REDUCTION SENSITIVE BIODEGRADABLE POLYESTERAMIDES

Abstract

The present invention relates to biodegradable polyesteramides (PEAs) comprising hydrophobic alpha-amino acids, diols, aliphatic dicarboxylic acids and optionally diamines whereby at least one of the dicarboxylic acids, diols or diamines comprises disulphide linkages. The present invention also relates to the use of the polyesteramides in medical applications such as cancer treatment, ophthalmic applications, therapeutic cardiovascular applications, veterinary applications, pain management applications, MSK applications and vaccine delivery compositions. The present invention also relates to a drug delivery composition comprising the PEA's and to a drug delivery system such as micro- or nanoparticles, micelles, liposomes, polymerosomes, micro- and nanogels, polymerosomes or nanotubes.

Claims

1.-13. (canceled)

14. A biodegradable polyesteramide comprising ester groups, amide groups, and disulphide linkages in the backbone of the PEA, wherein the polyesteramide is enzymatically degradable.

15. The biodegradable polyesteramide according to claim 14, wherein the 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.

16. A biodegradable polyesteramide comprising a residue of at least one of the structural formulas I, II or III: ##STR00012## wherein m is from 5 to 300; Y is a (C.sub.2-C.sub.20) aliphatic hydrocarbon or a (C.sub.2-C.sub.20) cycloaliphatic hydrocarbon; X is independently an aliphatic hydrocarbon, a cycloaliphatic hydrocarbon, or an aromatic hydrocarbon; and R is independently a side chain residue of an alpha amino acid with a positively charged group, a side chain residue of an amino acid with a negatively charged group, a side chain residue of an amino acid with an uncharged side group, or a side chain residue of an amino acid with a hydrophobic group.

17. The biodegradable polyesteramide according to claim 16 wherein the biodegradable polyesteramide 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 phenylalanine.

18. The biodegradable polyesteramide according to claim 16 wherein the biodegradable polyesteramide comprises a residue of structural formula II, wherein X is (C.sub.2-C.sub.20) alkylene or (C.sub.2-C.sub.20) alkenylene, 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, and R is a side chain residue of phenylalanine. ##STR00013##

19. The biodegradable polyesteramide according to claim 16, wherein the biodegradable polyesteramide comprises a residue of structural formula III, 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, and R is a side chain residue of phenylalanine.

20. The biodegradable polyesteramide according to claim 14, wherein the biodegradable polyesteramide has a Mw as measured by GPC in THF with polystyrene as standard of from 500 to 20,000 g/mol.

21. A drug delivery composition comprising the biodegradable polyesteramide according to claim 14 and a bioactive agent.

22. A drug delivery composition comprising the biodegradable polyesteramide according to claim 16 and a bioactive agent.

23. A drug delivery composition comprising the biodegradable polyesteramide according to claim 20 and a bioactive agent.

24. The drug delivery composition according to claim 21, wherein the bioactive agent comprises an anti-cancer agent.

25. The drug delivery composition according to claim 22, wherein the bioactive agent comprises an anti-cancer agent.

26. A drug delivery system comprising microparticles or nanoparticles comprising the drug delivery composition according to claim 21.

27. A drug delivery system comprising microparticles or nanoparticles comprising the drug delivery composition according to claim 22.

28. A drug delivery system comprising microparticles or nanoparticles comprising the drug delivery composition according to claim 23.

29. A drug delivery system comprising microparticles or nanoparticles comprising the drug delivery composition according to claim 24.

30. A drug delivery system comprising microparticles or nanoparticles comprising the drug delivery composition according to claim 25.

31. A drug delivery system comprising microparticles, nanoparticles, micelles, liposomes, polymerosomes, microgels, nanogels, or nanotubes comprising the drug delivery composition according to claim 21.

32. The drug delivery system according to claim 26, wherein the drug delivery system comprises particles having an average diameter of from 1 to 40 μm.

33. The drug delivery system according to claim 26, wherein the drug delivery system comprises particles having an average diameter of from 20 nm to 800 nm.

Description

FIGURES

[0046] FIG. 1: .sup.1H NMR spectrum of PEA-SS1 (400 MHz, DMSO-d.sub.6).

[0047] FIG. 2: Percentage of PEA (SS) film weight loss as a function of degradation time (days) in α-Chymotrypsin (0.1 mg/mL) and DTT (10 mM) in PBS at 37° C. and 120 rpm. PBS buffer serves as the control. (A) weight loss in 30 d; (B) weight loss in the first 3 d.

[0048] FIG. 3: .sup.1H NMR spectra of PEA(SS) before (A) and after treatment with DTT for 2.5 h (B), 5 h (C), 8 h (D), 11 h (E) and 23 h (F) (DMSO-d.sub.6, 400 MHz).

[0049] FIG. 4: .sup.1H NMR of PEA-SS(P2EG/Cys=88/12) in DMSO-d.sub.6 (400 MHz).

[0050] FIG. 5: .sup.1H NMR of PEA-SS(P2EG/Cys=78/22) in DMSO-d.sub.6 (400 MHz).

[0051] FIG. 6: .sup.1H NMR of PEA-SS(P2EG/Cys=57/43) in DMSO-d.sub.6 (400 MHz).

[0052] FIG. 7: .sup.1H NMR of PEA in DMSO-d.sub.6 (400 MHz).

[0053] FIG. 8: SEM images of PEA and PEA-SS(P2EG/Cys=78/22) films at. The bars represent 20 um. (A) Original film of non-reduction-sensitive PEA polymers; (B) incubation in PBS for 1 day; (C) incubation in PBS for 4 day. (D) Original film of reduction-sensitive PEA-SS polymers; (E) incubation in 10 mM DTT for 12 h; (F) incubation 10 mM DTT for 24 h.

[0054] FIG. 9: The size change of different ratios of nanoparticles upon the addition of 10 mM DTT in PBS (pH 7.4, 10 mM, NaCl 100 mM).

[0055] FIG. 10: FITC-BSA release from nanoparticles in the presence of 10 mM DTT.

[0056] FIG. 11: FITC-CC release from nanoparticles in the presence of 10 mM DTT.

[0057] FIG. 12: DOX release from nanoparticles.

[0058] FIG. 13: MTT assays of empty nanoparticles.

[0059] FIG. 14A+B: MTT assays of reduction-sensitive DOX-loaded PEA nanoparticles, (A) Hela cells; (B) MCF-7 cells. The incubation time was 48 h.

[0060] FIG. 15. 1H NMR of L-Arg-4 based reduction-sensitive poly (ester amide)s in D2O (400 MHz)

EXAMPLES

Example 1: Synthesis of Reduction-Sensitive Di-p-Toluenesulfonic Acid Salts of Bis-L-Phenylalanine Ester

[0061] ##STR00005##

[0062] L-Phenylalanine (L-Phe, 6.000 g, 0.0363 mol), bis(2-hydroxyethyl)disulfide (HES, 2.546 g, 0.0165 mol) and p-toluenesulfonic acid monohydrate (6.909 g, 0.0363 mol) in 92 mL of toluene (20 mL for Dean-Stark) were placed in a flask equipped with a magnetic stirrer, a Dean-Stark apparatus and a CaCl2 drying tube. The solid-liquid reaction mixture was heated to reflux for 24 h until 0.95 mL of water was evolved and the reaction mixture changed to ivory-white. 7 mL ethanol (1/10 v. of toluene) was added after the reaction mixture was cooled to 60° C. below and then cooled to r.t., filtered, washed twice using mixture of toluene and ethanol (10/1 v.) and dried in vacuum at r.t. The product Phe(SS)-2TsOH was purified by recrystallization from methanol/water (1:1) three times. Yield: 40%. 1H NMR (400 MHz, DMSO-d6): δ 2.29 (6H, CH3-Ph-SO3−), 2.86 (4H, —CH2−S—S—), 3.11 (4H, Ph-CH2−), 4.33 (6H, 4H of —COO—CH2−CH2−S—S— and 2H of +H3N—CH(CH2PH)—COO—), 7.10-7.49 (18H, Ph), 8.41 (6H, +H3N—CH(CH2PH)—). FTIR (cm-1): 3000 (—NH3+), 1735[—C(O)—], 1450, 1500 and 1600 (Ph), 1202 (—SO3−). DSC: Tm=231° C.

Example 2: Synthesis of Di-p-Nitrophenyl Ester of Adipic Acid

[0063] ##STR00006##

[0064] The monomer Di-NP-Adipate was obtained via reacting adipoyl dichloride with p-nitrophenol (FIG. 3). Briefly, to a solution of p-nitrophenol (16.777 g, 0.1206 mol) and triethylamine (16.810 mL, 0.1206 mol) in 200 mL of acetone, a solution of adipoyl dichloride (10.982 g, 8.715 mL, 0.0600 mol) in 80 mL acetone was added dropwise at 0° C. After completion of addition, the reaction mixture was continued stirring for 2 h, then warmed to r.t. and proceeded overnight. Finally, the resulting monomer was obtained by pouring the reaction mixture into 1.6 L of ultra-pure water, filtration, washing twice with water, drying in vacuum at 50° C. overnight and recrystallization from ethyl acetate three times. Yield: 71%. 1H NMR (400 MHz, DMSO-d6): δ 2.73 (4H, —OCO—CH2−), 1.76 (4H, —OCO—CH2−CH2−), 7.46 and 8.29 (4H, —O-Ph-NO2). DSC: Tm=124.1-124.5° C.

Example 3: Synthesis of Reduction-Sensitive L-Phenylalanine Based Polyesteramides (PEA-SS1)

[0065] PEA-SS1 having repeat disulfide bonds was synthesized via solution polycondensation of Phe(SS)-2TsOH and dinitrophenyl ester of adipic acid (Di-NP-adipate). The synthetic pathway is shown in reaction scheme 3. Take balanced PEA (SS) (1:1) synthesis as an example. Briefly, to a Schlenk bottle equipped with a magnetic stir bar was charged Phe(SS)-2TsOH (0.7098 g, 0.895 mmol), Di-NP-adipate (0.3473 g, 0.895 mmol), Et3N (0.275 mL, 1.969 mmol) and 0.471 mL of DMF. After 20 min degasing with nitrogen flow, the reaction vessel was sealed and immersed in an oil bath thermostated at 70° C. The polymerization was allowed to proceed for 48 h. The resulting polymer was isolated by dilution with DMF, precipitation in ethylacetate two times to remove nitrophenol, precipitation in water to remove Et3N-TsOH and freeze-drying for 2 days.

[0066] In order to verify the actual chemical structure of the new PEA(SS), its 1H NMR and FTIR spectra was obtained (FIG. 1). As shown in FIG. 1, peaks assignable to both Di-NP-adipate (b 2.00 and 1.33) and Phe(SS) (2.86, 3.01, 4.24, 4.45, 7.20 and 8.24) were present in the 1H NMR spectrum. Importantly, comparing the integrals of signals at δ 2.00 and 4.45 pointed to a 1:1 equivalent polycondensation of Di-NP-adipate and Phe(SS)-2TsOH. FTIR spectrum showed the characteristic absorption bands of amide groups (˜1638 cm-1), NH stretch of amide groups (3430 cm-1) and C═O stretch of ester groups (˜1735 cm-1).

##STR00007##

[0067] GPC curve showed a decreasing trend of PDI (Poly Dispersity Index) with the decrease of equivalent ratio as represented in below Table 1.

TABLE-US-00001 TABLE 1 M.sub.n,UV.sup.a/ M.sub.n,GPC/ M.sub.w,GPC/ Equivalent kDa kDa kDa PDI T.sub.g/° C. Yield/%  1.0 eqv — 12.1 33.0 2.73 — 70.9  1.0 eqv 58.3 22.8 47.7 2.10 39.49 82.5 0.98 eqv 24.5 22.3 44.1 1.97 35.22 65.7 0.96 eqv 13.1 21.8 36.6 1.68 37.03 63.0 0.93 eqv  9.3 16.6 24.0 1.45 37.54 62.9

Example 4: In Vitro Enzymatic and Reductive Biodegradation of Disulfide Containing Poly(Ester Amide)s

[0068] To study the biodegradability of PEA(SS) polymers, PEA(SS) films were drop-cast from a 40 mg/mL chloroform solution onto glass microscope slides (1 cm×1 cm), and the solvent was allowed to evaporate overnight at r.t. Then the coated slides were placed into the 24-well cell plate and further dried in vacuo at r.t. for 2 d.

[0069] The coated dried slides (each in duplicate) were immersed in 1 mL of PBS buffer (pH 7.4 0.2 g KCl, 0.2 g KH.sub.2PO.sub.4, 1.15 g Na.sub.2HPO.sub.4, 8.0 g NaCl in 1 L, containing 0.05% (w/v) sodium azide to inhibit bacterial growth), or PBS buffer with α-chymotrypsin (0.1 mg/mL), or PBS buffer with 10 mM DTT in 24-well cell plate and incubated at 37° C. and 120 rpm. The degradation medium was refreshed every 24 h. At predetermined intervals, the remaining polymer samples (on slides) were collected via aspiration of the incubation medium and following rinsing of the wells three times for 5 min with distilled water. The collected samples were then dried in vacuo at r.t. to a constant weight. The degree of the degradation was estimated from the weight loss of the PEA(SS) film based on the following formula:

[00001]$\mathrm{Weight}.Math..Math.\mathrm{loss}.Math..Math.\left(\%\right)=\hspace{1em}\left[1-\frac{\left(\mathrm{weight}.Math..Math.\mathrm{of}.Math..Math.\mathrm{the}.Math..Math.\mathrm{film}+\mathrm{slide}\right)-\left(\mathrm{weight}.Math..Math.\mathrm{of}.Math..Math.\mathrm{the}.Math..Math.\mathrm{slide}\right)}{\left(\mathrm{initial}.Math..Math.\mathrm{weight}.Math..Math.\mathrm{of}.Math..Math.\mathrm{the}.Math..Math.\mathrm{film}+\mathrm{slide}\right)-\left(\mathrm{weight}.Math..Math.\mathrm{of}.Math..Math.\mathrm{the}.Math..Math.\mathrm{slide}\right)}\right]\times 100.Math.\%$

[0070] The degradation kinetics of PEA(SS) films in PBS buffer (pH 7.4), α-chymotrypsin solution (0.1 mg/mL) or DTT solution (10 mM) were illustrated FIG. 2.

[0071] In addition, the molecular weight of the PEA(SS) films after degradation was monitored via GPC.

TABLE-US-00002 TABLE 2 Weight loss and molecular weight of PEA(SS) films before and after incubation in different media. Degradation Weight M.sub.n(GPC)/ M.sub.w(GPC)/ Polymer Condition loss/% kDa kDa PDI PEA(SS) Original polymer 0 23.6 56.6 2.4 PBS-12 d 14 20.0 48.7 2.4 0.1 mg/mL □α- 70 22.4 53.9 2.4 Chymotrypsin-1 d 10 mM DTT-1 d 12.5 No signal

[0072] .sup.1H NMR and GPC measurements confirmed successful cleavage of disulfide bonds of each repeating unit to yield small molecules after 23 h. The resonances at δ 4.24 and 2.86 attributable to the methylene protons neighboring to the ester (—COO—CH.sub.2—CH.sub.2—SS—) and to the disulfide bond (—CH.sub.2—SS—CH.sub.2—) shifted to δ 4.08 and 2.60 respectively, which due to the disulfide bond is cleaved to thiol end groups. In addition, a new peak at δ 2.44 characteristic of thiol protons was detected as shown in FIG. 3.

[0073] The disulfide cleavage ratios of PEA(SS) polymer after 2.5, 5, 8, 11 and 23 h are calculated to be 46%, 55%, 70.5%, 86.5 and 100% via compare the integral ratio of peak e or f to peak b, respectively. Moreover, GPC revealed no signal for the polymer after 23 h treatment with DTT, which may be because the PEA(SS) polymer has been disrupted into small molecules completely (Table 3). FTIR measurement showed the same spectrum with initial PEA(SS) (FIG. 3), which indicated only disulfide bond cleaved during the reduction process.

TABLE-US-00003 TABLE 3 GPC results of PEA(SS) before and after DTT treatment. Sample Mn (kDa) Mw (kDa) PDI PEA(SS) 22.8 47.7 2.1 PEA(SS) + DTT No signal

Example 5: Synthesis of Di-p-Toluenesulfonic Acid Salts of Bis-L-Phenylalanine Esters (II)

[0074] ##STR00008##

[0075] Typically, L-Phe (0.176 mol), p-toluenesulfonic acid monohydrate (0.176 mol), and diethylene glycol (0.08 mol) in 300 mL of toluene were placed in a flask equipped with a Dean-Stark apparatus, a CaCl.sub.2 drying tube, and a magnetic stirrer. The solid-liquid reaction mixture was heated (ca. 140° C.) to reflux for 16 h. The reaction mixture was then cooled to room temperature. After the solvent was removed by rotary evaporation, the mixture was dried in vacuo overnight and finally purified by recrystallization from water 3 times. Thermal properties of synthesized monomer were characterized by a DSC 2920 (TA Instruments, New Castle, Del.). The measurement was carried out from 0 to 300° C. at a scanning rate of 10° C./min and nitrogen gas flow rate of 25 mL/min. TA Universal Analysis software was used for thermal data analysis. The melting point was determined at the onset of the melting endotherm. The melting point is 245° C.

[0076] The structure of the di-p-toluenesulfonic acid salt monomer was confirmed by FTIR and NMR spectra. The .sup.1H NMR data of the monomer also showed characteristic signals of —CH.sub.2—O—CH.sub.2— (.sup.1H: δ˜3.50 ppm). The monomer was obtained as white powder, and the yield was about 63%.

[0077] FTIR (cm.sup.−1): 1736 [—C(O)—], 1177 (—O—), 1127 (—CH.sub.2—O—CH.sub.2—). .sup.1H NMR (DMSO-d.sub.6, ppm, δ): 2.29 (6H, H.sub.3C-Ph-SO.sub.3—), 3.05, 3.10 (4H, PhCH.sub.2—), 3.50 [4H, —(O)C—O—CH.sub.2—CH.sub.2—], 4.19 [2H, .sup.+H.sub.3NCH(CH.sub.2Ph)-], 4.31 [4H, —(O)C—O—CH.sub.2CH.sub.2—], 7.11 to −7.49 [18H, Ph], 8.39 [6H, .sup.+H.sub.3N—CH(CH.sub.2Ph)-].

Example 6: Solution Polycondensation of Toluene Sulfonate Di Ester of Phenylalanine, Di-Nitrophenyl Ester and Cystamine Dihydrochloride

[0078] PEAs were prepared by solution polycondensation of di-p-toluenesulfonic acid diester salt with di-p-nitrophenyl ester and cystamine dihydrochloride, which involved four different ratios of phenylalanine and cystamine dihydrochloride (SS: 0%, 10%, 20% and 40%, SS represents cystamine dihydrochloride). The combinations attempted in this work shown in below scheme 5 explaining the synthesis of L-Phenylalanine Based PEAs.

A: containing cystamine dihydrochloride (PEA-SS);
B: containing no cystamine dihydrochloride (PEAs).

##STR00009##

[0079] Table 4 summarizes the fundamental properties of the PEA-SS synthesized. All four PEA-SS were obtained in fairly good yields (61-72%). The number and weight averaged molecular weights (M.sub.n and M.sub.w) of synthesized PEA-SS were determined by GPC, and DMF was used as eluent.

TABLE-US-00004 TABLE 4 Fundamental Properties of PEA-SS Theoretical Experimental (molar ratio) (molar ratio).sup.a Polymer m:n-m.sup.b M.sub.n.sup.c PDI Yield (%) PEA-SS 90:10 88/12 48000 1.4 64 (P2EG/Cys = 88/12) PEA-SS 80:20 78/22 38500 1.5 66 (P2EG/Cys = 78/22) PEA-SS 60:40 57/43 21900 1.7 72 (P2EG/Cys = 57/43) PEA — — 25000 1.3 61 .sup.adetermined by .sup.1H NMR. .sup.bm:n-m is the molar ratio of di-p-toluenesulfonic acid salts of L-phenylalanine ester to cystamine. .sup.cDetermined by GPC (DMF as the eluent, 1.0 mL/min, 30° C., polystyrene standards).

[0080] The .sup.1H NMR spectra of four typical PEA-SS based on di-ethylene glycol are shown in FIGS. 4, 5, 6 and 7. The spectral data were fully in agreement with the anticipated chemical structure of the PEA polymers shown in Scheme 5.

Example 7: In Vitro Biodegradation of PEA-SS/PEA Copolymers

[0081] Biodegradation of PEA-SS/PEA copolymers were carried out in a small vial containing a small piece of dry PEA-SS/PEA film, (ca. 80 mg) and 10 mL of PBS buffer solution (pH 7.4, 10 mM,) consisting of 10 mM DTT or not. The vial was then incubated at 37° C. with a constant reciprocal shaking (100 rpm). At predetermined immersion durations, the film samples were removed from the incubation medium, washed gently with distilled water, and surface water was blotted by film paper and dry at room temperature. Scanning electron microscope (SEM) was employed to analyze the effect of biodegradation process on the surface morphology of PEA-SS/PEA polymers. The surface morphology changes of these PEA-SS/PEA film samples upon biodegradation are shown in FIG. 8. After 1 day and 4 days incubation in PBS buffer (pH 7.4, NaCl 100 mM), the PEA film samples showed little surface erosion. However, the PEA-SS film samples showed a significant biodegradation after 12 h and 24 h incubation in 10 mM DTT PBS buffer as evident by the appearance of rough or crater shaped eroded surface with more microscopic pores.

Example 8: Preparation of Nanoparticles

[0082] Nanoparticles were prepared by dialysis synthetic method with suitable size and narrow PDI (Table 5).

TABLE-US-00005 TABLE 5 Size and PDI results of nanoparticles. Nanoparticles Size (nm) PDI PEA-SS(P2EG/Cys = 88/12) 143 0.15 PEA-SS(P2EG/Cys = 78/22) 138 0.11 PEA-SS(P2EG/Cys = 57/43) 151 0.12 PEA 97 0.17 .sup.aSS represents Cystamine Dihydrochloride monomer.

[0083] The disulfide bonds containing PEA nanoparticles are reported to have reduction sensitivity in an intracellular mimicking environment. Here we investigated the responsiveness of the nanoparticles containing different ratios of disulfide bonds. We firstly followed the size change of the nanoparticles at different ratio in response to 10 mM DTT in PBS buffer (pH 7.4, 10 mM, NaCl 100 mM) by using DLS measurement. For the nanoparticles containing 10-40% SS, DTT (10 mM) treatment did not affect the size of the nanoparticles (FIG. 9).

Example 9: Preparation of Nanoparticles of PEA with and without Protein (FITC-BSA) Through Dialysis Synthetic Method

[0084] PEA nanoparticles were prepared by dialysis synthetic method. Briefly, the copolymer (4 mg) was first dissolved in DMSO (2 mL). This solution was then added dropwise to 4 mL of PBS (pH 7.4, 10 mM, NaCl 100 mM) buffer or protein solution and stirred using a magnetic stirrer at 25° C. The resulting PEA nanoparticle suspension was extensively dialyzed against PBS (pH 7.4, 10 mM, NaCl 100 mM) for 24 h (MWCO 500 kDa), and the dialysis medium was changed five times. The amount of protein was determined by fluorescence measurements (FLS920, excitation at 492 nm). For determination of protein loading content, protein loaded NPs were dissolved in DMSO and analyzed with fluorescence spectroscopy, wherein calibration curve was obtained with protein/DMSO solutions with different protein concentrations.

[0085] Protein loading content (PLC) and protein loading efficiency (PLE) were calculated according to the following formulas:

PLC (wt. %)=(weight of loaded protein/total weight of loaded protein and polymer×100%

PLE (%)=(weight of loaded protein/weight of protein in feed)×100%

[0086] The nanoparticles loaded FITC-BSA (bovine serum albumin) and FITC-CC (cytochrome C) have been prepared, table 6 and 7 represent the encapsulation results. The release of proteins from nanoparticles was investigated in the presence 10 mM DTT (FIG. 10+11). Remarkably, the more SS in the nanoparticles, the faster the protein was released. For example, 26, 39 and 80% of FITC-BSA was released in 44 h for PEA, PEA-SS (P2EG/Cys=88/12) and PEA-SS (P2EG/Cys=78/22) nanoparticles, respectively, moreover, almost 100% FITC-BSA was released in 12 h for PEA-SS (P2EG/Cys=57/43) nanoparticles.

TABLE-US-00006 TABLE 6 Encapsulation results FITC-BSA. Protein-loaded nanoparticles Size (nm)/ nanoparticles PDI PLC (wt. %) PLE (%) PEA 137/0.08 58 2.9 PEA-SS(P2EG/Cys = 88/12) 150/0.06 66 3.3 PEA-SS(P2EG/Cys = 78/22) 153/0.03 62 3.1 PEA-SS(P2EG/Cys = 57/43) 141/0.03 56 2.8 .sup.aFITC-BSA in feed was 5 wt. %.

TABLE-US-00007 TABLE 7 Encapsulation results FITC-CC. Protein-loaded nanoparticles Size (nm)/ nanoparticles PDI PLC (wt. %) PLE (%) PEA 130/0.08 55 2.8 PEA-SS(P2EG/Cys = 88/12) 145/0.08 68 3.4 PEA-SS(P2EG/Cys = 78/22) 147/0.06 65 3.3 PEA-SS(P2EG/Cys = 57/43) 132/0.07 60 3.0 .sup.aFITC-CC in feed was 5 wt. %.

Example 10—In Vitro Release of Proteins

[0087] The release of FITC-BSA and FITC-CC from nanoparticles was investigated using a dialysis release method (MWCO 500 kDa) at 37° C. with 0.5 mL of protein-loaded nanoparticle suspensions against 30 mL PBS (pH 7.4, 10 mM, NaCl 100 mM) with 10 mM DTT. At desired time intervals, 6 mL release media was taken out and replenished with an equal volume of fresh media. The amounts of released proteins as well as proteins remaining in the dialysis tube were determined by fluorescence measurements (FLS920, excitation at 492 nm). The release experiments were conducted in triplicate and are represented in FIGS. 10+11.

Example 11—Preparation of DOX-Loaded Nanoparticles

[0088] DOX-loaded PEA or PEA-SS nanoparticles were also prepared by dialysis synthetic method. Briefly, the copolymer (4 mg) was first dissolved in DMSO (2 mL), then predetermined DOX solution was added it. This solution was then added dropwise to 4 mL of PBS (pH 7.4, 10 mM, NaCl 100 mM) buffer and stirred using a magnetic stirrer at 25° C. The resulting PEA or PEA-SS nanoparticle suspension was extensively dialyzed against PBS (pH 7.4, 10 mM, NaCl 100 mM) for 24 h (MWCO 3.5 kDa), and the dialysis medium was changed five times.

[0089] The amount of DOX was determined by fluorescence measurements (FLS920, excitation at 480 nm). For determination of DOX loading content, DOX-loaded NPs were dissolved in DMSO and analyzed with fluorescence spectroscopy, wherein calibration curve was obtained with DOX/DMSO solutions with different DOX concentrations.

TABLE-US-00008 TABLE 8 The results of loading DOX. DOX DOX loading loading DOX feed content efficiency Size (nm)/ Nanoparticles ratio (wt. %) (wt. %) (%) PDI PEA 5 3.5 69.7  138/0.095 10 6.5 65.2 144/0.08 20 11.1 55.3 154/0.13 PEA-SS 5 3.4 67.8 142/0.11 (P2EG/Cys = 88/12) 10 6.4 63.8 146.5/0.10   20 11.0 54.8 160/0.14 PEA-SS 5 3.4 67.5 148/0.11 (P2EG/Cys = 78/22) 10 6.1 61.2 150/0.14 20 9.3 46.3 168/0.18 PEA-SS 5 3.1 62.8 140/0.10 (P2EG/Cys = 57/43) 10 5.6 56.4 145/0.12 20 8.2 40.9 157/0.17

Example 12—In Vitro DOX Release

[0090] In vitro release of DOX from the nanoparticles was studied using a dialysis tube (MWCO 12000, Spectrum) at 37° C. in PBS (pH 7.4, 10 mM, NaCl 100 mM) with or without 10 mM DTT. In order to acquire sink conditions, drug release studies were performed with 0.7 mL of micelle solution dialysis against 20 mL of the same medium. At desired time intervals, 6 mL release media was 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 as presented in FIG. 12 are the average data.

Example 13—MTT Assays of PEA and PEA-SS Nanoparticles

[0091] HeLa cells were plated in a 96-cell plate (5×10.sup.3 cells/well) using 10% fetal bovine serum, 1% .sub.L-glutamine, antibiotics penicillin (100 IU/mL) and streptomycin (100 μg/mL). After 24 h, prescribed amounts of PEA-SS/PEA nanoparticles were added and incubation for 48 h at 37° C. in an atmosphere containing % 5 CO.sub.2. Then 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) solution in PBS (5 mg/mL) was added and incubated for another 4 h. The medium was aspirated, the MTT-formazan generated by live cells was dissolved in 150 μL of DMSO for 20 min, and the absorbance at a wavelength of 490 nm of each well was measured using microplate reader (Bio-rad,ELX8081U). The cell viability (%) was determined by comparing the absorbance at 490 nm with control wells containing only cell culture medium. The experiments were performed four times each.

[0092] MTT assays showed that PEA-SS and PEA nanoparticles were practically nontoxic to Hela cells (cell viability >85%) up to a tested concentration of 2.4 mg/mL (FIG. 13).

Example 14—MTT Assays of DOX-Loaded PEA-SS/PEA Nanoparticles

[0093] Hela and MCF-7 cells were plated in a 96-well plate (5×10.sup.3 cells/well) using 10% fetal bovine serum, 1% .sub.L-glutamine, antibiotics penicillin (100 IU/mL) and streptomycin (100 μg/mL). After 24 h, prescribed amounts of DOX-loaded PEA-SS/PEA nanoparticles (5 μg/mL, 10 μg/mL) were added and incubation for 48 h at 37° C. in an atmosphere containing 5% CO.sub.2. Then 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) solution in PBS (5 mg/mL) was added and incubated for another 4 h. The supernatant was carefully aspirated, and the MTT-formazan generated by live cells was dissolved in 150 μL of DMSO for 20 min. The absorbance at a wavelength of 490 nm of each well was measured using microplate reader (Bio-rad,ELX8081U). The cell viability (%) was determined by comparing the absorbance at 490 nm with control wells containing only cell culture medium. The experiments were performed four times each.

[0094] The cytotoxicity of DOX-loaded PEA-SS/PEA nanoparticles was investigated in Hela and MCF-7 cells using MTT assays. The cells were incubated with DOX-loaded nanoparticles for 48 h at drug dosages of 5 and 10 μg DOX equiv. mL.sup.−1. Interestingly, there was an apparent dependency of anti-tumor activity on cystamine content, as well as DOX dosage (FIG. 14). For example, cell viability of 75.9, 69.7, 60.9 and 42.3% were observed for Hela cells treated for 48 h at a DOX dosage of 5 μg/mL and 69.3, 60.9, 45.1 and 20.7% at a DOX dosage of 10 μg/mL with DOX-loaded PEA, PEA-SS(P2EG/Cys=88/12), PEA-SS(P2EG/Cys=78/22) and PEA-SS(P2EG/Cys=57/43), respectively (FIG. 14A), indicating that the cytotoxicity of DOX-loaded nanoparticles intimately depends on disulfide content in the bioreducible environment. These results agree well with the in vitro as well as the intracellular DOX release profiles. In all cases, cell viabilities decreased with increasing drug dosages from 5 to 10 μg DOX equiv. mT.sup.−1.

Example 13—Synthesis of Di-p-Toluenesulfonic Acid Salts of L-Arqinine Ester

[0095] Di-p-toluenesulfonic acid salts of L-Arginine ester were prepared according to Scheme 3, Typically, L-Arginine (0.03 mol), p-toluenesulfonic acid monohydrate (0.06 mol), and Bis(2-hydroxyethyl) Disulfide (0.015 mol) in 60 mL toluene were placed in a flask equipped with a Dean-Stark apparatus, a CaCl2 drying tube and a magnetic stirrer. The solid-liquid reaction mixture was heated to reflux for 24 h until 1.65 mL (0.09 mol) of water was distilled. The reaction was cooled to room temperature. After the solvent was removed by rotary evaporation, the mixture was dried in vacuo overnight and finally purified by 3 times recrystallization in 2-propanol.

##STR00010##

[0096] 1H NMR (D2O, δ): 7.24-7.67 (16H, Ph-CH2−), 4.42 (4H, —C(O)OCH2CH2), 3.89 (4H, —NH—CH2−CH2−), 3.29 (4H, —NH—CH—), 2.76 (4H, —NH—CH2CH2SSCH2CH2−NH—), 2.56 (4.8H, Ph-CH3), 1.75-1.82 (8H, —CHCH2CH2CH2NH—).

Example 14—Synthesis of L-Arinine-4 Based Reduction-Sensitive Poly(Ester Amide)s

[0097] Polymerization reactions were carried out in DMA at 70° C. with excess triethylamine for 48 h (Scheme 1). 1.1 equiv. of di-p-toluenesulfonic acid salts of di-p-nitrophenyl ester of dicarboxylic acid was combined with L-Arginine-4 ester and cystamine mixture (molar ratio: 80/20). After polymerization, the reaction mixture was purified by precipitation and subsequent Soxhlet extraction. Then, L-Arginine-4 based reduction-sensitive poly(ester amide)s were modified by 1.8 kDa PEI, as shown in Scheme 7. The un-reacted PEI was removed by dialysis (MW 3500).

##STR00011##