CN103848995A - Method for preparing hyaluronic acid nanoparticles - Google Patents
Method for preparing hyaluronic acid nanoparticles Download PDFInfo
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- CN103848995A CN103848995A CN201210516294.4A CN201210516294A CN103848995A CN 103848995 A CN103848995 A CN 103848995A CN 201210516294 A CN201210516294 A CN 201210516294A CN 103848995 A CN103848995 A CN 103848995A
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Abstract
The invention discloses a method for preparing a hyaluronic acid nanoparticle material, which is used for crosslinking hyaluronic acid nanoparticles based on the effect of base pairing. By using the inverse emulsion crosslinking technology, crosslinking of the hyaluronic acid nanoparticles is realized through the effect of base pairing, so that the hyaluronic acid nanoparticle with a stable structure is obtained. The method avoids toxic chemical crosslinking agents, can keep the bioactivity of the embedded drug, and can remarkably improve the use security of the nanoparticle material. The method and equipment are simple and feasible, are safe to operate, have advantages of low preparation temperature, fast curing speed and short processing period, and have application value in embedding and transfer of active drug, protein or gene.
Description
Technical field
The invention belongs to field of biomedical polymer materials, be specifically related to a kind of crosslinked method of preparing hyaluronic acid nanometer microballoon of base pairing of utilizing.
Background technology
Natural saccharan Nano microsphere has excellent biocompatibility and adjustable biological degradability, can be used for embedding and the release of medicine, albumen or gene, has been widely used in the research in the fields such as medicine controlled releasing, organizational project and regenerative medicine at present.Conventionally, medicine in vivo metabolism is very fast, and frequent drug administration by injection can bring great misery to patient.Select suitable nano microsphere system, can realize medicine slow release in vivo, reach desirable result for the treatment of.In organizational project and regenerative medicine field, saccharan Nano microsphere is usually used in embedding cell somatomedin and gene, improves cytoskeletal biological activity, increase support in vivo organize inducibility.The saccharan that is commonly used to prepare Nano microsphere comprises: hyaluronic acid, Lalgine, chondroitin sulfate, chitosan, starch and Mierocrystalline cellulose etc.
Saccharan Nano microsphere must add chemical cross-linking agent processing in preparation engineering, and its structure could be stablized.These chemical cross-linking agents mainly contain paraformaldehyde, glutaraldehyde, water-soluble carbodiimide and genipin etc., and its effect is that the active group in saccharan molecule (amino, carboxyl and hydroxyl) is reached to crosslinked object through condensation.Chemical cross-linking agent has cytotoxicity, although can make Nano microsphere more stable in physical structure, but can cause activated protein class medicine (as cell growth factor etc.) sex change of embedding and inactivation, therefore be not suitable for the transmission of activated protein, linking agent also easily remains in material simultaneously.Avoiding using poisonous chemical cross-linking agent, be expected to obtain the saccharan nanospheres of high biological activity, is the effective way that reduces material toxicity and improve drug conveying efficiency.But, in prior art, still there is not the crosslinked method of preparing saccharan Nano microsphere of nontoxic chemical reagent that adopts.
Summary of the invention
The object of this invention is to provide a kind of method of preparing hyaluronic acid nanometer microballoon, avoid having used poisonous chemical cross-linking agent, can ensure by the biological activity of embedding medicinal.
The technical solution that realizes the object of the invention is: by base pairing effect, adopt reversed-phase emulsion crosslinking technological to prepare hyaluronic acid nanometer microballoon, specifically comprise the following steps:
Under step 1, room temperature, prepare hyaluronic acid aqueous solution, add polyphosphate, stir; Drip thymus pyrimidine hydrochloric acid soln, stirring reaction;
Step 2, hyaluronic acid solution after reaction is completed are cooled to the unreacted thymus pyrimidine of Precipitation, then with ammoniacal liquor, supernatant solution is adjusted to neutrality, extract by ethyl acetate subsequently, finally by organic solvent evaporate to dryness, obtain thymus pyrimidine functionalization hyaluronic acid;
Under step 3, room temperature, prepare hyaluronic acid aqueous solution, add polyphosphate, stir; Drip VITAMIN B4 hydrochloric acid soln, stirring reaction;
Step 6, emulsion after stirring is completed are poured in Virahol, after hyaluronic acid nanometer microballoon is separated out, carry out high speed centrifugation, clean respectively subsequently last room temperature vacuum-drying with Virahol, normal hexane and acetone.
The present invention's reagent source used: hyaluronic acid, thymus pyrimidine, VITAMIN B4, be purchased from Sigma company; Class of department 80, ethyl acetate, analytical pure, Nanjing Chemistry Reagent Co., Ltd.; Virahol, normal hexane, acetone, analytical pure, Solution on Chemical Reagents in Shanghai company limited; Hydrochloric acid, ammoniacal liquor, analytical pure, Solution on Chemical Reagents in Shanghai factory.Sodium-chlor, Repone K, analytical pure, Shanghai reagent three factories; Sodium phosphate dibasic, potassium primary phosphate, analytical pure, Hangzhou chemical reagent company limited.DMEM substratum, Giboco company; Calf serum, Hangzhou folium ilicis chinensis biomaterial Graduate School of Engineering; Penicillin, Streptomycin sulphate, Huabei Pharmaceutic Co., Ltd.
The present invention's instrument used: scanning electronic microscope (JSM-6330F, JEOL); Nano-particle size analysis instrument (Malvern Zetasizer Nano ZS); Microplate reader (Biorad, Model 550); Gold spraying instrument (Cressington 108 Auto).
Compared with prior art, the invention is characterized in: 1) avoid having used poisonous chemical cross-linking agent, therefore in Nano microsphere, do not have toxic residue, ensured the security of Nano microsphere; 2) related base pairing is based on intermolecular hydrogen bond action, not with by embedding active medicine generation chemical reaction, can effectively protect the biological activity of medicine, be specially adapted to embedding and the transmission of activated protein class medicine; 3) simple, the Yi Hang of technology and equipment of the present invention, operational safety, has that preparation temperature is low, curing speed is fast, treatment cycle is short, environment is not produced to the advantages such as pollution, is applicable to commercially producing.
Further illustrate the present invention below by embodiment.
Brief description of the drawings
Fig. 1 is that hyaluronic acid nanometer microballoon is prepared schematic diagram.
Fig. 2 is the relation of base pair proportioning in balling ratio and hyaluronic acid (1/2,1/1 and 2/1).
Fig. 3 is the diameter of Nano microsphere and the relation of hyaluronic acid solution concentration.
Fig. 4 is size distribution and the scanning electron microscopy of hyaluronic acid nanometer microballoon.
Fig. 5 is the weightlessness of hyaluronic acid nanometer microballoon in phosphate buffer soln.
Fig. 6 is the cytotoxicity of hyaluronic acid nanometer microballoon.
Embodiment
A kind of method of preparing hyaluronic acid nanometer microballoon provided by the invention, its step comprises:
Under step 1, room temperature, prepare mass/volume per-cent and be 0.5 ~ 2% hyaluronic acid aqueous solution, add the polyphosphate of 1 ~ 4 times of quality, stir; The thymus pyrimidine hydrochloric acid soln that to drip mass/volume per-cent be 0.04 ~ 0.2%, its volume is hyaluronic acid aqueous solution 1/5 ~ 1/2, then 40 ~ 60
ostirring reaction 16 ~ 24 hours under C;
Step 2, hyaluronic acid solution after reaction is completed are cooled to the unreacted thymus pyrimidine of Precipitation, then with ammoniacal liquor, supernatant solution is adjusted to neutrality, extract by ethyl acetate subsequently, finally by organic solvent evaporate to dryness, obtain thymus pyrimidine functionalization hyaluronic acid;
Under step 3, room temperature, prepare mass/volume per-cent and be 0.5 ~ 2% hyaluronic acid aqueous solution, add the polyphosphate of 1 ~ 4 times of quality, stir; The VITAMIN B4 hydrochloric acid soln that to drip mass/volume per-cent be 0.04 ~ 0.2%, its volume is hyaluronic acid solution 1/5 ~ 1/2, subsequently 40 ~ 60
ostirring reaction 16 ~ 24 hours under C;
Step 6, emulsion after stirring is completed are poured in 10 times of Virahols with upper volume, after hyaluronic acid nanometer microballoon is separated out, carry out high speed centrifugation, clean respectively subsequently last room temperature vacuum-drying with Virahol, normal hexane and acetone.
The wherein preparation of phosphate buffer soln (PBS): take 8 grams, analytical pure sodium-chlor, 0.2 gram, Repone K, 2.9 grams of Sodium phosphate dibasics, 0.2 gram of potassium primary phosphate, be dissolved in 1000 ml distilled waters, regulating pH is 7.4.
The preparation of polyphosphate: by 0.07mmol diethyl ether, 0.035mmol trichloromethane, 0.035mmol Vanadium Pentoxide in FLAKES mix and blend, 50
oreflux 12 hours under C, underpressure distillation obtains polyphosphate product.
The pattern of Nano microsphere and particle diameter detect: by dried Nano microsphere metal spraying (Cressington 108 Auto), at the upper microscopic appearance of observing of scanning electronic microscope (JSM-6330F, JEOL).The diameter of Nano microsphere is by nano-particle size analysis instrument (Malvern Zetasizer Nano ZS) measuring and calculating.
The weightlessness of Nano microsphere detects: get certain drying nano microballoon (W that weighs
0), be placed in 37
oin phosphate buffer soln under C, hatch.When test, Nano microsphere is centrifugal from solution, to clean respectively with distilled water and acetone, (W weighs after vacuum-drying
t), each sample parallel testing 5 times.The formula that calculates Nano microsphere weightlessness is 100% × (W
0– W
t)/W
0, it is 1 – 100% × (W that Nano microsphere retains quality
0– W
t)/W
0.
The cytotoxicity of Nano microsphere detects: the Nano microsphere after weighing is sterilized by 75% alcohol immersion for 2 hours, with sterilized phosphate buffer soln repeatedly rinsing remove ethanol, be placed in 24 well culture plate (Nunc
tM, Denmark) in, then inoculating quantity is 3 × 10
4human body inoblast, and add 1 milliliter of nutrient solution (DMEM substratum+10% calf serum+100 units per ml penicillin/streptomycin), be put in 37
ounder C, hatch.When test, every hole adds the phosphate buffer soln of 20 microlitre 0.5wt%MTT, is placed in 37
ounder C, hatch after 4 hours and add 200 microlitre dimethyl sulfoxide (DMSO), after vibration evenly, adopt microplate reader (Biorad, Model 550) to measure the absorbancy of purple material in 570 nanometers.
The present invention adopts ANOVA analysis of variance, significant difference value
pbe made as≤0.05.
Below in conjunction with embodiment, the present invention is done to further detailed description, but these examples are not used for limiting the present invention.
embodiment 1:
(1) under room temperature, prepare the hyaluronic acid aqueous solution that 50 milliliters of mass/volume concentration are 0.5%, add 0.5 gram of polyphosphate, stir and form homogeneous solution; Drip 25 milliliters of thymus pyrimidine hydrochloric acid solns that mass/volume concentration is 0.04%, 50
ostirring reaction 16 hours under C;
(2) hyaluronic acid solution after reaction is completed is cooled to 0
oc, the unreacted thymus pyrimidine of Precipitation, is then adjusted to neutrality with ammoniacal liquor by supernatant solution, extracts subsequently by ethyl acetate, finally by organic solvent evaporate to dryness, obtains thymus pyrimidine functionalization hyaluronic acid;
(3) adopt step 1) and 2) in same method, preparation VITAMIN B4 functionalization hyaluronic acid;
(4) respectively above-mentioned two kinds of base functionalization hyaluronic acids are dissolved in to 50 milliliters of paraffin oils, be mixed with respectively mass/volume concentration and be 0.5% solution, add separately 0.2 milliliter of class of department 80, ultrasonic volumetric 1/1 mixing and emulsifying 5 minutes, then stirs volatilization with the rotating speed of 800rpm and spends the night;
(5) emulsion after stirring is completed is poured in the Virahol of 10 times of volumes, carries out high speed centrifugation after Nano microsphere is separated out, and cleans respectively subsequently with Virahol, normal hexane and acetone, last room temperature vacuum-drying 24 hours.
embodiment 2:
(1) under room temperature, prepare the hyaluronic acid aqueous solution that 50 milliliters of mass/volume concentration are 2%, add 4 grams of polyphosphates, stir and form homogeneous solution; Drip 25 milliliters of thymus pyrimidine hydrochloric acid solns that mass/volume concentration is 0.2%, 60
ostirring reaction 24 hours under C;
(2) hyaluronic acid solution after reaction is completed is cooled to 0
oc, the unreacted thymus pyrimidine of Precipitation, is then adjusted to neutrality with ammoniacal liquor by supernatant solution, extracts subsequently by ethyl acetate, finally by organic solvent evaporate to dryness, obtains thymus pyrimidine functionalization hyaluronic acid;
(3) adopt step 1) and 2) in same method, preparation VITAMIN B4 functionalization hyaluronic acid;
(4) respectively above-mentioned two kinds of base functionalization hyaluronic acids are dissolved in to 50 milliliters of paraffin oils, be mixed with respectively mass/volume concentration and be 2.0% solution, add separately 0.5 milliliter of class of department 80, under ultrasonic, press thymus pyrimidine/VITAMIN B4 volume ratio 1/2 mixing and emulsifying 10 minutes, then stir volatilization with the rotating speed of 1000rpm and spend the night;
(5) emulsion after stirring is completed is poured in the Virahol of 10 times of volumes, carries out high speed centrifugation after Nano microsphere is separated out, and cleans respectively subsequently with Virahol, normal hexane and acetone, last room temperature vacuum-drying 24 hours.
embodiment 3:
(1) under room temperature, prepare the hyaluronic acid aqueous solution that 50 milliliters of mass/volume concentration are 1%, add 0.5 gram of polyphosphate, stir and form homogeneous solution; Drip 20 milliliters of thymus pyrimidine hydrochloric acid solns that mass/volume concentration is 0.08%, 40
ostirring reaction 18 hours under C;
(2) hyaluronic acid solution after reaction is completed is cooled to 0
oc, the unreacted thymus pyrimidine of Precipitation, is then adjusted to neutrality with ammoniacal liquor by supernatant solution, extracts subsequently by ethyl acetate, finally by organic solvent evaporate to dryness, obtains thymus pyrimidine functionalization hyaluronic acid;
(3) adopt step 1) and 2) in same method, preparation VITAMIN B4 functionalization hyaluronic acid;
(4) respectively above-mentioned two kinds of base functionalization hyaluronic acids are dissolved in to 50 milliliters of paraffin oils, be mixed with respectively mass/volume concentration and be 1.2% solution, add separately 0.3 milliliter of class of department 80, under ultrasonic, press thymus pyrimidine/VITAMIN B4 volume ratio 2/1 mixing and emulsifying 6 minutes, then stir volatilization with the rotating speed of 1200rpm and spend the night;
(5) emulsion after stirring is completed is poured in the Virahol of 10 times of volumes, carries out high speed centrifugation after Nano microsphere is separated out, and cleans respectively subsequently with Virahol, normal hexane and acetone, last room temperature vacuum-drying 24 hours.
embodiment 4:
(1) under room temperature, prepare the hyaluronic acid aqueous solution that 50 milliliters of mass/volume concentration are 1.5%, add 1.2 grams of polyphosphates, stir and form homogeneous solution; Drip 20 milliliters of thymus pyrimidine hydrochloric acid solns that mass/volume concentration is 0.13%, 50
o stirring reaction 20 hours under C;
(2) hyaluronic acid solution after reaction is completed is cooled to 0
oc, the unreacted thymus pyrimidine of Precipitation, is then adjusted to neutrality with ammoniacal liquor by supernatant solution, extracts subsequently by ethyl acetate, finally by organic solvent evaporate to dryness, obtains thymus pyrimidine functionalization hyaluronic acid;
(3) adopt step 1) and 2) in same method, preparation VITAMIN B4 functionalization hyaluronic acid;
(4) respectively above-mentioned two kinds of base functionalization hyaluronic acids are dissolved in to 50 milliliters of paraffin oils, be mixed with respectively mass/volume concentration and be 1.8% solution, add separately 0.4 milliliter of class of department 80, ultrasonic volumetric mixing and emulsifying 8 minutes, then stirs volatilization with the rotating speed of 1300rpm and spends the night;
(5) emulsion after stirring is completed is poured in the Virahol of 10 times of volumes, carries out high speed centrifugation after Nano microsphere is separated out, and cleans respectively subsequently with Virahol, normal hexane and acetone, last room temperature vacuum-drying 24 hours.
embodiment 5:
(1) under room temperature, prepare the hyaluronic acid aqueous solution that 50 milliliters of mass/volume concentration are 0.5%, add 0.5 gram of polyphosphate, stir and form homogeneous solution; Drip 15 milliliters of thymus pyrimidine hydrochloric acid solns that mass/volume concentration is 0.04%, 40
ostirring reaction 16 hours under C;
(2) hyaluronic acid solution after reaction is completed is cooled to 0
oc, the unreacted thymus pyrimidine of Precipitation, is then adjusted to neutrality with ammoniacal liquor by supernatant solution, extracts subsequently by ethyl acetate, finally by organic solvent evaporate to dryness, obtains thymus pyrimidine functionalization hyaluronic acid;
(3) adopt step 1) and 2) in same method, preparation VITAMIN B4 functionalization hyaluronic acid;
(4) respectively above-mentioned two kinds of base functionalization hyaluronic acids are dissolved in to 50 milliliters of paraffin oils, be mixed with respectively mass/volume concentration and be 0.5% solution, add separately 0.2 milliliter of class of department 80, under ultrasonic, press thymus pyrimidine/VITAMIN B4 volume ratio 1/2 mixing and emulsifying 5 minutes, then stir volatilization with the rotating speed of 1500rpm and spend the night;
(5) emulsion after stirring is completed is poured in the Virahol of 10 times of volumes, carries out high speed centrifugation after Nano microsphere is separated out, and cleans respectively subsequently with Virahol, normal hexane and acetone, last room temperature vacuum-drying 24 hours.
embodiment 6:
(1) under room temperature, prepare the hyaluronic acid aqueous solution that 50 milliliters of mass/volume concentration are 0.5%, add 0.5 gram of polyphosphate, stir and form homogeneous solution; Drip 10 milliliters of thymus pyrimidine hydrochloric acid solns that mass/volume concentration is 0.04%, 60
ostirring reaction 16 hours under C;
(2) hyaluronic acid solution after reaction is completed is cooled to 0
oc, the unreacted thymus pyrimidine of Precipitation, is then adjusted to neutrality with ammoniacal liquor by supernatant solution, extracts subsequently by ethyl acetate, finally by organic solvent evaporate to dryness, obtains thymus pyrimidine functionalization hyaluronic acid;
(3) adopt step 1) and 2) in same method, preparation VITAMIN B4 functionalization hyaluronic acid;
(4) respectively above-mentioned two kinds of base functionalization hyaluronic acids are dissolved in to 50 milliliters of paraffin oils, be mixed with respectively mass/volume concentration and be 0.5% solution, add separately 0.2 milliliter of class of department 80, under ultrasonic, press thymus pyrimidine/VITAMIN B4 volume ratio 2/1 mixing and emulsifying 5 minutes, then stir volatilization with the rotating speed of 800rpm and spend the night;
(5) emulsion after stirring is completed is poured in the Virahol of 10 times of volumes, carries out high speed centrifugation after Nano microsphere is separated out, and cleans respectively subsequently with Virahol, normal hexane and acetone, last room temperature vacuum-drying 24 hours.
Hyaluronic acid nanometer microballoon prepared by the present invention is evenly distributed, and mean diameter can be controlled in 65 ~ 120 nanometers, and Nano microsphere presents complete spherical.The diameter of Nano microsphere becomes large along with the increase of the concentration of hyaluronic acid solution.Under certain condition, in the time that the volume ratio of thymus pyrimidine functionalization hyaluronic acid and VITAMIN B4 functionalization hyaluronic acid solution is 1:1, balling ratio is the highest, reaches 87%.
The prepared saccharan Nano microsphere of the present invention is to cultivate after 7 days weightlessness very little, and approximately 92% Nano microsphere quality still retains, and this Nano microsphere Stability Analysis of Structures is described, is applicable to drug disposition treatment.Cell and this Nano microsphere are cultivated altogether, and result shows that cytoactive is uninfluenced, illustrates that this Nano microsphere does not have toxicity, good biocompatibility.
Above embodiment has been contained the most representative experimental data.
Claims (5)
1. a preparation method for hyaluronic acid nanometer microballoon, is characterized in that, comprises the following steps:
Under step 1, room temperature, prepare hyaluronic acid aqueous solution, add polyphosphate, stir; Drip thymus pyrimidine hydrochloric acid soln, stirring reaction;
Step 2, hyaluronic acid solution after reaction is completed are cooled to the unreacted thymus pyrimidine of Precipitation, then with ammoniacal liquor, supernatant solution is adjusted to neutrality, extract by ethyl acetate subsequently, finally by organic solvent evaporate to dryness, obtain thymus pyrimidine functionalization hyaluronic acid;
Under step 3, room temperature, prepare hyaluronic acid aqueous solution, add polyphosphate, stir; Drip VITAMIN B4 hydrochloric acid soln, stirring reaction;
Step 4, hyaluronic acid solution after reaction is completed are cooling, and the unreacted VITAMIN B4 of Precipitation, is then adjusted to neutrality with ammoniacal liquor by supernatant solution, extracts subsequently by ethyl acetate, finally by organic solvent evaporate to dryness, obtains VITAMIN B4 functionalization hyaluronic acid;
Step 5, respectively above-mentioned two kinds of base functionalization hyaluronic acids are dissolved in to paraffin oil, add separately class of department 80, after ultrasonic lower mixing and emulsifying, stir volatilization and spends the night;
Step 6, emulsion after stirring is completed are poured in Virahol, after hyaluronic acid nanometer microballoon is separated out, carry out high speed centrifugation, clean respectively subsequently last room temperature vacuum-drying with Virahol, normal hexane and acetone.
2. the preparation method of hyaluronic acid nanometer microballoon according to claim 1, is characterized in that, in described step 1, the mass/volume per-cent of hyaluronic acid aqueous solution is 0.5 ~ 2%, and the mass/volume per-cent of thymus pyrimidine hydrochloric acid soln is 0.04 ~ 0.2%; The polyphosphate quality wherein adding is hyaluronic acid aqueous solution 1 ~ 4 times, the volume of thymus pyrimidine hydrochloric acid soln is hyaluronic acid aqueous solution 1/5 ~ 1/2; 40 ~ 60
ostirring reaction 16 ~ 24 hours under C.
3. the preparation method of hyaluronic acid nanometer microballoon according to claim 1, is characterized in that, in described step 3, the mass/volume per-cent of hyaluronic acid aqueous solution is 0.5 ~ 2%, and the mass/volume per-cent of VITAMIN B4 hydrochloric acid soln is 0.04 ~ 0.2%; The polyphosphate quality wherein adding is hyaluronic acid aqueous solution 1 ~ 4 times, the volume of VITAMIN B4 hydrochloric acid soln is hyaluronic acid aqueous solution 1/5 ~ 1/2; 40 ~ 60
ostirring reaction 16 ~ 24 hours under C.
4. the preparation method of hyaluronic acid nanometer microballoon according to claim 1, is characterized in that, the base functionalization hyaluronic acid of preparing in described step 5 is dissolved in paraffin oil taking mass/volume per-cent as 0.5 ~ 2.0%; The class of department 80 adding is 0.2 ~ 0.5 milliliter; Two kinds of base functionalization hyaluronic acid solutions mix according to volume ratio 1/2 ~ 2/1; Emulsification times is 5 ~ 10 minutes; Mixing speed is 800 ~ 1500rpm.
5. the preparation method of hyaluronic acid nanometer microballoon according to claim 1, is characterized in that, in described step 6, the volume of Virahol is the more than 10 times of emulsion after having stirred.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106140040A (en) * | 2015-04-17 | 2016-11-23 | 南京理工大学 | A kind of preparation method clicking on crosslinked poly sugar microsphere without copper |
| CN106279726A (en) * | 2016-08-09 | 2017-01-04 | 北京大清生物技术有限公司 | Cross-linking sodium hyaluronate gel and preparation method thereof |
| CN107540763A (en) * | 2016-06-24 | 2018-01-05 | 宁夏妙朗生物科技有限公司 | A kind of method that the long-acting hyaluronic acid derivatives of injection-type are prepared using Biological cross-linker |
| CN109939243A (en) * | 2019-01-16 | 2019-06-28 | 深圳广行科学研究有限公司 | The hyaluronic acid and poly copper clusters, preparation method and application that copper clusters, thymidine are modified |
| CN111686307A (en) * | 2019-03-13 | 2020-09-22 | 南京理工大学 | Preparation method of biological conductive nanofiber tissue engineering scaffold |
-
2012
- 2012-12-04 CN CN201210516294.4A patent/CN103848995B/en not_active Expired - Fee Related
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| UTTAM MANNA ET AL.: ""Layer-by-Layer Self-Assembly of Modified Hyaluronic Acid/", 《BIOMACROMOLECULES》, vol. 10, 20 August 2009 (2009-08-20) * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106140040A (en) * | 2015-04-17 | 2016-11-23 | 南京理工大学 | A kind of preparation method clicking on crosslinked poly sugar microsphere without copper |
| CN106140040B (en) * | 2015-04-17 | 2019-01-18 | 南京理工大学 | A kind of no copper clicks the preparation method of crosslinked poly sugar microballoon |
| CN107540763A (en) * | 2016-06-24 | 2018-01-05 | 宁夏妙朗生物科技有限公司 | A kind of method that the long-acting hyaluronic acid derivatives of injection-type are prepared using Biological cross-linker |
| CN107540763B (en) * | 2016-06-24 | 2020-08-11 | 宁夏妙朗生物科技有限公司 | Method for preparing injection type long-acting hyaluronic acid gel by using biological cross-linking agent |
| CN106279726A (en) * | 2016-08-09 | 2017-01-04 | 北京大清生物技术有限公司 | Cross-linking sodium hyaluronate gel and preparation method thereof |
| CN106279726B (en) * | 2016-08-09 | 2019-04-05 | 北京大清生物技术股份有限公司 | Cross-linking sodium hyaluronate gel and preparation method thereof |
| CN109939243A (en) * | 2019-01-16 | 2019-06-28 | 深圳广行科学研究有限公司 | The hyaluronic acid and poly copper clusters, preparation method and application that copper clusters, thymidine are modified |
| CN109939243B (en) * | 2019-01-16 | 2022-03-15 | 武汉广行科学研究有限公司 | Copper cluster, thymine modified hyaluronic acid and poly-copper cluster, and preparation method and application thereof |
| CN114558029A (en) * | 2019-01-16 | 2022-05-31 | 武汉广行科学研究有限公司 | Copper cluster, thymine modified hyaluronic acid and poly-copper cluster, and preparation method and application thereof |
| CN111686307A (en) * | 2019-03-13 | 2020-09-22 | 南京理工大学 | Preparation method of biological conductive nanofiber tissue engineering scaffold |
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