Nanoparticles as delivery vehicles of active ingredients and methods for the production thereof

Abstract

A nanoparticle comprising a keratin polypeptide and at least one lipophilic active ingredient, wherein the at least one lipophilic active ingredient is non-covalently bound to the keratin polypeptide and can be e.g. a therapeutic or diagnostic agent, a nutraceutical, a cosmetic ingredients, a dye or a cosmeceutical, the keratin polypeptide being water-soluble; optionally, the nanoparticle further comprises at least one hydrophilic active ingredient non-covalently bound to the keratin polypeptide, which can be e.g. a therapeutic or diagnostic agent, a nutraceutical, a cosmetic ingredients, a dye or a cosmeceutical.

Claims

1. A nanoparticle comprising a keratin polypeptide and at least one lipophilic active ingredient, wherein said at least one lipophilic active ingredient is non-covalently bound to said keratin polypeptide, said keratin polypeptide is water-soluble, and said keratin polypeptide is a wild-type keratin polypeptide having a molecular weight higher than 14 kDa or a wild-type keratin polypeptide, having a molecular weight higher than 14 kDa which has been modified by chemical functionalization of the N-terminus with organic compounds and/or chemical functionalization of amino acid side chains therein with hydroxyl, amino and thiol groups.

2. The nanoparticle according to claim 1, wherein said at least one lipophilic active ingredient is selected from the group consisting of therapeutic agents, diagnostic agents, nutraceuticals, cosmetic ingredients, dyes and cosmeceuticals.

3. The nanoparticle according to claim 1, wherein said nanoparticle further comprises at least one hydrophilic active ingredient non-covalently bound to said keratin polypeptide.

4. The nanoparticle according to claim 3, wherein said at least one hydrophilic active ingredient is selected from the group consisting of therapeutic agents, diagnostic agents, nutraceuticals, cosmetic ingredients, dyes and cosmeceuticals.

5. The nanoparticle according to claim 2, wherein said at least one lipophilic active ingredient is a therapeutic agent selected from the group consisting of anticancer drugs, anti-inflammatory agents, antimicrobial agents, analgesics, hormones, anaesthetic agents, antianginals; anti-arrhythmic drugs; antibacterial and antiprotozoal agents; anti-coagulants; antidepressants; anti-diabetic drugs; anti-epileptic drugs; antifungal agents; antihistamines; anti-hypertensive drugs; anti-muscarinic agents; antineoplastic agents; anti-migraine drugs; anti-parasitic agents; anti-Parkinsonian drugs; antipsychotic, hypnotic and sedating agents; anti-stroke agents; anti-thrombotic agents; antitussives; antivirals; beta-adrenoceptor blocking agents; calcium channel blockers; cardiac inotropic agents; contraceptive agents; corticosteroids; dermatological agents; diuretics; gastro-intestinal agents; haemostatics; local anaesthetics; opioid analgesics; parasympathomimetics; peptides; steroids; stimulating agents; and vasodilators.

6. The nanoparticle according to claim 5, wherein said therapeutic agent is an anticancer drug, optionally selected from the group consisting of methotrexate, cisplatin; topotecan; 5-fluorouracil, thiotopotecan, thiocamptothecin, camptothecin, paclitaxel, docetaxel, doxorubicin, bicalutamide, MDV3100 enzalutamide, 9-hydroxyl stearic acid, curcumin, irinotecan hydrochloride, vinblastine, histone methyltransferases inhibitors, carboplatin, methylene blue, Azure A (N,N-dimethylthionine chloride), porphyrins and chlorins.

7. The nanoparticle according to claim 4, wherein said at least one hydrophilic active ingredient is a therapeutic agent selected from the group consisting of anticancer drugs, anti-inflammatory agents, antimicrobial agents, analgesics, hormones, anaesthetic agents, antianginals; anti-arrhythmic drugs; antibacterial and antiprotozoal agents; anti-coagulants; antidepressants; anti-diabetic drugs; anti-epileptic drugs; antifungal agents; antihistamines; anti-hypertensive drugs; anti-muscarinic agents; antineoplastic agents; anti-migraine drugs; anti-parasitic agents; anti-Parkinsonian drugs; antipsychotic, hypnotic and sedating agents; anti-stroke agents; anti-thrombotic agents; antitussives; antivirals; beta-adrenoceptor blocking agents; calcium channel blockers; cardiac inotropic agents; contraceptive agents; corticosteroids; dermatological agents; diuretics; gastro-intestinal agents; haemostatics; local anaesthetics; opioid analgesics; parasympathomimetics; peptides; steroids; stimulating agents; and vasodilators.

8. The nanoparticle according to claim 1, wherein said nanoparticle contains an amount of said at least one lipophilic active ingredient ranging from 5% to about 45% by weight of the weight of said nanoparticle.

9. The nanoparticle according to claim 1, wherein said nanoparticle has a diameter between 1 nm to 400 nm, or between 100-200 nm.

10. A pharmaceutical composition comprising the nanoparticles according to claim 5, and a pharmaceutically acceptable carrier.

11. A pharmaceutical formulation comprising the composition according to claim 10 for parenteral or oral administration.

12. A pharmaceutical composition comprising the nanoparticles according to claim 6, and a pharmaceutically acceptable carrier.

13. A pharmaceutical formulation comprising the composition according to claim 12 for parenteral or oral administration.

14. A cosmetic formulation for topical application, comprising the nanoparticles according to claim 4 and a cosmetically acceptable vehicle.

15. A cosmetic formulation for topical application, comprising the nanoparticles according to claim 2, and a cosmetically acceptable vehicle.

16. A functional food comprising the nanoparticles according to claim 1, wherein the functional food is selected from the group consisting of cereal bars, yogurt dairy products, bakery products, fruit juices and drinks.

17. A method for producing the nanoparticle according to claim 1, comprising: a) dissolving the keratin polypeptide in water or in a buffered aqueous solution at concentrations ranging from 1 to 5 mg/mL at room temperature, thereby obtaining a solution; b) filtering said solution; and c) adding said at least one lipophilic active ingredient previously dissolved in an organic solvent to said solution obtained in step b) under stirring.

18. A method of treating a patient suffering from a neoplastic disease, which comprises administering to the patient a therapeutically effective amount of the nanoparticle according to claim 6, wherein said neoplastic diseases are selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, colorectal cancer, pancreatic cancer, gastrointestinal stromal tumor, adrenal cancer, skin melanoma, non-skin melanoma, mouth cancer, eye tumor, nasal cavity and paranasal sinus cancer, penile cancer, bronchial carcinoma, heart cancer, uterine body cancer, cervical cancer, esophageal cancer, liver cancer, metastatic pancreatic cancer, lymphomas, Hodgkin lymphoma, non-Hodgkin lymphoma, cutaneous lymphoma, pediatric lymphomas, laryngeal cancer, pharyngeal cancer, malignant mesothelioma, leukemias, hairy cell leukaemia, chronic lymphocytic leukaemia, Acute lymphoblastic leukaemia, acute myeloid leukaemia, chronic myeloid leukaemia, pediatric leukemia, multiple myeloma, sarcomas, gliomas, renal cancer, testicular cancer, thyroid cancer, soft tissue sarcoma, bone sarcoma, Ewing sarcoma, Kaposi sarcoma, extraskeletal Ewing sarcoma, chondrosarcoma, osteosarcoma, metastatic bone cancer, choriocarcinoma, pineal gland cancer, salivary gland tumors, hypophysis cancer, primitive neuroectodermal tumour, multiple endocrine neoplasia type 1 (MEN1), Multiple Endocrine Neoplasia Type 2 (MEN2), bladder cancer, tumors of the pelvic organs, ureteral cancer, oral cavity cancer, anal cancer, vulvar cancer, vaginal cancer, spleen cancer, brain tumors, embryonal tumors, gall-bladder cancer, bile duct cancer, cancer cachexia, neuroblastoma cancer, pediatric neuroblastoma cancer, neuroendocrine cancer, pediatric tumors and Myeloproliferative neoplasms.

19. A method comprising administering the nanoparticle according to claim 5 by topical application to a patient.

20. A method comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition according to claim 10 to a patient by topical application.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The following figures form part of the present invention and are included to further demonstrate certain aspects of the invention. The invention may be better clarified if referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

(2) FIG. 1 shows the TEM images of taxol keratin nanoparticles at different magnifications. Images were recorded on a Gatan MSC794 Transmission Electron Microscope.

(3) FIG. 2 shows a graph which represents the correlation between taxol loading (expressed in percentage weight ratio between taxol and keratin) and keratin nanoparticles dimensions (nm).

(4) FIG. 3 represents a graph showing the stability of KNPs-PTX depending on the nanoparticles dimensions (nm), time (h) and polydispersity index (PDI), in physiological conditions.

(5) FIG. 4 represents a graph showing the percentage release of doxorubicin and taxol from keratin nanoparticles obtained by aggregation method.

(6) FIG. 5 shows a schematic representation of preparation of keratin nanoparticles loaded with lipophilic active ingredients through “aggregation induced by lipophilic compound”.

(7) FIG. 6 shows a schematic representation of preparation of keratin nanoparticles loaded with lipophilic and hydrophilic active ingredients through “aggregation induced by lipophilic compound”.

(8) FIGS. 7A and 7B represent bar graphs showing the IC.sub.50 values (A) and DOX accumulation levels (B) obtained on MCF7 and MDA-MB 231 cells following 72 h exposure to KNPs-DOX and DOX. (*p<0.05 vs MCF7 and DOX same cell line; *** p<0.0001 vs MCF7; ° p<0.01 vs DOX and KNPs-DOX same cell line, @ p<0.05 vs control; #p<0.05 vs all the others same cell line; § p<0.001 vs MCF7 and control.

(9) FIGS. 8A and 8B represent bar graphs showing the effects of PTX and KER-NPs-PTX on MCF-7 (A) and MDA MB 231 (B) cell proliferation in 2D model. Cell proliferation, after exposure to increasing concentrations of PTX (0.00002, 0.02, 5 μg/ml), was evaluated at 72 h by APH assay. Statistical significance versus untreated cells (represented by a dotted line): ** p<0.01, *** p<0.001. The y axis reports the percent of cell growth with respect to untreated cells (control), obtained by WST-1 absorbance.

(10) FIG. 9 shows a bar graph showing the number of alive HT29 cells after treatment with 9-HSA as free or loaded on keratin nanoparticles.

(11) FIG. 10 shows bar graphs showing the cell cycle analysis on HT29 cells treated with 9-HSA as free or loaded on keratin nanoparticles.

(12) FIG. 11 shows a graph representing the interaction between PTX with keratin, that was determined by real-time fluorescence spectroscopy. The excitation wavelength was 277 nm. The fluorescence spectra show the reduction in the fluorescence intensity of keratin by subsequent addition of PTX.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

(13) The present invention is based on the finding that keratin nanoparticles can be applied for the incorporation and delivery of at least one or more hydrophobic or of at least one or more hydrophobic and hydrophilic active ingredient. Moreover, keratin nanoparticles, as compared to other polypeptides and proteins, allow to stably incorporate high amount of the selected active ingredient, and to be easily re-suspended in green solvents such as water. As an example, taxol-keratin nanoparticles can efficiently cross tumour cells membrane and deliver the drug mainly at the target site. As a further example, vitamin A-keratin nanoparticles can be incorporated into cosmeceutical and nutraceuticals formulation in order to favour its uptake from cells and tissues.

(14) A. Keratin and Modified Keratin

(15) Keratin as used herein refers to a “polypeptide” comprising the term protein, which is constituted by a consecutive series of 2 or more amino acids and as used herein, and in some embodiments, the polypeptide comprises a consecutive series of at least 10 to 800 amino acids.

(16) The polypeptide has at 50% to 99% sequence homology to a known wild type keratin protein.

(17) The expression “modified keratin” as used herein refers to a keratin polypeptide including one or more additional amino acid residues at the C-terminus or N-terminus of the consecutive sequence of amino acids of the keratin polypeptide.

(18) In particular, the modified keratin polypeptide has at least 50% to 99% sequence homology to a known wild type keratin protein; the keratin polypeptide may include a chemical functionalization with organic compounds at the N-terminus or hydroxyl, amino and thiol groups of aminoacid side chains.

(19) B. Uses of Keratin and Keratin-Modified Nanoparticles

(20) In general, the nanoparticles of the present invention include one or more hydrophobic active ingredient or at least one or more hydrophobic and hydrophilic active ingredient. An active ingredient may be a therapeutic agent, a diagnostic agent, a nutraceutical compound, a cosmeceutical compound, a cosmetic ingredient, a dye, an organic or inorganic compounds or a combination thereof to be either delivered to cells and tissues, or to be included into composites and advanced functional materials.

(21) B.1. Hydrosoluble keratin and hydrosoluble modified-keratin nanoparticles for delivering lipophilic drugs and/or prodrugs (up to 4 different active ingredients): taxol, taxotere, doxorubicin, bicalutamide, 9-hydroxyl stearic acid, MDV3100, methotrexate, cisplatin; topotecan; 5-fluorouracil, thiotopotecan, thiocamptothecin, camptothecin, levodopa.

(22) B.2. Hydrosoluble keratin and hydrosoluble keratin-modified for delivering (formulating) lipophilic cosmeceuticals and cosmetic ingredients (up to 4 different cosmetic ingredients): enalapril, levodopa, pivampicillin, oseltamivir, tenofovir disoproxil, ximelagatran, MGS0210, fosfluconazole, propofol phosphate, valganciclovir, latanoprost, tazarotene.

(23) B.3. Hydrosoluble keratin and hydrosoluble keratin-modified for delivering (formulating) lipophilic nutraceuticals (up to 4 different nutraceuticals): vegetable/animal oils, vitamins, cholesterol, creatine, mineral salts, beta-carotene, flavonoids, vegetable or yeast extracts, hyaluronic acid, inositol, herbs.

(24) B.5 Hydrosoluble keratin and hydrosoluble keratin-modified for delivering (formulating) lipophilic cosmeceuticals (up to 4 different cosmeceuticals): vitamin A, lipoic acid, dimethyl amino ethanol, glycolic acid, salicylic acid, hyaluronic acid.

(25) B.4. Hydrosoluble keratin and hydrosoluble keratin-modified incorporating lipophilic dyes and active ingredients as additives for plastics and paints (up to 4 different dyes): porphyrins, phtalocyanines, fluorescent dyes, such as fluorescein, organic and inorganic pigments.

(26) B.5 Hydrosoluble keratin and hydrosoluble keratin-modified for delivering a combination of hydrophilic, such as doxorubicin hydrochloride, methylene blue or AzureA and lipophilic drugs and prodrugs (point B.1) (up to 4 different compounds)

(27) B.6 Hydrosoluble keratin and hydrosoluble keratin-modified for delivering a combination of products reported in B1 to B5.

(28) C. Methods

(29) The present patent application describes nanoparticles of hydrosoluble keratin or chemically modified hydrosoluble keratin for the incorporation of active ingredients through different techniques.

(30) According to the present invention, the nanoparticles include:

(31) i) a hydrosoluble keratin or a chemically modified hydrosoluble keratin polypeptide and ii) one or more lipophilic in combination or not with hydrophilic active ingredients, such as, but not limited to, a therapeutic compound, a prodrug, a nutraceutical, a cosmetic, an imaging agent, an antibacterial compound, a food supplement, a dye, an organic or inorganic compound, wherein the nanoparticles have a diameter of about 1 nm to about 500 nm, preferably between 100-200 nm.

(32) Hydrosoluble keratin may be obtained from different sources, but in some particular embodiments hydrosoluble keratin is obtained from raw wool.

(33) The method for incorporating the active ingredients into keratin nanoparticles may be selected among the following:

(34) i) Aggregation method induced by a lipophilic active ingredient: one or more lipophilic active ingredients (1 to 4) are dissolved into the minimal amount of a properly selected organic solvent and added to a water solution of hydrosoluble keratin, as shown in FIG. 5;

(35) ii) Aggregation method induced by a lipophilic active ingredient in presence hydrophilic active ingredients: one or more lipophilic active ingredients are dissolved into the minimal amount of a properly selected organic solvent and added to a water solution of hydrosoluble keratin possibly containing a hydrophilic active ingredient, as shown in FIG. 6;

(36) iii) Ionic gelation/aggregation: the hydrophilic ingredient is dissolved in water at a proper concentration and added to keratin solution in order to induce nanoparticles formation by ionic gelation; after that the lipophilic drug dissolved in proper solvent is added to the gelated nanoparticles in order to complete the nanoparticle formation by aggregation

(37) In particular, for methods i) and ii), depending on the concentration of the active ingredients and of the keratin solution, the formation of the hydrosoluble keratin nanoparticles is induced by the lipophilic active ingredient, which acts as aggregating element.

(38) All the described preparation methods allow to avoid dialysis, ultrafiltration and other possible purification procedures, while providing a maximum active ingredient loading efficiency.

EXAMPLES

Example 1

(39) Materials and Methods. Raw wool was kindly donated from Cariaggi Fine Yarns, S.p.A. Keratin was extracted from raw wool by sulphitolysis reaction. Briefly, a fiber sample, withdrawn from a combed sliver and cleaned by Soxhlet extraction with petroleum ether, was washed with distilled water and dried at 21° C. and 60% relative humidity overnight. Afterward, cleaned fibers (5 g) were cut into snippets and dispersed in 100 mL of aqueous solution containing urea (8 M), sodium metabisulphite (0.5 M) and sodium dodecyl sulfate (SDS, 0.1 M), under mechanical shaking at 65° C. overnight. The mixture was filtered with a vacuum filter (10-16 μm cut-off), dialyzed against distilled water using a cellulose tube (molecular weight cut-off 12-14 kDa) for 3 days at room temperature, changing the distilled water four times a day. The resulting aqueous solution was freeze-dried in order to obtain pristine keratin powder.

(40) General Method for the Preparation of Nanoparticles Through Aggregation Method.

(41) The lipophilic compound was used as aggregating agent, able to induce the formation of hydrosoluble keratin nanoparticles. Keratin was extracted from raw wool according to a previously reported literature procedure [Materials & Design 2016, 110, 475-484].

(42) In this method, keratin was dissolved in water (pH 6.5) or PBS (pH 5.5) or PBS (pH 7.4) or NaHCO.sub.3 buffer (pH 9) at concentrations ranging from 1 to 10 mg/mL at room temperature for at least 1 h.

(43) A desired amount of lipophilic compound dissolved in a proper solvent (compound concentration from 1 mg/mL to 10 mg/mL) was added to the keratin solution, under stirring at 750 rpm.

(44) Specific, although non-comprehensive, examples of lipophilic compounds used for the preparation of hydrosoluble keratin nanoparticles (h-KNPs) through aggregation method are listed in Table 1 along with nanoparticles sizes and poly-dispersity index (PDI), as well as the maximum drug loading expressed as % in weight of drug with respect to nanoparticles.

(45) TABLE-US-00001 TABLE 1 List of keratin nanoparticles obtained by aggregation and loaded with lipophilic active ingredients Loading Particle Starting Lipophilic (% drug/ size Entry solution drug keratin) (nm)/PDI 1 Keratin solution β-Carotene 5.66 180/0.15 1 mg/mL; pH = in THF 6.5 (1.5 mg/mL) 2 Keratin solution Vitamin E 10 115/0.11 1 mg/mL; pH = in EtOH 6.5 (4.5 mg/mL) 3 Keratin solution 9-HSA in EtOH 6.7 245/0.03 1 mg/mL; pH = (10 mg/mL) 9 4 Keratin solution PTX in EtOH 28.6 150/0.15 1 mg/mL; pH = (10 mg/mL) 6.5 or 7.4 5 Keratin solution PTX in EtOH 43 165/0.15 1 mg/mL; pH = (10 mg/mL) 6.5 or 7.4 6 Keratin solution PTX- 16.6 210/0.25 1 mg/mL; pH = F35 in EtOH 6.5 or 7.4 (6 mg/mL) 7 Keratin solution Doxorubicin 16.6 90/0.3 2 mg/mL; pH = in H.sub.2O 9 (1 mg/mL) 8 Keratin PTX in EtOH 16.6 155/0.15 functionalized (10 mg/mL) with Ce6 (Ker- Ce6 20 μg/mg) 9 Keratin PTX in EtOH 16.6 165/0.15 functionalized (10 mg/mL) with Ce6 (Ker- Ce6 30 μg/mg) 10 Keratin PTX- 8.8 260/0.22 functionalized F35 in EtOH with Ce6 (Ker- (10 mg/mL) Ce6 20 μg/mg) 11 Keratin solution PTX in EtOH PTX: 16.6 146/0.11 1 mg/mL; pH = (10 mg/mL) ICG: 2.8 6.5 or 7.4 Indocyanine green (ICG) 12 Keratin solution Doxorubicin DOXO: 10 170/0.25 1 mg/mL; pH = via gelation PTX: 10 or 6.5 or 7.4 PTX in EtOH 20 (10 mg/mL) 13 Keratin solution PTX in EtOH PTX: 9 138/0.19 1 mg/mL; pH = (10 mg/mL) IR780: 9 6.5 or 7.4 IR780 14 Keratin-FITC 9-HSA in EtOH FITCH: 1 180/0.2  1 mg/mL; pH = (10 mg/mL) 9-HSA: 6 6.5 or 7.4 15 Keratin solution Topotecan TPC: 9 158/0.25 1 mg/mL pH = (1 mg/mL PTX: 16.6 6.5 or 7.4 in H2O) PTX (10 mg/mL in EtOH) 16 Keratin solution Azure A AzA: 2.8 160/0.22 1 mg/mL; pH = (10 mg/mL) PTX: 13 6.5 or 7.4 PTX (10 mg/mL in EtOH)

Example 2—Preparation of Keratin Nanoparticles Loaded with Taxol Via Aggregation (KNPs-PTX)

(46) This example describes the process by which keratin nanoparticles loaded with taxol can be obtained. Keratin, was dissolved in mQ water or PBS at a concentration of 1.5 mg/mL and filtered using a 450 nm filter cut-off. Different amounts of taxol dissolved in ethanol (10 mg/mL) were added to the keratin solution, under stirring at 730 rpm. Taxol induces the aggregation of keratin into nanoparticles (KNPs-PTX), whose dimensions and polydispersity index depend on taxol loading. In particular, it was found that low drug concentration (5% wt) yielded particles having a mean diameter of about 110 nm; while, with increasing the drug amount from 10 to 30% wt, the nanoparticles dimensions increase to 138 nm. By using a taxol amount of 40% wt nanoparticles having a mean diameter of 158 nm were obtained (FIG. 2). Real-time fluorescence spectroscopy was used to monitor the loading/interaction between PTX and keratin. Fluorescence spectra, obtained by exciting the samples at 277 nm (tryptophan absorbance peak) show the reduction in the fluorescence intensity induced on keratin by subsequent addition of PTX (FIG. 11).

Example 3—Preparation of Keratin Nanoparticles Loaded with Doxorubicin Via Aggregation (KNPs-Doxorubicin)

(47) This example describes the process by which keratin nanoparticles loaded with doxorubicin can be obtained. Keratin is dissolved in carbonate buffer (pH 9) at a concentration of 4 mg/mL and filtered using a 450 nm filter cut-off. Different amounts of doxorubicin dissolved in water (1 mg/mL) were added to the keratin solution, under stirring at 730 rpm. In particular, the amount of added doxorubicin, with respect to keratin was of 15% and 30% wt. In basic environment, the hydrophobic doxorubicin induces the keratin aggregation into nanoparticles, involving the hydrophobic part of the protein. The particles dimensions depend on the doxorubicin loading. It was found that nanoparticles of about 80 and 100 nm were obtained with a doxorubicin loading of 15 and 20% wt, respectively.

Example 4—Stability of KNPs-Taxol Nanoparticles

(48) In order to evaluate the stability of KNPs-PTX in physiological conditions, 500 μg of keratin nanoparticles loaded with 16.6% wt of taxol, as obtained according to Example 2, were dissolved in 2 mL of PBS and maintained at 37° C. The nanoparticles dimension was controlled during time by dynamic light scattering measurements.

(49) A slight decrease of nanoparticles diameter from 114 nm to 124 nm occurs within the first 72 h; while no significant changes can be observed for the polydispersity index. These data suggest a good stability on KNPs-PTX nanoparticles in physiological conditions.

Example 5—Drug Release Mechanism of Keratin Nanoparticles Prepared by Aggregation Induced from Lipophilic Drug

(50) In order to test the drug release mechanism of lipophilic drug from keratin nanoparticles, the KNPs-PTX and KNPs-DOXO prepared as previously described were placed into a dialysis bag (cut-off 12-14 kDa), subsequently immersed in PBS solution at pH 7.4 at 37° C., under shaking. A little amount of ethanol was used in the case of taxol release study. Periodically, 1 mL of the outer solution was withdrawn and 1 mL of fresh buffer was added to the system. The drug concentration in the outer buffer was determined by UV-Vis spectrophotometer. It was found that, at pH 7.4, which corresponds to the physiological conditions of the blood stream, only 32% of taxol and 23% of doxorubicin were released in the first 24 h. These data indicate that, under physiological conditions no burst release of the drug occur, which might be highly beneficial in case of drug delivery where you need and elevated control over drug position and concentration. Moreover, the release processes were described with the Korsmeyer-Peppas model. In this model, the “n” value characterizes the release mechanism of the drug. As shown in FIG. 4, the “n” values for both nanoparticles are lower than 0.45 (0.31 for taxol and 0.32 for doxorubicin), indicating a Fickian diffusion mechanism of drugs. The kinetic constant related to the KNPs-PTX (k=12) is higher than that related to KNPs-DOX (k=9) indicating a faster release of taxol compared to doxorubicin.

Example 6—Preparation of Chlorine e6 (Ce6) Conjugated Keratin Nanoparticles Via Aggregation Induced by Taxol

(51) This example describes the process by which keratin powder conjugated with different amount of chlorine e6 Ce6 is used to prepared nanoparticles loaded with taxol as described in Example 2. Keratin powders covalently functionalized with different amounts of Ce6, e.g. 20, 30, 50 μg/mg, were dissolved in PBS at a concentration of 1.5 mg/mL. The aggregation is induced by adding 20% of taxol dissolved in ethanol. Dimensions of keratin nanoparticles were 155 nm for Ker-Ce6 20 μg/mg and 165 nm for Ker-Ce6 30 μg/mg.

Example 7—Preparation of Keratin Nanoparticles Loaded with Doxorubicin and Taxol by Combining the Aggregation and Ionic Gelation Methods Both Induced by Drugs

(52) This example describes the process by which keratin nanoparticles loaded with a hydrophilic drug as doxorubicin in water and a lipophilic drug as taxol can be obtained.

(53) Keratin was dissolved in mQ water at a concentration of 1 mg/mL and filtered using a 450 nm filter cut-off. Doxorubicin dissolved in water (1 mg/mL) was added to the keratin solution under stirring at 730 rpm as needed to obtain a final concentration of doxorubicin of 10% wt with respect to keratin. After stirring for 60 min, the solution was mildly centrifuged (400 g/5 min) in order to remove possible aggregates; afterwards, taxol dissolved in ethanol (10 mg/mL) was added to the keratin solution to obtain a final concentration of 20% wt with respect to keratin. The nanoparticles obtained by a combination of ionic gelation induced by doxorubicin and aggregation induced by taxol showed a final size of about 110 nm.

Example 8—In Vitro Internalization and Cytotoxicity Studies of KNPs Loaded with Doxorubicin

(54) The antiproliferative effect of the different DOX-loaded KNPs was assessed in human breast adenocarcinoma MCF7 and MDA-MB-231 cells by the MTT assay and compared to free DOX. Briefly, 3×10.sup.4 cells/ml were seeded onto 96-well plates and allowed to grow for 24 h prior to treatment with different concentrations of DOX or DOX-loaded KNPs (the range of DOX concentrations used was 5-2000 nM). After 72 h, MTT (5 DM in PBS) was added to the cells for 3 h at 37° C. Formazan crystals, formed by mitochondrial reduction of MTT, were dissolved in DMSO and the absorbance was read at 570 nm using a Universal Microplate Reader EL800 (BioTek Instruments). IC.sub.50 values were determined by using the median effect equation.

(55) Generally, IC.sub.50 is a pharmacokinetic measure of drug concentration at which 50% of the target is inhibited. Therefore, a drug with a lower IC.sub.50 value allows to achieve the desired effect using less quantity than that to be used if a drug with a higher IC.sub.50 value is provided.

(56) FIG. 7A clearly shows that doxorubicin loaded on keratin nanoparticles (DOX-KNPs) has a lower IC.sub.50 than that of free doxorubicin. This result indicates that the DOX-KNPs are more effective than free doxorubicin.

(57) Evaluation of DOX accumulation was performed on MCF-7 and MDA-MB-231 cells seeded at the density of 3×10.sup.5 cells/well in six-well plates and exposed to 1 μg/ml DOX and DOX-loaded KNPs for 2 h. Following this treatment, cells were rapidly washed with ice-cold PBS, detached with trypsin, re-suspended in ice-cold PBS, and analyzed by flow cytometry, using a Becton-Dickinson FACS Calibur equipped with a 15 mW, 488 nm, air-cooled argon ion laser. The fluorescence emission was collected through a 575 nm band-pass filter in log mode and DOX-fluorescence intensity was calculated from the flow cytometric profiles by the Cell Quest Pro software (Becton Dickinson). Control in FIG. 7B are untreated cells.

(58) FIG. 7B shows that doxorubicin loaded on keratin nanoparticles obtained according to the present invention accumulate in the targeted cell similarly to free doxorubicin. In particular, DOXO internalization results higher in MDA-MB 231 cells when loaded onto the nanoparticles as respect to free DOX

Example 9—In Vitro Activity of Keratin Nanoparticles Loaded with Paclitaxel

(59) The effect KNPs-PTX, on MCF-7 and MDA MB 231 cell growth in 2D model was evaluated by Acid phosphatase Assay Kit (APH, Sigma-Aldrich). Briefly 4.0×10.sup.3 MCF-7 and 5.0×10.sup.3 MDA MB 231 cells were seeded in 200 μl of growth medium in replicate (n=4) in a 48-well culture plate (TPP). After 48 h of cell growth, the medium was removed and the cells were incubated with the experimental medium containing KNPs-PTX at different PTX concentrations; e.g. 0.00002, 0.02 and 5 jg/ml. Seventy-two hours after treatment, APH assay was performed according manufacturer's instructions. Well absorbance was measured at 405 nm in a Synergy H1 Multi-Mode Reader (BioTek Instruments, Luzern, Switzerland).

(60) Cell death was evaluated using the Annexin V-APC and Propidium Iodide (PI) Apoptosis Detection Kit (Becton Dickinson, BD, Allschwil, Switzerland). Briefly, 1.0×10.sup.5 MCF-7 and MDA MB 231 in 2D model were treated with KER-NPs-PTX (PTX, 5 μg/mL). The cells were then washed twice with 1× annexin-binding buffer at 375 rcf for 5 min and were stained with APC-Annexin V and PI at 24 and 48 h. Samples were run on the flow cytometer at a 640 nm excitation to measure APC-Annexin V (FL4) and at 488 nm to measure PI (FL2), respectively. Any cell debris with low FSC and SSC was excluded from the analyses. Flow cytometry was carried out by a C6 flow cytometer (Accurri Cytometers, Milan, Italy) and the analysis was performed by a FCS Express 4 (BD Bioscience, Milan, Italy).

(61) As shown in FIG. 8, KER-NPs-PTX are able to efficiently delivery PTX in MCF-7 and MDA MB 231 cells. In particular, in MCF-7 cells a similar effect was observed on cellular growth induced by the two formulations at the highest concentration, i.e 5 μg/ml, 72 h after treatment. Interestingly, in MDA MB 231 cells, KER-NPs-PTX at the highest considered concentration, i.e. 5 μg/ml, were able to induced a significant reduction in cell growth (p<0.001) as compared to PTX as free (p<0.001) (FIG. 8B).

Example 10—In Vitro Activity of Keratin Nanoparticles Loaded with (R)-9-Hydroxyl Stearic Acid, (R)-9-HSA on HT29 Cells

(62) Briefly 5.0×10.sup.3 human colon adenocarcinoma cell line HT29 cells were seeded in 200 μl of growth medium in replicate (n=4) in a 48-well culture plate. After 24 h of cell growth, the medium was removed and the cells were incubated with the experimental medium containing KNPs-9HSA (50 μM of 9-HSA) and free 9-HSA. Twenty-four hours after treatment, cells were detached and counted.

(63) 9-hydroxystearic acid (9-HSA) belongs to a class of lipid peroxidation products identified in several human and murine cell lines. These products are greatly diminished in tumors compared to normal tissues and their amount is inversely correlated with the malignancy of the tumor. It is known that 9-HSA acts as a histone deacetylase 1 (HDAC1) inhibitor, thus resulting in an inhibition of proliferation together with an induction of differentiation of tumor cells.

(64) FIG. 9 shows that KNPs-9HSA as well as free 9-HSA inhibit tumor cell proliferation, thus showing that there is no lowering of the 9-HSA activity when it is loaded on keratin nanoparticles according to the present invention.

(65) Cell cycle analysis was performed as follows: 5.0×10.sup.3 HT29 cells were seeded in 200 μl of growth medium in a 48-well culture plate. After 24 h of cell growth, the medium was removed and the cells were incubated with the experimental medium containing KER-NPs-9HSA (50 μM of 9-HSA) and free 9-HSA. Twenty-four hours after treatment, cells were trypsinized, washed twice with PBS and then centrifuged. Cells pellet was re-suspended in 0.01% Nonidet-P40 (Sigma-Aldrich), 10 μg/mL of RNasi (Sigma-Aldrich), 0.1% of sodium citrate (Sigma-Aldrich), 50 μg/mL di propidium iodide (PI) (Sigma-Aldrich), and kept in the dark for 30 min at room temperature. Florescence was detected by a flow cytometry (Beckman Coulter Epics XL-MCL), while cell cycle analysis was performed with Cell Cycle program (MODEFIT 5.0).

(66) FIG. 10 shows that, conversely to the effect exerted by free 9-HSA that blocks the cell cycle in the G0/G1 phase, once loaded on to keratin nanoparticles a higher number of cells arrest in the S phase, thus providing evidence of the stronger cytotoxic activity of the keratin-9HSA formulation.

RELEVANT DOCUMENTS

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