CN103834053A - Injectable crosslinked hyaluronic acid gel and preparation method thereof - Google Patents
Injectable crosslinked hyaluronic acid gel and preparation method thereof Download PDFInfo
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- CN103834053A CN103834053A CN201410074850.6A CN201410074850A CN103834053A CN 103834053 A CN103834053 A CN 103834053A CN 201410074850 A CN201410074850 A CN 201410074850A CN 103834053 A CN103834053 A CN 103834053A
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 229
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 157
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 154
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- 238000004132 cross linking Methods 0.000 claims abstract description 37
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- 238000011049 filling Methods 0.000 claims abstract description 21
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- 239000007853 buffer solution Substances 0.000 claims abstract description 6
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- KUAUJXBLDYVELT-UHFFFAOYSA-N 2-[[2,2-dimethyl-3-(oxiran-2-ylmethoxy)propoxy]methyl]oxirane Chemical compound C1OC1COCC(C)(C)COCC1CO1 KUAUJXBLDYVELT-UHFFFAOYSA-N 0.000 claims description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 2
- 239000008157 edible vegetable oil Substances 0.000 claims description 2
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Images
Abstract
The invention relates to an injectable crosslinked hyaluronic acid gel and a preparation method thereof. The injectable crosslinked hyaluronic acid gel is prepared by blending hyaluronic acid gel granules and chlorinated sodium phosphate physiological buffer solution; the hyaluronic acid gel granules are prepared by comprising the steps of crosslinking treatment, emulsion crosslinking granulation, purification and drying and swelling, filling and sterilization technologies. The hyaluronic acid gel granules are uniform in granule size, the residual of the crosslinking agent is less than 0.2ppm, the injection pushing is proper, and the in-vivo degradation time can last more than 8-12 months. The implant has an excellent esthetical restoration effect, is applicable to being injected to and subcutaneous dermis deep layer to subcutaneous superficial layer, restoration of moderate to severe wrinkles or folds, and can satisfy the restoration demand of wrinkles or folds caused due to skin aging.
Description
Technical field
The invention belongs to medical cosmetology shaping technique, be specifically related to a kind of injectable cross-linked hyaluronic acid gel and preparation method thereof.
Background technology
Soft tissue filling agent has been widely used in aesthetic surgery to improve face contour, revise wrinkle, to fill and lead up depressed scar and packing volume (as lip) etc.Conventional injectable soft tissue compaction material is mainly hyaluronic acid series products both at home and abroad at present, this series products is as beauty treatment injection implant, there is obvious advantage, as it is stable lasting to inject rear wrinkle repairing effect, without excessive injection, if accidentally inject the excessive Unidasa processing that also can adopt immediately; Hyaluronic acid good biocompatibility, postoperative adverse reaction rate is very low, and the proper hyaluronic acid of crosslinking process can maintain long period action effect in vivo, general equal can reaching about 6～12 months.
Along with the development of technology and more and more ask beautiful person to approve and use injection cross-linked hyaluronic acid gel, country is also more and more stricter to the requirement of this series products, is the residual quantity etc. of degradation time, particle size distribution range, swelling ratio, injection pushing force and linking agent to the evaluation index of injection cross-linked hyaluronic acid gel product quality.For example, degradation time is the validity of products time length directly being determined by product degree of crosslinking; Particle size distribution range has determined the stability of hyaluronic acid derivatives validity and pushing force; Swelling ratio affects the hydrophilicity of product, determines that product is injected to the swelling volume size in body, thereby affects the judgement of doctor for injection volume size; And pushing force can characterize the degree of uniformity of product dispersion and the suitable chirality of clinical use; The residual quantity of linking agent has directly affected the final security of product.
The technology difficulties of hyaluronic acid derivatives is at present: how to guarantee suitable degree of crosslinking, make product have long-term anti-degradation capability and suitable swelling ratio; How to control particle size distribution range; How thoroughly to remove linking agent residual.These three aspects are the topmost control indexs that directly affect injection cross-linked hyaluronic acid gel validity of products, security.Therefore, current cross-linked-hyaluronic acid technology of preparing still has room for improvement.
Go on the market both at home and abroad and had and exceeded ten million example and ask beautiful person to use historical auspicious blue 2(Restylane) product, in its product description, point out that this product only has 80% size distribution in the scope of 80～1000 μ m, median size is 400 μ m, visible particle size distribution range is still comparatively wide in range, may cause the degradation rate of variant production to have different, thereby causing the difference of product result of use, so still having and optimizing space aspect control uniform particle diameter; Auspicious blue 2(Restylane in addition) hyaluronic acid concentration of product is 20mg/ml ± 3mg/ml, and product concentration limit of error is 15%, and this also can impact the stability of product result of use to a certain extent.Current technology of preparing and associated disadvantages below thereof make brief of the introduction.
In hyaluronic acid derivatives particle diameter control techniques, as Chinese patent 200810009194.6,201110004283.3,200610024700.X, 200810097847.0,201010117490.5 etc., adopt electric blender, Stirring or the pulverizing hyaluronic acid derivatives that sieves, thereby prepare gel particle, the size of gel particle is even not, and between median size batch, differ greatly, thereby while having a strong impact on product performance as injection, security and the validity etc. after pushing force, injection is stable.Chinese patent 200880005058.8 and 200880127125.3 adopts water and the organic phase of hyaluronic acid and linking agent to be mixed to get water-in-oil type emulsion in addition, makes hyaluronic acid and linking agent form nano-scale particle and complete crosslinking reaction simultaneously.The cross-linked hyaluronic acid gel grain diameter that these patents prepare does not all exceed 1 micron, and degradation in vivo speed too fast (1～3 month) is applied to injection beauty treatment and cannot reaches long-term repair effect.
Preparing the linking agent that cross-linked hyaluronic acid gel the most often uses has divinylsulfone, carbodiimide, glycidyl ether, aldehydes, polyoxyethylene glycol etc., wherein adopt divinylsulfone as Chinese patent 201110154939.X, 200580047101.3 etc.Linking agent divinylsulfone and polyoxyethylene glycol hybrid reaction are obtained polyethers vinyl sulphone and the unreacted divinylsulfone of part by 201110154939.X, again hyaluronic acid raw material is added wherein and reacted, prepare polyoxyethylene glycol and hyaluronic acid by the crosslinked polymkeric substance of divinylsulfone.The polyethers vinyl sulphone molecular chain that the method prepares is longer, and molecule space steric hindrance is larger, sets it as linking agent not good with hyaluronan molecule reaction effect again, and validity of products cannot meet the demands; It is residual to remove linking agent that patent 200580047101.3 is rinsed gel by damping fluid, then add sanitas, and the method cannot effectively be removed the linking agent of gel inside, and has added sanitas, is unfavorable for the biocompatibility of product.Chinese patent 200610152600.5,200810009194.6,200810097847.0 Deng adopt glycidyl ether as hyaluronic linking agent, wherein hyaluronic acid is dissolved in sodium hydroxide solution by 200610152600.5, add glycidyl ether crosslinking agent crosslinked, adopt NaCl solution soaking to remove linking agent, but verify through the inventor, remove in this way the poor effect of Racemic glycidol ether residue, cannot guarantee good product biocompatibility; Employing aldehydes cross-linked-hyaluronic acid, as Chinese patent 200610078103.5, the aldehyde compound that this patent is used is as larger in toxicity such as glutaraldehyde, and the hyaluronic biocompatibility preparing is not high, easily occurs the untoward reactions such as implant calcification.Also there is in addition patent to adopt repeatedly crosslinking technological to prepare cross-linked hyaluronic acid gel, as Chinese patent 200610152600.5, 00804642.5 etc., wherein 200610152600.5 disclose a kind of secondary crosslinking and prepared hyaluronic method: first adopted Racemic glycidol ethers linking agent to process hyaluronic acid, after again it being dissolved with alkaline solution, again adopt Racemic glycidol ethers linking agent crosslinked, when the method is crosslinked for the second time owing to having added the solvent of a times, cause space structure to widen, so the consumption of linking agent is increased to 2.5～6 times that are cross-linked for the first time for the second time, linking agent consumes and is residual more, be unfavorable for product cost and biocompatibility.
Chinese patent 200380111009.X adopts carbodiimide as hyaluronic linking agent, carbodiimide class linking agent is unstable in the aqueous solution, and verify through the inventor, the method adds after carbodiimide linking agent at hyaluronic acid raw material, in short period of time, form solid gel, linking agent is difficult to effectively mix, and cross-linking effect heterogeneity can exert an influence to the stability of product degradation time.200810097847.0 adopt hyaluronic acid at 15～35 ℃, to react and within 24 hours, obtain cross-linked hyaluronic acid gel with glycidyl ether, and its crosslinking temperature is lower, and cross-linking effect is not good, cannot guarantee product long-term effectiveness.
CN00804642.5 adopts multiple linking agent to be cross-linked simultaneously or sequentially hyaluronic acid, makes the multiple group generation crosslinking reaction in hyaluronan molecule.The shortcoming of the method is, the required chemical environment difference of optimum response of most linking agents, crosslinking reaction when being difficult to adopt same buffered environment to carry out multiple linking agent; Secondly, multiple linking agent order is crosslinked, may be due to after certain crosslinking reaction generation, and hyaluronic acid conformation and spatial distribution change, and therefore other crosslinking reactions are produced and hinder or disturb; In addition, adopt multiple linking agent, may cause removing the residual cost up of multiple linking agent, Product Safety increased risk.
Therefore at present all there is certain shortcoming in disclosed various cross-linked-hyaluronic acid patented technologies and preparation method, clearly control crosslinking degree, particle size distribution range, degradation cycle, linking agent residual aspect, still have optimization space.
Summary of the invention
For the defect of prior art, the object of this invention is to provide a kind of injectable cross-linked hyaluronic acid gel and preparation method thereof, this gel uniform particle diameter, the residual 0.2ppm that is less than of linking agent, biocompatibility is good; This preparation method can effectively control the homogeneity of hyaluronic acid derivatives particle, and injection pushing force is moderate, and the vivo degradation time reaches more than 8～12 months, can meet clinical demand of repairing for repairing wrinkle that skin aging causes or fold.
Injectable cross-linked hyaluronic acid gel prepared by the present invention, is formed by hyaluronic acid derivatives particle and phosphoric acid sodium-chlor physiological buffer solution preparation.Hyaluronic acid derivatives particle is take hyaluronic acid dry powder as raw material, makes by the technique of crosslinking Treatment, emulsification and cross linked granulation, purifying.
Described hyaluronic acid derivatives particle (pulverize and make again by the swelling rear gel forming of hyaluronic acid), its particle diameter mean size and particle size distribution range are 250 μ m ± 150 μ m, 500 μ m ± 150 μ m, 700 μ m ± 150 μ m or 850 μ m ± 150 μ m, hyaluronic acid concentration (being hyaluronic acid contents) is 15～25mg/mL, pushing force <15N(30G syringe needle), storage modulus is 100～600Pa, the residual 0.2ppm that is less than of linking agent.
Described phosphoric acid sodium-chlor physiological buffer is the mixing solutions of following three kinds of salt,, take water for injection as solvent, contains the sodium chloride solution that final concentration is 6～12g/L, the disodium phosphate soln of 0.01～0.1g/L potassium dihydrogen phosphate and 0.1～0.3g/L that is; Also can be take water for injection as solvent, contain the sodium chloride solution that final concentration is 6～12g/L, the potassium dihydrogen phosphate of 0.01～0.1g/L disodium phosphate soln and 0.1～0.3g/L.
Injectable cross-linked hyaluronic acid gel prepared by the present invention, can also contain single kind or multi-medicament or bioactive ingredients, as 0.1%～0.3% lignocaine, and 1%～10% collagen protein, 0.1%～0.5% Vc or Ve.
The preparation method of the present invention's injectable cross-linked hyaluronic acid gel, is take medical grade hyaluronic acid as raw material, and through crosslinking Treatment, emulsification and cross linked is granulated, purifying, and dry swelling, filling sterilizing is prepared from, and concrete steps are as follows:
Described basic solution A is the wherein a kind of of aqueous sodium hydroxide solution, potassium hydroxide aqueous solution or ammoniacal liquor.
Described Racemic glycidol ethers linking agent is BDDE, glycerin triglycidyl ether, resorcinol diglycidyl ether, glycidyl allyl ether, ethylene glycol diglycidylether, any one of 1,6-hexanediol diglycidyl ether or neopentylglycol diglycidyl ether.
This step is carried out preliminary crosslinking Treatment to hyaluronic acid derivatives, can improve the anti-degradation capability of hyaluronic acid derivatives, makes hyaluronic acid vivo degradation cycle after crosslinked compare natural hyaluronic acid and greatly extends, thereby have the potential quality that is applied to injection beauty treatment fields.
Described hyaluronic acid derivatives particle (pulverize and make again by the swelling rear gel forming of hyaluronic acid), its particle diameter mean size and particle size distribution range are 250 μ m ± 150 μ m, 500 μ m ± 150 μ m, 700 μ m ± 150 μ m or 850 μ m ± 150 μ m, hyaluronic acid concentration (being hyaluronic acid contents) is 15～25mg/mL, pushing force <15N(30G syringe needle), storage modulus is 100～600Pa, the residual 0.2ppm that is less than of linking agent.
Described vacuum and heating drying, its vacuum tightness is-0.08～0.1MPa, temperature is 40～60 ℃.
Described organic phase is any one of stable under normal temperature, liquid medicinal white oils, silicone oil or edible vegetable oil; In organic phase, can add one or both combinations of 0.1～0.5g/L cetyltrimethylammonium, 0.2～2g/L polyoxyethylene glycol, or add one or both combinations of the polyglycerol ester of alkyl-sulphate, the 0.2～2g/L of 0.1～0.5g/L, further strengthen emulsifying effect.
This step is by controlling water organic phase ratio, kinds of surfactants, concentration and stir speed (S.S.), can effectively control the emulsification degree of emulsion, water droplet size and degree of uniformity in the water-in-oil emulsion of formation be can effectively control, thereby median size and the particle size distribution range of hyaluronic acid derivatives effectively controlled.Compare physical pulverization and emulsification and prepare the technology of nano particle, hyaluronic acid derivatives prepared by the present invention can effectively be controlled particle diameter in micron level, and particle size distribution range is narrower, when guaranteeing the stable of validity of products and injecting, pushing force is stable, makes doctor more easily judge injection consumption and facilitate injection operation.
Described solution B refers to the mixed solution of any or both 1:1 of aqueous isopropanol, chloroform.
This step is by the processing that is separated of organic reagent and purified water, hyaluronic acid particles in emulsion can be separated with organic phase, obtain the uniform hyaluronic acid particles of particle diameter, also can partial cross-linked dose effectively be removed simultaneously, greatly reduce linking agent in product residual, guarantee that product has good security.
Step 4, dry swelling: the hyaluronic acid particles that step 3 is obtained regulates pH to neutral; By the vacuum and heating drying of the same terms in step 2, then add phosphoric acid sodium-chlor physiological buffer solution and carry out swelling treatment, repeat 2～5 times; After vacuum and heating drying, it is fully swelling that hyaluronic acid particles need adopt pressure method to carry out the last time.
In phosphoric acid physiological salt solution used, can add single medicine or bioactive ingredients or multi-medicament or bioactive ingredients of planting, as 0.1%～0.3% lignocaine, 1%～10% collagen protein, 0.1%～0.5% Vc or Ve etc., these compositions are directly dissolved in phosphoric acid sodium-chlor physiological buffer solution, complete interpolation in the time of hyaluronic acid water absorption and swelling.
The pressuring method of swelling treatment, thereby it is swelling to adopt piston-type container pressurized gas that hyaluronic acid particles and solution are carried out under 2～5 normal atmosphere, and temperature remains on normal temperature condition, and swelling time is 2～24 hours, final hyaluronic acid particles gel, its concentration is 15～25mg/mL.
This step is swelling by pressurization, when making hyaluronic acid particles absorb moisture, can mix mutually, thereby reaching uniform particles distributes, the uniform object of swelling rear gel, final assurance cross-linked hyaluronic acid gel keeps stable pushing force and suitable Young's modulus in the time of injection, avoid the rear phenomenon that spreads, is shifted in injecting body, can play good Moulding repair effect.In addition by dry swelling treatment repeatedly, can further remove linking agent in hyaluronic acid derivatives residual, make the residual level reaching lower than 0.2ppm of linking agent in hyaluronic acid derivatives that the present invention prepares.
It is filling that the hyaluronic acid particles gel that step 4 is prepared carries out pre-encapsulated injector, and under 95～110kPa and 115～125 ℃ of conditions, high pressure steam sterilization 10～30 minutes, prepares injectable cross-linked hyaluronic acid gel.
This step, by terminal sterilization, can guarantee that the injectable cross-linked hyaluronic acid gel preparing reaches aseptic level, effectively guarantees the security that finished product uses.
Injectable cross-linked hyaluronic acid gel prepared by the present invention, there is gel pattern homogeneous, without the feature of big or small uneven particle form, particle diameter mean size and particle size distribution range are 250 μ m ± 150 μ m, 500 μ m ± 150 μ m, 700 μ m ± 150 μ m or 850 μ m ± 150 μ m, hyaluronic acid concentration is 15～25mg/mL, pushing force <15N(30G syringe needle), storage modulus is 100～600Pa, the residual 0.2ppm that is less than of linking agent.This implant is applicable to be injected to subcutaneous skin corium deep layer to subcutaneous shallow-layer, to repair moderate to severe wrinkle or fold, has fabulous beauty treatment repairing effect.The present invention implants experiment by subcutaneous rat, and confirmation can at least maintain in subcutaneous rat the effect of 52 weeks.
Compared with prior art, the present invention has following beneficial effect:
(1) employing the present invention's technology of preparing, can be by the selection of each processing parameter in emulsification and cross linked granulation step, effectively control emulsification degree, thereby effectively control size and the distribution range (Fig. 1) of hyaluronic acid particles in hyaluronic acid derivatives, and distribute by the swelling hyaluronic acid derivatives uniform particles that can further make of the pressurization in dry swelling step, the complete homogeneous of gel form, maintains stable injection pushing force and repairs moulding effect.
(2) adopt technology of preparing of the present invention, can be by the vacuum-drying swelling treatment in the centrifugation in purification step and dry swelling step, remove that most organic reagents are residual and Racemic glycidol ethers linking agent is residual, guarantee that product has good biological safety (Fig. 2) and histocompatibility (Fig. 3).Rat intracutaneous is implanted experimental result and is shown, there is not any redness in implant site, fester, hemotoncus, histological stain section result shows: 4, the tissue inflammation reaction of 12,24,36 weeks is all lighter, the inflammatory cell quantity that around embedded material and inside infiltrates is all less, does not occur any carcinogenic reaction causing because linking agent is residual, has confirmed that hyaluronic acid derivatives particle prepared by the present invention has biocompatibility in good body.
(3) hyaluronic acid derivatives that prepared by the present invention, can effectively control size distribution in micron level by emulsification and cross linked granulation step, after being different from nano level gel particle and implanting, can be engulfed rapidly metabolism by scavenger cell etc., micron level hyaluronic acid derivatives particle prepared by the present invention, can effectively keep in vivo and can promote inoblast to synthesize collagen, thereby reaching the repairing effect of better wrinkle of skin and fold.Undertaken cultivating altogether with human fibroblasts by simulated in vivo environment in vitro, the ability of the synthetic collagen of inoblast is verified, result shows effective stimulus human fibroblasts to improve the synthetic ability of collagen.(Fig. 4)
(4) preparation method of the present invention has higher quality controllability, integrated artistic is reliable and stable, the every physical and chemical parameter of hyaluronic acid derivatives is stable, thereby the parameter such as degradation rate, kinetic viscosity that can control by parameters such as product crosslinking degree, median size and particle size distribution ranges in control product preparation process product, realizes the stable of quality product.In addition, many batches of hyaluronic acid derivatives concentration that prepare according to the present invention are also comparatively stable, and 90% above hyaluronic acid derivatives particle diameter can be controlled between the μ m of median size ± 150, in avoiding occurring batch or batch between the unsettled problem of quality, guaranteed that the technology of the present invention prepares the stable of hyaluronic acid derivatives result of use.(Fig. 1, Fig. 5)
(5) adopt the hyaluronic acids such as crosslinking Treatment of the present invention and emulsification and cross linked granulation to prepare gordian technique, can effectively improve the anti-degradation capability of hyaluronic acid derivatives, there is long-term good filling effect, evidence: its persistence is at least more than 12 months, effect is better than 6～12 months action times of like product, there is not the complication such as displacement, diffusion, thereby aspect the reinjected timed interval of needs, also be obviously longer than like product, can reduce the consumption expenditure of asking beautiful person, meet social demand widely.(Fig. 6)
Below in conjunction with the picture of embodiment and test, the present invention will be further described.
Accompanying drawing explanation
Fig. 1 is the detection curve of the hyaluronic acid derivatives size distribution prepared of embodiment.
Fig. 2 is the picture of the hyaluronic acid derivatives vitro cytotoxicity detected result prepared of embodiment.
Fig. 3 is that hyaluronic acid derivatives prepared by embodiment carries out the rat intracutaneous implantation picture of the result of histology afterwards.
Fig. 4 is that hyaluronic acid derivatives prepared by embodiment adopts RT-PCR method to detect the detection comparison diagram of type i collagen mrna expression amount.
Fig. 5 is hyaluronic acid concentration, median size, particle size distribution range, kinetic viscosity and the detected result of external degradation time of continuous three batches of hyaluronic acid derivatives of preparing of embodiment.
Fig. 6 is that hyaluronic acid derivatives prepared by embodiment carries out the rat intracutaneous implantation picture of outward appearance observations afterwards.
Embodiment
Below in conjunction with drawings and Examples, the present invention will be further described
The injectable cross-linked hyaluronic acid gel of following embodiment, by hyaluronic acid derivatives particle and the solution composition of phosphoric acid sodium-chlor physiological buffer; Hyaluronic acid derivatives particle, take hyaluronic acid dry powder as raw material, is made by the technique of crosslinking Treatment, emulsification and cross linked granulation, purifying.
Step 4, dry swelling: the hyaluronic acid particles that step 3 is obtained regulates pH to neutral; By the vacuum and heating drying of the same terms in step 2, then add phosphoric acid sodium-chlor physiological saline swelling treatment, repeat 2 times; The last time after vacuum and heating drying, adopt pressure method, hyaluronic acid particles is carried out fully swelling under 5 normal atmosphere of normal temperature, in phosphoric acid physiological salt solution used, add 0.1% lignocaine and 0.1% Vc, swelling time is 2 hours, and final hyaluronic acid particles concentration is 15mg/mL.
The hyaluronic acid derivatives that the present embodiment prepares, there is good cell compatibility, vitro cytotoxicity detected result as shown in Figure 2, completely not residual containing organic reagent in hyaluronic acid derivatives prepared by result demonstration the present embodiment, cytotoxicity is minimum, can Secure Application in facial wrinkles fill, the medical and beauty treatment fields such as the bridge of the nose increases, the modification of chin profile.
Step 4, dry swelling: the hyaluronic acid particles that step 3 is obtained regulates pH to neutral; By the vacuum and heating drying of the same terms in step 2, then add phosphoric acid sodium-chlor physiological saline swelling treatment, repeat 5 times; The last time after vacuum and heating drying, adopt pressure method, hyaluronic acid particles is carried out fully swelling under 5 normal atmosphere of normal temperature, in phosphoric acid physiological salt solution used, contain 0.3% lignocaine, 1% collagen protein, 0.5% Ve, swelling time is 24 hours, final hyaluronic acid particles concentration is 25mg/mL.
The hyaluronic acid derivatives that the present embodiment prepares, has good histocompatibility.Implant and detect through rat intracutaneous, detected result as shown in Figure 3, the inflammatory reaction of each time point hyaluronic acid implant site is all less, occur without acute and chronic untoward reaction, there is security in high body, can be applicable to the medical and beauty treatment fields such as facial wrinkles is filled, the bridge of the nose increases, the modification of chin profile.
Step 4, dry swelling: the hyaluronic acid particles that step 3 is obtained regulates pH to neutral; By the vacuum and heating drying of the same terms in step 2, then add phosphoric acid sodium-chlor physiological saline swelling treatment, repeat 3 times; After vacuum and heating drying, adopt pressure method the last time, under 3.5 normal atmosphere of normal temperature, carry out fully swelling, in phosphoric acid physiological salt solution used, contain 0.3% lignocaine, 0.2% Vc, swelling time is 12 hours, final hyaluronic acid particles concentration is 20mg/mL.
The hyaluronic acid derivatives that the present embodiment prepares, uniform particle diameter, gel pattern is even, median size and size distribution detected result are as shown in Figure 1, median size is 250 μ m, the microgroove that can be applicable to the positions such as facial canthus, the corners of the mouth, forehead is filled reparation, other 90% above size distribution is within the scope of 150～400 μ m, avoid occurring particle diameter heterogeneity and cause the unsettled problem of pushing force while injection, and guarantee that pushing force is less than 15N(30G syringe needle), easy to operate when injection, be easy to control injection level and hyaluronic acid injection volume.
Embodiment 4
Step 4, dry swelling: the hyaluronic acid particles that step 3 is obtained regulates pH to neutral; By the vacuum and heating drying of the same terms in step 2, then add phosphoric acid sodium-chlor physiological saline swelling treatment, repeat 4 times; The last time after vacuum and heating drying, adopt pressure method, under 4 normal atmosphere of normal temperature, hyaluronic acid particles is carried out fully swellingly, swelling time is 18 hours, and final hyaluronic acid particles concentration is 22mg/mL.
It is filling that the hyaluronic acid particles gel obtaining carries out pre-encapsulated injector, and under 102.5kPa and 121 ℃ of conditions, high pressure steam sterilization 18 minutes, prepares injectable cross-linked hyaluronic acid gel.
Continuous three batches of hyaluronic acid derivatives prepared by the present embodiment, every detected result of its hyaluronic acid concentration, median size, particle size distribution range, kinetic viscosity and external degradation time as shown in Figure 5, result shows detected result no significant difference between each batch, shows that the inventive method can effectively guarantee and control the stability of hyaluronic acid derivatives quality.The hyaluronic acid derivatives that adopts the present embodiment to prepare, be applied to rat intracutaneous and implant experiment, can effectively maintain 12 months above filling effects, as shown in Figure 6,4 weeks, 16 weeks, 32 weeks and 52 weeks each time points carry out outward appearance observation to injection site or injection material, the result matter acid gel that shows transparency has the moulding effect of good filling, and the cutaneous protuberance forming after injection still can obviously be seen from outward appearance after 12 months.
Step 4, dry swelling: the hyaluronic acid particles that step 3 is obtained regulates pH to neutral; By the vacuum and heating drying of the same terms in step 2, then add phosphoric acid sodium-chlor physiological saline swelling treatment, repeat 4 times; After vacuum and heating drying, adopt pressure method the last time, lower 2 normal atmosphere of normal temperature carry out fully swelling to hyaluronic acid particles, and swelling time is 20 hours, and final hyaluronic acid particles concentration is 18mg/mL.
The hyaluronic acid derivatives that the present embodiment prepares, there is the ability of the synthetic collagen of good promotion inoblast, itself and human fibroblasts are cultivated to the synthetic collagen level variation of rear detection the latter altogether, detected result as shown in Figure 5, shows that the hyaluronic acid derivatives that the present embodiment prepares can effectively promote fibroblastic propagation and collagen synthesis capability.The hyaluronic acid derivatives that the present embodiment is prepared carries out Unidasa external degradation, and degradation time approximately 120 hours is injected to rat intracutaneous and can maintains 14 months from the observable filling effect of outward appearance.
Embodiment 6
The present embodiment is substantially the same manner as Example 4, and difference is only: the mixing solutions described in step 2 and raisin seed oil, by 1:6 proportioning, have added the polyglycerol ester of 1.5g/L in raisin seed oil, further strengthen emulsifying effect.
The present embodiment is substantially the same manner as Example 4, and difference is only: the mixing solutions described in step 2 and soybean oil be by 1:8 proportioning, and in this soybean oil, adds the alkyl-sulphate of 0.1g/L, further strengthens emulsifying effect..
Embodiment 8
The present embodiment is substantially the same manner as Example 7, and difference is only: described in step 2, in soybean oil, add the cetyltrimethylammonium of 0.4g/L, further strengthen emulsifying effect.
For above-described embodiment, analyze in conjunction with experimental data, picture information etc. below:
Fig. 1 is the detection curve of the hyaluronic acid derivatives size distribution prepared of embodiment
Illustrate: hyaluronic acid derivatives prepared by the present invention can effectively be controlled product cut size distribution range (median size is 250 μ m, and 90% above size distribution is within the scope of 150～400 μ m)
Fig. 2 is the picture of the hyaluronic acid derivatives vitro cytotoxicity detected result prepared of embodiment
Four figure that this figure comprises are respectively Fig. 2-A, Fig. 2-B, Fig. 2-C, Fig. 2-D, Fig. 2-A is that median size is the body outer cell proliferation result picture of the hyaluronic acid derivatives of 250 μ m, Fig. 2-B is that median size is the body outer cell proliferation result picture of the hyaluronic acid derivatives of 800 μ m, the positive contrast body outer cell proliferation of Fig. 2-C result picture, the negative contrast body outer cell proliferation of Fig. 2-D result picture.
Result shows: apoptosis form appears in positive controls cell, and suspension dead cell quantity is more; Negative control group cell proliferation is good, and cellular form is normal; Median size prepared by the present invention be the hyaluronic acid derivatives group cell proliferation of 250 μ m and 800 μ m and cellular form all with negative control group there was no significant difference, and cellular form is better than negative control group.Illustrate that hyaluronic acid derivatives prepared by the present invention has fabulous cell compatibility, without any may cause Cytotoxic organic reagent or other objectionable constituent residual.
Fig. 3 is that hyaluronic acid derivatives prepared by embodiment carries out the rat intracutaneous implantation picture of the result of histology afterwards
Four figure that this figure comprises are respectively Fig. 3-A, Fig. 3-B, Fig. 3-C, Fig. 3-D, Fig. 3-A is 4 weeks injection site tissue section results after implanting, Fig. 3-B is 12 weeks injection site tissue section results after implanting, Fig. 3-C is 24 weeks injection site tissue section results after implanting, Fig. 3-D is 36 weeks injection site tissue section results after implanting, and the indicated region of hollow arrow is hyaluronic acid material.
Result shows: hyaluronic acid derivatives prepared by the present invention is within the different implantation phases, the tissue inflammation reaction that material causes is extremely light, and the inflammatory cell quantity that around material and inside infiltrates is little, foreign rejection, without long-term chronic untoward reactions such as granulomas, there is high histocompatibility.
Fig. 4, that hyaluronic acid derivatives prepared by embodiment is cultivated after 3,5,7 days altogether with human fibroblasts in vitro, get everyone inoblast in co-culture system, adopt RT-PCR method to detect the detection comparison diagram of type i collagen mrna expression amount after extracting mRNA wherein
Result shows: after 7 days, the type i collagen mrna expression amount of human fibroblasts under culture condition will have more 30% left and right than single culture human fibroblasts's expression amount altogether, hyaluronic acid derivatives prepared by the present invention is described, can plays the effect that promotes proliferation of fibroblasts.
Result shows: processing parameter of the present invention and method can effectively be controlled the stability of stating mass parameter on product.
Fig. 6 is that hyaluronic acid derivatives prepared by embodiment carries out the rat intracutaneous implantation picture of the result of outward appearance observation afterwards
Four figure that this figure comprises are respectively Fig. 6-A, Fig. 6-B, Fig. 6-C, Fig. 6-D, Fig. 6-A is 4 weeks injection site outward appearance observationss after implanting, Fig. 6-B is 32 weeks injection site outward appearance observationss after implanting for implanting latter 16 weeks injection site material shape observations Fig. 6-C, Fig. 6-D is 52 weeks injection site outward appearance observationss after implanting, and the indicated region of hollow arrow is hyaluronic acid material place.
Result shows: hyaluronic acid derivatives prepared by the present invention is within the different implantation phases, can rapid shaping after the implant that can guarantee preparation implants, there is long-term good filling effect, its persistence is at least more than 12 months, though in progressively degraded, be not shifted, the phenomenon such as diffusion.Within 12 months observation periods, there is not redness in injection site skin in addition, festers, infect, and the complication such as cutaneous necrosis, the biocompatibility of hyaluronic acid derivatives is good.
Claims (6)
1. an injectable cross-linked hyaluronic acid gel, is characterized in that: formed by hyaluronic acid derivatives particle and phosphoric acid sodium-chlor physiological buffer solution preparation; Hyaluronic acid derivatives particle is made take hyaluronic acid dry powder as raw material;
Described hyaluronic acid derivatives particle; its particle diameter mean size and particle size distribution range are 250 μ m ± 150 μ m, 500 μ m ± 150 μ m, 700 μ m ± 150 μ m or 850 μ m ± 150 μ m; hyaluronic acid concentration is 15～25mg/mL; pushing force <15N(30G syringe needle); storage modulus is 100～600Pa, the residual 0.2ppm that is less than of linking agent;
Described phosphoric acid sodium-chlor physiological buffer is the mixing solutions of following three kinds of salt: take water for injection as solvent, contain the sodium chloride solution that final concentration is 6～12g/L, the disodium phosphate soln of 0.01g/L～0.1g/L potassium dihydrogen phosphate and 0.1～0.3g/L, or, take water for injection as solvent, contain the sodium chloride solution that final concentration is 6～12g/L, the Rhodiaphos DKP hydrogen solution of 0.01g/L～0.1g/L disodium phosphate soln and 0.1～0.3g/L.
2. cross-linked hyaluronic acid gel according to claim 1, is characterized in that, contains following single kind or multi-medicament or bioactive ingredients: 0.1%～0.3% lignocaine, 1%～10% collagen protein, 0.1%～0.5% Vc or Ve.
3. cross-linked hyaluronic acid gel according to claim 1, it is characterized in that, in phosphoric acid physiological salt solution, be added with single medicine or bioactive ingredients or multi-medicament or bioactive ingredients of planting, as 0.1%～0.3% lignocaine, 1%～10% collagen protein, 0.1%～0.5% Vc or Ve.
4. a preparation method for injectable cross-linked hyaluronic acid gel, is characterized in that: take medical grade hyaluronic acid as raw material, through crosslinking Treatment, emulsification and cross linked is granulated, purifying, and dry swelling, filling sterilizing is prepared from, and concrete steps are as follows:
Step 1, crosslinking Treatment: the hyaluronic acid dry powder that is 300,000～3,000,000 Da by molecular weight is dissolved in basic solution A, the hyaluronic acid solution that formation concentration is 20～200g/L, pH is 9～13; The Racemic glycidol ethers linking agent that adds again 1～10% hyaluronic acid quality, carries out crosslinking Treatment 2-10 hour at 40 ℃-60 ℃, prepares hyaluronic acid derivatives;
Described basic solution A is the wherein a kind of of aqueous sodium hydroxide solution, potassium hydroxide aqueous solution or ammoniacal liquor;
Described Racemic glycidol ethers linking agent is BDDE; glycerin triglycidyl ether, resorcinol diglycidyl ether, glycidyl allyl ether; ethylene glycol diglycidylether, any one of 1,6-hexanediol diglycidyl ether or neopentylglycol diglycidyl ether;
Step 2, emulsification and cross linked are granulated: the hyaluronic acid derivatives that step 1 is obtained, and carry out vacuum and heating drying, then add basic solution A again to dissolve, then add and Racemic glycidol ethers linking agent identical in step 1, formation mixing solutions stirs; Again mixing solutions and organic phase are mixed according to the volume ratio of 1:5～1:10; at 40 ℃～60 ℃, continue stirring reaction 2～4 hours, stir speed (S.S.) is 10～100rpm, makes hyaluronic acid derivatives in organic phase, form emulsion; form hyaluronic acid particles, and be uniformly distributed in organic phase;
Described vacuum and heating drying, its vacuum tightness is-0.08～0.1MPa, temperature is 40～60 ℃;
Described organic phase is any one of stable under normal temperature, liquid medicinal white oils, silicone oil or edible vegetable oil;
Step 3, purifying: the emulsion that step 2 is obtained mixes with isopyknic solution B, concuss 5～10 minutes, then add and the purified water of 1～4 times of mixeding liquid volume ratio; finally centrifugal; get hyaluronic acid phase, then be placed in water for injection dialysis 12～48 hours, obtain hyaluronic acid particles;
Described solution B refers to the mixed solution of any or both 1:1 of aqueous isopropanol, chloroform;
Step 4, dry swelling: the hyaluronic acid particles that step 3 is obtained, regulates pH to neutral; By the vacuum and heating drying of the same terms in step 2, then add phosphoric acid sodium-chlor physiological buffer solution and carry out swelling treatment, repeat 2～5 times; After vacuum and heating drying, it is fully swelling that hyaluronic acid particles need adopt pressure method to carry out the last time;
Step 5, filling:
It is filling that the hyaluronic acid particles gel that step 4 is prepared carries out pre-encapsulated injector, and under 95～110kPa and 115～125 ℃ of conditions, high pressure steam sterilization 10～30 minutes, prepares injectable cross-linked hyaluronic acid gel.
5. the preparation method of cross-linked hyaluronic acid gel according to claim 4, it is characterized in that: in the organic phase in described step 2, can add one or both combinations of the cetyltrimethylammonium of 0.1～0.5g/L and the polyoxyethylene glycol of 0.2～2g/L; Or one or both combinations of the interpolation alkyl-sulphate of 0.1～0.5g/L and the polyglycerol ester of 0.2～2g/L, further strengthen emulsifying effect.
6. the preparation method of cross-linked hyaluronic acid gel according to claim 4, it is characterized in that: the pressuring method of the swelling treatment of described step 4, system adopts piston-type container pressurized gas, hyaluronic acid particles and solution are carried out under 2～5 normal atmosphere swelling, temperature remains on normal temperature condition, and swelling time is 2～24 hours.
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