CN106478841A

CN106478841A - The cysteine conjugates of hyaluronic acid prepared with mercaptan alkene clicking chemistry by lyophilization and its synthetic method and application

Info

Publication number: CN106478841A
Application number: CN201610834488.7A
Authority: CN
Inventors: 张响; 胡碧煌; 李雨欣
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2016-09-20
Filing date: 2016-09-20
Publication date: 2017-03-08
Anticipated expiration: 2036-09-20
Also published as: CN106478841B

Abstract

The invention discloses a kind of cysteine functionalization conjugates of hyaluronic acid prepared with mercaptan alkene clicking chemistry by lyophilization and its its synthetic method, also disclose that conjugates of hyaluronic acid, in the application preparing Injectable in-situ hydrogel, also discloses the application in culture islet cellss and insulin secretion for the hydrogel of Injectable in-situ formation.The present invention passes through lyophilization and mercaptan alkene clicking chemistry, obtains a series of cysteine functionalization conjugates of hyaluronic acid that different modifying rates have stable ehter bond, it has sulfhydryl reactive group, can functional modification further；The conjugates of hyaluronic acid of different modifying rate and polyethylene glycol conjugate lead to peroxy esters and mediate the hydrogel that nature chemistry coupled reaction obtains Injectable in-situ formation, there is good rheological property, wherein free sulfydryl can increase the cell adhesion of hyaluronic acid, can have wide biological medicine prospect as cell scaffold material in islet cell culture and the application that stimulates insulin secretion.

Description

Cysteine hyaluronic acid by lyophilization and mercaptan-alkene clicking chemistry preparation Conjugate and its synthetic method and application

Technical field

The present invention relates to a kind of lyophilization and mercaptan-alkene clicking modify the cysteine functionalization hyaluronic acid knot being formed Compound and its synthetic method, and its prepare Injectable in-situ formed hydrogel application, belong to biomedicine field.

Background technology

Biologic bracket material is organizational project and the key of regenerative medicine research.Because timbering material is not only cell and carries For structural support effect, and also act as template action, for guide tissue regeneration and control organizational structure.Wherein, hydrogel The biological support of class is particularly extensive in organizational project and regenerative medicine application.

Hydrogel is hydrophilic polymer network, can absorb substantial amounts of moisture, has good biocompatibility.Although Application in biological medicine for the hydrogel achieves huge progress, however, development meets clinical needs：In a mild condition not The hydrogel of the covalent cross-linking being formed using toxic reagent Injectable in-situ still suffers from challenging.Most of hydrogel is in tissue The major defect of the application of engineering aspect is exactly to need surgical operation to mediate, and the complexity of operation process is loaded down with trivial details undoubtedly to be brought to patient Very big misery and risk.Therefore, the research of injection aquagel comes into the picture, and it has significant advantage：Hydrogel It is formed in situ in vivo, is injected by needle tubing without surgical operation, minimally to invade to body tissue, not only rise To therapeutical effect, but also can reach preferably with the defect of various somatomedin, medicament mixed filling tissue arbitrary shape Therapeutic effect, thus can avoid the height in surgical procedures traumatic, especially when the tissue being used for repairing complicated shape When, injection aquagel has adaptivity.

The synthesis mechanism of mercaptan-alkene clicking chemistry be primarily referred to as mercaptan-alkene free radical addition (see accompanying drawing 1) (Kade M J, Burke D J, Hawker C J.The power of thiol-ene chemistry [J] .Journal of Polymer Science Part A Polymer Chemistry, 2010,48 (4)：743-750.).Under conditions of alkene exists, by heat Or so that the molecular hydrogen containing sulfydryl is sloughed thus obtaining the free radical of sulfur-bearing under the initiation of light, this free radical and carbon-carbon double bond (C= C) living radical centered on carbon is formed by anti-geneva (anti-Markovnikov) addition, with another dividing containing sulfydryl Son occurs chain transfer reaction to obtain addition compound product and another living radical.Therefore, had using mercaptan-alkene clicking chemistry reaction Simply, efficiently, reliably, selective C-S bonding, the advantage modified polysaccharide class compound tool that particularly can carry out in aqueous There is significant superiority.

Oxygen ester mediation nature be connected chemically (oxo-ester mediated native chemical ligation, OMNCL) it is the extension that nature is connected chemically (native chemical ligation) concept.It has been found that ester type compound After the compound of N- end cysteine in physiological conditions (phosphate buffered solution, pH 7-9) mixing, cysteine The alcohol of sulfydryl and ester type compound exchanges, and spontaneously resets after the intermediate forming five annulus, produce one new Amido link.This chemical reaction is referred to as oxygen ester mediation nature chemistry even (oxo-ester mediated native Chemical ligation, OMNCL, are shown in accompanying drawing 2).OMNCL has several clear advantages：1. there are chemo-selective, esters Only react with the compound of cysteine or N- terminal cysteine and form new amido link, do not deposited by other mercaptan and sulfydryl Interference；2. under mild conditions, do not use catalyst, initiator etc. may poisonous compound and highly effective reaction；③ It is connected chemically that the hydrogel that reaction formed is different from other using sulfydryl, chemistry coupled reaction is in formation new amido link naturally While, produce mercapto groups also on skeleton.This sulfydryl not only can be used for further in bone according to different purposes Functionalization hydrogel on frame, and, the polymer of sulfhydrylation (Thiolation) has more preferable bioadhesive.4. in addition, esters Alcohol in structure is discharged in solution when OMNCL reacts, then, it is possible to this alcohol is designed as when designing ester Hydroxylated medicine and the precursor of cell growth factor, thus while hydrogel is formed, discharge these medicines and growth because Son is in hydrogel.Therefore, the water of the covalent cross-linking being formed using the Injectable in-situ that feature and the advantage of OMNCL reaction obtain Gel has significant superiority.For example：Existing document based on Polyethylene Glycol, terminal ester or cysteine functionalization, lead to Peroxy esters mediation nature chemistry coupled reaction obtain polyethylene glycol hydrogel (Strehin I, Gourevitch D, Zhang Y, et al.Hydrogels Formed by Oxo-ester Mediated Native Chemical Ligation.[J] .Biomaterials Science, 2013,1 (6)：603-613.).

Hyaluronic acid (hyaluronic acid, HA) is distributed widely in the various tissues of animal, by N- acetyl-D- Portugal Grapes glucosamine and D-Glucose aldehydic acid disaccharidase recurring units composition, are applied to organizational project and (Allison D in regenerative medicine D, Grandeallen K J.Review.Hyaluronan：a powerful tissue engineering tool.[J] Tissue Engineering, 2006,12 (8)：2131-40.).Such as 20th century, the mid-80 HA was applied to medical science Beauty treatmentWound dressing And as arthritis treatment Medicine listsIn extracellular matrix, hyaluronic acid is the part of cell surface receptor CD44, and CD44 receptor ginseng With various kinds of cell process, such as cell adhesion, cell migration and increment.Due to its important physiological action, body or wound surface are straight The inflammatory reaction of tissue be can substantially mitigate and eliminate after connecing Injection of Hyaluronic Acid, wound surface and wound healing promoted.But, due to saturating Bright matter acid is rapidly removed by hyaluronidase in vivo, and the internal time of staying is short, the low deficiency of mechanical strength, such alleviation Effect is also of short duration, so the covalent modified hyaluronic acid gel forming Injectable in-situ covalent cross-linking of hyaluronic acid just shows Show its superiority.

Hyaluronic acid primary chemical is modified target spot and is included：The carboxyl of D-Glucose aldehydic acid, the hydroxyl of N-ACETYL-D-GLUCOSAMINE The N- acetyl group of base and N-ACETYL-D-GLUCOSAMINE.The HA derivant of Clinical practice mostly is and its carboxylic acid and hydroxyl is repaiied at present Decorations form, by being esterified (Esterification), amidatioon (Amidation), open loop (Ring Opening), crosslinking (Cross-linking), the various chemical modification methods such as grafting (Graft), prepare functionalization HA derivant.Wherein to transparent The research of the carboxyl modified of matter acid is relatively more, for example, have document to pass through the sumptuous modification of adipic acid diacid, and adds EDC and HOBt to make For couplant and catalyst, modify the carboxyl of hyaluronic acid, obtain conjugates of hyaluronic acid, then by being cross-linked to form hyaluronic acid Hydrogel (Yeo Y, Highley C B, Bellas E, et al.In situ, cross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions in a rabbit model [J] .Biomaterials, 2006,27 (27)：4698-705.).Also have and water-setting is formed by the hydroxyl reaction with hyaluronic acid The cross-linking chemistry reaction of glue, such as Seidlits is respectively adopted methacrylic anhydride and 4- pentenoic acid anhydride to hyaluronic acid C-6 position Primary hydroxyl group carry out esterification modify, finally under ultraviolet irradiation condition occur cross-linking reaction, obtain hydrogel (Seidlits SK, Khaing ZZ, Petersen RR, et al.The Effects of Hyaluronic Acid Hydrogels with Tunable Mechanical Properties on Neural Progenitor Cell Differentiation[J] .Biomaterials, 2010,31 (14)：3930-3940.).Also have first with cysteine-modifying glycidyl ether and hyalomitome The hydroxyl reaction of acid, then carry out nature chemical reaction formation hydrogel (Zhang X, Sun P, Huangshan L, et al.Improved method for synthesis of cysteine modified hyaluronic acid for in- Situ hydrogel formation [J] .Chemical Communications, 2015,51 (47)：9662-5.).Also have As Chinese patent CN 103910886A, its based on hyaluronic acid, using the polyethylene glycols after the monolateral modification of cysteine Compound and hyaluronic acid hydroxyl carry out ring-opening reaction and form ehter bond, are being combined by nature with multi-branched polyethyleneglycol derivative It is connected chemically and form the hyaluronic acid gel that Injectable in-situ is formed.

The hydrogel that hyaluronic acid is formed after crosslinking should be more satisfactory biologic bracket material.It overcomes sky So rapidly it is eliminated in hyaluronic acid body, the low deficiency of mechanical strength.But, by the crosslinking of the carboxyl preparation of hyaluronic acid Hydrogel, some side reactions may be produced.If there are some researches show, its carboxyl is modified by sulphation, and can lead to hyaluronic acid Melanoma cells, thus cause CD44 receptor can not normally identify.Cell CD44 receptor is presently considered to be negative by hyaluronic acid Electric charge acts on (Morra M.Engineering of biomaterials surfaces by with hyaluronic acid Hyaluronan. [J] .Biomacromolecules, 2005,6 (3)：1205-1223.), and reduce the negative charge of hyaluronic acid The effect of cell CD44 receptor and hyaluronic acid then can be affected, thus ability (the Herrera M of cytothesiss damage can be reduced B, Bussolati B, Bruno S, et al.Exogenous mesenchymal stem cells localize to the kidney by means of CD44 following acute tubular injury[J].Kidney International, 2007,72 (4)：430-441.), thus hyaluronic acid just to lose it superior as biomaterial Property.And hyaluronic acid gel is obtained by its hydroxyl reaction and generally requires to react by ultraviolet lighting or with additive method Through preformed hydrogel, thus do not enable in-situ injection.Therefore, the present invention is with hyaluronic acid as raw material, by cold The dry hydroxyl modifying hyaluronic acid N-ACETYL-D-GLUCOSAMINE with mercaptan-alkene clicking chemistry of lyophilizing, do not change its carboxyl carried negative Electric charge, obtains thering is stable ehter bond product, more logical peroxy esters mediation nature chemistry coupled reaction obtains Injectable in-situ and covalently hands over Connection hyaluronic acid gel has significantly superiority.

Content of the invention

First purpose of the present invention is to provide a kind of half Guang by lyophilization and mercaptan-alkene clicking chemistry preparation Propylhomoserin functionalization conjugates of hyaluronic acid.Second object of the present invention is to provide one kind to pass through lyophilization and mercaptan-alkene The synthetic method of the cysteine functionalization conjugates of hyaluronic acid of click chemistry preparation.Third object of the present invention is to carry Cysteine functionalization conjugates of hyaluronic acid for use by lyophilization and mercaptan-alkene clicking chemistry preparation is preparing injectable The method of the covalent cross-linking hyaluronic acid gel of being formed in situ property.Fourth object of the present invention is to provide Injectable in-situ The covalent cross-linking hyaluronic acid gel of formative is as cell scaffold material in islet cell culture with insulin secretion Application.

Technical scheme is as follows：

A kind of cysteine functionalization conjugates of hyaluronic acid by lyophilization and mercaptan-alkene clicking chemistry preparation, Structural formula is：

N=200-4000 in formula.

A kind of described cysteine functionalization hyaluronic acid prepared with mercaptan-alkene clicking chemistry by lyophilization is combined The preparation method of thing, comprises the following steps：

1) structural formula is taken to beCompound 1 hyaluronic acid with structural formula be's Compound 2, forming structural formula by cryodesiccated method isCompound 3；

2) structural formula is taken to beCompound 4 and structural formula be's Compound 5 forms structural formulaCompound 6；

3) compound 6 and disulfide bond reducing agent generation reduction reaction formation structural formula are's Compound 7；

4) take compound 3 and compound 7, draw in light and under agent and ultraviolet light, occur mercaptan-alkene clicking chemistry to be formed Structural formula isCompound 8；

5) tertbutyloxycarbonyl on compound 8 and acetyl aminomethyl protection group are sloughed respectively, obtaining structural formula isCompound 9, i.e. conjugates of hyaluronic acid product.

Step 1) in, the molecular weight of compound 1 hyaluronic acid is 8～1,600,000.

Step 1) in, its modification rate scope of compound 3 is 4.03～64.11%；Step 4) in, its modification rate of compound 8 Scope be 2.33～45.67%.

A kind of cysteine functionalization conjugates of hyaluronic acid by lyophilization and mercaptan-alkene clicking chemistry preparation exists Prepare the application of the hydrogel of Injectable in-situ formation.

Described Injectable in-situ hydrogel is by lyophilization and the half of mercaptan-alkene clicking chemistry preparation by described Cystine functionalization conjugates of hyaluronic acid, leads to peroxy esters mediation nature chemistry coupled reaction with polyethylene glycol conjugate and generates.

The preparation method of described Injectable in-situ hydrogel, comprises the following steps：

1) with described conjugates of hyaluronic acid and phosphate buffer wiring solution-forming；

2) Polyethylene Glycol oxygen ester and phosphate buffer wiring solution-forming are used；

3) two kinds of solution are mixed, by naturally chemical coupled reaction, prepare the hydrogel of Injectable in-situ formation.

Described Polyethylene Glycol its structural formula of oxygen ester isN in formula =50～500, m=2～8.

The preparation method of described Injectable in-situ hydrogel, step 1) and 2) in, the pH of phosphate buffer is 7.0～ 7.6, the solution concentration being made into is 1～20%；Step 3) in, the mol ratio 1 of conjugates of hyaluronic acid and Polyethylene Glycol oxygen ester: (0.1～1), the time range that hydrogel is formed is 5～8000 seconds.

A kind of described cysteine functionalization hyaluronic acid knot by lyophilization and mercaptan-alkene clicking chemistry preparation The hydrogel that the Injectable in-situ of compound preparation is formed is in the application of culture islet cellss and insulin secretion.

The invention has the beneficial effects as follows：First passage lyophilization and mercaptan-alkene clicking chemistry are modified and are obtained cysteine Functionalization conjugates of hyaluronic acid, its mercapto groups can occur chemical reaction with sulfhydryl reactive group, can rhetorical function further Change；Be found that first (lyophilization) under mild conditions according to different demand prepare different modifying rate (2.33～ 45.67%) method of functionalization conjugates of hyaluronic acid；This method should can be used for structural modification and the function of other polysaccharide Change；The cysteine functionalization conjugates of hyaluronic acid of different modifying rate and polyethylene glycol conjugate lead to peroxy esters mediation naturalization Learn and connect the hydrogel being prepared for Injectable in-situ formation, there is good rheological property；This kind of hydrogel has free Sulfydryl, can increase the cell adhesion of biomaterial, in islet cell culture and can stimulate insulin as cell scaffold material Secretion application, has wide biological medicine prospect.

Specific as follows：

The present invention carries out allyl using cryodesiccated method to the hydroxyl of hyaluronic acid N-ACETYL-D-GLUCOSAMINE first Base glycidyl ether functional modification, obtains different modifying rate pi-allyl derivatives of hyaluronic acids, its modification rate ratio is at normal temperatures Reaction modification rate in solution significantly improves (35.67%vs 3.3%)；Technique extends for heat-labile biological alive Property molecule such as enzyme, antibody, protide somatomedin etc. high molecular polymer modify such as Pegylation, have incomparable Advantage；With mercaptan-alkene clicking chemistry, serial cysteine functional modification is carried out to pi-allyl derivatives of hyaluronic acids, obtain To different modifying rate cysteine conjugates of hyaluronic acid.This route is different from other hyaluronic acid functional methods, is one Kind new innovation synthetic route, and for the first time using the structural modification in the modification chemistry of polysaccharide, being hyaluronic acid and Modifying of other polysaccharide provides new method.

Synthesize the cysteine conjugates of hyaluronic acid obtaining and four branch Polyethylene Glycol oxygen esters, logical peroxy esters mediate nature It is connected chemically the hydrogel forming active sulfydryl, increased the adhesion of hyaluronic acid, and also can be able to enter in cell culture One step carries out functional modification, is such as combined with medicine, plays the effect of medicine sustained and controlled release, in addition, the alcohol in ester structure exists Can be discharged in solution during OMNCL reaction, when designing ester, this alcohol can be designed as hydroxylated medicine or cell The precursor of somatomedin, thus while hydrogel is formed, discharging these medicines or somatomedin in hydrogel, increased Hyaluronic acid is in the application of biomedicine field.

The present invention will be applied to hydrogel by a series of cysteine conjugates of hyaluronic acid being chemically modified to obtain In formation, logical peroxy esters mediate nature be connected chemically obtain having controllability, Injectable in-situ formative covalent cross-linking transparent Matter acid hydrogel.By contrasting the rheologic behavio(u)r of hydrogel, being used as biologic bracket material in islet cell culture and insulin Secretion experimental result, probes into the optimal modification condition of hyaluronic acid.Cell culture neck can be used in as biologic bracket material Domain, the formation of hydrogel can be injected by needle tubing and with minimal damage, body be invaded, rather than passes through surgery handss in traditional sense Art, can be used for filling up the defect of any body shape, simultaneously also can packaging medicine or cytokine, be clinical medicine and group weaver The fields such as journey provide preferable biomaterial.

Therefore, the present invention, with hyaluronic acid as raw material, modifies its N- second by lyophilization and mercaptan-alkene clicking chemistry The hydroxyl of acyl-D-Glucose amine, and do not affect itself negative charge, it is connected chemically and poly- second two finally by oxygen ester mediation nature Alcohol compound reacts the hyaluronic acid gel that Injectable in-situ forms covalent cross-linking, and the hydrogel being formed has well Biocompatibility.The present invention has prominent substantive distinguishing features, for person of ordinary skill in the field, invention with respect to Prior art is non-obvious.

Brief description

Fig. 1 is mercaptan-alkene clicking chemistry mechanism figure；

Fig. 2 is oxygen ester mediation nature chemistry coupled reaction mechanism figure；

Fig. 3 is compound 6 hydrogen nuclear magnetic resonance spectrogram；

Fig. 4 is compound 7 hydrogen nuclear magnetic resonance spectrogram；

Fig. 5 is the hydrogen nuclear magnetic resonance spectrogram of the compound 3 (numbering 1) of synthesis in embodiment 1；

Fig. 6 is the hydrogen nuclear magnetic resonance spectrogram of the compound 3 (numbering 2) of synthesis in embodiment 1；

Fig. 7 is the hydrogen nuclear magnetic resonance spectrogram of the compound 3 (numbering 3) of synthesis in embodiment 1；

Fig. 8 is the hydrogen nuclear magnetic resonance spectrogram of the compound 3 (numbering 4) of synthesis in embodiment 1；

Fig. 9 is the hydrogen nuclear magnetic resonance spectrogram of the compound 3 (numbering 5) of synthesis in embodiment 1；

Figure 10 is the hydrogen nuclear magnetic resonance spectrogram of the compound 3 (numbering 6) of synthesis in embodiment 1；

Figure 11 is the hydrogen nuclear magnetic resonance spectrogram of the compound 3 (numbering 7) of synthesis in embodiment 1；

Figure 12 is hyaluronic acid raw material (compound 1) hydrogen nuclear magnetic resonance spectrogram；

Figure 13 is the hydrogen nuclear magnetic resonance spectrogram of the compound 8 (numbering 1) of synthesis in embodiment 1；

Figure 14 is the hydrogen nuclear magnetic resonance spectrogram of the compound 8 (numbering 2) of synthesis in embodiment 1；

Figure 15 is the hydrogen nuclear magnetic resonance spectrogram of the compound 8 (numbering 3) of synthesis in embodiment 1；

Figure 16 is the hydrogen nuclear magnetic resonance spectrogram of the compound 8 (numbering 4) of synthesis in embodiment 1；

Figure 17 is the hydrogen nuclear magnetic resonance spectrogram of the compound 8 (numbering 5) of synthesis in embodiment 1；

Figure 18 is the hydrogen nuclear magnetic resonance spectrogram of the compound 8 (numbering 6) of synthesis in embodiment 1；

Figure 19 is the hydrogen nuclear magnetic resonance spectrogram of the compound 8 (numbering 7) of synthesis in embodiment 1；

Figure 20 is the hyaluronic acid decorated rate of allyl glycidyl ether (◆) and Boc-Cys (Acm)-cysteamine sulfydryl is transparent Matter acid modification rate (▲) figure；

Figure 21 is dextran standard GPC calibration graph；

Figure 22 is the GPC chromatogram of Cys-HA1；

Figure 23 is the GPC chromatogram of Cys-HA2；

Figure 24 is the GPC chromatogram of Cys-HA3；

Figure 25 is the GPC chromatogram of Cys-HA4；

Figure 26 is the GPC chromatogram of Cys-HA5；

Figure 27 is the GPC chromatogram of Cys-HA6；

Figure 28 is the GPC chromatogram of Cys-HA7；

Figure 29 is the GPC chromatogram of HA (100kDa)；

Figure 30 is the rheology time sweep figure of the hydrogel that Cys-HA1 and 4-ARM-PEG-S-S is formed；

Figure 31 is the rheology frequency scanning figure of the hydrogel that Cys-HA1 and 4-ARM-PEG-S-S is formed；

Figure 32 is the rheology stress scans figure of the hydrogel that Cys-HA1 and 4-ARM-PEG-S-S is formed；

Figure 33 is the rheology time sweep figure of the hydrogel that Cys-HA2 and 4-ARM-PEG-S-S is formed；

Figure 34 is the rheology frequency scanning figure of the hydrogel that Cys-HA2 and 4-ARM-PEG-S-S is formed；

Figure 35 is the rheology stress scans figure of the hydrogel that Cys-HA2 and 4-ARM-PEG-S-S is formed；

Figure 36 is the rheology time sweep figure of the hydrogel that Cys-HA3 and 4-ARM-PEG-S-S is formed；

Figure 37 is the rheology frequency scanning figure of the hydrogel that Cys-HA3 and 4-ARM-PEG-S-S is formed；

Figure 38 is the rheology stress scans figure of the hydrogel that Cys-HA3 and 4-ARM-PEG-S-S is formed；

Figure 39 is the rheology time sweep figure of the hydrogel that Cys-HA4 and 4-ARM-PEG-S-S is formed；

Figure 40 is the rheology frequency scanning figure of the hydrogel that Cys-HA4 and 4-ARM-PEG-S-S is formed；

Figure 41 is the rheology stress scans figure of the hydrogel that Cys-HA4 and 4-ARM-PEG-S-S is formed；

Figure 42 is the rheology time sweep figure of the hydrogel that Cys-HA5 and 4-ARM-PEG-S-S is formed；

Figure 43 is the rheology frequency scanning figure of the hydrogel that Cys-HA5 and 4-ARM-PEG-S-S is formed；

Figure 44 is the rheology stress scans figure of the hydrogel that Cys-HA5 and 4-ARM-PEG-S-S is formed；

Figure 45 is the rheology time sweep figure of the hydrogel that Cys-HA6 and 4-ARM-PEG-S-S is formed；

Figure 46 is the rheology frequency scanning figure of the hydrogel that Cys-HA6 and 4-ARM-PEG-S-S is formed；

Figure 47 is the rheology stress scans figure of the hydrogel that Cys-HA6 and 4-ARM-PEG-S-S is formed；

Figure 48 is the rheology time sweep figure of the hydrogel that Cys-HA7 and 4-ARM-PEG-S-S is formed；

Figure 49 is the rheology frequency scanning figure of the hydrogel that Cys-HA7 and 4-ARM-PEG-S-S is formed；

Figure 50 is the rheology stress scans figure of the hydrogel that Cys-HA7 and 4-ARM-PEG-S-S is formed；

Figure 51 is the comparison rheology time sweep figure of the Cys-HA of 5% numbering 8；

Figure 52 is the time diagram that different modifying rate Cys-HA and 4-ARM-PEG-S-S are cross-linked to form hydrogel；

Figure 53 is the hyaluronic acid gel master drawing being formed；

Figure 54 is the RIN of different modifying rate Cys-HA and 4-ARM-PEG-S-S cross-linked hydrogel and interpolation active polypeptide M5f CCK lab diagram；

Figure 55 is HA hydrogel culture RIN m5f cell fluorescence figure (1 equivalent)；

Figure 56 is HA hydrogel culture RIN m5f cell fluorescence figure (1 equivalent) adding 1%GRGDSPG；

Figure 57 is HA hydrogel culture RIN m5f cell fluorescence figure (1 equivalent) adding 1%Exenatide；

Figure 58 be add 1%Exenatide+1%GRGDSPG HA hydrogel culture RIN m5f cell fluorescence figure (1 work as Amount)；

Figure 59 is insulin secretion canonical plotting；

Figure 60 is that 3.3mmol/l glucose sugar stimulates RIN m5f cell insulin secretion figure；

Figure 61 is that 16.7mmol/l glucose sugar stimulates RIN m5f cell insulin secretion figure.

Specific embodiment

N=200-4000 in formula.

Cysteine its synthetic route of functionalization conjugates of hyaluronic acid of mercaptan-alkene clicking chemistry preparation is as follows：

1) structural formula is taken to beCompound 1 hyaluronic acid with structural formula beCompound 2, by cryodesiccated method formed structural formula be's Compound 3；

Preferably, step 1) in, the molecular weight of compound 1 hyaluronic acid is 8～1,600,000.

Preferably, step 1) in, its modification rate scope of compound 3 is 4.03～64.11%；Step 4) in, compound 8 its The scope of modification rate is 2.33～45.67%.

Preferably, described Polyethylene Glycol its structural formula of oxygen ester isN in formula =50～500, m=2～8.

Preferably, the preparation method of described Injectable in-situ hydrogel, step 1) and 2) in, the pH of phosphate buffer For 7.0～7.6, the solution concentration being made into is 1～20%；Step 3) in, conjugates of hyaluronic acid and Polyethylene Glycol oxygen ester Mol ratio 1: (0.1～1), the time range that hydrogel is formed is 5～8000 seconds.

Below by way of specific embodiment, present disclosure is described in further detail.

Embodiment 1

1) synthesis of compound 3

The synthetic schemes of table 1 compound 3

Remarks：1g 100,000 hyaluronic acid contains 2.636mmol primary hydroxyl group；Allyl glycidyl ether molecular weight： 114.14g/mol density：0.962g/ml boiling point：154℃(lit.)

Take hyaluronic acid (100kDa) 14g, be dissolved in ultra-pure water 700ml and be configured to concentration in 1000ml round-bottomed flask and be The hyaluronic acid aqueous solution of 2% (w/v), 7 numberings of preparation are 1～7 250ml round-bottomed flask, and each flask loads 100ml 2% hyaluronic acid solution is standby, takes the allyl glycidyl ether volumetric flask of 9.76ml to add ultra-pure water to be settled to 100ml Mix homogeneously, adds, to 1～No. 7 round-bottomed flask, the allyl glycidyl ether aqueous solution corresponding to equivalent volume, is stirred at room temperature mixed Close uniformly, be transferred in culture dish and put into -80 DEG C of freezings, lyophilization, obtain a series of allyl glycidol of modification rates Ether conjugates of hyaluronic acid, and its modification rate is obtained by magnetic resonance detection.

Magnetic resonance detection is carried out to compound 3, in figure signal is analyzed belong to,¹H NMR(D₂O) it is shown in δ Pi-allyl (CH occurs at 5.88～5.98ppm₂=CH₂-CH₂- O-) signal, so can determine whether that this compound is compound 3, leads to Cross, at comparison δ 5.88～5.98ppm, pi-allyl (CH occurs₂=CH₂-CH₂- O-) hyaluronic acid at signal and δ 1.95ppm Methyl (- NH-COCH in middle acetylamino₃) signal ratio can calculate this step reaction modification rate be 4.03%～64.11%.

2) synthesis of compound 6

The synthetic schemes of table 2 compound 6

Take Boc-Cys (Acm)-OH 20g (compound 4,223.28g/mol, 68.41mmol) in 1000ml round-bottomed flask Middle use DCM 300ml dissolving, be followed by stirring for PyBOP 39.16g that additions DCM 200ml dissolves (520.40g/mol, 68.41mmol x1.1eg) solution, it is subsequently agitated for adding triethylamine 19.06ml (101.19g/mol, 68.41mmol x2eq), Addition solution with triethylamine can be changed into clarifying from muddiness, and next stir-activating 10 minutes is slowly added dropwise and uses DCM 200ml Dissolving has removed the cystamine 4.88ml (152.28g/mol, 68.41mmol x0.55eq) of dihydrochloride.2-aminoethyl disulfide dihydrochloride is joined According to Ivan S.Alferiev (Alferiev I S, Connolly J M, Levy R J.A novel mercapto- bisphosphonate as an efficient anticalcification agent for bioprosthetic Tissues [J] .Journal of Organometallic Chemistry, 2005,690 (10)：2543-2547.) described side It is standby that method removes hydrochlorate.After cystamine completion of dropping, reaction stirring reaction 3 hours under the conditions of room temperature, argon, course of reaction is used Silica gel 60 F254 silica gel thin-layer chromatography and acid ninhydrine detection, TLC developing solvent is methylene chloride-methanol-acetic acid (36%) (100: 6: 1), the dense removing solvent that reduces pressure after the completion of reaction, to being reduced to small size, is acidified with 0.1mol/l aqueous hydrochloric acid solution After pH to 6～7, with DCM, it is extracted, adopt 10mmol/1HCl, 5%NaHCO afterwards₃, H₂O extraction three each to organic layer Secondary, finally use saturation NaCl solution that organic layer is extracted once, 2h is dried to the organic layer anhydrous magnesium sulfate after extraction, decompression Concentrate and remove solvent, obtain oily compounds 6 (21.58g, yield 90%) and obtain white chunks after purification with silica gel chromatographic column Solid.¹H NMR (500MHz, DMSO)：δ 8.52 (s, 1H, CH₃CONH-), 8.02 (s, 1H ,-CONHCH₂-), 6.98 (d, J= 8.3Hz, 1H ,-OCONHCH-), 4.30-4.13 (m, 2H ,-NHCH ₂S-), 4.08 (d, J=5.3Hz, 1H ,-OCONHCH-), 3.31-3.16 (m, 2H ,-CONHCH ₂-), 2.74 (ddd, J=58.7,13.6,9.1Hz, 2H ,-SCH ₂CH-), 2.40 (d, J= 7.9Hz, 2H ,-CH ₂SH), 1.84 (s, 3H, CH ₃CONH-), 1.38 (s, 9H, (CH ₃ ) ₃OCO-) (see accompanying drawing 3).

3) compound 7 synthesizes

The synthetic schemes of table 3 compound 7

Take compound 620g (700.95g/mol, 28.53mmol) in the round-bottomed flask of 1000ml, molten with acetonitrile 400ml Solution, takes 9g tri- (2- carbonylethyl) microcosmic salt hydrochlorate (TCEP.HCl, 286.65g/mol, 28.53mmol x1.1eq) to be gone with 50ml Ion water dissolution adds above-mentioned acetonitrile solution, and reaction is stirred 2 hours under the conditions of room temperature, argon, course of reaction Silica Gel 60 F254 silica gel thin-layer chromatography chromatograph and acid ninhydrine, the detection of Ellman reagent, TLC developing solvent is dichloromethane-first Alcohol-acetic acid (36%) (100: 6: 1), reduce pressure after the completion of reaction dense removing solvent, is acidified pH to 3 with 0.1mol/l aqueous hydrochloric acid solution After～6, with DCM, it is extracted, adopt 5%NaHCO afterwards₃, 10mmol/l HCl, H₂O extraction each to organic layer three times, Finally with saturation NaCl solution, organic layer is carried out extracting once, concentrating under reduced pressure removes solvent and obtains oily compounds 7 (19.05g, yield 95%), obtains oily liquids after purification with silica gel chromatographic column.¹H NMR (500MHz, DMSO)：δ 8.52 (s, 1H, CH₃CONH-), 8.02 (s, 1H ,-CONHCH₂-), 6.98 (d, J=8.3Hz, 1H ,-OCONHCH-), 4.30-4.13 (m, 2H ,-NHCH ₂S-), 4.08 (d, J=5.3Hz, 1H ,-OCONHCH-), 3.31-3.16 (m, 2H ,-CONHCH ₂-), 2.74 (ddd, J=58.7,13.6,9.1Hz, 2H ,-SCH ₂CH-), 2.40 (d, J=7.9Hz, 2H ,-CH ₂SH), 1.84 (s, 3H, CH ₃CONH-), 1.50 (s, 1H ,-CH₂SH), 1.38 (s, 9H, (CH ₃ ) ₃OCO-) (see accompanying drawing 4).

4) synthesis of compound 8

The synthetic schemes of table 4 compound 8

Remarks：Compound 7 (Boc-Cys (Acm)-cysteamine-SH) molecular weight：351.13g/mol：Dimethoxybenzoin (DMPA) molecular weight：256.30g/mol.

The synthesis of compound 8 is to carry out (Killops K L, Campos L M, Hawker with reference to mercaptan-alkene clicking chemistry C J.Robust, Efficient, and Orthogonal Synthesis of Dendrimers via Thiol-ene " Click " Chemistry [J] .Journal of the American Chemical Society, 2008,130 (15)： 5062-4.).Take each 2g ultra-pure water of allyl glycidyl ether hyaluronic acid (compound 3) of above-mentioned seven kinds of different modifying rates It is configured to the hyaluronic acid aqueous solution that concentration is 2% (w/v), 7 numberings of preparation are the 250ml quartz beaker with cover of 1～No. 7, The corresponding modification rate allyl glycidyl ether hyaluronic acid of each flask loading is standby, takes compound 737.55g 78ml acetonitrile Dissolving, adds, to 1～No. 7 quartz beaker, compound 7 acetonitrile solution corresponding to equivalents and volume number, mixing is stirred at room temperature all Even, take dimethoxybenzoin 4.22g 39ml acetonitrile to dissolve, add corresponding equivalents and volume to above-mentioned 1～No. 7 quartz beaker Number dimethoxybenzoin (DMPA) acetonitrile solution, after mix homogeneously is stirred at room temperature, after 24h under ultraviolet light UV λ 365nm irradiates, Thiazolinyl blackout in nucleus magnetic hydrogen spectrum, points out mercaptan-alkene clicking chemistry complete.Carry out concentrating under reduced pressure removing organic after reaction completely Solvent, then the bag filter being 10kDa with molecular weight carry out dialysis remove small molecule, dialysis solution be 50% (v/v) ethanol water, The every 6h of dialysis treatment 48h changes a dialysis solution, and last dialysis, using ultra-pure water dialysis 6h, uses Silica during dialysis Gel 60 F254 silica gel thin-layer chromatography plate, acid ninhydrine and Ellman reagent carry out checking whether that dialysis finishes, dialysis completes After carry out freeze-drying process and obtain a series of compounds 8.Pass through its modification rate of magnetic resonance detection again.

Magnetic resonance detection is carried out to compound 8, in figure signal is analyzed belong to, 1H NMR (D₂O) it is shown in δ Tertbutyloxycarbonyl (Boc) signal occurs, so can determine whether that this compound is compound 8, by comparing δ 1.37ppm at 1.37ppm Methyl (- NH-COCH in acetylamino in hyaluronic acid at tertbutyloxycarbonyl (Boc) signal and δ 1.95ppm in place₃) signal Ratio can calculate the modification rate about 2.33%～45.67% of this step reaction.

5) synthesis of compound 9

The synthetic schemes of table 5 intermediate 8a

A. take off Boc protection group to take above-mentioned seven kinds of each 2g of different modifying rate compound 8 to number is 1～7, and each sample adds 20ml trifluoroacetic acid makes it dissolve, after stirring reaction 2h under conditions of being stirred at room temperature, solution carry out concentrating under reduced pressure dry in Mesosome 8a.

The synthetic schemes of table 6 compound 9

Remarks：2Na₂S₂O₃+I₂=Na₂S₄O₆+2NaI

B. take off Acm protection group to 1～No. 7 sample taking off the intermediate 8a obtaining after Boc protection group is processed through trifluoroacetic acid Product load in the round-bottomed flask of 250ml, adjust pH to neutral with 1%NaOH, take iodine 14.99g 168ml ethanol to dissolve, Add the iodohydrin solution of corresponding equivalents and volume number to the round-bottomed flask of 1～No. 7 250ml, reaction 4h, phase are stirred at room temperature Between, find in round-bottomed flask, have brown gel to produce, be added thereto to the mercaptoethanol aqueous solution of certain volume until gel molten Solution, after the completion of reaction, takes sodium thiosulfate (Na₂S₂O₃) 19.61g with after the ultrapure water dissolution of 84ml to above-mentioned 1～No. 7 round bottom Flask adds corresponding equivalents and the sodium thiosulfate solution of volume to be neutralized drop reaction, removes responseless iodine, After stirring reaction 2h, carry out concentrating under reduced pressure and remove solvent, then the bag filter dialysis being 10kDa with molecular weight removes small molecule, thoroughly Analysis liquid is 50% (v/v) ethanol water, and the every 6h of dialysis treatment 48h changes a dialysis solution, and last dialysis is using ultrapure Water dialysis 6h, is entered with Silica gel 60F254 silica gel thin-layer chromatography plate and acid ninhydrine and Ellman reagent during dialysis Row checks whether that dialysis finishes.Carry out lyophilization after the completion of dialysis, obtain a series of different modifying rate products 9.By to 1H NMR(D₂O) in collection of illustrative plates, signal is analyzed belonging to, on the basis of the proton nmr spectra of compound 8 at δ 1.37ppm Tertbutyloxycarbonyl signal (Boc) disappear, can determine whether this compound 8 deprotection completely, obtained compound is target product Compound 9.

Embodiment 2

Logical peroxy esters mediate the covalent cross-linking hyaluronic acid gel tool that nature chemistry coupled reaction Injectable in-situ is formed Body route is as follows：

Experimental procedure：

Solution A：Weigh a series of cysteine conjugates of hyaluronic acid (Cys-HA) of modification rates of embodiment 1 synthesis 35mg phosphate buffer (pH7.6) 350ul is dissolved in 1ml centrifuge tube.

B solution：Weigh 35mg 10k tetra- branch's Polyethylene Glycol oxygen ester conjugate (4-ARM-PEG-S-S) and use phosphate-buffered Liquid (pH7.6) 350ul is dissolved in 1ml centrifuge tube.

Solution A is mixed homogeneously using vortex mixer is rapid with B solution, adds and proceeds by test on flow graph.Advanced Row time sweep (Time sweep), performs a scan (Frequency sweep), finally when modulus curve no longer changes Carry out strain sweep (Strain sweep), parameter setting is as described below.

In rheology test, parameter is set to, scan pattern：Vibration；Rotor：PP25, PP spacing (gap) 1mm；Data takes Dot frequency：1/20s；Test temperature：20℃.

Time sweep (time sweep)：Frequency (f) is 1Hz, and stress (Strain) is 1%, and Time constant point fetches data；

Frequency sweep (frequency scans)：Frequency (f) is 10～0.01Hz, and stress (Strain) is 1%, takes altogether 19 points, No time Setting；

Strain sweep (stress scans)：Frequency (f) is 1Hz, and stress (Strain) is 0.1%～100%, takes 19 altogether Individual, No time Setting.

Embodiment 3

Cysteine conjugates of hyaluronic acid solution and four branch's Polyethylene Glycol oxygen ester conjugate (4-ARM-PEG-S-S) shapes Hydrogel mode is become to verify.

A. cysteine conjugates of hyaluronic acid (Cys-HA) 35mg weighing the maximum modification rate of synthesis in embodiment 1 uses Phosphate buffer (pH7.6) 700 μ l is dissolved in 1ml centrifuge tube, carries out time sweep (Time sweep) test of abundance, Time sweep (Time sweep) condition is consistent with embodiment 2, no gel formation.Result is shown in accompanying drawing 51.

B. the hydrogel that in embodiment 2, solution A is formed with B solution is put in enough TCEP.HCl solution and overnight see Examine, find or gel state.

Embodiment 4

With reference to embodiment 2, structural formula isN=50～500, m 10k tetra- branch's Polyethylene Glycol oxygen ester conjugate (4-ARM-PEG-S-S) in B solution in=2～8 compound 10 alternative embodiment 6.

Embodiment 5

RIN m5f cell CCK tests

CCK tests Cleaning Principle

Cell Counting Kit abbreviation CCK test kit, is a kind of based on WST-8 (chemical name：2- (2- methoxyl group -4- Nitre phenyl) -3- (4- nitre phenyl) -5- (2,4- disulfobenzene) -2H- tetrazolium monosodium salt) be widely used in cell proliferation and thin The fast high-sensitive degree detection kit of cellular toxicity.WST-8 belongs to the upgrading products of MTT, and operation principle is：In electronics coupled examination In the presence of agent, the orange-yellow first product generating high water soluble can be reduced by Intramitochondrial dehydrogenase.Face The depth of color is directly proportional to the propagation of cell, is inversely proportional to cytotoxicity.Measure OD value using microplate reader at 450nm wavelength, Reflection living cells quantity indirectly.

RIN m5f cell culture experiments

Numbering 2～7 cysteine conjugates of hyaluronic acid Cys-HA is made into concentration with the phosphate buffer of pH7.6 and is 10% (w/v) solution, every part of every hole 250ul is injected on 96 well culture plates in 5 holes totally, four branch's Polyethylene Glycol oxygen ester conjugates (4-ARM-PEG-S-S) also it is made into 10% (w/v) solution with same method, every part equally takes 250ul to mix with above-mentioned 2～No. 7 After closing uniformly, then inject 1%Ac-CGRGDSPG-NH to every hole is corresponding₂Or 1%Exenatide-NH₂, mix homogeneously, 30 minutes Washed with PBS 3 times, 250ulRIN m5f cell solution is injected in the most every hole, cell concentration is 5000/cm later², cell At the standard conditions (37 DEG C, 5%CO₂) cultivate 48 hours.

CCK experimental procedure

Add 10ul CCK solution in every group of corresponding 96 orifice plates, notice that the process adding must not generate gas in hole Bubble, they can affect the reading of OD value, add after finishing by culture plate be placed in incubator interior be incubated 1～4 hour (37 DEG C, 5% CO₂), and then measure the absorbance at 450nm with microplate reader, lived using the cell that below equation carries out calculating every group Power.

Cell viability (%)=[A (dosing)-A (blank)]/[A (0 dosing)-A (blank)] × 100

A (dosing)：There is the absorbance in the hole of cell, CCK solution and drug solution

A (blank)：There is culture medium and CCK solution without the absorbance in the hole of cell

A (0 dosing)：There is cell, CCK solution without the absorbance in the hole of drug solution

Embodiment 6

RIN m5f cell fluorescence is tested

Fluorescence experiments principle

Calcein-AM is that one kind can carry out fluorescently-labeled cell staining reagent to living cells, due in Calcein (calcium Yellowish green element) on the basis of strengthen hydrophobicity, therefore, it is possible to penetrate living cells film easily.After it enters into Cytoplasm, esterase Calcein (calcein) can be hydrolyzed to stay intracellular and complete membrane structure cannot be diffused out, so can illustrate Cell viability, can point out the integrity of cell again.Ethidium homodimer-1 is a kind of impermeable nucleic acid dye of film, it Damaged cell membrane and nuclear membrane can be entered and DNA is dyeed, therefore dead cell can be identified.Both dyestuffs are all in 495nm Place excites, Calcein-AM green light at 515nm, and Ethidiumhomodimer-1 glows at 635nm.

Fluorescence experiments step

RIN m5f cell culture experiments as described above, in every group of corresponding 96 orifice plates every hole add 2ul pH7.6 Phosphate buffer configuration 2mMCalcein-AM and 2mM Ethidium homodimer-1 solution, add finish after will Culture plate be placed in incubator interior incubation 0.5 hour (37 DEG C, 5%CO₂), and then with every group of cell life of fluorescence microscope Long situation.

Embodiment 7

RIN m5f cell insulin secretion is tested

Insulin secretion experimental principle

Experiment is using rat insulin (Insulin) water in double antibody sandwich method enzyme linked immunosorbent assay specimen Flat.It is coated microwell plate with rat anti-insulin (Insulin) antibody of purification, makes insolubilized antibody, toward the micropore being coated monoclonal antibody In sequentially add the pancreas of insulin (Insulin) and horseradish peroxidase (Horseradish Peroxidase, HRP) labelling Island element antibodies, form antibody-antigene-hrp-antibody complex, add substrate TMB colour developing after thorough washing.TMB exists Convert au bleu under the catalysis of HRP enzyme, and change into final yellow under the action of an acid.Pancreas in the depth and sample of color Island element (Insulin) is proportionate.With microplate reader under 450nm wavelength mensuration absorbance (OD value), calculated by standard curve Rat insulin (Insulin) concentration in sample.

Insulin secretion experimental procedure

1. the dilution of standard substance and sample-adding：It is coated on plate in enzyme mark and is marked with quasi- sample wells 10 hole, in first, second hole respectively Plus standard substance 100ul, then in first, second hole, add standard dilutions 50ul, mix；Then from the first hole, the second hole In respectively take 100ul to be added separately to the 3rd hole and the 4th hole, then add standard dilutions 50ul respectively in the three, the 4th holes, mix； Then first respectively take 50ul to discard in the 3rd hole and the 4th hole, respectively take 50ul to be added separately in the five, the 6th holes, then the 5th, add standard dilutions 50ul respectively in the 6th hole, mix；50ul is respectively taken to be added separately to from the five, the 6th holes after mixing 7th, in octal, then add standard dilutions 50ul respectively in the 7th, octal, divide from the 7th, octal after mixing Do not take 50ul to be added in the nine, the tenth holes, then add standard dilutions 50ul respectively in the 9th the tenth hole, from the nine the after mixing 50ul is respectively taken to discard in ten holes.After dilution, each hole sample-adding amount is all 50ul, and concentration is respectively 48mu/l, 24mu/l, 12mu/l, 6mu/l, 3mu/l, 0mu/l.

2. preparation of samples：Numbering 2～7 cysteine conjugates of hyaluronic acid Cys-HA phosphate buffer of pH7.6 Being made into concentration is 10% (w/v) solution, and every part of every hole 500ul is injected on 48 well culture plates in 5 holes totally, four branch's Polyethylene Glycol oxygen Ester conjugate (4-ARM-PEG-S-S) is also made into 10% (w/v) solution with same method, and every part equally takes 500ul and above-mentioned 2 After～No. 7 mix homogeneously, then inject 1%Ac-CGRGDSPG-NH to every hole is corresponding₂Or 1%Exenatide-NH₂, mix homogeneously, Wash 3 times after 30 minutes with PBS, the most every hole injection 500ulRIN m5f cell solution, cell concentration for 1000/ cm², cell standard conditions (37 DEG C, 5%CO₂) under after culture 48 hours, remove culture medium, then with Ke-woods Er Shi heavy carbonate Buffer (Krebs-Ringer bicarbonate buffer) washs 2 times.Use KRBB (Krebs-Ringer bicarbonate buffer) configuration concentration 3.3 and 16.7mmol/l glucose sugar juice are placed on incubator (37 DEG C, 5%CO₂) in culture 30 minutes, every group of cell is first 3.3mmol/l glucose sugar balance 30 minutes with 500ul concentration, so After add 500ul concentration be 3.3mmol/l glucose sugar and incubator (37 DEG C, 5%CO₂) in culture 60 minutes.Cultivate Bi Hou.Take 500ul solution ice bath in centrifuge tube to preserve from every group of corresponding aperture, be centrifuged 5 minutes at 2500rpm0 DEG C, every group takes The supernatant of 200ul is used for the mensure of insulin, removes remaining solution in every group of 48 orifice plates, adding 500ul concentration is 16.7mmol/l glucose sugar and incubator (37 DEG C, 5%CO₂) in culture 60 minutes, subsequently according to ELISA kit using saying Bright book is operated.

Here is that a kind of mercaptan-alkene clicking chemistry modifies the cysteine functionalization hyalomitome being formed to examples detailed above synthesis The rheological property of the hydrogel that acid conjugate, the Injectable in-situ of preparation are formed, islet cell culture is tested, insulin secretion Experiment detection.

First, mercaptan-alkene clicking chemistry modifies the cysteine functionalization conjugates of hyaluronic acid modification rate being formed

Contrast accompanying drawing 5～11 and accompanying drawing 12, the signal of the proton nmr spectra of compound 3 is analyzed belonging to.¹H NMR(D₂O) it is shown at δ 5.88～5.98ppm and pi-allyl (CH occurs₂=CH₂-CH₂- O-) signal, so can determine whether this change Compound is compound 3, pi-allyl (CH by comparing at δ 5.88～5.98ppm₂=CH₂-CH₂- O-) signal and δ Methyl (- NH-COCH in acetylamino in hyaluronic acid at 1.95ppm₃) signal ratio can calculate this step reaction modification rate About 4.03%～64.11% (see accompanying drawing 20)；

Accompanying drawing 13～19 is the proton nmr spectra of compound 8, in figure signal is analyzed belong to,¹H NMR(D₂O) show Show that tertbutyloxycarbonyl (Boc) signal occurs at δ 1.37ppm, so can determine whether that this compound is compound 8, by comparing δ Methyl (- NH- in acetylamino in hyaluronic acid at tertbutyloxycarbonyl (Boc) signal and δ 1.95ppm occurs at 1.37ppm COCH₃) signal ratio can calculate this step reaction modification rate about 2.33%～45.67% (see accompanying drawing 20).

2nd, mercaptan-alkene clicking chemistry modifies the cysteine functionalization conjugates of hyaluronic acid GPC result being formed

Table 7. dextran standard substance GPC data

The GPC data of table 8.Cvs-HA* and HA (compound 1)

Cys-HA*：Cysteine functionalization conjugates of hyaluronic acid (compound 9), surveys its molecular weight (see accompanying drawing with GPC 21, Figure 22～29), result show measured by the molecular weight of sample and the molecular weight of hyaluronate sodium (compound 1) compare, transparent Matter acid molecule does not diminish after standing the chemical modification of the present invention or degrades, and the molecular weight of some samples is slightly larger than hyaluronic acid Raw material.

3rd, lead to peroxy esters mediation be naturally connected chemically crosslinking Injectable in-situ formed hyaluronic acid gel and its Research on The Rheology

Table 9 different modifying rate Cys-HA and 4-ARM-PEG-S-S are cross-linked to form the time of hydrogel

Remarks：Lg 100,000 HA contains 2.636mmol primary alconol；Numbering 8 is controlled trial, the Cys- of the maximum modification rate of checking HA forms hydrogel not over disulfide bond crosslinking within the regular hour.

Can be seen that from the formation rheology scanning figure (see accompanying drawing 30～50) of hydrogel：2～No. 7 cysteine of numbering Conjugates of hyaluronic acid solution and four branch's Polyethylene Glycol oxygen ester conjugate (4-ARM-PEG-S-S) solution reactions were at 6 hours Intersection point in G ' and G in testing time ", and this puts as gel point, the time point that is, hydrogel is formed, gel time be 70s～ Between 8145s (see accompanying drawing 52), increase over time, G ' is in ascendant trend, is finally gradually up to the standard, illustrate crosslinked anti- Should be reacted complete.The time that 2～No. 7 gel points of numbering occur gradually successively decreases with the increase of modification rate, only allyl Base glycidyl ether is all not have gel point during 0.1 equivalent within the testing time of 5 hours, and the rheology being 8 from numbering Experiment is it can be seen that the cysteine conjugates of hyaluronic acid 6 hours 40 minutes under the same conditions of same concentrations maximum modification rate All there is no the appearance (see accompanying drawing 51) of gel point, the hydrogel that numbering 2～7 is formed overnight is still in TCEP.HCl solution Hydrogel state, can illustrate 2～No. 7 poly- second of cysteine conjugates of hyaluronic acid solution and four branches of numbering from these two aspects The hydrogel that glycol oxygen ester conjugate (4-ARM-PEG-S-S) is formed is logical peroxy esters mediation nature chemistry coupled reaction, and It is not the covalent cross-linking hydrogel that cysteine sulfydryl passes through that disulfide bond is formed.

4th, RIN m5f cell CCK experimental result

Accompanying drawing 53 is the hyaluronic acid gel master drawing being formed.

Carry out vertical coordinate contrast from accompanying drawing 54：We have found that under identical modification rate by CCK experimental cell, numbering 2～7 Cysteine conjugates of hyaluronic acid and four branch's Polyethylene Glycol oxygen ester conjugate (4-ARM-PEG-S-S) solution lead to peroxy esters and are situated between Lead nature be connected chemically formation covalent cross-linking hydrogel carry out in RIN m5f cell culture, (1) add cell adhesion molecules thin Born of the same parents' vigor substantially has close to 30%～55% cell rate of increase than do not add, illustrates cell adhesion molecules with hyalomitome Acid-polyethylene glycol hydrogel is to play positive role in RIN m5f cell culture in cell scaffold material.(2) adding cell Exenatide-NH in the experimental group of adhesion factor₂Compare Ac-CGRGDSPG-NH₂High close to RIN m5f cell rate of increase 20%, cell viability relatively, illustrates Exenatide-NH compared with both jointly interpolation group₂Compare Ac-CGRGDSPG-NH₂More RIN m5f cell growth can be stimulated to breed.

Abscissa contrasts：Same experimental group be we have found that by CCK experimental cell, numbering 2～7 half Guang under different modifying rate Propylhomoserin conjugates of hyaluronic acid and four branch's Polyethylene Glycol oxygen ester conjugate (4-ARM-PEG-S-S) solution lead to peroxy esters mediation certainly So being connected chemically formation covalent cross-linking hydrogel, to carry out cell viability in RIN m5f cell culture also variant, when pi-allyl contracting Water glycerin ether be during 1 equivalent of hyaluronic acid primary hydroxyl group (2.636mmol) every group have maximum cell vigor number, thin adding Born of the same parents adhesion factor Ac-CGRGDSPG-NH₂Group is particularly evident, and when modification rate is bigger, every group of cell viability number tends towards stability, explanation When allyl glycidyl ether is 1 equivalent, hyaluronic acid-poly ethylene glycol hydrogel is optimum to RIN m5f cell culture Modification rate.

5th, RIN m5f cell fluorescence experimental result

From accompanying drawing 55～58, RIN m5f cell fluorescence experimental result is similar to discussion to CCK experimental result, adds The hydrogel of active polypeptide can promote the growth of cell, polypeptide Exenatide-NH₂Especially pronounced in this respect, modifying Rate aspect fluorescence experiments can show mutual difference in number not as CCK experimental result, but also can find to contract when pi-allyl Water glycerin ether is that during 1 equivalent of hyaluronic acid primary hydroxyl group (2.636mmol), RIN m5f cell has preferable growth conditions.

6th, RIN m5f cell insulin secretion experimental result

Insulin secretion standard curve is shown in accompanying drawing 59, is tested by RIN m5f cell insulin secretion, we can be found that Under the same terms, concentration stimulates the insulin concentration to want than 3.3mmol/l glucose sugar juice for 16.7mmol/l glucose sugar juice Many.

Accompanying drawing 60～Figure 61, vertical coordinate contrasts：Under the sugared and identical modification rate of every group of same concentrations glucose, numbering 2～7 half Guang Propylhomoserin conjugates of hyaluronic acid and four branch's Polyethylene Glycol oxygen ester conjugate (4-ARM-PEG-S-S) solution lead to peroxy esters mediation certainly So it is connected chemically formation covalent cross-linking hydrogel RIN m5f cell insulin secretion experiment：(1) glucose sugar concentration is 3.3mmol/ L, 16.7mmol/l occur adding the cell secretory capacity of cell adhesion molecules than being eager to excel of not adding, and cell adhesion is described Factor RIN m5f cell insulin secretion experiment in hyaluronic acid-poly ethylene glycol hydrogel as cell scaffold material is played Positive role.(2) in two groups of glucose sugar concentration, add Exenatide-NH in the experimental group of cell adhesion molecules₂Compare Ac- CGRGDSPG-NH₂To stimulating RIN m5f cell insulin secretion to be eager to excel, cell viability compares compared with both jointly interpolation group Close, Exenatide-NH is described₂Compare Ac-CGRGDSPG-NH₂RIN m5f cell insulin secretion more can be stimulated.

Abscissa contrasts：Same experimental group be we have found that by RIN m5f cell insulin secretion, compile under different modifying rate The water that number 2～7 cysteine conjugates of hyaluronic acid and four branches' Polyethylene Glycol oxygen ester conjugate (4-ARM-PEG-S-S) are formed Gel carries out insulin concentration in RIN m5f cell insulin and has differences, when allyl glycidyl ether is hyaluronic acid primary During 1 equivalent of alcoholic extract hydroxyl group (2.636mmol), two groups of insulin secretions are with the presence of Cmax, when modification rate is bigger every group thin Born of the same parents' vigor number tends to downward trend, illustrates when allyl glycidyl ether is 1 equivalent, hyaluronic acid-poly ethylene glycol hydrogel It is optimal modification rate to RIN m5f cell insulin secretion.

In sum, the invention has the characteristics that：

1. the hydroxyl of directed modification hyaluronic acid, retain its free carboxyl, thus being conducive to it to be combined with cell CD44, Obtain having more preferable biocompatibility by the conjugates of hyaluronic acid stablizing ehter bond connection, and obtained hyaluronic acid spreads out Biology has cysteamine group, not only increase the adhesion of hyaluronic acid, and being also provided for can chemical modification further Functional group, and this cysteine conjugates of hyaluronic acid can be additionally used in formed Injectable in-situ formed covalent cross-linking saturating Bright matter acid hydrogel, and as timbering material the culture of islet cellss and the secretion of insulin application.

2. the present invention prepares the hydroxyl with the N-ACETYL-D-GLUCOSAMINE of hyaluronic acid using cryodesiccated method Base reaction forms a series of pi-allyl bright matter acid derivative of the modification rates stablizing ehter bond, and freeze-drying method is from now in polysaccharide And other macromole modification chemistry provides new thinking.

3. modified using mercaptan-alkene clicking chemistry and the cysteine derivative of synthesis be connected on pi-allyl hyaluronic acid, Finally obtain the cysteine conjugates of hyaluronic acid with different modifying rate, this route is different from other hyaluronic acids present Functional method is a kind of new innovation synthetic route, and for the first time using in the modification chemistry of polysaccharide, is hyaluronic acid Modification and the modification of other polysaccharide provide new method reference.

4. cysteine conjugates of hyaluronic acid carries cysteamine group, not only increases the adhesion of hyaluronic acid, also Be provided for can chemical modification further functional group, and lead to peroxy esters mediation naturalization with four branch Polyethylene Glycol oxygen esters Learn connection to prepare injectable, be formed in situ the new active sulfydryl of hyaluronic acid gel of covalent cross-linking, increased transparent The adhesion of matter acid is used for cell culture, also can carry out functional modification further, such as be combined with medicine, plays medicine and delays control The effect released.

5. the alcohol in ester structure can be discharged in solution when OMNCL reacts, when designing ester, can be this alcohol Be designed as the precursor of hydroxylated medicine or cell growth factor, thus while hydrogel is formed, discharge these medicines or Somatomedin is in hydrogel, thus increased the application in biomedicine field for the hyaluronic acid.

6. rheol test in, add reaction allyl glycidyl ether amount from 0.5～5 equivalent, hydrogel Being formed is all to react covalent cross-linking rather than disulfide bond by OMNCL, and modification rate is bigger, and the time needed for crosslinking also gets over Few, the hydrogel being formed has good rheological property, has the Optimalities such as gel time is controlled, mechanical performance is controlled Matter.

7., in the RIN m5f cell culture experiments of hydrogel and the experiment of RIN m5f cells secrete insulin, find hyalomitome Acid-polyethylene glycol hydrogel in cell culture as timbering material importance, and cell adhesion molecules cell give birth to Meaning played in growth process.

8. pass through to add allyl glycidyl ether 1 equivalent to modify the hyaluronic acid gel obtaining to RIN m5f cell Vigor and RIN m5f cell insulin secretion all obtain optimal result.Thus being that the cysteine being synthesized by the present invention is transparent Matter acid conjugate and with the hydrogel of Polyethylene Glycol covalent cross-linking islet cellss transplanting, the treatment of diabetes and other doctors The application in medicine field provides new direction.

Claims

1. a kind of cysteine functionalization conjugates of hyaluronic acid by lyophilization and mercaptan-alkene clicking chemistry preparation, its It is characterised by：Structural formula is：

N=200-4000 in formula.

2. described in claim 1, a kind of cysteine functionalization prepared by lyophilization and mercaptan-alkene clicking chemistry is transparent The preparation method of matter acid conjugate it is characterised in that：Comprise the following steps：

2) structural formula is taken to beCompound 4 and structural formula beChemical combination Thing 5 forms structural formulaCompound 6；

3) compound 6 and disulfide bond reducing agent generation reduction reaction formation structural formula areChange Compound 7；

4) take compound 3 and compound 7, draw in light and under agent and ultraviolet light, occur mercaptan-alkene clicking chemistry to form structure Formula isCompound 8；

3. a kind of cysteine function by lyophilization and mercaptan-alkene clicking chemistry preparation according to claim 2 Change conjugates of hyaluronic acid preparation method it is characterised in that：Step 1) in, the molecular weight of compound 1 hyaluronic acid is 8～ 1600000.

4. a kind of cysteine function by lyophilization and mercaptan-alkene clicking chemistry preparation according to claim 2 Change conjugates of hyaluronic acid preparation method it is characterised in that：Step 1) in, its modification rate scope of compound 3 be 4.03～ 64.11%；Step 4) in, the scope of its modification rate of compound 8 is 2.33～45.67%.

5. a kind of cysteine functionalization prepared by lyophilization and mercaptan-alkene clicking chemistry described in claim 1 is saturating Bright matter acid conjugate is in the application of the hydrogel preparing Injectable in-situ formation.

6. a kind of cysteine function by lyophilization and mercaptan-alkene clicking chemistry preparation according to claim 5 Change conjugates of hyaluronic acid in the application preparing Injectable in-situ hydrogel it is characterised in that：Described Injectable in-situ water-setting Glue is by the cysteine functionalization hyalomitome prepared with mercaptan-alkene clicking chemistry by lyophilization described in claim 1 Acid conjugate, leads to peroxy esters mediation nature chemistry coupled reaction with polyethylene glycol conjugate and generates.

7. the Injectable in-situ hydrogel described in claim 6 preparation method it is characterised in that：Comprise the following steps：

1) conjugates of hyaluronic acid described in claim 1 and phosphate buffer wiring solution-forming are used；

8. want the preparation method of Injectable in-situ hydrogel described in 7 according to right it is characterised in that：Described Polyethylene Glycol oxygen Its structural formula of ester isN=50～500 in formula, m=2～8.

9. Injectable in-situ hydrogel according to claim 8 preparation method it is characterised in that：Described step 1) and 2), in, the pH of phosphate buffer is 7.0～7.6, and the solution concentration being made into is 1～20%；Step 3) in, hyaluronic acid is tied Compound and the mol ratio 1 of Polyethylene Glycol oxygen ester:(0.1～1), the time range that hydrogel is formed is 5～8000 seconds.

10. a kind of cysteine functionalization prepared by lyophilization and mercaptan-alkene clicking chemistry described in claim 5 is saturating The hydrogel that the Injectable in-situ of bright matter acid conjugate preparation is formed is in the application of culture islet cellss and insulin secretion.