. The nanocapsule shell is made of biocompatible nanoporous gelatin and the nanopores on the shell are blocked by peptide strands tethered onto the nanocapsule surface to prevent drug release prior to arriving at targeted cells having overexpressed proteases. The peptides can be high-specificity peptide substrates targeting certain protease enzymes over-expressed by cancer cells such as matrix metalloproteinase (MMP) in breast cancer tissues and prostate specific antigen (PSA) in prostate cancer tissues. When the drug nanocapsules are delivered and arrive near the tumors through blood circulations, the peptides are partially cleaved by the protease enzymes in cancerous tissues and shortened, thereby unblocking the nanopores. The cargo molecules, such as drugs, are then released from the unprotected gelatin matrix and nanopores. In benign tissues where the concentration of the targeted protease is low, the peptides covering the nanopores remain intact and the drug remaining inside the nanocapsule is well contained.

For example, biodegradable gelatin micro and nanoparticles coated with Cathepsin D-specific peptide were developed as a vehicle for targeted delivery of chemotherapy drugs to treat breast cancer. These particles were tested on in-vitro cancer cell culture and in vivo mouse cancer models. In in vitro experiments where the cells were treated with doxorubicin-loaded gelatin nanoparticles conjugated with the Cathepsin D- specific peptides, cell viability was reduced significantly for human MCF7 and mouse 4T1 breast cancer cells, but was not reduced for non-targeted cells such as 3T3 cells or HeLa cells. Notably, the nanoparticle drug carriers delivered in xenograft 4T1 mouse breast cancer models were successfully visualized and tracked with both ultrasound and fluorescence imaging modalities to reveal real-time particle flow in the mouse body as well as the nanoparticle and drug distribution in the mouse body. The imaging results indicate primary drug distribution only in bladder and tumor sites and no significant systemic drug delivery was observed.

A schematic diagram of the gelatin chemo therapeutic drug delivery vehicle is shown in Figure 1. The nanoparticle core was fabricated by an Electric Field Assisted Precision Particle Fabrication (E-PPF) method using acidic gelatin, loaded with doxorubicin (DXR). The resulting nanospheres were coated with a high-density peptide layer, the hydrolysis of which is catalyzed by Cathepsin D, a specific biomarker protease secreted by breast cancer cells. Thus, the core is protected from general proteolysis, wherein DXR is safely contained, until the digestion of the peptide shell is catalyzed by Cathepsin D in the proximity of breast cancer cells.

As the peptide shield is removed, gelatin is exposed to general proteases abundant in all cell secretions, triggering the release of DXR. As a result, the drug is released only in the vicinity of the target cancer cells and its release dosage is controlled by the localized secretory proteases concentration. For the low presence of targeted protease in benign tissues, the peptides covering the nanoparticle surface remain intact and the drug inside the nanoparticle is well contained. By this method, highly effective chemotherapy can be achieved with minimal side effects.

The fabricated nanoparticles can be identified in high-resolution ultrasound images. The particles can also be made of or blended with material having distinctive acoustic impedance, such as metal nanoparticles, metal oxide nanoparticles, or air bubbles, and thus can be identified in high-resolution ultrasound images. These materials having distinctive acoustic impedance can be added at a level of about 0.1 wt.% to about 20 wt.% of the nanoparticles, for example, about 0.1 wt.% to about 10 wt.%, 0.5 wt.% to about 10 wt.%, 1 wt.% to about 5 wt.%, 2 wt.% to about 4 wt.%, or 1 wt.% to about 3 wt.%. In some embodiments, the drug molecules can be either fluorescent or can be labeled with fluorophores, so the drug release in a subject, such as in nude mouse models or in a human, can also be tracked by fluorescence imaging.

This disclosure thus provides methods for the fabrication of nanoporous biopolymer nanoparticles with encapsulated cancer drug molecules. Methods are also provided for conjugating the nanoparticle surface with the peptides that are specific to over-expressed secretory proteins, such as at mouse breast cancer (e.g., 4T1) sites. Additionally, in vitro and in vivo biomarker-specific nanoencapsulated chemotherapy drug delivery results are provided.

Definitions

As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley 's Condensed Chemical Dictionary 14^th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or use of a "negative" limitation.

The term "and/or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase "one or more" is readily understood by one of skill in the art, particularly when read in context of its usage. For example, one or more can refer to one or two, one to three, one to four, one to five, or one to ten or twenty, depending on the context of its usage.

The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and/or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.

As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percents or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non- limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group.

Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, as used in an explicit negative limitation.

The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

An "effective amount" or a "therapeutically effective amount" means an amount of a composition described herein that (i) treats or prevents the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. In the case of cancer, the therapeutically effective amount of the drug may inhibit the growth of cancer cells, reduce the number of cancer cells; reduce the tumor size; inhibit (e.g., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (e.g., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR).

The terms "treating", "treat" and "treatment" include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving the disease, pathologic or medical condition; and/or (iv) diminishing symptoms associated with the disease, pathologic or medical condition. Thus, the terms "treat", "treatment", and "treating" can extend to prophylaxis and include prevent, prevention, preventing, lowering, stopping, inhibiting, or reversing the progression or severity of the condition or symptoms being treated. As such, the term "treatment" can include medical, therapeutic, and/or prophylactic administration, as appropriate.

The terms "inhibit", "inhibiting", and "inhibition" refer to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells. The inhibition can be greater than about 20%, 40%, 60%>, 80%>, 90%>, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting.

A subject or a patient can be a mammal. The term "mammal" means a warm-blooded animal that has or is at risk of developing a disease described herein and includes, but is not limited to, guinea pigs, dogs, cats, rats, mice, hamsters, and primates, including humans. The term "subject at risk for cancer" is a person or patient having an increased chance of cancer (relative to the general population). Such subjects may, for example, be from families with a history of cancer. Additionally, subjects at risk may be individuals in whom there is a genetic history of a particular cancer associated with race, nationality or heritage or exposure to an environmental trigger.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by abnormal or unregulated cell growth. A "tumor" comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer. The term cancer may be used generically to include various types of cancer or specifically (as listed above).

The phrase "pharmaceutically acceptable" indicates that the substance or composition is compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.

Gelatin is a mixture of peptides and proteins produced by partial hydrolysis of collagen. The bonds of collagen are thereby broken down into a form that rearranges more easily, resulting in gelatin. Gelatin is typically 98-99% protein by dry weight. Gelatin is soluble in most polar solvents and forms a semi-solid colloid gel in water. The mechanical properties are sensitive to temperature variations, previous thermal history of the gel, and time. Gelatins of different isoelectric points (IEPs) can be obtained commercially from suppliers such as Nitta Gelatin Co. (Osaka, Japan).

The term "microparticle" refers to a particle having a diameter of about 1 μιη to about 999 μιη. The term "nanoparticle" refers to a particle having a diameter of about 1 nm to about 999 nm, or in some embodiments, up to about 2 μιη. In some embodiments, the terms can partially overlap, such as by about 10- 20% of a maximum or minimum diameter. Gelatin microspheres (GMS) are nonporous particles of gelatin having diameters of about 1 μιη to about 20 μιη. Gelatin particles can be either microparticles or nanoparticles. Nanoparticles can also be referred to as nanospheres. In some embodiments, Gelatin microspheres (GMS) are nonporous particles of gelatin having diameters of about 1-20 μιη. GMS generally refers to gelatin microspheres. Gelatin nanoparticles have been prepared as shown in Figure 2. When gelatin nanoparticles swell in water, the diameter can increase to about 1 -2 μιη, in which case they can be considered gelatin microspheres.

The term "amino acid," includes the residues of the natural amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Hyl, Hyp, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as unnatural amino acids (e.g. phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma- carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1,2,3,4,-tetrahydroisoquinoline- 3-carboxylic acid, penicillamine, ornithine, citruline, .alpha.-methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). The term also includes natural and unnatural amino acids bearing a conventional amino protecting group (e.g. acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g. as a (Ci-C6)alkyl, phenyl or benzyl ester or amide; or as an a-methylbenzyl amide). Other suitable amino and carboxy protecting groups are known to those skilled in the art (see for example, Greene and Wutz, "Protecting Groups In Organic Synthesis" 2^nd Ed., 1991, New York, John Wiley & Sons, Inc., and references cited therein).

The term "peptide" can refer to a polypeptide or a protein. A peptide is typically considered to have from 3 to 100 amino acids, often 4 to 35 amino acids. A protein is typically considered to have more than 100 amino acids. The sequence may be linear or cyclic. Peptide derivatives can be prepared, for example, as disclosed in U.S. Patent Nos. 4,612,302 (Szabo et al.); 4,853,371 (Coy et al.); and 4,684,620 (Hruby et al.). The phrase "peptide-coated" polymer carrier refers to a gelatin particle as described herein that has a plurality of peptides conjugated to the surface of the particle to an extent sufficient to inhibit deterioration of the gelatin particle in the absence of enzymes that cleave the peptide that is covalently bound to the gelatin particle surface. The peptide can be about 5-100 or about 10-100 amino acid residues in length, typically about 10-75 residues, even more typically about 10-50 residues, and more typically still about 10-25 residues in length. Peptide can be modified by the addition of a chemical moiety that facilitates cellular uptake or spectroscopic monitoring of the peptide, such as a fluorophore. Also, the peptide can include a quencher molecule, such that the fluorophore is not detectable until a portion of the peptide that includes the fluorophore is cleaved from a portion of the peptide conjugated to the gelatin particle.

A "cathepsin D-specific peptide" refers to an amino acid sequence that can be cleaved by cathepsin D.

Such sequences include Phe*Phe, Phe*Lys. Leu*Phe, and Leu*Tyr, where the sequence is cleaved at the site of the *. Examples of such sequences include but are not limited to amino acids containing the sequence Phe- Phe-Arg-Asp or Leu-Phe-Phe-Arg-Leu.

The term "cathepsin D" refers to a protein in humans that is encoded by the CTSD gene. The CTSD gene encodes a lysosomal aspartyl protease. The lysosomal aspartyl proteinase is a member of the peptidase Al family and has a specificity similar to, but narrower than, that of pepsin A. Mutations in the CTSD gene are involved in the pathogenesis of several diseases, including breast cancer and Alzheimer disease. The CTSD gene has been used as a breast cancer tumor marker. Cathepsin-D is an aspartic protease that depends on protonation of its active site Asp residue.

The terms "drug", "therapeutic agent" and "pharmaceutical agent" refer to a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Drugs include compounds used in "targeted therapy" and conventional chemotherapy. Treatment using various drugs includes, but is not limited to, administration of numerous anticancer agents, such as: agents that induce apoptosis; polynucleotides (e.g., ribozymes); polypeptides (e.g., enzymes); drugs; biological mimetics; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal antibodies conjugated with anticancer drugs, toxins, and/or radionuclides; biological response modifiers (e.g., interferons [e.g., IFN-a, etc.] and interleukins [e.g., IL-2, etc.], etc.); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g., all-trans-retinoic acid, etc.); gene therapy reagents;

antisense therapy reagents and nucleotides; tumor vaccines; inhibitors of angiogenesis, and the like. A drug can be an analgesic, an anesthetic, an antiacne agent, an antibiotic, an antibacterial, an anticancer, an anticholinergic, an anticoagulant, an antidyskinetic, an antiemetic, an antifibrotic, an antifungal, an antiglaucoma agent, an anti-inflammatory, an antineoplastic, an antiosteoporotic, an antipagetic, an anti- Parkinson's agent, an antisporatic, an antipyretic, an antiseptic, an antithrombotic, an antiviral, a calcium regulator, a keratolytic, or a sclerosing agent. Specific drugs that can be incorporated into the gelatin particles described herein are further described below.

The terms "encapsulated", "entrapped," and "impregnated", as used herein, refer to the incorporation or association of a drug or cargo molecule into the gelatin nanoporous matrix of a gelatin particle. Targeting Peptides

A variety of peptides can be conjugated to the surface of the gelatin particles described herein. The targeting peptides can be of any suitable and effective length but are typically about 8 amino acid residues to about 30 amino acid residues in length. These oligopeptides, peptide subunits and peptide derivatives ("peptides") described herein can be synthesized from their constituent amino acids, fluorophores, and quencher molecules by conventional peptide synthesis techniques, such as by using solid-phase technology. The peptides can then be purified by, for example, reverse-phase high performance liquid chromatography (HPLC). Standard methods of peptide synthesis are disclosed, for example, in the following works:

Schroeder et al., "The Peptides", Vol. I, Academic Press 1965; Bodansky et al., "Peptide Synthesis",

Interscience Publishers, 1966; McOmie (ed.) "Protective Groups in Organic Chemistry", Plenum Press, 1973; or Barony et al., "The Peptides: Analysis, Synthesis, Biology", 2, Chapter 1, Academic Press, 1980.

Conjugation techniques for linking peptides to proteins such as gelatin are well known in the art. Suitable techniques are described by, for example, Grant T. Hermanson in Bioconjugation Techniques, 2^nd Ed., Academic Press, New York, USA 2008. The peptides containing the enzyme cleavage site and their conjugation to gelatin particles may be carried out by techniques well known in the art of medicinal chemistry. For example, a free amine moiety on the targeting peptide may be covalently attached to the gelatin particle at a carboxyl terminus such that an amide bond is formed. Similarly, an amide bond may be formed by covalently coupling an amine moiety of the gelatin particle and a carboxyl moiety of the peptide. For these purposes a reagent such as a combination of 2-(lH-benzotriazol-l-yl)-l,3,3-tetramethyluronium

hexafluorophosphate (known as HBTU) and 1-hydroxybenzotriazole hydrate (known as HOBT), dicyclohexylcarbodiimide (DCC), N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide (EDC),

diphenylphosphorylazide (DPP A), benzotriazol- 1 -yl-oxy-tris-(dimethylamino)phosphonium

hexafluorophosphate (BOP) and the like may be utilized.

The targeting peptide can be specifically cleavable by an enzyme produced by a target cell. For example, certain protease recognition sites can be included into the targeting peptides that are conjugated to the surface of the gelatin particles to provide targeted delivery to tumor cites, where the peptides are then cleaved by proteases often overexpressed by tumor cells. Examples of such proteases and protease recognition sites are shown in Table 1 below. The cleavage of the targeting peptide from the gelatin particles allows for the release of the particle cargo, such as a drug, thereby providing a concentration of the drug at a tumor site that provides a desired therapeutic effect without systemic toxicity.

Table 1. Illustrative proteases and protease recognition sites.

Trp*, and His* at high pH

serine thrombin Arg* Occult Cancer serine prostate specific Prostate Cancer

antigen (PSA)

serine and peanut mottle Glu-Xaa-Xaa-Tyr- Prostate Cancer cysteine variants polyvirus Nla protease Gln*(Ser/Gly)

cysteine papaine Arg*, Lys*, Phe* Head, Neck, Breast cysteine bromelaine Lys*, Ala*, Tyr*, Gly* Stomach, Breast cysteine cathepsin B Arg*Arg, Phe*Arg Esophageal, Ovarian cysteine cathepsin L Phe*Arg Breast, Epithelial aspartyl HIV protease Phe*Pro Breast

aspartyl S. cerevisiae yapsin 2 Lys*, Arg* Breast, Pancreatic aspartyl cathepsin D Phe*Phe, Phe*Lys, Breast

Leu*Phe, Leu*Tyr

metallo- thermolysin *Tyr, *Phe, *Leu, *lle, *Val, Lung, Pancreatic

*Trp, and *His

metallo- peptidyl-Lys Xaa*Lys Prostate, Colorectal

metal loendopeptidase

metallo- peptidyl-Asp Xaa*Asp, Xaa*Glu, Xaa*Cys Prostate, Colorectal

metal loendopeptidase

metallo- coccolysin *Leu, *Phe, *Tyr, *Ala Prostate, Colorectal metallo- autolysin Leu-Trp-Met*Arg-Phe-Ala Breast

metallo- gelatinase A (MMP-2) Pro-Gln-Gly*lle-Ala-Gly-Gln Breast, Ovarian metallo- human neutrophil Gly-Leu-Ser-Ser-Asn- Breast

collagenase (MMP-8) Pro*lle-Gln-Pro

* indicates the peptide bond hydrolysable by a protease.

Xaa is any natural amino acid.

Additional amino acid sequences that are recognized and proteolytically cleaved by enzymes, such as prostate specific antigen (PSA) are described by U.S. Patent No. 5,599,686 (DeFeo-Jones et al.), which is incorporated herein by reference.

Fluorophores and Quenchers

Any chemical reaction that leads to a fluorescent or chemiluminescent signal may be used in the compositions and methods of the present invention. In some embodiments, fluorophores are used to measure enzymatic activity and, thus, detect the cleavage of targeting peptide bonds and the concomitant release of drugs from the gelatin particles. Essentially any fluorophore may be used, including BODIPY, fluorescein, fluorescein substitutes (Alexa Fluor dye, Oregon green dye), long wavelength dyes, and UV-excited fluorophores. These and additional fluorophores are listed in Fluorescent and Luminescent Probes for Biological Activity, A Practical Guide to Technology for Quantitative Real-Time Analysis, 2^nd Ed.; W. T. Mason, Ed. Academic Press (1999) (incorporated herein by reference). A quencher is a molecule that absorbs the energy of the excited fluorophore. Close proximity of a fluorophore and a quencher allow for the energy to be transferred from the fluorophore to the quencher. By absorbing this energy, the quencher prevents the fluorophore from releasing the energy in the form of a photon, thereby preventing fluorescence.

Quenchers may be categorized as non-fluorescent and fluorescent quenchers. Non-fluorescent quenchers are capable of quenching the fluorescence of a wide variety of fluorophores. Generally, non- fluorescent quenchers absorb energy from the fluorophore and release the energy as heat. Examples of non- fluorescent quenchers include 4-(4'-dimethylaminophenylazo)benzoic acid) (DABCYL), QSY-7, and QSY- 33.

Fluorescent quenchers tend to be specific to fluorophores that emit at a specific wavelength range.

Fluorescent quenchers often involve fluorescence resonance energy transfer (FRET). In many instances the fluorescent quencher molecule is also a fluorophore. In such cases, close proximity of the fluorophore and fluorescent quencher is indicated by a decrease in fluorescence of the "fluorophore" and an increase in fluorescence of the fluorescent quencher. Commonly used fluorescent fluorophore pairs

(fluorophore/fluorescent quencher) include fluorescein/tetramethylrhodamine, IAEDANS/fluorescein, fluorescein/fluorescein, and BODIPY FL/BODIPY FL.

Methods and devices for detecting fluorescence are well developed. Essentially any instrument or method for detecting fluorescent emissions may be used. For example, WO 99/27351 describes a monolithic bioelectrical device comprising a bioreporter and an optical application specific integrated circuit (OASIC). The device allows remote sampling for the presence of substances in solution. Furthermore, the fluorescence may be measured by a number of different modes. Examples include fluorescence intensity, lifetime, and anisotropy in either steady state or kinetic rate change modes (Lakowicz, J. R. in Principles of Fluorescence Spectroscopy; 2^nd Ed.; Kluwer Academic/Plenum: New York, 1999). Chemotherapeutic Drugs

A variety of can be used to treat cancers that are associated with the over-expression of certain biomarkers. In some embodiments, the drugs will be water soluble. Examples of drugs suitable for encapsulation into the gelatin particles described herein include the following:

Alkylating agents: Cisplatin, Carboplatin, Oxaliplatin, Mechlorethanmine, Cyclophosphamide, Chlorambucil, Ifosfamide.

Anti-metabolites: Azathioprine, Mercaptopurine, Pyrimidines.

Plant Alkaloids and Terpenoids: Vinca Alkaloids and Taxanes.

Taxanes: Paclitaxel (Taxol), Docetaxel

Topoisomerase inhibitors: Irinotecan, Topotecan, Amsacrine, Etoposide, Etoposide Phosphate, Teniposide.

Cytotoxic Antibiotics: Actinomycin, Anthracyclines (Doxorubicin, Daunorubicin, Valrubicin, Idarubicin, Epirubicin), Bleomycin, Plicamycin, Mitomycin. Examples of specific drugs that can be encapsulated into the gelatin particles described herein include Erlotinib (TARCEVA™, Genentech/OSI Pharm.), Bortezomib (VELCADE™, Millennium Pharm.), Fulvestrant (FASLODEX™, AstraZeneca), Sunitinib (SUTENT™, Pfizer), Letrozole (FEMARA™, Novartis), Imatinib mesylate (GLEEVEC™, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin

(Eloxatin™, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE™, Wyeth), Lapatinib (TYKERB™, GSK572016, Glaxo Smith Kline), Lonafarnib (SCH 66336), Sorafenib

(NEXAVAR™, Bayer), Irinotecan (CAMPTOSAR™), Pfizer) and Gefitinib (IRESSA™, AstraZeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN™,

cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and

trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (Angew Chem. Intl. Ed. Engl.

(1994) 33:183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCESf™ (doxorubicin), mo holino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone;

elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran;

spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g.,

TAXOL™ (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE™ (doxetaxel; Rhone-Poulenc Rorer, Antony, France); chloranmbucil; GEMZAR™ (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBESfE™

(vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA™); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

Additional useful drugs are: (i) anti -hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX™; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON™ (toremifme citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE™ (megestrol acetate), AROMASESf™ (exemestane; Pfizer), formestanie, fadrozole, RIVISOR™ (vorozole), FEMARA™ (letrozole; Novartis), and ARIMIDEX™ (anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME™) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTESf™, LEUVECTIN™, and VAXID™; PROLEUKIN™ rIL-2; a topoisomerase 1 inhibitor such as

LURTOTECAN™; ABARELIX™ rmRH; (ix) anti-angiogenic agents such as bevacizumab (AVASTIN™, Genentech); and (x) pharmaceutically acceptable salts, acids and derivatives of any of the above.

Preparation of Gelatin Particles

Gelatin micro- and nano-spheres (GMS) can be prepared using by the electric field assisted precision particle fabrication (E-PPF) method, for example, as described by Choy and coworkers (Macromolecular Bioscience 2008, 8, 758; Macromolecular Bioscience 2007, 7, 423), based on acoustic excitation and Coulombic repulsion. The gelatin particles can be prepared by passing a 5% w/v solution of warm DI water and gelatin through a nozzle having a 250 μιη orifice to generate hydrogel solution drops. The drop sizes can be separated by electrical charging followed by solvent removal. The size and size uniformity of the resulting dry spherical particles can be determined using SEM and a multisizer, respectively. Using these techniques, particles having precisely controlled sizes can be prepared, including particles of about 10 μηι to about 50 μηι in diameter. More than 90% of the particles are within 3 μιη of the average diameter.

The gelatin particles can be cross-linked using any suitable and effective crosslinker compound. Examples of crosslinkers include D,L-glyceraldehyde, various dialdehydes, and genipin. Glutaraldehyde has been found to be highly effective. Suitable amounts of a 25% aqueous solution of crosslinker include, e.g., 0.125, 0.375, 0.625 and 0.875%> w/v. Crosslinking can be carried out by mixing the particles and the crosslinker at about 4 °C for a period of time sufficient to provide substantial crosslinking, such as overnight or for about 24 hours. Any remaining glutaraldehyde can be deactivated by addition of glycine at room temperature (~23 °C). See Choy et al, Macromolecular Bioscience 2008, 8, 758. The resulting GMS can be washed with DI water and lyophilized for storage or before further processing.

Gelatin nanoparticles can also be prepared using the precipitation method by dissolving geletin in DI water at room temperature. Acetone can be added to the gelatin solution for purification. The supernatant can be discarded and the precipitated gelatin re-dissolved in DI water and stirred. Acetone can then be added drop-wise to form nanoparticles. For crosslinking, a glutaraldehyde solution can be added to the gelatin solution and stirred for a suitable period of time (e.g., 12 hours), followed by washing with acetone one or more times.

The gelatin particles can be loaded with various cargo molecules such as drugs, diagnostic agents, or combinations thereof, by swelling the gelatin particles in an aqueous solution or buffer followed by addition of a solution of the cargo molecules. The amount of cargo molecules added (e.g., the drug loading) can be tuned by adjusting the concentration of the cargo molecules in the solution added to the gelatin particles. The pH of the solution can be adjusted to increase the loading of the cargo into the gelatin matrix of a particle.

The gelatin particles can then be conjugated to various oligopeptides and targeting peptides. The targeting peptide can include a sequence of amino acids that can be recognized by an enzyme that is overexpressed at a cancer tumor site. The targeting peptide can also include a fluorophore, for example, at its C-terminus, and a quencher molecule near the Λ^-terminus. The proximity of the quencher molecule near the TV-terminus is not critical so long as a site cleaved by an enzyme separates the fluorophore and the quencher molecule.

In some embodiments, the fluorophore can be a fluorescent dye such as toluidine blue O (TBO), Alexa fluor 430, Rhodamine B, 6-carboxyfluorescein (FAM), BODIPY, fluorescein, fluorescein substitutes such as Oregon green dye, long wavelength dyes, UV-excited fluorophores, and the like. Such fluorophores are commercially available from suppliers such as Invitrogen and Sigma.

In some embodiments, the quencher molecule can be 2,4-dinitrophenyl (DNP), 4-(4'-(dimethylamino- phenylazo)benzoic acid) (DABCYL), QSY-7, QSY-33, and the like.

Standard amino acid conjugation techniques can be used to conjugate the peptide to the gelatin particle. Such techniques are well known in the art and are described in reference works such as

Bioconj ligation Techniques by G. T. Hermanson, 2^nd Ed., Academic Press, New York, USA 2008. For example, a solution of PBS, l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), and ^V- hydroxy succinimide (NHS) can be prepared to form a PBS-EDC-NHS solution as catalyst for the conjugation. Peptides that include a targeting sequence, such as one listed above in Table 1, a fluorophore molecule at the C-terminus, and a dark quencher molecule near the TV-terminus can be obtained from commercial suppliers such as BioMol International LP (Plymouth Meeting, PA, USA). To form the conjugates, the peptide and drug-loaded GMS are combined with the PBS-EDC-NHS solution. The mixture is stirred overnight and can be centrifuged and washed with dimethyl sulfoxide to collect the peptide-conjugated GMS.

Therapeutic Methods

The invention also provides a method of inhibiting the growth of tumors, both drug resistant and drug sensitive, by delivering a therapeutic or effective amount of the gelatin particles described herein, to a tumor, preferably in a mammal. Because dosage regimens for pharmaceutical agents are well known to medical practitioners, the amount of the gelatin particles that are effective or therapeutic for the treatment of the diseases or conditions recited herein in mammals, and particularly in humans, will be apparent to those skilled in the art. The optimal quantity and spacing of individual dosages of the formulations herein will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests

Inhibition of the growth of tumors associated with all cancers is contemplated by this invention, including multiple drug resistant cancer. Cancers for which the described gelatin particles may be particularly useful in inhibiting are ovarian cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), colorectal cancer, breast cancer, and head and neck cancer. In addition, it is contemplated that the formulations described and claimed herein can be used in combination with existing anticancer treatments. For example, the formulations described herein can be used in combination with taxanes such as (1) Taxol (paclitaxel) and platinum complexes for treating ovarian cancer; (2) 5FU and leucovorin or levamisole for treating colorectal cancer; and (3) cisplatin and etoposide for treating SCLC.

The gelatin particles containing therapeutic agents (e.g., antineoplastic agents) and the pharmaceutical formulations thereof can be used therapeutically in animals (including humans) in the treatment of infections or conditions which require: (1) repeated administrations, (2) the sustained delivery of the drug in its bioactive form, or (3) the decreased toxicity with suitable efficacy compared with the free drug in question. Such conditions include but are not limited to neoplasms such as those that can be treated with antineoplastic agents.

The mode of administration of the gelatin particles containing the pharmaceutical agents (e.g., antineoplastic agents) and the pharmaceutical formulations thereof can aid the determination of the sites and cells in the organism to which the compound will be delivered. The gelatin particles can be administered alone but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. The preparations may be injected parenterally, for example, intravenously. For parenteral administration, they can be used, for example, in the form of a sterile aqueous solution which may contain other solutes, for example, enough salts or glucose to make the solution isotonic. The doxorubicin gelatin particles, for example, may be given, as a 60 minute intravenous infusion at a dose of at least about 20 mg/m². They may also be employed for peritoneal lavage or intrathecal administration via injection. They may also be administered subcutaneously for example at the site of lymph node metastases. Other uses, depending on the particular properties of the preparation, may be envisioned by those skilled in the art.

For the oral mode of administration, the gelatin particle therapeutic drug (e.g., antineoplastic drug) formulations can be used in the form of tablets, capsules; lozenges, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like. In the case of tablets, carriers which can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.

For the topical mode of administration, the gelatin particle therapeutic drug (e.g., antineoplastic drug) formulations may be incorporated into dosage forms such as gels, oils, emulsions, and the like. Such preparations may be administered by direct application as a cream, paste, ointment, gel, lotion or the like.

For administration to humans in the curative, remissive, retardive, or prophylactic treatment of neoplastic diseases, the prescribing physician will ultimately determine the appropriate dosage of the neoplastic drug for a given human subject, and this can be expected to vary according to the age, weight, and response of the individual as well as the nature and severity of the patient's disease. The dosage of the drug in liposomal form will generally be about that employed for the free drug. In some cases, however, it may be necessary to administer dosages outside these limits.

The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

EXAMPLES

Example 1. Fabrication and Analysis of Drug Delivery Particles.

1.1. Micro and nanospheres fabrication, cross-linking and drug loading. Nano-porous polymer particles made of gelatin were created utilizing acoustic excitation, hydrodynamic force, and coulomb repulsion. Gelatin (225 g bloom, BioReagent) nano and microspheres (GMS) were prepared by the E-PPF method reported elsewhere (Choy et al., Macromolecular Bioscience 2007, 7, 423) and were cross-linked using 0.125, 0.375, 0.625 and 0.875% w/v glutaraldehyde (GA) (25% aqueous solution, Sigma-Aldrich) solutions at 4 °C for 24 hours, followed by the addition of glycine (Sigma-Aldrich) at room temperature to deactivate the remaining GA (Choy et al, Macromolecular Bioscience 2008, 8, 758). Additional techniques are described by Kulsharova et al. (IEEE Transactions on Nanobio science, 2013, 12(4), 304-310).

The resulting GMS were washed with DI water and were lyophilized. Morphology and uniformity of microparticles was studied by scanning electron microscopy (SEM, Hitachi S-4700). Five L/mg of toluidine blue O (TBO) (Sigma-Aldrich) or doxorubicin (DXR) ( Sigma- Aldrich) was impregnated into the GMS via swelling of GMS in a buffer solution with controlled pH values. A UV spectrometer (Varian Gary-5G) was used to measure drug loading efficiencies. Confocal laser scanning microscope (CLSM, Olympus Fluoview FV 300) was used to visualize the impregnation of DXR in GMS (excitation of 470 nm and emission of 580 nm). An SEM image of gelatin particles with diameter sizes of 200-800 nm is shown in Figure 2.

1.2. Zeta potential measurement. Each gelatin sample was prepared by cross-linking 5% w/v gelatin solution with a desired GA concentration at 50 °C for a day, followed by treating with glycine to remove unreacted GA. The cross-linked gelatin was lyophilized and filtered with a 0.22 μιη filter. Zeta potential was measured 5 times for each sample using a dynamic light scattering technique (NICOMP 380 ZLS Particle Sizer).

1.3. Swelling ratio measurement. The swelling ratio of GMS was calculated according to the equation: Swelling % = 100(^^ -7^ )/JT^ (l) where W_wet and W_dry are the weight of the wet and dry sample, respectively. Wet GMS samples were prepared by immersing dry GMS in a phosphate buffer saline (PBS) (Sigma-Aldrich) solution at room temperature for 24 hours.

1.4. Peptide conjugation. 500 L of 0.1 M PBS solution, ΙΟΟμί of 0.33 M l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride (EDC) (Sigma-Aldrich), and ΙΟΟμί of 0.5 mM N- hydroxysuccinimide (NHS) (Sigma-Aldrich) solution was added to form a PBS-EDC-NHS solution as catalyst for the conjugation (G. T. Hermanson, Bioconjugation Techniques, 2^nd Ed., Academic Press, New York, USA 2008). The peptide for targeting contains a Phe-Phe-Arg-Asp sequence, a blue fluorophore molecule at the C-terminus, and a darker quencher molecule near the Λ^-terminus (synthesized by BioMol). To this mixture, 200 of 100 nM peptide and drug-loaded GMS were added and kept overnight. The resulting mixture was centrifuged and washed with dimethyl sulfoxide to collect peptide-conjugated GMS. Fluorescence intensity (excitation of 328 nm and emission of 393 nm) from peptide on drug-loaded GMS incubated with purified Cathepsin D and culture media of MCF7, 3T3 and HeLa cells was measured at designed time intervals using the microplate reader (BioTek Synergy). A variety of peptides described herein can be conjugated to the particles using similar techniques.

2. In vitro experiments.

2.1. Cell culture. To test the effectiveness of fabricated micro and nanocarriers, two stages of cell culture experiments were overtaken. For the first set of experiments, MCF7 breast carcinoma, 3T3 mouse fibroblast and HeLa cervical carcinoma cells (ATCC) were cultured. ATCC-formulated Eagle's Minimum Essential Medium with 0.01 mg/mL bovine insulin, 10% fetal bovine serum was used as culture medium for MCF7. 3T3 Swiss mouse fibroblast cells (ATCC) were cultured using ATCC-formulated Dulbecco's Modified Eagle's Medium mixed with bovine calf serum to a final concentration of 10%. HeLa cells were cultured using ATCC recommended growth medium. All media were filtered using a 0.22 μιη vacuum filter for sterilization. The cells were added to the cultured media and then kept in 75 sq cm flasks for culturing in incubator 5% carbon dioxide at 37 °C.

For the second set of experiments, MCF7 breast carcinoma, 3T3 Swiss mouse fibroblast (ATCC) and 4T1 mouse breast cancer cells (ATCC) were cultured. ATCC-formulated Eagle's Minimum Essential

Medium and ATCC-formulated Dulbecco's Modified Eagle's Medium, each with 10% fetal bovine serum, were used for MCF7 and 3T3 cells, respectively. 4T1 mouse breast cancer cells were cultured using RPMI- 1640 media mixed with 10% FBS.

2.2. In vitro drug release profile. The in vitro release study of DXR from the drug-loaded microcarriers with and without the peptide surface coating was performed in the presence of Cathepsin D enzyme, MCF7 culture media, 3T3 culture media, HeLa culture media, or Collagenase 1A. Drug release was assessed by sampling the supernatant and measuring the fluorescence emission from the DXR molecules measured using a microplate reader (excitation of 470 nm and emission of 585 nm). Protease-, DNAs-, and RNAs- free water (Fisher Scientific) was used for the measurement.

2.3. In vitro chemotherapy on cancer cells. In a first set of in vitro experiments, MCF7, 3T3, and

HeLa cells were each cultured in a separate petri dish and grown until nearly confluent. Drug-loaded microparticles conjugated with peptides were then introduced into the cell culture media. Optical images were taken every two hours during a period of 10 hours. Trypsin-EDTA (ATCC) was used to trypsinize the cells for viable cell counting. Cell counting was done using a hemocytometer (Neubauer) with trypan blue stain (Sigma- Aldrich) for the cell viability tests.

Similarly, a second set of experiments was initiated by culturing MCF7, 3T3 and 4T1 cancer cells in separate petri dish. In an initial experiment, after reaching confluency, MCF7 and 3T3 cells were treated with fabricated drug-loaded and peptide-coated, this time, smaller sized nano-particles. Trypsin-EDTA (ATCC) was used for trypsanizing the cells and viable cells were count using a hemocytometer with trypan blue 2 hours for 6 hours total. The following experiment employed a more accurate technique, proliferation MTA assay (ATCC), for determining the effect of particles on viability of MCF7 and 4T1 cells.

3. In vivo experiments.

3.1. Sonication of micro- and nanoparticles. Because micro- and nano-particles tend to aggregate, dispersing the particles before loading with DXR or before imaging experiments can improve the loading and imaging results. Gelatin particles were sonicated for 1 minute with a 10 second pulse on and a 50 second pulse off for total of 6 minutes of processing time. The particles were placed in a tube in an ice bucket to avoid heating and preliminary degradation of the particles. Sonication was carried out at 20% power until the particles separated. The separation could be observed under a microscope or with naked eye.

3.2. In vivo ultrasound imaging. Double Passive Caviation Detection of GMS was carried out using a manually constructed ultrasound imaging system for controlled ultrasound imaging experiments. Three confocally aligned transducers were held in place during imaging (Figure 3). A 3 MHz transducer was used to insonify, while the other two flanking transducers were used to passively receive signals. A concentration of 2.2 x 10⁹ particles/mL of 0.5-2 μηι sized gelatin particles was gradually added to the detection system.

Additionally, preliminary control ultrasound imaging was carried out using a mouse imaging ultrasound system, the VisualSonics ultrasound system, VisualSonics Inc., Toronto, Ontario, Canada). For this system, a simple 6-well petri dish was used, where one well was filled with water, while another well was filled with the mix of bare gelatin particles and water. Foam absorbance material was placed on the bottom of the well to prevent reflection caused by the plastic of the well. A 55 MHz transducer was placed in each well and images were recorded.

3.3. In vivo chemotherapy on mice cancer models.

3.3.1. Mouse cancer models. All animal procedures performed in this study were conducted in accordance with the Institutional Animal Care and Use Committee at the University of Illinois Urbana- Champaign. Female 5 week old athymic nude mice (19-23 grams on arrival) were ordered from Harlan Laboratories and were individually housed in separate cages. For tumor inducement, mice were anesthetized under isoflurane and injected with lxlO⁵ 4T1 cells. Following injection, mice were monitored every 1-3 days. Tumors were allowed to grow up to a maximum size of 10 mm before exposure.

3.3.2. Ultrasound imaging of gelatin particles in mice models. In vivo control ultrasound imaging of cancer free mice models was carried out using a VisualSonics ultrasound imaging system. Particles ranging from 200 nm to 0.9 μιη sizes were sonicated in saline solution using the method described above. The mixture was brought to a concentration of 2 x 10⁹ particles/mL. The mouse was anesthetized and kept anesthetized throughout the imaging. The transducer of the VisualSonics system was focused on mouse heart's vena cava and 0.1 mL of the prepared mix of nanoparticles in saline solution was introduced into the body through tail-vein injection. Real-time video images were obtained starting from the injection time.

3.3.3. Fluorescence detection of drug-loaded peptide-conjugated nanoparticles.

3.3.3.1. Control fluorescence imaging experiments. In vivo fluorescence imaging experiments were run using Cri Maestro imaging system (CRi, Hopkinton, MA). In a first set of experiments, a chicken breast was used for preliminary fluorescence imaging. A 96-well plate was used as a substrate, within which 10 wells were filled with 150 of nanoparticles mixed in 2mg/mL DXR with saline solution. Particle concentrations varied from 10⁷ to 10⁹ particles per mL. A thin chicken breast slice was placed above to cover these wells. Additionally, to test the fluorescence concentration of DOX itself, a 2 mg/mL concentration of DOX in saline was injected into a piece of thick chicken breast and was imaged using a Maestro microscope.

3.3.3.2. In vivo fluorescence detection of nanocarrier distribution in mouse models. Mice cancer models with tumor site diameters of approximately 5 mm were used for imaging experiments. 0.1 mL of fabricated drug-loaded and peptide-conjugated nanoparticles mixed in saline solution were brought to a 2 x 10⁹ particles/mL concentration and were IV injected in the cancer mice models. Thirty minutes after particle injection, fluorescent images were recorded. Results and Discussion.

Characterization and drug loading of cross-linked GMS. The SEM and optical images in Figures 4A and 4B, respectively, show that the GMS fabricated by the E-PPF method are spherical and uniform in size: 30 ± 2 μιη in diameter dry, and 65 μιη in diameter wet. The confocal laser scanning microscopy image in Figure 4C shows the distribution of DOX impregnated in the GMS, measured at 580 nm. DOX (pKa = 8.2) interacts electrostatically with the gelatin matrix, the isoelectric point of which was measured to be 5, as shown in Figure 4D.

Drug loading efficiency. The DOX molecules immobilized by the gelatin are delivered to the target sites without being released while unbound 'free' DOX molecules would be released systemically via diffusion. It is therefore important to maximize the amount of drug complexed to the GMS matrix (i.e., the drug loading efficiency) in order to minimize the off-target release.

To study the factors affecting drug loading efficiency, toluidine blue O (TBO) was chosen as a model drug. TBO has a pKa value of 7.9, close to that of DOX (pKa = 8.2). The TBO was impregnated into the GMS cross-linked with 0.625 % w/v GA. Figure 4E shows the loading efficiency measured as a function of pH of the impregnation medium, exhibiting that the efficiency improved as the pH increased. In a basic environment, the negative charge of the gelatin microparticle (without peptide coating or drug loading) would be enhanced by the deprotonation of its carboxyl groups, which was supported by the decrease in zeta potential with pH (Figure 4D). On the other hand, the protonation of TBO would be promoted in the acidic media with pH lower than the pKa of the drug. Therefore, the increase in the loading efficiency observed in the basic media indicated that the former effect was more dominant than the latter on promoting electrostatic interactions between gelatin and TBO.

The effect of GMS cross-linking on drug loading efficiency was investigated using GA concentrations of 0.125 - 0.875 % w/v. Figure 4F shows that the loading amount increased with GA concentration until it reached about 0.625 %, above which the loading efficiency decreased. The acidity of gelatin increases with GA concentration, i.e., cross-linking, due to the consumption of gelatin amino groups. This effect was confirmed by the zeta potential of the gelatin microparticle (without peptide coating or drug loading) measured as a function of GA concentration (Figure 4G), leading to an increase in the loading efficiency. However, at a GA concentration of 0.875 %, the loading efficiency was observed to decrease, indicating that acidity was not the only factor affecting the efficiency.

With cross-linking, density of the gelatin matrix increases, which can slow inward drug diffusion during the loading process due to a decrease in water content in the GMS. Figure 4H shows the decreased swelling ratio, i.e., the water content, of the GMS as a function of GA concentration. The density of the gelatin matrix may not be a dominant factor affecting drug diffusion when it is low, but the density becomes a significant factor when it is high, offsetting the electrostatic effect. This can account for the decreased loading efficiency observed for the GMS cross-linked with 0.875 % GA.

Targeting specificity of peptide-coated GMS. The specificity of the designed peptide to cathepsin D enzyme was examined by measuring the fluorescence emission from the terminal methoxycoumarin (MCA) fluorophore molecule on the peptide strand, which is quenched by the 2,4-dinitrophenyl (DNP) molecule before the proteolytic reaction due to the near field fluorescence energy transfer. Figure 5A schematically illustrates GMS conjugated with the designed peptide containing a Leu-Phe-Phe-Arg-Leu sequence, which can be recognized by cathepsin D, an aspartic protease enzyme prominent in breast malignancy. Once hydrolysis of the peptide is catalyzed by cathepsin D, the peptide substrate fluoresces as an indicator of the proteolytic activity of the peptide coating layer on the microparticle surface.

The fluorescence intensity increases, as shown in Figure 5B, when the peptide-coated GMS loaded with DOX were incubated with purified cathepsin D and MCF7 breast cancer cell secretions, respectively, indicating successful proteolytic reactions on the particle surface. On the other hand, the blue fluorescence intensity remained unchanged when the particles were incubated with non -targeted protease enzyme, e.g. coUagenase 1 A and non -targeted human cell lines, e.g. HeLa cells, which strongly indicates the specificity of the peptide layer to the targeted cancer biomarker, in this case cathepsin D.

In the case of 3T3 mouse fibroblast cell secretions, possibly due to the nonspecific proteolysis of the peptides by the very minimum amount of cathepsin D or other potent protease produced by fibroblast cells, the peptide fluorescence intensity was also elevated although the elevation level and sustainability were lower than those for MCF7 breast cancer cells.

In vitro release profiles of DOX-loaded GMS. The release profiles of DOX from GMS, with and without the peptide coatings, were expressed as the fluorescence intensity of DOX (Figure 5C). The GMS were incubated in pure buffer solution, purified Cathepsin D solution, purified coUagenase 1A solution, secretion with culture media of MCF7 human breast cancer cells, 3T3 mouse fibroblast cells, and HeLa cells, respectively. The fluorescence intensity of DOX is proportional to the drug concentration. More DOX is released from GMS than from peptide-coated GMS when incubated with and without coUagenase.

CoUagenase is a common protease in the body that facilitates hydrolysis of gelatin and is over-secreted in several cancers.

The data of Figure 5C indicates that the peptide coating layer hinders the diffusive drug release and degradation of gelatin catalyzed by coUagenase. The release of DOX from GMS with the peptide coating was prohibited in most of the cases except for the purified Cathepsin D solution and MCF7 breast cancer cell secretion.

The drug release variation between the above two cases may be attributable to the different protease concentrations leading to different degradation rates or other proteases secreted by MCF7 cells, such as coUagenase. In a sharp contrast, the uncoated microparticle drug carrier had considerable natural diffusion- driven drug release and biodegradation even in the absence of cancer cells. Due to nonspecific proteolytic reactions in the case of 3T3 mouse fibroblast cell secretion, minor DOX release was observed but the amount was significantly lower than that in MCF7 human breast cancer cell secretions.

The enhancement of DOX release was also observed in both coated and uncoated GMS cultured with coUagenase, which is ascribable to collagenase-assisted hydrolysis of gelatin, leading to the dissociation of the drug carriers. Drug release, however, was still significantly suppressed compared to the nude GMS with or without the presence of collagenase. This evidence demonstrates that the peptide layer on the drug carrier particle surface dramatically diminishes the non-specific drug release and improves the specificity to the targeted cancer biomarkers.

Viability of cells cultured with peptide-coated micro- and nano-particles. The morphology of GMS with the peptide coating, cultured with three different cancer line cells, was examined and the number of resulting viable cells was determined. Microparticles incubated with MCF7 cells disintegrated after 6 hours (Figure 6A). However, the microparticles remained intact when incubated with the other two cells, 3T3 mouse fibroblast cells and HeLa cells (Figures 6 B and 6C). The red microspheres in the images are gelatin microparticles loaded with DOX, while the cells next to the microparticles are much smaller, yet are clearly visible. The number of both 3T3 fibroblast and HeLa cells increased due to cell proliferation, which indicates negligible cytotoxicity to these two non-targeted cells. In contrast, the number of MCF7 breast cancer cells dropped dramatically by more than 50% at 10 hours (Figure 6D), and this downward trend continued thereafter. These results show that the peptide coating enables the high specificity of the particle drug delivery system to only targeted cancer biomarkers and associated tumor cells.

To confirm the results obtained from microparticle sized carriers, experiments were conducted with nanoparticles on MCF7, 4T1 and 3T3 cells, as described above, and similar results were obtained. Cathepsin D secreted by mice breast cancer 4T1 triggered the release of doxorubicin immobilized by gelatin via the digestion of the peptide-coating layer. The peptide-coating layer remained intact when the particles were cultured with 3T3 mouse fibroblast cells, hindering the drug release by preventing the enzymatic degradation of gelatin. The viability was not reduced for mouse fibroblast cells, but was reduced significantly for 4T1 mouse cancer cells when treated with the doxorubicin-loaded gelatin spheres conjugated with the peptide. Figure 7 shows the initial viability experiment results obtained via hemocytometer for 3T3 and 4T1 cells. Viability of cells has been measured approximately every 2 hours for 7 hours.

To confirm the effect of fabricated peptide-conjugated particles on proliferation of mouse and human breast cancer cells, viability experiments were repeated using a more accurate MCT viability assay. Viability of 4T1 and MCF7 cells untreated and treated with nanoparticles (Figures 8A and 8B, respectively) was determined after incubation for 2 hours. Figure 8 shows that the viability of 4T1 and MCF7 cells was reduced for cells incubated with nanoparticles, while control cells that were untreated with nanoparticles continued their growth.

Control ultrasound and fluorescence imaging of bare and drug-loaded, peptide-conjugated micro- and nanoparticles. As a result of ultrasound detection, 1500 signals were collected. Among these, several responses were suitable for extrapolation. These signals were observed approximately every 20 seconds after injection of gelatin particles into the tank. Figure 9 shows unique signals that were caused by the detected gelatin particles. These signals, detected at high-pressure values, had weak principle responses with no post excitation and a strong fundamental frequency at 3 MHz with weaker harmonics.

Detection of gelatin particles using VisualSonics actual mouse imaging system led to successful and clear images of gelatin particles in a water solution. Figure 10 shows the setup and the obtained ultrasound images of imaged samples of water, without and with addition of nanoparticles. The nanoparticles in water were 1 -2 μιη in diameter. Nanoparticles are clearly visible from the picture (right-hand image).

Thus, gelatin nanoparticles were injected into control mice via the lateral tail vein and real-time video of the superior vena cava was taken immediately after the injection. Snapshots of the particles passing through the vein located near the mouse heart are shown in Figure 11. Figure 11(a) shows the vena cava before introducing the particles into the body, while Figure 11(b) shows gelatin particles passing through the vein. Results indicate that the gelatin nanoparticles can provide sufficient contrast to facilitate in vivo high- resolution ultrasound imaging. This observation may be a result of the swelling characteristic of the nanoparticles, which causes the formation of air gaps and free pores, giving them distinctive acoustic impedance. As a result, the particles can act as reflective mediums for ultrasound waves, allowing in vivo ultrasound detection, tracking of particle flow, and distribution in real time.

Control fluorescence imaging of chicken breast tissue with injected samples provided clear results of fluorescing DOX. Figure 12 shows 10 wells with a thin chicken breast layer on top (A) and a piece of chicken breast that is injected with DOX sample (B). Both clearly have strong fluorescence.

Figure 13 below shows the comparison of cancer free and cancerous mice models injected with 0.1 mL of gelatin nanoparticles in saline solution. The images show the distribution of particles within the mouse body and their concentration in the bladder. For cancerous mouse model (C), particles also were concentrated at tumor sites, which shows that the introduced particles are breast cancer specific due to the high specificity of the peptides that coat the particles to protease enzyme secreted in breast cancer sites.

In conclusion, peptide-coated micro- and nano-particles have been developed as a cancer-targeting drug carrier. Release of a drug, immobilized by cross-linked gelatin, is triggered only by the biomarker protease enzyme Cathepsin D secreted by breast cancer cells. The loading efficiency of the particles can be optimized by controlling the cross-linker concentration and pH of the drug medium during loading. In comparison to chemotherapy with free-form drugs or uncoated gelatin particles, the peptide-coated microspheres significantly improve the specificity of cancer chemotherapeutic drug delivery and mitigate adverse side-effects that result from off-target drug release. Microscale and nanoscale peptide-coated particle drug carriers were prepared and the particles were effective in both in vitro and in vivo studies.

The results obtained from in vitro and in vivo experiments show that the nanoparticles are effective tools for reducing the viability of cancer cells. In vivo experiments showed that the fabricated nanoparticles can readily be imaged with ultrasound imaging systems. The results also indicate that technologies originating from this concept can have a wide range of chemotherapeutic applications. The facile fabrication process of nanoparticles described herein allows for the preparation of favorable sized particles for cancer therapy. The in vitro experiment and imaging results indicate that the nanoparticles are suitable for the basis of a next generation cancer treatment technology. A variety of cancer biomarkers can be targeted by varying the peptide sequences for surface coating. Appropriate hybrid drug carriers may also be designed and synthesized to treat and cure different subtypes of cancers for personalized medicine and therapy. Example 2. Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceutical dosage forms that may be used for the therapeutic or prophylactic administration of the particles described herein. For example, the formulation can include a plurality of particles described herein in combination with a suitable diluent, excipient, or carrier, optionally in combination with other components. The particles described herein are referred to below 'Composition X'.

(i) Tablet 1 mg/tablet

'Composition X' 100.0

Lactose 77.5

Povidone 15.0

Croscarmellose sodium 12.0

Microcrystalline cellulose 92.5

Magnesium stearate 3.0

300.0

(if) Tablet 2 mg/tablet

'Composition X' 20.0

Microcrystalline cellulose 410.0

Starch 50.0

Sodium starch glycolate 15.0

Magnesium stearate 5.0

500.0

(iii) Capsule mg/capsule

'Composition X' 10.0

Colloidal silicon dioxide 1.5

Lactose 465.5

Pregelatinized starch 120.0

Magnesium stearate 3.0

600.0

(iv) Injection 1 (1 mg/mL) mg/mL

'Composition X' (free acid form) 1.0

Dibasic sodium phosphate 12.0

Monobasic sodium phosphate 0.7

Sodium chloride 4.5

1.0 N Sodium hydroxide solution q.s.

(pH adjustment to 7.0-7.5)

Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mg/mL) mg/mL

'Composition X' (free acid form) 10.0

Monobasic sodium phosphate 0.3

Dibasic sodium phosphate 1.1

Polyethylene glycol 400 200.0

0.1 N Sodium hydroxide solution q.s.

(pH adjustment to 7.0-7.5)

Water for injection q.s. ad 1 mL (vi) Aerosol mg/can

'Composition X' 20

Oleic acid 10

Trichloromonofluoromethane 5,000

Dichlorodifluoromethane 10,000

Dichlorotetrafluoroethane 5,000

(vi ) Topical Gel 1 wt.%

'Composition X' 5%

Carbomer 934 1.25%

Triethanolamine q.s.

(pH adjustment to 5-7)

Methyl paraben 0.2%

Purified water q.s. to 100;

(viii)Topical Gel 2 wt.%

'Composition X' 5%

Methylcellulose 2%

Methyl paraben 0.2%

Propyl paraben 0.02%

Purified water q.s. to 100;

(ix)Topical Ointment wt.%

'Composition X' 5%

Propylene glycol 1 %

Anhydrous ointment base 40%

Polysorbate 80 2%

Methyl paraben 0.2%

Purified water q.s. to lOOg

(x) Topical Cream 1 wt.%

'Composition X' 5%

White bees wax 10%

Liquid paraffin 30%

Benzyl alcohol 5%

Purified water q.s. to lOOg

(xi) Topical Cream 2 wt.%

'Composition X' 5%

Stearic acid 10%

Glyceryl monostearate 3%

Polyoxy ethylene stearyl ether 3%

Sorbitol 5%

Isopropyl palmitate 2 %

Methyl Paraben 0.2%

Purified water q.s. to lOOg

These formulations may be prepared by conventional procedures well known in the pharmaceutical art. It will be appreciated that the above pharmaceutical compositions may be varied according to well-known pharmaceutical techniques to accommodate differing amounts and types of active ingredient 'Composition X'. Aerosol formulation (vi) may be used in conjunction with a standard, metered dose aerosol dispenser. Additionally, the specific ingredients and proportions are for illustrative purposes. Ingredients may be exchanged for suitable equivalents and proportions may be varied, according to the desired properties of the dosage form of interest. While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

What is claimed is:

1. A gelatin particle comprising a gelatin core, one or more drugs or diagnostic agents impregnated into the gelatin core, and a layer of targeting peptides conjugated to the surface of the gelatin core;

wherein each targeting peptide independently comprises about 4 to about 100 amino acid residues including a sequence of amino acids cleavable by an enzyme overexpressed in cancer cells; the layer of targeting peptides inhibits or prevents release of the drugs or diagnostic agents from the gelatin core in the absence of the enzyme overexpressed in cancer cells; and

the diameter of the particle is about 50 nm to about 5 μιη.

2. The particle of claim 1 wherein the gelatin core is crosslinked.

3. The particle of claim 2 wherein the particle is crosslinked with a dialdehyde compound.

4. The particle of claim 1 wherein the targeting peptides comprise 5-25 amino acids.

5. The particle of claim 1 wherein the amino acids of the targeting peptide have a sequence of residues between the fluorophore and the quencher molecule that are cleavable by a serine protease, a cysteine protease, an aspartyl protease, or a metalloprotease.

6. The particle of claim 5 wherein the protease is factor Xa, trypsin, chymotrypsin, thrombin, protein specific antigen (PSA), peanut mottle, polyvirus Nla protease, papaine, bromelaine, cathepsin B, cathepsin L, HIV protease, S. cerevisiae yapsin 2, cathepsin D, thermolysin, peptidyl-Lys metalloendopeptidase, peptidyl-Asp metalloendopeptidase, coccolysin, autolysin, gelatinase A (MMP-2), or human neutrophil collagenase (MMP-8).

7. The particle of claim 6 wherein the targeting peptide comprises at least one protease recognition site sequence selected from Ile-Gly-Gly-Arg*; Lys*; Arg*; Tyr*; Phe*; Leu*; He*; Val*; Trp*; and His* at high pH; Arg*; Glu-Xaa-Xaa-Tyr-Gln*(Ser/Gly); Arg*; Lys*; Phe*; Lys*; Ala*; Tyr*; Gly*; Arg*Arg; Phe*Arg; Phe*Arg; Phe*Pro; Lys*; Arg*; Phe*Phe; Phe*Lys; Leu*Phe; Leu*Tyr; *Tyr; *Phe; *Leu; *Ile; *Val; *Trp; and *His; Xaa*Lys; Xaa*Asp; Xaa*Glu; Xaa*Cys; *Leu; *Phe; *Tyr; *Ala; Leu-Trp-Met*Arg-Phe-Ala; Pro-Gln-Gly*Ile-Ala-Gly-Gln; and Gly-Leu- Ser-Ser-Asn-Pro*Ile-Gln-Pro; wherein the asterisk represents the site of cleavage.

8. The particle of claim 1 wherein the targeting peptide comprises at least one protease recognition site sequence selected from Phe-Phe, Phe-Lys, Leu-Phe, and Leu-Tyr.

9. The particle of claim 1 wherein the targeting peptide is enzymatically cleavable by cathepsin D.

10. The particle of claim 9 wherein the targeting peptide cleavable by cathepsin D contains a Phe- Phe-Arg-Asp sequence or a Phe-Phe-Arg-Leu sequence.

11. The particle of claim 1 wherein the diameter of the particle is about 50 nm to about 75 μιη, about 100 nm to about 50 μιη, about 200 nm to about 20 μιη, about 200 nm to about 900 nm, about 1 μιη to about 2 μιη, about 1 μιη to about 20 μιη, or about 5 μιη to about 10 μιη.

12. The particle of claim 1 wherein one or more of the targeting peptides comprise a fluorophore at one terminus of the peptide, a quencher molecule near the other terminus of the peptide, and which peptides are enzymatically cleavable between the fluorophore and the quencher molecule.

13. The particle of claim 12 wherein the fluorophore is a blue fluorophore.

14. The particle of claim 13 wherein the fluorophore is methoxycoumarin (MCA).

15. The particle of claim 14 wherein the quencher molecule is 2,4-dinitrophenyl (DNP).

16. A pharmaceutical composition comprising a plurality of particles of any one of claims 1-15 and a pharmaceutically acceptable diluent or carrier.

17. A method of delivering a drug to a subject having a cancer tumor comprising administering a plurality of particles of claim 1 to a subject, wherein the location of the cancer tumor has elevated protease levels and the particles accumulate at cancer tumor, the proteases cleave the targeting peptides conjugated to the surface of the gelatin particles, thereby releasing the drug from the particles and delivering the drug to the cancer tumor, and treating the cancer tumor, or killing or inhibiting the growth of cancer cells in the tumor.

18. The method of claim 17 further comprising monitoring the cleavage of the targeting peptides by fluorescence microscopy or high-resolution ultrasound imaging.

19. A method comprising in-vivo imaging biomarker activated chemotherapy drug delivery by administering a plurality of particles of claim 1 to a subject having a cancer tumor and monitoring the particles upon cleavage of the targeting peptides by fluorescence microscopy or high-resolution ultrasound imaging.

20. A method of monitoring the progress of a therapeutic method comprising administering to a subject having a cancer tumor a plurality of particles of claim 1 and monitoring the area of the tumor for fluorescence or high-resolution ultrasound imaging, wherein the particles arrive at the tumor site, an enzyme at the tumor site cleaves the targeting peptide on the surface of the particles, thereby releasing the drug, diagnostic agent, or combination thereof.

21. A method of treating breast cancer comprising administering to a subject having breast cancer a plurality of particles of claim 1, wherein the location of the breast cancer has elevated protease levels and the particles accumulate at the location of the breast cancer, the proteases cleave the targeting peptides conjugated to the surface of the gelatin particles, thereby releasing the drug from the particles to the breast cancer and treating the breast cancer, or inhibiting the growth of cancer cells in the tumor.

22. A method of killing or inhibiting the growth of breast cancer cells comprising contacting breast cancer cells with a plurality of particles of claim 1 , wherein proteases associated with the breast cancer cells cleave the targeting peptides conjugated to the surface of the particles, thereby releasing the drug from the particles to the breast cancer cells and killing or inhibiting the growth of breast cancer cells.

23. Use of a composition of claim 1 for preparing a medicament for delivering a drug to a cancer tumor of a subject having a cancer tumor.

24. The use of claim 23 wherein the cancer is breast cancer, colon cancer, colorectal cancer, epithelial cancer, esophageal cancer, head and neck cancer, lung cancer, occult cancer, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer.

25. The use of claim 24 wherein the cancer is breast cancer and the drug is doxorubicin.

26. A gelatin particle comprising a gelatin core, one or more drugs or diagnostic agents impregnated into the gelatin core, and a layer of targeting peptides conjugated to the surface of the gelatin core;

wherein each targeting peptide independently comprises about 4 to about 100 amino acid residues including a sequence of amino acids cleavable by an enzyme overexpressed in cancer cells, a fluorophore at one terminus of the peptide, and a quencher molecule near the other terminus of the peptide; and

wherein the layer of targeting peptides inhibits or prevents release of the drugs or diagnostic agents from the gelatin core in the absence of the enzyme overexpressed in cancer cells; and

the diameter of the particle is about 50 nm to about 5 μιη.

27. The particle of claim 26 wherein the targeting peptide comprises at least one protease recognition site sequence selected from Phe-Phe, Phe-Lys, Leu-Phe, and Leu-Tyr.

28. The particle of claim 27 wherein one or more of the targeting peptides are enzymatically cleavable by cathepsin D.

29. The particle of claim 28 wherein the targeting peptide cleavable by cathepsin D contains a Phe-Phe-Arg-Asp sequence or a Phe-Phe -Arg-Leu sequence.

30. Use of the particle of a particle of any one of claims 26-29 for in-vivo imaging of the particles in a subject having a cancer tumor using high -resolution ultrasound imaging.