LACTIDE CONTAINING POLYESTER-POLYETHYLENE GLYCOL TRIBLOCK THERMORESPONSIVE COPOLYMERS

Abstract

The inventors of the technology disclosed herein have developed triblock copolymers of lactide-containing polyesters and poly(ethylene glycol), PEG, having thermoresponsive properties.

Claims

1. A triblock copolymer constructed of a lactide-containing polyester and poly(ethylene glycol), PEG, wherein in the triblock copolymer, the PEG is of a molecular weight between 1,000 and 3,000 Da.

2. The triblock copolymer according to claim 1, wherein the lactide-containing polyester is selected from D,L-PLA, L-PLA, D-PLA, PLGA and PCL.

3. The triblock copolymer according to claim 1, having a structure selected from D,L-PLA-PEG-D,L-PLA, D-PLA-PEG-D-PLA, L-PLA-PEG-L-PLA, PLGA-PEG-PLGA and PCL-PEG-PCL.

4. The triblock copolymer according to claim 1, being selected from hybrid triblocks containing PEG and one lactide-containing polyester segment.

5. The triblock copolymer according to claim 1, being selected from D,L-PLA-PEG-L-PLA, D,L-PLA-PEG-D-PLA, D-PLA-PEG-L-PLA, L-PLA-PEG-PLGA, D-PLA-PEG-PLGA, PLGA-PEG-PCL and PCL-PEG-D,L-PLA.

6. The triblock copolymer according to claim 1, wherein the lactide-containing polyester segment is PLGA.

7. The triblock copolymer according to claim 1, wherein the triblock is PLGA-PEG-PLGA.

8. A poly(D,L-lactic acid-co-glycolic acid)-b-poly(ethylene glycol)-b-poly (D,L-lactic acid-co-glycolic acid) (PLGA-PEG-PLGA) triblock copolymer, wherein in the PLGA-PEG-PLGA triblock copolymer, the PEG is of a molecular weight between 1,000 and 3,000 Da, the PLGA and PEG being present at a ratio of between 1 and 4, and wherein the triblock copolymer having a gelation temperature between 10 and 50° C.

9. A PLGA-PEG-PLGA triblock copolymer, wherein in the PLGA-PEG-PLGA triblock copolymer, the PEG is of a molecular weight between 1,000 and 3,000 Da, one or both of the PLGA segments being constructed of lactide and glycolide (LA:GA) moieties at a ratio (LA:GA) of about 6, and wherein the triblock copolymer having a gelation temperature between 10 and 50° C.

10. A PLGA-PEG-PLGA triblock, characterized by: in PLGA-PEG-PLGA triblock copolymer, the PEG is of a molecular weight between 1,000 and 3,000 Da, in the PLGA-PEG-PLGA triblock copolymer, the PLGA and PEG being present at a ratio of between 1 and 4, or one or both of the PLGA segments is constructed of lactide and glycolide (LA:GA) moieties at a ratio around about 6; and the triblock copolymer having a gelation temperature between 10 and 50° C.

11. The PLGA-PEG-PLGA triblock according to claim 8, characterized by: in PLGA-PEG-PLGA triblock copolymer, the PEG is of a molecular weight between 1,000 and 3,000 Da, in the PLGA-PEG-PLGA triblock copolymer, the PLGA and PEG being present at a ratio of between 1 and 4, in the PLGA-PEG-PLGA triblock copolymer, one or both of the PLGA segments is constructed of lactide and glycolide (LA:GA) moieties at a ratio around about 6; and the triblock copolymer having a gelation temperature between 10 and 50° C.

12. A PLGA-PEG-PLGA triblock copolymer material, the material being any one or more of materials in Table 1.

13. A method of manufacturing a PLGA-PEG-PLGA triblock copolymer having a gelation temperature between 10 and 50° C., the method comprising reacting PEG of a molecular weight between 1,000 and 3,000 Da with D,L-lactic acid (LA) and a glycolide (GA) at (1) a LA:GA ratio of about 6; and/or (2) with a D,L-lactic acid (LA) and a glycolide (GA) amounts sufficient to achieve a PLGA/PEG ratio of between 1 and 4; under conditions permitting formation of the triblock copolymer.

14.-30. (canceled)

31. A drug delivery vehicle comprising at least one active or non-active agent contained within a triblock copolymer according to claim 1.

32. The vehicle according to claim 31, for medicinal, cosmetic or veterinary use.

33. The vehicle according to claim 31, adapted for release of the at least one active agent over a predetermined period of time.

34.-37. (canceled)

38. A method of treatment of at least one disease or disorder in a subject, the method comprising administering to the subject a triblock copolymer according to claim 1 or a formulation comprising same.

39. A cosmetic method comprising topically administering to a subject a triblock according to claim 1 or a formulation comprising same.

40. A thermoresponsive article comprising two or more triblock copolymers according to claim 1, and at least one active or non-active agent, each of said two or more triblock copolymers liquefy at different temperatures, thereby permitting controlled release of said at least one active or non-active agent.

41. A controlled release article comprising two or more triblock copolymers according to claim 1, and at least one active or non-active agent, each of said two or more triblock copolymers gel at a different temperature, and comprise a different active or non-active agent, thereby permitting release of said at least one active or non-active agent.

42.-43. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0110] 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:

[0111] FIG. 1 depicts transition from a solution to a gel upon heating from room temperature to physiological temperatures.

[0112] FIG. 2 provides the structure of a PLGA-PEG-PLGA triblock copolymer. In the formula, x represents the number of PEG repeating units, y represents LA and z represents GA. Without limitation, X=22-69; Y=7-56; Z=0-28, where Z<Y/2.

[0113] FIG. 3 provides a representative .sup.1H NMR spectrum of PLGA-PEG-PLGA triblock copolymer (16, Table 1) with peak assignments. LA:GA ratios were calculated by comparing the integration of their respective peaks (peak C represents the CH of lactide and D the CH.sub.2 of glycolide), and overall polymer MW was determined by using a known integration of the PEG peak (A) and adding to it the total LA and GA content.

[0114] FIG. 4 provides a representative .sup.13C NMR spectrum of PLGA-PEG-PLGA triblock copolymer (16, Table 1) with peak assignments. Peak B represents PLGA block ester bonds and peak F represents the ester bridge between PEG and PLGA blocks.

[0115] FIG. 5 provides a representative IR spectrum of PLGA-PEG-PLGA triblock copolymer (16, Table 1). A strong band at 1750 cm.sup.−1 is observed for the formed polyester.

[0116] FIG. 6 provides a representative phase diagram of PLGA-PEG-PLGA (19, Table 1) aqueous solutions. As temperature increases the solution turns to a gel, and upon further heating a precipitate is formed.

[0117] FIG. 7 shows dependence of T.sub.gel on PLGA/PEG ratio. For each set of polymers based on a particular PEG MW, a linear relationship has been defined between the polymer's aqueous gelling temperature in a 20% solution and the polymer structure's PLGA/PEG ratio.

[0118] FIG. 8 shows a release profile of paracetamol from hydrogel of PLGA-PEG-PLGA 13. Media was exchanged at 16, 40, and 64 h after gel was formed and paracetamol content in media was tracked by UV absorbance at 243 nm.

[0119] FIG. 9 shows a PLGA-PEG-PLGA triblock copolymer modified by (1) extending the PEG block and (2) employing PCL sidechains.

[0120] FIG. 10 depicts an exemplary use of triblocks of the invention for tissue engineering.

DETAILED DESCRIPTION OF EMBODIMENTS

Experimental

[0121] Materials

[0122] PEG-1000 was purchased from Union Carbide Chemicals and Plastics Company Inc. PEG-1500 was purchased from BDH Chemicals Ltd. PEG-2000 and stannous octoate were purchased from Sigma Aldrich. Lactide and glycolide were purchased from Purac Biochem Bv. Dichloromethane was purchased from Bio-Lab Ltd.

[0123] Synthesis

[0124] Lactide-based triblocks according to the invention, amongst them PLA-PEG-PLA and PLGA-PEG-PLGA triblock copolymers were prepared by ring-opening polymerization (ROP) of poly(ethylene glycol) (PEG) in the presence of stannous octoate catalyst.

[0125] A sample synthesis for the preparation of PLGA-PEG-PLGA is as follows:

[0126] 50 μL of a 100 mg/mL solution of stannous octoate in dichloromethane (DCM) was added to a melt of PEG-1500 (595 mg, 0.397 mmol), D,L-lactide (696 mg, 4.83 mmol) and glycolide (94 mg, 0.81 mmol). Solvent was allowed to evaporate and the vial was purged with N.sub.2. The mixture was stirred at 120° C. for 2 h, followed by overnight stirring at 150° C. The crude polymer was taken up in DCM and precipitated into ether to afford polymer 8 in quantitative yield. .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 5.22-5.14 (m, LA), 4.92-4.67 (m, GA), 3.64 (s, PEG), 1.56 (d, J=6 Hz, LA); .sup.13C NMR (75 MHz, CDCl.sub.3, δ): 169 (C═O), 166 (C═O), 72 (LA, CH), 70 (PEG, CH.sub.2), 69 (PEG, CH.sub.2), 66 (LA, CH), 64 (GA, CH.sub.2), 61 (GA, CH.sub.2), 16 (LA, CH.sub.3); IR (NaCl): ν=1750 (s), 1452 (w), 1350 (w), 1184 (w), 1086 (s), 949 (w), 863 (w).

[0127] Nuclear Magnetic Resonance (NMR)

[0128] .sup.1H and .sup.13C NMR spectra were obtained on a Varian 300 MHz spectrometer with CDCl.sub.3 as the solvent and tetramethylsilane as shift reference.

[0129] Infrared (IR) Spectroscopy

[0130] Infrared spectroscopy (2000 FTIR; PerkinElmer) was performed on polymer samples cast onto NaCl plates.

[0131] Determination of Gelling Temperature

[0132] 20% w/v aqueous polymeric solutions were incubated at a given temperature for 10 minutes, and the vial was inverted to test for gelling. If the gel did not flow, the temperature was recorded as the gelling temperature of the solution (T.sub.gel). Results are accurate to +/−2.0° C.

[0133] Release Study

[0134] Paracetamol was dissolved in 1 mL of 20% aqueous PLGA-PEG-PLGA solution at a ratio of 5:100 paracetamol:polymer (w/w). The solution was heated to 37° C. until gel was formed. 4 mL 0.1 M phosphate-buffered saline solution (PBS) was added on top of the gel at 37° C. Paracetamol released was measured by UV absorbance at 243 nm.

[0135] Results and Discussion

[0136] Lactide-Containing Polyester—PEG Triblock Copolymers

[0137] Poly(D,L-lactic acid)-poly(ethylene glycol)-poly(D,L-lactic acid) (PDLLA-PEG-PDLLA) triblock copolymers were synthesized by ring-opening polymerization (ROP) of D,L-lactide by PEG (MW 1500 Da) in the presence of stannous octoate. In one example, stannous octoate (50 μL of a 10% solution in dichloromethane) was added to a heated mixture of PEG-1500 (216.06 mg, 0.144 mmol) and D,L-lactide (410.37 mg, 2.84 mmol) under N.sub.2 at 120° C. The mixture was stirred at 120° C. for 3 h, followed by overnight stirring at 150° C. to afford the polymer. A 20% w/v aqueous solution of the polymer formed a reversible thermoresponsive hydrogel with a sol-gel transition temperature of 40° C.

[0138] In another example, the same procedure was performed with PEG-1500 (297.9 mg, 0.199 mmol) and D,L-lactide (536.43, 3.72 mmol). A 20% w/v aqueous solution of the resultant polymer formed a reversible thermoresponsive hydrogel with a sol-gel transition temperature of 44° C.

[0139] Exemplary System 1: PLA-PEG-PLA Series

[0140] Poly(D,L-lactic acid)-poly(ethylene glycol)-poly(D,L-lactic acid) (PDLLA-PEG-PDLLA) triblock copolymers were synthesized by ring-opening polymerization (ROP) of D,L-lactide by PEG (MW 1500 Da) in the presence of stannous octoate. In one example, stannous octoate (50 μL of a 10% solution in dichloromethane) was added to a heated mixture of PEG-1500 (216.06 mg, 0.144 mmol) and D,L-lactide (410.37 mg, 2.84 mmol) under N.sub.2 at 120° C. The mixture was stirred at 120° C. for 3 h, followed by overnight stirring at 150° C. to afford the polymer. A 20% w/v aqueous solution of the polymer formed a reversible thermoresponsive hydrogel with a sol-gel transition temperature of 40° C.

[0141] In another example, the same procedure was performed with PEG-1500 (297.9 mg, 0.199 mmol) and D,L-lactide (536.43, 3.72 mmol). A 20% w/v aqueous solution of the resultant polymer formed a reversible thermoresponsive hydrogel with a sol-gel transition temperature of 40° C.

[0142] Exemplary System 2: PLGA-PEG-PLGA Copolymer Series

[0143] It has been demonstrated that the gelling temperature of aqueous solutions of PLGA-PEG-PLGA triblock copolymers was lowered by increasing either the lactide:glycolide (LA:GA) ratio in the PLGA block or polymer aqueous concentration. Here, a 6/1 LA:GA molar ratio and a 20% w/v aqueous polymer concentration were fixed in order to isolate the effect of PLGA/PEG weight ratio on gelling behaviour of the copolymer (FIG. 1). A series of such polymers (FIG. 2) were thereby synthesized with varying PEG and PLGA molecular weights and ratios, while keeping constant a 6:1 LA:GA molar ratio in the feed. Gelling temperature of 20% w/v aqueous solutions of each polymer were then tested (Table 1). Polymers with different molecular weight PLGA blocks were obtained by altering the ratio of combined LA and GA monomers relative to PEG in the feed.

[0144] In some embodiments of a triblock shown in FIG. 2, X=22-69; Y=7-56; Z=0-28 where Z<Y/2.

[0145] Characterization of PLGA-PEG-PLGA Copolymer Series

[0146] Polymer MW and experimental LA:GA ratio were defined by .sup.1H NMR (FIG. 3). To determine MW, the known integration value of the PEG peak (FIG. 3, peak A) was compared to the integrations of the lactide and glycolide peaks (FIG. 3, peaks C and D). The post-purification LA:GA ratio was also determined from the relative integration of the C (lactide CH) and D (glycolide CH.sub.2) peaks of the .sup.1H NMR spectrum. Ester formation was confirmed by .sup.13C NMR and IR. The carbon that experienced a chemical shift of 169 ppm corresponds to PLGA polyester and of 166 ppm to the ester connectivity between PEG and PLGA blocks (FIG. 4, peaks B and F). The IR ester band at 1750 cm.sup.−1 also indicates ester bond formation (FIG. 5). The spectroscopy and gelling results for all 26 synthesized polymers can be found in Table 1.

TABLE-US-00001 TABLE 1 Chemical properties of PLGA-PEG-PLGA triblock copolymers. Polymers 1-7 are based on PEG-1000, 8-20 on PEG-1500, and 21-26 on PEG-2000. Polymers were synthesized by ROP of D,L-lactide and glycolide by PEG in the presence of stannous octoate catalyst. PLGA/PEG and LA:GA ratios and polymer MW were determined by .sup.1H NMR (FIG. 3). PEG PLGA PLGA/ T.sub.gel Entry MW.sup.a MW.sup.b PEG.sup.c LA:GA.sup.d (C.) 1 1000 1077 1.08 6.2 50 2 1000 1526 1.53 5.6 40 3 1000 1894 1.89 5.3 32 4 1000 1946 1.95 5.8 35 5 1000 2159 2.16 6.6 20 6 1000 2176 2.18 6.6 24 7 1000 2468 2.47 5.7 15 8 1500 1789 1.19 6.1 50 9 1500 1872 1.25 6.1 50 10 1500 2049 1.37 5.9 50 11 1500 2408 1.61 6.1 45 12 1500 2485 1.66 6.6 45 13 1500 2819 1.88 6.7 40 14 1500 2861 1.91 5.7 40 15 1500 2983 1.99 6.6 40 16 1500 3006 2.00 6.2 40 17 1500 3182 2.12 6.6 40 18 1500 3314 2.21 5.7 35 19 1500 3510 2.34 5.8 35 20 1500 4529 3.02 6.8 25 21 2000 2406 1.20 5.8 60 22 2000 2640 1.32 5.7 58 23 2000 2670 1.34 6.5 58 24 2000 2727 1.36 5.3 60 25 2000 3163 1.58 5.7 58 26 2000 4016 2.01 6.1 55 .sup.acorresponds to X in the polymer structure (FIG. 1). .sup.bcorresponds to Y + Z in the polymer structure (FIG. 1). .sup.ccorresponds to (Y + Z)/X in the polymer structure (FIG. 1). .sup.dcorresponds to Y/Z in the polymer structure (FIG. 1).

[0147] The mechanism of PLGA-PEG-PLGA triblock copolymer gel formation has been described. In short, at temperatures of about 0-25° C., the inter-chain hydrogen bonding between PEG segments dominates the solution energy profile, and the polymer dissolves in water. As the temperature increases, these hydrogen bonds weaken, and inter-chain hydrophobic interactions between the PLGA segments of the copolymer strengthen, leading to a three-dimensional physically cross-linked system that does not exclude water, resulting in the hydrogel. As the temperature is further increased, the hydrophobic interactions are further strengthened and the polymer crashes out of solution (FIG. 6). The PLGA/PEG ratio is therefore crucial in determining the sol-gel transition temperature (T.sub.gel), as a low amount of hydrophobic inter-chain PLGA interactions relative to those of PEG would require a higher amount of energy to overcome the hydrophilic PEG-water and PEG-PEG interactions, and a high PLGA/PEG ratio would require less energy to overcome this barrier. Consequently, one would expect that a higher PLGA/PEG ratio would lead to a lower T.sub.gel.

[0148] By controlling the LA:GA ratio, PEG molecular weight, and polymer concentration, we were able to isolate the effect of PLGA block length on gelling temperature. As expected, increase of hydrophobic PLGA block lead to a lower gelling temperature, as the system required less energy for the PLGA-PLGA hydrophobic interactions to overcome the hydrogen bonding between hydrophilic PEG segments. Indeed, a linear relationship was found between descending PLGA block length and gelling temperature (FIG. 7).

[0149] Controlled Release of a Representative Drug

[0150] The controlled release of paracetamol as representative water soluble drug from within the gel to external physiological media was tested to prove the ability of a therapeutic agent to be released from the gel matrix. To this effect, paracetamol was dissolved in an aqueous solution containing 20% w/v PLGA-PEG-PLGA 13, then heated to 37° C. to form gel. PBS was added on top of the gel, and it was exchanged for fresh PBS each morning until almost 90% of paracetamol had been observed in the exchanged media. We chose paracetamol as a representative drug as its release profile was easy to follow by UV absorbance of the exchanged media. Over 50% of the paracetamol was released within 16 hours, and 90% released within 64 hours (FIG. 8). It should be noted that the gel maintained its robustness in the release media for over one week with almost no erosion or change in viscosity. These results are consistent with previously reported water-soluble drug release from PLGA-PEG-PLGA thermoresponsive hydrogels.

[0151] Due to the robustness of the hydrogel, and its optimized sol-gel sharp transition, it can be injected into a physiological environment at room temperature as a liquid, and gel in tissue. When representative therapeutic material was dissolved in the room-temperature solution, controlled release from the gel was achieved. This finding may allow for the targeted delivery and controlled release of any therapeutic material at an injectable site.

[0152] Comparative Data

[0153] Where equivalent PCL-PEG-PCL triblocks were used instead of PLGA-PEG-PLGA, hydrogels were similarly obtained. In this case, PCL-PEG-PCL is an example that forms a non-reversible hydrogel.

[0154] Where in the PCL-PEG-PCL triblocks the PEG had a MW of 4000 polymers formed hydrogels only when the following two conditions were met: [0155] i. PCL MW was in the range of 1,700-2,200 Da. [0156] ii. The suspended polymer was heated to 50° C. for 10 min.

[0157] Water soluble solutions of this polymer did not form gels and remained in solution at any temperature up to ˜40° C. When heated at 50° C. these polymers forms gels that were not reversible. The gels remained stable at room temperature for over one month. Similar results were obtained for PCL-PEG(8000)-PCL when the PCL MW was in the range of 3,000-4,400. Although logically this triblock should form a reversible hydrogel, it did not; but only at a narrow MW range it formed a non-reversible gel.

[0158] When PEG of MW over 3,000 Da was used in PLGA-PEG-PLGA triblocks, gels were never formed. PEG 4,000 and PEG 8,000 triblock PLGA polymers were either water soluble or insoluble without a gelling phase.

[0159] Experimental—High Molecular Weight PCL-PEG-PCL Triblock Copolymers

[0160] Hydrogel longevity and long-term stability may be enhanced by (1) replacing the PLGA sidechains with poly(caprolactone) (PCL), a more hydrophobic and hydrolytically stable polyester, and (2) using higher molecular weight (MW) PEG in order to afford higher MW biodegradable sidechains (FIG. 9).

[0161] The polymers were synthesized as follows, with relative amount of starting materials controlled to afford different MW PCL sidechains:

[0162] A 10% solution of stannous octoate (50 μL) was added to a melt of a 1:1 w/w mixture of PEG-4000 or PEG-8000 and ε-caprolactone under nitrogen atmosphere. The mixture was stirred at 120° C. for 2 h, followed by overnight stirring at 150° C.

[0163] Aqueous solutions of polymers were prepared at varying concentrations (10-30% w/w) and were tested for aqueous solubility and hydrogel stability. For a PEG-4000 starting material, PCL sidechains ranging from 1700-2200 afforded thermoresponsive hydrogels that formed gels upon heating to 50° C. Lower MW sidechains did not form gels up to 75° C., and higher MW sidechains were not dispersible in aqueous solution, even at 5% w/w. For triblock copolymers based on PEG-8000, a PCL MW range of 3000-3400 was shown to offer the same effect. In all cases, hydrogels were not reversible, so gel was maintained upon return to room temperature. Lower MW PEG-based polymers are ineffective for this application, as polymers thereof dissolve in aqueous solution and do not form gels. PLGA sidechains of PEG-4000 were either soluble (lower MW PLGA) or insoluble (as PLGA MW increased), and never formed gels.

[0164] Amongst the many possible applications, triblocks may be used for tissue engineering is illustrated in FIG. 10.

Example: Mixture of Polymers for Reaching a Certain Gelling Temperature

[0165] PLGA-PEG-PLGA triblock copolymers that one gels at 42° C. 20% w/v solution in deionized water (DDW) (Polymer A) and the second triblock copolymer that forms a reversible hydrogel at 34° C. (Polymer B) were used in this study.

[0166] A solution of both polymers A and B was prepared by dissolving 200 mg of polymer A and 200 mg of polymer B in 2 mL of DDW, so that each polymer content is 10% w/v. The aqueous solution of polymers A and B was incubated at a given temperature for 10 min, and the vial was inverted to test for gelling. If the gel did not flow, the temperature was recorded as the gelling temperature of the solution (Tgel). Polymer blend A+B formed a reversible hydrogel at 38° C.

[0167] A blend of polymers A and B was prepared by dissolving 100 mg of polymer A and 300 mg of polymer B in 2 mL of DDW, so that total polymer concentration was 20% w/v with a 1:3 w/w ratio of A:B. The aqueous solution of blended A and B was incubated at a given temperature for 10 min, and the vial was inverted to test for gelling. Polymer blend A+B at 1:3 w/w formed a reversible hydrogel at 36° C.

[0168] A blend of polymers A and B was prepared by dissolving 300 mg of polymer A and 100 mg of polymer B in 2 mL of DDW, so that total polymer concentration was 20% w/v with a 3:1 w/w ratio of A:B. The polymer blend A+B 3:1 formed a reversible hydrogel at 40° C.

[0169] Similar mixtures of individual polymers that gel at different temperatures where prepared and showed an in between gelling temperature. The intermediate temperature was affected by the different polymers used as well as the relative w/w ratio of the polymers used to form the gel.