CN106478841B

CN106478841B - By being freeze-dried and the cysteine conjugates of hyaluronic acid of mercaptan-alkene clicking chemistry preparation and its synthetic method and application

Info

Publication number: CN106478841B
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: 2019-02-19
Anticipated expiration: 2036-09-20
Also published as: CN106478841A

Abstract

The invention discloses a kind of cysteine functionalization conjugates of hyaluronic acid and its its synthetic method by being freeze-dried with mercaptan-alkene clicking chemistry preparation, also disclose that conjugates of hyaluronic acid in the application for preparing Injectable in-situ hydrogel, also discloses application of the hydrogel in culture islet cells and insulin secretion of Injectable in-situ formation.For the present invention by freeze-drying and mercaptan-alkene clicking chemistry, obtaining a series of different modifying rates has the cysteine functionalization conjugates of hyaluronic acid for stablizing ehter bond can further functional modification with sulfhydryl reactive group；The conjugates of hyaluronic acid of different modifying rate leads to peroxy esters with polyethylene glycol conjugate and nature is mediated to be connected chemically the hydrogel for reacting to obtain Injectable in-situ and being formed, with good rheological property, wherein free sulfydryl can increase the cell adhesion of hyaluronic acid, cell scaffold material be can be used as in islet cell culture and the application that stimulates insulin secretion, there is wide biological medicine prospect.

Description

By being freeze-dried the cysteine hyaluronic acid with mercaptan-alkene clicking chemistry preparation Conjugate and its synthetic method and application

Technical field

The present invention relates to a kind of freeze-dryings to modify the cysteine functionalization hyaluronic acid knot to be formed with mercaptan-alkene clicking It closes object and its synthetic method and it prepares the application that Injectable in-situ forms hydrogel, belong to biomedicine field.

Background technique

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

Hydrogel is hydrophilic polymer network, can absorb a large amount of moisture, has good biocompatibility.Although Application of the hydrogel in biological medicine achieves huge progress, however, development meets clinical needs: in a mild condition not The hydrogel of the covalent cross-linking formed using toxic reagent Injectable in-situ still suffers from challenge.Most of hydrogel is being organized The major defect of application in terms of engineering is exactly that surgical operation is needed to mediate, and the complexity of surgical procedure is cumbersome undoubtedly to be brought to patient Very big pain and risk.Therefore, the research of injection aquagel comes into the picture, with significant advantage: hydrogel It is formed in situ in vivo, is injected without surgical operation by needle tubing, minimally to invade body tissue, not only risen To therapeutic effect, but also the defect of filling tissue arbitrary shape can be mixed with various growth factors, drug, reach better Therapeutic effect, the height that thus can avoid in surgical procedures is traumatic, especially when the tissue for being used to repair 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 attached 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 the conditions of existing for the alkene, pass through heat Or slough the molecular hydrogen containing sulfydryl under the initiation of light to obtain the free radical of sulfur-bearing, the free radical and carbon-carbon double bond (C= C the living radical centered on carbon) is formed by (anti-Markovnikov) addition of anti-geneva, with another point containing sulfydryl Son occurs chain transfer reaction and obtains addition product and another living radical.Therefore, have using mercaptan-alkene clicking chemistry reaction Simply, efficiently, reliably, the C-S bonding of selectivity, particularly modified polysaccharide class compound tool the advantages of can carry out in aqueous solution There is significant superiority.

Oxygen ester mediate nature be connected chemically (oxo-ester mediated native chemical ligation, OMNCL) it is extension that nature is connected chemically (native chemical ligation) concept.It has been found that ester type compound After the compound of the end N- cysteine in physiological conditions (phosphate buffer solution, pH 7-9) mixing, cysteine The alcohol of sulfydryl and ester type compound exchanges, and spontaneously resets after forming the intermediate of five annulus, generate one it is new Amido bond.This chemical reaction is referred to as oxygen ester and nature chemistry is mediated to connect (oxo-ester mediated native Chemical ligation, OMNCL, are shown in attached drawing 2).OMNCL has several clear advantages: 1. having chemo-selective, esters It only reacts to form new amido bond with the compound of cysteine or N- terminal cysteine, not deposited by other mercaptan and sulfydryl Interference；2. under mild conditions, the highly effective reaction without using the possible toxic compound such as catalyst, initiator；③ From it is other using sulfydryl to be connected chemically the hydrogel that reaction is formed different, be connected chemically reaction naturally in the new amido bond of formation While, a mercapto groups are generated also on skeleton.This sulfydryl can not only be used to according to different purposes further in bone Functionalization hydrogel on frame, moreover, the polymer of sulfhydrylation (Thiolation) has better bioadhesive.4. in addition, esters Alcohol in structure is discharged into solution when OMNCL reacts, then, when designing ester, it is possible to which this alcohol is designed as The precursor of hydroxylated drug and Porcine HGF, thus hydrogel formation while, discharge these drugs and growth because Son is into hydrogel.Therefore, the water for the covalent cross-linking that the Injectable in-situ that the characteristics of being reacted using OMNCL and advantage are obtained is formed Gel has significant superiority.Such as: existing document based on polyethylene glycol, lead to by terminal ester or cysteine functionalization Peroxy esters mediate nature be connected chemically 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 the Portugal N- acetyl-D- Grapes glucosamine and D-Glucose aldehydic acid disaccharide recurring unit composition, are applied to (Allison D in organizational project and regenerative medicine D, Grandeallen K J.Review.Hyaluronan:a powerful tissue engineering tool. [J] Tissue Engineering, 2006,12 (8): 2131-40.).Such as the 1980s, mid-term HA was applied to medicine BeautyWound dressing And as arthritis treatment Medicine listingIn extracellular matrix, hyaluronic acid is the ligand of cell surface receptor CD44, and CD44 receptor is joined With various kinds of cell process, such as cell adhesion, cell migration and increment.Due to its important physiological action, body or the surface of a wound are straight Connecing can inflammatory reaction that is substantially reduced and eliminating tissue, the promotion surface of a wound and wound healing after injecting hyaluronic acid.But due to saturating The deficiencies such as bright matter acid is rapidly removed by hyaluronidase in vivo, and the internal residence time is short, mechanical strength is low, such alleviation Effect is also of short duration, so the covalent modified hyaluronic acid gel for forming Injectable in-situ covalent cross-linking of hyaluronic acid is just shown Show its superiority.

Hyaluronic acid primary chemical modification target spot includes: the hydroxyl of the carboxyl of D-Glucose aldehydic acid, N-ACETYL-D-GLUCOSAMINE The N- acetyl group of base and N-ACETYL-D-GLUCOSAMINE.The HA derivative of clinical use is mostly and repairs to its carboxylic acid and hydroxyl at present It adorns, by esterification (Esterification), amidation (Amidation), open loop (Ring Opening), is crosslinked (Cross-linking), various chemical modification methods such as (Graft) are grafted, functionalization HA derivative is prepared.Wherein to transparent The research of the carboxyl modified of matter acid is relatively more, such as has document by the sumptuous modification of adipic acid diacid, and EDC and HOBt is added and makees For couplant and catalyst, the carboxyl of hyaluronic acid is modified, obtains 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 react to form water-setting by the hydroxyl with hyaluronic acid The cross-linking chemistry of glue reacts, for example methacrylic anhydride and 4- pentenoic acid anhydride is respectively adopted to hyaluronic acid C-6 in Seidlits Primary hydroxyl group carries out esterification modification, and cross-linking reaction finally occurs under ultraviolet irradiation condition, 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.).There are also first with cysteine-modifying glycidol ether and hyalomitome The hydroxyl reaction of acid, then carry out nature and chemically react to form 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 Such as Chinese patent CN 103910886A, based on hyaluronic acid, using the polyethylene glycols after the unilateral modification of cysteine Compound and hyaluronic acid hydroxyl carry out ring-opening reaction and form ehter bond, are passing through nature in conjunction with multi-branched polyethyleneglycol derivative It is connected chemically the hyaluronic acid gel to form Injectable in-situ formation.

The hydrogel that hyaluronic acid is formed after crosslinking should be more satisfactory biologic bracket material.It overcomes day It is rapidly removed in right hyaluronic acid body, the deficiencies such as mechanical strength is low.But it is crosslinked by prepared by the carboxyl of hyaluronic acid Hydrogel, some side reactions may be generated.If some researches show that its carboxyls to be modified by sulphation, hyaluronic acid will lead to Melanoma cells, to cause CD44 receptor that cannot normally identify.Cell CD44 receptor is presently considered to be through the negative of hyaluronic acid Charge and hyaluronic acid act on (Morra M.Engineering of biomaterials surfaces by Hyaluronan. [J] .Biomacromolecules, 2005,6 (3): 1205-1223.), and reduce the negative electrical charge of hyaluronic acid It then will affect the effect of cell CD44 receptor and hyaluronic acid, so that ability (the Herrera M of cell repair 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.), so hyaluronic acid just loses it as the superior of biomaterial Property.And by its hydroxyl react to obtain hyaluronic acid gel generally require to react by ultraviolet lighting or with other methods Through preformed hydrogel, in-situ injection thus can not achieve.Therefore, the present invention is using hyaluronic acid as raw material, by cold The dry hydroxyl with mercaptan-alkene clicking chemistry modification hyaluronic acid N-ACETYL-D-GLUCOSAMINE is lyophilized, it is negative not change its carboxyl institute band Charge obtains having and stablizes ehter bond product, then logical peroxy esters mediate nature to be connected chemically reaction and obtain Injectable in-situ covalently to hand over Joining hyaluronic acid gel has significant ground superiority.

Summary of the invention

The first purpose of this invention is to provide a kind of half Guang by being freeze-dried with mercaptan-alkene clicking chemistry preparation Propylhomoserin functionalization conjugates of hyaluronic acid.Second object of the present invention is to provide a kind of by freeze-drying 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 mention Injectable is being prepared for use by the cysteine functionalization conjugates of hyaluronic acid of freeze-drying and mercaptan-alkene clicking chemistry preparation 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 and insulin secretion Using.

Technical scheme is as follows:

A kind of cysteine functionalization conjugates of hyaluronic acid by being freeze-dried with mercaptan-alkene clicking chemistry preparation, Structural formula are as follows:

N=200-4000 in formula.

It is described a kind of by being freeze-dried in conjunction with cysteine functionalization hyaluronic acid prepared by mercaptan-alkene clicking chemistry The preparation method of object, comprising the following steps:

1) structural formula is taken to be1 hyaluronic acid of compound be with structural formulaCompound 2, forming structural formula by the method for freeze-drying is Compound 3；

2) structural formula is taken to beCompound 4 and structural formula be Compound 5 formed structural formula beCompound 6；

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

4) compound 3 and compound 7 are taken, mercaptan-alkene clicking chemistry occurs in the case where light draws agent and ultraviolet light and is formed Structural formula isCompound 8；

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

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

In step 1), its modification rate range of compound 3 is 4.03~64.11%；In step 4), its modification rate of compound 8 Range be 2.33~45.67%.

It is a kind of to be existed by being freeze-dried the cysteine functionalization conjugates of hyaluronic acid prepared with mercaptan-alkene clicking chemistry Prepare the application of the hydrogel of Injectable in-situ formation.

The Injectable in-situ hydrogel is by described by being freeze-dried half with mercaptan-alkene clicking chemistry preparation Cystine functionalization conjugates of hyaluronic acid leads to peroxy esters with polyethylene glycol conjugate and nature is mediated to be connected chemically and react generation.

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

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

2) with polyethylene glycol oxygen ester and phosphate buffer wiring solution-forming；

3) two kinds of solution are mixed, by being connected chemically reaction naturally, prepares the hydrogel of Injectable in-situ formation.

Described its structural formula of polyethylene glycol oxygen ester isIn formula N=50~500, m=2~8.

The preparation method of the 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%；In step 3), the molar ratio 1 of conjugates of hyaluronic acid and polyethylene glycol oxygen ester: (0.1~1), the time range that hydrogel is formed are 5~8000 seconds.

A kind of cysteine functionalization hyaluronic acid knot by being freeze-dried with mercaptan-alkene clicking chemistry preparation Close application of the hydrogel in culture islet cells and insulin secretion of the Injectable in-situ formation of object preparation.

The beneficial effects of the present invention are: first passage freeze-drying and mercaptan-alkene clicking chemistry are modified to obtain cysteine Functionalization conjugates of hyaluronic acid, mercapto groups can be chemically reacted with sulfhydryl reactive group, can further rhetorical function Change；Have found for the first time the different modifying rate for preparing according to different needs that (is freeze-dried) under mild conditions (2.33~ 45.67%) method of functionalization conjugates of hyaluronic acid；This method should can be used for the structural modification and function of other polysaccharide Change；The cysteine functionalization conjugates of hyaluronic acid and polyethylene glycol conjugate of different modifying rate lead to peroxy esters and mediate naturalization The hydrogel that connection is prepared for Injectable in-situ formation is learned, there is good rheological property；This kind of hydrogel has free Sulfydryl can increase the cell adhesion of biomaterial, can be used as cell scaffold material in islet cell culture and stimulation insulin Secretion application, has wide biological medicine prospect.

It is specific as follows:

The present invention carries out allyl using hydroxyl of the method for freeze-drying to hyaluronic acid N-ACETYL-D-GLUCOSAMINE for the first time Base glycidol ether functional modification obtains different modifying rate allyl derivatives of hyaluronic acids, and modification rate ratio is at normal temperature Reaction modification rate in solution significantly improves (35.67%vs 3.3%)；Technique extends living for the biology of thermally labile Property molecule such as enzyme, antibody, protide growth factor etc. high molecular polymer modify such as Pegylation, have incomparable Advantage；Serial cysteine functional modification is carried out to allyl derivatives of hyaluronic acids with mercaptan-alkene clicking chemistry, is obtained 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 of polysaccharide chemistry, being hyaluronic acid and The modification modification of other polysaccharide provides new method.

Obtained cysteine conjugates of hyaluronic acid and four branch polyethylene glycol oxygen esters is synthesized, leads to peroxy esters and mediates nature It is connected chemically the hydrogel to form active sulfydryl, increases the adhesion of hyaluronic acid, and can be in cell culture, it can also be into One step carries out functional modification, for example in conjunction with drug, plays the role of medicine sustained and controlled release, in addition, the alcohol in ester structure exists OMNCL can be discharged into solution when reacting, and when designing ester, this alcohol can be designed as hydroxylated drug or cell The precursor of growth factor, to discharge these drugs or growth factor while hydrogel formation into hydrogel, increase Application of the hyaluronic acid in 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 mediation nature, which is connected chemically to obtain, has controllability, the covalent cross-linking of Injectable in-situ formative transparent Matter acid hydrogel.By comparing the rheologic behavio(u)r of hydrogel, as biologic bracket material in islet cell culture and insulin Experimental result is secreted, the optimal modification condition of hyaluronic acid is probed into.Cell culture neck can be used in as biologic bracket material Domain, the formation of hydrogel can be injected by needle tubing invades body with minimal damage, rather than passes through surgery hand in traditional sense Art, the defect that can be used for filling up any body shape, at the same can also packaging medicine or cell factor, be clinical medicine and group weaver The fields such as journey provide ideal biomaterial.

Therefore, the present invention modifies its N- second with mercaptan-alkene clicking chemistry by freeze-drying using hyaluronic acid as raw material Acyl-D-Glucose amine hydroxyl mediates nature to be connected chemically and poly- second two without influencing itself negative electrical charge finally by oxygen ester Alcohol compound reacts the hyaluronic acid gel that Injectable in-situ forms covalent cross-linking, and the hydrogel formed has well Biocompatibility.The present invention have substantive distinguishing features outstanding, for person of ordinary skill in the field, invention relative to The prior art is non-obvious.

Detailed description of the invention

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

Fig. 2 is that oxygen ester mediates nature to be connected chemically reaction mechanism figure；

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Figure 20 is that the hyaluronic acid decorated rate (◆) of allyl glycidyl ether and Boc-Cys (Acm)-cysteamine sulfydryl are 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 for the hydrogel that Cys-HA1 and 4-ARM-PEG-S-S is formed；

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Figure 51 is the control rheology time sweep figure of the Cys-HA of 5% number 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 to be formed；

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

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

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

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

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

Figure 59 is insulin secretion canonical plotting；

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

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

Specific embodiment

N=200-4000 in formula.

Its synthetic route of the cysteine functionalization conjugates of hyaluronic acid of mercaptan-alkene clicking chemistry preparation is as follows:

1) structural formula is taken to be1 hyaluronic acid of compound be with structural formulaCompound 2, forming structural formula by the method for freeze-drying is's Compound 3；

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

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

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

Preferably, described its structural formula of polyethylene glycol oxygen ester is N=50~500 in formula, m=2~8.

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

The contents of the present invention are described in further detail below by way of specific embodiment.

Embodiment 1

1) synthesis of compound 3

The synthetic schemes of 1 compound 3 of table

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

Hyaluronic acid (100kDa) 14g is taken, is dissolved in ultrapure water 700ml and is configured to concentration in 1000ml round-bottomed flask and is The hyaluronic acid aqueous solution of 2% (w/v), prepares the 250ml round-bottomed flask that 7 numbers are 1~7, and each flask is packed into 100ml 2% hyaluronic acid solution is spare, takes the allyl glycidyl ether of 9.76ml that ultrapure water is added with volumetric flask and is settled to 100ml It is uniformly mixed, the allyl glycidyl ether aqueous solution of corresponding equivalent volume is added to 1~No. 7 round-bottomed flask, is stirred at room temperature mixed It closes uniformly, is transferred in culture dish and is put into -80 DEG C of freezings, be freeze-dried, 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, analysis ownership is carried out to signal in figure,¹H NMR(D₂O) it is shown in δ Occurs allyl (CH at 5.88~5.98ppm₂=CH₂-CH₂- O-) signal, so can determine whether that the compound is compound 3, it is logical It crosses at 5.88~5.98ppm of comparison δ and 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 2 compound 6 of table

Take Boc-Cys (Acm)-OH 20g (compound 4,223.28g/mol, 68.41mmol) in 1000ml round-bottomed flask It is middle with DCM 300ml dissolve, be followed by stirring for be added with DCM 200ml dissolution PyBOP 39.16g (520.40g/mol, 68.41mmol x1.1eg) solution, it is subsequently agitated for that triethylamine 19.06ml (101.19g/mol, 68.41mmol x2eq) is added, As the addition solution of triethylamine can be become clarifying from muddiness, stir-activating 10 minutes, next it is slowly added dropwise with DCM 200ml Dissolution has removed the cystamine 4.88ml (152.28g/mol, 68.41mmol x0.55eq) of dihydrochloride.2-aminoethyl disulfide dihydrochloride ginseng 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.) side It is spare that method removes hydrochloride.After cystamine is added dropwise, reaction is stirred to react 3 hours under the conditions of room temperature, argon gas, and reaction process is used 60 F254 silica gel thin-layer chromatography of Silica gel and acid ninhydrine detection, TLC solvent are methylene chloride-methanol-acetic acid (36%) (100: 6: 1) are depressurized dense removing solvent to small size is reduced to after the reaction was completed, are acidified with 0.1mol/l aqueous hydrochloric acid solution After pH to 6~7, it is extracted with DCM, uses 10mmol/1HCl, 5%NaHCO later₃, H₂O respectively extracts three to organic layer It is secondary, finally organic layer is extracted once with saturation NaCl solution, to the organic layer extracted dry 2h of anhydrous magnesium sulfate, decompression Concentration removal solvent, obtains oily compounds 6 (21.58g, yield 90%) with silica gel chromatographic column and obtains white chunks after purification 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 ,-NHCH₂ S-), 4.08 (d, J=5.3Hz, 1H ,-OCONHCH), 3.31-3.16 (m, 2H ,-CONHCH₂ ), 2.74 (ddd, J=58.7,13.6,9.1Hz, 2H ,-SCH₂ CH-), 2.40 (d, J= 7.9Hz, 2H ,-CH₂ SH), 1.84 (s, 3H, CH₃ CONH-), 1.38 (s, 9H, (CH₃)₃ OCO-) (see attached drawing 3).

3) compound 7 synthesizes

The synthetic schemes of 3 compound 7 of table

Take compound 620g (700.95g/mol, 28.53mmol) in the round-bottomed flask of 1000ml, it is 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 Above-mentioned acetonitrile solution is added in ionized water dissolution, and reaction is stirred 2 hours under the conditions of room temperature, argon gas, reaction process Silica 60 F254 silica gel thin-layer chromatography chromatography of gel and acid ninhydrine, the detection of Ellman reagent, TLC solvent are methylene chloride-first Alcohol-acetic acid (36%) (100: 6: 1), depressurizes dense removing solvent after the reaction was completed, is acidified pH to 3 with 0.1mol/l aqueous hydrochloric acid solution After~6, it is extracted with DCM, uses 5%NaHCO later₃, 10mmol/l HCl, H₂O respectively extracts organic layer three times, Finally organic layer extract once with saturation NaCl solution, removing solvent is concentrated under reduced pressure and obtains oily compounds 7 (19.05g, yield 95%), obtains oily liquids with silica gel chromatographic column after purification.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 ,-NHCH₂ S-), 4.08 (d, J=5.3Hz, 1H ,-OCONHCH), 3.31-3.16 (m, 2H ,-CONHCH₂ ), 2.74 (ddd, J=58.7,13.6,9.1Hz, 2H ,-SCH₂ CH-), 2.40 (d, J=7.9Hz, 2H ,-CH₂ SH), 1.84 (s, 3H, CH₃ CONH-), 1.50 (s, 1H ,-CH₂SH), 1.38 (s, 9H, (CH₃)₃ OCO-) (see attached drawing 4).

4) synthesis of compound 8

The synthetic schemes of 4 compound 8 of table

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 referring 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 ultrapure 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), prepares the 250ml quartz beaker with cover that 7 numbers are 1~No. 7, Each corresponding modification rate allyl glycidyl ether hyaluronic acid of flask loading is spare, takes compound 737.55g 78ml acetonitrile 7 acetonitrile solution of compound of corresponding equivalents and volume number is added to 1~No. 7 quartz beaker, it is equal that mixing is stirred at room temperature for dissolution It is even, it takes dimethoxybenzoin 4.22g 39ml acetonitrile to dissolve, corresponding equivalents and volume is added to above-mentioned 1~No. 7 quartz beaker Number dimethoxybenzoin (DMPA) acetonitrile solutions, be stirred at room temperature after mixing, ultraviolet light UV λ 365nm irradiation under for 24 hours after, Alkenyl blackout in nucleus magnetic hydrogen spectrum prompts mercaptan-alkene clicking chemistry complete.It is organic that reduced pressure removing is carried out after fully reacting Solvent, then the bag filter for being 10kDa with molecular weight carry out dialysis removing small molecule, and dialyzate is 50% (v/v) ethanol water, The every 6h of dialysis treatment 48h replaces a dialyzate, and last time dialysis uses Silica during dialysis using ultrapure water dialysis 6h 60 F254 silica gel thin-layer chromatography plate of gel, acid ninhydrine and Ellman reagent carry out checking whether that dialysis finishes, and dialysis is completed Freeze-drying process is carried out afterwards obtains a series of compounds 8.Pass through its modification rate of magnetic resonance detection again.

Magnetic resonance detection is carried out to compound 8, analysis ownership, 1H NMR (D are carried out to signal in figure₂O) it is shown in δ Occurs tertbutyloxycarbonyl (Boc) signal at 1.37ppm, so can determine whether that the compound is compound 8, by comparing δ 1.37ppm There is tertbutyloxycarbonyl (Boc) signal and methyl (- NH-COCH in acetylamino in hyaluronic acid at 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 5 intermediate 8a of table

A. taking off Boc protecting group to take each 2g of above-mentioned seven kinds of different modifying rates compound 8 number is 1~7, and each sample is added 20ml trifluoroacetic acid makes it dissolve, and after being stirred to react 2h under conditions of being stirred at room temperature, solution be concentrated under reduced pressure in dry Mesosome 8a.

The synthetic schemes of 6 compound 9 of table

Remarks: 2Na₂S₂O₃+I₂=Na₂S₄O₆+2NaI

B. 1~No. 7 sample of the Acm protecting group to the intermediate 8a obtained after trifluoroacetic acid takes off the processing of Boc protecting group is taken off Product are fitted into the round-bottomed flask of 250ml, with 1%NaOH adjusting pH to neutrality, iodine 14.99g 168ml ethyl alcohol are taken to dissolve, The iodohydrin solution of corresponding equivalents and volume number is added to the round-bottomed flask of 1~No. 7 250ml, reaction 4h, phase is stirred at room temperature Between, it finds there is the generation of brown gel in round-bottomed flask, the mercaptoethanol aqueous solution of certain volume is added thereto until gel is molten Solution, after the reaction was completed, takes sodium thiosulfate (Na₂S₂O₃) after the 19.61g ultrapure water of 84ml dissolution to above-mentioned 1~No. 7 round bottom Corresponding equivalents is added in flask and the sodium thiosulfate solution of volume carries out acid-base titration reaction, removes responseless iodine, It after being stirred to react 2h, carries out that removing solvent is concentrated under reduced pressure, then the bag filter dialysis for 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 replaces a dialyzate, and last time dialysis is using ultrapure Water is dialysed 6h, during dialysis with Silica gel 60F254 silica gel thin-layer chromatography plate and acid ninhydrine and Ellman reagent into Row checks whether that dialysis finishes.It is freeze-dried after the completion of dialysis, obtains a series of different modifying rate products 9.By to 1H NMR(D₂O) signal carries out analysis ownership in map, on the basis of the nuclear magnetic resonance spectroscopy of compound 8 at δ 1.37ppm Tertbutyloxycarbonyl signal (Boc) disappear, can determine whether that the compound 8 is deprotected completely, obtained compound be target product Compound 9.

Embodiment 2

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

Experimental procedure:

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

B solution: it weighs tetra- branch's polyethylene glycol oxygen ester conjugate (4-ARM-PEG-S-S) of 35mg 10k and uses phosphate-buffered Liquid (pH7.6) 350ul is dissolved in 1ml centrifuge tube.

Solution A is uniformly mixed rapidly with B solution using vortex mixer, is added on rheometer and is started to be tested.It is advanced Row time sweep (Time sweep), performs a scan (Frequency sweep), finally when modulus curve no longer changes It carries out strain sweep (Strain sweep), parameter setting is as described below.

Parameter is set as in rheology test, scan pattern: oscillation；Rotor: PP25, PP spacing (gap) 1mm；Data take Dot frequency: 1/20s；Test temperature: 20 DEG C.

Time sweep (time sweep): frequency (f) is 1Hz, and stress (Strain) is 1%, Time constant point access evidence；

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

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

Embodiment 3

Cysteine conjugates of hyaluronic acid solution and four branch polyethylene glycol oxygen ester conjugate (4-ARM-PEG-S-S) shapes Is verified at hydrogel mode

A. cysteine conjugates of hyaluronic acid (Cys-HA) 35mg for weighing the maximum modification rate synthesized in embodiment 1 is used 700 μ l of phosphate buffer (pH7.6) is dissolved in 1ml centrifuge tube, carries out sufficient time sweep (Time sweep) test, It is consistent in time sweep (Time sweep) condition and embodiment 2, no gel-forming.The results are shown in attached figure 51.

B. in embodiment 2, solution A is put into enough TCEP.HCl solution with the hydrogel that B solution is formed and sees overnight It examines, discovery or gel state.

Embodiment 4

Referring to embodiment 2, it is by structural formulaN=50~500, m Tetra- branch's polyethylene glycol oxygen ester conjugate (4-ARM-PEG-S-S) of 10k in B solution in 10 alternative embodiment 6 of=2~8 compound.

Embodiment 5

RIN m5f cell CCK experiment

CCK tests testing principle

Cell Counting Kit abbreviation CCK 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, working principle are as follows: tries in electronics coupled In the presence of agent, the orange-yellow formazan product for generating high water soluble can be restored by Intramitochondrial dehydrogenase.Face The depth of color and the proliferation of cell are directly proportional, are inversely proportional with cytotoxicity.OD value is measured at 450nm wavelength using microplate reader, Reflection living cells quantity indirectly.

RIN m5f cell culture experiments

2~7 cysteine conjugates of hyaluronic acid Cys-HA of number is made into concentration with the phosphate buffer of pH7.6 and is 10% (w/v) solution, every part of every hole 250ul are injected on 96 well culture plates in 5 holes totally, four branch's polyethylene glycol oxygen ester conjugates (4-ARM-PEG-S-S) is also made into 10% (w/v) solution with same method, and every part equally takes 250ul mixed with above-mentioned 2~No. 7 After closing uniformly, then is corresponded to every hole and inject 1%Ac-CGRGDSPG-NH₂Or 1%Exenatide-NH₂, it is uniformly mixed, 30 minutes It is washed 3 times with PBS later, finally injects 250ulRIN m5f cell solution to every hole, cell concentration is 5000/cm², cell At the standard conditions (37 DEG C, 5%CO₂) culture 48 hours.

CCK experimental procedure

10ul CCK solution is added in every group of corresponding 96 orifice plate, pays attention to not generating gas in hole in the process of addition Bubble, they will affect the reading of OD value, after addition by culture plate put in the incubator in be incubated for 1~4 hour (37 DEG C, 5% CO₂), the absorbance at 450nm and then is measured with microplate reader, the cell for be calculated every group using following formula is living Power.

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

A (dosing): the absorbance in the hole with cell, CCK solution and drug solution

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

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

Embodiment 6

The experiment of RIN m5f cell fluorescence

Fluorescence experiments principle

Calcein-AM is a kind of cell staining reagent that fluorescent marker can be carried out to living cells, due in Calcein (calcium Yellowish green element) on the basis of strengthen hydrophobicity, therefore living cells film can be penetrated easily.After it enters cytoplasm, esterase Calcein (calcein) can be hydrolyzed to stay in intracellular and complete membrane structure can not be diffused out, so can illustrate Cell viability, and the integrality of cell can be prompted.Ethidium homodimer-1 is a kind of impermeable nucleic acid dye of film, it It can enter damaged cell membrane and nuclear membrane and DNA dyed, therefore can identify dead cell.Both dyestuffs are all in 495nm Place's excitation, Calcein-AM green light at 515nm, Ethidiumhomodimer-1 glow at 635nm.

Fluorescence experiments step

RIN m5f cell culture experiments are as described above, 2ul pH7.6 is added in every hole in every group of corresponding 96 orifice plate Phosphate buffer configuration 2mMCalcein-AM and 2mM Ethidium homodimer-1 solution, after addition will Culture plate put in the incubator in be incubated for 0.5 hour (37 DEG C, 5%CO₂), it is and then raw with every group of cell of fluorescence microscope Long situation.

Embodiment 7

The experiment of RIN m5f cell insulin secretion

Insulin secretion experimental principle

Experiment is using rat insulin (Insulin) water in double antibody sandwich method enzyme linked immunosorbent assay sample It is flat.It is coated with microwell plate with rat anti-insulin (Insulin) antibody of purifying, solid phase antibody is made, toward the micropore of coating monoclonal antibody In sequentially add insulin (Insulin) and horseradish peroxidase (Horseradish Peroxidase, HRP) label pancreas Island element antibody combines, and forms antibody-antigene-hrp-antibody complex, and after thoroughly washing plus substrate TMB develops the color.TMB exists Au bleu is converted under the catalysis of HRP enzyme, and is converted to final yellow under the action of an acid.Pancreas in the depth and sample of color Island element (Insulin) is positively correlated.It is measured under 450nm wavelength with microplate reader absorbance (OD value), is calculated by standard curve Rat insulin (Insulin) concentration in sample.

Insulin secretion experimental procedure

1. dilution and the sample-adding of standard items: being marked with quasi- 10 hole of sample wells on enzyme mark coating plate, in the first, second hole respectively Add standard items 100ul, standard dilutions 50ul is then added in the first, second hole, mixes；Then from the first hole, the second hole In respectively take 100ul to be added separately to third hole and the 4th hole, then add standard dilutions 50ul respectively in third, the 4th hole, mix； Then first 50ul is respectively taken to discard in third hole and the 4th hole, then 50ul is respectively taken to be added separately in the five, the 6th holes, then the Five, add standard dilutions 50ul in the 6th hole respectively, mix；Respectively 50ul is 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 It does not take 50ul to be added in the nine, the tenth holes, then adds standard dilutions 50ul respectively in the 9th the tenth hole, from the nine the after mixing Respectively 50ul is taken to discard in ten holes.Each hole sample-adding amount is all 50ul after dilution, and concentration is respectively 48mu/l, 24mu/l, 12mu/l, 6mu/l, 3mu/l, 0mu/l.

2. preparation of samples: the phosphate buffer of 2~7 cysteine conjugates of hyaluronic acid Cys-HA pH7.6 of number 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 ~No. 7 after mixing, then injects 1%Ac-CGRGDSPG-NH to every hole is corresponding₂Or 1%Exenatide-NH₂, it is uniformly mixed, Wash 3 times with PBS after 30 minutes, finally injects 500ulRIN m5f cell solution to every hole, cell concentration for 1000/ cm², cell standard conditions (37 DEG C, 5%CO₂) under cultivate 48 hours after, remove culture medium, then with Ke-woods Er Shi heavy carbonate Buffer (Krebs-Ringer bicarbonate buffer) washs 2 times.Use G-Lin 's bicarbonate buffer (Krebs-Ringer bicarbonate buffer) configuration concentration 3.3 and 16.7mmol/l glucose sugar juice are placed on incubator (37 DEG C, 5%CO₂) middle 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) middle culture 60 minutes.It has cultivated Bi Hou.It takes 500ul solution ice bath in centrifuge tube to save from every group of corresponding aperture, is centrifuged 5 minutes at 2500rpm0 DEG C, every group takes The supernatant of 200ul is used for the measurement of insulin, removes solution remaining in every group of 48 orifice plates, adding 500ul concentration is 16.7mmol/l glucose sugar and incubator (37 DEG C, 5%CO₂) middle culture 60 minutes, subsequent use according to ELISA kit is said Bright book is operated.

The cysteine functionalization hyalomitome to be formed is modified below to synthesize a kind of mercaptan-alkene clicking chemistry to examples detailed above Acid conjugate, preparation Injectable in-situ formed hydrogel rheological property, islet cell culture experiment, insulin secretion Experiment detection.

One, mercaptan-alkene clicking chemistry modifies the cysteine functionalization conjugates of hyaluronic acid modification rate to be formed

Attached drawing 5~11 and attached drawing 12 are compared, the signal of the nuclear magnetic resonance spectroscopy of compound 3 is subjected to analysis ownership.¹H NMR(D₂O it) is shown at 5.88~5.98ppm of δ and allyl (CH occurs₂=CH₂-CH₂- O-) signal, so can determine whether the change Conjunction object is compound 3, allyl (CH occurs at 5.88~5.98ppm of δ by comparing₂=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 attached drawing 20)；

Attached drawing 13~19 is the nuclear magnetic resonance spectroscopy of compound 8, carries out analysis ownership to signal in figure,¹H NMR(D₂O it) shows Show at δ 1.37ppm tertbutyloxycarbonyl (Boc) signal occur, so can determine whether that the compound is compound 8, by comparing δ Occur tertbutyloxycarbonyl (Boc) signal and methyl (- NH- in acetylamino in hyaluronic acid at δ 1.95ppm at 1.37ppm COCH₃) signal ratio can calculate this step reaction modification rate about 2.33%~45.67% (see attached drawing 20).

Two, mercaptan-alkene clicking chemistry modifies the cysteine functionalization conjugates of hyaluronic acid GPC result to be formed

7. dextran standard items GPC data of table

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 attached drawing with GPC 21, Figure 22~29), the measured molecular weight of sample and the molecular weight of Sodium Hyaluronate (compound 1) compare as the result is shown, transparent Matter acid molecule is subjected to not becoming smaller or degrading after chemical modification of the invention, and the molecular weight of some samples is slightly larger than hyaluronic acid Raw material.

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

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

Remarks: 100,000 HA of lg contains 2.636mmol primary alconol；Number 8 is check experiment, verifies the Cys- of maximum modification rate HA forms hydrogel not over disulfide bond crosslinking within the regular hour.

It can be seen that 2~No. 7 cysteines of number from the formation rheology scanning figure (see attached drawing 30~50) of hydrogel 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 There is intersection point in G ' and G " in testing time, this point be gel point, i.e., hydrogel formed time point, gel time be 70s~ (see attached drawing 52) between 8145s, as time increases, G ' is in ascendant trend, finally reaches horizontal gradually, illustrates that crosslinking is anti- Answer fully reacting.The time that 2~No. 7 gel points of number occur gradually successively decreases with the increase of modification rate, only allyl All without gel point within 5 hours testing times when base glycidol ether is 0.1 equivalent, and the rheology for being 8 from number Experiment can be seen that cysteine conjugates of hyaluronic acid 40 minutes 6 hours under the same conditions of same concentrations maximum modification rate All the appearance (see attached drawing 51) of gel point, number 2~7 are not formed by hydrogel and are still overnight in TCEP.HCl solution Hydrogel state can illustrate 2~No. 7 cysteine conjugates of hyaluronic acid solution of number and the poly- second of four branches from these two aspects Glycol oxygen ester conjugate (4-ARM-PEG-S-S), which is formed by hydrogel, is that logical peroxy esters mediate nature to be connected chemically reaction, and It is not that cysteine sulfydryl by disulfide bond is formed by covalent cross-linking hydrogel.

Four, RIN m5f cell CCK experimental result

Attached drawing 53 is the hyaluronic acid gel master drawing formed.

From progress ordinate comparison in attached drawing 54: by CCK experimental cell we have found that under identical modification rate, number 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 It leads nature to be connected chemically to be formed in the progress RIN m5f cell culture of covalent cross-linking hydrogel, (1) adds the thin of cell adhesion molecules Born of the same parents' vigor obviously has than what is do not added close to 30%~55% cell growth rate, illustrates cell adhesion molecules with hyalomitome Acid-polyethylene glycol hydrogel is to play positive effect in RIN m5f cell culture in cell scaffold material.(2) in addition cell Exenatide-NH in the experimental group of adhesion factor₂Compare Ac-CGRGDSPG-NH₂High close is wanted to RIN m5f cell growth rate 20%, cell viability relatively, illustrates Exenatide-NH compared with the two jointly addition group₂Compare Ac-CGRGDSPG-NH₂More RIN m5f cell growth and breeding can be stimulated.

Abscissa comparison: by CCK experimental cell we have found that same experimental group, number 2~7 half Guangs 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 and mediate certainly So be connected chemically to be formed covalent cross-linking hydrogel carry out RIN m5f cell culture in cell viability it is also variant, when allyl contract There is maximum cell vigor number for every group when water glycerin ether is 1 equivalent of hyaluronic acid primary hydroxyl group (2.636mmol), it is thin in addition 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.

Five, RIN m5f cell fluorescence experimental result

From attached drawing 55~58 as it can be seen that RIN m5f cell fluorescence experimental result is similar to discussion to CCK experimental result, addition The hydrogel of active peptides can promote the growth of cell, polypeptide Exenatide-NH₂It is especially pronounced in this respect, it is modifying Fluorescence experiments can show mutual difference not as good as CCK experimental result in number in terms of rate, but can also find to contract when allyl RIN m5f cell possesses preferable growth conditions when water glycerin ether is 1 equivalent of hyaluronic acid primary hydroxyl group (2.636mmol).

Six, RIN m5f cell insulin secretion experimental result

Insulin secretion standard curve is shown in attached drawing 59, is tested by RIN m5f cell insulin secretion, we can be found that Concentration is that the insulin concentration ratio 3.3mmol/l glucose sugar juice that 16.7mmol/l glucose sugar juice stimulates out is wanted under the same terms It is more.

60~Figure 61 of attached drawing, ordinate comparison: under every group of same concentrations glucose sugar and identical modification rate, number 2~7 half Guangs Propylhomoserin conjugates of hyaluronic acid and four branch's polyethylene glycol oxygen ester conjugate (4-ARM-PEG-S-S) solution lead to peroxy esters and mediate certainly So be connected chemically to form the experiment of covalent cross-linking hydrogel RIN m5f cell insulin secretion: (1) glucose concentration is 3.3mmol/ L, the cell secretory capacity that cell adhesion molecules occurs adding in 16.7mmol/l is eager to excel than what is do not added, illustrates cell adhesion The factor is played by RIN m5f cell insulin secretion experiment in cell scaffold material of hyaluronic acid-poly ethylene glycol hydrogel Positive effect.(2) in two groups of glucose concentrations, Exenatide-NH in the experimental group of cell adhesion molecules is added₂Compare Ac- CGRGDSPG-NH₂It is eager to excel to stimulation RIN m5f cell insulin secretion, compared with addition group is compared to cell viability jointly with the two It is close, illustrate Exenatide-NH₂Compare Ac-CGRGDSPG-NH₂RIN m5f cell insulin secretion can more be stimulated.

Abscissa comparison: it by RIN m5f cell insulin secretion we have found that same experimental group, is compiled under different modifying rate The water that number 2~7 cysteine conjugates of hyaluronic acid and four branch's polyethylene glycol oxygen ester conjugates (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 Two groups of insulin secretions are every group thin when modification rate is bigger with the presence of maximum concentration when 1 equivalent of alcoholic extract hydroxyl group (2.636mmol) 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 best modification rate to RIN m5f cell insulin secretion.

In conclusion the invention has the characteristics that:

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

2. the hydroxyl with the N-ACETYL-D-GLUCOSAMINE of hyaluronic acid is prepared using the method for freeze-drying in the present invention Base reacts the bright matter acid derivative of allyl to form a series of modification rates of stable ehter bond, and freeze-drying method is from now in polysaccharide And other macromoleculars modification chemistry provides new thinking.

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

4. cysteine conjugates of hyaluronic acid has cysteamine group, the adhesion of hyaluronic acid is not only increased, also Be provided for can further chemical modification functional group, and with four branch polyethylene glycol oxygen esters lead to peroxy esters mediate naturalization The active sulfydryl of new hyaluronic acid gel that connection prepares injectable, covalent cross-linking is formed in situ is learned, is increased transparent The adhesion of matter acid is used for cell culture, can also further progress functional modification play that drug is slow to be controlled such as with drug ining conjunction with The effect released.

5. the alcohol in ester structure can be discharged into solution when OMNCL reacts, can be this alcohol when designing ester Be designed as the precursor of hydroxylated drug or Porcine HGF, thus hydrogel formation while, discharge these drugs or Growth factor is into hydrogel, to increase hyaluronic acid in the application of biomedicine field.

6. in the test of rheology, the amount of the allyl glycidyl ether of reaction is added from 0.5~5 equivalent, hydrogel Formation is all to react covalent cross-linking rather than disulfide bond by OMNCL, and modification rate is bigger, is crosslinked the required time and also gets over It is few, hydrogel is formed by with good rheological property, and there are the Optimalities such as gel time is controllable, mechanical performance is controllable Matter.

7. finding hyalomitome in RIN m5f cell culture experiments and RIN m5f the cells secrete insulin experiment of hydrogel Acid-polyethylene glycol hydrogel is raw in cell as the importance of timbering material and cell adhesion molecules in cell culture Meaning played in growth process.

8. the hyaluronic acid gel modified by addition 1 equivalent of allyl glycidyl ether is to RIN m5f cell Vigor and RIN m5f cell insulin secretion all obtain optimal result.Thus transparent for the cysteine synthesized through the invention Matter acid conjugate and transplanting with the hydrogel of polyethylene glycol covalent cross-linking in islet cells, the treatment of diabetes and other doctors The application in medicine field provides new direction.

Claims

1. a kind of system by being freeze-dried with the cysteine functionalization conjugates of hyaluronic acid of mercaptan-alkene clicking chemistry preparation Preparation Method, it is characterised in that: the following steps are included:

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

3) compound 6 is with disulfide bond reducing agent generation reduction reaction formation structural formulaChange Close object 7；

4) compound 3 and compound 7 are taken, mercaptan-alkene clicking chemistry occurs under photoinitiator and ultraviolet light and forms structure Formula isCompound 8；

5) tertbutyloxycarbonyl and acetyl aminomethyl protecting group on compound 8 are sloughed respectively, and obtaining structural formula isCompound 9, i.e. cysteine functionalization conjugates of hyaluronic acid, n=200 in formula ~4000.

2. a kind of cysteine function by being freeze-dried with mercaptan-alkene clicking chemistry preparation according to claim 1 Change the preparation method of conjugates of hyaluronic acid, the modification rate range of compound 3 is 4.03%~64.11% in step 1)；Step 4) range of the modification rate of compound 8 is 2.33%~45.67% in.

3. the cysteine functionalization hyaluronic acid that preparation method of any of claims 1-2 is prepared combines Object.

4. cysteine functionalization conjugates of hyaluronic acid as claimed in claim 3 is in the hydrogel for preparing Injectable in-situ formation Application.

5. cysteine functionalization conjugates of hyaluronic acid according to claim 4 is in the water for preparing Injectable in-situ formation The application of gel, it is characterised in that: the hydrogel that the Injectable in-situ is formed is by cysteine functionalization hyaluronic acid Conjugate is connected chemically with the logical peroxy esters mediation nature of polyethylene glycol oxygen ester reacts generation.

6. cysteine functionalization conjugates of hyaluronic acid according to claim 5 is in the water for preparing Injectable in-situ formation The application of gel, it is characterised in that: Injectable in-situ formed hydrogel preparation method the following steps are included:

1) with cysteine functionalization conjugates of hyaluronic acid and phosphate buffer wiring solution-forming A；

2) with polyethylene glycol oxygen ester and phosphate buffer wiring solution-forming B；

3) leading to peroxy esters mediates nature to be connected chemically reaction, prepares the hydrogel of Injectable in-situ formation.

7. cysteine functionalization conjugates of hyaluronic acid according to claim 5 or 6 is formed preparing Injectable in-situ Hydrogel application, it is characterised in that: the structural formula of the polyethylene glycol oxygen ester isN=50~500 in formula, m=2~4.

8. cysteine functionalization conjugates of hyaluronic acid according to claim 6 is in the water for preparing Injectable in-situ formation The application of gel, it is characterised in that: the step 1) and 2) in, the pH of phosphate buffer is 7.0~7.6, is made into Solution concentration is 1%~20%；In step 3), the molar ratio of conjugates of hyaluronic acid and polyethylene glycol oxygen ester be 1:(0.1~ 1) time range that, hydrogel is formed is 5 seconds~8000 seconds.

9. the water-setting formed by Injectable in-situ prepared by cysteine functionalization conjugates of hyaluronic acid as claimed in claim 3 Application of the glue in culture islet cells or insulin secretion.