CN114656652A

CN114656652A - Preparation method of low-swelling temperature-sensitive injectable chitosan-based hydrogel

Info

Publication number: CN114656652A
Application number: CN202011532492.0A
Authority: CN
Inventors: 魏长征; 姜芳; 王意波; 蒋丽霞
Original assignee: Shanghai Qisheng Biological Preparation Co ltd
Current assignee: Shanghai Qisheng Biological Preparation Co ltd
Priority date: 2020-12-23
Filing date: 2020-12-23
Publication date: 2022-06-24

Abstract

The invention relates to a preparation method for regulating and controlling swelling characteristics of temperature-sensitive chitosan-based hydrogel. The charge modification is carried out on the hydroxyl and amino groups of the chitosan and the derivative hydroxybutyl chitosan thereof, so that the characteristics of hydrophilic and hydrophobic interaction and hydrogen bond interaction are changed, and the swelling performance of the temperature-sensitive hydrogel is regulated and controlled. The invention carries out structural modification on the temperature-sensitive chitosan-based hydrogel, has simple process and easy purification, thereby widening the application of the temperature-sensitive hydroxybutyl chitosan in the biomedical field, in particular to the low-swelling injectable hydrogel.

Description

Preparation method of low-swelling temperature-sensitive injectable chitosan-based hydrogel

Technical Field

The invention relates to a preparation method of a low-swelling temperature-sensitive injectable chitosan-based hydrogel, belonging to the field of macromolecules and biomedical materials.

Background

The intelligent hydrogel is a hydrogel which can realize different functions through certain external stimulation, such as pH, temperature, magnetic field, inorganic salt and the like, and has important application in the field of biomedical materials. The temperature-sensitive hydrogel is an intelligent hydrogel which is concerned, can realize the transformation of gel-sol through the triggering of temperature, generally speaking, the temperature-sensitive gel is in a solution state under the condition of low temperature, and can become gel when the temperature is raised to the body temperature of 37 ℃. The characteristic endows the hydrogel with excellent injectability, mild use conditions and strong operability, and a potentially toxic cross-linking agent is not required to be added, so the hydrogel is widely applied to the aspects of drug slow release, cell delivery, gel filling and the like.

Chitosan is a natural alkaline polysaccharide with abundant natural resources, can be extracted from shells of shrimps, crabs and insects, and has lower price. In the previous patent, hydroxyl and amino sites of chitosan are subjected to hydroxybutylation modification (patent CN103172762B), so that the hydroxybutyl chitosan hydrogel which can be injected with temperature sensitivity is obtained, and the hydroxybutyl chitosan hydrogel has important applications in the aspects of nerve blocking, drug slow release and the like.

However, the hydroxybutyl chitosan hydrogel will shrink in volume under physiological conditions to a certain extent due to its amphiphilicity (hydrophilicity and hydrophobicity) and polyelectrolyte properties, and is susceptible to changes in swelling performance caused by pH, salt concentration and kind, and temperature (patent CN 106421899B). However, in the use process of the temperature-sensitive hydrogel, especially as an injectable hydrogel, excessive swelling or shrinkage occurs after the hydrogel is implanted into the body, which limits the application of the hydrogel, for example, the mechanical strength of the hydrogel is reduced when the swelling rate of the hydrogel is too high under physiological conditions, and the growth of peripheral tissues is possibly influenced, or the body indexes are abnormal; if the hydrogel shrinks in volume, the implant material may fail, especially as a filler material. Therefore, obtaining controlled low swelling hydrogel materials is of paramount importance.

In order to obtain hydrogels with low swelling properties, it is currently possible to adjust the concentration of the hydrogel, the hydrophilic-hydrophobic balance, the crosslinking pattern and the degree of crosslinking.

A decrease in hydrogel concentration may decrease the swelling ratio. The literature (Nat Biomed Eng,2017,1, DOI:10.1038/s41551-017-0044) indicates that the four-arm double-bond functionalized polyethylene glycol and the four-arm sulfhydryl functionalized polyethylene glycol are pre-crosslinked to form an oligomeric polyethylene glycol aggregate, and then are further crosslinked to realize the gel forming property and low swelling property under the condition of extremely low polymer concentration (4g/L), thereby having important application in artificial vitreous body materials.

The proper crosslinking pattern and degree of crosslinking of the hydrogel can achieve its low swelling ratio. Generally, an increase in the degree of physical or chemical crosslinking decreases the swelling ratio of the hydrogel. Patent CN106674544B prepares an injectable temperature-sensitive gel material of an injectable multi-arm poly (lactide-co-glycolide) polyethylene glycol block copolymer with temperature responsiveness. The hydrogel molecular structure has high hydrogen bond crosslinking density, and can form a compact network structure, so that water molecules are difficult to enter the gel, the low swelling rate of the hydrogel is ensured, and the swelling rate is lower than 20%. In addition, patent CN109021256A dissolves sodium silicate, acrylamide and sodium alginate in water to initiate acrylamide polymerization, and at the same time, releases calcium ions in situ to crosslink calcium ions with alginate molecular chains, thereby forming organic-inorganic hybrid reinforced gels, and these hybrid structures enhance the "entanglement" between alginate networks and polyacrylamide networks, thereby reducing the swelling of hydrogel in physiological environment. Patent US9308301B2 forms hydrogels by crosslinking of polydiamines and polyglycidyl ethers, achieving low swelling by controlling the degree of crosslinking of the hydrogel. Similarly, patent WO2015017340A2 proposes cross-linking oxidized dextran with multi-arm PEG with amino groups to give hydrogels of low swelling properties.

In addition, the polymer with high swelling ratio and the temperature-sensitive polymer with volume contractibility are chemically crosslinked by controlling the hydrophilic and hydrophobic segments and the crosslinking degree of the hydrogel, and the low swelling can be achieved by accurately controlling the proportion of the two (Biomacromolecules,2017,18,3, 757-. Although the method can realize in-situ gel formation, a large amount of synthetic work is required, the ratio of groups for covalent crosslinking is strictly controlled, the gel formation time and the swelling ratio are coordinated, and the operation difficulty is high.

Disclosure of Invention

The invention provides a method for preparing a low-swelling temperature-sensitive chitosan-based hydrogel material, which aims to solve the problem of volume shrinkage of an injectable temperature-sensitive hydroxybutyl chitosan hydrogel.

In order to achieve the purpose, the technical scheme adopted by the experiment is as follows:

a preparation method of a low-swelling temperature-sensitive injectable chitosan-based hydrogel comprises the following steps:

the temperature-sensitive chitosan hydrogel pre-solution with low swelling property is obtained by performing charge modification on amino and hydroxyl sites of chitosan or hydroxybutyl chitosan, and the hydrogel is formed along with the temperature rise, so that the temperature-sensitive chitosan hydrogel pre-solution is obtained. The deacetylation degree of the chitosan is 50% -98%, preferably more than 85%; weight average molecular weight 5X 10⁴Da-100×10⁴Da, preferably 30-60X 10⁴Da；

The temperature sensitivity mainly comes from modification of hydroxybutylation of hydroxyl and amino sites of chitosan, and the used reagent is 1, 2-epoxybutane, and the modification degree is 0.5-3, preferably 1.5-2.5; the degree of modification of said charge change is in the range of 0.2 to 1, preferably 0.3 to 0.6.

The charge modification refers to the modification of amino and hydroxyl sites of chitosan or hydroxybutyl chitosan, so that the charge density (positive charge density) and the charge type (positive charge, negative charge or no charge) of the chitosan or hydroxybutyl chitosan are changed compared with those of the chitosan or hydroxybutyl chitosan without modification under specific conditions. The charge modification reaction comprises amidation reaction, etherification reaction, esterification reaction, click chemical reaction, Schiff base reaction and the like.

One implementation approach of the low-swelling temperature-sensitive injectable chitosan-based hydrogel comprises the following steps:

(1) a charge modification compound or polymer is further introduced into the active amino or hydroxyl site of the side chain of the temperature-sensitive hydroxybutyl chitosan;

(2) the pre-solution of the low-swelling temperature-sensitive injectable chitosan-based hydrogel with the concentration of 0.5mg/ml-40mg/ml is obtained by a freeze-drying re-dissolving or concentrating mode, and the solvent can be water or aqueous solution containing salts such as sodium chloride, phosphoric acid and the like. In general, the solvent contains sodium chloride and a low concentration of buffer salt such as phosphate, etc., and adjustment of osmotic pressure and pH is carried out, the pH of the solution being not less than 6.3, preferably 6.8 to 7.5. Then, the pre-solution is triggered to solidify into gel by increasing the temperature in vivo (37 ℃), the gel forming temperature is 10-37 ℃, preferably 20-37 ℃, and the gel forming time is within 1min at 37 ℃.

The further charge modification of the active amino or hydroxyl site of the side chain of the temperature-sensitive hydroxybutyl chitosan can be the introduction of a compound or a polymer; or one or more compounds or polymers may be introduced as intermediates for modification prior to final charge modification. In addition, only amino or hydroxyl site modifications may be made, both sites may be modified, and if both sites are charge modified, the same or different compounds or polymers may be used.

One implementation way of the low-swelling temperature-sensitive injectable chitosan-based hydrogel comprises the following steps:

(1) a charge modification compound or polymer is further introduced into the active amino or hydroxyl site of the chitosan;

(2) reacting the charge-modified chitosan in the step (1) with 1, 2-butylene oxide under an alkaline condition to obtain low-swelling temperature-sensitive chitosan;

(3) the pre-solution of the low-swelling temperature-sensitive injectable chitosan-based hydrogel with the concentration of 0.5mg/ml to 40mg/ml is obtained by a freeze-drying re-dissolving or concentrating mode, and the solvent can be water or aqueous solution containing salts such as sodium chloride, phosphoric acid and the like. In general, the solvent contains a salt such as sodium chloride and a low-concentration buffer salt to adjust the osmotic pressure and pH, and the pH of the solution is not less than 6.3, preferably 6.8 to 7.5. Then, the pre-solution is triggered to solidify into gel by increasing the temperature in vivo (37 ℃), the gel forming temperature is 10-37 ℃, preferably 20-37 ℃, and the gel forming time is within 1min at 37 ℃.

The chitosan is firstly subjected to charge modification and then subjected to hydroxybutylation modification to obtain the low-swelling temperature-sensitive injectable chitosan-based hydrogel, and considering that the step (3) needs to be carried out under a strong alkali condition, the stability of a terminal group and a connecting compound or a polymer structure needs to be considered when the group is modified in the step (2), and the compound or the polymer with unstable groups such as sulfydryl, ester bond and the like is not suitable, so that the group can be modified after the compound or the polymer reacts with 1, 2-epoxybutane. When the charge modifying compound and the polymer are introduced, the compound or the polymer can be introduced; or one or more compounds or polymers may be introduced as intermediates for modification prior to final charge modification. In addition, only amino or hydroxyl site modification may be performed, both sites may be modified, and if both sites are charge modified, the same or different compounds or polymers may be used.

The method for adjusting the swelling property of the hydrogel system through charge modification generally comprises the following common methods:

(1) the charge modification of chitosan or hydroxybutyl chitosan is realized by reducing the density of amino groups, namely, part of amino groups are blocked by reacting with amino sites of chitosan, and the modified terminal group has no charge. The term "uncharged" as used herein means under specific conditions, but it is possible to charge the substrate under electric field, magnetic field, salt, pH, etc., and the term also includes the same;

(2) the charge modification is realized by introducing a new charge type, namely, the modified end is negatively charged or forms a salt form (such as sodium salt and potassium salt) by reacting with amino and hydroxyl sites of chitosan or hydroxybutyl chitosan;

(3) simultaneously reduces the charge density and introduces new charge species, namely, changes of the charge density and the charged species are realized by reacting with amino or hydroxyl sites of the chitosan or the hydroxybutyl chitosan. For example, carboxyl groups, sulfonic acid groups, and zwitterionic species are introduced by reaction at the amino site.

Among the types of chemical reactions used are: amidation (amino group and carboxyl group, amino group and acid anhydride, etc.), etherification (amino group and epoxy, etc.), esterification (hydroxyl group and carboxyl group, hydroxyl group and acid anhydride, etc.), click chemistry (amino group and alkynyl group, amino group and carbon-carbon double bond, mercapto group and carbon-carbon double bond, etc.), schiff base reaction (amino group and aldehyde group and reduction reaction for stabilizing schiff base), etc. The modified end group includes hydroxyl, carboxyl, sulfhydryl, aromatic ring, alkyl, double bond, etc. The terminal modification can be grafted with a compound or a polymer, or a certain functional group such as double bonds, carboxyl, sulfydryl, hydroxyl and the like can be firstly modified at an amino or hydroxyl site through multiple grafting reactions, and then the terminal modification is further carried out, so that the purpose of regulating and controlling the charge is achieved. Some of the compounds or polymers with charge-modulating properties are described in the following structures: :

wherein: (1) chloroacetic acid; (2) chloropropanol; (3) bromopropanol; (4) n, N-dimethyl (methacryloyloxyethyl) ammonio propanesulfonic acid inner salt; (5) n, N-dimethyl-N-acrylamidopropyl-N-propanesulfonic acid inner salt; (6) n, N-dimethyl-N-methacrylamidopropyl-N-propanesulfonic acid inner salt; (7) betaine methacrylate; (8) alkyl diacids (succinic acid, adipic acid); (9) malic acid; (10) lactobionic acid; (11) sodium 3-carboxybenzenesulfonate; (12) sodium allyl sulfonate; (13) succinic anhydride; (14) glutaric anhydride; (15) adipic anhydride; (16) - (25) functionalized PEG; the number of structural units is defined as i in structures (16) to (24).

In the invention, the obtained low-swelling injectable chitosan-based hydrogel can achieve the sterilization effect through terminal sterilization (moist heat sterilization), does not influence the gel forming capability, and is suitable for industrial production.

In the invention, the obtained low-swelling temperature-sensitive injectable chitosan-based hydrogel has controllable low-swelling performance, but the strength and time of gel formation can be changed due to different solvents and pH values. Under a slightly acidic environment, no gel formation is caused, or the gel formation is poor, so that it is necessary to ensure that the pH of the solution is not less than 6.3, preferably 6.8 to 7.5, and particularly preferably 7.1 to 7.4. In addition, the existence of salt has certain influence on the properties of the hydrogel, for example, the strength of the low-swelling temperature-sensitive chitosan-based hydrogel taking physiological saline (0.9% sodium chloride) as a solvent is obviously higher than that of the hydrogel taking water as the solvent at the same concentration.

In the invention, the obtained low-swelling injectable chitosan-based hydrogel is a natural polysaccharide-based polymer, has lower gel forming concentration, can form gel at the concentration of less than 1%, and the gel forming concentration of the temperature-sensitive hydrogel of the synthetic polymer is 1% or even more than 10% (J.Mater.chem.B,2013,1, 1249-1255; J.Mater.chem.2011, 21, 2246-2254), and the use of high concentration may cause the increase of the swelling rate. In the invention, the light transmittance of the obtained low-swelling temperature-sensitive injectable chitosan-based hydrogel is also obviously improved, most of the temperature-sensitive hydrogels are opaque white or milk white under physiological conditions, the main reason is that the refractive index is changed due to overlarge size of micelles in the gelling process of the temperature-sensitive hydrogel, and the refractive index of the low-swelling temperature-sensitive injectable chitosan-based hydrogel is close to that of water through charge modification, so that the field with the refractive index or light transmittance requirements can be widened.

In the invention, the principle of changing hydrophilicity and hydrophobicity, hydrogen bond interaction and the like to obtain controllable low swelling property is realized by the low swelling temperature-sensitive injectable chitosan-based hydrogel. The low-swelling temperature-sensitive injectable chitosan base realizes gelation and low swelling property through a physical process, and has the advantages of strong operability, no by-product, safety and degradability.

The invention has the advantages of

(1) The hydrogel provided by the invention has the characteristic of reversible gel-sol at low temperature, is a solution at low temperature, has low viscosity, is easy to inject, can quickly realize gelation due to the increase of temperature when being injected into a body, is particularly suitable for injection of irregular and narrow gaps, and has small damage to the body.

(2) The hydrogel provided by the invention has good biocompatibility, can form gel under low concentration, does not need a cross-linking agent, can be completely degraded into sugar units in vivo, is discharged along with metabolism, and is harmless to human bodies.

(3) The hydrogel provided by the invention has a swelling ratio of-10% -20%, can realize in-vivo non-swelling through process optimization, reduces the damage of swelling to an organism and the reduction of mechanics, and can be used in the biological fields of cell delivery, medical and aesthetic gel filling, in-vivo cavity blocking, skin repair, adhesion prevention, artificial vitreous body and the like.

(4) The hydrogel provided by the invention is simple to operate, mild in reaction conditions, high in repeatability and suitable for large-scale industrial production.

Drawings

FIG. 1 is a diagram of swollen material before and after modification: a is the sample before modification, B is the sample of example 1, C is the sample of example 2

FIG. 2 modified gel transition temperature and modulus of elasticity at 37 ℃

Detailed Description

The features of the present invention and other related features are further described in detail below by way of examples to facilitate understanding by those of ordinary skill in the art, but are not intended to limit the invention in any way. Those skilled in the art should also realize that such changes, modifications, additions and substitutions can be made without departing from the spirit and scope of the invention.

Example 1

Dissolving 1.0g of hydroxybutyl chitosan in 100ml of water, adjusting the pH to 5 by acetic acid or sodium hydroxide, and stirring uniformly at 20 ℃. Subsequently, 0.35g of succinic anhydride was added, and the pH of the whole was adjusted to 7 by a sodium hydroxide or sodium carbonate solution, and reacted for 3 hours. Finally, the pH was adjusted to neutral and dialyzed against 0.01M PBS7.2 for 3 days, against deionized water for 1 day, and lyophilized. The obtained sample is stored at a low temperature of-20 ℃. This was redissolved in PBS7.2 at a concentration of 2% and had a swelling ratio of 5.2%. Compared with unmodified hydroxybutyl chitosan, the volume shrinkage in the swelling experiment was significantly improved, as shown in fig. 1.

Example 2

Dissolving 1.5g of hydroxybutyl chitosan in 100ml of water, adjusting the pH to 6.5 by acetic acid or sodium hydroxide, and stirring uniformly at 25 ℃. Subsequently, 1.7g of succinic anhydride was added, and the pH of the whole was adjusted to 6 by a sodium hydroxide or sodium carbonate solution, followed by reaction for 12 hours. Finally, the pH was adjusted to neutral and dialyzed against 0.01M PBS7.2 for 3 days, against deionized water for 1 day, and lyophilized. The obtained sample is stored at a low temperature of-20 ℃. This was redissolved in PBS7.2 at a concentration of 1.5% and had a swelling ratio of 17.3%.

Example 3

Dissolving 1.3g of hydroxybutyl chitosan in 100ml of water, adjusting the pH to 6.5 by acetic acid or sodium hydroxide, and stirring uniformly at 10 ℃. Subsequently, 0.7g of succinic anhydride was added, and the pH of the whole was adjusted to 9 by a sodium hydroxide or sodium carbonate solution, and reacted for 4 hours. Finally, the pH was adjusted to neutral and precipitated with acetone, dialyzed against 0.01M PBS7.2 for 3 days, against deionized water for 1 day, and freeze-dried. The obtained sample is stored at a low temperature of-20 ℃. This was redissolved in PBS7.2 at a concentration of 2%, and the gel transition temperature was 15.53 ℃ and the gel strength was 488Pa, as shown in FIG. 2.

Swelling capacity test method: 2.0g of the cooled gel pre-solution was poured into a cylindrical mold (d.h.. 16 mm.16 mm) and placed in a constant temperature and humidity oven at 37 ℃ for 30min to fully gel. It was then removed and weighed as Wi, then soaked in 37 ℃ aqueous PBS7.2, removed at intervals and weighed, wiped dry until no further mass increase, i.e., swelling equilibrium was reached, at which time the mass was Wt. Swelling ratio (%) - (Wt-Wi)/Wi × 100%.

Rheology test methods: 0.5g of the cooled gel pre-solution was added to the rheometer for testing. The change in the elastic modulus (G ') and loss modulus (G ″) with temperature, corresponding to the gel formation temperature G' ═ G ″, was obtained using a 1 ° cone plate test, the plate diameter was 35mm, the test temperature was from 4 ℃ to 40 ℃, and the elastic modulus at 37 ℃ was the elastic modulus corresponding to the temperature in the curve.

Claims

1. A preparation method of a low-swelling temperature-sensitive injectable chitosan-based hydrogel is characterized in that chitosan is used as a raw material, and the low-swelling temperature-sensitive injectable chitosan-based hydrogel is obtained through hydroxybutylation modification and charge modification, wherein the structural formula of the low-swelling temperature-sensitive injectable chitosan-based hydrogel is shown in the following figure, wherein m + n is 1, and n is the deacetylation degree.

Wherein, the temperature sensitive group is grafted 1, 2-butylene oxide; -X is a swelling-regulating compound or polymer.

2. The method according to claim 1, wherein the swelling ratio of the low-swelling temperature-sensitive injectable chitosan-based hydrogel is between-10% and 20%.

3. The method of claim 1, wherein the chitosan has a degree of deacetylation of 50% to 98%; weight average molecular weight 5X 10⁴Da-100×10⁴Da。

4. The method according to claim 1, wherein the thermosensitivity of the low-swelling thermosensitivity injectable chitosan-based hydrogel is mainly derived from hydroxybutylation modification of chitosan, and the used reagent is 1, 2-butylene oxide, which has a modification degree of 0.5-3.

5. The method of claim 1, wherein the charge modification is performed at the amino group at position 2, the hydroxyl group at position 4 or 6 of chitosan, and the degree of modification is 0.2-1.

6. The method of claim 1, wherein the modification method comprises amidation, etherification, esterification, click chemistry, schiff base reaction, etc.

7. The method of claim 1, wherein the compound or polymer used for charge modification comprises: chloroacetic acid, chloropropanol, bromopropanol, N-dimethyl (methacryloyloxyethyl) ammonio propanesulfonic acid inner salt, N-dimethyl-N-methacrylamidopropyl-N-propanesulfonic acid inner salt, N-dimethyl-N-acrylamidopropyl-N-propanesulfonic acid inner salt, betaine methacrylate, alkyl diacids (succinic acid, adipic acid), malic acid, acid anhydrides (succinic anhydride, glutaric anhydride, adipic anhydride), lactobionic acid, sodium 3-carboxybenzenesulfonate, sodium allylsulfonate, functionalized PEG (such as COOH-PEG-COOH, COOH-PEG-OH, N-propyl bromide, N-dimethyl-N-methacrylamidopropyl-N-propanesulfonic acid inner salt, N-dimethyl-N-acrylamidopropyl-N-propanesulfonic acid inner salt, sodium allylsulfonate, functionalized PEG (such as COOH, COOH-PEG-OH, COOH, N-dimethyl-methacrylamidopropyl-N-acrylamidopropyl-N-propanesulfonic acid inner salt, N₃PEG, COOH-PEG-SH, double bond-PEG-COOH, CHO-PEG-SH, PEG-CHO), amino acid polypeptide derivatives (at least containing 1 carboxyl), aldehyde group compounds.

8. The method of claim 1, wherein the hydroxybutylation modification and the charge modification are performed by performing the charge modification and then the hydroxybutylation modification on the chitosan or performing the hydroxybutylation modification and then the charge modification.

9. The method according to claims 1 to 8, wherein the solvent of the low-swelling temperature-sensitive injectable chitosan-based hydrogel is water, or an aqueous solution containing salts such as sodium chloride and phosphate, and the pH of the solution is not lower than 6.3, and the concentration is 0.5mg/ml to 40mg/ml (w/v).

10. The method according to claims 1 to 9, wherein the low-swelling temperature-sensitive injectable chitosan-based hydrogel has a gelation transition temperature of 10 to 37 ℃.

11. The method according to claims 1 to 10, wherein the low-swelling temperature-sensitive chitosan-based hydrogel can be used as an injectable gel material in the biological fields of cell delivery, medical gel filling, body lumen occlusion (e.g. lacrimal duct embolism), skin repair, adhesion prevention, artificial vitreous body and the like.