CN108186607B

CN108186607B - Preparation method of breast cancer targeted chitosan graft polymer drug-loaded composite material

Info

Publication number: CN108186607B
Application number: CN201810148511.6A
Authority: CN
Inventors: 朱利民; 钱倩倩; 牛世伟
Original assignee: Donghua University
Current assignee: Hebei Yingzhi Medical Device Research Co ltd
Priority date: 2018-02-13
Filing date: 2018-02-13
Publication date: 2021-01-05
Anticipated expiration: 2038-02-13
Also published as: CN108186607A

Abstract

The invention relates to a preparation method of a breast cancer targeted chitosan graft polymer drug-loaded composite material, which comprises the following steps: dissolving CS-g-PNIPAM in HAc solution, stirring until the CS-g-PNIPAM is dissolved, adjusting the pH value to 4-6, adding activated K237 peptide solution, continuously stirring for reaction, dialyzing, freeze-drying to obtain K237-CS-g-PNIPAM, dissolving the K237-CS-g-PNIPAM in DMF, adding PTX, stirring for reaction, dialyzing, freeze-drying to obtain K237-CS-g-PNIPAM/PTX. The novel drug-loaded material has the characteristics of pH sensitive release and breast cancer cell targeting, can be used for anti-tumor research, and has good practical value.

Description

Preparation method of breast cancer targeted chitosan graft polymer drug-loaded composite material

Technical Field

The invention belongs to the field of nano materials, and particularly relates to a preparation method of a breast cancer targeted chitosan graft polymer drug-loaded composite material.

Background

The chitosan as a natural cationic polysaccharide has the advantages of wide source, low price, no toxicity, good biocompatibility, biodegradability and the like, and can be widely applied to the fields of medicines, foods, chemical industry and the like. However, chitosan has many disadvantages, and the chitosan has high crystallinity due to hydrogen bonds among chitosan molecules, so that the chitosan is insoluble in common organic solvents, thereby greatly limiting the application of chitosan. Therefore, the modification research of chitosan is carried out and many advances are made. At present, along with the development of controllable/active free radical polymerization in the aspects of definite synthetic structure and controllable molecular weight of macromolecules, controllable grafting modification of chitosan achieves good results. The synthetic polymer which is bonded, hydrophilic or has a certain special function in the chitosan molecule can improve certain performances of the chitosan and improve the application range of the chitosan. In addition, CS is also considered a natural polymer with pH sensitive properties. The graft polymer of chitosan not only has the characteristics of chitosan, but also has the advantages of synthetic polymer. Has been widely used in the pharmaceutical industry due to its potential in the development of drug delivery systems.

Paclitaxel is a natural antitumor drug extracted from the trunk and bark of Taxus genus plant Taxus, and has obvious therapeutic effect on many cancers. The water solubility of the paclitaxel is very low, and in order to increase the solubility of the paclitaxel, the paclitaxel is usually dissolved by polyoxyethylene castor oil and ethanol and then administered, but after administration, serious anaphylactic reaction, neutropenia and other toxic and side effects exist, so that the clinical application of the paclitaxel is limited. In order to eliminate toxic and side effects and improve the dispersibility of paclitaxel in water, related scholars explore a plurality of administration technologies, such as preparing liposome, embedding cyclodextrin, preparing emulsion, preparing microspheres, polymer nanoparticles and the like, so as to improve the administration mode and curative effect of paclitaxel.

The K237 peptide (HTMYYHHYQHHL-NH2) is a 12-mer polypeptide isolated from a phage display library and has high affinity and specificity for binding KDR/Flk-1 tyrosine kinase, one of the two receptors of vascular endothelial growth factor overexpressed on tumor neovasculature. The excellent characteristics of the nanotechnology are expected to inhibit the metastatic spread of the tumor, especially different treatment strategies can be combined on one platform, the novel tumor protein targeting K237 peptide is conjugated to the surface of the nanoparticle through the EDC-NHS technology, and the chitosan graft polymer modified by the K237 peptide has good research prospects in the targeting treatment of the breast cancer cells.

Disclosure of Invention

The invention aims to solve the technical problem of providing a preparation method of a breast cancer targeted chitosan graft polymer drug-loaded composite material, which is simple to operate and mild in reaction conditions. The particle size of the chitosan graft polymer nano particles is less than 100nm, the chitosan graft polymer nano particles can reach the selective metabolism of specific tissues or cells to the maximum extent, and the chitosan graft polymer nano particles have the characteristics of pH sensitive release and breast cancer targeting.

The invention relates to a preparation method of a breast cancer targeted chitosan graft polymer drug-loaded composite material, which comprises the following steps:

(1) dissolving CS-g-PNIPAM in HAc solution, stirring until the CS-g-PNIPAM is dissolved, adjusting the pH to 4-6, adding activated K237 peptide solution, stirring for reaction, dialyzing, and freeze-drying to obtain chitosan graft polymer K237-CS-g-PNIPAM; wherein the mass ratio of the K237 peptide to the CS-g-PNIPAM is 1: 1-2;

(2) dissolving the K237-CS-g-PNIPAM obtained in the step (1) in DMF, adding paclitaxel PTX, stirring for reaction, dialyzing, and freeze-drying to obtain the chitosan graft polymer drug-loaded composite material K237-CS-g-PNIPAM/PTX targeted by the breast cancer; wherein the mass ratio of PTX to K237-CS-g-PNIPAM is 1: 2-5.

The CS-g-PNIPAM in the step (1) is prepared by dissolving chitosan CS in an acetic acid solution with the concentration of 1%, adding methanol, stirring until the solution is clear, then dropwise adding an acetic anhydride solution, stirring at room temperature for reaction, then precipitating and precipitating by using a NaOH solution with the concentration of 10%, filtering and drying to obtain acetylated chitosan, dissolving in dimethylformamide DMF, stirring and dissolving, adding a chain transfer agent 2- (dodecyl trithiocarbonate) -2-methylpropionic acid DDACT, a catalyst 4- (N, N-dimethylamino) pyridine DMAP and a dehydrating agent dicyclohexylcarbodiimide DCC, stirring at room temperature for reaction, dissolving an RAFT reagent for obtaining chitosan through purification and freeze-drying in dimethylformamide DMF, adding NIPAm and an initiator azobisisobutyronitrile AIBN under the protection of nitrogen, performing oil bath reaction, and purifying.

The concentration of the HAc solution in the step (1) is 1%.

In the step (1), NaOH solution with the concentration of 1% is adopted for adjusting the pH value.

In the step (1), a dialysis bag with MWCO of 3500 is adopted for dialysis.

The K237 peptide solution activated in the step (1) is prepared by dissolving K237 peptide in HAc-NaAc solution with the concentration of 1%, adding EDC and NHS, and magnetically stirring for 8-12 h; wherein the mass ratio of the K237 peptide to the EDC to the NHS is 20-22: 40-110: 20-70.

And (2) stirring in the step (1) is magnetic stirring, the stirring activation time is 8-12 h, and the continuous stirring reaction time is 22-26 h.

And (3) stirring and reacting for 22-26 h in the step (2).

And (3) the freeze drying time in the step (2) is 2-4 days.

Advantageous effects

(1) According to the invention, by utilizing the physical adsorption effect between chitosan and paclitaxel, a large amount of therapeutic drugs can be loaded on the hydrophobic core of the nanoparticle, so that the breast cancer targeted chitosan graft polymer drug-loaded composite material containing paclitaxel is synthesized, and the preparation method is simple to operate and mild in experimental conditions.

(2) The particle size of the chitosan graft polymer nano-particles is less than 100nm, so that the breast cancer can be selectively metabolized by specific tissues or cells to the maximum extent.

(3) The breast cancer targeted chitosan graft polymer drug-loaded composite material of paclitaxel can be slowly released for a long time. And has pH sensitive conveying, high release rate in a lower pH value environment, suitability for the microenvironment of tumor tissues and potential for subsequent related experimental analysis by applying the pH sensitive conveying agent.

(4) The K237 peptide in the breast cancer targeted chitosan graft polymer can realize the active targeting effect on breast cancer cells, and can further study the in-vivo circulation and metabolic distribution of the breast cancer cells.

Drawings

FIG. 1 shows the DLS results of CS-g-PNIPAM and K237-CS-g-PNIPAM in example 1.

FIG. 2 shows the TGA results for CS-RAFT (a), CS (b), CS-g-PNIPAM (c), K237-CS-g-PNIPAM (d) in example 1.

FIG. 3 is a TEM image of CS-g-PNIPAM (left) and K237-CS-g-PNIPAM (right) in example 1.

FIG. 4A is the drug release profile of K237-CS-g-PNIPAM/PTX in example 3 at different pH environments.

FIG. 4B is the drug release profile of K237-CS-g-PNIPAM/PTX in example 4 under different temperature environments.

FIG. 5 shows the results of MTT cytotoxicity of L929 cells with different concentrations of PTX, K237-CS-g-PNIPAM and K237-CS-g-PNIPAM/PTX in example 5.

FIG. 6 shows MTT cytotoxicity results of different concentrations of PTX, K237-CS-g-PNIPAM and K237-CS-g-PNIPAM/PTX on MCF-7 cells in example 5.

FIG. 7 shows confocal microscope results of K237-CS-g-PNIPAM, K237-CS-g-PNIPAM/PTX on MBA-MD-231 and L929 cells in example 6.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

(1) Dissolving 5g of chitosan CS in 250mL of 1% acetic acid solution, stirring to completely dissolve the chitosan CS, slowly adding 250mL of methanol, stirring until the mixture is clear, dropwise adding 1mL of acetic anhydride solution, stirring at room temperature for reaction for 3.5 hours, adding 10% NaOH solution to separate out chitosan in the solution, filtering and drying to obtain acetylated chitosan, and grinding into fine powder for later use. Dissolving 0.298g of the obtained acetylated chitosan in 5mL of DMF, stirring and dissolving, adding 0.370g of DDACT, 0.015g of DMAP and 0.205g of DCC, stirring and reacting for 48h at room temperature, pouring into ice water, carrying out ice-water bath overnight, dialyzing for three days by using a dialysis bag with MWCO of 3500, and freeze-drying to obtain the RAFT reagent of chitosan. Dissolving 0.047g of the obtained chitosan RAFT reagent in 5mL of DMF, magnetically stirring for 4.5h under nitrogen atmosphere to completely dissolve the chitosan RAFT reagent, adding 0.0016g of AIBN as an initiator, carrying out oil bath reaction at 60 ℃ under the protection of 0.5g of NIPAm nitrogen for 24h, adding 50mL of diethyl ether for precipitation for 3 times, and purifying to obtain the chitosan temperature-sensitive block copolymer CS-g-NIPAM.

(2) Dissolving 20mg of K237 peptide in 2ml of 1% HAc-NaAc solution, adding 40mg of EDC and 20mg of NHS, and magnetically stirring and activating for 8 hours to obtain an activated K237 solution; dissolving 25mg of CS-g-PNIPAM in 1% HAc buffer solution, stirring until the CS-g-PNIPAM is dissolved, adding 1% NaOH to adjust the pH value to 6, slowly adding the activated K237 solution, continuously stirring for reaction for 24 hours, dialyzing by using a dialysis bag with MWCO of 3500, and freeze-drying to obtain the chitosan graft polymer K237-CS-g-PNIPAM.

(3) Dissolving 25mg of K237-CS-g-PNIPAM in 5ml of DMF, adding 10mg of paclitaxel PTX, stirring and reacting for 24h, dialyzing to remove the unconnected PTX, and freeze-drying for 3 days to obtain the chitosan graft polymer drug-loaded composite material K237-CS-g-PNIPAM/PTX targeted by the breast cancer.

In the present example, Zeta potentials of CS, N-acetyl, CS-RAFT, CS-g-PNIPAM and K237-CS-g-PNIPAM are shown in Table 1.

TABLE 1 Zeta potential values of the respective compounds of example 1

Compound (I)	CS	N-acetyl CS	CS-RAFT	CS-g-PNIPAM	K237-CS-g-PNIPAM
						Zeta potential (mv)	-2.2±1.6	-22.7±2.4	-27.4±1.3	-8.1±2.1	-12.0±2.5

The DLS results of CS-g-PNIPAM and K237-CS-g-PNIPAM in this example are shown in FIG. 1, and it can be seen that the particle sizes of the two nanoparticles are about 70 nm.

The TGA results of CS, CS-RAFT, CS-g-PNIPAM, and K237-CS-g-PNIPAM in this example are shown in FIG. 2, and it can be seen that 40%, 5%, 12%, and 10% by weight of the CS, CS-RAFT, CS-g-PNIPAM, and K237-CS-g-PNIPAM remained when the temperature reached 900 ℃.

In the present example, TEM images of CS-g-PNIPAM (left) and K237-CS-g-PNIPAM (right) are shown in FIG. 3, which shows that the particle size of the chitosan grafted polymer nanoparticle is less than 100nm and the particle size of the targeting peptide nanoparticle becomes smaller.

Example 2

(1) Dissolving 5g of chitosan CS in 250mL of 1% acetic acid solution, stirring to completely dissolve the chitosan CS, slowly adding 250mL of methanol, stirring until the mixture is clear, dropwise adding 1mL of acetic anhydride solution, stirring at room temperature for reaction for 3.5 hours, adding 10% NaOH solution to separate out chitosan in the solution, filtering and drying to obtain acetylated chitosan, and grinding into fine powder for later use. Dissolving 0.298g of the obtained acetylated chitosan in 5ml DMF, stirring and dissolving, adding 0.370g of DDACT, 0.015g of DMAP and 0.205g of DCC, stirring and reacting for 48h at room temperature, pouring ice water, carrying out ice water bath overnight, dialyzing for three days by using a dialysis bag with MWCO of 3500, and freeze-drying to obtain the RAFT reagent of chitosan. Dissolving 0.047g of the obtained chitosan RAFT reagent in 5ml DMF, magnetically stirring for 4.5h under nitrogen atmosphere to completely dissolve the chitosan RAFT reagent, adding 0.0032g of AIBN serving as an initiator, carrying out oil bath reaction at 60 ℃ under the protection of 1g of NIPAm nitrogen for 24h, adding 50ml of diethyl ether for precipitation for 3 times, and purifying to obtain the chitosan temperature-sensitive block copolymer CS-g-NIPAM.

(2) Dissolving 21.42mg of K237 peptide in 2ml of 1% HAc-NaAc solution, adding 104mg of EDC and 68mg of NHS, and magnetically stirring and activating for 12h to obtain an activated K237 solution; dissolving 25mg of CS-g-PNIPAM in 1% HAc buffer solution, stirring until the CS-g-PNIPAM is dissolved, adding 1% NaOH to adjust the pH value to 6, slowly adding the activated K237 solution, continuously stirring for reaction for 24 hours, dialyzing by using a dialysis bag with MWCO of 3500, and freeze-drying to obtain the chitosan graft polymer K237-CS-g-PNIPAM.

(2) Dissolving 60mg of K237-CS-g-PNIPAM in 10ml of DMF, adding 30mg of paclitaxel PTX, stirring and reacting for 24h, dialyzing to remove the unconnected PTX, and freeze-drying for 3 days to obtain the chitosan graft polymer drug-loaded composite material K237-CS-g-PNIPAM/PTX targeted by the breast cancer.

Example 3

The drug-loaded composite material K237-CS-g-PNIPAM/PTX prepared in example 1 is subjected to a drug release test under different pH conditions:

weighing 2mg of drug-loaded compound K237-CS-g-PNIPAM/PTX, fully dissolving in pure water, respectively putting into two dialysis bags (MW 3500), respectively, putting into a dissolution instrument, respectively supplementing 20ml of PBS solution (pH 7.4) and acetic acid solution (pH 5.0), setting the temperature of the dissolution instrument at 37 ℃, rotating at 90 r/min, respectively taking out 1ml of dialysate in 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 20h, 32h, 44h, 56h and 75h, and supplementing 1ml of corresponding PBS buffer solution and acetic acid buffer solution. And carrying out ultraviolet detection on the collected dialysate, setting the absorption wavelength to be 227nm, collecting data and calculating the drug release condition.

The release curves of PTX in two pH environments are shown in fig. 4A, and it can be seen that after 75h, PTX is released by about 91% in an acetic acid sustained release solution with pH 5.0; and only 62% of the release is released in PBS environment with pH 7.4, and the release of the two is obviously different. The pH value of the tumor tissue is lower than that of normal tissue cells, so that the release of the drug-loaded composite material just meets the characteristic, and the drug-loaded composite material is a pH sensitive material which can be used for treating tumors.

Example 4

The drug-loaded composite material K237-CS-g-PNIPAM/PTX prepared in example 2 is subjected to a drug release test under different temperature conditions:

weighing 2mg of drug-loaded compound K237-CS-g-PNIPAM/PTX, fully dissolving in pure water, respectively putting into two dialysis bags (MW 3500), putting into a dissolution instrument, supplementing 20ml of PBS solution (pH 7.4), setting the temperature of the dissolution instrument at 37 ℃ and 25 ℃, rotating at 90 r/min, respectively taking out 1ml of dialysate at 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 20h, 32h, 44h, 56h and 75h, and supplementing back 1ml of corresponding PBS buffer solution. And carrying out ultraviolet detection on the collected dialysate, setting the absorption wavelength to be 227nm, collecting data and calculating the drug release condition.

The release profiles of PTX in two temperature environments are shown in fig. 4B, and it can be seen that after 75 hours, PTX is released about 70% in a sustained release solution at 37 ℃. And only 46% of the release rate is released in an environment at 25 ℃, and the release rate is remarkably different. Compared with normal tissue cells, the temperature of the tumor tissue is about 0.5 ℃ higher than that of the normal cell tissue, so that the release of the drug-loaded composite material just meets the characteristic, and the drug-loaded composite material is a temperature-sensitive material which can be used for tumor treatment.

Example 5

The drug-loaded composite material K237-CS-g-PNIPAM/PTX prepared by the invention is subjected to cytotoxicity test:

l929 and MCF-7 cells were seeded in 96 well cell culture plates, respectively, at a cell density of about 10,000 per well, and 200. mu.L of medium per well was replenished at 5% CO₂And culturing in an incubator for 24h under the conditions of (1). The next day the old medium was decanted, 20. mu.L of PBS containing different concentrations of PTX, K237-CS-g-PNIPAM and K237-CS-g-PNIPAM/PTX were added and 180. mu.L of fresh medium was replenished to a total volume of 200. mu.L per well. After 24h incubation, 20. mu.L of 0.5% MTT solution was added to each well, the mixture was kept at rest for 4h in a 37 ℃ incubator, the culture medium in the wells was aspirated, 200. mu.L DMSO was added, the mixture was placed on a shaker under dark conditions and low-speed shaking for 15-20min, and the UV absorbance of each well at 450nm was measured using an ELISA.

MTT cytotoxicity results of PTX, K237-CS-g-PNIPAM and K237-CS-g-PNIPAM/PTX on L929 cells are shown in figure 5, MTT cytotoxicity results on MCF-7 cells are shown in figure 6, and it can be known that PTX concentration in K237-CS-g-PNIPAM/PTX is 0.001-10 mu M, and the results show that the toxicity on L929 and MCF-7 cells is larger, and further verify that the killing effect of the composite drug-loaded material on the cells is only related to PTX in the composite drug-loaded material, and MTT detection is carried out on the drug carrier K237-CS-g-PNIPAM, and the drug-loaded material with the same concentration as the K237-CS-g-PNIPAM in the drug-loaded composite does not generate cytotoxicity, and also verify that the composite drug-loaded composite kills only related to PTX in the composite drug-loaded composite.

Example 6

The drug-loaded composite material K237-CS-g-PNIPAM/PTX prepared by the invention is subjected to a targeting verification test:

put cover glass into 24-well cell culture plate, and seed MBA-MD-231 cells, the cell density of each well is about 30,000, and make up 1mL of culture solution per well5％CO₂And culturing in an incubator for 24h under the conditions of (1). The next day the old medium was decanted, 100. mu.L of PBS solution containing 5. mu.M PTX K237-CS-g-PNIPAM/PTX was added and 900. mu.L of fresh medium was replenished and incubated for 2 h. The medium containing the material was aspirated and washed with PBS and fixed for 15min with 1ml of 2.5% glutaraldehyde. Glutaraldehyde is aspirated and washed with PBS, stained with 1ml Hoescht 33342 for 15 min. Hoescht 33342 was aspirated and rinsed with PBS, the coverslip was removed, a drop of fluorescent blocking agent was applied, and the slide was mounted for confocal laser microscopy. In order to detect the targeting of the material, non-targeted cells L929 are selected, and the operation process is the same as that of MBA-MD-231.

The results of the K237-CS-g-PNIPAM, K237-CS-g-PNIPAM/PTX confocal laser microscopy on MBA-MD-231 and L929 cells are shown in FIG. 7, and it can be found that FITC fluorescence intensity of MBA-MD-231 cells after incubation of the material is higher than that of L929 cells, which indicates that the material has certain targeting property on breast cancer cells.

Claims

1. A preparation method of a breast cancer targeted chitosan graft polymer drug-loaded composite material comprises the following steps:

(1) dissolving chitosan graft polymer CS-g-PNIPAM in HAc solution, stirring until the chitosan graft polymer CS-g-PNIPAM is dissolved, adjusting the pH to 4-6, adding activated K237 peptide solution, stirring for reaction, dialyzing, and freeze-drying to obtain chitosan graft polymer K237-CS-g-PNIPAM; wherein the mass ratio of the K237 peptide to the CS-g-PNIPAM is 1: 1-2; wherein the particle size of the chitosan graft polymer K237-CS-g-PNIPAM nano-particles is less than 100 nm;

2. The preparation method of the breast cancer targeted chitosan graft polymer drug-loaded composite material according to claim 1, which is characterized in that: the concentration of the HAc solution in the step (1) is 1%.

3. The preparation method of the breast cancer targeted chitosan graft polymer drug-loaded composite material according to claim 1, which is characterized in that: in the step (1), NaOH solution with the concentration of 1% is adopted for adjusting the pH value.

4. The preparation method of the breast cancer targeted chitosan graft polymer drug-loaded composite material according to claim 1, which is characterized in that: the dialysis in the step (1) adopts a dialysis bag with MWCO = 3500.

5. The preparation method of the breast cancer targeted chitosan graft polymer drug-loaded composite material according to claim 1, which is characterized in that: the K237 peptide solution activated in the step (1) is prepared by dissolving K237 peptide in HAc-NaAc solution with the concentration of 1%, adding EDC and NHS, and magnetically stirring for 8-12 h; wherein the mass ratio of the K237 peptide to the EDC to the NHS is 20-22: 40-110: 20-70.

6. The preparation method of the breast cancer targeted chitosan graft polymer drug-loaded composite material according to claim 1, which is characterized in that: and (2) stirring in the step (1) is magnetic stirring, and the stirring reaction time is 22-26 h.

7. The preparation method of the breast cancer targeted chitosan graft polymer drug-loaded composite material according to claim 1, which is characterized in that: and (3) stirring and reacting for 22-26 h in the step (2).

8. The preparation method of the breast cancer targeted chitosan graft polymer drug-loaded composite material according to claim 1, which is characterized in that: and (3) the freeze drying time in the step (2) is 2-4 days.