Disclosure of Invention
The invention aims to provide a gel material, and a preparation method and application thereof. The preparation method can control the crosslinking sites of endogenous polyamine and hyaluronic acid, influences various performance parameters of the prepared crosslinked hyaluronic acid gel by controlling the crosslinking reaction sites, controls the release speed of endogenous polyamine such as spermidine in the degradation process of the hyaluronic acid gel, and continuously plays the physiological activity role of endogenous polyamine such as spermidine.
The technical scheme provided by the invention is as follows:
in one aspect, the present invention provides a gel material derived primarily from crosslinking endogenous polyamines with hyaluronic acid, the endogenous polyamines including spermine (tetramino compounds) and/or spermidine (triamino compounds); crosslinking of the endogenous polyamine with the hyaluronic acid comprises two-site crosslinking, three-site crosslinking, or four-site crosslinking. In particular, the invention realizes that the endogenous polyamine and the hyaluronic acid are crosslinked at three sites or four sites for the first time to form a multi-site crosslinked active star-shaped network structure.
According to the invention, spermine or spermidine of endogenous polyamine is selected as a multi-site crosslinking agent to form a compact network structure, so that the thermal stability of the formed amide bond crosslinked hyaluronic acid gel is increased. The reason why endogenous diamine such as putrescine is not selected to be used in the invention is that diamine such as putrescine has higher toxicity, and the crosslinking site is the same as amino acid such as lysine and arginine, so that the possibility of realizing multi-site crosslinking is not provided.
In one embodiment, the present invention provides a gel material obtained by crosslinking an endogenous polyamine with hyaluronic acid, the endogenous polyamine comprising spermine and/or spermidine; crosslinking of the endogenous polyamine with the hyaluronic acid comprises two-site crosslinking, three-site crosslinking, or four-site crosslinking.
Further, the amino residue in the gel obtained by the two-site crosslinking is lower than 20%; preferably, the amino residue in the gel obtained by crosslinking in the two-site crosslinking is less than 15%; more preferably, the amino residue in the gel obtained by crosslinking in the double sites accounts for less than 10 percent.
Furthermore, the residual quantity of amino groups and imino groups in the gel obtained by the three-site crosslinking or the four-site crosslinking is lower than 20 percent.
Furthermore, the amino residue in the gel obtained by crosslinking in the three-site crosslinking or four-site crosslinking is less than 15%; preferably, the amino residue in the gel obtained by crosslinking in the three-site crosslinking or four-site crosslinking accounts for less than 10 percent.
Furthermore, the imino residue in the gel obtained by crosslinking in the three-site crosslinking or four-site crosslinking is lower than 15%; preferably, the imino residue in the gel obtained by crosslinking in the three-site crosslinking or four-site crosslinking is less than 10%.
Further, the crosslinking reaction efficiency of the double-site crosslinking, the three-site crosslinking or the four-site crosslinking in the crosslinking reaction of the endogenous polyamine and the hyaluronic acid is higher than 75%; preferably, the crosslinking reaction efficiency is higher than 80%; more preferably, the crosslinking reaction efficiency is higher than 85%.
Further, the elastic modulus loss rate (G' loss rate) of the gel obtained by the crosslinking is lower than 22%; preferably, the modulus of elasticity loss (G' loss) is less than 15%; more preferably, the modulus of elasticity loss ratio (G' loss ratio) is less than 10%.
In another aspect, the present invention provides a method of preparing a gel material, the method comprising the steps of:
Adjusting the pH=4.50-6.50 of a mixed solution of hyaluronic acid and endogenous polyamine, and adding an activating agent to enable the hyaluronic acid and the endogenous polyamine to generate double-site, three-site or four-site crosslinking reaction to obtain the gel material;
wherein the endogenous polyamine comprises spermine and/or spermidine.
The spermidine and spermine have amino groups and imino groups; wherein spermidine contains one imino group and two amino groups, and spermine contains two amino groups and two imino groups. The invention discovers that the reaction sites of spermine or spermidine and hyaluronic acid can be controlled by adjusting the pH value of the mixed solution of hyaluronic acid and endogenous polyamine after dissolution, and double-site crosslinked hydrogel and three-site or four-site crosslinked active star-shaped network gel can be obtained. During the reaction, the amino site reaction of spermine or spermidine (i.e., the two-site reaction) or the amino group and imino group may be co-reacted (i.e., the multi-site reaction) may be adjusted by the pH of the solution. The double-site crosslinking refers to that when hyaluronic acid is crosslinked with spermine or spermidine, amino groups in the spermine or spermidine are taken as main reaction sites, and particularly, the amino residue in gel obtained by the double-site crosslinking is less than 20%; the three-site or four-site crosslinking means that amino and imino in spermine or spermidine are taken together as reaction sites when hyaluronic acid is crosslinked with spermine or spermidine, and particularly, the residual quantity of amino and imino in gel obtained by the three-site crosslinking or four-site crosslinking is less than 20 percent.
The invention discovers that the reaction activity of imino can be improved by controlling the pH value of the solution to be between 5.00 and 5.49, and three-site or four-site reaction can be relatively stably realized; the pH value of the solution is controlled between 4.50 and 4.99 or between 5.50 and 6.50, which can provide imino reaction activity and relatively stably realize the joint participation of multiple sites in the reaction, thus preparing the active star-shaped network gel. Therefore, the invention obtains hyaluronic acid hydrogel taking imino groups as crosslinking sites for the first time, and discovers that hydrogels with different crosslinking sites can obtain different gel properties under the condition of the same crosslinking degree.
In the present invention, controlling the pH of the mixed solution between 5.00 and 5.49, including but not limited to 5.00, 5.10, 5.20, 5.30, 5.40 or 5.49, allows for a relatively stable three-or four-site reaction. Controlling the pH of the mixed solution between 4.50 and 4.99 or 5.50 and 6.50, including but not limited to 4.50, 4.60, 4.70, 4.80, 4.90, 4.99, 5.50, 5.60, 5.70, 5.80, 5.90, 6.00, 6.10, 6.20, 6.30, 6.40 or 6.50, allows a two-site cross-linking reaction to be achieved relatively stably. The experimental results are not ideal outside the pH defined by the present invention, and the pH range of the present invention is a pH condition suitable for the method of the present invention, which is found through a great number of experiments.
The method is a multi-site active reaction technology (Spermidine/Spermine Multisite Active Reaction Technology, short SMART) of endogenous polyamines (spermine and Spermidine). The method can control the crosslinking site of endogenous polyamine and hyaluronic acid, and further influence various performance parameters of crosslinked hyaluronic acid gel by controlling the crosslinking reaction site. In addition, the release rate of endogenous polyamines such as spermidine in the degradation process of hyaluronic acid gel can be controlled, and the physiological activity of endogenous polyamines such as spermidine can be continuously exerted.
In one embodiment, an activator is added during the two-site crosslinking reaction, three-site crosslinking reaction, or four-site crosslinking reaction;
preferably, the activator comprises one or more of water-soluble carbodiimides, phosphonium bromide salts of triphenylphosphine with bromide, carbonium salts, and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMTMM).
In one embodiment, the water-soluble carbodiimide activator includes 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide, 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide, 1, 3-bis [ bis (methoxymethyl) methyl ] carbodiimide, and the like, or salts thereof, and mixtures of one or more thereof.
In one embodiment, the phosphonium bromide salt of triphenylphosphine with bromide includes phosphonium salts of triphenylphosphine with carbon tetrabromide, phosphonium salts of triphenylphosphine with N-bromosuccinimide, and the like. The phosphonium bromide salt is obtained by known methods from triphenylphosphine and bromide in methylene chloride.
In one embodiment, the carbonium salt comprises one or a mixture of several of O- (7-azabenzotriazol-1-yl) -bis (dimethylamino) carbonium Hexafluorophosphate (HATU), O- (benzotriazol-1-yl) -bis (dimethylamino) carbonium Hexafluorophosphate (HBTU), O- (5-chlorobenzotriazol-1-yl) -bis (dimethylamino) carbonium Hexafluorophosphate (HCTU), O- (benzotriazol-1-yl) -bis (dimethylamino) carbonium tetrafluoroborate (TBTU), O- (N-succinimidyl) -bis (dimethylamino) carbonium tetrafluoroborate (TSTU), 2- (5-norbornene-2, 3-dicarboximido) -1, 3-tetramethyluronium tetrafluoroborate (TNTU).
Among the activators, when a water-soluble carbodiimide activator is used, it is necessary to use it simultaneously in combination with an auxiliary agent to improve the crosslinking reaction efficiency. Preferably, the adjuvant comprises any one or more of N-hydroxysuccinimide (NHS), sulphonated N-hydroxysuccinimide (sulpho-NHS), t-butanol, 1-hydroxybenzotriazole (HOBt); more preferably, the adjuvant is added in an amount of 10 to 30% of the mass of the carbodiimide, including but not limited to 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% and 30%. The water-soluble carbodiimide and the auxiliary agent are combined to ensure that the crosslinking reaction efficiency can reach about 70 percent.
Among three types of activators, namely phosphonium bromide salt, carbonium salt and 4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride, formed by triphenylphosphine and bromide, the highest crosslinking efficiency of the carbonium salt is 80-95%, wherein when HATU catalyst is adopted, the crosslinking efficiency is as high as 95%; the crosslinking efficiency of phosphonium bromide and DMTMM is inferior, and the crosslinking efficiency is in the range of 70-85%.
In a preferred embodiment, HATU of a carbonium salt is used as reaction activator. The molecular formula structure of HATU is shown below (1):
the mechanism by which HATU participates in the amide condensation reaction is as follows:
in the crosslinking reaction, when double-site crosslinking is carried out (the main reaction site of spermine or spermidine is amino), the addition amount of the activator is 200-280% of the amount of the endogenous polyamine substance; including but not limited to 210%, 220%, 230%, 240%, 250%, 260%, 270%, and 280%; when three-site and/or four-site crosslinking is performed (imino sites also participate in the reaction), the activator is added in an amount of 300 to 550% of the amount of the endogenous polyamine species; including but not limited to 320%, 350%, 370%, 390%, 400%, 420%, 450%, 470%, 500%, 520% and 550%, preferably, when the endogenous polyamine is spermine, the activator is added in an amount of 400 to 550% of the amount of the spermine substance, and when the endogenous polyamine is spermidine, the activator is added in an amount of 300 to 400% of the amount of the spermidine substance.
The amount of the activator added is related to the amino crosslinking site of the crosslinking agent spermine or spermidine, and when the activator is used, each molecule of the activator can activate one carboxyl group and carry out an amide coupling reaction with one amino (or imino).
In one embodiment, in the crosslinking reaction, when the endogenous polyamine is spermine, the spermine is added in an amount of 0.3-35% of the mass of the hyaluronic acid, including but not limited to 0.4%, 0.5%, 0.8%, 1%, 3%, 5%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% and 35%; when the endogenous polyamine is spermidine, the adding amount of the spermidine accounts for 0.5-40% of the mass of the hyaluronic acid; including but not limited to 0.5%, 0.8%, 1%, 3%, 5%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 34%, 38%, and 40%.
Further, the mass concentration of hyaluronic acid in the aqueous reaction system solution is 10-150 mg/mL, including, for example, but not limited to, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150mg/mL.
Further, in the finally obtained crosslinked hyaluronic acid hydrogel, the mass concentration of hyaluronic acid is 1-50mg/mL, including but not limited to 1, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50mg/mL.
In one embodiment, the hyaluronic acid has a molecular weight of 100KDa (kilodaltons) to 3000KDa; preferably, the hyaluronic acid is hyaluronic acid prepared by a microbial fermentation method.
In one embodiment, in the crosslinking reaction, when the activator comprises a phosphonium bromide salt of a carbonium salt and/or triphenylphosphine with bromide, the temperature of the crosslinking reaction is 10 to 60 ℃ and the time of the crosslinking reaction is 14 to 24 hours;
when the activator comprises water-soluble carbodiimide and/or 4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride, the temperature of the crosslinking reaction is 40-60 ℃, and the time of the crosslinking reaction is 14-24 h.
In the reaction process, the double-site crosslinked gel and the three-site or four-site crosslinked active star-shaped network gel can be controlled by different reaction temperatures and the proportion of the activator and spermine or spermidine. Because the imino is influenced by steric hindrance effect, the reactivity of the imino is lower than that of the primary amino, and therefore, when only the primary amino participates in the double-site reaction, the crosslinking reaction can be completed only by lower temperature and lower addition amount of an activating agent; however, when the imino group is required to participate in the reaction, it is necessary to raise the reaction temperature and to lengthen the reaction time in order to raise the crosslinking reaction efficiency of the imino group in order to increase the reactivity of the imino group.
The crosslinking reaction of spermidine with hyaluronic acid and Spermine with hyaluronic acid is shown below, wherein (3) is the crosslinking reaction of hyaluronic acid with Spermine (specmine) (4-site star network). (4) Is a cross-linking reaction (3-site star network) of hyaluronic acid with Spermidine (spearmine).
The invention adopts the SMART multi-site active reaction technology to prepare the hyaluronic acid gel, fully utilizes the different activities of amino and imino in spermine or spermidine molecules, and can respectively obtain the two-site cross-linked gel and the three-site or four-site cross-linked active star-shaped network gel by controlling one or more of reaction conditions such as pH, temperature, type of active agent and addition amount.
In one embodiment, the gel material is further subjected to an eluent elution, crushing, drying treatment;
preferably, the eluent is an organic solvent; more preferably, the organic solvent is a soluble alcohol or a soluble ketone; more preferably, the organic solvent is ethanol or acetone;
further, the volume ratio of gel to eluent in the reaction system during crushing is 1:1 to 5;
further, the particle size of the gel after pulverization is 10 to 500. Mu.m.
In one embodiment, the method of preparation further comprises re-dissolving, wet heat sterilizing the dried gel material;
Preferably, the reconstituted solution is a phosphate buffered solution; more preferably, the mass concentration of phosphate buffer salt in the phosphate buffer solution is 5-40 mg/mL;
further, the temperature of the wet heat sterilization is 120-130 ℃, and the time of the wet heat sterilization is preferably 15-45 min.
In a specific embodiment, the gel is eluted with an organic solvent and the gel particles are crushed, and the washing may be repeated a plurality of times to remove the residual activator. The drying can be vacuum drying, and the drying can remove the organic solvent. The dissolution efficiency of the residual activator in the multiple cleaning process can be ensured by crushing the gel into a lower particle size.
In a specific embodiment, the gel particles after vacuum drying are reconstituted with phosphate buffer solution, filled into pre-filled syringes and subjected to wet heat sterilization to obtain the final product gel. The pH value of the phosphate buffer solution ranges from 6.8 to 7.6. The concentration of the hyaluronic acid gel of the final product is 1-35 mg/mL.
In a specific embodiment, the method comprises the steps of:
(a) Directly dissolving hyaluronic acid and endogenous polyamine in water, and regulating the pH value of the solution;
(b) Adding an activating agent to complete the crosslinking reaction of spermidine and/or spermine and hyaluronic acid;
(c) Adding an organic eluting agent, crushing gel particles, washing for multiple times to remove residual activating agent, and drying in vacuum to remove organic solvent;
(d) And re-dissolving the gel particles after vacuum drying by using a phosphate buffer solution, filling the re-dissolved gel particles in a pre-filling and sealing syringe, and carrying out damp-heat sterilization to obtain the final product gel.
After the hyaluronic acid gel prepared by the method is subjected to wet heat sterilization, the loss rate of the elastic modulus is low, the minimum loss rate can be controlled within 10%, the rheological property of the gel before sterilization can be effectively maintained, and the thermal stability of the hyaluronic acid gel is improved to a great extent.
The invention designs polyamine such as spermidine as a hyaluronic acid cross-linking agent, and simultaneously takes the whole hyaluronic acid gel as a slow-release gel of polyamine such as spermidine, namely the gel can continuously and prototypically release polyamine substances such as spermidine in the natural degradation process in vivo. The three-site or four-site active star network gel can stably release spermidine monomers under the enzymatic initiation of hyaluronidase, and continuously plays a role in bioactivity of spermidine.
The invention also provides the gel material obtained by the preparation method.
In another aspect, the invention also provides the use of the gel material or the gel material prepared by the preparation method in preparing a tissue filling and repair material or a drug carrier. For example, in the preparation of products for pharmaceutical, medical cosmetic and cosmetic applications, such as soft tissue filling, soft tissue repair, or cosmetic injection for eliminating facial skin wrinkles, etc.
The beneficial effects are that:
(1) The gel material provided by the invention realizes multi-site crosslinking of endogenous polyamine and hyaluronic acid, and forms a more compact network structure;
(2) After the obtained hyaluronic acid gel is subjected to wet heat sterilization, the loss rate of elastic modulus is low (the minimum elastic modulus can be controlled within 10 percent), the rheological property of the gel before sterilization can be effectively maintained, and the thermal stability of the hyaluronic acid gel is greatly improved;
(3) The preparation method can realize the control of the number of crosslinking reaction sites, and further can influence the performance of the prepared crosslinked hyaluronic acid gel;
(4) The invention can control the release rate of endogenous polyamines such as spermidine in the degradation process of hyaluronic acid gel, and continuously exert the physiological activity of endogenous polyamines such as spermidine;
(5) The invention improves the usability of the gel in the fields of soft tissue filling, soft tissue repair, medical cosmetology and the like, and is suitable for popularization and application.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: preparation of double-site crosslinked hydrogel of HATU catalytic spermine and hyaluronic acid
3.0g of sodium hyaluronate (molecular weight: 900kDa, containing 7.4mmol of repeating structural units of hyaluronic acid) was weighed, then 98mL of purified water was added, and after complete dissolution, at this time the concentration of hyaluronic acid was 30mg/mL, 0.03g (mole number: 0.15 mmol) of spermine was added to the hyaluronic acid solution, at this time the spermine content was 1% of the mass of hyaluronic acid (mole number of repeating structural units of hyaluronic acid: 2%). The pH value of the hyaluronic acid solution is regulated to about 6.20 by using a hydrochloric acid solution with the concentration of 6mol/L, then the hyaluronic acid solution is added and stirred uniformly, 0.32mmol of HATU and 213 percent of spermine mol are added, the mixture is stirred uniformly continuously, and the mixture is placed in a constant temperature oven with the temperature of 25 ℃ for reaction for 24 hours in a sealing way. After the reaction is finished, 40mL of absolute ethyl alcohol is added, and an IKA T25 high shear disperser is adopted to pulverize gel particles, wherein the pulverizing speed is 10000 revolutions per minute, and the duration is 5 minutes. After the completion of the pulverization, 200mL of absolute ethyl alcohol was continuously added to completely dehydrate the gel. Separating the precipitate, continuously cleaning the precipitate with 200mL of absolute ethyl alcohol for 5 times, and vacuum drying the precipitate in a vacuum oven at 40 ℃ under the vacuum degree of-0.09 MPa for 24 hours. And after the gel is completely dried, taking 1.0g of xerogel, adding 50mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL to prepare hydrogel with the concentration of 20mg/mL of hyaluronic acid, filling the gel into a prefilled syringe after the gel is completely swelled, and carrying out damp-heat sterilization at the temperature of 121 ℃ for 15min to obtain the final product gel. The gel particle diameter D0.5 was 158 μm and D0.9 was 196. Mu.m.
Example 2: preparation of three-site crosslinked hydrogel of HATU catalytic spermidine and hyaluronic acid
3.0g of sodium hyaluronate (molecular weight: 900kDa, containing 7.4mmol of repeating structural units of hyaluronic acid) was weighed, then 98mL of purified water was added, and after complete dissolution, at this time the concentration of hyaluronic acid was 30mg/mL, 0.022g (molar number: 0.15 mmol) of spermidine was added to the hyaluronic acid solution, at this time the content of spermidine was 0.7% of the mass of hyaluronic acid (molar number of repeating structural units of hyaluronic acid: 2%). Adding 0.48mmol of HATU, namely 320 percent of spermidine mole number, uniformly stirring, adjusting the pH value of the hyaluronic acid solution to about 5.40 by using a 6mol/L hydrochloric acid solution, then adding and uniformly stirring, and sealing and placing into a 50 ℃ incubator for reaction for 14h. After the reaction is finished, 40mL of absolute ethyl alcohol is added, and an IKA T25 high shear disperser is adopted to pulverize gel particles, wherein the pulverizing speed is 10000 revolutions per minute, and the duration is 5 minutes. After the completion of the pulverization, 200mL of absolute ethyl alcohol was continuously added to completely dehydrate the gel. Separating the precipitate, continuously cleaning the precipitate with 200mL of absolute ethyl alcohol for 5 times, and vacuum drying the precipitate in a vacuum oven at 40 ℃ under the vacuum degree of-0.09 MPa for 24 hours. And after the gel is completely dried, taking 1.0g of xerogel, adding 50mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL to prepare hydrogel with the concentration of 20mg/mL of hyaluronic acid, filling the gel into a prefilled syringe after the gel is completely swelled, and carrying out damp-heat sterilization at the temperature of 121 ℃ for 15min to obtain the final product gel. The gel particle diameter D0.5 was 143. Mu.m, and D0.9 was 201. Mu.m.
Example 3: preparation of four-site crosslinked hydrogel of HATU catalytic spermine and hyaluronic acid
3.0g of sodium hyaluronate (molecular weight: 900kDa, containing 7.4mmol of repeating structural units of hyaluronic acid) was weighed, then 98mL of purified water was added, and after complete dissolution, at this time the concentration of hyaluronic acid was 30mg/mL, 0.03g (mole number: 0.15 mmol) of spermine was added to the hyaluronic acid solution, at this time the spermine content was 1% of the mass of hyaluronic acid (mole number of repeating structural units of hyaluronic acid: 2%). Adding 0.65mmol HATU and 433% of spermine mole number, stirring, adjusting pH of hyaluronic acid solution to about 5.30 with 6mol/L hydrochloric acid solution, stirring, sealing, and reacting in a 60 deg.C incubator for 14h. After the reaction, 40mL of acetone is added, and the gel particles are crushed by an IKAT25 high shear dispersing machine, wherein the crushing speed is 10000 revolutions per minute, and the crushing lasts for 5min. After the completion of the pulverization, 200mL of acetone was further added to completely dehydrate the gel. The precipitate was separated, washed successively with 200mL of acetone for 5 times, and then placed in a vacuum oven and dried under vacuum at 40℃for 24 hours at a vacuum of-0.09 MPa. And after the gel is completely dried, taking 1.0g of xerogel, adding 50mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL to prepare hydrogel with the concentration of 20mg/mL of hyaluronic acid, filling the gel into a prefilled syringe after the gel is completely swelled, and carrying out damp-heat sterilization at the temperature of 121 ℃ for 15min to obtain the final product gel. The gel particle diameter D0.5 was 139. Mu.m, and D0.9 was 186. Mu.m.
Example 4: preparation of double-site crosslinked hydrogel of spermidine and hyaluronic acid catalyzed by succinic imide triphenyl phosphonium bromide
Dissolving 0.2mol of triphenylphosphine and 0.2mol of N-bromosuccinimide in 1000mL of dichloromethane, stirring for 24h at 20-25 ℃, removing the dichloromethane by using a rotary evaporator after the reaction is finished to obtain bromosuccinimide triphenyl phosphonium salt, and sealing for later use.
3.0g of sodium hyaluronate (molecular weight: 900kDa, containing 7.4mmol of repeating structural units of hyaluronic acid) was weighed, then 98mL of purified water was added, and after complete dissolution, at this time the concentration of hyaluronic acid was 30mg/mL, 0.022g (molar number: 0.15 mmol) of spermidine was added to the hyaluronic acid solution, at this time the content of spermidine was 0.7% of the mass of hyaluronic acid (molar number of repeating structural units of hyaluronic acid: 2%). Adding 0.32mmol of the succinic imide triphenyl phosphonium bromide obtained in the previous step, namely 213 percent of spermidine mole number, uniformly stirring, adjusting the pH value of the hyaluronic acid solution to about 6.00 by using a 6mol/L hydrochloric acid solution, then adding and uniformly stirring, sealing and placing into a constant temperature oven at25 ℃ for reaction for 24 hours. After the reaction, 40mL of acetone is added, and the gel particles are crushed by an IKAT25 high shear dispersing machine, wherein the crushing speed is 10000 revolutions per minute, and the crushing lasts for 5min. After the completion of the pulverization, 200mL of acetone was further added to completely dehydrate the gel. The precipitate was separated, washed successively with 200mL of acetone for 5 times, and then placed in a vacuum oven and dried under vacuum at 40℃for 24 hours at a vacuum of-0.09 MPa. And after the gel is completely dried, taking 1.0g of xerogel, adding 50mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL to prepare hydrogel with the concentration of 20mg/mL of hyaluronic acid, filling the gel into a prefilled syringe after the gel is completely swelled, and carrying out damp-heat sterilization at the temperature of 121 ℃ for 15min to obtain the final product gel. The gel particle diameter D0.5 was 141 μm and D0.9 was 191. Mu.m.
Example 5: preparation of three-site crosslinked hydrogel of spermidine and hyaluronic acid catalyzed by succinic imide triphenyl phosphonium bromide
3.0g of sodium hyaluronate (molecular weight: 900kDa, containing 7.4mmol of repeating structural units of hyaluronic acid) was weighed, then 98mL of purified water was added, and after complete dissolution, at this time the concentration of hyaluronic acid was 30mg/mL, 0.022g (molar number: 0.15 mmol) of spermidine was added to the hyaluronic acid solution, at this time the content of spermidine was 0.7% of the mass of hyaluronic acid (molar number of repeating structural units of hyaluronic acid: 2%). 0.48mmol of the triphenylphosphonium bromide which is prepared according to the method in the example 4, namely 320 percent of spermidine mole number, is added, the mixture is stirred uniformly, the pH value of the hyaluronic acid solution is regulated to about 5.20 by using a hydrochloric acid solution with the concentration of 6mol/L, then the mixture is added, stirred uniformly, and the mixture is sealed and placed into a constant temperature box with the temperature of 40 ℃ for reaction for 24 hours. After the reaction, 40mL of acetone is added, and the gel particles are crushed by an IKAT25 high shear dispersing machine, wherein the crushing speed is 10000 revolutions per minute, and the crushing lasts for 5min. After the completion of the pulverization, 200mL of acetone was further added to completely dehydrate the gel. The precipitate was separated, washed successively with 200mL of acetone for 5 times, and then placed in a vacuum oven and dried under vacuum at 40℃for 24 hours at a vacuum of-0.09 MPa. And after the gel is completely dried, taking 1.0g of xerogel, adding 50mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL to prepare hydrogel with the concentration of 20mg/mL of hyaluronic acid, filling the gel into a prefilled syringe after the gel is completely swelled, and carrying out damp-heat sterilization at the temperature of 121 ℃ for 15min to obtain the final product gel. The gel particle diameter D0.5 was 133. Mu.m, and D0.9 was 178. Mu.m.
Example 6: preparation of DMTMM catalyzed three-site crosslinked hydrogel of spermidine and hyaluronic acid
3.0g of sodium hyaluronate (molecular weight: 900kDa, containing 7.4mmol of repeating structural units of hyaluronic acid) was weighed, then 98mL of purified water was added, and after complete dissolution, at this time the concentration of hyaluronic acid was 30mg/mL, 0.022g (molar number: 0.15 mmol) of spermidine was added to the hyaluronic acid solution, at this time the content of spermidine was 0.7% of the mass of hyaluronic acid (molar number of repeating structural units of hyaluronic acid: 2%). Adding DMTMM 0.48mmol, namely 320 percent of spermidine mole number, uniformly stirring, adjusting the pH value of the hyaluronic acid solution to about 5.10 by using a hydrochloric acid solution with the concentration of 6mol/L, then adding the hyaluronic acid solution, uniformly stirring, sealing and placing the hyaluronic acid solution into a constant temperature box with the temperature of 40 ℃ for reaction for 24 hours. After the reaction is finished, 40mL of absolute ethyl alcohol is added, and an IKA T25 high shear disperser is adopted to pulverize gel particles, wherein the pulverizing speed is 10000 revolutions per minute, and the duration is 5 minutes. After the completion of the pulverization, 200mL of absolute ethyl alcohol was continuously added to completely dehydrate the gel. Separating the precipitate, continuously cleaning the precipitate with 200mL of absolute ethyl alcohol for 5 times, and vacuum drying the precipitate in a vacuum oven at 40 ℃ under the vacuum degree of-0.09 MPa for 24 hours. And after the gel is completely dried, taking 1.0g of xerogel, adding 50mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL to prepare hydrogel with the concentration of 20mg/mL of hyaluronic acid, filling the gel into a prefilled syringe after the gel is completely swelled, and carrying out damp-heat sterilization at the temperature of 121 ℃ for 15min to obtain the final product gel. The gel particle diameter D0.5 was 144. Mu.m, and D0.9 was 206. Mu.m.
Example 7: preparation of four-site crosslinked hydrogel of EDC catalytic spermine and hyaluronic acid
3.0g of sodium hyaluronate (molecular weight: 900kDa, containing 7.4mmol of repeating structural units of hyaluronic acid) was weighed, then 98mL of purified water was added, and after complete dissolution, at this time the concentration of hyaluronic acid was 30mg/mL, 0.03g (mole number: 0.15 mmol) of spermine was added to the hyaluronic acid solution, at this time the spermine content was 1% of the mass of hyaluronic acid (mole number of repeating structural units of hyaluronic acid: 2%). Adding 0.65mmol of EDC, 433% of spermine mole number, adding 20% of NHS of EDC mass, stirring uniformly, regulating pH value of hyaluronic acid solution to about 5.45 with 6mol/L hydrochloric acid solution, stirring uniformly continuously, sealing and placing into a 60 ℃ constant temperature oven for reaction for 14h. After the reaction is finished, 40mL of absolute ethyl alcohol is added, and an IKA T25 high shear disperser is adopted to pulverize gel particles, wherein the pulverizing speed is 10000 revolutions per minute, and the duration is 5 minutes. After the completion of the pulverization, 200mL of absolute ethyl alcohol was continuously added to completely dehydrate the gel. Separating the precipitate, continuously cleaning the precipitate with 200mL of absolute ethyl alcohol for 5 times, and vacuum drying the precipitate in a vacuum oven at 40 ℃ under the vacuum degree of-0.09 MPa for 24 hours. And after the gel is completely dried, taking 1.0g of xerogel, adding 50mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL to prepare hydrogel with the concentration of 20mg/mL of hyaluronic acid, filling the gel into a prefilled syringe after the gel is completely swelled, and carrying out damp-heat sterilization at the temperature of 121 ℃ for 15min to obtain the final product gel. The gel particle diameter D0.5 was 125. Mu.m, and D0.9 was 193. Mu.m.
Example 8: preparation of multi-site crosslinked hydrogel by combining HATU catalytic spermidine and spermine and participating in hyaluronic acid
3.0g of sodium hyaluronate (molecular weight: 900kDa, containing 7.4mmol of repeating structural units of hyaluronic acid) was weighed, then 98mL of purified water was added, and after complete dissolution, at this time, the concentration of hyaluronic acid was 30mg/mL, 0.022g (mole number: 0.15 mmol) of spermidine and 0.03g (mole number: 0.15 mmol) of spermine were added to the hyaluronic acid solution, respectively, at this time, the content of spermidine or spermine each accounted for 2% of the mole number of repeating structural units of hyaluronic acid. Adding 0.48mmol of HATU, namely 320 percent of spermidine mole number, adding 0.65mmol of HATU, namely 433 percent of spermine mole number, uniformly stirring, adjusting the pH value of the hyaluronic acid solution to about 5.00 by using a 6mol/L hydrochloric acid solution, then adding and uniformly stirring, sealing and placing into a 50 ℃ incubator for reaction for 14h. After the reaction is finished, 40mL of absolute ethyl alcohol is added, and an IKA T25 high shear disperser is adopted to pulverize gel particles, wherein the pulverizing speed is 10000 revolutions per minute, and the duration is 5 minutes. After the completion of the pulverization, 200mL of absolute ethyl alcohol was continuously added to completely dehydrate the gel. Separating the precipitate, continuously cleaning the precipitate with 200mL of absolute ethyl alcohol for 5 times, and vacuum drying the precipitate in a vacuum oven at 40 ℃ under the vacuum degree of-0.09 MPa for 24 hours. And after the gel is completely dried, taking 1.0g of xerogel, adding 50mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL to prepare hydrogel with the concentration of 20mg/mL of hyaluronic acid, filling the gel into a prefilled syringe after the gel is completely swelled, and carrying out damp-heat sterilization at the temperature of 121 ℃ for 15min to obtain the final product gel. The gel particle diameter D0.5 was 134. Mu.m, and D0.9 was 236. Mu.m.
Example 9: EDC catalyzes three-site crosslinking of spermidine and hyaluronic acid
6g of sodium hyaluronate (molecular weight 120 kDa) was weighed, then 35mL of purified water was added thereto, and after complete dissolution, at this time the concentration of hyaluronic acid was 150mg/mL, 2.4g (molar number 16.6 mmol) of spermidine was added to the hyaluronic acid solution, at this time the content of spermidine was 40% of the mass of hyaluronic acid. Under the precondition of three-site crosslinking, the pH value of the hyaluronic acid solution is regulated to about 5.2 by using a 6mol/L hydrochloric acid solution, then the mixture is added and stirred uniformly, 66.4mmol EDC (that is 400% of the mole number of spermidine) is added, 2.4g of Sulfo-NHS (20% of the mass of EDC) is added at the same time, the mixture is stirred uniformly continuously, and the mixture is sealed and placed into a blast drying box at 60 ℃ for reaction for 24 hours. After the reaction is finished, 40mL of absolute ethyl alcohol is added, and an IKA T25 high shear disperser is adopted to pulverize gel particles, wherein the pulverizing speed is 10000 revolutions per minute, and the duration is 5 minutes. After the completion of the pulverization, 200mL of absolute ethyl alcohol was continuously added to completely dehydrate the gel. Separating the precipitate, continuously cleaning the precipitate with 200mL of absolute ethyl alcohol for 5 times, and vacuum drying the precipitate in a vacuum oven at 40 ℃ under the vacuum degree of-0.09 MPa for 24 hours. After complete drying, 1.0g of xerogel is taken, 20mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL is added, after the gel is completely swelled, the gel is filled into a prefilled syringe, and moist heat sterilization is carried out at 121 ℃ for 15min, thus obtaining the final product gel. The gel particle diameter D0.5 was 145. Mu.m, and D0.9 was 213. Mu.m.
Example 10: preparation of four-site crosslinked hydrogel of HATU catalytic spermine and hyaluronic acid
0.5g of sodium hyaluronate (molecular weight 2800 kDa) was weighed, then 38.5mL of purified water was added, and after complete dissolution, the concentration of hyaluronic acid was 13.3mg/mL, and 1.5mg (mole number: 7.4. Mu. Mol) of spermine was added to the hyaluronic acid solution, wherein the content of spermine was 0.3% of the mass of hyaluronic acid. On the premise of four-site crosslinking, the pH value of the hyaluronic acid solution is regulated to about 5.2 by using a 6mol/L hydrochloric acid solution, then the hyaluronic acid solution is added and stirred uniformly, 29.6 mu mol of HATU, namely 400% of spermine mole number, is added, and the mixture is continuously stirred uniformly, and is sealed and placed into a blast drying oven at 30 ℃ for reaction for 24 hours. After the reaction, 40mL of acetone is added, and the gel particles are crushed by an IKA T25 high shear disperser, wherein the crushing speed is 10000 revolutions per minute, and the crushing lasts for 5min. After the completion of the pulverization, 200mL of acetone was further added to completely dehydrate the gel. The precipitate was separated, washed successively with 200mL of acetone for 5 times, and then placed in a vacuum oven and dried under vacuum at 40℃for 24 hours at a vacuum of-0.09 MPa. After complete drying, taking 0.4g of xerogel, adding 400mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL, filling the gel into a prefilled syringe after the gel is completely swelled, and carrying out damp-heat sterilization at 121 ℃ for 15min to obtain the final product gel. The gel particle diameter D0.5 was 156. Mu.m, and D0.9 was 231. Mu.m.
Example 11: preparation of three-site crosslinked hydrogel of HATU catalytic spermidine and hyaluronic acid
0.5g of sodium hyaluronate (molecular weight 2800 kDa) was weighed, then 38.5mL of purified water was added, and after complete dissolution, at this time the concentration of hyaluronic acid was 13.3mg/mL, 2.5mg (molar number: 17.2. Mu. Mol) of spermidine was added to the hyaluronic acid solution, at this time the content of spermidine was 0.5% of the mass of hyaluronic acid. On the premise of three-site crosslinking, the pH value of the hyaluronic acid solution is regulated to about 5.2 by using a 6mol/L hydrochloric acid solution, then the hyaluronic acid solution is added and stirred uniformly, 51.6 mu mol of HATU, namely 300% of the mole number of spermidine, is added, and the mixture is continuously stirred uniformly, sealed and placed into a blast drying oven at 30 ℃ for reaction for 24 hours. After the reaction, 40mL of acetone is added, and the gel particles are crushed by an IKAT25 high shear dispersing machine, wherein the crushing speed is 10000 revolutions per minute, and the crushing lasts for 5min. After the completion of the pulverization, 200mL of acetone was further added to completely dehydrate the gel. The precipitate was separated, washed successively with 200mL of acetone for 5 times, and then placed in a vacuum oven and dried under vacuum at 40℃for 24 hours at a vacuum of-0.09 MPa. After complete drying, 0.4g of xerogel is taken, 200mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL is added, after the gel is completely swelled, the gel is filled into a prefilled syringe, and moist heat sterilization is carried out at 121 ℃ for 15min, thus obtaining the final product gel. The gel particle diameter D0.5 was 125. Mu.m, and D0.9 was 201. Mu.m.
Example 12: EDC catalyzes four-site crosslinking of spermine and hyaluronic acid
6g of sodium hyaluronate (molecular weight 120 kDa) was weighed, then 35mL of purified water was added thereto, and after complete dissolution, at this time the concentration of hyaluronic acid was 150mg/mL, 2.1g (molar number: 10.4 mmol) of spermine was added to the hyaluronic acid solution, at this time the content of spermine was 35% of the mass of hyaluronic acid. Under the precondition of four-site crosslinking, the pH value of the hyaluronic acid solution is regulated to about 5.2 by using a hydrochloric acid solution with the concentration of 6mol/L, then the mixture is added and stirred uniformly, 57.2mmol EDC (namely 550% of spermine mole number) is added, meanwhile, 2.0g of HOBt (20% of EDC mass) is added, the mixture is stirred uniformly continuously, and the mixture is sealed and placed into a blast drying oven with the temperature of 60 ℃ for reaction for 24 hours. After the reaction is finished, 40mL of absolute ethyl alcohol is added, and an IKA T25 high shear disperser is adopted to pulverize gel particles, wherein the pulverizing speed is 10000 revolutions per minute, and the duration is 5 minutes. After the completion of the pulverization, 200mL of absolute ethyl alcohol was continuously added to completely dehydrate the gel. Separating the precipitate, continuously cleaning the precipitate with 200mL of absolute ethyl alcohol for 5 times, and vacuum drying the precipitate in a vacuum oven at 40 ℃ under the vacuum degree of-0.09 MPa for 24 hours. After complete drying, 1.0g of xerogel is taken, 20mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL is added, after the gel is completely swelled, the gel is filled into a prefilled syringe, and moist heat sterilization is carried out at 121 ℃ for 15min, thus obtaining the final product gel. The gel particle diameter D0.5 was 148 μm and D0.9 was 197. Mu.m.
Example 13: preparation of double-site crosslinked hydrogel under HATU catalytic spermidine and hyaluronic acid meta-acid conditions
3.0g of sodium hyaluronate (molecular weight: 900kDa, containing 7.4mmol of repeating structural units of hyaluronic acid) was weighed, then 98mL of purified water was added, and after complete dissolution, at this time the concentration of hyaluronic acid was 30mg/mL, 0.022g (molar number: 0.15 mmol) of spermidine was added to the hyaluronic acid solution, at this time the content of spermidine was 0.7% of the mass of hyaluronic acid (molar number of repeating structural units of hyaluronic acid: 2%). Adding 0.48mmol of HATU, namely 320 percent of spermidine mole number, uniformly stirring, adjusting the pH value of the hyaluronic acid solution to about 4.60 by using a 6mol/L hydrochloric acid solution, then adding and uniformly stirring, and hermetically placing into a 50 ℃ incubator for reaction for 14h. After the reaction is finished, 40mL of absolute ethyl alcohol is added, and an IKA T25 high shear disperser is adopted to pulverize gel particles, wherein the pulverizing speed is 10000 revolutions per minute, and the duration is 5 minutes. After the completion of the pulverization, 200mL of absolute ethyl alcohol was continuously added to completely dehydrate the gel. Separating the precipitate, continuously cleaning the precipitate with 200mL of absolute ethyl alcohol for 5 times, and vacuum drying the precipitate in a vacuum oven at 40 ℃ under the vacuum degree of-0.09 MPa for 24 hours. And after the gel is completely dried, taking 1.0g of xerogel, adding 50mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL to prepare hydrogel with the concentration of 20mg/mL of hyaluronic acid, filling the gel into a prefilled syringe after the gel is completely swelled, and carrying out damp-heat sterilization at the temperature of 121 ℃ for 15min to obtain the final product gel. The gel particle diameter D0.5 was 138 μm and D0.9 was 210. Mu.m.
Comparative example 1: preparation of HATU-catalyzed spermidine-hyaluronic acid crosslinked hydrogel under strong acid conditions 3.0g of sodium hyaluronate (molecular weight 900kDa, containing 7.4mmol of repeated structural units of hyaluronic acid) was weighed, then 98mL of purified water was added, after complete dissolution, at which time the concentration of hyaluronic acid was 30mg/mL, 0.022g (mole number 0.15 mmol) of spermidine was added to the hyaluronic acid solution, at which time the spermidine content was 0.7% of the mass of hyaluronic acid (mole number of repeated structural units of hyaluronic acid was 2%). Adding 0.48mmol of HATU, namely 320 percent of spermidine mole number, uniformly stirring, adjusting the pH value of the hyaluronic acid solution to about 3.20 by using a 6mol/L hydrochloric acid solution, then adding and uniformly stirring, and sealing and placing into a 50 ℃ incubator for reaction for 14h. After the reaction is finished, 40mL of absolute ethyl alcohol is added, and an IKA T25 high shear disperser is adopted to pulverize gel particles, wherein the pulverizing speed is 10000 revolutions per minute, and the duration is 5 minutes. After the completion of the pulverization, 200mL of absolute ethyl alcohol was continuously added to completely dehydrate the gel. Separating the precipitate, continuously cleaning the precipitate with 200mL of absolute ethyl alcohol for 5 times, and vacuum drying the precipitate in a vacuum oven at 40 ℃ under the vacuum degree of-0.09 MPa for 24 hours. And after the gel is completely dried, taking 1.0g of xerogel, adding 50mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL to prepare hydrogel with the concentration of 20mg/mL of hyaluronic acid, filling the gel into a prefilled syringe after the gel is completely swelled, and carrying out damp-heat sterilization at the temperature of 121 ℃ for 15min to obtain the final product gel. The gel particle diameter D0.5 was 143. Mu.m, and D0.9 was 201. Mu.m.
Comparative example 2: EDC catalyzes spermine and hyaluronic acid to carry out crosslinking reaction
This comparative example was gel prepared according to the method disclosed in US9907739B 2.
3.0g of sodium hyaluronate (molecular weight: 900kDa, containing 7.4mmol of repeating structural units of hyaluronic acid) was weighed, then 98mL of purified water was added, and after complete dissolution, at this time the concentration of hyaluronic acid was 30mg/mL, 0.03g (mole number: 0.15 mmol) of spermine was added to the hyaluronic acid solution, at this time the spermine content was 1% of the mass of hyaluronic acid (mole number of repeating structural units of hyaluronic acid: 2%). Adjusting the pH value of the hyaluronic acid solution to about 6.2 by using a 6mol/L hydrochloric acid solution, uniformly stirring, adding 7.4mmol EDC (electro-chemical vapor deposition), namely 100% of the mole number of the hyaluronic acid structural unit and 49 times of the mole number of spermine, simultaneously adding HOBt with 20% of the mass of the EDC, continuously stirring uniformly, sealing and placing into a constant temperature box at 25 ℃ for reaction for 24 hours. After the reaction is finished, 40mL of absolute ethyl alcohol is added, and an IKA T25 high shear disperser is adopted to pulverize gel particles, wherein the pulverizing speed is 10000 revolutions per minute, and the duration is 5 minutes. After the completion of the pulverization, 200mL of absolute ethyl alcohol was continuously added to completely dehydrate the gel. Separating the precipitate, continuously cleaning the precipitate with 200mL of absolute ethyl alcohol for 5 times, and vacuum drying the precipitate in a vacuum oven at 40 ℃ under the vacuum degree of-0.09 MPa for 24 hours. And after the gel is completely dried, taking 1.0g of xerogel, adding 50mL of pH7.0 phosphate buffer solution with the concentration of 10mg/mL to prepare hydrogel with the concentration of 20mg/mL of hyaluronic acid, filling the gel into a prefilled syringe after the gel is completely swelled, and carrying out damp-heat sterilization at the temperature of 121 ℃ for 15min to obtain the final product gel. The gel particle diameter D0.5 was 155. Mu.m, and D0.9 was 221. Mu.m.
Example 14: spermine or spermidine crosslinking efficiency detection
2.0ml of each of the hyaluronic acid gels obtained in examples 1 to 8 and comparative examples 1 to 2 was taken out, and 50ml of absolute ethanol was added to each gel, and the mixture was stirred for 30 minutes until the gel became a white precipitate. Separating the precipitate, and vacuum drying at 40 ℃ under the vacuum degree of-0.09 MPa for 24 hours to remove ethanol. The gel was taken out, 10mL of sulfuric acid solution of 0.5mol/L was added thereto, and after 2 hours of treatment at 90℃the gel was neutralized to pH=7 with sodium hydroxide solution of 6mol/L, respectively. The solution is placed into a vacuum drying oven, and is dried for 24 hours under the vacuum degree of-0.09 MPa at the temperature of 60 ℃ to remove redundant water. Dissolved in deuterated water at a concentration of 10mg/mL and subjected to 1H spectroscopy by a 400M nuclear magnetic resonance spectrometer.
By aligning1 The key characteristic peaks in the H NMR spectrum are integrated, the actual molar ratio of spermidine to hyaluronic acid is obtained through integrating the peak areas, and the crosslinking efficiency is obtained through the ratio of the actual molar ratio to the theoretical molar ratio (calculated according to actual feeding). Wherein, the characteristic H atoms selected from spermidine are alpha-H of amino or imino, the number of hydrogen atoms is 8, the characteristic H atoms selected from spermine are alpha-H of amino or imino, the number of H atoms is 12, the integral range of the H atoms of spermine and spermidine is the same, and the integral range of the H atoms of spermine and spermidine is 2.5-2.85ppm; the characteristic H atoms selected from hyaluronic acid are alpha-H of carbonyl in acetamido, wherein the number of H atoms is 3, and the integral range is 1.85-2.05ppm; in the integration, the integrated peak area of carbonyl alpha-H in hyaluronic acid is set to be 3, and thus, the integrated peak area of spermidine or amino and imino alpha-H in spermine can be obtained. The calculation formula is as follows:
Actual molar ratio of spermidine cross-linking = integrated peak area of amino groups and imino groups α -H in spermidine/8;
actual crosslinking molar ratio of spermine = integrated peak area of amino and imino α -H in spermine/12;
crosslinking efficiency= (actual crosslinking molar ratio/theoretical crosslinking molar ratio) ×100%.
The crosslinking efficiencies of examples 1-8 are shown in Table 1, and the nuclear magnetic spectra are shown in FIG. 1:
TABLE 1 actual crosslinking efficiency
Analysis from the data in table 1 found that when double site crosslinking, the crosslinking efficiency was lower than that of multi site crosslinking. When comparing examples 1, 4 and examples 2, 3, 5 for two-site crosslinking, it was found that the efficiency of two-site crosslinking is generally lower than that of multi-site crosslinking, probably due to the combined effects of the three conditions of activator usage, pH and temperature, resulting in lower crosslinking efficiency at two-site crosslinking; in the same multi-site crosslinking, the efficiency of activation by each activator is in the order of: HATU > triphenylphosphine salt > DMTMM > EDC + promoter. Among them, HATU activation efficiency can be as high as approximately 95%, while edc+nhs activation efficiency is only 75%. Under the same conditions, the crosslinking efficiency of spermine and spermidine is close, the efficiency of spermine is slightly lower, and the possibility is caused by higher content of imino groups in spermine, the steric hindrance of the imino groups is larger, and the reactivity is lower than that of primary amino groups. In addition, by analysis of the comparative example, the reactivity of amino groups with imino groups was inhibited outside the multi-site pH range, and thus the crosslinking efficiency was low; at a temperature lower than the test temperature of multi-site crosslinking, even if the reaction is carried out according to the conditions of multi-site crosslinking, the finally obtained crosslinking efficiency is similar to that of double-site crosslinking, and the reaction efficiency is lower.
Example 15: effect of spermidine Cross-Linked hyaluronic acid gel on cell proliferation
L-929 cells were seeded in cell culture medium, and 1% penicillin-streptomycin solution and 10% fetal bovine serum solution were added. L-929 cells were incubated in a humidified 5% carbon dioxide cell incubator at 37℃for 3 days. Then, the spermidine three-site crosslinked hyaluronic acid gel obtained in example 2 was transferred into a 96-well plate, cured by an ultraviolet lamp and sterilized. Again, L-929 cell culture broth was added to wells containing spermidine crosslinked hyaluronic acid gel, and 1mL of trypsin solution containing 0.1% EDTA was added, the number of L-929 cells in each well being 1X 105 And each. And continue to be placed in the cell incubator to promote cell growth.
The proliferation rate of spermidine crosslinked hyaluronic acid gel cells was determined by MTT method. After 24, 48 and 72 hours of incubation, 100. Mu.L of MTT aqueous solution (5 mg. Multidot. ML-1) was added to each well and incubation was continued for 4 hours in an incubator. The MTT solution was then removed, 150. Mu.L of dimethylsulfoxide was added to dissolve formazan crystals, and the absorbance of the solution was measured at 490nm wavelength using a microplate reader to determine the extent of cell proliferation of the gel on L-929. Cell viability was calculated according to the following formula:
Cell viability (%) = (As/Ac) ×100%;
where As is the absorbance of the sample solution at 570nm and Ac is the absorbance of the blank at 570 nm.
The cell proliferation results are shown in FIG. 2: from the figure, it can be seen that the cell viability of spermidine cross-linked hyaluronic acid gel at 24, 48 and 72 hours was 112%, 123% and 138%, respectively. From the cell viability of these two types of gels, it can be seen that spermidine crosslinked hyaluronic acid hydrogel has no cytotoxicity, and the phenomenon that the cell viability increases with time indicates that the hydrogel plays a role in promoting cell growth to a certain extent.
Example 16: rheological property detection of spermine or spermidine cross-linked hyaluronic acid gel
Among the hyaluronic acid gels obtained in examples 1 to 13 and comparative example 2, 2.0ml was taken out of each of the two types before sterilization and after sterilization, the elastic modulus (G') of the gel was measured using a TA DHR-2 type flat plate rheometer, and the elastic modulus loss rate was calculated. Wherein the G' loss rate is calculated from the following formula:
g 'loss rate= (pre-sterilization G' -post-sterilization G ')/pre-sterilization G';
rheometer parameters are, operating gap: 1000mm, loading gap: 45000m, operating temperature: 37 ℃, deformation amount: 1%, frequency: 0.9Hz, run time: 60s. The rheological data for each gel are shown in table 2:
TABLE 2 rheological data of gels
The G 'loss rate can be considered as an indicator of the thermal stability of the gel, with lower G' loss rates leading to higher thermal stability of the gel. As can be seen from the data in table 2, taking examples 1-7 as examples, the G' loss rate has a certain relationship with the crosslinking efficiency in example 14, wherein under the condition of similar crosslinking degree and crosslinking efficiency (examples 2 and 3), the thermal stability of the hyaluronic acid crosslinked by spermidine and the hyaluronic acid crosslinked by spermine is similar, but the elastic modulus of spermine is higher and the thermal stability is higher, probably because the crosslinked network structure formed by 4-site crosslinking of spermine is more compact, and the same theory can be applied to the three-site crosslinked spermidine gel and the two-site crosslinked spermidine gel, namely the thermal stability of the two-site crosslinked gel is worse than that of the three-site crosslinked gel. The four activators are compared, and the thermal stability sequence of the gel is as follows: HATU > triphenylphosphine salt > DMTMM > EDC + adjuvant in the same order as in example 14. In addition, the concentration of hyaluronic acid in the crosslinked hyaluronic acid hydrogels also had a large influence on the thermal stability of the gel, and examples 10 and 11 employed the same crosslinking sites as examples 2 and 3, but since the concentrations of hyaluronic acid in the crosslinked hyaluronic acid hydrogels of examples 10 and 11 were 1mg/mL and 2mg/mL, respectively, and the corresponding concentrations of hyaluronic acid in examples 2 and 3 were 20mg/mL, a large difference in G 'was caused, and the decrease value of G' was increased. In addition, the amount of activator added also had a greater effect on the hydrogel properties, and example 1 and example 13 were otherwise identical, with a 28.6% decrease in HATU activator added in example 13 (i.e., 164.8. Mu. Mol) compared to example 1, resulting in a significant decrease in G 'in example 13 compared to example 1 and a significant increase in the G' decrease ratio compared to example 1.
Example 17: release detection of spermidine in gel along with degradation process of hyaluronic acid under thermal degradation condition
Taking 2mL of finished gel in example 2, sealing each sample in a penicillin bottle, placing the sample in a forced air drying oven at 125 ℃ for accelerated hydrolysis, taking out 3 parallel samples every 15min, cooling to about 25 ℃, respectively adjusting the pH to 12 by sodium hydroxide, respectively adding 20mL of chromatographic grade acetone, filtering by a 0.2 mu m filter membrane, and then introducing acetone filtrate into an Agilent 7890B type gas chromatograph, and adopting an SGE H2 capillary column (30 m multiplied by 0.53mm multiplied by 1.0 mu m), wherein the chromatographic conditions are as follows: the temperature of the sample inlet is 220 ℃, the pressure is 40, the separation flow rate is 30, the split ratio is 10:1, the separation time is 0.8, and the column temperature is: 100 ℃ for 0.5min, at 20 ℃/min up to 180 ℃ for 1min, detector (FID): the temperature is 250 ℃, the air flow rate is 350mL/min, the H2 flow rate is 35mL/min, and the tail blowing N2 flow rate is 30mL/min.
The total degradation time is 90min, 6 detection points are set, and the detection result is shown in figure 3.
The purpose of this experiment was to simulate the release of spermidine monomers during thermal degradation of hyaluronic acid due to hydrolysis of the crosslinking sites. However, under normal physiological environment, the degradation process of the crosslinked hyaluronic acid is longer, the period is shorter than several months, and the period is longer than 1 year, so that the experiment adopts the release amount detection experiment of spermidine in the hydrolysis process of spermidine crosslinked hyaluronic acid gel under the high temperature condition higher than 100 ℃, and the degradation process of the hyaluronic acid and the release process of the spermidine can be observed in a shorter time. As shown in fig. 3, the release of spermidine is slow along with the degradation process of hyaluronic acid, the initial release speed is slow, the release speed of spermidine is obviously accelerated along with the disintegration of the crosslinked hyaluronic acid network in the later period, and the accumulated release amount is also obviously improved, so that the release of spermidine in the gel is a slow-first-fast-second process. The experiment can also prove that the crosslinking technology in the invention is an active reaction technology, and can continuously release spermidine monomers in a prototype manner in the continuous degradation process of crosslinked hyaluronic acid, so that the gel can continuously release spermidine in vivo and exert the specific physiological activity of the spermidine.
Example 18: release detection of spermidine in gel with degradation of hyaluronic acid under enzymatic conditions
Taking 2mL of finished gel in example 2, sealing each sample in a penicillin bottle, adding 1000u of hyaluronidase into each sample for accelerated hydrolysis, taking out 3 parallel samples every 30min, cooling to about 25 ℃, taking out 0.5mL of supernatant, respectively adjusting pH to 12 by sodium hydroxide, respectively adding 20mL of chromatographic grade acetone, filtering by using a 0.2 μm filter membrane, and feeding acetone filtrate into an Agilent 7890B type gas chromatograph, and adopting an SGE H2 capillary column (30 m multiplied by 0.53mm multiplied by 1.0 μm), wherein chromatographic conditions are as follows: the temperature of the sample inlet is 220 ℃, the pressure is 40, the separation flow rate is 30, the split ratio is 10:1, the separation time is 0.8, and the column temperature is: 100 ℃ for 0.5min, at 20 ℃/min up to 180 ℃ for 1min, detector (FID): the temperature is 250 ℃, the air flow rate is 350mL/min, the H2 flow rate is 35mL/min, and the tail blowing N2 flow rate is 30mL/min.
The total degradation time is 180min, 6 detection points are set, and the detection result is shown in figure 4.
The purpose of this experiment was to simulate the release of spermidine monomers during enzymatic degradation of hyaluronic acid due to gel degradation. As shown in fig. 4, the release of spermidine is slowly released along with the degradation process of hyaluronic acid, and the release rate of spermidine is almost unchanged in the whole degradation process of hyaluronic acid gel, which indicates that spermidine can be uniformly and stably released when the hyaluronic acid gel obtained by crosslinking with spermidine is enzymatically degraded. The experiment can also prove that the crosslinking technology in the invention is an active reaction technology, and can continuously release spermidine monomers in a prototype manner in the continuous degradation process of crosslinked hyaluronic acid, so that the gel can continuously release spermidine in vivo and exert the specific physiological activity of the spermidine.
Example 19: detection of remaining active sites in double-site and multiple-site cross-linked gels
Taking about 2g of ninhydrin, adding 100ml of purified water to dissolve the ninhydrin, and uniformly mixing the dissolved ninhydrin with the purified water; and (5) keeping the materials away from light at the temperature of 2-8 ℃ for standby. 54.6g of sodium acetate is taken, 20ml of acetic acid solution with the concentration of 1mol/L is added for dissolution, and then water is added for dilution to 500ml for standby. Respectively taking spermidine standard substance and spermine standard substance, placing into a 10ml measuring flask, diluting to scale with purified water, shaking uniformly to prepare 500 μg/ml spermidine standard use solution, and 500ug/ml spermine standard use solution; the standard solution was diluted to 10, 50, 100, 200. Mu.g/ml and used as a standard curve at the time of use.
Taking the hydrogels before wet heat sterilization in examples 1-8 and comparative examples 1 and 2 and the spermine and spermidine series standard solutions, respectively taking 1mL, sequentially adding 2.0mL of the acetic acid-sodium acetate buffer solution and 2.0mL of the ninhydrin solution, adding a plug, fully mixing uniformly, heating in a water bath at 70 ℃ for 30min, taking out, rapidly cooling to room temperature, adding purified water to dilute to 25mL, and uniformly mixing; measuring the absorbance of the amino group derivative at 565nm wavelength of the mixed solution, and simultaneously detecting the absorbance of the amino group derivative at 400nm wavelength; blank correction was performed in the same way with purified water.
The amino or imino residue ratio (%) is calculated as follows:
residual amount ratio= (a0 -Ai )/A0 ×100％
Wherein A0 is the absorbance value measured by the control solution, and Ai is the absorbance value measured by the sample.
Wherein one spermidine molecule contains one imino group and two amino groups, and one spermine molecule contains two imino groups and two amino groups.
The actual detection results are shown in table 3:
TABLE 3 amino or imino residue ratio in gels
| Examples | Residual amount of amino group | Residual amount of imino groups |
| Example 1 | 10.34％ | 86.61％ |
| Example 2 | 7.31％ | 8.02％ |
| Example 3 | 8.53％ | 9.45％ |
| Examples4 | 18.13％ | 89.45％ |
| Example 5 | 10.16％ | 12.31％ |
| Example 6 | 13.35％ | 13.43％ |
| Example 7 | 17.41％ | 14.31％ |
| Example 8 | 6.32％ | 7.88％ |
| Comparative example 1 | 39.25％ | 97.13％ |
| Comparative example 2 | 40.35％ | 98.31％ |
As is clear from the results of Table 3, examples 1 and 4 were two-site crosslinking, in which the main reactive group was amino group, the reaction efficiency was as high as 89.66% (100% -amino residue, the same applies hereinafter), the lowest amino group reaction efficiency was also more than 80%, and the imino group reaction efficiency was not more than 15%, so that the two-site crosslinking reaction involving the amino group was mainly performed under this condition. In examples 2, 3 and 8, the reaction efficiency of the imino group and the imino group of spermidine or spermine was 90% or more, and in examples 5 to 7, the reaction efficiency of the imino group was 85% or more. Indicating that under this condition, most of spermidine undergoes three-site crosslinking and spermine undergoes four-site crosslinking. The authenticity of the three-site and four-site cross-linking in the present invention was further confirmed. By examination of the comparative example, it was found that the imino reaction efficiency in spermidine or spermine was reduced to not more than 10% and the reaction efficiency of amino groups was also reduced outside the conditions defined in the present invention. Among them, although the total activator of comparative example 2 was added in an amount 49 times that of the crosslinking agent, the imino group in spermine failed to participate in the reaction due to unsuitable reaction conditions such as pH, reaction temperature, etc., and the reaction efficiency of the amino group did not exceed 60%.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.