CN114736317B

CN114736317B - Modified chitosan gel and preparation method and application thereof

Info

Publication number: CN114736317B
Application number: CN202210465051.6A
Authority: CN
Inventors: 姜涛
Original assignee: Hunan Bade Medical Technology Co ltd
Current assignee: Hunan Bade Medical Technology Co ltd
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2023-04-18
Anticipated expiration: 2042-04-29
Also published as: CN114736317A

Abstract

The invention provides modified chitosan of acetyl tyrosine and a preparation method thereof. The invention also provides modified chitosan gel obtained by crosslinking the modified chitosan, and a preparation method and application thereof.

Description

Modified chitosan gel and preparation method and application thereof

Technical Field

The invention relates to the field of medical gel, in particular to modified chitosan gel and a preparation method and application thereof.

Background

Liver cancer, a malignant tumor of the liver, can be divided into primary and secondary types. The primary liver malignant tumor originates from the epithelium or mesenchymal tissue of the liver, and the primary liver malignant tumor is called primary liver cancer and is a malignant tumor with great harm; the latter is called sarcoma, and is less common than primary liver cancer. Secondary or metastatic liver cancer refers to the invasion of malignant tumors of multiple organ origins in the whole body to the liver. Liver metastases, which are common in other organ malignancies, are common.

The early stage of liver cancer can be treated by surgical removal of tumor tissue, however, the tumor cells are difficult to completely remove during surgical removal, and therefore, patients need to undergo chemotherapy to remove the unresectable tumor cells after surgery. Conventional chemotherapy requires intravenous drip, which is injected into the patient. This method of systemic administration has very high side effects and often the patient suffers from very many and serious adverse reactions. After operation, local and targeted administration is carried out at the tumor resection part, so that adverse reactions of patients can be well reduced. However, how to realize the sustained release and controlled release of the drug in the local administration becomes a problem to be solved urgently.

In view of the above, a new technical solution is needed to solve the above technical problems.

Disclosure of Invention

The first purpose of the invention is to provide modified chitosan which can perform controlled release and sustained release on drugs after crosslinking.

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

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

The fourth purpose of the invention is to provide a modified chitosan gel which can control or slowly release the drug.

The fifth purpose of the invention is to provide a preparation method of the modified chitosan gel.

The invention realizes the purpose through the following technical scheme:

a modified chitosan having the structure shown in the following formula:

wherein n is more than or equal to 100 and less than or equal to 300,4 percent and less than or equal to 8 percent.

The preparation method of the modified chitosan comprises the following steps:

s1, dissolving chitosan in an MES solution to obtain a solution A;

s2, putting acetyl tyrosine in a MES solution to obtain a solution B;

s3, mixing the solution A and the solution B to obtain a solution C, adjusting the pH value to 4-5 by adopting an inorganic alkali solution, adding HOBT and HBTU into the solution C, and performing amidation reaction to obtain the modified chitosan.

The concentration of the MES solution is 125-175mmol/L;

the inorganic alkali solution comprises sodium hydroxide or potassium hydroxide.

In the solution A, the content of the chitosan is 2-5wt%;

in the solution B, the content of the acetyl tyrosine is 2-5wt%.

The content of HOBT in the solution C is 0.1-0.3mol/L;

the content of HBTU in the solution C is 0.1-0.5mol/L.

The application of the modified chitosan is applied to the preparation of controlled-release medicaments for treating liver cancer.

A modified chitosan gel, wherein the modified chitosan gel is obtained by crosslinking the modified chitosan; the modified chitosan gel comprises a medicament for treating liver cancer.

The medicament for treating liver cancer comprises adriamycin, capecitabine or gemcitabine;

in the modified chitosan gel, the release speed of the medicine for treating liver cancer is 5-6%/day.

The preparation method of the modified chitosan gel comprises the following steps:

dissolving the modified chitosan into an ethanol water solution, and sequentially adding the medicine for treating liver cancer and tyrosinase to obtain the modified chitosan gel.

In the ethanol water solution, the content of the modified chitosan is 10-20wt%.

Compared with the prior art, the invention has the following technical effects:

1. the modified chitosan provided by the invention can spontaneously form modified chitosan gel in the presence of tyrosinase. Because the tyrosinase content in cancer cells is high, the modified chitosan can be used for targeting to form gel in the vicinity of the cancer cells.

2. The preparation method of the modified chitosan provided by the invention is simple in preparation process, and can realize mass production of the modified chitosan.

3. The modified chitosan provided by the invention has good affinity to chemotherapeutic drugs for treating cancers, and can be used for preparing controlled-release drugs for treating liver cancers.

4. The modified chitosan gel provided by the invention has good affinity to chemotherapeutic drugs, and the monomers of the modified chitosan gel can release the chemotherapeutic drugs to cancer cells at a certain rate after forming gel on the surfaces of the cancer cells. Compared with systemic administration, the local administration mode of the modified chitosan gel can reduce the adverse reaction of patients, improve the compliance of the patients and improve the living quality of the patients.

5. The preparation method of the modified chitosan gel provided by the invention has the advantages of simple process and high reliability.

Drawings

FIG. 1 shows the modified chitosan prepared in example 1 ¹ H-NMR spectrum;

FIG. 2 shows the rheology profile of the modified chitosan of example 4 before and after cross-linking; wherein FIG. 2 (a) is a rheological profile before crosslinking and FIG. 2 (b) is a rheological profile after crosslinking;

FIG. 3 shows UV spectra of the modified chitosan of example 4 before and after crosslinking;

FIG. 4 shows the drug release profile of the modified chitosan gel prepared in example 4;

FIG. 5 shows the drug release profile of the modified chitosan gel prepared in example 5;

FIG. 6 shows the drug release profile of the modified chitosan gel prepared in example 6;

FIG. 7 shows the drug release profile of the modified chitosan gel prepared in comparative example 1;

FIG. 8 shows the drug release profile of the modified chitosan gel prepared in comparative example 2;

FIG. 9 shows the drug release profile of the modified chitosan gel prepared in comparative example 3;

FIG. 10 shows the drug release profile of the modified chitosan gel prepared in comparative example 4;

FIG. 11 shows the drug release profile of the modified chitosan gel prepared in comparative example 5;

FIG. 12 is a graph showing the drug release profile of the modified chitosan gel prepared in comparative example 6;

FIG. 13 shows TEM photographs of modified chitosan gels in the culture broth of HepG2 cells after cross-linking in the culture broth of HepG2 cells, and then removing the HepG2 cells in example 7.

Detailed Description

The present invention will be further described with reference to the following examples.

The invention provides a modified chitosan, wherein acetyl tyrosine is modified on an amino group of the chitosan. In the environment with tyrosinase, the modified chitosan is crosslinked under the action of tyrosinase to form modified chitosan gel. In particular, in use, a solution of the modified chitosan including a chemotherapeutic drug may be applied to the resection site after resection of the hepatoma tumor. The modified chitosan can form gel around the tumor cells because the expression level of tyrosinase in the tumor cells is higher than that of normal cells. The chitosan which does not form gel can be decomposed by the body. During the formation of the gel, the chemotherapeutic drug is encapsulated in the modified chitosan gel. However, the amino group of the modified chitosan is modified with the acetyl tyrosine, so that in space, a plurality of gaps can exist in the modified chitosan gel, and the gaps can provide diffusion channels for chemotherapeutic drugs, so that the drugs can be uniformly and stably diffused, and therefore, the modified chitosan gel can have a better controlled and sustained release effect. In the unmodified chitosan gel, the chemotherapeutic drug is confined in the gel, and relatively diffuses more slowly and is unstable. Of course, the tyrosinase can also be directly added into the solution of the modified chitosan containing the chemotherapeutic drug in vitro to obtain the modified chitosan gel, and then the modified chitosan gel is directly coated on the tumor resection part, so that the chemotherapeutic drug can be uniformly released on the whole resection part. The polymerization degree of the modified chitosan is not too large. When the polymerization degree is more than 300, the particle size of the gel formed by the polymerization degree is also increased, which results in an increase in the release area of the drug and thus increases adverse effects of normal cells affected by the chemotherapeutic drug. When the polymerization degree is less than 100, the particle size of the gel is too small, so that the drug release amount is too small to play a role in inhibiting the growth of tumor cells.

Specifically, the preparation method of the modified chitosan comprises the following steps:

s1, dissolving chitosan in an MES solution to obtain a solution A;

s2, putting the acetyl tyrosine in an MES solution to obtain a solution B;

In the method, MES represents morpholine ethanesulfonic acid, HOBT represents 1-hydroxybenzotriazole, HBTU represents O-benzotriazole-tetramethyluronium hexafluorophosphate.

In certain embodiments of the present invention, the MES solution has a concentration of 125 to 175mmol/L. The MES solution can be used as a buffer solution to adjust the pH value of the reaction system. It will be appreciated by those skilled in the art that the MES solution concentration can be in the vicinity of the above concentration range, and the present invention can be practiced.

In certain embodiments of the present invention, the inorganic base solution may be a sodium hydroxide solution or a potassium hydroxide solution. It will be appreciated by those skilled in the art that the invention may be practiced with other types of inorganic bases.

In some embodiments of the invention, the chitosan content in the solution a is 2-5wt%; in the solution B, the content of the acetyl tyrosine is 2-5wt%. The content of HOBT in the solution C is 0.1-0.3mol/L; the content of HBTU in the solution C is 0.1-0.5mol/L.

The modified chitosan can relatively uniformly release chemotherapeutic drugs after crosslinking, so the modified chitosan can be applied to the preparation of controlled-release drugs for treating liver cancer.

Specifically, the modified chitosan can be crosslinked to obtain modified chitosan gel; the chitosan can contain a medicament for treating liver cancer. Preferably, the drug for treating liver cancer may be sorafenib, ranvatinib, doraninib or apatinib; in the modified chitosan gel, the release speed of the medicine for treating liver cancer is 5-6%/day.

specifically, the modified chitosan is dissolved in an ethanol water solution, and the medicine for treating liver cancer and tyrosinase are sequentially added to obtain the modified chitosan gel. Preferably, the content of the modified chitosan in the ethanol aqueous solution is 10-20wt%.

The invention will be further illustrated by the following specific examples

Example 1

Preparation of modified chitosan

3g of chitosan having a polymerization degree of 200 was dissolved in 100mL of MES (150 mM) to obtain a solution A. 3 grams of acetyl tyrosine was dissolved in 100mL of MES (150 mM) solution to give solution B. And mixing the solution A and the solution B to obtain a solution C. The pH was then adjusted back to 4.5 with aqueous NaOH in solution C. The reaction was continued for 2 hours with stirring. The modified chitosan with polymerization degree of 200 and m of 6 percent is obtained.

Then, 5g of NaCl was added and dialyzed against a NaCl solution containing 20mM for 3 days. And (5) after dialysis, freeze-drying to obtain the modified chitosan freeze-dried powder. Taking a small amount of lyophilized powder ¹ H-NMR analysis of the resulting ¹ The H-NMR spectrum is shown in FIG. 1. As can be seen from the figure, the modified chitosan prepared in example 1 was modified with acetyltyrosine.

Example 2

Preparation of modified chitosan

3g of chitosan having a degree of polymerization of 300 was dissolved in 100mL of MES (150 mM) to obtain solution A. 3 grams of acetyl tyrosine was dissolved in 100mL of MES (150 mM) solution to give solution B. And mixing the solution A and the solution B to obtain a solution C. The pH was then adjusted back to 4 to aqueous NaOH in solution C. The reaction was continued for 2 hours with stirring. The modified chitosan with the polymerization degree of 300 and the m of 4 percent is obtained.

Then, 5g of NaCl was added and dialyzed against a solution containing 20mM NaCl for 3 days. And (5) after dialysis, freeze-drying to obtain the modified chitosan freeze-dried powder.

Example 3

Preparation of modified chitosan

3g of chitosan having a degree of polymerization of 100 was dissolved in 100mL of MES (175 mM) to obtain solution A. 3 grams of acetyl tyrosine was dissolved in 100mL MES (175 mM) to give solution B. And mixing the solution A and the solution B to obtain a solution C. The pH was then adjusted back to 5 to aqueous NaOH in solution C. The reaction was continued for 2 hours with stirring. The modified chitosan with the polymerization degree of 100 and the m of 8 percent is obtained. And (3) modifying the chitosan.

Then, 5g of NaCl was added and dialyzed against a solution containing 20mM NaCl for 3 days. And (5) after dialysis, freeze drying to obtain the modified chitosan freeze-dried powder.

Example 4

Preparation of modified chitosan gel

Dissolving the modified chitosan prepared in the embodiment 1 in ethanol water to obtain ethanol water solution with the content of 15%, then adding the raw material medicines of adriamycin, uniformly mixing, then slowly dripping 80U/mL tyrosinase solution into the ethanol water solution, and obtaining the modified chitosan gel after complete reaction.

The inventor uses a HAAKE MARS 60 rheometer to analyze the rheological properties of the modified chitosan ethanol aqueous solution and the modified chitosan gel prepared in example 4. The inventors tested the law of change of the viscous modulus (G ") of the solution system by means of a rheometer. The specific parameters of the rheometer are as follows: the strain is 4-6%, the scanning frequency is 0.1Hz, and the testing temperature is room temperature. Fig. 2 (a) is a rheological profile before crosslinking, and fig. 2 (b) is a rheological profile after crosslinking. As can be seen from fig. 2 (a) and from fig. 2 (b), the rheology curve of G "after crosslinking is significantly lower than before crosslinking. The results also indicate that tyrosinase is able to catalyze the crosslinking reaction of the modified chitosan solution to form a gel.

FIG. 3 shows UV spectra of modified chitosan before and after crosslinking in example 4. From the figure, it can be seen that the ultraviolet absorption peak of the modified chitosan after crosslinking is obviously higher than that before crosslinking. This shows that tyrosinase can catalyze the crosslinking of the modified chitosan prepared in example 1.

The chitosan has broad-spectrum antibacterial activity, can be quaternized in an acidic environment to form macromolecules with positive charges, the cell wall of bacteria is negatively charged, and the chitosan can be bonded with the negatively charged cell wall to destroy the normal function of the cell membrane so as to play an antibacterial role. Furthermore, tyrosine can also affect Extracellular Polymeric Substances (EPS) of bacteria, so that the introduction of acetyltyrosine into chitosan can enable the material to have better inhibition effect on escherichia coli and staphylococcus aureus.

The inventors also tested the drug release of the modified chitosan gel prepared in example 4 using a high performance liquid chromatograph.

Chromatographic conditions are as follows:

mobile phase: 0.1% phosphoric acid-methanol-acetonitrile (40;

a chromatographic column: agilentC ₁₈ Columns (250 mm. Times.4.6 mm,5 μm);

flow rate: 1.0 mL/min ^-1 ；

Column temperature: 30 ℃;

detection wavelength: 237nm;

sample introduction amount: 20 μ L.

The release rate was determined as follows:

the modified chitosan gel prepared in example 4 was collected by keeping out of the light and the release profile was measured in dissolution medium at pH 6.8. The rotation speed is 75 r.min according to dissolution test method ^-1 The volume of the medium was 900mL. 5mL samples were taken at 0.25d, 0.5d, ld, 1.5d, 2d, 3d, 4d, 6d, 8d, and 12d, respectively, and the samples were measured by HPLC to plot the release curve.

preparation of pH6.8 Medium

1.7g of monopotassium phosphate and 1.775g of anhydrous disodium hydrogen phosphate are taken, a proper amount of water is added for dissolution, and then 3.5mL of Tween-60 is added to reach a constant volume of 1000mL.

The drug release profile of the modified chitosan gel prepared in example 4 is shown in fig. 4. As can be seen from the figure, the release speed of the adriamycin is slow and uniform, which indicates that the modified chitosan gel prepared in example 4 can be used for preparing controlled and sustained release medicines for treating liver cancer.

Example 5

Preparation of modified chitosan

Dissolving the modified chitosan prepared in the embodiment 1 in ethanol water to obtain ethanol water solution with the content of 15%, then adding the raw material medicine of capecitabine, uniformly mixing, then slowly dropwise adding 80U/mL tyrosinase solution into the ethanol water solution, and obtaining the modified chitosan gel after complete reaction.

The drug release profile of the modified chitosan gel prepared in example 5 is shown in fig. 5. As can be seen from the figure, the release speed of capecitabine is slow and uniform, which indicates that the modified chitosan gel prepared in example 5 can be used for preparing controlled and sustained release medicines for treating liver cancer.

Example 6

Preparation of modified chitosan

Dissolving the modified chitosan prepared in the embodiment 1 in ethanol water to obtain ethanol water solution with the content of 15%, then adding gemcitabine raw material medicine, uniformly mixing, then slowly dropwise adding 80U/mL tyrosinase solution into the ethanol water solution, and obtaining the modified chitosan gel after complete reaction.

The drug release profile of the modified chitosan gel prepared in example 6 is shown in fig. 6. As can be seen from the figure, the gemcitabine release slowly and uniformly, which indicates that the modified chitosan gel prepared in example 6 can be used for preparing controlled and sustained release drugs for treating liver cancer.

Comparative example 1

5g of chitosan having a degree of polymerization of 50 was dissolved in 100mL of MES (175 mM) to obtain solution A. 3 grams of acetyl tyrosine was dissolved in 100mL of MES (175 mM) solution to give solution B. And mixing the solution A and the solution B to obtain a solution C. The pH was then adjusted back to 5 to aqueous NaOH in solution C. The reaction was continued for 2 hours with stirring. Obtaining the modified chitosan. Dissolving the modified chitosan in ethanol water to obtain ethanol water solution with the content of 15%, then adding the raw material drug of adriamycin, uniformly mixing, then slowly dropwise adding 80U/mL tyrosinase solution into the ethanol water solution, and obtaining the modified chitosan gel after complete reaction.

The drug release profile of the modified chitosan gel prepared in comparative example 1 is shown in fig. 7. As can be seen from the figure, the release rate of capecitabine is too fast.

Comparative example 2

5g of chitosan having a degree of polymerization of 350 was dissolved in 100mL of MES (175 mM) to obtain solution A. 3 grams of acetyl tyrosine was dissolved in 100mL MES (175 mM) to give solution B. And mixing the solution A and the solution B to obtain a solution C. The pH was then adjusted back to 5 to aqueous NaOH in solution C. The reaction was continued for 2 hours with stirring. Obtaining the modified chitosan. Dissolving the modified chitosan in ethanol water to obtain ethanol water solution with the content of 15%, then adding the raw material drug of adriamycin, uniformly mixing, then slowly dropwise adding 80U/mL tyrosinase solution into the ethanol water solution, and obtaining the modified chitosan gel after complete reaction.

The drug release profile of the modified chitosan gel prepared in comparative example 2 is shown in fig. 8. As can be seen from the figure, the release rate of capecitabine is too slow.

Comparative example 3

5g of chitosan having a degree of polymerization of 50 was dissolved in 100mL of MES (175 mM) to obtain solution A. 3 grams of acetyl tyrosine was dissolved in 100mL of MES (175 mM) solution to give solution B. And mixing the solution A and the solution B to obtain a solution C. The pH was then adjusted back to 5 with aqueous NaOH in solution C. The reaction was continued for 2 hours with stirring. Obtaining the modified chitosan. Dissolving the modified chitosan in ethanol water to obtain ethanol water solution with the content of 15%, then adding the raw material medicines of capecitabine, uniformly mixing, then slowly dropwise adding 80U/mL tyrosinase solution into the ethanol water solution, and obtaining the modified chitosan gel after complete reaction.

The drug release profile of the modified chitosan gel prepared in comparative example 3 is shown in fig. 9. As can be seen from the figure, the release rate of capecitabine is too fast.

Comparative example 4

5g of chitosan having a degree of polymerization of 350 was dissolved in 100mL of MES (175 mM) to obtain solution A. 3 grams of acetyl tyrosine was dissolved in 100mL of MES (175 mM) solution to give solution B. And mixing the solution A and the solution B to obtain a solution C. The pH was then adjusted back to 5 to aqueous NaOH in solution C. The reaction was continued for 2 hours with stirring. Obtaining the modified chitosan. Dissolving the modified chitosan in ethanol water to obtain ethanol water solution with the content of 15%, then adding the raw material drug of adriamycin, uniformly mixing, then slowly dropwise adding 80U/mL tyrosinase solution into the ethanol water solution, and obtaining the modified chitosan gel after complete reaction.

The drug release profile of the modified chitosan gel prepared in comparative example 4 is shown in fig. 10. As can be seen from the figure, the release rate of capecitabine is too slow.

Comparative example 5

The drug release profile of the modified chitosan gel prepared in comparative example 5 is shown in fig. 11. As can be seen from the graph, the release rate of gemcitabine is too fast.

Comparative example 6

The drug release profile of the modified chitosan gel prepared in comparative example 6 is shown in fig. 12. As can be seen from the graph, the release rate of gemcitabine is too slow.

Example 7

Cross-linking of modified chitosan in HepG2 cell culture fluid

Culture solution: DMEM +10% fetal bovine serum.

And (3) passage: the culture medium was aspirated. Washed once with PBS, added with 0.25% pancreatin without EDTA, and placed in an incubator at 37 ℃ for 2 minutes. And then putting the mixture into a 1.5ml conical centrifuge tube, centrifuging the mixture for 3 minutes at 800 rpm to ensure that the cells are cleaner. Then the supernatant in the centrifuge tube is sucked out, only the precipitate is left, and then fresh complete culture medium is added, and the mixture is added into a cell bottle after being blown uniformly.

And (3) crosslinking: the modified chitosan prepared in example 1 was dissolved in ethanol water to obtain an aqueous solution with a content of 15%, and then the above aqueous solution was added to a cell flask and left to stand for 2 days.

FIG. 13 shows TEM photographs of the modified chitosan after cross-linking in the culture solution of HepG2 cells and after removing the HepG2 cells in example 7. As can be seen from the figure, darker and smaller gel particles appear in the culture broth. This indicates that tyrosinase expressed in HepG2 cells can catalyze the crosslinking of the modified chitosan prepared in example 1.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A modified chitosan having a structure represented by the following formula:

，

wherein n is more than or equal to 100 and less than or equal to 300,4 percent and more than or equal to m and less than or equal to 8 percent;

the modified chitosan is applied to the preparation of controlled-release drugs for treating liver cancer.

2. The method for preparing modified chitosan according to claim 1, comprising the steps of:

s1, dissolving chitosan in an MES solution to obtain a solution A;

s2, putting the acetyl tyrosine in an MES solution to obtain a solution B;

3. The method for producing a modified chitosan according to claim 2, wherein:

the concentration of the MES solution is 125-175mmol/L;

the inorganic alkaline solution comprises sodium hydroxide or potassium hydroxide.

4. The method for producing a modified chitosan according to claim 2, wherein:

in the solution A, the content of the chitosan is 2-5wt%;

in the solution B, the content of the acetyl tyrosine is 2-5wt%.

5. The method for preparing modified chitosan according to claim 2, wherein:

the content of HOBT in the solution C is 0.1-0.3mol/L;

the content of HBTU in the solution C is 0.1-0.5mol/L.

6. Use of the modified chitosan of claim 1, wherein: is applied to preparing controlled and sustained release medicines for treating liver cancer.

7. A modified chitosan gel, which is characterized in that:

the modified chitosan gel is obtained by crosslinking the modified chitosan of claim 1;

the modified chitosan gel comprises a drug for treating liver cancer.

8. The modified chitosan gel of claim 7, wherein:

in the modified chitosan gel, the release speed of the medicine for treating liver cancer is 6-9%/day.

9. The method of claim 7, comprising the steps of:

dissolving the modified chitosan in an ethanol water solution, and sequentially adding the medicine for treating liver cancer and tyrosinase to obtain the modified chitosan gel.

10. The method of claim 9, wherein the modified chitosan gel is prepared by: