CN107158410B

CN107158410B - A kind of folic acid-chitosan-Cy7 polymer with tumor targeting and preparation method thereof

Info

Publication number: CN107158410B
Application number: CN201710111439.5A
Authority: CN
Inventors: 陈海军; 张英英; 高瑜
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2017-02-28
Filing date: 2017-02-28
Publication date: 2020-05-08
Anticipated expiration: 2037-02-28
Also published as: CN107158410A

Abstract

The invention discloses a novel folic acid-chitosan-Cy7 polymer (CF7) used as a diagnosis and treatment agent and a preparation method thereof. The derivatives are 6-azido-6-deoxy-N-phthalimido-chitosan and alkynyl-modified folic acid (ALK-FA) and alkynyl-modified heptamethine dye Cy7 (ALK‑Cy7) reaction. The polymer CF7 of the present invention can self-assemble to form nanoparticles, and can be used as a nano-diagnostic agent for tumors. The folic acid molecules coupled on the skeleton can target and recognize tumor cells with high expression of folic acid receptors, and the Cy7 molecules can be used for near-infrared fluorescence imaging. and photodynamic therapy.

Description

Folic acid-chitosan-Cy 7 polymer with tumor targeting property and preparation method thereof

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a folic acid-chitosan-Cy 7 polymer (CF 7), a preparation method and application thereof, and nanoparticles formed by the polymer, and a preparation method and application thereof.

Background

Cancer is one of the leading causes of health threats to humans today. Traditional pharmacotherapy causes damage to normal cells and tissues, and becomes the biggest difficulty in tumor treatment. Because the pathological and physiological characteristics of the tumor tissue and the normal tissue are obviously different, the vascular permeability of the tumor part is enhanced, and macromolecular drugs, nano-carriers and the like can easily penetrate through vascular endothelial cells to enterWhen introduced into Tumor tissue, and due to clearance disorders, accumulate at the Tumor site at high concentrations for a long time, which is known as the penetration and retention-Enhancing (EPR) effect (Ghaz-Jahanian, M.A.; Abbasour-Aghdam, F.; Anaarjan, N.; Berenjian, A.; Jafariadeh-Malmerir, H., Application of Chitosan-base nanovehicles in Tumor-Targeted Drug Delivery).Molecular Biotechnology2015,57, (3), 201-218.). In another aspect, a plurality of specific receptors, such as folate receptors, are present on the cell membrane of tumor tissue. Targeted therapy of tumor cells is a therapeutic approach that utilizes specific antigens and receptors on the surface of tumor cells as targets. The Folate Receptor (FR) is highly expressed on the surface of most tumor cells, while it is underexpressed on the surface of normal cells. The folic acid can be coupled with anticancer drugs to achieve the purpose of actively targeting tumor cells.

Near Infrared (NIR) fluorescent dyes can detect fluorescence signals from deep tissues because of their good tissue penetration, the greater penetration depth of absorbed NIR light into biological tissues, and the lesser influence of excited fluorescence on the biological tissues themselves. The dye has good application prospect in the early detection of cancer as a non-invasive molecular imaging reagent. The most representative of the dyes are near infrared cyanine dyes. Heptamethine cyanine dye (Cy 7) is one of the excellent fluorescent labeling dyes, the molar absorption coefficient is the highest among the fluorescent dyes, and has been widely used for labeling and detecting proteins, antibodies, nucleic acids and other biomolecules (Xiao, L.; Zhang, Y.; Berr, S.; Chordia, M. D.; Pramonojamo, P.; Pu, L.; Pan, D., A novel near-infrared fluorescence labeling probe for in vivo nuclear pharmaceutical packaging, Molecular imaging2012, 11, (5), 372-82.). Photodynamic Therapy (PDT) is a new technology for diagnosing and treating diseases by using Photodynamic effect. The basis of action is the photodynamic effect. The process is that the laser irradiation with specific wavelength excites the photosensitizer absorbed by the tissue, and the excited photosensitizer transfers the energy to the surrounding oxygen to generate singlet oxygen with strong activity, and the singlet oxygen and the adjacent biological macromolecules generate oxidation reaction to generate cytotoxicity, thereby causing cell damage and death. There have been many studies using Cy7 as a photosensitizer for photodynamic therapy. We chose Cy7 for near infrared fluorescence imaging and photodynamic therapy.

Chitosan (chemical name: β - (1 → 4) -2-amino-2-deoxy-D-glucose) is obtained by deacetylation of chitin (chitin) widely existing in the nature, has the characteristics of good biocompatibility, biodegradability, low toxicity and the like, has amino and hydroxyl in the structure, is easy to chemically modify, and is a novel drug carrier with wide application prospect.

Sharpless, the Nobel prize-winning chemist in 2001 proposed a "Click" chemical reaction. The reaction is represented by reacting a monomer having an alkynyl group and an azide group at the terminal to form a compound having a triazole ring structure. The reaction is fast, efficient and highly selective, and has been widely used for synthesizing various materials.

Based on the above background, the present invention designs a polymer, which is coupled by Click chemistry reaction from an azide chitosan derivative and an alkynylated folic acid (ALK-FA) and an alkynylated Cy7 (ALK-Cy 7), and can form nanoparticles by self-assembly. By connecting FA and Cy7, the fluorescent probe not only has the capability of efficiently identifying target tumor cells, but also can be used for near-infrared fluorescence imaging and photodynamic therapy.

Disclosure of Invention

Based on the above research background, the present inventors synthesized a folate-chitosan-Cy 7 polymer (CF 7) by reacting ALK-FA and ALK-Cy7 with 6-azido-6-deoxy-N-phthalimido-chitosan using a "Click" reaction. The polymer CF7 can be self-assembled to form nanoparticles, can be used as a nano diagnosis and treatment agent for tumors, folic acid molecules coupled on a skeleton of the polymer CF7 can be used for identifying tumor cells with high folic acid receptor expression in a targeted manner, and Cy7 molecules can be used for near-infrared fluorescence imaging and photodynamic therapy.

Therefore, the invention aims to provide a polymer CF7 and a preparation method thereof. The invention also aims to provide nanoparticles formed by the polymer, and a preparation method and application thereof.

The invention provides a polymer CF7, which has the structural formula:

wherein n is the number of repeating units of the chitosan derivative.

The polymer CF7 of the present invention can be prepared by the following method, the reaction formula is as follows:

wherein n is the number of repeating units of the chitosan derivative.

In the reaction formula, 1 is chitosan; 2 is N-phthalimidyl chitosan; 3 is 6-bromo-6-deoxy-N-phthalimido-chitosan; 4 is 6-azido-6-deoxy-N-phthalimido-chitosan; and 5 is polymer CF 7.

The synthesis method of CF7 comprises the following steps:

step a: weighing chitosan 1, and reacting with phthalic anhydride to replace amino to obtain N-phthalimide chitosan 2;

step b: performing bromine substitution reaction on the 6-hydroxyl on the product 2 to obtain a product 6-bromine-6-deoxidation-N-phthalimide group-chitosan 3;

step c: 6-bromine on the product 3 is subjected to azide substitution reaction to obtain 6-azido-6-deoxy-N-phthalimide-chitosan 4;

step d: carrying out Click reaction on the product 4, ALK-FA and ALK-Cy7 under the catalytic action of anhydrous copper sulfate and vitamin C sodium salt to obtain a product 5; wherein ALK-FA is obtained by reacting folic acid and propynylamine (Guo, Z.; Zhang, P.; Song, M.; Wu, X.; Liu, C.; ZHao), Z.; Lu, J.; Zhang, X., Synthesis andpreliminary evaluation of novel Tc-99m-labeled folate derivative via clickreaction for SPECT imaging.Applied Radiation And Isotopes2014, 91, 24-30), ALK-Cy7 is obtained by a series of reactions of phenylhydrazine and 3-methyl-2-butanone (Yang, Z.; Lee, J. H.; Jeon, H. M.; Han, J. H.; Park, N.; He, Y.; Lee, H.; Hong, K. S.; Kang, C.; Kim, J.S., Folate-Based Near-implanted Fluorescent therapeutic Gemcitabine Delivery.J Am Chem Soc2013, 135, (31), 11657-11662）。

The weight average molecular weight of chitosan 1 (Cs) used in the present invention is 10-1000 kilodaltons.

The polymer CF7 of the invention has the following specific reaction steps:

step a: weighing chitosan 1, dissolving in anhydrous DMF, adding phthalic anhydride, protecting with nitrogen, and heating in 120 deg.C oil bath under stirring. When the reaction is finished, pouring the reaction liquid into ice water to separate out a yellow-white precipitate. Carrying out suction filtration, washing the solid with diethyl ether and acetone, and drying to obtain N-phthalimide chitosan 2;

step b: product 2 was weighed out, dissolved in N-methylpyrrolidone (NMP) and N-bromosuccinimide (NBS) and Triphenylphosphine (TPP) were added. The reaction was carried out at 80 ℃ for two hours under nitrogen. After the reaction, the reaction solution was poured into ethanol to precipitate a solid. Centrifuging, collecting a product, washing the product with ethanol and acetone respectively for three times, and drying to obtain a brownish red solid 3;

step c: 3 was weighed and dissolved in N-methylpyrrolidone. Adding sodium azide (NaN)₃) Reacting for 4 hours at 80 ℃ under the protection of nitrogen. After the reaction, the reaction solution was poured into ethanol to precipitate a solid. Centrifuging, collecting the product, and washing the product with ethanol, secondary water and acetone respectively for three times. Drying to obtain brown solid 4;

step d: dissolve product 4 in dimethyl sulfoxide (DMSO), then add ALK-FA and ALK-Cy7 under nitrogen protection, then dissolve anhydrous copper sulfate and vitamin C sodium salt in water, slowly drop into beaker. The reaction was carried out at 80 ℃ for 72 hours. After the reaction is finished, adding the reaction solution into a dialysis bag, dialyzing for 72 hours by pure water, and freeze-drying to obtain a product 5 (CF 7); wherein the ALK-FA is obtained by the reaction of folic acid and propynylamine, and the ALK-Cy7 is obtained by the reaction of phenylhydrazine and 3-methyl-2-butanone in a series of reactions.

The molecular weight of the polymer CF7 in the invention is 100-1000 kilodaltons.

In the step d, the mass ratio of the product 4 to the ALK-FA and ALK-Cy7 is as follows: 2: 1. The cut-off molecular weight of the dialysis bag is 10000-14000.

The polymer can form nanoparticles and a preparation method thereof. The method comprises the steps of dissolving polymer CF7 in dimethyl sulfoxide, then slowly dripping the polymer CF7 into a beaker filled with pure water by a syringe, stirring and mixing the mixture, and standing the mixture at room temperature. The polymer forms nanoparticles by self-assembly. The method comprises the following specific steps: the polymer is prepared into 0.1-1 mg/ml solution by using dimethyl sulfoxide, then 1 ml of solution is absorbed by using an injector, the solution is slowly dripped into a beaker filled with 20-50 ml of pure water, the mixture is stirred and stands for 0.5-1 hour at room temperature, and the polymer forms nanoparticles through self-assembly.

The polymer CF7 is used for near infrared fluorescence imaging and photodynamic therapy of tumor cells.

The invention has the following action principle: 1, improving the solubility of chitosan; 2, Cy7 was targeted to cancer cells and used for near infrared fluorescence imaging and photodynamic therapy.

The invention has the beneficial effects that:

1, the polymer of the invention is self-assembled to form nanoparticles, the particle size is less than 300 nanometers, and the nanoparticles can be delivered to tumor sites in a targeted manner through intravenous administration.

The polymer and the nanoparticles formed by the polymer overcome the defect of poor solubility of chitosan, selectively concentrate on tumor cells by utilizing the active targeting effect of folic acid on the surface of a nano carrier and folic acid receptors on the surface of the tumor cells, and can also utilize Cy7 molecules in the nanoparticles to perform near-infrared fluorescence imaging and photodynamic therapy on the tumor cells.

Drawings

FIG. 1 Infrared spectra of Cs-N3, C7, and CF7 prepared in example 1 and example 2.

FIG. 2 UV spectra of Cs-N3, CF7, and CF prepared in example 1, example 3.

FIG. 3 fluorescence spectra of Cs-N3, CF7, and C7 prepared in example 1, example 2.

Fig. 4 is a confocal image of the CF7Ns nanoparticles and the C7Ns nanoparticles prepared in example 4 and example 5.

Figure 5 in vitro cytotoxicity of the nanoparticles CF7Ns and C7Ns prepared in example 4, example 5.

Detailed Description

The present invention will be further described with reference to the following examples, but the invention is not limited to these examples, and various changes and equivalents may be made within the scope of the invention as set forth in the claims.

Chitosan 1 was purchased from Bo and ao Biotechnology, Inc. of Shanghai, with a molecular weight of 60 kilodaltons and a degree of deacetylation of 90%.

Example 1

Synthesis of Polymer CF 7:

step a: 800 mg of chitosan was weighed out and dissolved in 60 mL of anhydrous DMF, followed by addition of 1.6 g of phthalic anhydride, nitrogen blanketing, and heating with stirring in a 120 ℃ oil bath. When the reaction solution became clear, the reaction was terminated. The reaction solution was poured into an appropriate amount of ice water to precipitate a white precipitate. And (3) carrying out suction filtration, washing the solid with diethyl ether and acetone for 3 times respectively, removing redundant phthalic anhydride, and drying to obtain a product 2.

Step b: 100 mg of product 2 was weighed, 10 mL of N-methylpyrrolidone (NMP) was added, and the mixture was dissolved by heating with stirring. When the solution was cooled and placed in ice water, 616 mg of N-bromosuccinimide (NBS), 902 mg of Triphenylphosphine (TPP) were added. The reaction was carried out at 80 ℃ for two hours under nitrogen. After the reaction was completed, the mixture was poured into 100 mL of ethanol to precipitate a solid. The product was collected by centrifugation (5000 r/min) and washed three times with ethanol, acetone each. After drying, a brown solid 3 was obtained.

Step c: 50 mg of product 3 are weighed out and dissolved in 5 mL of N-methylpyrrolidone, and 50 mg of sodium azide (NaN) are added₃) Stirring and heating for 4 hours at 80 ℃ under the protection of nitrogen. Reaction ofAfter completion, the reaction mixture was poured into 50 mL of ethanol to precipitate a solid. The product was collected by centrifugation (8000 r/min) and washed three times with ethanol, secondary water and acetone, respectively. Drying gave a brown solid 4 (Cs-N)₃). And 6-position bromine on the product 3 is replaced by azido by infrared spectrum analysis. By IR spectroscopy analysis, product 4 (Cs-N) is shown in FIG. 1₃) At 2100 cm^-1There is an infrared absorption peak indicating that the azide group has successfully replaced the bromine at the 6-position.

Step d: 20mg of product 4 was weighed out, dissolved in 5 mL of dimethyl sulfoxide, and 10mg of ALK-FA and 10mg of ALK-Cy7 were added. The flask was sealed with a rubber stopper, evacuated and then protected with nitrogen. To the flask, 2.5 mg of copper sulfate pentahydrate (dissolved in 100. mu.L of secondary water) was added dropwise followed by 2 mg of sodium ascorbate (dissolved in 100. mu.L of secondary water) using a 1 mL syringe. The reaction was left to react at 50 ℃ for 72h in the absence of light. After the reaction, the reaction solution was dialyzed for 72 hours in a dialysis bag having a standard of 14000. After dialysis, the solids in the dialysis bag were lyophilized. And (3) analyzing by an infrared spectrogram, reacting 6-position azide in the product 4 with alkynyl in ALK-FA and ALK-Cy7 to generate a triazole ring. By IR spectroscopy analysis, as shown in FIG. 1, CF7 was at 2100 cm^-1There was no infrared absorption peak, indicating that the azide group had successfully reacted with the alkyne group to form a triazole ring. And at 1531 cm^-1、1638 cm^-1There were two absorption peaks indicating successful attachment of folic acid to the chitosan scaffold. The product was dissolved in dimethyl sulfoxide and the UV absorbance measured, as shown in FIG. 2, with CF7 having UV absorbance at 280nm, indicating that folic acid was successfully attached to the chitosan scaffold. The product is dissolved in dimethyl sulfoxide, the excitation wavelength is 633nm, and the fluorescence intensity is measured. As shown in FIG. 3, CF7 has a characteristic peak of ALK-Cy7 at 801nm, indicating that ALK-Cy7 has also been successfully attached to the chitosan scaffold.

Example 2

Synthesis of Chitosan-Cy 7 Polymer (C7):

10mg of product 4 from example 1 was weighed out, dissolved in 5 mL of dimethyl sulfoxide, and 10mg of ALK-Cy7 was added. The flask was sealed with a rubber stopper, evacuated and then protected with nitrogen. 2.5 mg of copper sulfate pentahydrate (dissolved in 100. mu.L of secondary water) was added dropwise to the flask using a 1 mL syringe, followed by dropwise additionAdd 2 mg sodium ascorbate (dissolved in 100 μ L of secondary water). The reaction was left to react at 50 ℃ for 72h in the absence of light. After the reaction, the reaction solution was dialyzed for 72 hours in a dialysis bag having a standard of 14000. After dialysis, the product was lyophilized. And (3) analyzing by an infrared spectrogram, reacting 6-position azide in the product 4 with alkynyl in ALK-Cy7 to generate the triazole ring. As shown in FIG. 1, the chitosan-Cy 7 polymer (C7) was at 2100 cm^-1There was no infrared absorption peak, indicating that the azide group had successfully reacted with the alkyne group in ALK-Cy7 to form the triazole ring. The product is dissolved in dimethyl sulfoxide, the excitation wavelength is 633nm, and the fluorescence spectrum is measured. As shown in FIG. 3, CF7 has a characteristic peak of ALK-Cy7 at 801nm, indicating that ALK-Cy7 has also been successfully attached to the chitosan scaffold.

Example 3

Synthesis of Chitosan-FA Polymer (CF):

10mg of product 4 of example 1 was weighed out, dissolved in 5 mL of dimethyl sulfoxide, and 10mg of ALK-FA was added. The flask was sealed with a rubber stopper, evacuated and then protected with nitrogen. To the flask, 2.5 mg of copper sulfate pentahydrate (dissolved in 100. mu.L of secondary water) was added dropwise followed by 2 mg of sodium ascorbate (dissolved in 100. mu.L of secondary water) using a 1 mL syringe. The reaction was left to react at 50 ℃ for 72h in the absence of light. After the reaction, the reaction solution was dialyzed for 72 hours in a dialysis bag having a standard of 14000. After dialysis, the product was lyophilized. And (3) analyzing by an infrared spectrogram, reacting 6-azido in the product 4 with alkynyl in ALK-FA to generate the triazole ring. As shown in FIG. 1, the chitosan-FA polymer (CF) was at 2100 cm^-1There was no infrared absorption peak, indicating that the azide group had successfully reacted with the alkynyl group in ALK-FA to form a triazole ring. And at 1531 cm^-1、1638 cm^-1There are two absorption peaks, indicating successful attachment of ALK-FA to the chitosan scaffold. The product was dissolved in dimethyl sulfoxide and UV absorbance was measured, as shown in FIG. 2, with CF7 having UV absorbance at 280nm, indicating that ALK-FA had successfully attached to the chitosan scaffold.

Example 4

The polymer CF7 is used for the preparation method of the drug nanoparticles:

CF7 obtained in example 1 was dissolved in dimethyl sulfoxide, and then added dropwise slowly to a beaker containing pure water with a syringe, stirred and mixed, and allowed to stand at room temperature. The polymer forms nanoparticles by self-assembly. The method comprises the following specific steps: preparing 0.1-1 mg/ml solution of CF7 by using dimethyl sulfoxide, then sucking 1 ml by using an injector, slowly dripping the solution into a beaker filled with 20-50 ml of pure water, stirring, standing at room temperature for 0.5-1 hour, and forming the nano-particles (CF 7 Ns) by self-assembly of the polymer.

Example 5

The chitosan-Cy 7 polymer (C7) is used for the preparation method of the drug nanoparticles:

c7 was dissolved in dimethyl sulfoxide, and then added slowly dropwise to a beaker containing pure water with a syringe, stirred and mixed, and allowed to stand at room temperature. The polymer forms nanoparticles by self-assembly. The method comprises the following specific steps: preparing 0.1-1 mg/ml solution of C7 by using dimethyl sulfoxide, then sucking 1 ml by using an injector, slowly dripping the solution into a beaker filled with 20-50 ml of pure water, stirring, standing at room temperature for 0.5-1 hour, and forming the nanoparticles (C7 Ns) by self-assembly of the polymer.

Example 6

Human cervical cancer cell line Hela cells (folate receptor over-expression cells) and human liver cancer cell line HepG2 cells (folate receptor under-expression) are used as test cell lines (the cells are purchased from the cell resource center of Shanghai Life science research institute of Chinese academy of sciences).

The cell culture method comprises the following steps: taking out Hela cell seed-preserving tube from liquid nitrogen tank, rapidly melting and thawing in 37 deg.C water bath, centrifuging at 1000 rpm for 5min, removing supernatant, taking 1 mL DMEM complete culture solution, blowing cell precipitate uniformly, transferring to culture bottle to make culture medium in bottle 4 mL, placing at 37 deg.C and 5% (v/v) CO₂Culturing in an incubator. Taking out HepG2 cells frozen in liquid nitrogen, unfreezing in water at 37 ℃, transferring the cell suspension into a 1.5 mL centrifuge tube, centrifuging for 5min at 1000 rpm, removing the supernatant, adding 1 mL RPMI 1640 complete culture solution, gently blowing and beating uniformly, transferring the cell suspension into a culture bottle, supplementing 3 mL RPMI 1640 complete culture solution, placing the culture bottle in 5% (v/v) CO₂And cultured in an incubator at 37 ℃.

Confocal imaging experiments: hela cells and HepG2 cells were digested, plated in 24-well plates overnight, washed 2 times with PBS after the cells were completely attached, and incubated with 20 μ g/mL of CF7Ns and C7Ns of example 4 and example 5, respectively, at 37 ℃ for 4 h. Then washed 2 times with PBS, incubated with 4wt% paraformaldehyde for 10min, washed 2 times with PBS, followed by addition of DAPI, incubated at room temperature in the dark for 10min, and then washed 2 times with PBS. Laser confocal imaging.

The results of confocal imaging of nanoparticles are shown in fig. 4. As can be seen from FIG. 4, in Hela cells, the fluorescence intensity of CF7Ns is stronger than that of C7Ns, while in HepG2 cells, the fluorescence intensity of CF7Ns is equivalent to that of C7Ns, indicating that the folic acid modification can increase the uptake of nano-drugs by folate receptor over-expression tumor cell lines. And CF7Ns can be targeted to and delivered to a cell strain with high expression of a folate receptor for imaging, thereby providing an effective means for tumor treatment.

Example 7

Cytotoxicity experiments: selecting good Hela or HepG2 cells with log phase growth, trypsinizing to obtain cell concentration of 0.5-1 × 10⁵And (4) preparing a cell suspension. Amount of 100 μ L cell suspension per wellAfter being inoculated into a 96-well plate and cultured for 24 hours, 40. mu.g/mL Cs-N of example 1 was added₃40 ug/mL of CF7Ns and CFNs of examples 4 and 5, and a solvent control and a blank control were additionally provided, and in order to examine the photodynamic therapy effect, the experimental components to which CF7Ns and CFNs were added were divided into four groups, two of which were irradiated with infrared light (NIR) and the other two of which were not irradiated with infrared light. And after 24h of incubation, removing the old culture medium by suction, washing the old culture medium by PBS for 3 times, adding 90 mu L of 1640 culture medium without serum and phenol red and 10 mu L of MTT solution into each hole, continuing to incubate for 4h, carefully removing the supernatant, adding 150 mu L of dimethyl sulfoxide into each hole, oscillating for 10min in a dark place to completely dissolve the bluish purple crystals, measuring the absorbance of each hole at the wavelength of 570 nm by using a multifunctional microplate reader, and calculating the survival rate of the cells according to the following formula. Survival (%) = (experimental absorbance-solvent control absorbance)/(blank absorbance-solvent control absorbance).

The cytotoxicity results of the nanoparticles are shown in fig. 5. As can be seen from FIG. 5, Cs-N₃Both CF7Ns and C7Ns were less toxic to both cells and killed the cells to varying degrees. In Hela cells, CF7Ns was more toxic than C7Ns, CF7Ns + NIR (irradiation of infrared light) was more toxic than CF7Ns and C7Ns + NIR was also more toxic than C7Ns when the cells were exposed to infrared light; in HepG2 cells, C7Ns was comparable in toxicity to CF7Ns, CF7Ns + NIR and C7Ns + NIR. This shows that folic acid modification can increase the toxicity of nano medicine to folic acid receptor over-expression tumor cells, so as to improve antitumor selectivity. Also, photodynamic therapy was shown to enhance the antitumor effect.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. A preparation method of a folic acid-chitosan-Cy 7 polymer with tumor targeting is characterized by comprising the following steps: the structural formula is as follows:

；

wherein n is the number of repeating units of the chitosan derivative; the molecular weight of the folic acid-chitosan-Cy 7 polymer is 100-1000 kilodaltons; the reaction formula is as follows:

；

the method comprises the following specific steps:

step b: carrying out bromine substitution reaction on the 6-position hydroxyl on the product 2 to obtain a product 3;

step c: carrying out azido substitution reaction on the 6-position bromine on the product 3 to obtain a product 4;

step d: carrying out Click reaction on the product 4, folic acid ALK-FA modified by alkynyl and heptamethine cyanine dye ALK-Cy7 modified by alkynyl under the catalytic action of anhydrous copper sulfate and vitamin C sodium salt to obtain a product 5; wherein the ALK-FA is obtained by the reaction of folic acid and propynylamine, and the ALK-Cy7 is obtained by the reaction of phenylhydrazine and 3-methyl-2-butanone in a series of reactions.

2. The method of claim 1 for preparing folic acid-chitosan-Cy 7 polymer, wherein: the weight average molecular weight of the chitosan 1 is 10-1000 kilodaltons.

3. The method of claim 1 for preparing folic acid-chitosan-Cy 7 polymer, wherein: the mass ratio of the product 4 to the ALK-FA and ALK-Cy7 is 2: 1.

4. The folic acid-chitosan-Cy 7 polymer drug nanoparticles prepared by the preparation method of claim 1.

5. A drug nanoparticle according to claim 4, wherein: the preparation method comprises the following steps: the folic acid-chitosan-Cy 7 polymer is prepared into 0.1-1 mg/ml solution by using dimethyl sulfoxide, then 1 ml of solution is sucked by using a syringe, the solution is dropwise added into a beaker filled with 20-50 ml of pure water at a speed of one drop per second, the mixture is stirred and stands at room temperature for 0.5-1 hour, and the polymer forms nanoparticles through self-assembly.