CN111686664B - Emulsified cross-linked sodium hyaluronate gel microsphere for injection and preparation method thereof - Google Patents
Emulsified cross-linked sodium hyaluronate gel microsphere for injection and preparation method thereof Download PDFInfo
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/06—Making microcapsules or microballoons by phase separation
- B01J13/14—Polymerisation; cross-linking
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
Abstract
The invention provides a preparation method of emulsified cross-linked sodium hyaluronate gel microspheres for injection, which comprises the following steps of firstly preparing emulsion of sodium hyaluronate microsphere particles; and then adding the cross-linking agent and sodium hyaluronate into the emulsion according to a certain mass ratio, stirring and uniformly mixing in an ice bath, then carrying out primary reaction under stirring at room temperature, finally reacting under stirring in a water bath at 35-42 ℃, obtaining microsphere emulsion after the reaction is finished, and stirring and cleaning by using normal hexane, then ethyl acetate and then absolute ethyl alcohol during cleaning. The method ensures that uniform and ordered crosslinking reaction is carried out in a single sodium hyaluronate colloid particle, obtains proper viscous modulus, elastic modulus and extrusion force, does not carry out crosslinking reaction among particles, improves the yield of qualified gel microspheres, is suitable for injection, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to an emulsified cross-linked sodium hyaluronate gel microsphere for injection and a preparation method thereof.
Background
Sodium Hyaluronate (SH) is an acidic mucopolysaccharide. As early as 1930-1940, meyer extracted sodium hyaluronate from animal synovial fluid, skin, rooster comb and human umbilical cord, and Kendell, 1937, extracted SH from bacteria. SH obtained from different sources and different purification methods have different molecular weights but no species differences. The hyaluronic acid preparation has a long history of medical use, and is generally used in ophthalmology and orthopaedics, and in 1962, a sodium hyaluronate preparation (Healon xi Lang) is approved by FDA to be applied to aqueous humor and vitreous body substitutes in ophthalmic surgery and a base of ophthalmic external drugs. Thereafter, it has also been applied to joint cavity injections for arthritis and to vocal cords for vocal insufficiency. In recent years, sodium hyaluronate has been reported to be used as an external medicine for treating skin wounds so as to achieve the effects of absorbing seepage, resisting inflammation and promoting wound healing. Most of these products are not crosslinked and therefore their residence time in the body is typically only a few days. Because sodium hyaluronate is rapidly degraded, sodium hyaluronate for ophthalmic and intra-articular injection cannot be applied to skin augmentation, and in order to prolong the retention time, it is necessary to crosslink these small sodium hyaluronate particles into large molecular weight aggregates by chemical bonds by means of a crosslinking method to delay the degradation thereof. The cross-linking technique of sodium hyaluronate was used since 1980, and sodium hyaluronate after cross-linking forms a three-dimensional gel-like structure, but its physicochemical properties and tissue compatibility remained unchanged. The concentration of the sodium hyaluronate preparation commonly used at present reaches 5.5-20 mg/ml, the particle diameter can reach 125-1000 mu m according to requirements, and the degradation time can reach more than 6 months after the sodium hyaluronate preparation is injected into tissues. The sodium hyaluronate has the advantages of no anti-immune prototype, no need of skin test and refrigeration, non-animal source (extracted from streptococcus), absorbability, long curative effect, no toxicity and the like. Sodium hyaluronate shows various important physiological functions in organisms by virtue of unique molecular structure and physicochemical properties, such as joint lubrication, regulation of permeability of blood vessel walls, regulation of protein and water electrolyte diffusion and operation, promotion of wound healing and the like.
Because of the action of hydrogen bonds between monosaccharides on a sodium hyaluronate straight chain shaft, sodium hyaluronate molecules are in a rigid spiral columnar structure in space, strong hydrophilicity is generated due to the existence of a large number of hydroxyl groups on the inner side of the column, and the water molecules combined by the hyaluronic acid molecules are locked in the double-spiral columnar structure, so that the water molecules are not easy to run off, and the sodium hyaluronate water retention agent has a special water retention effect, has the water retention capacity theoretically as high as 500ml/mg, and is known as an ideal natural moisture retention factor. With the age and the reduction of hyaluronic acid in the body, the health care product containing hyaluronic acid can supplement hyaluronic acid in the body by oral administration, has the effects of delaying senility, moistening skin and the like, and is widely used in countries such as Japan, america and the like. At present, many methods for preparing cross-linked hyaluronic acid at home and abroad are available, the cross-linked hyaluronic acid and a cross-linking agent are generally stirred and mixed in an aqueous solution, the cross-linking agent is added to synthesize chemical bonds between high molecular chains of the hyaluronic acid, the obtained massive gel is generally mechanically crushed or pressed and passes through a screen to obtain different particle size specifications, but the prepared cross-linked hyaluronic acid gel has the problems of non-uniform cross-linking degree, non-standard particle shape, non-uniform particle size and the like. Patent publication No. EP2225281 describes a method for preparing crosslinked hyaluronic acid microbeads in emulsion, the microspheres obtained by the method have uniform shape and particle size distribution between 1nm and 1 μm, but the finally obtained crosslinked hyaluronic acid microbeads have no effective measures for controlling oil phase residue and crosslinking agent residue, and have the problems of poor biocompatibility and water retention, unstable product and the like, and cannot well meet the market demand. Patent publication No. CN 10333333351A provides a process for preparing cross-linked sodium hyaluronate microspheres capable of being used as an embolic agent by using sodium hyaluronate as a raw material. The emulsifying speed is 500 rpm-2000 rpm, the crosslinking reaction process is stirring for 4-6 h at room temperature, and the particle size of the obtained microsphere is 80-2000 mu m.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the preparation method of the emulsified cross-linked sodium hyaluronate gel microspheres for injection, and the method has the advantages of mild reaction conditions, simple preparation steps, convenience in operation, no harmful substances, good biocompatibility, acid and alkali resistance and the like. Therefore, the invention adopts the following technical scheme:
a preparation method of emulsified cross-linked sodium hyaluronate gel microspheres for injection is characterized by comprising the following steps: the method comprises the following steps:
(1) Preparing an emulsion of sodium hyaluronate microsphere particles;
(2) Adding a cross-linking agent and sodium hyaluronate into the emulsion according to a certain mass ratio, stirring and uniformly mixing in an ice bath, then carrying out a preliminary reaction under stirring at room temperature, finally carrying out a reaction under stirring in a water bath at 35-42 ℃, obtaining a microsphere emulsion after the reaction is finished, cooling, and then adjusting the pH to 4-6;
(3) Firstly, stirring and cleaning the microsphere emulsion with the pH value of 4-6 by using normal hexane for several times, standing and layering, removing an upper oil phase, stirring and cleaning the microsphere emulsion by using ethyl acetate for several times after the completion, standing and layering, and removing the upper oil phase; after the completion, stirring and cleaning the mixture for a plurality of times by using absolute ethyl alcohol, standing and layering the mixture, and removing an upper oil phase;
(4) Placing the microspheres washed by the organic solvent in the step (3) into a buffer solution, standing and swelling for overnight, and pouring out supernate;
(5) And (4) sieving the product obtained in the step (4), and removing gel microspheres which are not in the range by sieving to obtain the emulsified cross-linked sodium hyaluronate gel microspheres for injection.
In a preferred embodiment, in the emulsion in step (1), the oil phase is one of liquid paraffin, vegetable oil, mineral oil, silicone oil or synthetic oil, preferably liquid paraffin; the mass percentage of the emulsifier in the oil phase is 2-8%.
In a preferred embodiment, sodium hyaluronate is prepared by a microbial fermentation method, sodium hyaluronate is prepared into sodium hyaluronate alkaline solution gel with the concentration of 8-20 g/ml, an alkaline solution is NaOH solution or KOH solution, the sodium hyaluronate alkaline solution gel is obtained by strong stirring, then the sodium hyaluronate alkaline solution gel is added into an oil phase containing an emulsifier and is emulsified at high speed by a shearing machine to form an emulsion of sodium hyaluronate microsphere particles, and the weight ratio of the sodium hyaluronate alkaline solution gel to the oil phase is 1. Further, the molecular weight of the sodium hyaluronate gel is 90 to 150 ten thousand daltons, preferably 110 to 130 ten thousand daltons.
In a preferred embodiment, the emulsion prepared in step (1) is prepared at a shear speed of 4000 to 5000rpm for 30 to 60min.
In a preferred embodiment, the crosslinking agent in step (2) is selected from divinyl sulfone (DVS), 1, 4-butanediol diglycidyl ether (BDDE), preferably divinyl sulfone (DVS); the cross-linking agent is added into the emulsion according to the mass ratio of 10-30 percent of sodium hyaluronate.
In a preferred embodiment, in step (2), stirring in ice bath is carried out for 50min to 70min, then reaction is carried out for 80min to about 100min under stirring at room temperature, and finally reaction is carried out for 50min to 70min under stirring in water bath at 35-42 ℃.
In a preferable embodiment, in the step (3), the emulsion is stirred and washed twice by using normal hexane, the mixture is kept stand for layering, and the upper oil phase is discarded, and the washing time is 5-10 min each time; after the completion, stirring and cleaning twice by using absolute ethyl alcohol, standing and layering, removing an upper oil phase, and cleaning for 5-10 min each time; stirring and cleaning twice with ethyl acetate, standing and layering, removing an upper oil phase, and cleaning for 5-10 min each time; and then stirring and cleaning twice with absolute ethyl alcohol, standing and layering, removing an upper oil phase, and cleaning for 5-10 min each time.
In a preferred embodiment, the buffer solution in step (4) is a PBS buffer solution, the mass ratio of disodium hydrogen phosphate is 0.035%, the mass ratio of sodium dihydrogen phosphate is 0.014%, the mass ratio of sodium chloride is 0.7%, and the pH value of the buffer solution is 6.0-8.0.
In a preferred embodiment, gel microspheres with different particle sizes and different water absorption times and swelling ratios, wherein the particle sizes of the gel microspheres range from 60 to 330 mu m, are prepared by controlling the emulsification speed, the emulsification time, the sodium hyaluronate alkali solution concentration in the water phase, the weight ratio of the water phase to the oil phase and the dosage of the cross-linking agent in the step (1).
In a preferred embodiment, the prepared gel microspheres are stored in a syringe for sealing storage after being subjected to moist heat sterilization.
The invention also relates to an emulsified cross-linked sodium hyaluronate gel microsphere for injection, which is prepared by any one of the preparation methods.
The preparation method of the emulsified and crosslinked sodium hyaluronate gel microspheres for injection is different from the conventional crosslinking method, sodium hyaluronate alkaline gel is added into an oil phase containing an emulsifier, a W/O type emulsion is obtained by high-speed shearing through an emulsifying shearing machine, the surface tension of two phases is reduced under the action of the emulsifier, so that sodium hyaluronate colloid is uniformly distributed in the emulsion in a microsphere form, the added crosslinking agent and the colloid particles are subjected to crosslinking reaction through a three-step method, uniform and ordered crosslinking reaction inside the single sodium hyaluronate colloid particle is ensured, proper viscous modulus, elastic modulus and extrusion force are obtained, the crosslinking reaction among the particles is avoided, the yield of qualified gel microspheres is improved, and the emulsified and crosslinked sodium hyaluronate gel microspheres for injection are suitable for injection and have wide application prospects.
Detailed Description
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.
Example 1
(1) Weighing 6.39g of sodium hyaluronate with a molecular weight of 110 to 130 ten thousand daltons, and preparing sodium hyaluronate lye gel with a mass percentage concentration of 8 g/ml with 1% NaOH solution;
(2) Uniformly mixing 300.05g of liquid paraffin and 18.03g of span to obtain an oil phase, slowly adding the sodium hyaluronate gel obtained in the step (1) into the oil phase, and emulsifying at a high speed of 4000rpm for 30min by using a stirrer;
(3) After emulsification in the step (2) is finished, adding 1.06g of cross-linking agent divinyl sulfone, stirring in an ice bath for 1h, stirring in a water bath at the room temperature for 1.5h, stirring in a water bath at the temperature of 40 ℃ for 1h, and adjusting the pH value to 5.0 by using glacial acetic acid after cooling;
(4) Pouring an upper oil phase, adding n-hexane, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding n-hexane, stirring and cleaning for the second time for 6min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the second time for 6min, pouring the upper oil phase after standing and layering, adding ethyl acetate, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding ethyl acetate, stirring and cleaning for the second time for 6min, adding absolute ethyl alcohol, stirring and cleaning for the third time for 6min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the fourth time for 5min, and pouring the upper oil phase after standing and layering;
(5) Preparing a PBS buffer solution, wherein the mass ratio of disodium hydrogen phosphate is 0.035%, the mass ratio of sodium dihydrogen phosphate is 0.014%, and the mass ratio of sodium chloride is 0.7%, placing the microspheres washed by the organic solvent in the last step into the buffer solution, standing and swelling for overnight, and pouring out the supernatant;
(6) And (4) sieving the product to remove gel microspheres out of range.
(7) And storing the prepared gel in an injector for sealing and storing after moist heat sterilization.
Observing the prepared gel microsphere under a particle size analyzer, wherein the microsphere is in a regular spherical shape, and the particle size is 80-280 mu m.
Precisely weighing a certain amount of the microsphere particles W1, immersing the microsphere particles into physiological saline, weighing W2 again after the microspheres reach a water absorption saturation state, and calculating to obtain the water absorption multiple W2/W1=33.85 times of the microspheres.
Measuring the average value D1 of the particle diameters of part of the microspheres, immersing the microspheres in physiological saline until the microspheres reach a water absorption saturated state, measuring the average value D2 of the particle diameters again, and calculating the swelling ratio D2-D1/D1 = 100% =23.37% of the microspheres.
Taking a proper amount of microspheres, and measuring the viscous modulus of the microspheres to be 296Pa and the elastic modulus to be 875Pa by using a rheometer. Taking a proper amount of microspheres, installing a 27G needle, pushing at the speed of 20mm/min, recording the average pushing force of the syringe handle as 14N, and being suitable for injection.
Example 2
(1) Weighing 6.45g of sodium hyaluronate with the molecular weight of 110-130 kilodaltons, and preparing the sodium hyaluronate into sodium hyaluronate alkali lye gel with the mass percentage concentration of 8 g/ml by mixing the sodium hyaluronate alkali lye gel with 1-percent NaOH solution;
(2) Uniformly mixing 300.05g of liquid paraffin and 18.03g of span to obtain an oil phase, slowly adding the sodium hyaluronate gel obtained in the step (1) into the oil phase, and emulsifying at a high speed of 4500rpm for 30min by using a stirrer;
(3) After emulsification in the step (2) is finished, adding 1.07g of cross-linking agent divinyl sulfone, stirring for 1h in ice bath, stirring for 1.5h in room temperature, stirring for 1h in water bath at 40 ℃, and adjusting the pH value to 5.0 by using glacial acetic acid after cooling;
(4) Pouring an upper oil phase, adding n-hexane, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding n-hexane, stirring and cleaning for the second time for 6min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the second time for 6min, pouring the upper oil phase after standing and layering, adding ethyl acetate, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding ethyl acetate, stirring and cleaning for the second time for 6min, adding absolute ethyl alcohol, stirring and cleaning for the third time for 6min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the fourth time for 5min, and pouring the upper oil phase after standing and layering;
(5) Preparing a PBS buffer solution, wherein the mass ratio of disodium hydrogen phosphate is 0.035%, the mass ratio of sodium dihydrogen phosphate is 0.014%, and the mass ratio of sodium chloride is 0.7%, placing microspheres washed by the organic solvent in the last step into the buffer solution, standing and swelling for overnight, and pouring out supernatant;
(6) And (4) sieving the product to remove gel microspheres out of range.
(7) And storing the prepared gel in an injector for sealing and storing after moist heat sterilization.
The morphology of the microspheres was observed in the same manner as in example 1, and the particle size of the microspheres obtained in this example was 70 to 250 μm.
The water absorption capacity of the microspheres was 34.26 times as measured in the same manner as in example 1.
The swelling ratio of the microspheres was 25.61% by the same method as in example 1.
The microspheres had a viscous modulus of 263Pa and an elastic modulus of 847Pa, which were measured in the same manner as in example 1. The syringe handle pushing force was measured to be 13N in the same manner as in example 1, and was suitable for injection.
From the comparison of the experimental results of example 1 and example 2, the microsphere particles with different particle size distributions can be prepared by controlling the emulsification speed. When the emulsifying speed is higher, the acting force applied to the water phase is increased, the dispersity of the microspheres is also increased, the particle size is smaller, and vice versa.
Example 3
(1) Weighing 6.40g of sodium hyaluronate with the molecular weight of 110-130 kilodaltons, and preparing the sodium hyaluronate into sodium hyaluronate alkali lye gel with the mass percentage concentration of 16 percent g/ml by mixing with 1 percent of NaOH solution;
(2) Uniformly mixing 300.07g of liquid paraffin and 18.02g of span 80 to obtain an oil phase, slowly adding the sodium hyaluronate gel obtained in the step (1) into the oil phase, and emulsifying at a high speed of 4000rpm for 30min by using a stirrer;
(3) After emulsification in the step (2) is finished, adding 1.06g of cross-linking agent divinyl sulfone, stirring in an ice bath for 1h, stirring in a water bath at the room temperature for 1.5h, stirring in a water bath at the temperature of 40 ℃ for 1h, and adjusting the pH value to 5.0 by using glacial acetic acid after cooling;
(4) Pouring an upper oil phase, adding n-hexane, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding n-hexane, stirring and cleaning for the second time for 6min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the second time for 6min, pouring the upper oil phase after standing and layering, adding ethyl acetate, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding ethyl acetate, stirring and cleaning for the second time for 6min, adding absolute ethyl alcohol, stirring and cleaning for the third time for 6min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the fourth time for 5min, and pouring the upper oil phase after standing and layering;
(5) Preparing a PBS buffer solution, wherein the mass ratio of disodium hydrogen phosphate is 0.035%, the mass ratio of sodium dihydrogen phosphate is 0.014%, and the mass ratio of sodium chloride is 0.7%, placing microspheres washed by the organic solvent in the last step into the buffer solution, standing and swelling for overnight, and pouring out supernatant;
(6) And (4) sieving the product to remove gel microspheres out of range.
(7) And storing the prepared gel in an injector for sealing and storing after moist heat sterilization.
The morphology of the microspheres was observed in the same manner as in example 1, and the particle size of the microspheres obtained in this example was 100 to 310. Mu.m.
The water absorption capacity of the microspheres was measured to be 30.74 times in the same manner as in example 1.
The swelling ratio of the microspheres was measured to be 21.55% by the same method as in example 1.
The microspheres had a viscous modulus of 313Pa and an elastic modulus of 891Pa, which were measured in the same manner as in example 1. The syringe handle pushing force was measured to be 15N in the same manner as in example 1, and was suitable for injection.
From the comparison of the experimental results of example 1 and example 3, it is concluded that gel microspheres with different particle size distributions can be prepared by controlling the concentration of the hyaluronic acid sodium lye gel. The low concentration of the sodium hyaluronate in the solution ensures that the water content of the sodium hyaluronate microspheres is higher, the dispersibility of the sodium hyaluronate microspheres in the emulsion is improved, and the particle size of the obtained microspheres is reduced.
Example 4
(1) Weighing 6.38g of sodium hyaluronate with a molecular weight of 110 to 130 ten thousand daltons, and preparing sodium hyaluronate lye gel with a mass percentage concentration of 8 g/ml with 1% NaOH solution;
(2) Uniformly mixing 300.03g of liquid paraffin and 80.04g of span to obtain an oil phase, slowly adding the sodium hyaluronate gel obtained in the step (1) into the oil phase, and emulsifying at a high speed of 4000rpm for 30min by using a stirrer;
(3) After emulsification in the step (2) is finished, adding 1.59g of cross-linking agent divinyl sulfone, stirring in an ice bath for 1h, stirring in a water bath at the temperature of 40 ℃ for 1h, cooling, and adjusting the pH value to 5.0 by using glacial acetic acid;
(4) Pouring an upper oil phase, adding n-hexane, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding n-hexane, stirring and cleaning for the second time for 6min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the second time for 6min, pouring the upper oil phase after standing and layering, adding ethyl acetate, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding ethyl acetate, stirring and cleaning for the second time for 6min, adding absolute ethyl alcohol, stirring and cleaning for the third time for 6min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the fourth time for 5min, and pouring the upper oil phase after standing and layering;
(5) Preparing a PBS buffer solution, wherein the mass ratio of disodium hydrogen phosphate is 0.035%, the mass ratio of sodium dihydrogen phosphate is 0.014%, and the mass ratio of sodium chloride is 0.7%, placing the microspheres washed by the organic solvent in the last step into the buffer solution, standing and swelling for overnight, and pouring out the supernatant;
(6) Sieving the product to remove gel microspheres out of range.
The morphology of the microspheres was observed in the same manner as in example 1, and the particle size of the microspheres obtained in this example was 90 to 280 μm.
The microspheres had a water absorption capacity of 26.48 times as measured in the same manner as in example 1.
The swelling ratio of the microspheres was measured to be 18.60% in the same manner as in example 1.
The microspheres had a viscous modulus of 286Pa and an elastic modulus of 894Pa as measured in the same manner as in example 1. The syringe handle pushing force was measured to be 15N in the same manner as in example 1, and was suitable for injection.
From the comparison of the experimental results of example 1 and example 4, it is concluded that gel microspheres with different water absorption times and swelling ratios can be prepared by controlling the amount of the cross-linking agent. The water absorption capacity and swelling ratio of the crosslinked sodium hyaluronate depend on the crosslinking degree of the crosslinked sodium hyaluronate, and when solvent molecules enter a network formed by crosslinking polymers, the solvent molecules cause the stretching of a three-dimensional molecular network to expand a polymer crosslinking system. When the crosslinking degree is smaller, the polymer chain between crosslinking points in the crosslinking network is longer, more solvent molecules enter the crosslinking network, and the system expands more; when the crosslinking degree is larger, the polymer chains among crosslinking points in the crosslinking network are shorter and the crosslinking points are dense, so that less solvent molecules enter and the system expands less.
From the examples 1 to 4, it can be seen that the emulsified cross-linked sodium hyaluronate gel microspheres for injection prepared by the preparation method of the present invention have the water absorption multiple of 26 to 34 times, the swelling ratio of 18 to 26%, the viscous modulus of 263 to 313Pa, the elastic modulus of 847 to 894Pa, and the extrusion force of 13 to 15N, have good water absorption performance and swelling performance, and are moderate in viscous modulus, elastic modulus and extrusion force, suitable for injection, and have wide application prospects.
Comparative example 1 (crosslinking method Using patent publication No. CN 103333351A)
(1) Weighing 6.41g of sodium hyaluronate with a molecular weight of 110 to 130 ten thousand daltons, and preparing sodium hyaluronate lye gel with a mass percentage concentration of 8 g/ml with 1% NaOH solution;
(2) Uniformly mixing 300.04g of liquid paraffin and 80.00g of span to obtain an oil phase, slowly adding the sodium hyaluronate gel obtained in the step (1) into the oil phase, and emulsifying at a high speed of 4000rpm for 30min by using a stirrer;
(3) After the emulsification in the step (2) is finished, adding 1.06g of cross-linking agent divinyl sulfone, stirring at room temperature for 4h, cooling, and adjusting the pH value to 5.0 by using glacial acetic acid;
(4) Pouring an upper oil phase, adding n-hexane, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding n-hexane, stirring and cleaning for the second time for 6min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the second time for 6min, pouring the upper oil phase after standing and layering, adding ethyl acetate, stirring and cleaning for the first time for 8min, pouring the upper oil phase after standing and layering, adding ethyl acetate, stirring and cleaning for the second time for 6min, adding absolute ethyl alcohol, stirring and cleaning for the third time for 6min, pouring the upper oil phase after standing and layering, adding absolute ethyl alcohol, stirring and cleaning for the fourth time for 5min, and pouring the upper oil phase after standing and layering;
(5) Preparing a PBS buffer solution, wherein the mass ratio of disodium hydrogen phosphate is 0.035%, the mass ratio of sodium dihydrogen phosphate is 0.014%, and the mass ratio of sodium chloride is 0.7%, placing the microspheres washed by the organic solvent in the last step into the buffer solution, standing and swelling for overnight, and pouring out the supernatant;
(6) And (4) sieving the product to remove gel microspheres out of range.
(7) And storing the prepared gel in an injector for sealing and storing after moist heat sterilization.
The morphology of the microspheres was observed in the same manner as in example 1, and the particle size of the microspheres obtained in this example was 120 to 350. Mu.m.
The water absorption capacity of the microspheres was measured to be 26.92 times in the same manner as in example 1.
The swelling ratio of the microspheres was measured to be 20.13% in the same manner as in example 1.
The microspheres had a viscous modulus of 356Pa and an elastic modulus of 992Pa as measured in the same manner as in example 1. The syringe handle pushing force was measured to be 18N in the same manner as in example 1.
Comparative example 2 (crosslinking method using patent publication No. CN 103333351A)
(1) Weighing 6.40g of sodium hyaluronate with the molecular weight of 110-130 kilodaltons, and preparing the sodium hyaluronate into sodium hyaluronate alkali lye gel with the mass percentage concentration of 8 g/ml by mixing the sodium hyaluronate alkali lye gel with the concentration of 1-percent NaOH solution;
(2) Uniformly mixing 300.08g of liquid paraffin and 18.02g of span 80 to obtain an oil phase, slowly adding the sodium hyaluronate gel obtained in the step (1) into the oil phase, and emulsifying at a high speed of 4000rpm for 30min by using a stirrer;
(3) After the emulsification in the step (2) is finished, adding 1.06g of cross-linking agent divinyl sulfone, stirring at room temperature for 6h, cooling, and adjusting the pH value to 5.0 by using glacial acetic acid;
(4) Pouring an upper oil phase, adding n-hexane, stirring and cleaning for the first time for 8min, standing for layering, pouring the upper oil phase, adding n-hexane, stirring and cleaning for the second time for 6min, standing for layering, pouring the upper oil phase, adding absolute ethyl alcohol, stirring and cleaning for the first time for 8min, standing for layering, pouring the upper oil phase, adding absolute ethyl alcohol, stirring and cleaning for the second time for 6min, standing for layering, pouring the upper oil phase, adding ethyl acetate, stirring and cleaning for the first time for 8min, standing for layering, pouring the upper oil phase, adding ethyl acetate, stirring and cleaning for the second time for 6min, adding absolute ethyl alcohol, stirring and cleaning for the third time for 6min, standing for layering, pouring the upper oil phase, adding absolute ethyl alcohol, stirring and cleaning for the fourth time for 5min, standing for layering, and pouring the upper oil phase;
(5) Preparing a PBS buffer solution, wherein the mass ratio of disodium hydrogen phosphate is 0.035%, the mass ratio of sodium dihydrogen phosphate is 0.014%, and the mass ratio of sodium chloride is 0.7%, placing the microspheres washed by the organic solvent in the last step into the buffer solution, standing and swelling for overnight, and pouring out the supernatant;
(6) And (4) sieving the product to remove gel microspheres out of range.
(7) The prepared gel is stored in an injector for sealing and preservation after being sterilized by moist heat.
The morphology of the microspheres was observed in the same manner as in example 1, and the particle size of the microspheres obtained in this example was 140 to 380. Mu.m.
The water absorption capacity of the microspheres was measured to be 25.50 times in the same manner as in example 1.
The swelling ratio of the microspheres was measured to be 19.81% by the same method as in example 1.
The microsphere was measured to have a viscous modulus of 374Pa and an elastic modulus of 1028Pa in the same manner as in example 1. The syringe handle pushing force was measured to be 19N in the same manner as in example 1.
Through the comparison of the experimental results of example 1 and comparative example 1 with comparative example 2, it is found that when the reaction is carried out by the crosslinking process in the patent publication No. CN 10333333351A (i.e. room temperature crosslinking is carried out for 4-6 h), the microsphere has larger particle size, larger viscous modulus and elastic modulus, larger pushing force of the syringe handle and is not suitable for injection. This is because the crosslinking of sodium hyaluronate with a crosslinking agent such as divinyl sulfone is a rapid process, and the present invention adopts a stepwise method to allow the two to react, i.e., first ice bath, then room temperature, and then water bath. Firstly, adopting an ice bath, under high-speed stirring, leading divinyl sulfone to enter a water-in-oil water phase and be mixed with sodium hyaluronate uniformly in preference to reaction, leading divinyl sulfone to be slowly subjected to cross-linking reaction from a lower temperature to room temperature, and if the divinyl sulfone is directly subjected to cross-linking at room temperature, leading the reaction of the sodium hyaluronate and the divinyl sulfone to be more violent, prolonging the molecular chain and increasing the particle size of microspheres. The particle size of the microspheres is increased, the viscous modulus and the elastic modulus are increased, and the extrusion force is also increased.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art; the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A preparation method of emulsified cross-linked sodium hyaluronate gel microspheres for injection is characterized by comprising the following steps: the method comprises the following steps:
(1) Preparing an emulsion of sodium hyaluronate microsphere particles;
(2) Adding a cross-linking agent and sodium hyaluronate into the emulsion according to a certain mass ratio, stirring and uniformly mixing in an ice bath, then carrying out a primary reaction under stirring at room temperature, finally reacting under stirring in a water bath at 35-42 ℃, obtaining a microsphere emulsion after the reaction is finished, cooling firstly, and then adjusting the pH to 4-6; wherein, stirring in ice bath for 50min to 70min, reacting at room temperature for 80min to 100min under stirring, and reacting in water bath at 35-42 ℃ for 50min to 70min;
(3) Firstly, stirring and cleaning the microsphere emulsion with the pH value of 4-6 by using normal hexane for a plurality of times, standing and layering, removing an upper oil phase, stirring and cleaning the microsphere emulsion by using ethyl acetate for a plurality of times after finishing, standing and layering, and removing the upper oil phase; after the completion, stirring and cleaning the mixture for a plurality of times by using absolute ethyl alcohol, standing and layering the mixture, and removing an upper oil phase;
(4) Placing the microspheres washed by the organic solvent in the step (3) into a buffer solution, standing and swelling for one night, and pouring supernatant;
(5) Sieving the product obtained in the step (4) to remove gel microspheres out of range, thereby obtaining the emulsified cross-linked sodium hyaluronate gel microspheres for injection;
in the emulsion in the step (1), the oil phase is one of liquid paraffin, vegetable oil, mineral oil, silicone oil or synthetic oil; the mass percentage of the emulsifier in the oil phase is 2-8%;
preparing sodium hyaluronate into sodium hyaluronate alkaline solution gel with the concentration of 8-20% g/ml by adopting a microbial fermentation method, wherein the alkaline solution is NaOH solution or KOH solution, strongly stirring to obtain the sodium hyaluronate alkaline solution gel, adding the sodium hyaluronate alkaline solution gel into an oil phase containing an emulsifier, and emulsifying at a high speed by using a shearing machine to form an emulsion of sodium hyaluronate microsphere particles, wherein the weight ratio of the sodium hyaluronate alkaline solution gel to the oil phase is 1.
2. The method of claim 1, wherein: the oil phase is liquid paraffin.
3. The production method according to claim 1, characterized in that: when the emulsion is prepared in the step (1), the rotating speed of a shearing machine is 4000-5000 rpm, and the time is 30-60 min.
4. The method of claim 1, wherein: sodium hyaluronate prepared by a microbial fermentation method in the step (1); the molecular weight of the sodium hyaluronate gel is 90-150 ten thousand daltons.
5. The method of claim 4, wherein: the molecular weight of the sodium hyaluronate gel is 110-130 ten thousand daltons.
6. The process according to claim 1, wherein the crosslinking agent in the step (2) is selected from the group consisting of divinyl sulfone (DVS), 1, 4-butanediol diglycidyl ether (BDDE); the cross-linking agent is added into the emulsion according to the mass ratio of 10-30 percent of sodium hyaluronate.
7. The method according to claim 6, wherein the crosslinking agent in the step (2) is divinyl sulfone (DVS).
8. The preparation method according to claim 1, wherein gel microspheres with different particle sizes and different water absorption times and swelling ratios, which have particle sizes ranging from 60 to 330 μm, are prepared by controlling the emulsification speed, the emulsification time, the sodium hyaluronate alkaline solution concentration in the aqueous phase, the weight ratio of the aqueous phase to the oil phase, and the amount of the cross-linking agent in step (1).
9. The method of claim 1, wherein the gel microspheres are stored in a syringe after being sterilized by moist heat and then hermetically stored.
10. An emulsified cross-linked sodium hyaluronate gel microsphere for injection is characterized in that: the emulsified and crosslinked sodium hyaluronate gel microspheres for injection are prepared by the preparation method of any one of claims 1 to 9.
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