AMPHIPHILIC BLOCKCOPOLYMERS COMPRISING REDUCTION SENSITIVE BIODEGRADABLE POLYESTERAMIDES

Abstract

The present invention relates to amphiphilic block copolymers comprising reduction sensitive biodegradable polyesteramides. The present invention also relates to micelles comprising the amphiphilic block copolymers. The invention also relates to drug delivery systems comprising the micelles. The amphiphilic block copolymers comprise hydrophilic and hydrophobic blocks whereby the hydrophobic blocks comprise a biodegradable polyesteramide based on alpha-amino acids, diols, aliphatic dicarboxylic acids and optionally diamines whereby at least one of the dicarboxylic acids, diols or diamines comprises disulphide linkages.

Claims

1.-17. (canceled)

18. An amphiphilic block copolymer comprising hydrophilic bocks and hydrophobic blocks, wherein the hydrophobic blocks comprise a biodegradable polyesteramide comprising disulphide linkages in the backbone of the biodegradable polyesteramide.

19. The amphiphilic block copolymer according to claim 18, wherein the biodegradable polyesteramide comprise residues of alpha -amino acids, diols, aliphatic dicarboxylic acids, and optionally diamines, wherein at least one of the diols, aliphatic dicarboxylic acids, or diamines comprises a disulphide linkage.

20. The amphiphilic block copolymer according to claim 18, wherein the biodegradable polyesteramide comprises a residue of at least one of the following structural formulas I, II or III, or a combination thereof: ##STR00004## wherein m is from 5 to 300; Y is a (C.sub.2-C.sub.20) aliphatic hydrocarbons or a (C.sub.2-C.sub.20) cycloaliphatic hydrocarbons; X is independently an aliphatic hydrocarbon, a cycloaliphatic hydrocarbon, or an aromatic hydrocarbon; and R is independently a side chain residue of arginine, histidine lysine, aspartic acid, glutamic acid, serine, threonine, asparginine, glutamines, cysteine, seleno cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, tyrosine, tryptophan, or phenylalanine.

21. The amphiphilic block copolymer according to claim 20, wherein X is an aliphatic C.sub.2-C.sub.8 hydrocarbon.

22. The amphiphilic block copolymer according to claim 20, wherein Y is (C.sub.2-C.sub.20)alkylene, (C.sub.2-C.sub.20) alkenylene, bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural Formula (IV), or combinations thereof. ##STR00005##

23. The amphiphilic block copolymer according to claim 20, wherein the hydrophobic block comprises a residue of structural Formula I, wherein X is a (C.sub.2-C.sub.20) alkylene or (C.sub.2-C.sub.20) alkenylene and R is a side chain residue of arginine or phenylalanine.

24. The amphiphilic block copolymer according to claim 22, wherein the hydrophobic block comprises a residue of structural Formula I, wherein X is a (C.sub.2-C.sub.20) alkylene or (C.sub.2-C.sub.20) alkenylene and R is a side chain residue of arginine or phenylalanine.

25. The amphiphilic block copolymer according to claim 20, wherein the hydrophilic blocks comprise poly(ethylene oxide), polyvinylpyrrolidone, or polyoxazoline.

26. The amphiphilic block copolymer according to claim 24, wherein the hydrophilic blocks comprise poly(ethylene oxide).

27. The amphiphilic block copolymer according claim 18, wherein the amphiphilic block copolymer is a triblock copolymer.

28. The amphiphilic block copolymer according to claim 20, wherein the amphiphilic block copolymer is a triblock copolymer, the hydrophilic block comprises poly(ethylene oxide), and the hydrophobic block comprises a residue of structural formula I wherein R is a side chain residue of arginine or phenylalanine.

29. The amphiphilic block copolymer according to claim 24, wherein the amphiphilic block copolymer is a triblock copolymer and the hydrophilic block comprises poly(ethylene oxide).

30. A drug delivery system comprising micelles comprising the amphiphilic block copolymer according to claim 18.

31. A drug delivery system comprising micelles comprising the amphiphilic block copolymer according to claim 20.

32. A drug delivery system comprising micelles comprising the amphiphilic block copolymer according to claim 29.

33. The drug delivery system according to claim 30, wherein the micelles further comprise a hydrophobic drug.

34. The drug delivery system according to claim 31, wherein the micelles further comprise an anticancer drug.

35. The drug delivery system according to claim 33, wherein the hydrophobic drug is an anticancer drug.

36. The drug delivery system according to claim 34, wherein the average particle size of the micelles is from 10 to 400 nm.

37. The drug delivery system according to claim 35, wherein the average particle size of the micelles is from 10 to 400 nm.

Description

FIGURES

[0063] FIG. 1: .sup.1H NMR spectrum (400 MHz, DMSO-d.sub.6) of NP-PEA(SS)-NP.

[0064] FIG. 2: .sup.1H NMR spectrum (400 MHz, DMSO-d.sub.6) of PEG-PEA(SS)-PEG triblock copolymer.

[0065] FIG. 3: Size distribution of PEG-PEA(SS)-PEG micelles in PB (10 mM, pH 7.4) determined by DLS

[0066] FIG. 4: The fluorescence intensity ratio I.sub.1/I.sub.3 of pyrene as a function of PEG-PEA(SS)-PEG concentration. The critical micelle concentration (CMC) in aqueous medium is determined to be ca. 9.9×10.sup.−3 mg/mL.

[0067] FIG. 5: The size change of PEG-PEA(SS)-PEG micelles in response to 10 mM DTT in PB buffer (10 mM, pH 7.4) determined by DLS

[0068] FIG. 6: Count rate/PDI change of PEG-PEA(SS)-PEG micelles in response to 10 mM DTT in PB buffer (10 mM, pH 7.4) determined by DLS.

[0069] FIG. 7: Reduction-triggered release of DOX from PEG-PEA(SS)-PEG micelles in PB (10 mM, 7.4) at 37° C. in the presence or absence of DTT.

[0070] FIG. 8: Cytotoxicity of PEG-PEA(SS)-PEG micelles determined via MTT assays in HeLa and MCF-7 cells. The cells are incubated with micelles for 24 h. Data are presented as the average±standard deviation (n=4).

[0071] FIG. 9: The anti-tumor activity of DOX-loaded PEG-PEA(SS)-PEG micelles versus free DOX in HeLa and MCF-7 cells. DOX dosage was 12.5 μg/mL. The cells were incubated with DOX-loaded micelles or free DOX for 24 h or 48 h.

[0072] FIG. 10: Intracellular drug release and anti-tumor activity of drug-loaded PEG-PEA-SS-PEG micelles.

EXAMPLES

Example 1

[0073] Synthesis of PEG-NH2 from PEG-NPC (Nitro-phenylcarbonate) Followed by End Modification with Ethylenediamine

[0074] Part 1. Synthesis of PEG-NPC

[0075] Synthesis of PEG NPC is a described in Sun H L, Guo B N, Cheng R, et al. Biomaterials 2009; 30(31):6358-66. PEG-NPC is an amino (—NH.sub.2) reactive PEG derivative. In brief, under a nitrogen atmosphere, to a DCM solution (70 mL) of mPEG (10.0 g, 2 mmol) and pyridine (0.791 g, 10 mmol), a solution of p-Nitrophenylcarbonate (p-NPC) (1.614 g, 8 mmol) in 15 mL DCM was added dropwise at 0° C. After completion of addition, the reaction mixture was warmed to r.t. and the reaction proceeded for another 20 h. The resulting polymer PEG-NPC was isolated by precipitation in cold diethylether and dried in vacuum. Yield: 94.9%. .sup.1H NMR showed NPC functionality close to 100%.

[0076] Part 2 Synthesis of PEG-NH.sub.2

[0077] Under a nitrogen atmosphere, to a DCM solution (10 mL) of ethylenediamine (0.465 g, 7.74 mmol), a solution of PEG-NPC (2.000 g, 0.387 mmol) in DCM (10 mL) was added dropwise at r.t. The reaction was allowed to continue for 24 h. The resulting PEG-NH.sub.2 was isolated by precipitation in cold diethylether twice and dried in vacuum. Yield: 78%.

[0078] 1H NMR (400 MHz, CDCl3), δ (ppm): 5.32 (—NH—C(O)—); 4.22 (—CH2-O—C(O)—NH2); 3.64 (PEG); 3.38 (CH3O—); 3.24 (—CH2-NH—C(O)—O—); 2.82 (—CH2-NH2); 2.75 (NH2-CH2-).

Example 2

[0079] Synthesis of PEG-PEA(SS)-PEG Triblock Copolymer.

[0080] Amphiphilic triblock copolymer PEG-PEA (SS)-PEG were synthesized via coupling reaction between mPEG-NH.sub.2 of example 1 and NP-PEA (SS)-NP (Scheme 1). In brief, under a nitrogen atmosphere, to a DMF solution (0.7 mL) of mPEG-NH.sub.2 (0.183 g, 0.036 mmol), a solution of NP-PEA(SS)-NP (0.04 g, 0.0036 mmol) in 0.8 mL DMF was added dropwise at r.t. The reaction is allowed to continue for 3 d at 40° C. The resulting PEG-PEA(SS)-PEG copolymer was isolated by precipitation in cold ethyl acetate, filtrated and washed using ultra-pure water twice and freeze-dried for 2 d.

##STR00003##

Example 3

[0081] Micelle formation and critical micelle concentration (CMC).

[0082] Micelles of PEG-PEA(SS)-PEG were prepared under stirring by dropwise addition of 0.8 mL PB (10 mM, pH 7.4) to 0.2 mL of block copolymer solution (2 mg/mL) in DMF at r.t. followed by extensive dialysis against PB for 24 hr.

[0083] The critical micelle concentration (CMC) was determined using pyrene as a fluorescence probe. The concentration of block copolymer was varied from 3.0×10.sup.−4 to 0.15 mg/mL and the concentration of pyrene was fixed at 0.6 μM. The fluorescence spectra were recorded using FLS920 fluorescence spectrometer with the excitation wavelength of 330 nm. The emission fluorescence at 372 and 383 nm was monitored. The CMC was estimated as the cross-point when extrapolating the intensity ratio/.sub.372//.sub.383 at low and high concentration regions.

[0084] Micelles of PEG-PEA(SS)-PEG block copolymer were prepared by dialysis method. DLS measurements show an average micelle size of 158 nm with a narrow size distribution (PDI=0.07) (FIG. 3). The critical micelle concentration (CMC), determined using pyrene as a probe, was estimated to be approximately 9.9 mg/L (FIG. 4).

Example 4

[0085] Reduction-triggered destabilization of PEG-PEA(SS)-PEG micelles

[0086] The size change of micelles in response to 10 mM DTT in PB buffer (10 mM, pH 7.4) was followed by DLS measurement. Briefly, to 1.5 mL solution of PEG-PEA(SS)-PEG micelles in PB (10 mM, pH 7.4) previously degassed with nitrogen for 20 min, was added 10 mM DTT. The solution was placed in a shaking bed at 37° C. with a rotation speed of 200 rpm. At different time intervals, the size was determined using DLS. The size change of PEG-PEA(SS)-PEG micelles in response to 10 mM DTT in PB (10 mM, pH 7.4) was followed by DLS measurement. Notably, fast aggregation was observed for the micelles following addition of 10 mM DTT, in which micelle size increased from 160 nm to 325 nm (44%) and 1230 nm (54%) in 0.5 h, reaching over 1900 nm (62%) after 12 h (FIG. 5). Concomitantly, PDI increased from 0.07 to 0.64 after 12 h while the count rate decreased from 483 kcps to 64 kcps in 4 h (FIG. 6). Aggregates were formed due to most probably reductive cleavage of the repeated disulfide bonds, which results in water insoluble small molecules. In contrast, little change in micelle size, PDI and count rate is discerned after 12 h in the absence of DTT under otherwise the same conditions.

Example 5

[0087] Loading and reduction-triggered release of DOX from PEG-PEA(SS)-PEG micelles.

[0088] DOX was loaded into micelles by dropwise addition of 0.8 mL PB (10 mM, pH 7.4) to a mixture of 0.2 mL PEG-PEA(SS)-PEG copolymer solution in DMF (2 mg/mL) and DOX solution in DMSO (5 mg/mL) at varying DOX/polymer weight ratios (5-30 wt %) under stirring at r.t., followed by dialysis against PB (10 mM, pH 7.4) for 24 h at r.t. (MWCO 3500). The dialysis medium was changed five times. The whole procedure was performed in the dark. The amount of DOX was determined using fluorescence (FLS920) measurement (excitation at 480 nm and emission at 555 nm). For determination of drug loading content, lyophilized DOX loaded micelles were dissolved in DMSO and analyzed with fluorescence spectroscopy, wherein calibration curve was obtained with DOX/DMSO solutions with different DOX concentrations.

[0089] Drug loading content (DLC) and drug loading efficiency (DLE) are calculated according to the following formula:

DLC (wt. %)=[weight of loaded drug /(weight of polymer+drug)]×100%

DLE (%)=(weight of loaded drug/weight of drug in feed)×100%

[0090] The release profiles of DOX from PEG-PEA(SS)-PEG micelles were studied using a dialysis tube (MWCO 12000) at 37° C. in three different media, i.e. PB (10 mM, pH 7.4) only, PB (10 mM, pH 7.4) with 2 mM DTT or 5 mM DTT. In order to acquire sink conditions, drug release studies were performed at low drug loading contents (ca. 1.0 wt. %) and with 0.6 mL of micelle solution dialysis against 20 mL of the same medium. At desired time intervals, 6 mL release media is taken out and replenished with an equal volume of fresh media. The amount of DOX released was determined by using fluorescence (FLS920) measurement (excitation at 480 nm). The release experiments were conducted in triplicate. The results presented are the average data with standard deviations.

[0091] DOX is loaded into micelles by dialysis of a polymer/DOX solution in DMF against PB buffer. The theoretical drug loading content is set at 5, 10, 20 and 30 wt. %. The results show that PEG-PEA(SS)-PEG micelles encapsulated DOX efficiently, affording a drug loading efficiency between ca. 52% and 68% (Table 1).

TABLE-US-00001 TABLE 1 DOX loading content and loading efficiency with PEG-PEA(SS)-PEG micelles. Theoretical Micelle DOX loading DOX loading drug loading size.sup.a content.sup.b efficiency Entry content (wt. %) (nm) PDI.sup.a (wt. %) (%) 1 0 157.8 0.07 — — 2 5 139.2 0.09 3.30 68.10 3 10 125.9 0.07 5.55 58.79 4 20 118.2 0.10 9.85 54.66 5 30 129.8 0.07 13.53  52.16 .sup.aThe average size and polydispersity index (PDI) of micelles determined by DLS measurements. .sup.bDetermined by fluorescence measurement.

[0092] The release of DOX form PEG-PEA(SS)-PEG micelles is studied using a dialysis tube (MWCO 12000) in pH 7.4 PB buffer at 37° C. in the presence or absence of DTT. Remarkably, the results show that PEG-PEA(SS)-PEG micelles released DOX rapidly in the presence of 2 or 5 mM DTT, a reductive environment analogous to that of the intracellular compartments such as cytosol and the cell nucleus and 83% or 90% DOX is released after 11 h, respectively. In contrast, minimal drug release (<30%) is observed within 12 h in the absence of DTT (FIG. 7)

[0093] It is observed that fast drug release from PEG-PEA(SS)-PEG micelles is triggered by reduction. This is in line with the previous observation that PEG-PEA(SS)-PEG micelles are destabilized and form aggregates quickly in response to 10 mM DTT. It should be further noted that no burst release is observed and DOX is released from PEG-PEA(SS)-PEG micelles in a zero order manner up to 70% release. This constant release rate indicates that release of DOX is controlled most likely by a combination of diffusion and degradation.

Example 6

[0094] Cellular Uptake and Intracellular Release of DOX

[0095] HeLa cells are plated on microscope slides in a 24-well plate (5×10.sup.4 cells/well) under 5% CO.sub.2 atmosphere at 37° C. using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% L-glutamine, antibiotics penicillin (100 IU/mL), and streptomycin (100 μg/mL). After 24 h, 50 μL of PB (10 mM, pH 7.4) solution of DOX-loaded PEG-PEA(SS)-PEG micelles or free DOX (DOX dosage: 12.5 μg/mL) is added. After incubation at 37° C. and a humidified 5% CO.sub.2-containing atmosphere for 0.5, 2 or 4 h, the culture media are removed and the cells on microscope plates are washed three times with PBS. The cells are then fixed with 4% formaldehyde for 15 min and washed with PBS 3 times. The cell nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI, blue) for 15 min and washed with PBS 3 times.

[0096] The cellular uptake and intracellular release behaviors of DOX-loaded PEG-PEA(SS)-PEG micelles are followed with CLSM using HeLa cells. Remarkably, strong DOX fluorescence in the cells is clearly observed in the cytosol as well as the cell nucleus after just 0.5 h incubation with DOX-loaded PEG-PEA(SS)-PEG micelles, indicating fast internalization of micelles and rapid release of DOX inside cells. This is in accordance with the expectation that disulfide bonds are cleaved in the intracellular compartments such as the cytosol and the cell nucleus due to presence of comparatively high concentrations of reducing glutathione tripeptides (2˜10 mM). The fluorescence intensity of DOX inside cells and the amount of DOX transported into the cell nucleus increased further when increasing the incubation time to 2 and 4 h. In comparison, free DOX is mainly accumulated in cell nucleus.

Example 7

Cell Viability Assay

[0097] HeLa and MCF-7 cells are plated in a 96-well plate (1×10.sup.4 cells/well) using DMEM medium supplemented with 10% fetal bovine serum, 1% L-glutamine, antibiotics penicillin (100 IU/mL), and streptomycin (100 μg/mL) and incubation for 24 h. The cells are incubated with blank PEG-PEA(SS)-PEG micelles, DOX loaded PEG-PEA(SS)-PEG micelles, or free DOX (DOX dosage: 12.5 or 25 μg/mL) for 24 and 48 h at 37° C. in a humidified 5% CO.sub.2-containing atmosphere. Then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) solution in PBS (10 μL, 5 mg/mL) are added and incubated for another 4 h. The medium is aspirated, the MTT-formazan generated by live cells is dissolved in 150 μL of DMSO, and the absorbance at a wavelength of 490 nm of each well is measured using a microplate reader. The relative cell viability (%) is determined by comparing the absorbance at 490 nm with control wells containing only cell culture medium. Data are presented as average±SD (n=4).

[0098] The cytotoxicity of blank PEG-PEA(SS)-PEG micelles is evaluated in HeLa and MCF-7 cells using MTT assays. The results revealed that these PEG-PEA(SS)-PEG micelles are practically nontoxic to HeLa (cell viability: 81.5-102.0%) and MCF-7 cells (cell viability: 97.8-103.2%) at varying concentrations from 0.1 to 1.2 mg/mL (FIG. 8), indicating that PEG-PEA(SS)-PEG micelles possess excellent biocompatibility.

[0099] The anti-tumor activity of DOX-loaded PEG-PEA(SS)-PEG nanoparticles is also investigated via MTT assays. HeLa and MCF-7 cells are treated for 24 or 48 h with free DOX or DOX-loaded micelles (DOX dosage: 12.5 μg/mL). It should be noted that, after 24 h incubation, DOX-loaded PEG-PEA(SS)-PEG micelles exhibited lower toxicity to both the HeLa and MCF-7 cells than free DOX (cell viabilities of 31.8% and 47.4%, respectively) (FIG. 9). However, comparable cell viabilities are observed following 48 h incubation with DOX-loaded PEG-PEA(SS)-PEG micelles and free DOX to HeLa cells (cell viabilities 28.1% versus 19.1%) as well as MCF-7 cells (cell viabilities 48.4% versus 37.5%).