WO2015161814A1

WO2015161814A1 - Method for homogeneously preparing hydroxypropyl modified chitin with low degree of deacetylation

Info

Publication number: WO2015161814A1
Application number: PCT/CN2015/077262
Authority: WO
Inventors: 蒋序林; 刘慧�; 杨奇志; 卓仁禧
Original assignee: 武汉大学
Priority date: 2014-04-25
Filing date: 2015-04-23
Publication date: 2015-10-29
Also published as: CN103951764A; CN103951764B

Abstract

Disclosed is a method for homogeneously preparing a hydroxypropyl modified chitin with a low degree of deacetylation, the method comprising: first chitin is added into a mixed aqueous solution of soluble alkali and urea to prepare a homogeneous chitin solution; then hydroxypropyl reagent or an aqueous solution thereof is added, and stirred and reacted; after post-treatments and drying, the hydroxypropyl modified chitin with a low degree of deacetylation is obtained. The hydroxypropyl modified chitin with a low degree of deacetylation has temperature sensitivity, and can be used as an injectable hydrogel and a drug carrier material.

Description

Method for preparing low-deacetylation hydroxypropyl modified chitin by homogeneous phase

Technical field

The invention relates to a method for uniformly preparing low-deacetylation hydroxypropyl modified chitin, belonging to the field of natural polymer modification and material science.

Background technique

Chitin is the second largest renewable natural polymer after cellulose and has a linear structure. Chitin and its derivatives have excellent biocompatibility, biodegradability, low toxicity and various biological activities, and have great research and application value. Under normal conditions, chitin itself has high crystallinity due to hydrogen bonding, is difficult to dissolve in water and low-concentration acid-base solution, and is not easily soluble in common organic solvents, thereby limiting its research and development and application.

The chitin and the carboxylation reagent are reacted in a sodium hydroxide-urea system to obtain a carboxy chitin having pH sensitivity and temperature sensitivity. A homogeneous preparation is reported in the Chinese Patent Application Publication No. CN201310641249.6. A method of deacetylating carboxy chitin. The reaction of chitin with hydroxyethylating agent (mainly chlorohydrin and ethylene oxide) can produce hydroxyethyl chitin which is soluble in water [a method for preparing water-soluble hydroxyethyl chitin, China Patent Application Publication No. CN200610116655.0]. Hou Chunlin et al. reported the preparation of temperature-sensitive hydroxybutyl chitosan [Chinese Patent Application Publication No. CN200810033699.6] and temperature-sensitive hydroxypentyl chitosan [Chinese Patent Application Publication No. CN201210220246.0] and its clinical application Research [Zhou Lin, temperature-sensitive hydroxybutyl chitosan as a basis for the prevention and treatment of cerebrospinal fluid leakage materials, clinical research, 2012, Second Military Medical University Changzheng Hospital Master's Thesis]. However, there are few reports on the preparation of hydroxypropyl modified chitin, especially low deacetylated hydroxypropyl chitin. Wang Aiqin et al reported the application of water-soluble hydroxypropyl chitin in artificial tears [Wang Aiqin et al. Sexuality and its application in artificial tears, Chinese Marine Medicine, 1997,16,14-17], but the modified chitin has a higher degree of deacetylation of 0.4 and is reacted in a heterogeneous medium of isopropanol. The reaction process is difficult to control.

Summary of the invention

The invention aims at the limitation of the prior art technology for preparing hydroxypropyl chitin, and provides a simple method for preparing low-deacetylation hydroxypropyl modified chitin by homogeneous process: the reaction medium uses a lower alkali concentration and does not use an organic solvent. The reaction conditions are mild, which is favorable for large-scale industrial production; chitin is basically not degraded, the degree of acetylation of chitin is reduced, the distribution of product substitution degree is easy to control, and the yield is high. The prepared hydroxypropyl chitin hydrogel has good injectability and can be used as a carrier material for drugs (including polypeptides, proteins and cells), and is widely used in the fields of medical cosmetic care and tissue engineering minimally invasive repair.

The technical solution provided by the present invention is as follows:

A method for homogeneously preparing a low degree of deacetylation hydroxypropyl modified chitin, comprising the steps of:

The chitin with a degree of deacetylation of less than 25% is prepared into a 0.5% to 8% by weight aqueous solution of homogeneous chitin, and a hydroxypropylating agent is added to the aqueous solution of the homogeneous chitin, and the hydroxypropylating agent is added. The amount is 1 to 40 times the number of moles of the chitin structural unit, and the homogeneous reaction is carried out at 0 to 50 ° C for 10 to 105 hours, and the reaction solution is post-treated to obtain a hydroxypropyl modified chitin having a degree of deacetylation of less than 30%. .

The chitin is preferably a powdery chitin having a degree of deacetylation of less than 10%, and preferably has a weight average molecular weight of from 5 × 10 ⁴ to 5 × 10 ⁶ .

The hydroxypropylating agent is preferably a propylene oxide ring, a chloropropanol, a bromopropanol or any mixture thereof.

The post-treatment method comprises the steps of: adjusting the pH of the reaction solution to 6.9-7.6 with an acid solution, precipitating the product with an organic solvent, and washing or pure water dialysis to remove small molecules such as urea and salt, and drying is obtained. Hydroxypropyl-modified chitin with an acetyl degree of less than 30% can even obtain hydroxypropyl-modified chitin with a degree of deacetylation of less than 20%.

The acid is preferably one or more of hydrochloric acid, sulfuric acid, nitric acid, formic acid, acetic acid, and citric acid.

The organic solvent is preferably one or more of methanol, ethanol, isopropanol, acetone, and tetrahydrofuran.

The preparation method of the homogeneous chitin aqueous solution comprises the following steps: firstly preparing a mixed liquid having a mass ratio of soluble alkali: urea: pure water=6-20:3-12:68-91; the degree of deacetylation is lower than 25% of chitin is dissolved in the above mixture, and after being uniformly mixed, it is allowed to stand at -30 to -12 ° C for 24 to 96 hours, and after thawing, it is sufficiently stirred at 1 to 25 ° C to obtain a homogeneous aqueous solution of chitin.

The soluble base is sodium hydroxide, potassium hydroxide and/or lithium hydroxide.

The above process produces a low degree of deacetylation hydroxypropyl modified chitin with temperature sensitivity.

The above method can produce hydroxypropyl modified chitin having a degree of deacetylation of less than 15%.

A temperature-sensitive low-deacetylated hydroxypropyl chitin prepared by the above method.

The hydroxypropyl modified chitin prepared by the above method is used as an artificial tear, an injectable hydrogel and a drug carrier material.

The process of the present invention produces a temperature sensitive low degree of deacetylation hydroxypropyl modified chitin having a degree of deacetylation of less than 15%. The low deacetylation degree hydroxypropyl modified chitin prepared by the invention can be used as an artificial tear, an injectable hydrogel and a drug carrier material; and because of its low cost and biodegradability, it can be widely used in other medical health care and foods. Processing and other fields have high application development value. The reaction medium of the invention is a mixed aqueous solution of a soluble alkali and urea with a lower alkali concentration, without an organic solvent, the reaction conditions are mild, the damage to the chitin molecule is small, the product substitution degree distribution is easy to control, and the yield is high. Conducive to large-scale industrial production.

Compared with the prior art, the invention has significant technological progress and characteristics:

First, the invention prepares low-deacetylation hydroxypropylated chitin in a homogeneous aqueous solution of chitin, has simple preparation process, mild reaction condition, easy control process, high yield, and can be used for large-scale industrial production;

Secondly, the invention adopts a lower concentration of alkali liquid (6-20% by weight) as a reaction medium, the reaction process does not involve an organic solvent, is non-toxic and non-polluting, and has a low cost;

Third, during the reaction of the present invention, chitin is substantially not degraded, the degree of acetylation of chitin is reduced, and the degree of hydroxypropyl substitution of the product is uniform.

DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a nuclear magnetic resonance spectrum of 10 wt% DCl/D ₂ O at 25 ° C of hydroxypropyl modified chitin obtained by using propylene oxide as a hydroxypropylating agent in Example 1.

2 is a nuclear magnetic resonance spectrum of 20 wt% DCl/D ₂ O at 25 ° C of hydroxypropyl modified chitin synthesized by using bromopropanol as a hydroxypropylating reagent at different reaction times in Example 9.

Figure 3 is a graph showing the rheological behavior of hydroxypropyl modified chitin HPCH-8 (solution concentration 2 wt%, pH 7.4) at elevated temperature and temperature drop in Example 10 of the present invention.

4 is a graph showing changes in storage modulus and loss modulus of a hydroxypropyl modified chitin HPCH-8 solution (1 wt% to 3 wt%, pH 7.4) at 37 ° C in Example 10 according to the present invention.

Figure 5 is a graph showing the release profile of bovine serum albumin in PBS buffer and lysozyme (1.5 mg/L) aqueous solution in hydroxypropyl modified chitin HPCH-8 hydrogel.

Figure 6 is a graph showing the toxicity of cells (293T) in different concentrations of HPCH-4 hydroxypropyl modified chitin PBS aqueous solution.

Figure 7 is a graph showing the change in the number of three-dimensional cell proliferation cultures (CCK-8 method) of COS-7 cells (250,000/mL) in HPCH-4 hydroxypropyl modified chitin hydrogel.

detailed description

The present invention will be further described in detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

Example 1

First, prepare a mixture of sodium hydroxide: urea: pure water = 11:4:85 (mass ratio); dissolve 2.0 g of chitin (deacetylation degree less than 25%) in 100 g of the above mixture, and mix well After standing at -30 ° C for 24 hours, thawing and stirring well at 2 to 5 ° C to obtain 2 wt% of a homogeneous aqueous solution of chitin; adding 11.4 g of propylene oxide as a hydroxypropyl group to a homogeneous aqueous solution of chitin The reagent is stirred at a temperature of 4 to 6 ° C for 24 hours, and then the temperature is raised to 9 to 11 ° C to stir the reaction. The reaction solution is sampled at 6, 12, and 24 hours, respectively, and the obtained sample solution is respectively 1 M. The hydrochloric acid solution was adjusted to a pH of 7.0 to obtain a uniform transparent solution; then, it was dialyzed against distilled water for 7 days to remove small molecules such as urea and salt in the solution, and lyophilized to obtain a sponge-like hydroxypropyl-modified chitin. The reaction was obtained in 6, 12 and 24 hours. The three products were recorded as HPCH-7, HPCH-8 and HPCH-9, respectively, and the reaction yields were 87%, 91% and 87%, respectively, and their nuclear magnetic resonance spectra are shown in Fig. 1. It can be seen from Fig. 1 that the obtained product modified chitin has a hydroxypropyl group introduced therein, and these products have a high degree of acetylation (>85%), and are calculated by nuclear magnetic resonance spectroscopy: the acetylation degree DA of HPCH-7 is 0.92. , its hydroxypropyl molar substitution MS is 0.53; HPCH-8 has an acetylation degree DA of 0.89, its hydroxypropyl molar substitution MS is 0.77; HPCH-9 has an acetylation DA of 0.87, and its hydroxypropyl molar substitution degree The MS is 0.80.

Example 2

First, prepare a mixture of sodium hydroxide: urea: pure water = 11:4:85 (mass ratio); then dissolve 2.0 g of chitin (deacetylation degree less than 25%) in 100 g of the above mixture, and mix After homogenization, it was allowed to stand at -30 ° C for 24 hours, and after thawing, it was thoroughly stirred at 2 to 5 ° C to obtain 2 wt% of a homogeneous aqueous solution of chitin; and 11.4 g of propylene oxide was added as a hydroxypropyl group to a homogeneous aqueous solution of chitin. The reagent is stirred at a temperature of 4 to 6 ° C for 24 hours, and then the temperature is raised to 14 to 16 ° C to stir the reaction. The reaction is sampled at 6 and 12 hours, respectively, and the reaction solution is a homogeneous transparent system. The obtained sample solution was adjusted to pH 7.5 with a 1 M hydrochloric acid solution, and then 1.2 liters of acetone was added to precipitate the product. After filtration, the precipitate was washed with an aqueous ethanol solution (80%, v/v), and the urea and salts were washed away. Small molecule material; the precipitate was then dried at 50 ° C to obtain a powdery hydroxypropyl modified chitin. The products obtained in the reaction for 6 and 12 hours were all dissolved in pure water. These two products were designated as HPCH-4 and HPCH-5, respectively, and the yields were 83% and 86%, respectively. The NMR degree of HPCH-4 was 0.90, the hydroxypropyl molar substitution MS was 0.99, the acetyl degree DA of HPCH-5 was 0.86, and the hydroxypropyl molar substitution MS was 0.99.

Example 3 Effect of Different Feed Ratios

First, prepare a mixture of sodium hydroxide: urea: pure water = 11:4:85 (mass ratio); then, 3.0 g of chitin (deacetylation degree less than 25%) is dissolved in 150 g of the above mixture, and mixed. After homogenization, it was placed at -30 ° C for 24 h and stirred at the same time. After thawing, 2 wt% of the homogeneous chitin aqueous solution was prepared by thoroughly stirring at 2 to 5 ° C; the homogeneous chitin aqueous solution was divided into 3 equal parts of volume, respectively. Proportion of propylene oxide (1.43g, 2.85g and 4.28g) as hydroxypropylation reagent, the rest of the steps are exactly the same; the temperature is controlled at 4 ~ 6 ° C, the reaction is stirred for 24 hours, and then the temperature is raised at 14 ~ 16 ° C The reaction was stirred for 6 hours, and the reaction liquid was a homogeneous transparent system; then the pH of the reaction liquid was adjusted to 7.2 with a 1 M hydrochloric acid solution, and then 0.8 liter of acetone was added to precipitate the reaction product, and the precipitate was filtered with an aqueous ethanol solution (80%). , v/v) washing, washing away small molecular substances such as urea and salt; then drying the precipitate at 50 ° C to obtain a powdery hydroxypropyl modified chitin. The three products obtained by adding 1.43 g, 2.85 g and 4.28 g of propylene oxide were designated as HPCH-1, HPCH-2 and HPCH-3, respectively, and the yields were 87%, 85% and 86%, respectively. The NMR degree of HPCH-1 was 0.89, the hydroxypropyl molar substitution MS was 0.34, the acetylation degree DA of HPCH-2 was 0.89, and the hydroxypropyl molar substitution MS was 0.58. HPCH was calculated by nuclear magnetic resonance spectroscopy. The acetylation degree DA of -3 was 0.88, and the hydroxypropyl molar substitution MS was 0.83.

Example 4

First, prepare a mixture of sodium hydroxide: urea: pure water = 11:4:85 (mass ratio); then dissolve 2.0 g of chitin (deacetylation degree less than 25%) in 100 g of the above mixture, and mix After homogenization, it was placed in a freezer compartment of -30 ° C for 24 hours and stirred at appropriate time. After thawing, it was thoroughly stirred at 2 to 5 ° C to obtain 2 wt% of a homogeneous aqueous solution of chitin; then 0.57 g of a homogeneous chitin aqueous solution was added. Propylene oxide as a hydroxypropylation reagent, adjust the reaction temperature to 4 ~ 6 ° C to stir the reaction for 24 hours, then increase the temperature to 14 ~ 16 ° C to stir the reaction for 33 hours; then adjust the pH of the reaction solution with 1M hydrochloric acid solution to 7.2, a clear solution was obtained; then, 2 L of an aqueous ethanol solution (85%, v/v) was added to precipitate a reaction product, and after filtration, the precipitate was dried at 50 ° C to obtain 1.9 g of a powdery hydroxypropyl-modified chitin.

Except that the time of stirring the reaction at 14-16 ° C was extended from 33 hours for another 3 days, that is, the reaction time was stirred at 14-16 ° C for 105 hours, and the other operation steps were identical, and the obtained powdery hydroxypropyl group was obtained. The base modified chitin is completely dissolved in pure water.

Example 5

First, prepare a mixture of sodium hydroxide: urea: pure water = 6:3:91 (mass ratio); then dissolve 0.5 g of chitin (deacetylation degree less than 25%) in 100 g of the above mixture, and mix After homogenization, it was allowed to stand at -12 to -20 ° C for 72 hours, and after thawing, it was thoroughly stirred at 1 to 12 ° C to obtain a 0.5 wt% homogeneous solution of chitin; and 5.7 g of propylene oxide was added to the homogeneous solution of chitin. As a hydroxypropylation reagent, the reaction is carried out at 0 to 50 ° C (low temperature first, then slowly warmed up, the temperature rise process is not strictly controlled, the whole process is controlled to be a homogeneous system), and the reaction is stirred for 10 hours, and then treated with 1 M hydrochloric acid solution. The pH of the reaction solution was adjusted to 6.9, and dialyzed against distilled water for 7 days to remove small molecules such as urea and salt in the reaction liquid, followed by lyophilization to obtain a water-soluble hydroxypropyl-modified chitin in the form of a sponge powder.

Example 6

First, prepare a mixture of sodium hydroxide: urea: pure water = 11:4:85 (mass ratio); then dissolve 1.0 g of chitin (deacetylation degree less than 25%) in 100 g of the above mixture, and mix After homogenization, it was placed in a freezer compartment of -28 ° C for 24 hours, and stirred at the same time; after thawing, it was thoroughly stirred at 12-25 ° C to obtain a 1 wt% homogeneous solution of chitin; and 5.8 g of chitin homogeneous solution was added. Propylene oxide as a hydroxypropylation reagent, the reaction temperature is controlled at 8 ~ 12 ° C to stir the reaction for 15 hours, then increase the temperature to 18 ~ 22 ° C to stir the reaction, control the reaction liquid is a homogeneous transparent system; the reaction liquid in 18 ~ 22 The reaction was sampled at 6 ° C and 6 hours, respectively, and the obtained sample solution was adjusted to pH 7.4 with a 1 M hydrochloric acid solution to obtain a clear solution; then, 2 L of an aqueous ethanol solution (85%, v/v) was added to precipitate the reaction product. After filtration, the precipitate was dried at 50 ° C to obtain a powdery hydroxypropyl modified chitin.

Calculated by nuclear magnetic resonance spectroscopy: the reaction solution was reacted at 18-22 ° C for 6 hours to obtain hydroxypropyl modified chitin acetylation DA of 0.88, and its hydroxypropyl molar substitution MS was 0.44; the reaction solution was 18-22 Reaction at °C for 37 hours to obtain hydroxypropyl The base modified chitin has a degree of acetylation DA of 0.85 and a hydroxypropyl molar substitution MS of 0.55.

Example 7

First, prepare a mixture of sodium hydroxide: urea: pure water = 8:4:88 (mass ratio); then dissolve 1.2 g of chitin in 30 g of the above mixture, mix well and place at -30 ° C for 36 h. And stirring at the same time; the solution is thawed and thoroughly stirred at 2-8 ° C to obtain a 4 wt% homogeneous solution of chitin; then 3.4 g of propylene oxide is added as a hydroxypropylation reagent to the chitin homogeneous solution. The reaction was stirred at 5 ° C for 6 hours, at 8 to 12 ° C for 12 hours, at 14 to 16 ° C for 12 hours, at 8 to 22 ° C for 12 hours, and at 30 to 40 ° C for 12 hours. The system was homogeneously transparent; then the pH of the reaction solution was adjusted to 7.6 with a 1 M hydrochloric acid solution, and then 1 L of acetone was added to precipitate the reaction product, and the filtered precipitate was washed with an aqueous ethanol solution (80%, v/v), and the urea was washed away. And a small molecular substance such as a salt; the precipitate is then dried at 50 ° C to obtain a powdery water-soluble hydroxypropyl-modified chitin.

Example 8

First, prepare a mixture of sodium hydroxide: urea: pure water = 20:12:68 (mass ratio); then dissolve 8.0 g of chitin in 100 g of the above mixture, and mix well at -20 to -30 ° C. The refrigerator was placed in the freezer for 96 hours, and stirred at the appropriate time. After thawing, the mixture was thoroughly stirred at 2 to 5 ° C to obtain a 8 wt% homogeneous solution of chitin; 22.8 g of propylene oxide was added to the homogeneous solution of chitin. The hydroxypropylation reagent was stirred and homogenized at 2 to 20 ° C for 48 hours, then the pH of the reaction solution was adjusted to 7.2 with a 2 M hydrochloric acid solution, and a large amount of ethanol-acetone mixture (1/1, v/v was added). The reaction product was precipitated 3 times to remove small molecules such as urea and salt therein; and then the precipitate was vacuum dried to obtain a powdery water-soluble hydroxypropyl-modified chitin.

Example 9 different hydroxypropylation reagents

First, prepare a mixture of sodium hydroxide: urea: pure water = 11:4:85 (mass ratio); then add 1.0 g of chitin to 100 g of the above mixed aqueous solution, mix well and freeze in a refrigerator at -26 ° C. Place in the chamber for 24 hours, stir at appropriate time, thaw after thawing at 20 ° C to obtain a 1 wt% homogeneous solution of chitin; add 6.9 g of 3-bromopropanol as a hydroxypropylation reagent to the homogeneous solution of chitin The reaction temperature was further adjusted at 8 to 12 ° C for 48, 72 and 84 hours, respectively; the obtained sample solution was adjusted to pH with a 1 M hydrochloric acid solution to obtain a transparent solution, using 1 liter of aqueous ethanol solution (85%). , v / v) precipitation of the reaction product, after filtration, the precipitate was dried at 50 ° C to obtain a powdery water-soluble hydroxypropyl modified chitin, the nuclear magnetic resonance spectrum of these three products is shown in Figure 2, at 8 The bromopropanol modified chitin obtained by stirring the reaction at ～12 ° C for 48 h has an acetylation degree DA of 0.92 and a hydroxypropyl molar substitution MS of 0.34; the bromopropanol modified by stirring the reaction at 8 to 12 ° C for 72 hours Chitin has an acetylation degree DA of 0.89 and a hydroxypropyl molar substitution MS of 0.39; The reaction bromopropanol 84h modified chitin obtained was stirred at 12 ℃ DA acetylation degree of 0.89, which is a hydroxypropyl molar substitution MS of 0.44.

Similarly, the other steps are identical, using 3-chloropropanol instead of 3-bromopropanol, wherein the molar ratio of 3-chloropropanol to chitin structural unit is 20:1, and the reaction temperature is 13-17 ° C. A water-soluble hydroxypropyl-modified chitin was also obtained after a reaction time of 84 hours.

Example 10 Temperature Sensitivity

100 mg of the hydroxypropyl modified chitin CHCH-8 prepared in Example 1 was weighed, and 5 mL of 0.15 M PBS buffer salt having a pH of 7.4 was added to completely dissolve, and the hydroxypropyl modified chitin was heated and cooled using a rheometer. When the storage modulus and loss modulus change, the temperature rise (cooling) rate is 0.5 ° C / min, the frequency is 1 Hz, the strain is 5%, and the result is shown in FIG. 3 . As can be seen from Fig. 3, the hydroxypropyl modified chitin is temperature sensitive, and its gelation transition temperature is 18 ° C at elevated temperature, and can be rapidly solidified into a gel state at a physiological temperature of 37 ° C; gelation The transition temperature was 11.5 ° C when the temperature was lowered. Thus, the hydroxypropyl-modified chitin produced by the present invention is temperature sensitive and reversible and is expected to be used as a temperature sensitive injectable hydrogel material. The higher the concentration of hydroxypropyl modified chitin, the lower the gelation transition temperature and the greater the gel storage modulus formed, as shown in FIG. As can be seen from the figure, these aqueous solutions of hydroxypropyl-modified chitin can be rapidly gelled (less than 30 seconds) at 37 °C. Hydroxypropyl modified chitin with different molar substitution degree, different concentration of hydroxypropyl modified chitin can form hydrogels with different morphologies, which can be used for drug loading, and the release rate can be adjusted according to needs, so Such hydroxypropyl modified chitin is expected to be used as a pharmaceutical carrier material.

Example 11 Injectability of Hydroxypropyl Modified Chitin

100 mg of the hydroxypropyl-modified chitin CHCH-8 obtained in Example 1 was weighed out and completely dissolved in 5 mL of a 0.15 M PBS buffer salt having a pH of 7.4. 1 mL of the solution was directly injected into a PBS buffered saline solution at 37 ° C through a syringe equipped with a 25 G needle to rapidly become a gel; 1 mL of the solution was injected into a mold of different shapes and rapidly became a gel at 37 ° C; 1mL of the solution was sterilized and packaged in a steam autoclave and placed in a refrigerator at 4 ° C overnight. Then, 0.5 mL of C57BL/6j mice were subcutaneously injected through a syringe equipped with a 25G needle, and it was found that the gel could be rapidly formed under the skin of the mouse. The gel shape remained essentially unchanged after one week. It shows that the hydrogel has good injectability and can be used for cosmetic beauty care and minimally invasive tissue repair.

Example 12 Application of Hydroxypropyl Modified Chitin as Drug (Protein) Carrier

Using bovine serum albumin (BSA) as a model drug, an aqueous solution of BSA aqueous solution (20 mg/mL) and hydroxypropyl modified chitin CHCH-8 (2%) was placed, mixed and placed at 37 ° C to form a gel, that is, all BSA Load into the hydrogel. The protein release profile was tested in a 37 ° C water bath, as shown in Figure 5, indicating that the protein could be released slowly, but the release rate of the protein was significantly accelerated in the presence of lysozyme (1.5 mg/L), indicating that the hydroxypropyl group was modified. The chitin hydrogel can be used as a drug (protein) carrier.

Example 13 Hydroxypropyl Modified Chitin Cytocompatibility

Using the hydroxypropyl modified chitin CHCH-4 prepared in Example 2, PBS solutions of different polymer concentrations were prepared, and 293T cells were selected as representatives, and these hydroxypropyl modified chitin aqueous solutions were co-cultured with cells in two dimensions. The relative amount of viable cells was tested by MTT method for 48 hours, and the results are shown in Fig. 6. As can be seen from the figure, the hydroxypropyl modified chitin has no significant cytotoxicity in the range of polymer concentrations tested (0.1-2000 mg/L). The hydroxypropyl modified chitin PBS aqueous solution was mixed with the COS-7 cell suspension to prepare a polymer hydrogel. The three-dimensional cell culture was carried out at 37 ° C, and the cell growth and proliferation were found to be good, as shown in FIG. 7 . It is indicated that the hydroxypropyl modified chitin hydrogel can be used as a cell carrier without affecting the three-dimensional growth and proliferation of cells, and can be used for tissue engineering minimally invasive repair.

In the embodiment of the present invention, the aqueous sodium hydroxide solution may be replaced by other soluble alkali solution such as potassium hydroxide aqueous solution, lithium hydroxide or a mixture thereof, and the acid whose pH is adjusted may be replaced by sulfuric acid, nitric acid, formic acid, acetic acid or citric acid. The solvent used for the precipitation may be methanol, ethanol, isopropanol, acetone or tetrahydrofuran instead of acetone and an aqueous ethanol solution, and the effects of the present invention are not affected.

The specific embodiments of the present invention have been described in detail with reference to the preferred embodiments of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

A method for uniformly preparing low-deacetylation hydroxypropyl modified chitin, comprising the steps of: preparing chitin with a degree of deacetylation of less than 25% to form a homogeneous shell of 0.5 wt% to 8 wt% In the aqueous solution of the solution, a hydroxypropylating agent is added to the aqueous solution of the homogeneous chitin, and the hydroxypropylating agent is added in an amount of from 1 to 40 times the number of moles of the chitin structural unit, and the homogeneous phase is stirred at 0 to 50 ° C. The reaction is carried out for 10 to 105 hours, and the reaction solution is post-treated to obtain a hydroxypropyl-modified chitin having a degree of deacetylation of less than 30%.
The method according to claim 1, wherein said raw material chitin is a powdery chitin having a degree of deacetylation of less than 10%, and has a weight average molecular weight of from 5 × 10 4 to 5 × 10 6 ; The hydroxypropylating agent is a propylene oxide ring, chloropropanol, bromopropanol or any mixture thereof.
The method according to claim 1 or 2, wherein the method for preparing a homogeneous aqueous solution of chitin comprises the following steps: firstly preparing a mass ratio of soluble alkali: urea: pure water = 6 to 20:3 a mixture of 12:68 to 91; the chitin having a degree of deacetylation of less than 25% is dissolved in the above mixture, and after being uniformly mixed, it is allowed to stand at -30 to -12 ° C for 24 to 96 hours, and after thawing at 1 to 25 Stir well at °C to obtain a homogeneous aqueous solution of chitin.
The method of claim 3 wherein said soluble base is sodium hydroxide, potassium hydroxide and/or lithium hydroxide.
The method according to claim 1 or 2, wherein the post-treatment method comprises: adjusting the pH of the reaction solution with an acid solution to 6.9 to 7.6, precipitating the product with an organic solvent, and dialysis by washing or pure water. The urea and the salt are removed, and after drying, a hydroxypropyl-modified chitin having a degree of deacetylation of less than 30% is obtained.
The method according to claim 5, wherein the dilute acid used is one or more of hydrochloric acid, sulfuric acid, nitric acid, formic acid, acetic acid, and citric acid; and the organic solvent is methanol, ethanol, or isopropyl. One or more of alcohol, acetone, and tetrahydrofuran.
The method according to claim 1 or 2, wherein the produced hydroxypropyl modified chitin is temperature sensitive.
The method according to claim 1 or 2, wherein the obtained hydroxypropyl-modified chitin has a degree of deacetylation of less than 15%.
A temperature-sensitive low-deacetylation hydroxypropyl-modified chitin prepared by the method of any one of claims 1-8.
Use of the hydroxypropyl modified chitin of any one of claims 1 to 8 as an artificial tear, an injectable hydrogel and a pharmaceutical carrier material.