Gel of sodium hyaluronate cross-linked by polyethylene glycol epoxy derivative for injection and preparation method thereof

Abstract

The present invention discloses polyglycol epoxide crosslinked sodium hyaluronate gel for injection and a preparation method thereof. A polyglycol epoxide is a compound with single molecular weight preferably; a plurality of ether bonds are present in the molecule of the polyglycol epoxide, the water solubility is good, and thus, the polyglycol epoxide is more easily subjected to a crosslinking reaction with polysaccharides; and meanwhile, polyglycol is relatively easy in adjustment of the number of repeating units and relatively easy in control of length, and thus, the sodium hyaluronate gel prepared by taking the polyglycol epoxide as a crosslinker is relatively easy in regulation and control of properties. The crosslinked sodium hyaluronate gel is low in toxicity, little in residual, small in squeezing and pushing force, good in shaping performance, good in enzyme resistance and long in in-vivo retention time. The present invention further discloses a mild crosslinker deactivation technology. Unreacted epoxide groups in the gel are subjected to a hydrolysis reaction in a carbonate buffer system with a pH of 8-9, so that the difficulty of impurity removal of the crosslinked sodium hyaluronate gel can be effectively lowered, and the problem of toxicity in the prior art due to the fact that BDDE is used in a crosslinking method is avoided.

Claims

1. A preparation method of a biocompatible polyglycol epoxide, comprising a step of catalyzing a reaction between EPOX—X and polyglycol with a catalyst in a solvent; the polyglycol has a structure of ##STR00015## and n is an integer of 10-30; in the EPOX—X, —X is Cl; EPOX is ##STR00016## wherein Y is (CR.sub.1R.sub.2).sub.m; m is 1; R.sub.1 and R.sub.2 are H, wherein each of the polyglycol epoxide is a compound with single molecular weight and the biocompatiblity of the polyglycol epoxide is obtained by detecting a lower cytotoxicity than those of lower molecular weight.

2. The preparation method according to claim 1, wherein the method is characterized in that the n is one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; and/or, the catalyst is an inorganic base.

3. The preparation method according to claim 2, wherein the method is characterized in that the catalyst comprises sodium carbonate or potassium carbonate; and/or, the solvent comprises one or more agents selected from the group consisting of 1,4-dioxane, tetrahydrofuran, toluene, acetone, ethyl acetate, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide and water; and/or, a mole ratio of mono-hydroxyl on the polyglycol to EPOX—X is 1: (2-10).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a squeezing and pushing force curve of dodecaethylene glycol diglycidyl ether crosslinked sodium hyaluronate gel provided in embodiment 10 of the present invention.

(2) FIG. 2 is a squeezing and pushing force curve of polyethylene glycol PEG1000 diglycidyl ether crosslinked sodium hyaluronate gel provided in embodiment 10 of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(3) Unless otherwise defined, all scientific and technical terms used in the present invention have meanings the same as those generally understood by technical personnel in the technical field involved in the present invention, for example: ‘alkyl’ means a linear or branched hydrocarbon chain free radical free of an unsaturated bond; in the present invention, C1-C6 alkyl means alkyl containing 1-6 carbon atoms, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tertiary butyl, n-amyl, n-hexyl, etc., preferably C1-C3 alkyl (such as methyl, ethyl, n-propyl and isopropyl); ‘cycloalkyl’ means an alicyclic hydrocarbon, and typical cycloalkyl contains 1 to 4 monocyclic rings and/or condensed rings and contains 3 to about 18 carbon atoms; and in the present invention, C3-C6 cycloalkyl means cycloalkyl containing 3-6 carbon atoms, such as cyclopropyl, cyclopentyl and cyclohexyl.

(4) Technical schemes in embodiments of the present invention will be described clearly and completely below with reference to drawings in the embodiments of the present invention. Apparently, the embodiments described are only part of the embodiments of the present invention, rather than all embodiments. All other embodiments achieved by those having ordinary skill in the art on the premise of not making inventive labor on the basis of the embodiments of the present invention all fall within the protection scope of the present invention.

Embodiment 1: Synthesis of tetraethylene Glycol Diglycidyl Ether (Ia)

(5) Synthesis of tetraethylene glycol diglycidyl ether with a structure as follows:

(6) ##STR00011##

(7) Tetraethylene glycol (0.1 mol), tetrahydrofuran (100 mL) and potassium hydroxide (0.4 mol) are added into a three-necked flask, stirring is performed in a water bath, then, chloroepoxy propane (0.6 mol) is dropwise added into the reaction system, the reaction temperature is controlled not to exceed 35° C., and a reaction is carried out at room temperature overnight. Reaction liquor is filtered after the reaction ends up, filter residues are washed with dichloromethane, then, the obtained filtrate is collected, and rotary evaporation is performed to remove dichloromethane, so as to obtain a crude product. The crude product is subjected to molecular distillation, thereby obtaining pure tetraethylene glycol diglycidyl ether.

(8) .sup.1H-NMR (DMSO-d6): 2.52-2.55 (m, 2H), 2.70-2.73 (m, 2H), 3.07-3.11 (m, 2H), 3.22-3.28 (m, 2H), 3.52-3.56 (m, 16H), 3.68-3.73 (m, 2H);

(9) HPLC detection: product purity 99.6%;

(10) Mass spectrum ESI: 329.6 [M+Na].

Embodiment 2: Synthesis of Dodecaethylene Glycol Ddiglycidyl Ether (Ib)

(11) Synthesis of dodecaethylene glycol diglycidyl ether with a structure as follows:

(12) ##STR00012##

(13) Dodecaethylene glycol (0.1 mol), tetrahydrofuran (100 mL) and potassium hydroxide (0.4 mol) are added into a three-necked flask, stirring is performed in a water bath, then, chloroepoxy propane (0.6 mol) is dropwise added into the reaction system, the reaction temperature is controlled not to exceed 35° C., and a reaction is carried out at room temperature overnight. Reaction liquor is filtered after the reaction ends up, filter residues are washed with dichloromethane, then, the obtained filtrate is collected, and rotary evaporation is performed to remove dichloromethane, so as to obtain a crude product. The crude product is subjected to molecular distillation, thereby obtaining pure dodecaethylene glycol diglycidyl ether.

(14) .sup.1H-NMR (DMSO-d6): 2.51-2.55 (m, 2H), 2.70-2.73 (m, 2H), 3.07-3.11 (m, 2H), 3.22-3.29 (m, 2H), 3.51-3.57 (m, 48H), 3.68-3.73 (m, 2H);

(15) HPLC detection: product purity 99.3%;

(16) Mass spectrum ESI: 681.9 [M+Na].

Embodiment 3: Synthesis of Tetracosaethylene Glycol Diglycidyl Ether (Ic)

(17) Synthesis of tetracosaethylene glycol diglycidyl ether with a structure as follows:

(18) ##STR00013##

(19) Tetracosaethylene glycol (0.1 mol), tetrahydrofuran (100 mL) and potassium hydroxide (0.4 mol) are added into a three-necked flask, stirring is performed in a water bath, then, chloroepoxy propane (0.6 mol) is dropwise added into the reaction system, the reaction temperature is controlled not to exceed 35° C., and a reaction is carried out at room temperature overnight. Reaction liquor is filtered after the reaction ends up, filter residues are washed with dichloromethane, then, the obtained filtrate is collected, and rotary evaporation is performed to remove dichloromethane, so as to obtain a crude product. The crude product is subjected to column separation, thereby obtaining pure tetracosaethylene glycol diglycidyl ether.

(20) .sup.1H-NMR (DMSO-d6): 2.51-2.55 (m, 2H), 2.70-2.73 (m, 2H), 3.07-3.11 (m, 2H), 3.22-3.28 (m, 2H), 3.51-3.56 (m, 96H), 3.68-3.73 (m, 2H);

(21) HPLC detection: product purity 99.2%;

(22) Mass spectrum MALDI-TOF: 1210.0 [M+Na].

Embodiment 4: Synthesis of PEG1000 Diglycidyl Ether (Id)

(23) Synthesis of PEG1000 diglycidyl ether with a structure as follows:

(24) ##STR00014##

(25) Di-HO-PEG1000-OH (0.1 mol), tetrahydrofuran (100 mL) and potassium hydroxide (0.4 mol) are added into a three-necked flask, stirring is performed in a water bath, then, chloroepoxy propane (0.6 mol) is dropwise added into the reaction system, the reaction temperature is controlled not to exceed 35° C., and a reaction is carried out at room temperature overnight. Reaction liquor is filtered after the reaction ends up, filter residues are washed with dichloromethane, then, the obtained filtrate is collected, and rotary evaporation is performed to remove dichloromethane, so as to obtain a crude product. The crude product is subjected to column separation, thereby obtaining pure PEG1000 diglycidyl ether.

(26) .sup.1H-NMR (DMSO-d6): 2.52-2.55 (m, 2H), 2.70-2.73 (m, 2H), 3.06-3.11 (m, 2H), 3.23-3.29 (m, 2H), 3.45-3.69 (m, H in —(CH2CH2O)—), 3.72-3.75 (m, 2H);

(27) Mass spectrum MALDI-TOF: 901.8, 945.9, 989.9, 1033.9, 1078.0, 1122.0, 1166.1, 1210.1, 1245.1, 1298.2, 1342.2 [M+Na].

Embodiment 5: Tetraethylene Glycol Diglycidyl Ether Crosslinked Sodium Hyaluronate Gel (IIa)

(28) A crosslinker, i.e., tetraethylene glycol diglycidyl ether (0.2 mol) is dissolved into a NaOH solution, the solution with the crosslinker dissolved is added into sodium hyaluronate powder (1 mol of polymeric unit), and a reaction is carried out at a temperature of 40° C. with stirring to form crosslinked sodium hyaluronate gel; secondly, a proper amount of hydrochloric acid is added into the gel to adjust pH to 7.0, and then, a proper amount of PBS buffer solution is added for gel swelling; then, the gel is screened with a standard pharmacopoeia screen, gel particles are collected, then, the crosslinked sodium hyaluronate gel is washed with a carbonate buffer solution at a high temperature; and finally, the gel is subjected to repeated dialysis, then, screening is performed with the standard pharmacopoeia screen, and compounding, filling and steam sterilizing are performed, thereby obtaining modified sodium hyaluronate gel for injection.

(29) .sup.1H-NMR (D20, NaOD): 1.80 (s, 3H), 3.26 (s, 3H), 3.41 (s, H), 3.56 [s, (HEG4+5H)], 3.79 (s, 1H), 4.33-4.36 (d, 2H).

Embodiment 6: Dodecaethylene Glycol Diglycidyl Ether Crosslinked Sodium Hyaluronate Gel (IIb)

(30) A crosslinker, i.e., dodecaethylene glycol diglycidyl ether (0.2 mol) is dissolved into a NaOH solution, the solution with the crosslinker dissolved is added into sodium hyaluronate powder (1 mol of polymeric unit), and a reaction is carried out at a temperature of 40° C. with stirring to form crosslinked sodium hyaluronate gel; secondly, a proper amount of hydrochloric acid is added into the gel to adjust pH to 7.0, and then, a proper amount of PBS buffer solution is added for gel swelling; then, the gel is screened with a standard pharmacopoeia screen, gel particles are collected, then, the crosslinked sodium hyaluronate gel is washed with a carbonate buffer solution at a high temperature; and finally, the gel is subjected to repeated dialysis, then, screening is performed with the standard pharmacopoeia screen, and compounding, filling and steam sterilizing are performed, thereby obtaining modified sodium hyaluronate gel for injection.

(31) .sup.1H-NMR (D.sub.2O, NaOD): 1.81 (s, 3H), 3.28 (s, 3H), 3.40 (s, H), 3.56 [s, (H.sub.EG12+5H)], 3.78 (s, 1H), 4.32-4.35 (d, 2H).

Embodiment 7: Tetracosaethylene Glycol Diglycidyl Ether Crosslinked Sodium Hyaluronate Gel (IIc)

(32) A crosslinker, i.e., tetracosaethylene glycol diglycidyl ether (0.2 mol) is dissolved into a NaOH solution, the solution with the crosslinker dissolved is added into sodium hyaluronate powder (1 mol of polymeric unit), and a reaction is carried out at a temperature of 40° C. with stirring to form crosslinked sodium hyaluronate gel; secondly, a proper amount of hydrochloric acid is added into the gel to adjust pH to 7.0, and then, a proper amount of PBS buffer solution is added for gel swelling; then, the gel is screened with a standard pharmacopoeia screen, gel particles are collected, then, the crosslinked sodium hyaluronate gel is washed with a carbonate buffer solution at a high temperature; and finally, the gel is subjected to repeated dialysis, then, screening is performed with the standard pharmacopoeia screen, and compounding, filling and steam sterilizing are performed, thereby obtaining modified sodium hyaluronate gel for injection.

(33) .sup.1H-NMR (D.sub.2O, NaOD): 1.80 (s, 3H), 3.27 (s, 3H), 3.40 (s, H), 3.56 [s, (H.sub.EG24+5H)], 3.79 (s, 1H), 4.32-4.36 (d, 2H).

Embodiment 8: PEG1000 Diglycidyl Ether Crosslinked Sodium Hyaluronate Gel (IId)

(34) A crosslinker, i.e., PEG1000 diglycidyl ether (0.2 mol) is dissolved into a NaOH solution, the solution with the crosslinker dissolved is added into sodium hyaluronate powder (1 mol of polymeric unit), and a reaction is carried out at a temperature of 40° C. with stirring to form crosslinked sodium hyaluronate gel; secondly, a proper amount of hydrochloric acid is added into the gel to adjust pH to 7.0, and then, a proper amount of PBS buffer solution is added for gel swelling; then, the gel is screened with a standard pharmacopoeia screen, gel particles are collected, then, the crosslinked sodium hyaluronate gel is washed with a carbonate buffer solution at a high temperature; and finally, the gel is subjected to repeated dialysis, then, screening is performed with the standard pharmacopoeia screen, and compounding, filling and steam sterilizing are performed, thereby obtaining modified sodium hyaluronate gel for injection.

(35) .sup.1H-NMR (D.sub.2O, NaOD): 1.80 (s, 3H), 3.26 (s, 3H), 3.41 (s, H), 3.55 [s, (H.sub.PEG1000+5H)], 3.80 (s, 1H), 4.32-4.35 (d, 2H).

Embodiment 9: Cytotoxicity Experiment

(36) A cytotoxicity experiment is achieved through detecting the propagation rate of cells referring to standards for biological evaluation-in vitro cell toxicity test of medical instruments. In the experiment, an extraction method is adopted, a cell toxicity test is performed by adopting cells L929, a relative growth rate (RCR) of the cells is calculated through assaying the absorbance value by using an enzyme-linked immunoassay instrument, and the higher the RCR, the better the biocompatibility and the lower the toxicity. By taking BDDE as a control, cytotoxicity test results of polyglycol diglycidyl ether crosslinkers prepared in embodiments 1-4 are shown in a table 1; meanwhile, median inhibitory concentrations of crosslinker compounds (polyglycidyl ether prepared in embodiments 1-3 and BDDE) are tested by adopting the cells L929, and experimental results are shown in a table 2; and moreover, by taking BDDE crosslinked sodium hyaluronate gel (BDDE-HA) as a control, cytotoxicity test results of polyglycol diglycidyl ether crosslinked sodium hyaluronate gel prepared in embodiments 5-8 are shown in a table 3.

(37) TABLE-US-00001 TABLE 1 Cytotoxicity test results of polyglycol diglycidyl ether crosslinkers Test Group Ia Ib Ic Id BDDE RCR 62% 89% 89% 87% 30%

(38) TABLE-US-00002 TABLE 2 Test results of median inhibitory concentrations (IC50) of polyglycidyl ether and BDDE Test Group Compound Cell line IC50 (μM) 1 Ia L929 127.3 2 Ib L929 2303 3 Ic L929 1883 4 BDDE L929 97.95

(39) TABLE-US-00003 TABLE 3 Cytotoxicity test results of polyglycol diglycidyl ether crosslinked sodium hyaluronate gel Test Group IIa IIb IIc IId BDDE-HA RCR 88% 91% 92% 89% 85%

(40) Seen from the table 1 and the table 2, the biocompatibility of the polyglycol diglycidyl ether crosslinkers is obviously superior to that of the BDDE; and seen from the table 3, the biocompatibility of the polyglycol diglycidyl ether crosslinked sodium hyaluronate gel is superior.

Embodiment 10: Squeezing and Pushing Force Experiment

(41) The condition of the crosslinked sodium hyaluronate gel during actual use is understood as one of indexes of evaluation on the product quality through a squeezing and pushing force experiment by using a multi-purpose mechanical tester. In the present invention, a pushing rod is pushed at a constant speed of 30 mm/min, and a sample in a syringe is squeezed out by a syringe needle of 29G to obtain a squeezing and pushing force curve, so that changes of a squeezing and pushing force of the sample during squeezing can be seen. The sample is easily squeezed out if the squeezing and pushing force is small, and the sample is not easily squeezed out if the squeezing and pushing force is large; in addition, if the size difference of the squeezing and pushing force is large, the condition that the sample is subjected to a non-uniform dispersion or gathered concentration phenomenon is shown, and the applicability during injection will be affected. A table 4 records change conditions of the squeezing and pushing force during gel squeezing and pushing, experimental data of the squeezing and pushing force of the dodecaethylene glycol diglycidyl ether crosslinked sodium hyaluronate gel are shown in FIG. 1, and a squeezing and pushing force curve is relatively smooth, which indicates that the gel is relatively homogeneous; experimental data of the squeezing and pushing force of the polyethylene glycol PEG1000 diglycidyl ether crosslinked sodium hyaluronate gel with a high molecular weight are shown in FIG. 2, and a squeezing and pushing force curve is relatively large in fluctuation, which indicates that the gel is relatively poor in homogeneity.

(42) TABLE-US-00004 TABLE 4 Test results of squeezing and pushing force of polyglycol diglycidyl ether crosslinked sodium hyaluronate gel Test Group IIa IIb IIc IId BDDE-HA Squeezing and 7-8N 7-8N 8-9N 5-9N 7-8N pushing force

Embodiment 11: In-Vitro Enzymolysis Stability Experiment

(43) 0.5 g of crosslinked sodium hyaluronate gel is taken, 2 mL of 300 U/mL hyaluronidase solution is added, heat-preserving degradation is performed for 40 hours at a temperature of 37° C., PBS is added until a volume is 5 mL, 1 mL of the mixture is taken, 4 mL of anhydrous ethanol is added, centrifugation is performed for 15 min at a revolving speed of 10,000 r/min, 2 mL of supernatant is taken, and volume metering is performed with PBS until the volume is 5 mL to obtain a solution I; additionally, 0.5 g of crosslinked sodium hyaluronate gel is taken, 10 mL of 0.5 mol/L sulfuric acid solution is added, hydrolysis is performed in a boiling water bath for 15 min, and dilution is performed with water until a volume is 100 mL to obtain a solution II. 1 mL of solution I and 1 mL of solution II are separately taken, and the glucuronic acid content is measured by an improved carbazole development process. In-vitro enzyme degradation resistance of the gel is represented by a coefficient R, R=1−0.625A/B, wherein A represents the glucuronic acid content of the solution I, and B represents the glucuronic acid content of the solution II. The higher the R value, the better the in-vitro enzyme degradation resistance, and the more stable the crosslinked gel. Test results are shown in a table 5.

(44) TABLE-US-00005 TABLE 5 Test results of in-vitro enzymolysis stability of polyglycol diglycidyl ether crosslinked sodium hyaluronate gel Test Group IIa IIb IIc IId BDDE-HA Enzyme degradation 78% 82% 81% 69% 71% resisting coefficient

(45) The above embodiments are only the preferred embodiments of the present invention and not intended to limit the present invention, and any modification, equivalent replacement and the like made within the spirit and principle of the present invention shall fall within the scope of protection of the present invention.