WO2010059963A2 - Préparation et méthodologie de nanoparticules de fibroïne de soie - Google Patents

Préparation et méthodologie de nanoparticules de fibroïne de soie Download PDF

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WO2010059963A2
WO2010059963A2 PCT/US2009/065374 US2009065374W WO2010059963A2 WO 2010059963 A2 WO2010059963 A2 WO 2010059963A2 US 2009065374 W US2009065374 W US 2009065374W WO 2010059963 A2 WO2010059963 A2 WO 2010059963A2
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nanoparticle
cancer
drug
tree
vitamin
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PCT/US2009/065374
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WO2010059963A3 (fr
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Anshu B. Mathur
Carmen N. Rios
Vishal Gupta
Abraham Aseh
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The Board Of Regents Of The University Of Texas System
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Priority to US13/129,370 priority Critical patent/US20110305765A1/en
Publication of WO2010059963A2 publication Critical patent/WO2010059963A2/fr
Publication of WO2010059963A3 publication Critical patent/WO2010059963A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P23/00—Anaesthetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates generally to the fields of drug delivery, nutraceuticals, nanoparticles, molecular biology, and therapeutics. More particularly, the invention concerns fibroin nanoparticles, such as silk fibroin nanoparticles, for the delivery of therapeutic agents, diagnostic agents, or nutraceuticals into cells and tissues, and compositions, methods, and kits that involve the fibroin nanoparticles of the present invention.
  • the invention also concerns nanoparticles that include a blend of silk fibroin and one or more additional agents, such as a chitosan or a proteoglycan such as decorin.
  • Cancer is one of the most common causes of morbidity and mortality in the world.
  • treatment of cancer using pharmaceutical agents is often limited by reduced bioavailability, lack of tissue specificity, and tissue toxicity of the therapeutic agent.
  • curcumin is a yellow polyphenol extracted from the rhizome of turmeric, which has shown good activity as an anti-cancer agent by inhibiting proliferation of various cancers (Kunnumakkara et al., 2008).
  • chemotherapeutic agents such as curcumin is limited by the lack of bioavailability and tissue specificity (Anand et al, 2007).
  • curcumin nanoparticles To enhance the bioavailability of curcumin, several approaches have been taken including curcumin nanoparticles.
  • copolymers that are biodegradable, biocompatible, and non-toxic has been employed in the formulation of drugs.
  • Recent studies demonstrated various formulations of curcumin nanoparticles using polymeric materials (Sahu et al, 2008; Bisht et al, 2007), solid lipids (Tiyaboonchai et al, 2007), and liposomes (Sou et al, 2008; Li et al, 2005; Kunwar et al., 2006).
  • none of the above formulations were derived from natural polymers to eliminate tissue toxicity completely.
  • liposomes reduced the toxicity, there is no tissue specificity associated with them.
  • Curcumin has been shown to suppress many tumerogenic pathways including Her2 pathway in breast cancer cells (Kunnumakkara et al., 2008; Hong et al., 1999). However, none of the nanoparticle studies demonstrated the efficacy of nanocurcumin on breast cancer cells or tumors.
  • Silk fibroin is a protein created by Bombyx mori (silkworms) in the production of silk.
  • Silk consists of two main proteins - sericin and SF.
  • SF consists of layers of antiparallel beta sheets that contribute to the tensile strength of silk.
  • SF is also highly elastic.
  • the removal of sericin coating from silk ensures non-inflammatory or non-toxic response (Santin et al., 1999).
  • the SF show strong affinity towards polysaccharides and possess high strength and flexibility (Roden et al, 1985; Altman et al, 2003).
  • SF coating has been shown to increase retention of emodin in breast cancer cells thereby increasing the efficacy of therapeutic (Cheema et al, 2007).
  • Silk microspheres Wang et al, 2007; Wenk et al, 2008
  • nanolayers Wang et al, 2007
  • coatings on drug-loaded liposomes Gobin et al. , 2006
  • the coating of SF on drug-loaded liposomes increases the adhesion and residence time, and brings the drug in close proximity to the cell (Cheema et al., 2007; Gobin et al., 2006).
  • Loading the drug in liposomes and then coating with SF is a two-step process, which makes the particle size larger than 100 nm (Gobin et al., 2006).
  • CS chitosan
  • CS contains high charge density, non toxic, sequester growth factors and have been shown to have wound healing properties (VandeVord et al., 2002).
  • Chitosans have been evaluated as carriers for drugs in view of their biocompatilibity and biodegradability (Bayomi et al., 1998; Genta et al., 1998; Ko et al., 2003; Katas and Alpar, 2006). There have been limited reports concerning nanoparticles that include chitosan and TPP for delivery of siRNA (Katas and Alpar, 2006; Liu et al., 2007). Scaffolds that include a blend of SF and CS have been previously described for musculo fascial regeneration in ventral hernia repair (Gobin et al., 2006). Thus, there is the need for alternative drug delivery formulations to enhance drug bioavailability, target specific cells, and minimize risk of tissue toxicity while optimizing therapeutic outcomes.
  • the present invention is in part based on the finding that incorporation of a drug and/or nutraceutical into a nanoparticle that includes a fibroin results in enhanced bioavailability and improved efficacy of the drug and/or neutraceutical. Further, there may be a reduction in tissue toxicity. Further, the nanoparticles possess excellent cell adhesion characteristics with a low likelihood of inflammatory and thrombogenic responses. For example, the inventors have prepared silk fibroin (SF) nanoparticles (with or without chitosan) that include curcumin and the HIV drug Rl 5K peptide and have found that these nanoparticles provide more enhanced delivery of therapeutic agent to diseased cells.
  • SF silk fibroin
  • the method of targeting cells is related to the ability of SF to self-assemble into beta sheet structures.
  • the beta sheet structure of amyloid proteins of the prions has been shown to be the main driving force for the infection of cells in mad cow disease, as these structures are able to cross the blood-brain barrier.
  • the present invention generally provides for nanoparticles or complexes for delivery of a drug and/or nutraceutical.
  • the nanoparticle includes (a) a fibroin polypeptide and (b) a drug and/or nutraceutical, wherein the nanoparticle or complex has a diameter of about 1 nm to about 500 nm.
  • the fibroin may be any type of fibroin, but in particular embodiments it is silk fibroin polypeptide from the silkworm Bombyx mori (hereinafter "silk fibroin" abbreviated as SF; SEQ ID NO:1; GenBank Accession No. AAL83649).
  • Other examples of fibroin include spider silk fibroin and other insect silk fibroins.
  • fibroins include fibroin from Antipaluria urichi (GenBank Accession No. ACJ04053; SEQ ID NO:2); fibroin from Oecophytta smaragdina (GenBank Accession No. ABW21705; SEQ ID NO:3); fibroin from Oecophylla smaragdina (GenBank Accession No. ABW21703; SEQ ID NO:4); fibroin from Mymecia forficata (GenBank Accession No. ABW21701; SEQ ID NO:5); and fibroin from Bombus terrestris (GenBank Accession No. ABW21697; SEQ ID NO:6).
  • the fibroin may be genetically engineered, chemically synthesized, or obtained from natural sources.
  • the fibroin may be produced from genetically engineered cells in vivo or in vitro.
  • a "polypeptide” as used herein refers to a consecutive series of 2 or more amino acids, and as used herein encompasses the terms "peptide” and "protein.” Therefore, in some embodiments, the polypeptide comprises a consecutive series of at least 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,
  • a SF polypeptide may comprise between 10 and 262, between 20 and 250, between 30 and 220, between 40 and 200, between 50 and 180, or between 60 and 120 consecutive amino acids of SEQ ID NO:1.
  • the fibroin polypeptide may include one or more additional amino acid residues at the C-terminus or N-terminus of the consecutive sequence of amino acids of the fibroin polypeptide.
  • the fibroin polypeptide has at least 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or greater sequence homology to a known fibroin protein, such as SF.
  • the fibroin may or may not encapsulte the drug.
  • the nanoparticle or complex has a diameter of about 1 nm to about 300 nm. In more specific embodiments, the drug delivery nanoparticle has a diameter of about 1 nm to about 100 nm.
  • the fibroin polypeptide may coat a core that includes the drug and/or nutraceutical.
  • the fibroin polypeptide and the drug/nutraceutical are admixed together to form nanoparticles without a defined core.
  • the nanoparticles set forth herein may include a single drug/nutraceutical, or more than one drug/nutraceutical.
  • a "drug" as used herein references to any therapeutic or diagnostic agent.
  • Therapeutic agents include agents that can be applied in the treatment or prevention of a disease or health-related condition. Diagnostic agents include agents that can be applied in determining the presence or absence of a disease or health-related condition in a subject.
  • the drug may be a small molecule, a peptide, a polypeptide, a protein, a lipid, a carbohydrate, an antibody, an antibody fragment, a DNA a RNA, or a combination thereof (such as a lipoprotein).
  • the drug is a therapeutic agent.
  • Any therapeutic agent is contemplated for inclusion in the nanoparticles of the present invention.
  • Non-limiting examples of types of therapeutic agents include an anticancer agent, an anti-inflammatory agent, an antimicrobial agent, an analgesic, a hormone, an agent that can be applied in the treatment of a degenerative disease, and an anesthetic agent.
  • the therapeutic agent is an anticancer agent. Any agent that can find application in the treatment or prevention of cancer is contemplated as an anticancer agent.
  • Non-limiting examples of anticancer agents include curcumin, emodin, thiotepa, cyclosphosphamide, busulfan, topotecan, chlorambucil, melphalan, carmustine, lomustine, actinomycin, bleomycin, dactinomycin, daunorubicin, mitomycin C, methotrexate, 5-fluorouracil (5-FU), 6- mercaptopurine, thioguanine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, doxetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, cisplatin, oxaliplatin, carboplatin, vinblastine, etoposide, vincristine, retinoic acid, cisplatin (CDDP), carboplatin, procarbazine, and mechlore
  • the drug is a curcuminoid.
  • curcuminoids include curcumin, demethoxycurcumin, and bisdemethoxycurcumin.
  • the drug may be a derivative of a curcuminoid.
  • the drug may be a tetrahydrocurcuminoid.
  • the drug is emodin.
  • the therapeutic agent is an agent that can be applied in the treatment of HIV infection.
  • the therapeutic agent is I15K peptide.
  • the drug may also be an agent that can be applied in the diagnosis of disease (i.e., a diagnostic agent).
  • the diagnostic agent may be an agent that can be applied in imaging, such as CT, MRI, PET, SPECT, ultrasound, or other type of imaging.
  • the diagnostic agent is a quantum dot. For example, coating a quantum dot with fibroin as set forth herein may reduce toxicity of the quantum dot and facilitate tissue- targeting.
  • a "nutraceutical” as used herein includes any agent, food, or part of a food, that provides medical or health benefits to a subject. Included in this definition are dietary supplements. Non-limiting examples of nutraceuticals include vitamins, minerals, herbs or other bonanicals, amino acids, and so forth. They may aid in the treatment and/or prevention of a disease and/or disorder, or may aid in maintaining a state of health or well-being.
  • Nutraceuticals include, for example, vitamins and dietary supplements.
  • Non-limiting examples of vitamin and dietary supplements include vitamin A, vitamin D, acetyl carnitine, vitamin B- 12, vitamin B-3, vitamin B-6, C ester, calcium citrate, cholesterol, chromium, CLA (tonalin), cod liver oil, creatine, vitamin D, CHEA, Dong Quai, vitamin E, fish oil, gaba, vitamin A, acidophilus, alpha lipoic, vitamin B-I, vitamin B-150, vitamin B-5, vitamin B-9, biotin, vitamin C, calcium, chlorella, choline, coenzyme Q-IO, cranberry extract, D-ribose, EDTA, flaxseed oil, acai, arginine, aspirin, vitamin B-IOO, vitamin B-2, B-complex, beta- carotene, brewers yeast, magnesium, chia, chlorophyll, chondroitin, glucosamine, DHA, DM
  • Non-limiting examples of herbs include buta, aligator yam, alfalfa, almond, amalaki, ashwagandha, asoka tree, ambrette plant, apricot, arecanut palm, arjuna, aloe vera, arnica, ash gourd, ashoka tree, asparagus, babchi seed, bacopa, bael tree, bahama grass, banyan tree, barbados aloe, buddhist bauhinia, belliric myrobalan, bengal gram, bermuda grass, betelnut palm, bindweed, bishop's weed, billilotan, bitter melon, black nightshade, boerhavia, bitter gourd, black cumin, black nightshade, black-oil plant, black pepper, black plum, bonduc fruit, boswellia, bread wheat, butea gum tree, cinnamon, cajuput tree, calendula, cal mint, camphor, caper, cayenne, cal mint
  • the nanoparticles may also include additional ingredients other than drug/nutraceutical and fibroin.
  • the particle further comprises a chitosan. Chitosans, and examples of chitosans, are discussed in the specification below.
  • the chitosan has a deacetylation degree of greater than about 70%.
  • the chitosan has a deacetylation degree of greater than about 80%.
  • the chitosan has a deacetylation degree of between 70% and 99%.
  • the weight ratio of the SF polypeptide to the chitosan in the nanoparticle may be any ratio.
  • the weight ratio of SF polypeptide to chitosan in the nanoparticle is about 1 to about 6. In more specific embodiments, the weight ratio of SF to chitosan is about 2 to about 4.
  • the nanoparticles include a fibroin and a proteoglycan. In particular embodiments the proteoglycan is decorin.
  • the present invention also concerns compositions that include the a drug delivery nanoparticle of the present invention and a carrier.
  • the composition further comprises a chitosan.
  • the drug may be a therapeutic agent or a diagnostic agent.
  • the drug is an anticancer agent.
  • anticancer agents include any of the aforementioned agents.
  • the anticancer agent is a curcuminoid or emodin.
  • the present invention also generally concerns methods of delivering a drug and/or nutraceutical to a subject involving administering to the subject an effective amount of a composition that includes a nanoparticle as set forth herein.
  • the subject may be any subject, but in particular embodiments the subject is a mammal. Non-limiting examples of mammals contemplated include mice, rats, rabbits, cats, dogs, sheep, goats, horses, cows, primates, and humans. In specific embodiments, the subject is a human.
  • the methods of delivering a drug set forth herein may involve delivery of a diagnostic agent or a therapeutic agent.
  • the therapeutic agent for example, may be delivered for the purpose of treating or preventing a disease or health-related condition in a subject.
  • the subject is a human with a disease
  • the drug is delivered for the purpose of treating a disease in the subject.
  • the disease may be any disease.
  • Non-limiting examples of types of diseases include cancer, premalignancies, infectious diseases, inflammatory diseases, and degenerative diseases.
  • Non-limiting examples of types of cancer include breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer cell, liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, intestinal cancer, lymphoma, and leukemia.
  • the cancer is breast cancer.
  • any therapeutic agent is contemplated for inclusion in the nanoparticles of the present invention.
  • the therapeutic agent is an anticancer agent.
  • anticancer agents are set forth elsewhere in this specification.
  • the drug is a curcuminoid.
  • the drug is emodin.
  • the present invention also concerns methods of preparing a nanoparticle of the present invention that includes the steps of: (a) preparing a composition that includes a SF polypeptide, one or more drugs and/or nutraceuticals, and a carrier; (b) dispensing droplets of the composition of (a) on a surface; (c) lyophilizing the surface of (b) to remove the carrier, wherein dried dots of a composition that include a SF polypeptide and drug and/or nutraceutical are formed on the surface; (d) removing the composition of (c) from the surface, wherein the composition of (c) includes nanoparticles having a diameter of about 1 nm to about 500 nm.
  • the method includes freezing the surface following dispensing of the droplets of the composition of (a) on the surface, and prior to step (c).
  • the nanoparticles that are obtained in step (d) may optionally be washed in a buffered solution.
  • the dots may be of any size, but in particular embodiments are 500 micrometer to 3 mm in diameter.
  • the nanoparticles that are obtained may be frozen for later use.
  • The may be stored in a dried form at room temperature or in a pharmaceutical carrier.
  • the surface may be composed of any material but in particular embodiments the surface is a glass surface.
  • the drug may be any of the aforementioned agents.
  • the drug is an anticancer agent, such as a curcuminoid or emodin.
  • the composition of step (a) further includes a chitosan
  • the nanoparticles that are formed include a chitosan, a silk fibroin polypeptide, and one or more drugs.
  • the method of preparing the nanoparticle further comprises sonicating the composition of (a). Sonication can be performed for any duration. For example, it can be performed for not greater than 72 hours.
  • kits that include at least a first sealed container that includes a fibroin polypeptide and a drug and/or nutraceutical.
  • the kit may include any of the aforementioned nanoparticles of the present invention.
  • the kit incudes a nanoparticle in the first sealed container that includes an anticancer agent, such as any of the anticancer agents set forth above.
  • the anticancer agent is curcumin or emodin.
  • the kit may optionally include one or more additional items, such as instructions, a syringe, a catheter, a needle, a second sealed container that includes an aqueous solvent such as phosphate buffered saline or normal saline.
  • any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention.
  • any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
  • the use of the term "or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or.”
  • any embodiment of any of the present methods, compositions, nanoparticles, and kits may consist of or consist essentially of — rather than comprise/include/contain/have — the described features and/or steps.
  • FIG. 1 Curcumin nanoparticle sizes as measured from TEM images. Between 22 to 50 nanoparticles were measured from TEM images for each formulation. *p ⁇ 0.001 vs. 0.1% SF, V ⁇ -001 vs. 0.1% 25:75 SFCS, ⁇ p ⁇ 0-05 V s. 10% 25:75 SFCS, J p ⁇ 0-(XM vs. 0.1% 50:50 SFCS, # p ⁇ 0.001 vs . io% 75:25 SFCS.
  • 5A MCF-7, *p ⁇ 0.01 vs. 0.1% SF, tpO.OOl V s. io% SF
  • 5B MDA-MB-453, J p ⁇ 0-001 vs. 0.1% SF, V0.001 vs. 10% SF, # p ⁇ 0.01 V s. io% SF.
  • FIG. 7 Nanoparticle Size of nanocurcumin particles, ⁇ indicates p ⁇ 0.001 as compared to 75:25 SFCS and 0.1% SF with curcumin; @ indicates p ⁇ 0.001 as compared to
  • FIG. 8 Enhanced curcumin entrapment with 0.1% SF. *P ⁇ 0.05 vs. 0.1% SF.
  • FIG. 9 Curcumin release from nanoparticles. Burst release observed with SFCS blends; slower but higher release for 0.1% SF.
  • FIG. 10 Intracellular uptake of nanocurcumin into breast cancer cells - absorbance. Only 0.1% SF showed significant uptake of curcumin; uptake was similar for MCF-7 and MDA-MB-453 cells. *P ⁇ 0.001, 0.1% SF vs. all other blends.
  • FIG. 11 Intracellular uptake of nanocurcumin into breast cancer cells - fluorescence. Only 0.1% SF showed significant update of curcumin; uptake was similar for MCF-7 and MDA-MB-453 cells. *P ⁇ 0.001, 0.1% SF vs. all other blends.
  • FIG. 12. Standards for cell viability assay of breast cancer cells incubated with nanocurcumin. Seeded cells in 96-well plate; incubated for 4 days; performed MTT assay for cell viability.
  • FIG. 13 Efficacy of nanocurcumin on cells. MTT assay used for cell viability; 0.1% SF showed significant decrease in cell number compared to other blends; efficacy was similar for MCF-7 and MDA-MB-453 cells. PO.01 vs. 0.1% SF,
  • FIG. 14 Reduced liposome size by sonication.
  • FIG. 15. Higher emodin entrapment and release with sonication.
  • the present invention is based on the finding that silk fibroin nanoparticles can be applied in the delivery of therapeutic agents, diagnostic agents, and/or nutraceuticals to a subject.
  • the nanoparticles result in improved bioavailability, improved efficacy, and reduced risk of toxicity.
  • curcumin nanoparticles can be delivered to the site of a tumor in a manner that allows for long-term drug delivery and retention of the drug in the diseased cells.
  • Silk fibroin provides a delivery mechanism for any therapeutic or diagnostic agent to a cell of interest. It is a carrier system that delivers the pharmaceutical or nutraceutical to the respective cells and retains it at the diseased site so that bioavailability at the site of disease is increased with resulting improved efficacy and reduced systemic toxicity.
  • Silk as the term is generally known in the art, means a filamentous fiber product secreted by an organism.
  • Non-limiting examples of such organisms include a silkworm or a spider.
  • Silks produced from insects namely (i) Bombyx mori silkworms, and (ii) the glands of spiders, typically Nephilia clavipes, are the most often studied forms of the material; however, hundreds to thousands of natural variants of silk exist in nature. Fibroin is produced and secreted by a silkworm's two silk glands.
  • Silkworm silk has been used in biomedical applications for over 1,000 years. The
  • SF silkworm fibroin.
  • SF may be obtained from any source known to those of ordinary skill in the art.
  • SF may be obtained from a solution containing a dissolved silkworm silk from Bombyx mori.
  • the SF suitable for use in the present invention can be obtained from a solution containing a genetically engineered silk.
  • the SF can be prepared by any conventional method known to one skilled in the art.
  • B. mori cocoons may be boiled in an aqueous solution.
  • the cocoons are rinsed, for example, with water to extract the sericin proteins and the extracted silk is dissolved in an aqueous salt solution.
  • the salt is consequently removed using, for example, dialysis.
  • the SF may be produced using organic solvents. Such methods have been described, for example, in Li et al. (2001); Nazarov et al. (2004). SF may also be obtained from any of a number of commercial sources known to those of ordinary skill in the art.
  • the present invention concerns nanoparticles comprising at least one SF polypeptide.
  • polypeptide refers to a consecutive series of two or more amino acids.
  • the size of at least SF polypeptide may comprise, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
  • amino acid residue refers to any naturally occurring amino acid, any amino acid derivative or any amino acid mimic known in the art.
  • residues of the protein or peptide are sequential, without any non-amino acid interrupting the sequence of amino acid residues.
  • sequence may comprise one or more non-amino acid moiety.
  • sequence of residues of the protein or peptide may be interrupted by one or more non-amino acid moieties.
  • polypeptide encompasses amino acid sequences comprising at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid, including but not limited to Aad, 2-Aminoadipic acid; EtAsn, N-Ethylasparagine; Baad, 3- Aminoadipic acid, HyI, Hydroxylysine; BaIa, ⁇ -alanine, ⁇ -Amino-propionic acid; AHyI, allo-Hydroxylysine; Abu, 2-Aminobutyric acid; 3Hyp, 3-Hydroxyproline; 4Abu, 4- Aminobutyric acid, piperidinic acid; 4Hyp, 4-Hydroxyproline; Acp, 6-Aminocaproic acid, Ide, Isodesmosine; Ahe, 2-Aminoheptanoic acid; AIIe, allo-Isoleucine; Aib, 2-Amino
  • Proteins or peptides may be made by any technique known to those of skill in the art, including the expression of polypeptides through standard molecular biological techniques, the isolation of polypeptides from natural sources, or the chemical synthesis of polypeptides. Alternatively, various commercial preparations of SF polypeptides are known to those of skill in the art.
  • the nanoparticles of the present invention include one or more drugs.
  • a “drug” as used herein refers to a diagnostic or therapeutic agent.
  • a “therapeutic agent” as used herein refers to any agent that can be administered to a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.
  • nanoparticles that include a therapeutic agent may be administered to a subject for the purpose of reducing the size of a tumor, reducing or inhibiting local invasiveness of a tumor, or reducing the risk of development of metastases.
  • a “diagnostic agent” as used herein refers to any agent that can be administered to a subject for the purpose of diagnosing a disease or health-related condition in a subject. Diagnosis may involve determining whether a disease is present, whether a disease has progressed, or any change in disease state.
  • the drug may be a small molecule, a peptide, a protein, a polypeptide, an antibody, an antibody fragment, a DNA, or an RNA.
  • the therapeutic agent may be a siRNA, miRNA, or shRNA.
  • the therapeutic agent or diagnostic agent can be any such agent known to those of ordinary skill in the art.
  • the therapeutic agent may be an anti-inflammatory agent, an anti-infective agent, an agent that can be applied in the treatment of a hyperproliferative disease such as cancer, an agent that can be applied in the treatment of a degenerative disease, and so forth.
  • therapeutic agents include, but are not limited to, agents for the prevention of restenosis, agents for treating renal disease, agents used for intermittent claudication, agents used in the treatment of hypotension and shock, angiotensin converting enzyme inhibitors, antianginal agents, anti-arrhythmics, anti-hypertensive agents, antiotensin ii receptor antagonists, antiplatelet drugs, b-b lockers bl selective, beta blocking agents, botanical product for cardiovascular indication, calcium channel blockers, cardiovascular/diagnostics, central alpha-2 agonists, coronary vasodilators, diuretics and renal tubule inhibitors, neutral endopeptidase/angiotensin converting enzyme inhibitors, peripheral vasodilators, potassium channel openers, potassium salts, anticonvulsants, antiemetics, antinauseants, anti-parkinson agents, antispasticity agents, cerebral stimulants, agents that can be applied in the treatment of trauma, agents that can be applied in the treatment of Alzheimer disease or dementia, agents that
  • diagnostic agents include, but are not limited to, magnetic resonance image enhancement agents, positron emission tomography products, radioactive diagnostic agents, radioactive therapeutic agents, radio-opaque contrast agents, radiopharmaceuticals, ultrasound imaging agents, angiographic diagnostic agents, and other reporter agents.
  • the therapeutic agent is a chemotherapeutic agent.
  • chemotherapeutic agents may be used in accordance with the present invention.
  • the term “chemotherapy” refers to the use of drugs to treat cancer.
  • a “chemotherapeutic agent” (or “anticancer agent”) is used to connote a compound or composition that is administered in the treatment of cancer.
  • agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle.
  • an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
  • Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.
  • Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; du
  • CPT-I l CPT-I l
  • topoisomerase inhibitor RFS 2000 difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; a curcuminoid, emodin, and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • DMFO difluoromethylornithine
  • retinoids such as retinoic acid
  • capecitabine a curcuminoid, emodin, and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the therapeutic agent is a curcuminoid.
  • curcuminoids are those compounds which due to their structural similarity to curcumin, exhibit anti-proliferative or pro-apoptotic effects on cancer cells similar to that of curcumin.
  • curcumin is also known as diferuloylmethane or (E,E)-l,7-bis(4-hydroxy-3- methoxyphenyl)-l,6-heptadiene-3,5-dione.
  • Curcuminoids, or analogs of curcumin, which may have anti-cancer effects similar to curcumin include, for example, Ar-tumerone, methylcurcumin, demethoxy curcumin, bisdemethoxycurcumin, sodium curcuminate, dibenzoylmethane, acetylcurcumin, feruloyl methane, tetrahydrocurcumin, l,7-bis(4- hydroxy-3-methoxyphenyl)-l,6-heptadiene-3,5-dione (curcumin 1), l,7-bis(piperonyl)-l,6- heptadiene-3,5-dione (piperonyl curcumin) l,7-bis(2-hydroxy naphthyl)-l,6-heptadiene-2,5- dione (2-hydroxyl naphthyl curcumin), l,l-bis(phenyl)-l,3,8,10 undecatetraene
  • Curcumin analogs may also include isomers of curcumin, such as the (Z,E) and (Z,Z) isomers of curcumin.
  • curcumin metabolites which have anti-cancer effects similar to curcumin can also be used in the present invention.
  • curcumin metabolites include glucoronides of tetrahydrocurcumin and hexahydrocurcumin, and dihydroferulic acid.
  • curcumin analogs or metabolites can be formulated as metal chelates, especially copper chelates.
  • Other appropriate derivatives of curcumin, curcumin analogues and curcumin metabolites appropriate for use in the present invention will be apparent to one of skill in the art.
  • Curcuminoids may be derived from a natural source, such as the perennial herb
  • Curcuma longa L. which is a member of the Zingiberaceae family.
  • the spice turmeric is extracted from the rhizomes of Curcuma longa L. and has long been associated with traditional-medicine treatments used in Malawi and Chinese medicine. Turmeric was administered orally or topically in these traditional treatment methods. Curcuminoids may also be chemically synthesized or obtained from commercial sources.
  • the nanoparticles of the present invention include chitosan as a component.
  • chitosans are a family of cationic, binary hetero-polysaccharides composed of (l ⁇ 4)-linked 2-acetamido-2-deoxy- ⁇ -D-glucose (GIcNAc, A-unit) and 2-amino-2-deoxy- ⁇ - D-glucose, (GIcN; D-unit) (Varum et al, 1991).
  • the chitosan has a positive charge, stemming from the de-acetylated amino group (-NH 3 + ).
  • Chitosan, chitosan derivatives or salts ⁇ e.g., nitrate, phosphate, sulphate, hydrochloride, glutamate, lactate or acetate salts) of chitosan may be used and are included within the meaning of the term "chitosan.”
  • chitosan derivatives is intended to include ester, ether, or other derivatives formed by bonding of acyl and/or alkyl groups with -OH groups, but not the NH 2 groups, of chitosan. Examples are O-alkyl ethers of chitosan and O-acyl esters of chitosan. Modified chitosans, particularly those conjugated to polyethylene glycol, are also considered “chitosan derivatives.” Many chitosans and their salts and derivatives are commercially available ⁇ e.g., SigmaAldrich, Milwaukee, WI).
  • Chitosans may be obtained from any source known to those of ordinary skill in the art.
  • chitosans may be obtained from commercial sources.
  • Chitosans may be obtained from chitin, the second most abundant biopolymer in nature.
  • Chitosan is prepared by N-deacetylation of chitin.
  • Chitosan is commercially available in a wide variety of molecular weight (e.g., 10-1000 kDa) and usually has a degree of deacetylation ranging between 70%-90%.
  • the chitosan (or chitosan derivative or salt) used preferably has a molecular weight of 4,000 Dalton or more, preferably in the range 25,000 to 2,000,000 Dalton, and most preferably about 50,000 to 300,000 Dalton.
  • Chitosans of different molecular weights can be prepared by enzymatic degradation of high molecular weight chitosan using chitosanase or by the addition of nitrous acid. Both procedures are well known to those skilled in the art and are described in various publications (Allan and Peyron, 1995; Domard and Cartier, 1989).
  • the chitosan is water-soluble and may be produced from chitin by deacetylation to a degree of greater than 40%, preferably between 50% and 98%, and more preferably between 70% and 90%.
  • chitosan Some methods of producing chitosan involve recovery from microbial biomass, such as the methods taught by U.S. Pat. No. 4,806,474 and U.S. Patent Application No. 20050042735, herein incorporated by reference. Another method, taught by U.S. Pat. No. 4,282,351, teaches only how to create a chitosan-beta-glucan complex. Chitosan derivatives are also suitable for use in this invention. Suitable chitosan derivatives include, without limitation, esters, ethers or other derivatives formed by bonding acyl and/or alkyl groups with the hydroxyl groups, but not the amino groups of chitosan.
  • Examples include O-alkyl ethers of chitosan and O-acyl esters of chitosan.
  • the chitosan, chitosan derivative or salt used in the present invention may be water soluble.
  • Chitosan glutamate is water soluble.
  • water soluble it is meant that the chitosan, chitosan derivative or salt dissolves in water at an amount of at least 10 mg/ml at room temperature and atmospheric pressure.
  • Silk fibroin polypeptide-containing nanoparticles may be formulated by mixing one or more therapeutic agents with a SF-containing solution. Alternatively, a drug containing particle can be coated onto the pre-formed drug-containing particle. Any pharmaceutical carrier can be used that does not dissolve the silk fibroin. Examples of methods for formation of silk fibroin nanoparticles are discussed in the Example section below.
  • raw silk can be obtained and degummed using any method known to those of ordinary skill in the art.
  • the raw silk may be degummed in 0.25% sodium carbonate and 0.25% solidium dodecylsulfate, and then dissolved in calcium nitrate tetrahydrate and methanol solution.
  • Drug can be suspended in a composition that includes the silk fibroin polypeptide.
  • the nanoparticles that form in the composition can be extracted using any method known to those of ordinary skill in the art.
  • the drug suspension can be dispersed on a glass slide, the slide can be frozen, and then lyophilized. Dry dots containing SFCS coated drug nanoparticles can then be scrated off the slides and collected in an appropriate container. The nanoparticles can then be washed, and stored for later use.
  • the nanoparticles are formed of a combination of a silk fibroin polypeptide and a chitosan.
  • Information regarding SF has been previously discussed.
  • raw silk can be obtained and degummed using any method known to those of ordinary skill in the art.
  • the preferred process for preparing the nanoparticles of the invention is by mixing together the ingredients.
  • Information regarding preparation of compositions that include SF and a chitosan can be found in Gobin et al. (2005).
  • the raw silk may be degummed in 0.25% sodium carbonate and 0.25% solidium dodecylsulfate, and then dissolved in calcium nitrate tetrahydrate and methanol solution.
  • Chitosan can be dissolved in a suitable solvent, such as 2% acetic acid, and then blended with a mixture of SF in an appropriate solvent, such as calcium nitrate tetrahydrate and methanol solution.
  • chitosan such as a powder of chitosan or a derivative thereof or a salt of chitosan or a salt of a derivative of chitosan
  • a suitable solvent for example, the solvent may be water, acetic acid, or hydrochloric acid.
  • the chitosan-containing solution that is formed may optionally be centrifuged to remove contaminants, although removal of all contaminants is not required.
  • the pH of the chitosan solution may then be adjusted such that the pH is in a range of about 3.5 to about 5.5.
  • a blend of silk fibroin polypeptide and chitosan may then be created.
  • One or more drugs can optionally be added to the chitosan-silk fibroin blend.
  • the silk fibroin - chitosan (SFCT) blend can then be dialyzed against water and filtered.
  • the blend can then be combined with a composition that includes one or more drugs to form nanoparticles, and the nanoparticles can be isolated from the composition using any method known to those of ordinary skill in the art.
  • Additives suitable for use with the present invention include biologically or pharmaceutically active compounds.
  • biologically active compounds include, but are not limited to: cell attachment mediators, such as collagen, elastin, fibronectin, vitronectin, laminin, proteoglycans, or peptides; biologically active ligands; and substances that enhance or exclude particular varieties of cellular or tissue ingrowth.
  • Biocompatible polymers can be added to the silk fibroin containing nanoparticles of the present invention.
  • Biocompatible polymers useful in the present invention include, for example, polyethylene oxide (PEO) (U.S. Pat. No. 6,302,848), polyethylene glycol (PEG) (U.S. Pat. No. 6,395,734), collagen (U.S. Pat. No. 6,127,143), fibronectin (U.S. Pat. No. 5,263,992), keratin (U.S. Pat. No. 6,379,690), polyaspartic acid (U.S. Pat. No. 5,015,476), polylysine (U.S. Pat. No. 4,806,355), alginate (U.S. Pat. No.
  • PEO polyethylene oxide
  • PEG polyethylene glycol
  • collagen U.S. Pat. No. 6,127,143
  • fibronectin U.S. Pat. No. 5,263,992
  • keratin U.S. Pat. No.
  • the nanoparticles can be purified using any method known to those of ordinary skill in the art.
  • the nanoparticles may be purified by centrifugation and removal of supernatant.
  • centrifugation may be at 12000 rpm for about 30 min to about 60 min. Centrifugation may be repeated once, or more than once. In particular embodiments, centrifugation is repeated three times.
  • Nanoparticles that are formed by the present methods can be analyzed using any method and technique known to those of ordinary skill in the art. For example, particle size may be measured by dynamic light scattering or by transmission electron microscopy. In some embodiments, nanoparticle size is heterogeneous and poorly defined. If desired, nanoparticle size may be reduced using any method known to those of ordinary skill in the art. The nanoparticle size can be controlled using standard techniques such as sieving.
  • the nanoparticles may be stored using any method known to those of ordinary skill in the art.
  • the nanoparticles may be stored at 4 0 C until ready for use. In some embodiments, the nanoparticles are stored at room temperature.
  • Nanoparticles of the present invention may be stored in a dry form and reconstituted for later use, or they can be stored dispersed in a solution.
  • the particles of the present invention may optionally include one or more additional ingredients.
  • additional ingredients include, but are not limited to, sugars such as sucrose and trehalose; polyols such as mannitol and sorbitol; and surfactants such as polysorbates; amino acids such as glycine; and polyethylene glycol.
  • the total amount of additional ingredients may be up to a total of about 10% by weight of the nanoparticle.
  • the nanoparticle further includes a targeting moiety.
  • a "targeting moiety” is a term that encompasses various types of affinity reagents that may be used to enhance the localization or binding of a substance to a particular location in an animal, including organs, tissues, particular cell types, diseased tissues or tumors.
  • a targeting moiety is still considered to selectively bind even if it also binds to other proteins that are not substantially homologous with the target so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that target moiety binding to the target is still selective despite some degree of cross-reactivity.
  • Targeting moieties may include peptides, peptide mimetics, polypeptides, antibodies, antibody-like molecules, nucleic acids, aptamers, and fragments thereof. Targeting moieties also include small molecules.
  • the targeting moiety may be covalently bound to the nanoparticle or noncovalently bound to the nanoparticle.
  • Other materials or substances which may serve as targeting ligands include, for example, proteins, including antibodies, antibody fragments, hormones, hormone analogues, glycoproteins and lectins, peptides, polypeptides, amino acids, sugars, saccharides, including monosaccharides and polysaccharides, carbohydrates, vitamins, steroids, steroid analogs, hormones, cofactors, and genetic material, including nucleosides, nucleotides, nucleotide acid constructs, petptide nucleic acids (PNA), aptamers, and polynucleotides.
  • proteins including antibodies, antibody fragments, hormones, hormone analogues, glycoproteins and lectins, peptides, polypeptides, amino acids, sugars, saccharides, including monosaccharides and polysaccharides, carbohydrates, vitamins, steroids, steroid analogs, hormones, cofactors, and genetic material, including nucleosides, nucleotides, nucleotide acid constructs
  • targeting ligands in the present invention include cell adhesion molecules (CAM), among which are, for example, cytokines, integrins, cadherins, immunoglobulins and selectin.
  • CAM cell adhesion molecules
  • the pharmaceutical formulations of the present invention may also encompass precursor targeting ligands.
  • a precursor to a targeting ligand refers to any material or substance which may be converted to a targeting ligand. Such conversion may involve, for example, anchoring a precursor to a targeting ligand.
  • Exemplary targeting precursor moieties include maleimide groups, disulfide groups, such as ortho-pyridyl disulfide, vinylsulfone groups, azide groups, and [agr]-iodo acetyl groups.
  • the nanoparticle includes a lipid.
  • the nanoparticle may be a liposome that is coated with a silk fibroin polypeptide or a composition that includes a silk fibroin polypeptide.
  • Lipid complexes and liposomes can be formed using any method known to those of ordinary skill in the art. Selection of the appropriate lipids for such composition is governed by the factors of: (1) liposome stability, (2) phase transition temperature, (3) charge, (4) non- toxicity to mammalian systems, (5) encapsulation efficiency, (6) lipid mixture characteristics. It is expected that one of skill in the art who has the benefit of this disclosure could formulate liposomes according to the present invention which would optimize these factors.
  • the vesicle-forming lipids of this type are preferably ones having two hydrocarbon chains, typically acyl chains, and a head group, either polar or nonpolar.
  • the hydrocarbon chains may be saturated or have varying degrees of unsaturation.
  • synthetic vesicle-forming lipids and naturally-occurring vesicle-forming lipids including the sphingo lipids, ether lipids, sterols, phospholipids, particularly the phosphoglycerides, and the glyco lipids, such as the cerebrosides and gangliosides.
  • Phosphoglycerides include phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, phosphatidylserine phosphatidylglycerol and diphosphatidylglycerol (cardiolipin), where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation.
  • PC stands for phosphatidylcholine
  • PS stand for phosphatidylserine.
  • Lipids containing either saturated and unsaturated fatty acids are widely available to those of skill in the art. Additionally, the two hydrocarbon chains of the lipid may be symmetrical or asymmetrical. The above-described lipids and phospholipids whose acyl chains have varying lengths and degrees of saturation can be obtained commercially or prepared according to published methods.
  • Exemplary phosphatidylcholines include dilauroyl phophatidylcholine, dimyristoylphophatidylcholine, dipalmitoylphophatidylcholine, distearoylphophatidyl- choline, diarachidoylphophatidylcholine, dioleoylphophatidylcholine, dilinoleoyl- phophatidylcholine, dierucoylphophatidylcholine, palmitoyl-oleoyl-phophatidylcholine, egg phosphatidylcholine, myristoyl-palmitoylphosphatidylcholine, palmitoyl-myristoyl- phosphatidylcholine, myristoyl-stearoylphosphatidylcholine, palmitoyl-stearoyl- phosphatidylcholine, ste
  • Assymetric phosphatidylcholines are referred to as 1-acyl, 2-acyl-sn- glycero-3-phosphocho lines, wherein the acyl groups are different from each other.
  • Symmetric phosphatidylcholines are referred to as l,2-diacyl-sn-glycero-3-phosphocho lines.
  • PC refers to phosphatidylcholine.
  • the phosphatidylcholine 1,2- dimyristoyl-sn-glycero-3-phosphocholine is abbreviated herein as "DMPC.”
  • the phosphatidylcholine l,2-dioleoyl-sn-glycero-3-phosphocholine is abbreviated herein as "DOPC.”
  • the phosphatidylcholine l,2-dipalmitoyl-sn-glycero-3-phosphocholine is abbreviated herein as "DPPC.”
  • saturated acyl groups found in various lipids include groups having the trivial names propionyl, butanoyl, pentanoyl, caproyl, heptanoyl, capryloyl, nonanoyl, capryl, undecanoyl, lauroyl, tridecanoyl, myristoyl, pentadecanoyl, palmitoyl, phytanoyl, heptadecanoyl
  • the corresponding IUPAC names for saturated acyl groups are trianoic, tetranoic, pentanoic, hexanoic, heptanoic, octanoic, nonanoic, decanoic, undecanoic, dodecanoic, tridecanoic, tetradecanoic, pentadecanoic, hexadecanoic, 3,7,11,15- tetramethylhexadecanoic, heptadecanoic, octadecanoic, nonadecanoic, eicosanoic, heneicosanoic, docosanoic, trocosanoic and tetracosanoic.
  • Unsaturated acyl groups found in both symmetric and asymmetric phosphatidylcholines include myristoleoyl, palmitoleyl, oleoyl, elaidoyl, linoleoyl, linolenoyl, eicosenoyl and arachidonoyl.
  • the corresponding IUPAC names for unsaturated acyl groups are 9-cis-tetradecanoic, 9-cis-hexadecanoic, 9-cis- octadecanoic, 9-trans-octadecanoic, 9-cis-12-cis-octadecadienoic, 9-cis-12-cis-15-cis- octadecatrienoic, 11-cis-eicosenoic and 5-cis-8-cis-l l-cis-14-cis-eicosatetraenoic.
  • Exemplary phosphatidylethanolamines include dimyristoyl- phosphatidylethanolamine, dipalmitoyl-phosphatidylethanolamine, distearoyl- phosphatidylethanolamine, dioleoyl-phosphatidylethanolamine and egg phosphatidylethanolamine.
  • Phosphatidylethanolamines may also be referred to under IUPAC naming systems as l,2-diacyl-sn-glycero-3-phosphoethanolamines or l-acyl-2-acyl-sn- glycero-3-phosphoethanolamine, depending on whether they are symmetric or assymetric lipids.
  • Exemplary phosphatidic acids include dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid and dioleoyl phosphatidic acid.
  • Phosphatidic acids may also be referred to under IUPAC naming systems as 1 ,2-diacyl-sn-glycero-3 -phosphate or l-acyl-2-acyl-sn- glycero-3 -phosphate, depending on whether they are symmetric or assymetric lipids.
  • Exemplary phosphatidylserines include dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, dioleoylphosphatidylserine, distearoyl phosphatidylserine, palmitoyl- oleylphosphatidylserine and brain phosphatidylserine.
  • Phosphatidylserines may also be referred to under IUPAC naming systems as l,2-diacyl-sn-glycero-3-[phospho-L-serine] or 1- acyl-l-acyl-sn-glycero-S-fphospho-L-serine], depending on whether they are symmetric or assymetric lipids.
  • PS refers to phosphatidylserine.
  • Exemplary phosphatidylglycerols include dilauryloylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoyl- phosphatidylglycerol, dimyristoylphosphatidylglycerol, palmitoyl-oleoyl- phosphatidylglycerol and egg phosphatidylglycerol.
  • Phosphatidylglycerols may also be referred to under IUPAC naming systems as l,2-diacyl-sn-glycero-3-[phospho-rac-(l- glycerol)] or l-acyl-2-acyl-sn-glycero-3-[phospho-rac-(l-glycerol)], depending on whether they are symmetric or assymetric lipids.
  • the phosphatidylglycerol 1 ,2-dimyristoyl-sn- glycero-3-[phospho-rac-(l-glycerol)] is abbreviated herein as "DMPG”.
  • DMPG phosphatidylglycerol l,2-dipalmitoyl-sn-glycero-3-(phospho-rac-l -glycerol) (sodium salt) is abbreviated herein as "DPPG”.
  • Suitable sphingomyelins might include brain sphingomyelin, egg sphingomyelin, dipalmitoyl sphingomyelin, and distearoyl sphingomyelin.
  • Suitable lipids include glycolipids, sphingolipids, ether lipids, glycolipids such as the cerebrosides and gangliosides, and sterols, such as cholesterol or ergosterol.
  • sterols such as cholesterol or ergosterol.
  • cholesterol is sometimes abbreviated as "Choi.”
  • lipids suitable for use in liposomes are known to persons of skill in the art and are cited in a variety of sources, such as 1998 McCutcheon's Detergents and Emulsifiers, 1998 McCutcheon's Functional Materials, both published by McCutcheon Publishing Co., New Jersey, and the Avanti Polar Lipids, Inc. Catalog.
  • Suitable lipids for use in the present invention will have sufficient long-term stability to achieve an adequate shelf-life.
  • Factors affecting lipid stability are well-known to those of skill in the art and include factors such as the source (e.g. synthetic or tissue-derived), degree of saturation and method of storage of the lipid.
  • Treatment and “treating” as used herein refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.
  • nanoparticles that include a therapeutic agent may be administered to a subject for the purpose of reducing the size of a tumor, reducing or inhibiting local invasiveness of a tumor, or reducing the risk of development of metastases.
  • therapeutic benefit or “therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition.
  • a reduction in the frequency or severity of the signs or symptoms of a disease For example, reduction in the size of a tumor.
  • Prevention and “preventing” are used according to their ordinary and plain meaning to mean “acting before” or such an act.
  • those terms refer to administration or application of an agent, drug, or remedy to a subject or performance of a procedure or modality on a subject for the purpose of blocking the onset of a disease or health-related condition.
  • a subject at risk of developing cancer may be administered an effective amount of a composition comprising nanoparticles of the present invention to reduce the risk of development of the cancer compared to the risk in a subject that did not receive nanoparticles.
  • Determining prognosis refers to predicting the likelihood that a subject with have a certain course or outcome of a disease. For example, in some embodiments determining prognosis involves determining likelihood of reduced survival or likelihood of tumor growth.
  • Certain embodiments of the present invention concern methods of treating or preventing disease in a subject involving administration of nanoparticles of the present invention.
  • the disease may be any disease that can affect a subject.
  • diseases include hyperproliferative disease, an inflammatory disease, a degenerative disease, or an infectious disease.
  • the disease is a hyperproliferative disease.
  • the disease is cancer.
  • the cancer can be any cancer.
  • the cancer may be a solid tumor, metastatic cancer, or non-metastatic cancer.
  • the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer is human ovarian cancer.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • compositions comprising nanoparticles of the present invention.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (Remington's, 1990). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
  • compositions used in the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions, and these are discussed in greater detail below.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • compositions comprising nanoparticles may be extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate.
  • the active compounds will then generally be formulated for administration by any known route, such as parenteral administration.
  • compositions that are sterile solutions for intravascular injection or for application by any other route as discussed in greater detail below.
  • a person of ordinary skill in the art would be familiar with techniques for generating sterile solutions for injection or application by any other route.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients familiar to a person of skill in the art.
  • compositions may vary depending upon the route of administration.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • compositions for parenteral administration include, formulations for administration via an implantable drug delivery device, and any other form.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. A person of ordinary skill in the art would be familiar with well-known techniques for preparation of oral formulations.
  • pharmaceutical composition includes at least about 0.1% by weight of the drug.
  • the composition may include, for example, about 0.01% to about 0.1% by weight of the drug.
  • the pharmaceutical composition includes about
  • compositions set forth herein may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • parabens e.g., methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal or combinations thereof.
  • the composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that exotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
  • a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof.
  • polyol e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.
  • lipids e.g., triglycerides, vegetable oils, liposomes
  • isotonic agents such as, for example, sugars, sodium chloride or combinations thereof.
  • nasal solutions or sprays, aerosols or inhalants in the present invention.
  • Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays.
  • Sterile injectable solutions are prepared by incorporating the nanoparticles in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by sterilization.
  • nanoparticles Upon formulation, nanoparticles will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the nanoparticles can be administered to the subject using any method known to those of ordinary skill in the art.
  • a pharmaceutically effective amount of a composition comprising nanoparticles may be administered intravenously, intracerebrally, intracranially, intrathecally, into the substantia nigra or the region of the substantia nigra, intradermally, intraarterially, intraperitoneally, intralesionally, intratracheally, intranasally, topically, intramuscularly, intraperitoneally, subcutaneously, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination
  • a pharmaceutically effective amount of the nanoparticles is determined based on the intended goal, for example inhibition of cell death.
  • the quantity to be administered depends on the subject to be treated, the state of the subject, the protection desired, and the route of administration. Precise amounts of the therapeutic agent also depend on the judgment of the practitioner and are peculiar to each individual.
  • a dose of the drug may be about 0.0001 milligrams to about 1.0 milligrams, or about 0.001 milligrams to about 0.1 milligrams, or about 0.1 milligrams to about 1.0 milligrams, or even about 10 milligrams per dose or so. Multiple doses can also be administered.
  • a dose is at least about 0.0001 milligrams.
  • a dose is at least about 0.001 milligrams.
  • a dose is at least 0.01 milligrams.
  • a dose is at least about 0.1 milligrams.
  • a dose may be at least 1.0 milligrams.
  • a dose may be at least 10 milligrams.
  • a dose is at least 100 milligrams or higher.
  • a dose of a drug may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
  • the dose can be repeated as needed as determined by those of ordinary skill in the art.
  • a single dose is contemplated.
  • two or more doses are contemplated.
  • the time interval between doses can be any time interval as determined by those of ordinary skill in the art.
  • the time interval between doses may be about 1 hour to about 2 hours, about 2 hours to about 6 hours, about 6 hours to about
  • Certain embodiments of the present invention provide for the administration or application of one or more secondary forms of therapies for the treatment or prevention of a disease.
  • the disease may be a hyperproliferative disease, such as cancer.
  • the secondary form of therapy may be administration of one or more secondary pharmacological agents that can be applied in the treatment or prevention of cancer. If the secondary therapy is a pharmacological agent, it may be administered prior to, concurrently, or following administration of the nanoparticles.
  • the interval between the administration of the nanoparticles and the secondary therapy may be any interval as determined by those of ordinary skill in the art.
  • the interval may be minutes to weeks.
  • the agents are separately administered, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that each therapeutic agent would still be able to exert an advantageously combined effect on the subject.
  • the interval between therapeutic agents may be about 12 h to about 24 h of each other and, more preferably, within about 6 hours to about 12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several d (2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
  • the timing of administration of a secondary therapeutic agent is determined based on the response of the subject to the nanoparticles.
  • any compound or therapy of the present invention to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described therapy.
  • a standard therapy will include chemotherapy, radiotherapy, immunotherapy, surgical therapy or gene therapy and may be employed in combination with the inhibitor of gene expression therapy, anticancer therapy, or both the inhibitor of gene expression therapy and the anti-cancer therapy, as described herein.
  • chemotherapeutic agents may be used in accordance with the present invention.
  • the term "chemotherapy” refers to the use of drugs to treat cancer.
  • a "chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer.
  • agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle.
  • an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
  • Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • SERMs selective estrogen receptor modulators
  • aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole
  • anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3- diox
  • Radiotherapy Other factors that cause DNA damage and have been used extensively include what are commonly known as ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • Other forms of DNA damaging factors are also contemplated such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287) and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • the terms "contacted" and "exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing, for example, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing. 3.
  • immunotherapeutics In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • Trastuzumab (HerceptinTM) is such an example.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • toxin chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells. The combination of therapeutic modalities, i.e., direct cytotoxic activity and inhibition or reduction of ErbB2 would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.
  • Immune stimulating molecules also exist including: cytokines such as IL-2, IL-4, IL- 12, GM-CSF, gamma-IFN, chemokines such as MIP-I, MCP-I, IL-8 and growth factors such as FLT3 ligand.
  • cytokines such as IL-2, IL-4, IL- 12, GM-CSF, gamma-IFN
  • chemokines such as MIP-I, MCP-I, IL-8 and growth factors such as FLT3 ligand.
  • Combining immune stimulating molecules, either as proteins or using gene delivery in combination with a tumor suppressor has been shown to enhance anti-tumor effects (Ju et al, 2000).
  • antibodies against any of these compounds can be used to target the anti-cancer agents discussed herein.
  • immunotherapies currently under investigation or in use are immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998;
  • cytokine therapy e.g., interferons ⁇ , ⁇ and ⁇ ; IL-I, GM-CSF and TNF (Bukowski et al, 1998; Davidson et al, 1998; Hellstrand et al, 1998) gene therapy, e.g., TNF, IL-I, IL-2, p53 (Qin et al, 1998; Austin-Ward and Villaseca, 1998; U.S. Patents
  • anti-ganglioside GM2 anti-HER- 2, anti-pl85
  • anti-cancer therapies may be employed with the gene silencing therapies described herein.
  • an antigenic peptide, polypeptide or protein, or an autologous or allogenic tumor cell composition or "vaccine” is administered, generally with a distinct bacterial adjuvant (Ravindranath and Morton, 1991; Morton et al, 1992; Mitchell et al, 1990; Mitchell et al, 1993).
  • the patient's circulating lymphocytes, or tumor infiltrated lymphocytes are isolated in vitro, activated by lymphokines such as IL-2 or transduced with genes for tumor necrosis, and readministered (Rosenberg et al, 1988; 1989). 4. Surgery
  • Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well. 5.
  • Other Agents may be of varying dosages as well. 5.
  • agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment.
  • additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents.
  • Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-I, MIP-lbeta, MCP-I, RANTES, and other chemokines.
  • cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyerproliferative efficacy of the treatments.
  • Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention.
  • Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.
  • hyperthermia is a procedure in which a patient's tissue is exposed to high temperatures (up to 106 0 F).
  • External or internal heating devices may be involved in the application of local, regional, or whole-body hyperthermia.
  • Local hyperthermia involves the application of heat to a small area, such as a tumor. Heat may be generated externally with high-frequency waves targeting a tumor from a device outside the body. Internal heat may involve a sterile probe , including thin, heated wires or hollow tubes filled with warm water, implanted microwave antennae, or radio frequency electrodes.
  • a patient's organ or a limb is heated for regional therapy, which is accomplished using devices that produce high energy, such as magnets.
  • some of the patient's blood may be removed and heated before being perfused into an area that will be internally heated.
  • Whole-body heating may also be implemented in cases where cancer has spread throughout the body. Warm- water blankets, hot wax, inductive coils, and thermal chambers may be used for this purpose.
  • Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described.
  • the use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.
  • a kit is envisioned containing nanoparticles or ingredients for the formation of nanoparticles of the present invention in one or more suitable container means.
  • a suitable container means is a container that will not react with components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube.
  • the container may be made from sterilizable materials such as plastic or glass.
  • the kit includes a composition comprising nanoparticles in one or more container means.
  • the kit includes a single container means that comprises an SF polypeptide or a solution comprising an SF polypeptide, and a separate container means that comprises a drug.
  • the kit includes one or more therapeutic or diagnostic agents.
  • the one or more therapeutic or diagnostic agents may be in the same container means with the polyphosphate and/or chitosan.
  • the kit may further comprise a lipid in a suitable container means.
  • the kit may further include an instruction sheet that outlines the procedural steps of the methods, and will follow substantially the same procedures as described herein or are known to those of ordinary skill.
  • raw silk was degummed in 0.25% (w/v) sodium carbonate and 0.25% (w/v) sodium dodecylsulfate (Sigma-Aldrich) for 1 hour at 100 0 C and then dissolved in calcium nitrate tetrahydrate and methanol solution (molar ratio of 1 :4:2 Ca:H 2 O:MeOH) at 65°C.
  • Chitosan was dissolved separately in 2% acetic acid solution at the same concentration as silk fibroin solution and was mixed together to prepare a particular blend. For example, three parts of SF were mixed with one part of CS to prepare 75:25 SFCS. The SFCS solution was then dialyzed against ultra-pure water for 4 days and filtered. The final solution was 10% (w/v) of SF or SFCS and diluted 100 times to make 0.1% solutions.
  • the SFCS coating was crystallized by suspending nanoparticles in 0.5 ml of 50:50 methanol and IN sodium hydroxide solution (for SF coating, only methanol was used) for 15 minutes (Gobin et al, 2006). The suspension was centrifuged at 10,000 rpm for 10 minutes and supernatant removed. The pellet containing nanoparticles was rinsed with PBS twice to remove remaining methanol and sodium hydroxide. PBS rinses were performed by adding 0.5 ml of PBS to the nanoparticles and centrifuging at 10,000 rpm for 10 minutes. After the second rinse, the SFCS coated curcumin nanoparticles were suspended in PBS for further analysis.
  • TEM Transmission electron microscopy
  • SFCS coated curcumin samples suspended in PBS were imaged using a JEM 1010 transmission electron microscope (TEM; JEOL USA Inc., Peabody, MA). Samples were placed on formver-coated and carbon-coated, copper grids treated with poly-L-lysine for 1 hour. The samples were then blotted dry and imaged. The size of the nanoparticles from TEM images was measured using ImageJ software. Curcumin entrapment efficiency and release. After collecting the nanoparticles from glass slides, the methanol/sodium hydroxide rinse and 2 subsequent PBS rinses (as defined above) were collected to calculate entrapment efficiency.
  • the curcumin that was not entrapped (not coated with SFCS) was completely soluble in organic solvent and hence was collected in methanol rinses.
  • the amount of curcumin in the samples was measured by reading the absorbance at 450 nm using UV spectrophotometer (ThermoSpectronics, Rochester, NY) and calculated from curcumin standard in ethanol (Bisht et al., 2007).
  • the nanoparticle suspension was centrifuged at 10,000 rpm for 10 minutes and absorbance was measured in the supernatant. The pellet was again suspended in PBS for the next time point and kept at 37°C shaker. Cell Culture.
  • MCF-7 Breast cancer cell lines MCF-7 (Her2-) and MDA-MB-453 (Her2+) were obtained from American Type Culture Collection (ATCC, Manassas, VA). MCF-7 cells were cultured in Dulbecco Modified Eagles Medium with F- 12 (Invitrogen, Grand Island, NY) and MDA-MB-453 cells in Leibovitz's L-15 medium (ATCC). Both culture mediums were supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and 1% antibiotics (Invitrogen).
  • MCF-7 and MDA-MB 453 cells were seeded in 96-well plates (2000 cells/well) and incubated overnight.
  • SFCS or SF coated nanocurcumin was added to each well at a concentration of 83.3 ⁇ g curcumin/well and incubated for 4 days.
  • the nanoparticles containing medium were removed from the wells.
  • Cells were lysed in 100 ⁇ l of dimethyl sulfoxide (DMSO, Fisher Scientific, Pittsburg, PA). The 50 ⁇ l cell lysis suspension was reserved for absorbance measurement and the other 50 ⁇ l for fluorescence measurements using a VersaFluor fluorometer (Bio-Rad Laboratories, Hercules, CA).
  • Curcumin has auto-fluorescence properties (Kunwar et al., 2006), therefore, fluorescence assays were used in conjunction with absorbance to confirm the curcumin uptake measurements.
  • Cell viability assay To measure the efficacy of nanocurcumin on MCF-7 and MDA- MB 453, the cells were seeded in 96-well plates (2000 cells/well) and incubated overnight. Nanocurcumin coated with SFCS or SF was added to each well at a concentration of 83.3 ⁇ g curcumin/well and incubated for 4 days. The nanoparticles containing medium were removed from the wells and a CellQuanti-MTT cell viability assay kit (BioAssay Systems, Hayward, CA) was used to determine the viability of cells remaining in each well. Briefly, 80 ⁇ l of culture medium and 15 ⁇ l of MTT reagent were added to each well and incubated for 4 hours.
  • MTT solubilization solution 100 ⁇ l was added to each well and plates were placed on a shaker for 1 hour. The absorbance readings were taken at 570 nm using MRX Microplate Reader (Dynex Technologies, Guernsey, Channel Islands, Great Britain).
  • Nanoparticle Size Measurement SF or SFCS coated curcumin particles were imaged using TEM to characterize the size. The shape of the nanoparticles was mostly round and square. Size measurements of curcumin particles from TEM images show that all formulations created particles of size less than 100 nm except 0.1% 50:50 SFCS (130 ⁇ 4.2 nm) as shown in FIG. 2. Particle size of curcumin coated with 0.1% SF was significantly lower than 0.1% 50:50 SFCS (p ⁇ 0.001) and higher than 0.1% 25:75 SFCS and 0.1% 75:25 SFCS (p ⁇ 0.001) coated particles. The particle size for 10% 75:25 SFCS was significantly higher than 10% SF and 10% 25:75 and 10% 50:50 SFCS (p ⁇ 0.001).
  • Curcumin Entrapment Efficiency The entrapment of curcumin was more than 96% for SF coated nanoparticles for both 0.1% and 10% SF. The entrapment efficacy decreased to 64-73% for SFCS coated nanoparticles regardless of concentration of SF and CS (FIG. 2).
  • Curcumin entrapment was significantly higher for 0.1% SF and 10% SF compared to all
  • M t and M 00 are the amounts of drug at any time t and at infinite time, respectively, k and t are the constants and exponent dependent upon the composition/structure of the coating and release mechanism.
  • Table 1 showed the values of n and k for all the nanoparticle formulations. For n ⁇ 0.5, the drug release mechanism is diffusion based (Seipmann and
  • Curcumin Nanoparticle Efficacy on Breast Cancer Cells The efficacy of curcumin nanoparticle formulations was measured on both MCF-7 and MDA-MB-453 cells using MTT assay as shown in FIG. 7. The number of cells in control samples (no nanoparticles) increased from 2000 (initial density) to 3563 ⁇ 215 (MCF-7) and 3267 ⁇ 864 (MDA-MB-453) over a period of 4 days. Exposure of 0.1% SF and 10% SF nanocurcumin to MCF-7 and MDA-MB- 453 cells significantly decreased number of viable cells compared to controls (p ⁇ 0.01).
  • the objective of this study was to prepare nanoparticles for embedding within silk f ⁇ broin/chitosan-tissue flap composite used to fill defects after tumor resection in cancer patients. Emodin loaded liposomes were prepared, and these liposomes were entrapped within an SFCS composite. Liposome size release and drug release were examined.
  • Emodin is a selective receptor tyrosine kinase inhibitor. It is effective for HER-2/neu over expressing breast cancer cells. It functions to sensitize cells to other chemotherapeutic agents.
  • Liposomes are phospholipid bilayer microscopic vesicles. Drugs may be incorporated within the lipid bilayer or in the hydrophilic core. Liposomes are biodegradable, have high tissue targeting, lack immunogenicity and have low toxicity.
  • Emodin loaded liposomes were sonicated at different times (0 min, 5 min, 15 min, 30 min, and 60 min) to reduce particle size. Sonication was found to be effective for reducing liposome size (FIG. 14). Liposome size was found to be less than 100 nm after 5 minutes of sonication. There was found to be a higher emodin entrapment and release with sonication (FIG. 15). Entrapment efficiency was significantly lower without sonication (*p ⁇ 0.01 vs. all sonication time points). A higher drug release was also associated with sonication (FIG. 15). * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

Abstract

La présente invention concerne des nanoparticules pour l’administration de médicaments et/ou nutraceutiques qui comprennent un polypeptide de fibroïne et un médicament ou nutraceutique, où la nanoparticule a un diamètre d’environ 1 nm à environ 500 nm, et des compositions des nanoparticules. Les nanoparticules peuvent comprendre en outre un chitosane, ou un protéoglycane tel que la décorine. La présente invention concerne en outre des procédés d’administration d’un médicament et/ou nutraceutique à un sujet qui mettent en œuvre l’administration au sujet de nanoparticules de la présente invention. La présente invention concerne en outre des procédés de production des nanoparticules de l’invention, et des trousses qui comprennent des nanoparticules de l’invention.
PCT/US2009/065374 2008-11-21 2009-11-20 Préparation et méthodologie de nanoparticules de fibroïne de soie WO2010059963A2 (fr)

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