SELF-ASSEMBLED PEPTIDE NANOFIBERS AND USE THEREOF FOR TARGETED DRUG DELIVERY

Abstract

Provided are peptide-based nanostructures, as well as diagnostic and therapeutic methods using same.

Claims

1. A nanostructure comprising a plurality of peptides, at least a portion of the plurality of peptides comprises peptides having at least one pi-pi (-) interacting moiety and at least one protease interacting moiety, wherein the pi-pi interacting moiety and the at least one protease interacting moiety are different, wherein the at least one protease interacting moiety comprises at least one protease cleavable bond; and wherein the plurality of peptides is free of dipeptides.

2. The nanostructure according to claim 1, wherein each of said peptides in said plurality of peptides comprises between 3 and 10 amino acids.

3. The nanostructure according to claim 1, wherein at least a portion of the plurality of peptides has an amino acid sequence of the general formula X.sub.2(m)-X.sub.3(p)-X.sub.4(q) (SEQ ID NO:1), wherein each of X.sub.2, X.sub.3 and X.sub.4 is independently an amino acid and each of m, p and q is an integer independently selected from 1 to 3, wherein at least one of the amino acids or a combination of two or more amino acids constitutes a protease interacting moiety and at least one amino acid or a combination of two or more amino acids constitutes a pi-pi interacting moiety.

4. The nanostructure according to claim 1, wherein the at least one pi-pi interacting moiety comprises at least one aromatic moiety, being optionally an aromatic amino acid.

5. The nanostructure according to claim 1, being a cathepsin cleavable nanostructure, wherein the at least one protease interacting moiety comprises at least one cathepsin cleavable bond.

6. The nanostructure according to claim 1, being a nanofiber.

7. The nanostructure according to claim 1, wherein the at least one protease interacting moiety comprises at least one aromatic amino acid and at least one hydrophilic amino acid.

8. The nanostructure according to claim 7, wherein the hydrophilic amino acid is a positively charged amino acid selected from lysine, arginine and histidine.

9. The nanostructure according to claim 8, wherein the positively charged amino acid is lysine.

10. The nanostructure according to claim 1, wherein the at least one pi-pi interacting moiety is an aromatic amino acid selected from phenylalanine, tryptophan, tyrosine and histidine.

11. The nanostructure according to claim 10, wherein the aromatic amino acid is phenylalanine.

12. The nanostructure according to claim 1, wherein the protease is cathepsin B or cathepsin L.

13. The nanostructure according to claim 1, associated with at least one agent.

14. The nanostructure according to claim 13, wherein the at least one agent is associated to the surface of the nanostructure, encapsulated within the nanostructure or present inside a void in the nanostructure.

15. The nanostructure according to claim 1, wherein the peptide is selected from peptides having a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.

16. A drug delivery system comprising a nanostructure and at least one drug associated therewith, wherein the nanostructure comprises a plurality of peptides, at least a portion of the plurality of peptides comprises peptides having at least one pi-pi (-) interacting moiety and at least one protease interacting moiety, wherein the pi-pi interacting moiety and the at least one protease interacting moiety are different, and wherein the at least one protease interacting moiety comprises at least one protease cleavable bond.

17. A method selected from the group consisting of: a. A method for treating a pathology associated with increased protease presence or for preventing onset of a pathology associated with increased protease presence in a subject, the method comprising administering to the subject a drug delivery system according to claim 16; and b. A method for diagnosis of a pathology associated with increased protease presence in a subject, the method comprising administering to the subject a diagnostically effective amount of a drug delivery system according to claim 16 associated with an agent comprising an imaging moiety, and imaging the subject or a body region of the subject to thereby identify body regions in which said nanostructure has been localized.

18. The method according to claim 17 for treating a pathology associated with increased cathepsin presence or for preventing onset of a pathology associated with increased cathepsin presence in a subject.

19. The method according to claim 18, wherein the pathology is a proliferative disorder or an inflammatory disorder.

20. The method according to claim 17 for diagnosis of a pathology associated with increased cathepsin presence in a subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0106] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0107] FIGS. 1A-C are representative HPLC chromatograms showing cleavage of a tetra-peptide substrate (TPS) by cathepsins, each TPS was treated with cathepsin B, cathepsin L or a vehicle at 37 C. and cleavage products were analyzed by liquid chromatography-MS (LC-MS). FIG. 1ATPS3 absorbance at 215 nm, FIG. 1BTPS3 after cathepsin B treatment, FIG. 1CTPS3 after cathepsin L treatment.

[0108] FIG. 2A-K show structure of exemplary TPS peptides utilized in accordance with the invention. FIGS. 2A-2I show structures of TPSs based on the FFKF scaffold, each carrying a unique N-termini chemical group and a variation in the C-termini charge: TPS1 (FIG. 2A), TPS2 (FIG. 2B), TPS3 (FIG. 2C), TPS4 (FIG. 2D), TPS6 (FIG. 2E) and TPS7 (FIG. 2F), TPS9 (FIG. 2G), TPS8 (FIG. 2H) and TPS5 (FIG. 2I). FIG. 2J provides a schematic representation of protease binding pockets (S) and corresponding substrate amino acid (P), the cleavage site is the scissile bond between the P1 (N-termini to the scissile bond) and P1 (C-termini to the scissile bond) residues. FIG. 2K shows exemplary synthesis of TPSs carried out using Fmoc based solid phase peptide synthesis. In the scheme: Fmoc is 9-fluorenylmethylcarbonyl; Boc is t-butyloxycarbonyl; Cbz is carboxybenzyl; TFA is trifluoroacetic acid.

[0109] FIG. 3 is a schematic representation of an exemplary nanostructure and an agent according with some embodiments.

[0110] FIGS. 4A-I are TEM micrographs showing self-assembly of TPSs triggered by solvent-switching between DMSO and water, final concentration of 5 mg/ml of TPS1 (FIG. 4A), TPS2 (FIG. 4B), TPS3 (FIG. 4C), TPS4 (FIG. 4D), TPS5 (FIG. 4E), TPS6 (FIG. 4H) and TPS7 (FIG. 4I) and 1 mg/ml of TPS8 (FIG. 4F) and TPS9 (FIG. 4G) were analyzed. TEM analysis revealed self-assembled nanofibers with different morphologies for all TPSs, excluding TPS6 and TPS7 (FIGS. 4H and 4I, respectively).

[0111] FIGS. 5A-C provide a kinetic profile of TPS4 self-assembly at different concentrations, evaluated by turbidity measurements. after solvent switching from DMSO to water, absorbance at 350 nm was measured every two minutes during the first 5 hours, monitoring the self-assembly at 37 C. A significant increase in turbidity was observed in all concentrations tested (up to 2.80.6 folds), demonstrating continuous assembly of the nanofibers.

[0112] FIGS. 6A-E show TPS4 nanofibers degradation by cathepsin B, FIGS. 6A-C are graphs showing TPS4 assembly behavior, following assembly of TPS4 at the indicated peptide concentrations, and treatment with cathepsin B or a vehicle. The degradation of the assembled TPS4 was evaluated by turbidity measurements monitoring the absorbance at 350 nm, the addition of cathepsin B led to decreased turbidity due to enzyme-mediated degradation of the nanofibers. FIG. 6D is a bar representation showing quantification of absorbance at 350 nm indicating significant reduction in turbidity in cathepsin B treated samples the results described with standard deviation, ** p<0.01, *** p<0.001. FIG. 6E provides TEM images of TPS4 nanofibers degraded by cathepsin B, the nanofibers were analyzed at the indicated concentrations by TEM before and after cathepsin B treatment. A substantial reduction in the number of detected nanofibers was found in the treated samples (lower panel). Scale bar is 5 M.

[0113] FIG. 7 is a bar representation of TPS4 nanofibers cytotoxicity. MDA-MB 231 cells were cultured in 96 well plate one day prior to treatment, triplicate samples of cells were incubated with 0.5 mg/ml TPS4 nanofibers in growth medium or with 0.1% DMSO vehicle control, after 24 or 48 hours treatment, cell survival was determined by standard methylene blue assay, the graphs shows that TPS4 nanofibers show no growth inhibition of MDA-MB 231 cells.

[0114] FIGS. 8A-C show TPS4 nanofibers as a targeted drug-release carrier. The assembly of premixed solution of Doxorubicin (Dox) and TPS4 in DMSO was triggered by solvent change and allowed for overnight self-assembly in the dark. FIG. 8A provides a bright field and FIG. 8B fluorescent microscopy (excitation 53550 nm, emission 61075 nm) images of the nanofibers. FIG. 8C is a graph representation showing the effect of cathepsin B or a vehicle added to samples from FIGS. 8A and 8B and released drug was collected for 4 hours by dialysis, the amount of released Dox was extrapolated from a fluorescent Dox calibration curve, results described with standard deviation.

[0115] FIGS. 9A-G show an improved drug release profile. Dox was precipitated by ammonium sulfate to generate Dox particles (DPs), TPS4 was added and allowed to assemble overnight. FIG. 9A and FIG. 9B are bright field and fluorescent microscopy images, respectively, of DP-TPS4. FIG. 9C and FIG. 9D are SEM and TEM images, respectively, of DPs alone showing that DPs form unstructured aggregates as well as fibers (white arrow). FIG. 9E and FIG. 9F are SEM and TEM images, respectively, of DPs-TPS4 showing defined nanofibers. FIG. 9G is a graph representation showing the release profile of Dox from DP-TPS4 nanostructure in the presence and absence of cathepsin B. After 8 hours, 91.80.3% of the drug was released from DP-TPS4 following cathepsin B treatment. Results are described with standard error, ** p<0.01, *** p<0.001, **** p<0.0001.

[0116] FIG. 10 is a bar representation showing degradation of TPS4 nanofibers by tissue lysates. Mice 4T1 tumor lysates or vehicle were mixed with TPS4 assemblies and after overnight treatment, the amount of remaining nanofibers relative to vehicle was determined by absorbance measurement at 350 nm, less than 20% TPS4 nanofibers remained following lysate treatments in all concentrations tested and reached full degradation at the lowest concentration.

[0117] FIG. 11 is a graph representation showing degradation of TPS4 nanofibers by tissue lysates, following 0.5 mg/ml TPS4 assembly, lysates from 4T1 tumors or from mice muscles were added and the degradation of the assembled TPS4 was evaluated by turbidity measurements, monitoring the absorbance at 350 nm, addition of 4T1 tumor lysates led to dramatic decreased turbidity resulting from enzyme-mediated degradation of the nanofibers, results described with standard error, * p<0.05, ** p<0.01, *** p<0.001.

DETAILED DESCRIPTION OF EMBODIMENTS

Non-Limiting Examples

Results and Discussion

[0118] To target the delivery of therapeutics to cancerous tissues with elevated cathepsin activity, a series of TPSs based on the FFKF scaffold was designed. To optimize the TPSs self-assembly, the peptide designs contain unique N-termini chemical groups and charge variation at the C-termini (FIGS. 2A-2I). Aromatic chemical groups were introduced at the N- and C-termini to ensure that the addition of the lysine to the FF variant will not impair the self-assembly process. A tri-peptide fragment FFK was the expected cleavage product since the target site for cleavage by these cathepsins is postulated to be the amide bond after the P1 Lys, recognized by the protease S1 pocket (FIG. 2J). Following synthesis of these peptides (FIG. 2K), their ability to be recognized and cleaved by cathepsin proteases was studied. The peptides were incubated with either cathepsin B or cathepsin L and their cleavage products were analyzed using mass spectroscopy (MS). Results obtained from MS analysis revealed that all TPSs were recognized and cleaved by both enzymes. In addition to the expected tri-peptide cleavage product, di-peptides and/or single amino acids were detected (FIG. 1, Table 1), as accounted for by the known highly promiscuous nature of the cathepsin proteases. It is widely accepted that the sequence determines the substrate specificity to a protease, in some cases the inventors found correlations between the cleavage pattern and the peptide N-termini capping group. In cathepsin B for example, a charged amine group lead to removal of a single amino acid while bulky hydrophobic groups (Fmoc or Cbz) led to removal of two or three amino acids.

TABLE-US-00001 TABLE 1 Number of amino acids removed from each TPS after cathepsin treatment according to the fragment analysis by LC-MS. TPS Cat B Cat L TPS1 1 2 NH.sub.2-FFKF-OH TPS2 2 2 Ac-FFKF-OH TPS3 2, 3 2 Fmoc-FFKF-OH TPS4 2 2 Cbz-FFKF-OH TPS5 1 1 Fmoc-FKF-amide TPS6 1 2 NH.sub.2-FFKF-amide TPS7 1 2 Ac-FFKF-amide TPS8 ND* 2 Cbz-FFKF-amide TPS9 2, 3 1, 2 Fmoc-FFKF-amide Cat; cathepsin, ND; not determined, *detected fragments identity could not be determined.

[0119] Next, the inventors investigated the ability of the different TPSs to self-assemble into ordered nanostructures using a solvent-mediated approach to trigger the assembly. Nanostructures formation was verified by transmission electron microscopy (TEM) and revealed that all TPSs self-assembled into ordered structures with morphology of elongated nanofibers network, excluding TPS6 and 7 (FIG. 4). In most cases the addition of a charged amino acid, Lys, to the fundamental FF structure did not prohibit structure formation, most likely because of the many aromatic rings within the TPSs. The inventors observed variance in the nanofibers diameters of the different TPSs (Table 2) that was attribute to the chemical modifications at the N- and C-termini Aromatic moieties (such as Fmoc and Cbz) and carbonyl/amide groups (FIG. 2) can contribute to the total - and hydrogen bond interactions, respectively. These additional interactions may enable stronger stacking forces yielding well-packed nanostructures with relatively smaller diameter. Interestingly, upon assembly initiation TPS4 stood out since it instantly formed fibers, while most other TPSs took over two hours to assemble, as was confirmed by microscopic examination (data not shown).

TABLE-US-00002 TABLE 2 Summary of TPSs diameters TPS1 TPS2 TPS3 TPS4 TPS5 TPS6 TPS7 TPS8 TPS9 Diameter 310 80 12 1 14 2 24 6 66 13 30 6 13 4 (nm)

[0120] Considering its immediate self-assembly, the inventors continued with TPS4 (Cbz-FFKF-OH), as a self-assembling peptide-substrate model for cleavage by cathepsin B. TPS4 was found to assemble into ordered nanofibers, forming an opaque peptide-solution upon assembly initiation. To evaluate the assembly kinetics of TPS4 the inventors examined the turbidity changes of the self-assembled peptide at different peptide concentrations by monitoring the absorbance at 350 nm. Absorbance was already detected at the initial time point followed by a significant increase in turbidity during the first five hours (FIGS. 5A-C) indicating continuous assembly of the nanofibers over the course of the experiment. This increase in turbidity is in line with assembly of Boc-Phe-Phe, where nanofibers formation was associated with the increase in turbidity. Upon addition of cathepsin B to the assembled TPS4 a decrease in turbidity was obtained, due to the degradation of the nanofibers by the enzyme (FIG. 6A-C). To validate that the decrease in turbidity was a result of nanofiber degradation, samples of nanofibers with and without cathepsin B treatment were analyzed by TEM at the final time point. While very few nanofibers were found in the samples treated with cathepsin B, a substantial number of nanofibers were easily found in non-treated samples (FIG. 6E). This validated that cathepsin B can access TPS4 and degrade it even as assembled nanofibers.

[0121] The inventors further evaluated nanofiber degradation by cathepsin B at various TPS4 concentrations to examine the optimum peptide concentration for best assembly and degradation when the assembly process reached equilibrium. As expected, under the tested conditions a significant and robust degradation of TPS4 assemblies was obtained for all tested peptide concentrations. Most dramatic turbidity reduction was found at the lowest concentration tested, 0.5 mg/ml. At the higher TPS concentration, however, only partial degradation was observed (FIG. 6D). The inventors speculate that at higher peptide concentrations more assemblies are present in the solution that might physically limit the accessibility of the enzyme to the substrate. Another possibility is that the degradation products are quickly recovered and assembled to the remaining TPS structures that are present at a high concentration in the surrounding environment. Overall these results suggest that nanofibers degradation is concentration dependent and that TPS4 assemblies should be optimized to generate suitable drug delivery systems.

[0122] To further investigate the ability of TPS4 to serve as a cathepsin targeted drug delivery vehicle the inventors first evaluated growth inhibition by TPS4 and found no cytotoxicity of MDA-MB 231 cells by 0.5 mg/ml, (FIG. 7). Then the inventors assessed the ability of assemblies obtained from 0.5 mg/ml TPS4 to serve as a carrier for the anti-cancerous drug Doxorubicin (Dox). Dox was chosen as a model drug because of its intrinsic fluorescent properties which allow easy monitoring of the drug. Initially, Dox was encapsulated in nanofibers of TPS4 by solvent switching of the mixture from DMSO to water, generating fluorescent nanofibers as visualized by fluorescent microscopy (FIG. 8B). Exposure of the Dox containing nanofibers to cathepsin B resulted in drug release due to nanofibers degradation. Unfortunately, the inventors found only a 15% difference in the release profile when comparing the amount of drug released from the nanofibers with or without the enzyme (FIG. 8C). The inventors suspected that Dox was only coating the nanofibers by weak interactions and was naturally released thus leading to the small differences observed upon enzyme addition. To improve the release profile upon enzyme treatment, the inventors attempted to first generate small Dox particles and then coat them with peptide nanofibers. The Dox particles were inspired by the doxil liposome in which drug retention was achieved by base change of a weak-base-drug with sulfate ions in an intra-liposome aqueous phase. In that process, after accumulation in a liposome filled with ammonium sulfate, (doxorubicin).sub.2SO.sub.4 (doxorubicin sulfate) precipitated. To investigate the hypothesis, the inventors first generated Dox particles (DPs) by precipitation with ammonium sulfate and then coated these particles with peptide nanofibers triggering the assembly of TPS4 by solvent exchange, Dox loading efficiency was found to be 485%. The new assembly, Dox particles-TPS4 (DP-TPS4), formed highly fluorescence structures, as observed by fluorescent microscopy (FIGS. 9A and 9B). Scanning electron microscopy (SEM) and TEM analysis of the DPs revealed differences in the structures formed with and without TPS4. DPs alone formed unstructured aggregates as well as fibers with 10-20 nm diameter (FIGS. 9C and 9D, marked by a white arrows). DPs-TPS4 formed defined nanofibers throughout, with fibers diameter ranging from 40 to 60 nm (FIGS. 9E and 9F). The inventors then turned to investigate the release profile of Dox from DP-TPS4 assemblies in the presence or absence of cathepsin B. As expected, in the presence of the enzyme a significant increase in Dox release was obtained that reached 91.80.3% after eight hours, as compared to the spontaneous, non-specific, drug release from DPs-TPS4 structures without enzyme treatment (55.00.2%) (FIG. 9G).

[0123] Further, the inventors analyzed the expression and activity levels of cathepsins in 4T1 murine breast cancer cells. Similar to other cancers, this cell line naturally expresses high levels of various cathepsins, especially cathepsin B. Therefore, the inventors evaluated the ability of 4T1 tumor lysates from tumors-bearing mice to degrade TPS4 nanofibers. The inventors found that the tumor lysates degraded TPS4 nanofibers in a concentration-dependent manner (FIG. 10). The inventors then evaluated TPS4 nanofiber degradation over time by tissue lysates, while 4T1 tumor lysates degraded the majority of TPS4 nanofibers within two hours, the lysates generated from mice muscles had limited effect in the first ten hours tested (FIG. 11). Taking these results together, the inventors foresee that tetra-peptide substrates that form nanostructures could serve as a promising platform for targeted drug delivery to cancers that exhibit highly elevated protease activities.

[0124] In conclusion, the inventors applied a substrate-based approach to generate a library of self-assembled tetra-peptides to serve as carriers for therapeutics to pathogenic tissues characterized by elevated protease activity. The inventors have demonstrated that in most cases elongation of the FF variant by two additional amino acids, including a charged lysine, did not impair the substrates self-assembling into ordered nanofibers. Furthermore, the inventors show the capability of the cathepsin proteases to process their substrate both in solution and within nanostructures. Generation of Dox particles-TPS4 led to an improved release profile of Dox from the nanostructures by cathepsin B activity. Finally, the inventors demonstrated that the intrinsic high cathepsins activity of tumor lysates can fully degrade TPS4 nanofibers. The findings described herein suggest a new platform for drug-delivery, targeted to pathologies with high cathepsins activity.

Materials and Methods

Chemical Synthesis

[0125] Unless otherwise noted, all resins and reagents were purchased from commercial suppliers and used without further purification. Tetra peptides were synthesis on solid phase using 2-Chlorotrityl chloride resin (peptide 1-5) or Rink-amide resin (peptide 6-9) with Fmoc based chemistry. Peptide elongation was performed with HOBT (1-hydroxybenzotriazole) and PyBOP (benzotriazole-1-yl-oxy) tris (pyrrolidino) phosphonium hexafluorophosphate) as coupling reagents and DIEA (diisopropylethylamine) Cleavage of peptide from the resin and Boc (t-butyloxycarbonyl) deprotection were performed using TFA (trifluoroacetic acid) in DCM (dichloro methane). Peptides were purified by C18 reverse phase HPLC in acetonitrile/water gradient supplemented with 0.1% trifluoroacetic acid (TFA) or by precipitation. Final peptides were characterized by a Liquid Chromatography Mass Spectrometer (LCMSThermo Scientific MSQ-Plus attached to an Accela UPLC system) to more than 95% purity. Final peptides were lyophilized and kept at 20 C. until use.

Cleavage of Substrate Peptides by Cathepsins

[0126] Each peptide (1 nmole) was treated with 0.9 mole cathepsin B or cathepsin L (generated by transformation of E. coli BL21(DE3)pLysS strain with the pET3a/procathepsin B or procathepsin L vectors) or acetate buffer vehicle (50 mM acetate, 5 mM MgCl.sub.2, 2 mM DTT, adjusted to pH 5.5) for 3 hours at 37 C. Next, enzymes were precipitated by an hour incubation at 80 C. in 80% cold methanol followed by 10 minutes of 6000 g centrifugation at 4 C. Supernatant volume was reduced to about 15 l under vacuum, adjusted to a final volume of 30 l with methanol and analyzed by LCMS equipped with C18 reverse phase.

Assembly Preparation

[0127] Lyophilized peptides were dissolved in DMSO to a concentration of 100 mg/ml and then diluted in DDW to the indicated final concentration.

Transmission Electron Microscopy Analysis

[0128] Transmission electron microscopy (TEM) analysis was performed by applying 10 l of samples to a 200 or 400-mesh copper grids covered by carbon-stabilized Formvar film (SPI, West Chester, Pa.). The samples were allowed to adsorb for 2 minutes before excess fluid was blotted off. Samples were negatively stained by depositing 10 l of 2% uranyl acetate on the grid and allowing it to adsorb for 2 minutes before excess fluid was blotted off. Except the sample in FIG. 9E, all samples were negatively stained. TEM micrographs were recorded using a JEOL 1200EX or JEOL JEM-1400Plus electron microscope operating at different kV.

Scanning Electron Microscopy

[0129] Scanning Electron Microscopy (SEM) samples were prepared as described above for TEM on a 400-mesh copper grids, without uranyl acetate staining, and viewed using FBI Magellan 400L system.

Kinetics of TPS4 Self-Assembly

[0130] Freshly prepared aliquots of 0.5, 1 or 2 mg/ml of TPS4 were placed in triplicates in a 96-well plate, 100 ul/well. Plate was placed at 37 C. in a Biotek Cytation 3 plate reader (Winooski, Vt., USA) and absorbance at 350 nm was measured every 2 minutes during 5 hours.

Turbidity Analysis of TPS4 Degradation by Cathepsin B

[0131] Turbidity analysis for TPS4 solutions was conducted using triplicates of freshly prepared solutions as described above in a 96 well plate. Aliquots of 1, 2 or 4 mg/ml were allowed to assemble for 4-5 hours at room temperature. Next, 0.064, 0.128 or 0.255 M of cathepsin B or acetate buffer vehicle were added, diluting the samples to 0.5, 1, and 2 mg/ml, respectively. The plate was placed at 37 C. in a Biotek Synergy HT plate reader (Winooski, Vt., USA) and absorbance was measured for 12 hours as described above. After 24 hours since enzyme addition, a color picture was taken and the turbidity was measured again.

TPS4 Nanofibers Cytotoxicity

[0132] MDA-MB 231 human breast adenocarcinoma cells were cultured in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin in a humidified atmosphere of 5% CO.sub.2 at 37 C. One day prior to treatment MDA-MB 231 cells (710.sup.3 cells/well) were cultured in a 96-well plate. The media of TPS4 that was assembled in DMEM was removed by centrifugation (14,000 rpm, 4 C.) and the nanofibers were resuspended in DMEM supplemented with 10% PBS, 1% penicillin and 1% streptomycin. Cells in triplicates were incubated with 0.5 mg/ml of TPS4 nanofibers (final concentration of 0.1% DMSO was kept constant) in growth medium at 37 C. After 24 or 48 hours of treatment, cell survival was determined by standard methylene blue assay.

TPS4 Assembly in the Presence of Doxorubicin (Dox)

[0133] Assembly of TPS4 in the presence of Dox was achieved by premixing the peptide and the drug in DMSO followed by a dilution in DDW to final concentration of 1 mg/ml TPS4 and 5% w/w Dox. The assembly was preformed over-night in the dark.

Preparation of Doxorubicin Particles

[0134] Doxorubicin.HCl (Sigma Aldrich), 1.06 mg, was dissolved in 2 l dry DMSO. Then, 69 l of 20 mM ammonium sulfate aqueous solution were added followed by a short sonication. Doxorubicin particles were precipitated by 10 minutes centrifugation at 14,000 rpm, 4 C. to obtain a red pellet and a light red supernatant. Supernatant was removed and re-precipitated by ammonium sulfate aqueous solution. Pellets were combined and resuspended in DDW to a final concentration of 10 mg/ml Doxorubicin particles (DP).

DP-TPS4 Assembly

[0135] Assembly of TPS4 in the presence of DP was achieved by dilution while stirring of TPS4 in a solution of DP suspended in 20 mM ammonium sulfate. The assembly was preformed overnight in the dark. Next, access of Dox or DP were washed off by centrifugation (10 min, 14,000 rpm or 5 min, 8200 rpm, 4 C. respectively) and the resulting DP-TPS4 was resuspended in DDW to a final concentration of 1 mg/ml. For fluorescent characterization, 5 l of each assembly were placed on a coverslip and allowed for complete dryness in the dark. Fluorescent images were taken at 60 magnification using an Olympus inverted fluorescent microscope IX51 equipped with a TritC filter (ex/em 53550/61075 nm).

Determination of Dox Loading in DP-TPS4

[0136] Following DP-TPS4 assembly as described above, the supernatant of access Dox was collected by centrifugation (10 min, 14,000 rpm, 4 C.) and the resulting precipitation of DP-TPS4 was dissolved in DMSO. Dox fluorescence intensity in supernatant and precipitation was read at excitation/emission of 480/595 nm by a Cytation 3 plate reader and the amount of Dox was obtained from the calibration curve of Dox in 20 mM ammonium sulfate or DMSO respectively. The loading efficiency of DP in DP-TPS4 assembly was determined by the ratio of amount obtained from DP-TPS4 precipitation to the total amount obtained from supernatant and DP-TPS4 precipitation. Loading efficiency was determined in triplicate.

Release of Dox by Cathepsin B

[0137] 20% w/w DP or 5% w/w Dox in 1 mg/ml assembly (as described above) were placed in a dialysis unit (1 kDa molecular weight cut-off, Slide-A-Lyzer MINI Dialysis Devices, ThermoFisher Scientific). Next, cathepsin B was added to generate a final concentration of 0.5 mg/ml nanofibers assembly and 2 M cathepsin B. The samples were placed at 37 C. and dialyzed while gently shaking, with dialysis replaced at indicated time points. At the end of the experiment, samples in dialysis units were collected and recovered. All collected samples were lyophilized and resuspended in DMSO and the fluorescence intensity of Dox was read at excitation/emission of 480/595 nm by a Cytation 3 plate reader. The amount of released Dox was obtained from the calibration curve of Dox in DMSO.

Kinetics of Nanofibers Degradation by Tissue Lysates

[0138] Tissue lysates were prepared as previously described. Assembly of TPS4 in triplicates was performed for 4 hours in acetate buffer as described in FIG. 6B. Next, 100 g of lysates from 4T1 tumor or muscles, or RIPA vehicle control (1% Tergitol-type NP-40 (nonyl phenoxypolyethoxylethanol), 0.1% SDS, 0.5% sodium deoxycholate) were added in acetate buffer. The absorbance was read at 350 nm after gentle shaking at 37 C. for indicated times using a BioTek plate reader.