CN104130424A - Preparation method for hyaluronic acid/bacterial cellulose composite hydrogel - Google Patents
Preparation method for hyaluronic acid/bacterial cellulose composite hydrogel Download PDFInfo
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- CN104130424A CN104130424A CN201410241845.XA CN201410241845A CN104130424A CN 104130424 A CN104130424 A CN 104130424A CN 201410241845 A CN201410241845 A CN 201410241845A CN 104130424 A CN104130424 A CN 104130424A
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Abstract
The invention relates to a preparation method for a hyaluronic acid/bacterial cellulose composite hydrogel. The method includes: taking out a bacteria fermented bacterial cellulose membrane, mashing the bacterial cellulose membrane and performing high-pressure homogenization to obtain a bacterial cellulose suspension solution, dissolving hyaluronic acid in the solution, adjusting the pH value to acidity, conducting continuous stirring to obtain an acidic gel, then washing the gel to neutral, and carrying out vacuum drying, thus obtaining the hyaluronic acid/bacterial cellulose composite dry hydrogel. The gel prepared by the method has a three-dimensional network structure, and the material has good light transmission. Fig. 2 shows that the rehydrated dry gel retains the good light transmission and high water content of the hyaluronic acid/bacterial cellulose composite hydrogel, and Fig. 3 shows that the shape of the composite hydrogel has typical gel characteristics, and the average pore size is dozens of micrometers. Figure 4 shows that under a shearing rate of 0.1-100Hz, the material behaves as an elastomer, thus proving that the material has good elastic properties in the range.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of preparation method of hyaluronic acid/bacteria cellulose composite aquogel.
Technical background
In recent years, due to the demand of energy scarcity and environment-friendly material, Mierocrystalline cellulose, as a kind of abundant reproducible natural resources, more and more receives fervent concern in exploitation nature renewable resources field.Bacteria cellulose (bacterial cellulose is called for short BC) is the synthetic Mierocrystalline cellulose of some bacterium.Although the Mierocrystalline cellulose that BC and plant produce has identical molecular structure unit, BC, unlike plant cellulose, without association products such as xylogen, pectin and hemicelluloses, has high chemical purity, high-crystallinity and high-polymerization degree.It also has superfine reticulated structure, high elastic coefficient, very strong water-holding power.BC is easy to regulation and control in biosynthetic process.Its excellent biocompatibility, adaptability and biodegradability makes it receive much concern at bio-medical material and field of tissue engineering technology.
But the concrete Application Areas function of single bacteria cellulose is more single, comes with some shortcomings, as better in the water retention property of bacteria cellulose, but it is difficult to return to the original form after drying out, i.e. difficult rehydration after dehydration.Although the light transmission of bacteria cellulose is good, if as corneal material, its light transmission does not meet the requirement of material.Therefore, for the bacteria cellulose of different purposes, can utilize other material to modify bacteria cellulose, to be met the bacteria cellulose composite material of demand.At present, bacteria cellulose is modified, made it there is good application prospect at aspects such as tissue engineering bracket, artificial blood vessel, artificial skin and medical accessories.
Hyaluronic acid is the biological mucopolysaccharide with good performance of keeping humidity and viscoelasticity, hyaluronic acid is very extensive at medicine and cosmetic field application at present, because it has higher transparency and water retention property, so select it and bacteria cellulose compound, to expect to obtain having the matrix material of good elasticity performance, rehydration performance and light transmission.
Patent documentation CN102212208A discloses " a kind of preparation method of bacteria cellulose/hyaluronic acid composite ", and this patent is flooded bacteria cellulose film after absorption in hyaluronic acid solution, freeze-drying.Then make bacteria cellulose and hyaluronic acid crosslinked under the effect of carbodiimide, form bacteria cellulose/hyaluronic acid composite, be mainly used in the purposes such as dressing and beauty mask.Although the tensile strength of bacteria cellulose film is very high, its modulus of compression is lower, and compressed rear moisture is easily extruded, and the moisture losing is difficult to recover; And the necessary cryopreservation of bacterial cellulose wet-coating, also needs to consider to prevent microbial contamination.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, provide that a kind of technique is simple, good biocompatibility, have good lock water function and be easy to the preparation method of hyaluronic acid/bacteria cellulose composite aquogel of storage, film prepared by the present invention can be used as biomedical material and tissue engineering bracket material.
The object of the invention is to be achieved through the following technical solutions:
A kind of preparation method of hyaluronic acid/bacteria cellulose composite aquogel, bacteria cellulose film after fermentation using bacteria is taken out, the bacterial cellulose suspension that bacteria cellulose film is smashed to pieces, obtained after high-pressure homogeneous, getting hyaluronic acid is dissolved in wherein, it is acid regulating pH value, lasting stirring obtains acid gel, then gel is cleaned to neutrality and be get final product.
And described bacterium is acetobacter xylinum (Gluconacetobacter xylinum), be preserved in Chinese common micro-organisms culture presevation administrative center, deposit number is CGMCC No.2955.
And the concentration of described bacterial cellulose suspension is 0.2-0.3%, the g/mL of unit.
And the concentration that hyaluronic acid is dissolved in bacterial cellulose suspension is 20-40g/L.
And mean fibre length is 10-100 micron in described bacterial cellulose suspension.
And described adjusting pH value acidity is pH=2～2.5.
And described gel cleans to neutral final vacuum dry, makes hyaluronic acid bacteria cellulose composite drying gel.
And described high-pressure homogeneous pressure is 10Bar.
And, described hyaluronan molecule amount 120-150 ten thousand.
Hyaluronic acid/bacteria cellulose composite aquogel is in the application of preparing in slow releasing pharmaceutical auxiliary agent.
Advantage of the present invention and positively effect are as follows:
1, the method for preparing hyaluronic acid/bacteria cellulose composite aquogel provided by the invention adopts physical principle that the two is organically combined, relate to the step such as homogeneous, mixing, technique is simple, production cost is lower, and the finished product are preserved after drying, and preservation condition relatively easily meets, normal temperature storage, dried product, because water ratio is lower, makes film itself be difficult for microbial contamination, extends product preservation period and valid period.
2, gel prepared by present method has tridimensional network, can find out by accompanying drawing the performance that it is superior, wherein, after desiccant gel rehydration, retain as seen from Figure 2 the good light permeability of hyaluronic acid/bacteria cellulose composite aquogel, high-moisture percentage, find out that by Fig. 3 its pattern is typical gel feature, mean pore size is tens microns, visible it retained the good light permeability of hyaluronic acid/bacteria cellulose composite aquogel, high-moisture percentage, as seen from Figure 4, under the shearing rate of 0.1～100Hz, material shows as elastomerics, prove that it has good elasticity performance in this scope.
3, present method only relates to the soda acid that regulates pH value to use in the process of preparing composite aquogel, do not relate to harmful composition, after preparation, film is washed till neutrality the most at last, make present method obtain the needs that film adapts to most occasions, can be used as biomedical material and tissue engineering bracket material, as aspect tissue engineering comea support, have a good application prospect.
4, the composite aquogel of preparing in the present invention is pH reversible gel, regulates the pH value of gel for neutral, and fully stirs, gel can be exchanged into colloidal sol, regulating its pH is 2～2.5 again, and colloidal sol changes again gel into, and pH sensitive aquagel can be used as medicine control and discharges.For example, the pH value of stomach is 1.5, is gel state, can avoid medicine to be damaged under one's belt, in the pH6.8 of enteron aisle environment, changes colloidal sol into, and drug release out.
Brief description of the drawings
Fig. 1 is hyaluronic acid/bacteria cellulose composite aquogel that the present invention is dry;
Fig. 2 is the hyaluronic acid/bacteria cellulose composite aquogel after the dry rehydration of the present invention;
Fig. 3 is the stereoscan photograph after hyaluronic acid/bacteria cellulose composite aquogel of the present invention freeze-drying;
Fig. 4 is the rheometer of the present invention storage modulus of measuring hyaluronic acid/bacteria cellulose composite aquogel (G ') and out-of-phase modulus (the G ") change curve with frequency.
Concrete embodiment
In order to understand the present invention, below in conjunction with embodiment, the invention will be further described: following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
A kind of preparation method of hyaluronic acid/bacteria cellulose composite aquogel, utilize acetobacter xylinum (Gluconacetobacter xylinum CGMCC No.2955) static cultivation bacteria cellulose film, by bacteria cellulose film through smashing to pieces, after repeatedly high-pressure homogeneous, obtain bacterial cellulose suspension, hyaluronic acid is dissolved in wherein, it is acid regulating pH value, constantly stir and obtain acid gel (SNJ), used again PBS buffered soln (pH=6.86, concentration is 0.05mol/L) clean to neutral, dry, make hyaluronic acid bacteria cellulose composite drying gel.
Concrete grammar can be with reference to following steps:
(1) spawn culture
By in acetobacter xylinum (Gluconacetobacter xylinum CGMCC No.1.812) the inclined-plane seed access HS substratum 1 of a ring or two ring activation, cultivate 24h in 30 DEG C, 160rpm.
(2) fermentation
With the inoculum size of 5-20% (v/v), access is equipped with in the container of HS substratum, after 4-6d, take out bacteria cellulose film, wash substratum and the impurity on film surface with distilled water, then film is soaked in to the NaOH solution of 0.1M, boil 30min, can further remove a large amount of thalline and residual substratum in BC film, finally, repeatedly rinse with the acetum of distilled water or 0.1M, approximate 7.0 until record the pH value of film.
(3) bacterial cellulose suspension
Pure BC film is smashed to pieces through tissue refiner, and then adopted high pressure homogenizer to carry out high-pressure homogeneous (pressure is 10Bar), can be uniformly dispersed for high-pressure homogeneous ten times and staple length is the BC suspension of tens microns later.
(4) hyaluronic acid/bacterial cellulose gel
The hyaluronic acid that takes certain mass is dissolved in BC suspension, after hyaluronic acid dissolves completely, add HCl that 1～2 concentration is 6M in the suspension mixing to pH=2～2.5, constantly stir, to forming the jelly with good viscoelasticity, be hyaluronic acid/bacterial cellulose gel.Cleaned the neutrality to pH with PBS buffered soln, for subsequent use after dry, obtain " desiccant gel ".
When use, " desiccant gel " soaked in distilled water, until its swollen no longer takes out from distilled water, cut suitable thickness, obtain light transmission extraordinary " rehydration gel ".
It should be noted that, prepared gel is pH reversible gel, and the pH value that regulates gel is for neutral, and fully stirs, and gel can be exchanged into colloidal sol, then to regulate its pH be 2～2.5, and colloidal sol changes again gel into.
Described HS medium component is: in 1L water, contain glucose 20g, yeast extract paste 5g, peptone 5g, Na
2hPO
42.7g, citric acid 1.15g, pH6.0.
The disodium phosphate soln that described PBS buffered soln is 49.5 milliliters of 0.2M mixes with the sodium dihydrogen phosphate of 50.5 milliliters of 0.2M, obtains 100 milliliters of PBS buffered soln.
The thickness of gel-film can be determined according to his purposes.If do not need stored dry directly to use, PBS cleans extremely neutral after-applied certain pressure makes gel reach the thickness needing.If need stored dry, thus can be in reconstitution process the period control its product thickness and water ratio, or after rehydration, cut corresponding thickness completely.Dry film is 20～60 times of left and right with the ratio of complete rehydration film thickness, and the time that rehydration needs is completely about 60～85 minutes.Hyaluronic acid/bacteria cellulose compound water congealing glued membrane prepared by the present invention rehydration 8 times repeatedly, does not affect performance and transparency.
In order to illustrate the preparation method of above-mentioned gel, the invention provides two and specifically prepare example, the correlation detection data of the gel that respective instance prepares are provided simultaneously.
Embodiment 1:
A preparation method for hyaluronic acid/bacteria cellulose composite aquogel, step is as follows:
(1) by acetobacter xylinum CGMCC No.2955 (be stored in Chinese common micro-organisms culture presevation administrative center) inclined-plane seed, access is equipped with in the Erlenmeyer flask of 100mL seed culture medium, after 30 DEG C of shaking culture 24h, obtains acetobacter xylinum seed.By acetobacter xylinum seed with 5% inoculum size, in access fermention medium (fermention medium is preparation like this: contain glucose 20g in 1L water, yeast extract paste 5g, peptone 5g, Na2HPO42.7g, citric acid 1.15g, pH6.0).After 6d, with the inoculum size of 5% (V/V), access is equipped with in the container of HS substratum, takes out bacteria cellulose film after 6d.
(2) wash substratum and the impurity on film surface with distilled water, then film is soaked in to the NaOH solution of 0.1M, boil 30min, further remove a large amount of thalline and the residual substratum in BC film, finally, repeatedly rinse with the acetum of distilled water or 0.1M, approximate 7.0 until record the pH value of film.
(3) pure BC film is smashed to pieces through tissue refiner, and then adopted high pressure homogenizer to carry out high-pressure homogeneous (pressure is 10Bar).Be uniformly dispersed for high-pressure homogeneous ten times and staple length is the BC suspension of tens microns later.
(4) the hyaluronic acid that takes certain mass is dissolved in 0.24% (g/mL) BC suspension, makes hyaluronic mass concentration reach 30g/L.After hyaluronic acid dissolves completely, add HCl that 1～2 concentration is 6M in the suspension mixing to pH=2～2.5, constantly stir, to forming the jelly with good viscoelasticity, be hyaluronic acid/bacterial cellulose gel.Cleaned the neutrality to pH with PBS buffered soln, the water ratio of this gel exceedes 90%.Through Haake torque rheometer (MarsII) test, this gel is elastomerics in certain range of shear rate.
(5) be dried rear for subsequent usely, obtain " desiccant gel ".
When use, " desiccant gel " soaked in distilled water, until its swollen no longer takes out from distilled water, transmittance that should " rehydration gel " is 90%, and in water, after complete swelling, water suction becomes 17.4 times of self dry weight.Thickness is the xerogel film of 40 μ m, and after complete swelling, thickness becomes 1.8cm.
Embodiment 2:
A preparation method for hyaluronic acid/bacteria cellulose composite aquogel, step is as follows:
(1), while fermentation, acetobacter xylinum seed is with 10% inoculum size, in access HS substratum.
(2), while preparing gel, make the mass concentration of hyaluronic acid in 0.24% (g/mL) BC suspension reach 40g/L.The water ratio of this gel exceedes 90%.Through Haake torque rheometer (MarsII) test, this gel is elastomerics in certain range of shear rate.
(3) it is dry rear for subsequent use, obtains " desiccant gel ".
When use, " desiccant gel " soaked in distilled water, until its swollen no longer takes out from distilled water, transmittance that should " rehydration gel " is 91%, and in water, swelling rear water suction becomes 29.9 times of self dry weight.Thickness is the xerogel film of 20 μ m, and after complete swelling, thickness becomes 1.2cm.
Claims (10)
1. the preparation method of hyaluronic acid/bacteria cellulose composite aquogel, it is characterized in that: the bacteria cellulose film after fermentation using bacteria is taken out, the bacterial cellulose suspension that bacteria cellulose film is smashed to pieces, obtained after high-pressure homogeneous, getting hyaluronic acid is dissolved in wherein, it is acid regulating pH value, lasting stirring obtains acid gel, then gel is cleaned to neutrality and be get final product.
2. the preparation method of hyaluronic acid/bacteria cellulose composite aquogel according to claim 1, it is characterized in that: described bacterium is acetobacter xylinum (Gluconacetobacterxylinum), be preserved in Chinese common micro-organisms culture presevation administrative center, deposit number is CGMCCNo.2955.
3. the preparation method of hyaluronic acid/bacteria cellulose composite aquogel according to claim 1, is characterized in that: the concentration of described bacterial cellulose suspension is 0.2-0.3%, the g/mL of unit.
4. the preparation method of hyaluronic acid/bacteria cellulose composite aquogel according to claim 1, is characterized in that: the concentration that hyaluronic acid is dissolved in bacterial cellulose suspension is 20-40g/L.
5. the preparation method of hyaluronic acid/bacteria cellulose composite aquogel according to claim 1, is characterized in that: in described bacterial cellulose suspension, mean fibre length is 10-100 micron.
6. the preparation method of hyaluronic acid/bacteria cellulose composite aquogel according to claim 1, is characterized in that: described adjusting pH value is acid is pH=2～2.5.
7. the preparation method of hyaluronic acid/bacteria cellulose composite aquogel according to claim 1, is characterized in that: described gel cleans to neutral final vacuum dry, makes hyaluronic acid bacteria cellulose composite drying gel.
8. the preparation method of hyaluronic acid/bacteria cellulose composite aquogel according to claim 1, is characterized in that: described high-pressure homogeneous pressure is 10Bar.
9. the preparation method of hyaluronic acid/bacteria cellulose composite aquogel according to claim 1, is characterized in that: described hyaluronan molecule amount 120-150 ten thousand.
10. hyaluronic acid/bacteria cellulose composite aquogel that prepared by the described method of one of claim 1-9 is in the application of preparing in slow releasing pharmaceutical auxiliary agent.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106084255A (en) * | 2016-06-15 | 2016-11-09 | 河南省科学院能源研究所有限公司 | A kind of preparation method of cellulose nano-fibrous/hyaluronic acid derivatives composite |
| CN106667791A (en) * | 2015-11-06 | 2017-05-17 | 乐天精密化学株式会社 | Method for producing dry hydrogel sheet, and the dry hydrogel sheet manufactured by the method |
| CN108744054A (en) * | 2018-06-15 | 2018-11-06 | 北京水元生生物科技有限公司 | A kind of injection-type beauty and shaping facial bulking agent compositions gel and preparation method thereof |
| CN114854806A (en) * | 2022-05-26 | 2022-08-05 | 华中科技大学 | Method for preparing tubular bacterial cellulose hydrogel by using super-hydrophobic mold |
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| CN102210888A (en) * | 2010-04-08 | 2011-10-12 | 上海交通大学医学院附属第九人民医院 | Agarose-enhanced three-dimensional nanometer porous bacterial cellulose stent and application thereof |
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| CN102210888A (en) * | 2010-04-08 | 2011-10-12 | 上海交通大学医学院附属第九人民医院 | Agarose-enhanced three-dimensional nanometer porous bacterial cellulose stent and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106667791A (en) * | 2015-11-06 | 2017-05-17 | 乐天精密化学株式会社 | Method for producing dry hydrogel sheet, and the dry hydrogel sheet manufactured by the method |
| CN106084255A (en) * | 2016-06-15 | 2016-11-09 | 河南省科学院能源研究所有限公司 | A kind of preparation method of cellulose nano-fibrous/hyaluronic acid derivatives composite |
| CN108744054A (en) * | 2018-06-15 | 2018-11-06 | 北京水元生生物科技有限公司 | A kind of injection-type beauty and shaping facial bulking agent compositions gel and preparation method thereof |
| CN114854806A (en) * | 2022-05-26 | 2022-08-05 | 华中科技大学 | Method for preparing tubular bacterial cellulose hydrogel by using super-hydrophobic mold |
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Inventor after: Jia Yuanyuan Inventor after: Wang Yan Inventor after: Wu Zhimin Inventor after: Chang Dongmei Inventor after: Huo Mingming Inventor after: Wang Man Inventor before: Jia Yuanyuan Inventor before: Huo Mingming Inventor before: Wang Man Inventor before: Wang Yan Inventor before: Fu Wei Inventor before: Jia Shiru |
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