A kind of resistance to enzymolysis cross-linking sodium hyaluronate gel and preparation method thereof and preparationTechnical field
The invention belongs to biomedicine field, be related to a kind of resistance to enzymolysis cross-linking sodium hyaluronate gel and preparation method thereof andPreparation.
Background technology
Hyaluronic acid (also known as sodium hyaluronate) is that one kind is connected by glucuronic acid and N-acetylglucosamine by glycosidic bondThe linear macromolecule polysaccharide connect.Under physiological status, hyaluronic acid usually exists with sodium-salt form.Sodium Hyaluronate is thinThe important component of extracellular matrix has the transhipment for adjusting immune, barrier protection, adjusting osmotic pressure, regulating and controlling macromolecular substancesEtc. a variety of effects.Sodium Hyaluronate can be used for the auxiliary treatment of ophthalmologic operation adjuvant drug and osteoarthritis, and be repaiied in woundMultiple, tissue generation etc. has important physiological function.
Conventional method for improving cross-linking hyaluronic acid sodium resistance to enzymolysis performance is by increasing crosslinker concentration, improving and hand overIt is realized the methods of starting transparent matter acid na concn during connection, but frequently can lead to cross-linking hyaluronic acid sodium transparency reduce, is biologicalPoor compatibility injects the shortcomings of poor performance, is unfavorable for the clinical practice of product.
Invention content
For the resistance to enzymolysis performance of cross-linking sodium hyaluronate gel of the prior art it is bad and for improving crosslinking it is solidifyingThe method of glue resistance to enzymolysis performance can lead to problems such as gel biological compatibility poor, the present invention is intended to provide a kind of novel resistance to enzymolysis is handed overJoin hyaluronic acid sodium gel and preparation method thereof and preparation.The present invention is made using two kinds of Sodium Hyaluronates with different molecular weightIt for raw material, is crosslinked in alkaline environment, after organic solvent deposit, washing, drying, swelling, it is saturating to obtain resistance to enzymolysis crosslinkingBright matter acid sodium gel.The cross-linking sodium hyaluronate gel has many advantages, such as that controllable concentration, low cross-linking agent residual, resistance to enzymolysis are strong.Entire preparation method is easy to operation, easily controllable.
Specifically, the present invention adopts the following technical scheme that：
A kind of preparation method of resistance to enzymolysis cross-linking sodium hyaluronate gel, includes the following steps：
1) Sodium Hyaluronate is dissolved using lye：
The mixture being made of high molecular weight sodium hyaluronate and low molecular weight sodium hyaluronate is dissolved in lye, is obtainedTo the alkaline solution of Sodium Hyaluronate；
2) crosslinking of Sodium Hyaluronate：
Under agitation, crosslinking agent is added in the alkaline solution of the Sodium Hyaluronate obtained into step 1), in 25~55 DEG C are reacted 1~6 hour, obtain the alkaline solution of cross-linking hyaluronic acid sodium；
3) precipitation of cross-linking hyaluronic acid sodium, washing and drying：
The pH value of the alkaline solution of the cross-linking hyaluronic acid sodium obtained in step 2) is adjusted to 6.5~7.5 using acid, thenAdd in precipitating solvent, until until being no longer precipitated and precipitating, then using precipitating solvent by the washing of precipitate 5 times of precipitation withOn, low-temperature vacuum drying obtains cross-linking hyaluronic acid sodium powder；
4) swelling of cross-linking hyaluronic acid sodium powder：
Aseptically, the cross-linking hyaluronic acid sodium powder obtained in step 3) is added to phosphate buffer (PBSBuffer solution) in, it is fully stirred evenly after swelling, obtains resistance to enzymolysis cross-linking sodium hyaluronate gel.
In a preferred technical solution, by weight percentage, mixture is by 10%~50% described in step 1)The high molecular weight sodium hyaluronate and 50%~90% the low molecular weight sodium hyaluronate composition.
In a preferred technical solution, high molecular weight sodium hyaluronate described in step 1) and the low molecular weight are saturatingBright matter acid sodium is made by bacterial fermentation process.
In a preferred technical solution, the molecular weight of high molecular weight sodium hyaluronate described in step 1) for 1.0 ×106~4.0 × 106Dalton, preferably 1.5 × 106~3.0 × 106Dalton.
In a preferred technical solution, the molecular weight of low molecular weight sodium hyaluronate described in step 1) for 1.0 ×105~1.0 × 106Dalton, preferably 2.0 × 105~5.0 × 105Dalton.
In a preferred technical solution, lye described in step 1) is water-soluble for sodium hydrate aqueous solution or potassium hydroxideLiquid.
In a preferred technical solution, the molar concentration of lye described in step 1) is 0.1~0.5 mol/L, excellentSelect 0.25 mol/L.
In a preferred technical solution, the amount ratio of mixture described in step 1) and the lye is 1~2 gram:10 milliliters, preferably 1 gram:10 milliliters.
In a preferred technical solution, crosslinking agent described in step 2) is 1,4-butanediol diglycidyl ether(BDDE) or divinylsulfone (DVS).
In a preferred technical solution, the weight ratio of crosslinking agent described in step 2) and mixture described in step 1)It is 1:5~100, preferably 1:10~50.
In a preferred technical solution, acid described in step 3) is hydrochloric acid.
In a preferred technical solution, precipitating solvent described in step 3) is alcohol or its aqueous solution, wherein describedAlcohol is methanol, ethyl alcohol or isopropanol.
In a preferred technical solution, the temperature of low-temperature vacuum drying described in step 3) is 0~4 DEG C.
In a preferred technical solution, under phosphate buffer described in step 4) includes by weight percentageRow ingredient：0.65%~0.9% sodium chloride (NaCl), 0.05%~0.2% disodium hydrogen phosphate (Na2HPO4), 0.005%~0.03% sodium dihydrogen phosphate (NaH2PO4) and surplus purified water, the pH value of the phosphate buffer is 6.5~7.5.
In a preferred technical solution, cross-linking hyaluronic acid sodium powder described in step 4) and the phosphate-bufferedThe amount ratio of liquid is 15~30 milligrams:1 milliliter, preferably 20 milligrams:1 milliliter.
A kind of resistance to enzymolysis cross-linking sodium hyaluronate gel is made by above-mentioned preparation method.
A kind of resistance to enzymolysis cross-linking sodium hyaluronate gel preparation is passed through by above-mentioned resistance to enzymolysis cross-linking sodium hyaluronate gelIt dispenses and sterilizes and be made.
In a preferred technical solution, the detailed process of the packing is as follows：Aseptically, using full-automaticThe cross-linking sodium hyaluronate gel is distributed into prefilled syringe, and vacuum is jumped a queue by vacuum bottle placer of jumping a queue.
In a preferred technical solution, for high pressure steam sterilization, temperature is 110~130 DEG C for the sterilizing, and the time is10~15 minutes.
Compared with prior art, using above-mentioned technical proposal the invention has the advantages that：
(1) present invention uses the high and low molecular weight hyaluronic acid sodium cross-linking hyaluronic acid sodium that crosslinked mode obtains mutuallyGel compared with existing cross-linked gel preparation process, reduces processing step, saves production cost；
(2) cross-linking sodium hyaluronate gel of the invention has stable three-dimensional net structure, with passing through two-phase gel systemThe product that Preparation Method obtains is compared, and heat resistanceheat resistant solution performance, resistance to enzymic degradation and is injected fluency and is improved significantly；
(3) method that the present invention is precipitated using organic solvent ethyl alcohol or isopropanol and combines low-temperature vacuum drying obtainsCross-linking hyaluronic acid sodium dry powder so that the gel for preparing any concentration is achieved, with taking in two-phase gel preparation methodDialysis technique is compared, and has the advantages that easy to operate, concentration is controllable.
Description of the drawings
Fig. 1 be embodiment 1 to 5 in cross-linking sodium hyaluronate gel before sterilization after elasticity modulus comparison diagram.
Fig. 2 is the resistance to enzymolysis performance comparison figure of the cross-linking sodium hyaluronate gel and commercial product in embodiment 1 to 5.
Fig. 3 for different rinsing times to crosslinking agent in cross-linking sodium hyaluronate gel remaining impact effect figure.
Specific embodiment
Further description is made to the present invention below with reference to the drawings and specific embodiments.Unless otherwise indicated, it is followingInstrument, reagent, material used in embodiment etc. can be obtained by routine business means.
Embodiment 1：The preparation of resistance to enzymolysis cross-linking sodium hyaluronate gel and gel preparation.
Weigh the high molecular weight sodium hyaluronate as made from bacterial fermentation process (0.2g, 3.0 × 106Da) and low molecular weight is saturatingBright matter acid sodium (1.8g, 4.0 × 105Da), it is dissolved in after mixing in 0.25M sodium hydrate aqueous solutions (20mL), obtains hyalomitomeThe alkaline solution of sour sodium.
Under mechanical agitation, BDDE (0.2g) is added in into the alkaline solution of above-mentioned Sodium Hyaluronate, in 40 DEG C of water3h is reacted in bath, obtains the alkaline solution of cross-linking hyaluronic acid sodium.
0.1M hydrochloric acid is added in into the alkaline solution of above-mentioned cross-linking hyaluronic acid sodium and is adjusted to pH=7.0, is slowly added to95% ethyl alcohol is precipitated completely to white precipitate.The precipitation of precipitation is rinsed 5 times with 95% ethyl alcohol, in 4 DEG C of vacuum drying, is handed overJoin Hyal powder 1.84g.
According to 20mg:Above-mentioned dried powder is added to PBS buffer solution and (included with weight percent by the amount ratio of 1mLThan the following ingredients of meter：0.65%~0.9% NaCl, 0.05%~0.2% Na2HPO4, 0.005%~0.03%NaH2PO4With the purified water of surplus) in, it is fully stirred evenly after swelling, obtains resistance to enzymolysis cross-linking sodium hyaluronate gel.
Aseptically, above-mentioned single-phase cross-linking sodium hyaluronate gel is dispensed using fully automatic vacuum bottle placer of jumping a queueEnter in prefilled syringe, and vacuum is jumped a queue, then in 121 DEG C of high pressure steam sterilization 15min, obtain resistance to enzymolysis cross-linked transparent matterSour sodium gel preparation.
Embodiment 2：The preparation of resistance to enzymolysis cross-linking sodium hyaluronate gel and gel preparation.
Weigh the high molecular weight sodium hyaluronate as made from bacterial fermentation process (1.0g, 3.0 × 106Da) and low molecular weight is saturatingBright matter acid sodium (1.0g, 4.0 × 105Da), it is dissolved in after mixing in 0.25M sodium hydrate aqueous solutions (20mL), obtains hyalomitomeThe alkaline solution of sour sodium.
Under mechanical agitation, BDDE (0.04g) is added in into the alkaline solution of above-mentioned Sodium Hyaluronate, in 40 DEG C of water3h is reacted in bath, obtains the alkaline solution of cross-linking hyaluronic acid sodium.
0.1M hydrochloric acid is added in into the alkaline solution of above-mentioned cross-linking hyaluronic acid sodium and is adjusted to pH=6.5, is slowly added to95% ethyl alcohol is precipitated completely to white precipitate.The precipitation of precipitation is rinsed 5 times with 95% ethyl alcohol, in 4 DEG C of vacuum drying, is handed overJoin Hyal powder 1.82g.
According to 20mg:Above-mentioned dried powder is added to PBS buffer solution and (included with weight percent by the amount ratio of 1mLThan the following ingredients of meter：0.65%~0.9% NaCl, 0.05%~0.2% Na2HPO4, 0.005%~0.03%NaH2PO4With the purified water of surplus) in, it is fully stirred evenly after swelling, obtains resistance to enzymolysis cross-linking sodium hyaluronate gel.
Aseptically, above-mentioned single-phase cross-linking sodium hyaluronate gel is dispensed using fully automatic vacuum bottle placer of jumping a queueEnter in prefilled syringe, and vacuum is jumped a queue, then in 121 DEG C of high pressure steam sterilization 15min, obtain resistance to enzymolysis cross-linked transparent matterSour sodium gel preparation.
Embodiment 3：The preparation of resistance to enzymolysis cross-linking sodium hyaluronate gel and gel preparation.
Weigh the high molecular weight sodium hyaluronate as made from bacterial fermentation process (0.5g, 3.0 × 106Da) and low molecular weight is saturatingBright matter acid sodium (1.5g, 4.0 × 105Da), it is dissolved in after mixing in 0.25M sodium hydrate aqueous solutions (20mL), obtains hyalomitomeThe alkaline solution of sour sodium.
Under mechanical agitation, BDDE (0.1g) is added in into the alkaline solution of above-mentioned Sodium Hyaluronate, in 50 DEG C of water2h is reacted in bath, obtains the alkaline solution of cross-linking hyaluronic acid sodium.
0.1M hydrochloric acid is added in into the alkaline solution of above-mentioned cross-linking hyaluronic acid sodium and is adjusted to pH=7.5, is slowly added toIsopropanol is precipitated completely to white precipitate.The precipitation of precipitation is rinsed 5 times with isopropanol, in 4 DEG C of vacuum drying, it is saturating to obtain crosslinkingBright matter acid sodium powder end 1.83g.
According to 20mg:Above-mentioned dried powder is added to PBS buffer solution and (included with weight percent by the amount ratio of 1mLThan the following ingredients of meter：0.65%~0.9% NaCl, 0.05%~0.2% Na2HPO4, 0.005%~0.03%NaH2PO4With the purified water of surplus) in, it is fully stirred evenly after swelling, obtains resistance to enzymolysis cross-linking sodium hyaluronate gel.
Aseptically, above-mentioned single-phase cross-linking sodium hyaluronate gel is dispensed using fully automatic vacuum bottle placer of jumping a queueEnter in prefilled syringe, and vacuum is jumped a queue, then in 121 DEG C of high pressure steam sterilization 15min, obtain resistance to enzymolysis cross-linked transparent matterSour sodium gel preparation.
Embodiment 4：The preparation of resistance to enzymolysis cross-linking sodium hyaluronate gel and gel preparation.
Weigh the high molecular weight sodium hyaluronate as made from bacterial fermentation process (1.0g, 1.5 × 106Da) and low molecular weight is saturatingBright matter acid sodium (1.0g, 4.0 × 105Da), it is dissolved in after mixing in 0.25M potassium hydroxide aqueous solutions (20mL), obtains hyalomitomeThe alkaline solution of sour sodium.
Under mechanical agitation, DVS (0.2g) is added in into the alkaline solution of above-mentioned Sodium Hyaluronate, in 50 DEG C of water-bathsMiddle reaction 2h, obtains the alkaline solution of cross-linking hyaluronic acid sodium.
0.1M hydrochloric acid is added in into the alkaline solution of above-mentioned cross-linking hyaluronic acid sodium and is adjusted to pH=7.0, is slowly added toIsopropanol is precipitated completely to white precipitate.The precipitation of precipitation is rinsed 5 times with isopropanol, in 4 DEG C of vacuum drying, it is saturating to obtain crosslinkingBright matter acid sodium powder end 1.86g.
According to 20mg:Above-mentioned dried powder is added to PBS buffer solution and (included with weight percent by the amount ratio of 1mLThan the following ingredients of meter：0.65%~0.9% NaCl, 0.05%~0.2% Na2HPO4, 0.005%~0.03%NaH2PO4With the purified water of surplus) in, it is fully stirred evenly after swelling, obtains resistance to enzymolysis cross-linking sodium hyaluronate gel.
Aseptically, above-mentioned single-phase cross-linking sodium hyaluronate gel is dispensed using fully automatic vacuum bottle placer of jumping a queueEnter in prefilled syringe, and vacuum is jumped a queue, then in 121 DEG C of high pressure steam sterilization 15min, obtain resistance to enzymolysis cross-linked transparent matterSour sodium gel preparation.
Embodiment 5：The preparation of resistance to enzymolysis cross-linking sodium hyaluronate gel.
Weigh the high molecular weight sodium hyaluronate as made from bacterial fermentation process (0.2g, 1.5 × 106Da) and low molecular weight is saturatingBright matter acid sodium (1.8g, 3.0 × 105Da), it is dissolved in after mixing in 0.25M potassium hydroxide aqueous solutions (20mL), obtains hyalomitomeThe alkaline solution of sour sodium.
Under mechanical agitation, DVS (0.1g) is added in into the alkaline solution of above-mentioned Sodium Hyaluronate, in 50 DEG C of water-bathsMiddle reaction 2h, obtains the alkaline solution of cross-linking hyaluronic acid sodium.
0.1M hydrochloric acid is added in into the alkaline solution of above-mentioned cross-linking hyaluronic acid sodium and is adjusted to pH=7.0, is slowly added to95% ethyl alcohol is precipitated completely to white precipitate.The precipitation of precipitation is rinsed 5 times with 95% ethyl alcohol, in 4 DEG C of vacuum drying, is handed overJoin Hyal powder 1.85g.
According to 20mg:Above-mentioned dried powder is added to PBS buffer solution and (included with weight percent by the amount ratio of 1mLThan the following ingredients of meter：0.65%~0.9% NaCl, 0.05%~0.2% Na2HPO4, 0.005%~0.03%NaH2PO4With the purified water of surplus) in, it is fully stirred evenly after swelling, obtains resistance to enzymolysis cross-linking sodium hyaluronate gel.
Aseptically, above-mentioned single-phase cross-linking sodium hyaluronate gel is dispensed using fully automatic vacuum bottle placer of jumping a queueEnter in prefilled syringe, and vacuum is jumped a queue, then in 121 DEG C of high pressure steam sterilization 15min, obtain resistance to enzymolysis cross-linked transparent matterSour sodium gel preparation.
Embodiment 6：The elastic modulus detection experiment of resistance to enzymolysis cross-linking sodium hyaluronate gel.
Resistance to enzymolysis cross-linking sodium hyaluronate gel obtained in Example 1 to 5 respectively, uses TA AR2000ex rheologiesInstrument opens air valve to 30bar, and set temperature is 25 DEG C, frequency 0.25Hz, the front and rear elasticity modulus of detection sterilizing, resultAs shown in Figure 1.
As shown in Figure 1, the elasticity modulus of sample only than being declined slightly before sterilizing, is substantially protected in embodiment 1 to 5 after sterilizingIt is fixed to keep steady, and illustrates the structure of sample and has higher heat resistanceheat resistant solution performance.
Embodiment 7：The resistance to enzymolysis performance measurement experiment of resistance to enzymolysis cross-linking sodium hyaluronate gel.
Cross-linking sodium hyaluronate gel 4mg obtained in Example 1 to 5 respectively is added in saturating according to the ratio of 10U/mgBright matter acid enzyme solutions, 37 DEG C of water-bath 48h, 100 DEG C are heated 30min, are then centrifuged for taking supernatant, are detected according to carbazole development processAbsorbance value at 530nm, and calculate resistance to enzymolysis rate.Meanwhile commercial product Restylane is taken as reference substance, same treatmentAfter detect, the results are shown in Figure 2.
As shown in Figure 2, the uronic acid release rate of sample is significantly lower than commercialized product in embodiment 1 to 5Restylane illustrates that sample has higher resistance to enzymolysis performance.
Embodiment 8：Crosslinking agent residues detecton experiment in resistance to enzymolysis cross-linking sodium hyaluronate gel.
The white precipitate being precipitated after 95% ethyl alcohol is added in using in embodiment 1 as detection sample, investigates different washing timesThe crosslinking agent in gel (BDDE) remains afterwards, and the results are shown in Figure 3.
From the figure 3, it may be seen that after 5 rinsings, the crosslinking agent in gel is remained already below 2ppm, meets gel productsRelated request.
Embodiment 9：The power of injecting of resistance to enzymolysis cross-linking sodium hyaluronate gel measures experiment.
Cross-linking sodium hyaluronate gel obtained uses universal testing machine as detection sample using in embodiment 1 to 5With the driving velocity of 30mm/min, the syringe needle detection product of 27 1/2G injects power, and the results are shown in Table 1.
1 gel products of table inject power measurement result
| Embodiment is numbered | Inject power (N) |
| 1 | 16 |
| 2 | 15 |
| 3 | 17 |
| 4 | 16 |
| 5 | 14 |
By the result in table 1 it is found that the power of injecting of sample is respectively less than 20N in embodiment 1 to 5, illustrate have during Clinical practiceThere is preferable feel, it is easily operated.