阅读说明：本技术 一种改性壳聚糖微凝胶及其制备方法与应用 (Modified chitosan microgel and preparation method and application thereof ) 是由 倪才华 张丽萍 桑欣欣 于 2020-12-08 设计创作，主要内容包括：本发明涉及一种改性壳聚糖微凝胶及其制备方法与应用,属于壳聚糖衍生物合成技术领域。本发明通过ε-己内酯与3-羟基己二酸3,6-内酯的无规共聚物对壳聚糖改性,其无规共聚物结构单元中侧链悬挂的带负电荷的羧基与壳聚糖分子中带正电荷的氨基进行复合形成改性壳聚糖微凝胶,制备方法简单、安全可靠、容易控制；且所制得的改性壳聚糖微凝胶稳定性好、载药率高、无细胞毒性、生物相容性较好,可作为抗癌药物载体。此外,本发明采用的壳聚糖和ε-己内酯与3-羟基己二酸3,6-内酯的无规共聚物都是可生物降解的聚合物,适合作为生物材料用于人体内。(The invention relates to a modified chitosan microgel and a preparation method and application thereof, belonging to the technical field of synthesis of chitosan derivatives. According to the invention, the chitosan is modified by the random copolymer of epsilon-caprolactone and 3-hydroxy adipic acid 3, 6-lactone, and the carboxyl with negative charge suspended on the side chain in the structural unit of the random copolymer is compounded with the amino with positive charge in the chitosan molecule to form the modified chitosan microgel, so that the preparation method is simple, safe, reliable and easy to control; the prepared modified chitosan microgel has good stability, high drug loading rate, no cytotoxicity and good biocompatibility, and can be used as an anticancer drug carrier. In addition, the chitosan and the random copolymer of epsilon-caprolactone and 3-hydroxy adipic acid 3, 6-lactone adopted by the invention are biodegradable polymers and are suitable for being used as biological materials in human bodies.)

1. The preparation method of the modified chitosan microgel is characterized by comprising the following steps: dissolving LC-HA of a random copolymer of epsilon-caprolactone and 3-hydroxyadipic acid 3, 6-lactone in toluene or tetrahydrofuran to form an LC-HA solution, dropwise adding the LC-HA solution into the chitosan suspension under the condition of stirring, uniformly mixing, and dialyzing and purifying to obtain modified chitosan microgel; wherein the LC-HA is synthesized by ring-opening polymerization reaction of epsilon-caprolactone and 3-hydroxy adipic acid 3, 6-lactone in toluene under the action of a catalyst.

2. The method of claim 1, wherein the concentration of the LC-HA solution is 1.0 wt%; the concentration of the chitosan suspension is 1.0 wt%; the volume ratio of the LC-HA solution to the chitosan suspension is 1: 3-1: 6.

3. The method for preparing modified chitosan microgel of claim 1, wherein the random copolymer of epsilon-caprolactone and 3-hydroxyadipate 3, 6-lactone LC-HA is synthesized by the following steps: adding toluene, epsilon-caprolactone, 3-hydroxyadipic acid 3, 6-lactone and a catalyst into a reaction container, heating for reflux reaction, cooling to room temperature, precipitating by using an organic solvent A to obtain a crude product of a random copolymer of the epsilon-caprolactone and the 3-hydroxyadipic acid 3, 6-lactone, and purifying the crude product to obtain the random copolymer LC-HA of the epsilon-caprolactone and the 3-hydroxyadipic acid 3, 6-lactone; wherein the organic solvent A is one of petroleum ether, diethyl ether, methanol or ethanol.

4. The method for preparing the modified chitosan microgel of claim 3, wherein the molar ratio of the epsilon-caprolactone to the 3, 6-hydroxyadipate is 1:1 to 1: 3; the catalyst is one of stannous chloride or stannous octoate, and the dosage of the catalyst is 0.5-1.0% of the total weight of epsilon-caprolactone and 3-hydroxyadipic acid 3, 6-lactone; the dosage of the toluene is 3-5 times of the total weight of the epsilon-caprolactone and the 3-hydroxy adipic acid 3, 6-lactone.

5. The method for preparing a modified chitosan microgel as claimed in claim 3, wherein the heating reflux reaction method comprises: heating to 85-95 ℃ for reflux reaction for 2-4 h, and then continuously heating to 105-110 ℃ for reflux reaction for 2-3 h.

6. The method of claim 3, wherein the crude product is purified by the following steps: dissolving the crude product of the random copolymer of epsilon-caprolactone and 3-hydroxy adipic acid 3, 6-lactone in toluene or tetrahydrofuran, precipitating the crude product by using an organic solvent A, circularly dissolving and precipitating the crude product for three times in the way, and finally drying the precipitate in vacuum.

7. The method for preparing a modified chitosan microgel as claimed in claim 2, wherein the chitosan suspension is prepared by the following steps: preparing an acetic acid solution of chitosan with the concentration of 0.02-0.04 g/mL, precipitating the chitosan under an alkaline condition, filtering to obtain a chitosan precipitate, repeatedly washing until the filtrate is neutral, soaking in an organic solvent B, filtering, drying in vacuum to constant weight, grinding into powder, and preparing a chitosan suspension with the concentration of 1.0 wt% by using ultrapure water; wherein the concentration of the acetic acid solution is 2 wt%; the organic solvent B is one of ethanol or isopropanol.

8. The method for preparing modified chitosan microgel as claimed in claim 1, wherein the cut-off molecular weight of the dialysis bag used in dialysis is 14000, and the conditions of dialysis purification are as follows: the dialysis time is 72h, and the dialysate is replaced every 8 h; the dialysate is deionized water.

9. The modified chitosan microgel prepared by the method for preparing the modified chitosan microgel of any one of claims 1 to 8.

10. Use of the modified chitosan microgel of claim 9 in anticancer drug carriers.

Technical Field

The invention relates to a modified chitosan microgel and a preparation method and application thereof, belonging to the technical field of synthesis of chitosan derivatives.

Background

The chitosan is a natural polycation polysaccharide obtained by deacetylating chitin, has no toxicity, irritation, sensitization and mutation, has good biocompatibility and biodegradability, and can be dissolved in acidic aqueous solution. The chitosan can be prepared into hydrogel, chitosan film, nano micelle and other forms and is widely used as medical materials. The hydrogel prepared from chitosan can be used as a drug carrier to control drug slow release, prolong drug curative effect, reduce drug toxic and side effects, improve permeability of hydrophobic drug to cell membrane and drug stability, change drug delivery route, and greatly enhance targeted drug delivery capability of the preparation. The chitosan has wide sources, has the advantages of mature preparation technology, simple and easy working procedures, no odor, heat resistance, low price and the like, and is widely used in the fields of medicines and the like at present.

However, chitosan can only be dissolved in an acidic solution, and pure chitosan is difficult to prepare into hydrogel and has low drug loading rate for hydrophobic drugs. Therefore, it is necessary to modify chitosan so that the modified chitosan derivative has hydrophilic and hydrophobic amphiphilic structures or forms micro-regions with local hydrophobicity. Detchprohm et al, synthetic of a Novel Chitin Derivative in Aqueous Reaction Media in the presence of water as a medium, published in 2001, Macromolecular Chemistry and Physics, 202, 3560, 3570, teach the preparation of chitosan-graft-polycaprolactone by ring-opening polymerization of chitosan and caprolactone monomers in the presence of stannous octoate as a catalyst and water as a swelling agent. On the basis, researchers in the field carry out a series of improvement and development researches, for example, the invention patent with the Chinese patent publication number of CN 100384883C provides a method for preparing chitosan-graft-polycaprolactone, the invention patent with the publication number of CN 101580556B provides a method for preparing a temperature-sensitive amphiphilic graft copolymer with chitosan as a main chain, and the invention patent with the publication number of CN 111187416A provides a method for preparing a chitosan-g-poly epsilon-caprolactone derivative drug-carrying nano micelle and application thereof. These patents all adopt epsilon-caprolactone as a monomer to improve the synthesis of the amphiphilic chitosan derivative. The epsilon-caprolactone contains a lactone ring structure and can be subjected to ring-opening polymerization under the action of a catalyst to form hydrophobic polyester. Polyester is a biodegradable polymer suitable for use as a biomaterial in the human body. However, in the prior art, when hydrogel is prepared on the basis of the above concept, a chemical crosslinking method is usually adopted, and a small molecule containing aldehyde group is used as a crosslinking agent and chemically bonded with amino group of chitosan to form hydrogel, for example, the invention patent of chinese patent publication No. CN 106046398A discloses a hydrogel and a preparation method thereof, although chitosan hydrogel with good biocompatibility can be prepared under mild conditions, the preparation steps are too complicated, the biological safety of hydrogel cannot be completely ensured due to the existence of aldehyde group substances, and in addition, the drug loading rate of the hydrogel is also to be improved.

The polyelectrolyte complex is formed by mixing polyelectrolytes with opposite charges, namely, the polycation and the polyanion are combined through Coulomb force. After the polyelectrolyte forms a complex, the polyelectrolyte has certain stability in aqueous solution, and microgel can be formed due to the existence of the micro-hydrophobic region. Thus, the present inventors have shown that, after a large number of experimental studies, a microgel is formed by modifying chitosan with a random copolymer of epsilon-caprolactone and 3, 6-hydroxyadipate. The 3-hydroxy adipic acid 3, 6-lactone molecule contains a lactone ring structure, and can be subjected to ring-opening polymerization under the action of a catalyst to form hydrophobic polyester. Meanwhile, after ring-opening polymerization of 3-hydroxy adipic acid 3, 6-lactone, a side chain contains free negative charge carboxyl, and the free negative charge carboxyl can form a compound microgel with positive charge amino in chitosan. Because the monomers epsilon-caprolactone and 3-hydroxy adipic acid 3, 6-lactone both contain 6 carbon atoms, the obtained polymer main chain is more flexible and has certain hydrophobicity, thereby being beneficial to the formation of microgel and the improvement of drug loading rate.

Disclosure of Invention

The first purpose of the invention is to provide a preparation method of modified chitosan microgel.

The second purpose of the invention is to provide a modified chitosan microgel prepared by the method.

The third purpose of the invention is to provide an application of the modified chitosan microgel.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

the first aspect of the invention provides a preparation method of modified chitosan microgel, which comprises the following steps:

step 1: preparation of random copolymers of epsilon-caprolactone and 3-hydroxyadipic acid 3, 6-lactone

Adding toluene, epsilon-caprolactone, 3-hydroxyadipic acid 3, 6-lactone and a catalyst into a reaction vessel, heating to 85-95 ℃ for reflux reaction for 2-4 h, then continuing heating to 105-110 ℃ for reflux reaction for 2-3 h, cooling to room temperature, precipitating by using an organic solvent A to obtain a crude product of the epsilon-caprolactone and 3-hydroxyadipic acid 3, 6-lactone random copolymer, and purifying the crude product to obtain the epsilon-caprolactone and 3-hydroxyadipic acid 3, 6-lactone random copolymer, which is marked as LC-HA;

wherein the molar ratio of the added epsilon-caprolactone to the 3, 6-lactone of the 3-hydroxyadipic acid is 1: 1-1: 3; the dosage of the added catalyst is 0.5-1.0% of the total weight of the epsilon-caprolactone and the 3-hydroxy adipic acid 3, 6-lactone.

Step 2: preparation of Chitosan suspensions

Preparing an acetic acid solution of chitosan with the concentration of 0.02-0.04 g/mL (the acetic acid concentration is 2 wt%), precipitating the chitosan under an alkaline condition, filtering to obtain a chitosan precipitate, repeatedly washing the chitosan precipitate with deionized water until the filtrate is neutral, soaking the chitosan precipitate in a small amount of an organic solvent B, filtering, drying in vacuum until the weight is constant, grinding the chitosan precipitate into powder, and preparing a chitosan suspension with the concentration of 1.0 wt% with ultrapure water.

And step 3: preparation of modified chitosan microgel

Dissolving the random copolymer of epsilon-caprolactone and 3-hydroxy adipic acid 3, 6-lactone prepared in the step 1 in tetrahydrofuran or toluene to prepare an LC-HA solution with the concentration of 1.0 wt%, dropwise adding the solution into the chitosan suspension prepared in the step 2 under the stirring condition, uniformly mixing, transferring the mixture into a dialysis bag, and dialyzing and purifying to obtain the modified chitosan microgel;

wherein the volume ratio of the LC-HA solution to the chitosan suspension is 1: 3-1: 6.

Further, in step 1, the catalyst is one of stannous chloride or stannous octoate.

Further, in the step 1, the amount of the toluene is 3-5 times of the total weight of the epsilon-caprolactone and the 3-hydroxyadipic acid 3, 6-lactone.

Further, in step 1, the purification method is as follows: dissolving the crude product of the random copolymer of epsilon-caprolactone and 3-hydroxy adipic acid 3, 6-lactone in toluene or tetrahydrofuran, precipitating the crude product by using an organic solvent A, circularly dissolving and precipitating the crude product for three times in the way, and finally drying the precipitate in vacuum.

Furthermore, the organic solvent A is one of petroleum ether, diethyl ether, methanol or ethanol.

Further, in step 2, the organic solvent B is one of ethanol or isopropanol.

Further, in step 3, the cut-off molecular weight of the dialysis bag is 14000.

Further, in step 3, the dialysis purification conditions are as follows: the dialysis time was 72h, and the dialysate was changed every 8 h.

Further, the dialysate is deionized water.

The second aspect of the invention provides a modified chitosan microgel prepared by the preparation method, wherein the average diameter of the modified chitosan microgel is 175-230 nm, and the drug loading rate is 14.2-18.5%.

The third aspect of the invention provides an application of the modified chitosan microgel in an anticancer drug carrier.

Compared with the prior art, the invention has the advantages and beneficial effects that due to the adoption of the technical scheme:

1. the main chain of the random copolymer of epsilon-caprolactone and 3-hydroxy adipic acid 3, 6-lactone is more flexible and has certain hydrophobicity, and the product obtained by modifying chitosan by using the random copolymer has amphipathy, thereby being beneficial to the formation of stable microgel and the improvement of drug loading rate;

2. the microgel is formed by compounding the carboxyl with negative charge suspended on the side chain in the structural unit of the random copolymer and the amino with positive charge in the chitosan molecule, and the preparation method is simple, safe, reliable and easy to control;

3. chitosan and random copolymers of epsilon-caprolactone and 3-hydroxyadipate 3, 6-lactone are biodegradable polymers suitable for use as biomaterials in the human body.

Drawings

FIG. 1 is an infrared spectrum of a random copolymer of ε -caprolactone and 3-hydroxyadipic acid 3, 6-lactone, prepared in step 1 of example 1 of the present invention;

FIG. 2 is a transmission electron microscope image of modified chitosan microgel prepared in example 1 and example 4 of the present invention, wherein, FIG. A and FIG. B represent samples from example 1 and example 4, respectively;

FIG. 3 is a graph showing the viability of Hela cells in the modified chitosan gel of example 1 of the present invention at different concentrations by mass.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in detail below with reference to the embodiments and the accompanying drawings.

Example 1

A preparation method of modified chitosan microgel comprises the following steps:

step 1: preparation of random copolymers of epsilon-caprolactone and 3-hydroxyadipic acid 3, 6-lactone

100mL of toluene is placed in a 250mL three-neck flask with a reflux device, 5.7g of epsilon-caprolactone, 14.4g of 3-hydroxyadipic acid 3, 6-lactone and 0.1g of stannous octoate are added, the flask is placed in an oil bath pot to be heated, the temperature is raised to 95 ℃, the temperature is raised to 105 ℃ after 3 hours of reaction, the reaction is continued to be raised for 2 hours, the reaction is cooled to room temperature, the reaction liquid is dripped into excessive petroleum ether for precipitation, a crude product of a random copolymer of the epsilon-caprolactone and the 3-hydroxyadipic acid 3, 6-lactone is obtained by filtering, the crude product is circularly dissolved by the toluene and precipitated by the petroleum ether for three times, and finally the precipitate is dried in vacuum, so that the random copolymer of the epsilon-caprolactone and the 3-hydroxyadipic acid 3, 6-lactone is obtained and is marked as LC-HA.

Performing infrared spectrum measurement on the prepared LC-HA by using a Fourier infrared spectrometer (FTLA2000-104, ABB BOMEN Corporation), and setting a scanning wavelength range to be 500-4000 cm^-1Resolution of 4cm^-1. The measurement results are shown in FIG. 1, and are 3300-3530 cm^-1A large broad peak appearing in the range, belonging to the overlapped stretching vibration peak of-OH and-COOH; 2850cm^-1The minor peaks at the left and right are methylene CH₂The stretching vibration absorption peak of (1); 1740cm^-1The peak is the stretching vibration peak of C ═ O, 1710cm^-1The position is a stretching vibration peak of C ═ O in the carboxyl; 950cm^-1The absorption peak at (a) was out-of-plane deformation vibration of oh.. O, and the presence of carboxyl groups was confirmed. These characteristic peaks indicate the successful synthesis of random copolymers of epsilon-caprolactone and 3-hydroxyadipic acid 3, 6-lactone.

Step 2: preparation of Chitosan suspensions

Weighing 2g of chitosan, dissolving the chitosan in 100mL of 2 wt% acetic acid solution, uniformly stirring, slowly dropping the acetic acid solution of chitosan into 5 wt% excessive sodium hydroxide solution, magnetically stirring, continuously stirring for 2h after dropping, filtering to obtain chitosan precipitate, repeatedly washing the chitosan precipitate with deionized water until the filtrate is neutral, soaking the chitosan precipitate after the last filtration in a small amount of ethanol for 3h, filtering again, drying in vacuum to constant weight, grinding into powder, and preparing chitosan suspension with the concentration of 1.0 wt% by using ultrapure water.

And step 3: preparation of modified chitosan microgel

Dissolving 2g of the LC-HA prepared in the step 1 in 200mL of tetrahydrofuran to prepare an LC-HA solution with the concentration of 1.0% (m/v, g/mL), taking 10mL of the LC-HA solution out of the solution, slowly dropwise adding the solution into 30mL of chitosan suspension under the condition of magnetic stirring, fully stirring to ensure that the solution is uniformly compounded, then transferring the solution into a dialysis bag with the molecular weight cutoff of 14000, dialyzing the solution in deionized water for 72h, replacing the deionized water in the dialysis bag every 8h to obtain the modified chitosan microgel, and freeze-drying the modified chitosan microgel for later use, wherein a sample is marked as MCS-1.

Example 2

The only difference compared to example 1 is: the amount of the chitosan suspension added in the step 3 is 40mL, and the rest is the same as that in the example 1; the modified chitosan microgel obtained in example 2 was labeled as MCS-2.

Example 3

The only difference compared to example 1 is: the amount of the chitosan suspension added in the step 3 is 50mL, and the rest is the same as that in the example 1; the modified chitosan microgel obtained in example 3 was labeled as MCS-3.

Example 4

The only difference compared to example 1 is: the amount of the chitosan suspension added in the step 3 is 60mL, and the rest is the same as that in the example 1; the modified chitosan microgel obtained in example 4 was labeled as MCS-4.

Measurement of Performance

1. Particle size testing of modified chitosan microgels

The modified chitosan microgel particles prepared in examples 1 to 4 were measured for particle size using a nanometer particle size analyzer (ZetaPALS model, Brookharen, USA) at a sample concentration of 0.15mg/mL and a measurement temperature of 25 ℃ as shown in Table 1.

TABLE 1 raw Material composition and Properties of modified Chitosan microgel

As can be seen from Table 1, the microgel particles of examples 1 to 4 have an average diameter that continuously increases with the increase in the content of the chitosan suspension, because the hydrophilicity of the composite increases with the increase in the chitosan component, resulting in an increase in the volume of the microgel.

2. Morphology observation of modified chitosan microgel

Taking the modified chitosan microgel samples prepared in examples 1 and 4 as an example, the morphology of the microgel was observed by a transmission electron microscope (model JEM-2100, JEOL Ltd., Japan). The specific operation method comprises the following steps: the modified chitosan microgel samples prepared in example 1 and example 4 were dropped on a copper mesh, dried at room temperature, and placed in a sample chamber of a transmission electron microscope, an accelerating voltage of 100kV was set, and the morphology of the microgel was observed under a high vacuum state, as shown in fig. 2, wherein, fig. a and B represent transmission electron microscope images of the modified chitosan microgel prepared in example 1 and example 4, respectively. As can be seen from FIG. 2, the samples of examples 1 and 4 both have regular spherical morphology and uniform particle size distribution, which indicates that the modified chitosan microgel obtained in examples 1 and 4 is relatively stable. In addition, the particle size of the sample in example 4 is larger than that in example 1, which is consistent with the rule obtained from the analysis result of the nanometer particle analyzer.

3. Determination of drug loading rate of modified chitosan microgel

The method for measuring the drug loading rate of the modified chitosan microgel prepared in the embodiment 1-4 comprises the following specific steps:

precisely weighing 30mg of camptothecin, dissolving the camptothecin in a mixed solvent of 5mL of methanol and 5mL of ultrapure water, adjusting the pH to 12, dropwise adding the camptothecin into 50mL of aqueous solution of modified chitosan microgel with the concentration of 1.2mg/mL, stirring uniformly, slowly adjusting the pH to 5 by using 0.1M hydrochloric acid, filling the mixture into a dialysis bag with the molecular weight cutoff of 3500, dialyzing the mixture in deionized water for 12 hours to remove small molecular substances, and freeze-drying the mixture to obtain the drug-loaded microgel; and dispersing the drug-loaded microgel in methanol, magnetically stirring for 24h, centrifuging, taking the supernatant, measuring the absorbance at 395nm, and calculating the drug-loaded amount according to a standard curve, thereby calculating the drug loading rate (LR,%) of the microgel. The calculation formula of the drug loading rate of the microgel is shown as the following formula 1:

in the formula 1, W_DIs the mass of drug loaded in the sample, mg; w_SIs the mass of the sample, mg.

The results of measuring the drug loading rates of the modified chitosan microgels of examples 1 to 4 are shown in table 1. As can be seen from Table 1, the loading rates of the microgels of examples 1 to 4 are in a continuously decreasing state with the increase of the content of the chitosan suspension, because the LC-HA component is relatively decreased with the increase of the chitosan component, and the loading rate for the hydrophobic drug is decreased.

4. Cytotoxicity determination of modified chitosan microgel

Taking the modified chitosan microgel prepared in example 1 as an example, the cytotoxicity of the microgel is determined by an MTT method, which specifically comprises the following steps:

collecting Hela cells (human cervical cancer cells) growing in logarithmic phase, trypsinizing, diluting with RPMI-1640 culture medium containing fetal bovine serum (10 wt%) to obtain cell suspension (concentration of 6 × 104/mL), inoculating to 96-well culture plate, inoculating 100 μ L per well, culturing at 37 deg.C with 5% CO₂After 24h of incubation in the incubator, the medium was discarded.

Respectively setting a negative control group, a positive control group and an experimental group, respectively adding 100 μ L of RPMI-1640 complete culture medium, 0.64 wt% phenol-containing RPMI-1640 complete culture medium, and different concentrations of modified chitosan microgel-1640 complete culture medium into each well of the negative control group, the positive control group and the experimental group, setting 4 multiple wells in each group, and continuously performing at 37 deg.C and 5% CO₂After 24h incubation in the incubator, 20. mu.L of MTT solution was added to each well, and after further incubation for 4h, the solution in the plate was discarded, 150. mu.L of dimethyl sulfoxide solution was added to each well, and the absorbance (OD) was measured at 570nm with a microplate reader (Infine 200PRO NanoQuant type, Tecan, Switzerland) and the cell viability (percentage) was calculated.

In the formula 2, the first step is,experimental group OD_570nmRepresents the absorbance at 570nm of the experimental group and the OD of the negative control group_570nmThe absorbance at 570nm of the negative control group is shown.

According to calculation, the survival rate of Hela cells in the positive control group was 0, the survival rate of Hela cells in the negative control group was 100%, and the survival rate of Hela cells in the experimental group (i.e., the modified chitosan microgel prepared in example 1 at different mass concentrations) is shown in fig. 3. As shown in FIG. 3, the survival rate of Hela cells in the modified chitosan microgel solutions with different mass concentrations is more than 90%, and the modified chitosan microgel prepared by the invention has no cytotoxicity and better biocompatibility according to pharmacopoeia standards.

The above description is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto. All equivalent changes, simplifications and modifications which do not depart from the spirit and scope of the invention are intended to be covered by the scope of the invention.