Hydrophilic polyester and a block copolymer thereof

Abstract

The invention belongs to the field of macromolecules and biomedical materials, and relates to a polymer, a block copolymer comprising the polymer as a segment, methods for preparing the polymer and for preparing the block copolymer, a micelle particle or a vesicle particle prepared from the block copolymer, and a composition comprising the polymer, the block copolymer, the micelle particle and/or the vesicle particle. The polymer provided in the invention can be used as a novel biomedical material in in the fields such as pharmaceutical formulations, immunological formulations, and gene delivery reagents.

Claims

1. A polymer comprising repeat units of Formula (I); ##STR00018## wherein, X is SO or SO.sub.2; m is 1-100; and n is 1-10.

2. The polymer according to claim 1 having a structure of Formula (II): ##STR00019## wherein, X, m and n have the same meanings as defined in claim 1; k is 10-1000; and W is a terminal group.

3. A method for preparing the polymer according to claim 1, comprising the step of carrying out a polymerization reaction using Compound 1 as a monomer; wherein, Compound 1 has a structure according to Formula (III); ##STR00020## wherein, X and m have the same meanings as defined in claim 1; and t=1-10.

4. A block polymer, comprising a segment consisting of the repeat units of Formula (I).

5. A method for preparing the block copolymer according to claim 4, comprising the step of using a polymer having a structure of Formula (II) to initiate a polymerization reaction of a second monomer.

6. A micelle particle, comprising the block copolymer according to claim 4.

7. A vesicle particle, comprising the block copolymer according to claim 4.

8. A pharmaceutical composition, comprising the polymer according to claim 1, a block polymer comprising a segment consisting of the repeat units of Formula (I) in claim 1, a micelle particle comprising the block polymer and/or a vesicle particle comprising the block polymer, and a drug.

9. A composition, comprising the polymer according to claim 1, a block polymer comprising a segment consisting of the repeat units of Formula (I) in claim 1, a micelle particle comprising the block copolymer and/or a vesicle particle comprising the block copolymer.

10. The polymer according to claim 1, wherein the polymer has one or more features selected from: (1) the polymer is a homopolymer; (2) the polymer has a number-average molecular eight of 400-300000; (3) m=3; and (4) n=3.

11. The block polymer according to claim 4, which is a diblock copolymer having a structure of Formula (IV): ##STR00021## Wherein X, W, m, n and k have the same meanings as defined in claim 2; h is 2-10; and j is 10-1000.

12. The micelle particle according to claim 6, having one or more features selected from: 1) the micelle particle has a particle size of 90-110 nm; 2) the micelle particle is loaded with a drug; 3) the micelle particle is prepared by a method comprising the following steps of: (1) dissolving the block copolymer in an organic solvent to obtain a solution; (2) adding the solution obtained in the step (1) dropwise to water, so as to obtain a mixture; and (3) placing the mixture obtained in the step (2) in a dialysis bag, and carrying out dialysis in water.

13. The vesicle particle according to claim 7, having one or more features selected from: 1) the vesicle particle has a particle size of 150-250 nm; 2) the vesicle particle is loaded with a drug; 3) the vesicle particle is prepared by a method comprising the following steps: (1) dissolving the block copolymer according to claim 4 in an organic solvent to obtain a solution; (2) injecting the solution obtained in step (1) into water, and stirring until the organic solvent volatilizes completely; and (3) subjecting the product obtained in step (2) to centrifugation, and then filtration.

14. The pharmaceutical composition according to claim 8, wherein the drug is selected from the group consisting of a polypeptide, DNA, RNA and a small molecule compound.

15. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition is a pharmaceutical formulation or an immunological formulation.

16. The composition according to claim 9, which is a pharmaceutically acceptable supplementary material, a gene delivery reagent or an immunoadjuvant.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the .sup.1H NMR spectrum of the homopolymer of the functional monomer 1b in Example 3 of the invention. The homopolymer has a polymerization degree of 100. In the figure, a-k represent the positions of hydrogen atoms, and in the structural formula, n represents a polymerization degree, which is different from the meaning of n in Formula (I), (II) and (IV).

(2) FIG. 2 is the .sup.1H NMR spectrum of the homopolymer of the functional monomer 1a in Example 4 of the invention. The homopolymer has a polymerization degree of 10. In the figure, a-k represent the positions of hydrogen atoms, and in the structural formula, n represents a polymerization degree, which is different from the meaning of n in Formula (I), (II) and (IV).

(3) FIG. 3 is the .sup.1H NMR spectrum of an amphiphilic diblock copolymer (P1b.sub.50-b-PCL.sub.50) in Example 5 of the invention. In the figure, a and b represent the positions of hydrogen atoms, and in the structural formula, n represents a polymerization degree, which is different from the meaning of n in Formula (I), (II) and (IV).

(4) FIG. 4 shows the GPC curves of the hydrophilic segment P1b.sub.50 (Curve 1) and its amphiphilic diblock copolymer (P1b.sub.50-b-PCL.sub.50) (Curve 2) in Example 5 of the invention. In the figure, the abscissa represents the elution time, and the ordinate represents the signal intensity of detector. As shown in the figure, P1b.sub.50-b-PCL.sub.50 had a shorter elution time than P1b.sub.50, indicating an increase in molecular weight.

(5) FIG. 5 is the experimental result of the water contact angle of a non-modified gold surface (upper figure, a water contact angle of 641), an mPEG-SH (M.sub.n is 6500)-modified gold surface (middle figure, a water contact angle of 441) and a hydrophilic polyester-modified gold surface (bottom figure, a water contact angle of) 531) in Experimental example 1 of the invention. The result shows that the hydrophilicity of the gold surface modified by the hydrophilic polyester of the invention is comparable to that of the gold surface modified by PEG with a similar molecular weight.

(6) FIG. 6 is the experimental result of the in vitro cytotoxicity of polymers in Experimental example 2 of the invention. In the figure, the abscissa represents the concentration of polymer, and the ordinate represents cell viability. The result shows that the polymer of the invention had a cell viability comparable to that of PEG, i.e. had good biocompatibility.

(7) FIG. 7 is the experimental result of the in vitro inhibition of cell adhesion by polymers in Experimental example 3 of the invention. FIG. a is the fluorescent microscopic photograph of the gold surface modified by the polymer of the invention, and FIG. b is the fluorescent microscopic photograph of the gold surface modified by mPEG-SH. FIG. c shows the cell density on the gold surfaces modified by the polyester of the invention and mPEG-SH, respectively, wherein the cell density on both the two surfaces was very low. The experiment shows that the polyester material having a hydrophilic side chain according to the invention was comparable to PEG in terms of the property of cell adhesion inhibition.

(8) FIG. 8 is the GPC curves of polymer before degradation (Curve 1) and after degradation (Curve 2) in Experimental example 4 of the invention. As shown in the figure, after polymer degradation, the elution time of GPC increases, and the molecular weight of the polymer decreases. The result demonstrates that the polymer could be degraded under alkaline condition.

(9) FIG. 9 shows the particle size distribution of the micelle particle of an amphiphilic diblock copolymer (P1b.sub.50-b-PCL.sub.100) as measured by a dynamic laser scattering apparatus in Experimental example 6 of the invention, wherein the micelle particle has a particle size of 90-110 nm.

(10) FIG. 10 shows the particle size distribution of the vesicle particle of an amphiphilic diblock copolymer (P1b.sub.50-b-PCL.sub.50) as measured by a dynamic laser scattering apparatus in Experimental example 7 of the invention, wherein the vesicle particle has a particle size of 150-250 nm.

SPECIFIC MODES FOR CARRYING OUT THE INVENTION

(11) The embodiments of the invention are illustrated in detail by reference to the following examples. However, it is understood by those skilled in the art that the examples are used only for the purpose of illustrating the invention, rather than limiting the protection scope of the invention. In the case where the concrete conditions are not indicated in the examples, the examples are carried out according to conventional conditions or the conditions recommended by the manufacturer. The agents or instruments of which the manufacturer are not indicated are regular products that can be purchased on the market.

EXAMPLE 1

Preparation of a Functional Lactone Monomer 1a

(12) ##STR00013##

(13) Compound 4 was prepared by the method as described in CN patent ZL201310169131.8.

(14) To a 25 mL round-bottom flask, 10 mL aqueous hydrogen peroxide solution (at a concentration of 30%) was added, and then Compound 4 (0.292 g, 1 mmol) was added. After stirring for 10 min, the water phase was extracted with 20 mL dichloromethane for three times, and the obtained organic phase was dried with anhydrous sodium sulfate. The drying agent was removed by filtration, and the solvent was removed by evaporation under reduced pressure, to obtain a functional lactone monomer 1a, as colorless oil (Yield: 84%).

(15) .sup.1H NMR (CDCl.sub.3, 400 MHz) 4.25-4.17 (m, 2H), 2.85-2.77.91 (m, 3H), 3.41-3.871 (s, 8H), 3.35-3.18 (m, 7H), 2.54-2.49 (m, 1H), 1.99-1.93 (m, 2H), 1.85-1.71 (m, 1H). .sup.13C NMR (100 MHz, CDCl3) 168, 70.69, 70.10, 70.11, 70.23, 67.36, 64.68, 58.83, 55.68, 55.10, 34.69, 24.97, 21.76. ESI MS calculated value: 308.4, measured value: [M+Na.sup.+]=331.3.

EXAMPLE 2

Preparation of a Functional Lactone Monomer 1b

(16) ##STR00014##

(17) Compound 4 was dissolved in dichloromethane, and stirred in an ice-water bath, and then metachloroperbenzoic acid (mCPBA) was added slowly. After reacting at room temperature for 2 h, the resultant mixture was filtrated, and the filtrate was washed with a saturated sodium carbonate solution for more than three times. The organic phases were combined, and the solvent was removed by evaporation under reduced pressured, to obtain a functional monomer 1b, as light yellow oil (Yield: 87%).

(18) .sup.1H NMR (CDCl.sub.3, 400 MHz) 4.39-4.36 (m, 2H), 3.94-3.91 (m, 3H), 3.65-3.51 (s, 8H), 3.35-3.18 (m, 7H), 2.54-2.49 (m, 1H), 1.99-1.93 (m, 2H), 1.85-1.71 (m, 1H). .sup.13C NMR (100 MHz, CDCl3) 172, 71.76, 70.56, 70.31, 70.18, 68.41, 64.68, 58.83, 55.68, 55.10, 34.69, 24.97, 21.76. ESI MS calculated value: 324.1, measured value: [M+Na.sup.+]=347.3.

EXAMPLE 3

Synthesis of a Homopolymer of Monomer 1b

(19) ##STR00015##

(20) In a glove box, substantially free of water and oxygen, under the protection of argon, to a 25 mL round-bottom flask, the monomer 1b (0.324 g, 1.0 mmol) and benzyl alcohol (1.1 mg, 0.01 mmol) were added, and after mixing homogeneously, 2.5 mg catalyst diphenyl phosphate (2.5 mg, 0.01 mmol) was added. After further stirring at room temperature for 24 h, 20 mL dichloromethane and triethylamine (0.3 g) were added, and the solvent was removed by rotary evaporation. The crude product was transferred to a dialysis bag (molecular weight cutoff of 1 kDa), and was dialyzed in a tetrahydrofuran solution (containing 40% water) for 1-7 d, during which the solvent was renewed twice. Finally, the dialysis solution was freeze-dried to obtain a polyester homopolymer (0.24 g), as yellowish oily liquid. As measured by Gel Permeation Chromatography (GPC), M.sub.n=7325, M.sub.w=7856, PDI=1.11, and polymerization degree k was 100. The .sup.1H NMR spectrum of the product was shown in FIG. 1.

EXAMPLE 4

Synthesis of a Homopolymer of Monomer 1a

(21) ##STR00016##

(22) In a glove box, substantially free of water and oxygen, under the protection of argon, to a 25 mL round-bottom flask, the monomer 1a (0.68 g, 0.45 mmol), benzyl alcohol (0.05 mmol), diphenyl phosphate (12.5 mg, 0.05 mmol) and 4-dimethylaminopyridine (DMAP) (6 mg, 0.05 mmol) were added, and stirred at 65 C. for 72 h. After cooling to room temperature, 10 mL dichloromethane and triethylamine (0.15 g) were added, and the solvent was removed by rotary evaporation. The crude product was transferred to a dialysis bag (molecular weight cutoff of 1 kDa), and dialyzed in a tetrahydrofuran solution (containing 20% water) for 1-7 d, during which the solvent was renewed twice. Finally, the dialysis solution was freeze-dried to obtain a polyester product (0.51 g), as light yellow oily liquid. As measured by Gel Permeation Chromatography (GPC), M.sub.n=1106, M.sub.w=1117, PDI=1.01, and polymerization degree k was 10. The .sup.1H NMR spectrum of the product was shown in FIG. 2.

EXAMPLE 5

Synthesis of a Block Copolymer of Monomer 1b and -caprolactone

(23) ##STR00017##

(24) In a glove box, substantially free of water and oxygen, under the protection of argon, to a 25 mL round-bottom flask, the monomer 1b (0.324 g, 1 mmol) and benzyl alcohol (2.08 L, 0.02 mmol) were added. Under stirring at room temperature, diphenyl phosphate (5 mg, 0.02 mmol) was added to the reaction flask. After further reaction for 48 h (the conversion percent of monomer 1b was greater than 95%), the hydrophilic segment P1b was obtained. 2 mL toluene and g-caprolactone (CL, 106.6 L, 1 mmol) were added. After stirring for 12 h (the conversion percent of caprolactone was 100%), 15 mL dichloromethane and triethylamine (0.3 g) were added, and the solvent was removed by rotary evaporation. The crude product was transferred to a dialysis bag (molecular weight cutoff of 1 kDa), and was dialyzed in a tetrahydrofuran solution (containing 20% water) for 1-7 d, during which the solvent was renewed for three times. Finally, the dialysis solution was freeze-dried to obtain an amphiphilic diblock copolymer P1b-b-PCL, wherein, P1b was a hydrophilic segment, and PCL was a hydrophobic segment. P1b-b-PCL was a semitransparent white solid. As measured by Gel Permeation Chromatography (GPC), M.sub.n=7350, M.sub.w=7791, PDI=1.06. The .sup.1HNMR spectrum of P1b.sub.50-b-PCL.sub.50 was shown in FIG. 3. It was determined by calculation that P1b had a polymerization degree of 50, and PCL had a polymerization degree of 50. The polymerization process as monitored by GPC was illustrated in FIG. 4. In the figure, Curve 1 was the GPC curve of the hydrophilic segment P1b.sub.50, and Curve 2 was the GPC curve of the P1b.sub.50-b-PCL.sub.50. As shown in the figure, P1b.sub.50-b-PCL.sub.50 had a shorter elution time than P1b.sub.50, indicating an increase in molecular weight.

EXPERIMENTAL EXAMPLE 1

Experiment on Hydrophilicity of Polymers

(25) According to the method described in Example 3, polymerization was carried out by using 1b as monomer and using an alcohol compound having a thiol functional group (OHCH.sub.2CH.sub.2CH.sub.2CH.sub.2SH) as initiator, to obtain a polyester with a hydrophilic side chain (M.sub.n=6880, PDI=1.11) wherein the terminal group was a thiol group. The polyester was linked to a gold surface, and a water contact angle assay was carried out. The result was shown in FIG. 5. The water contact angle of polyester-modified gold surface was very close that of mPEG-SH (M.sub.n=6500)-modified gold surface. The experiment indicated that the polyester compound having a hydrophilic side chain was substantially comparable to PEG with a similar molecular weight in terms of hydrophilicity.

EXPERIMENTAL EXAMPLE 2

In Vitro Cytotoxic Assay of Polymers

(26) The in vitro cytotoxity of polymers was determined by MTT method, so as to verify the biocompatibility of polymers.

(27) Human foreskin fibroblasts (HFF) growing well in logarithmic growth phase were used, and the medium was pipetted off and discarded. The residual medium was washed off with 1 mL 1PBS, and PBS was pipetted off and discarded. A 2 mL 25% digestion solution (0.05% trypsin+0.02% EDTA) was added, and after digestion in an incubator for 4 min, when it was observed that the digestion solution turned yellow, the cells shrank and turned round, and most of the cells detached from the wall, the culture dish was shaken gently to have the cells almost completely detached from the wall, without blowing individual undetached cells. To the cell suspension, a 2 mL complete medium (DMEM high glucose medium+10% fetal bovine serum+1 Penicillin-Streptomycin) was added to stop digestion, and the culture dish could be shaken gently to mix the solution homogeneously. The cell suspension was transferred to a 15 mL centrifuge tube, and centrifuged at 900 rpm for 5 min. The supernatant solution was discarded. A 2 mL complete medium (DMEM high glucose medium+10% fetal bovine serum+1 Penicillin-Streptomycin) was added for washing, centrifugation was performed, and the supernatant solution was discarded. A 2 mL double antibodies-free medium (DMEM high glucose medium+10% fetal bovine serum) was added, and the cells were blew up gently, counted by a cell counting chamber, diluted to a desired density, and seeded to a 96-well plate. The 96-well plate was incubated in a 37 C., 5% CO.sub.2 incubator for 24 h. The medium in each well was pipetted off, and a 100 L aqueous solution of the polymer prepared in Example 3 was added to each well. The 96-well plate was incubated in a 37 C., 5% CO.sub.2 incubator for 24 h. The sample solution in each well was pipetted off, and a 100 L complete medium (DMEM high glucose medium+10% fetal bovine serum+1 Penicillin-Streptomycin) was added to each well, followed by the addition of a 20 L MTT solution. The 96-well plate was incubated in a 37 C., 5% CO.sub.2 incubator for 4 h. The sample solution in each well was pipetted off, and 150 L dimethyl sulfoxide (DMSO) was added to each well to dissolve formazan crystal. The optical density value (OD value) at 490 nm for each well was measured by ELISA instrument. The plate wells containing no cells, to which DMSO was added, were used as blank control, and the OD value finally used in calculation should be the value obtained by subtracting the measured DMSO blank control value from the directly measured OD value of the experimental group. Cell viability was calculated in accordance with the following formula:

(28) $V=\frac{{\mathrm{OD}}_{\mathrm{polymer}}-{\mathrm{OD}}_{\mathrm{DMSO}}}{{\mathrm{OD}}_{\mathrm{control}}-{\mathrm{OD}}_{\mathrm{DMSO}}}100\%$

(29) The experimental result was shown in FIG. 6. In the figure, the abscissa represents the concentration of polymer, and the ordinate represents cell viability. The experimental result showed that the polyester having a hydrophilic side chain as disclosed in the invention was comparable to PEG in terms of cell viability, and had good biocompatibility as PEG did.

EXPERIMENTAL EXAMPLE 3

(30) Cell Adhesion Inhibition Assay

(31) Raw 264.7 cells were taken out from a culture bottle, digested, and floating in a medium. The cells were counted by a hemocytometer, and then the cells were diluted to 510.sup.5 cells/mL. Gold-coated glass slides were modified by the polyester having a thiol group as terminal group as prepared in Experimental example 1, and mPEG-SH (M.sub.n=6500), respectively. The modified gold-coated glass was divided into three identical parts, placed in sterile culture dishes, and soaked in 4 mL medium. 1 mL diluted cell suspension was added to each culture dish, so that the final density of cells was 110.sup.5 cells/mL. The samples were incubated in an incubator (37 C., 5% CO.sub.2) for 24 h, and the samples were rinsed with a medium to ensure that the unabsorbed cells were removed. The cells absorbed on the gold slides were stained with Calcein-AM. The viability of cells were then measured by fluorescent microscopic photography, and cell counting was also carried out under microscope. The experimental result was shown in FIG. 7, wherein FIG. a and FIG. b were the fluorescent microscopic photographs of the gold surface modified by the polyester of the invention and the gold surface modified by mPEG-SH, respectively. As shown in the figure, there were a very small number of cells on the gold surfaces modified by polyester and mPEG-SH (M.sub.n=6500). FIG. c showed the cell density on the gold surfaces modified by the polyester of the invention and mPEG-SH, and the cell density on the two surfaces was very low. The assay showed that the polyester material having a hydrophilic side chain according to the invention was comparable to PEG with respect to the property of cell adhesion inhibition.

EXPERIMENTAL EXAMPLE 4

(32) Polymer Degradation Experiment

(33) According to the method described in Example 3, polymerization was carried out by using 1b as monomer and using benzyl alcohol as initiator, so as to obtain a polyester (M.sub.w=5423, 10 mg). The polyester was dissolved in methanol (5 mL), and a methanol solution of sodium methoxide (50 mg, 30 wt %) was added to the solution. The polyester was degraded under stirring at room temperature for 30 min, and the pH of solution was adjusted to a neutral pH by using a suitable concentration of HCl (2 M). The solvent was removed by rotary evaporation. The residue was dissolved in tetrahydrofuran (2 mL), and the resultant solution was analyzed by GPC. The result was compared with the GPC result before degradation. GPC curves were shown in FIG. 8. Curve 1 was the GPC curve before polyester degradation, and Curve 2 was the GPC curve after polyester degradation. As shown in the figure, after polymer degradation, the elution time of GPC increased, and the molecular weight of the polymer decreased. The experiment showed that the polyester material having a hydrophilic side chain according to the invention could be degraded under alkaline condition.

EXPERIMENTAL EXAMPLE 5

(34) According to the steps in Example 5, a block copolymer (P1b.sub.50-b-PCL.sub.400) of monomer 1b and -caprolactone was synthesized. 10 mg copolymer (P1b.sub.50-b-PCL.sub.100) was dissolved in 1 mL N,N-dimethylformamide, and the solution was added dropwise to 1 mL purified water in an ice bath under ultrasonic condition, and then the mixed solution was placed in a dialysis bag having a molecular weight cutoff of 1000, and dialyzed for 1 day, so as to obtain a micelle solution of the copolymer. The particle size of the micelle particle was measured, and the particle size distribution was shown in FIG. 9, wherein the particle size was 90-110 nm.

EXPERIMENTAL EXAMPLE 6

(35) According to the steps in Example 5, a block copolymer (P1b.sub.50-b-PCL.sub.50) of monomer 1b and -caprolactone was synthesized. 15 mg the copolymer (P1b.sub.50-b-PCL.sub.50) was dissolved in 1 mL N,N-dimethylformamide. The solution was injected to 30 mL purified water at a temperature of 25 C. at a speed of 0.1 mL per min, and was stirred until ethanol volatilized completely. A milk-white solution was obtained. The solution was centrifuged at 1000 rpm for 1 min, and filtrated through 0.8 m, 0.45 m, and 0.22 m microfiltration membrane in order, so as to obtain a vesicle solution of the copolymer. The particle size of the vesicle particle was measured, and the particle size distribution was shown in FIG. 10, wherein the particle size was 150-250 nm.

(36) Although the embodiments of the invention have been described in detail, a person skilled in the art would understand that according to all the disclosed teachings, details can be amended and modified, and these alterations all fall into the protection scope of the invention. The scope of the invention is defined by the attached claims and any equivalent thereof.