CN107158410A - A kind of folic acid chitosan Cy7 polymer with tumor-targeting and preparation method thereof - Google Patents

Abstract

The invention discloses a novel folic acid-chitosan-Cy7 polymer (CF7) used as a diagnostic agent and a preparation method thereof. The derivative is composed of 6-azide-6-deoxy-N-phthalimido-chitosan with alkyne-modified folic acid (ALK-FA) and alkyne-modified heptamethine cyanine dye Cy7 (ALK‑Cy7) reaction synthesis. The polymer CF7 of the present invention can be self-assembled to form nanoparticles, which can be used as a nano-diagnosis and treatment agent for tumors. The folic acid molecules coupled to its skeleton can target and recognize tumor cells with high expression of folic acid receptors, and the Cy7 molecule can be used for near-infrared fluorescence imaging. and photodynamic therapy.

Description

A folic acid-chitosan-Cy7 polymer with tumor targeting and its preparation method

technical field

The invention belongs to the technical field of biomedicine, and relates to a folic acid-chitosan-Cy7 polymer (CF7) and its preparation method and application, as well as nanoparticles formed by the polymer, its preparation method and application.

Background technique

Cancer is one of the main causes of threats to human health today. Traditional drug chemotherapy can cause damage to normal cells and tissues, which has become the biggest difficulty in tumor treatment. Due to the significant difference in pathological and physiological characteristics between tumor tissue and normal tissue, the permeability of blood vessels in tumor sites is enhanced, and macromolecular drugs and nanocarriers can easily penetrate vascular endothelial cells into tumor tissue. Long-term, high-concentration accumulation at the tumor site, this effect is called the enhanced penetration and retention (EPR) effect (Ghaz-Jahanian, MA; Abbaspour-Aghdam, F.; Anarjan, N.; Berenjian, A.; Jafarizadeh-Malmiri , H., Application of Chitosan-Based Nanocarriers in Tumor-Targeted Drug Delivery. Molecular Biotechnology 2015,57, (3), 201-218.). On the other hand, there are a variety of specific receptors on the cell membrane of tumor tissue, such as folate receptor. Targeted therapy of tumor cells is a treatment method that uses specific antigens and receptors on the surface of tumor cells as targets. Folate receptors (FR) are highly expressed on the surface of most tumor cells, while folate receptors are low expressed on the surface of normal cells. We can use folic acid to couple anticancer drugs to actively target tumor cells.

Due to the good tissue permeability of near-infrared (NIR) fluorescent dyes, the absorbed near-infrared light has a greater penetration depth in biological tissue, and the excited fluorescence is less affected by the biological tissue itself, so it can detect deep Tissue fluorescence signal. This kind of dye has a good application prospect in the early detection of cancer as a non-invasive molecular imaging reagent. Among them, the most representative ones are near-infrared cyanine dyes. Heptamethine cyanine (Cy7) is one of the fluorescent labeling dyes with excellent performance. Its molar absorptivity is the highest among fluorescent dyes. It has been widely used in the labeling and detection of proteins, antibodies, nucleic acids and other biomolecules. (Xiao, L.; Zhang, Y.; Berr, S. S.; Chordia, M. D.; Pramoonjago, P.; Pu, L.; Pan, D., A novel near-infrared fluorescence imaging probe for in vivo neutrophil tracking. Molecular imaging2012, 11, (5), 372-82.). Photodynamic therapy (Photodynamic Therapy, PDT) is a new technology that uses photodynamic effect for disease diagnosis and treatment. Its basis is the photodynamic effect. The process is that the photosensitizer absorbed by the tissue is excited by the laser irradiation of a specific wavelength, and the photosensitizer in the excited state transfers energy to the surrounding oxygen to generate highly active singlet oxygen, and the singlet oxygen and adjacent biological Oxidation of macromolecules produces cytotoxicity, which in turn leads to cell damage and even death. At present, many studies have used Cy7 as a photosensitizer for photodynamic therapy. So we choose 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, which exists widely in nature. Chitosan has good biocompatibility, biodegradability and low toxicity. There are both amino groups and hydroxyl groups in its structure, which is easy to chemically modify and is a new type of drug carrier with broad application prospects. In addition, chitosan is also widely used in the field of biomedicine, such as tissue engineering, wound healing, bioimaging and drug delivery. However, due to the poor solubility of chitosan, it needs to be chemically modified. Therefore, most of the current literature introduces functional groups on its 2-position to increase its solubility.

The 2001 Nobel Prize winner in Chemistry Sharpless proposed the "Click" chemical reaction. Its representative reaction is to react a monomer with an alkynyl group and an azide group at the end to form a compound with a triazole ring structure. The reaction is fast, efficient and highly selective, and has been widely used in the synthesis of various materials.

Based on the above background, the present invention designs a kind of polymer, which is composed of azidated chitosan derivatives, alkynylated folic acid (ALK-FA) and alkynylated Cy7 (ALK-Cy7) through Click Chemical reactions couple and can form nanoparticles through self-assembly. By linking FA and Cy7, it not only has the ability to efficiently identify target tumor cells, but also can be used for near-infrared fluorescence imaging and photodynamic therapy.

Contents of the invention

Based on the above research background, the inventors used the "Click" reaction to react ALK-FA and ALK-Cy7 with 6-azido-6-deoxy-N-phthalimide-chitosan to synthesize folic acid- Chitosan-Cy7 polymer (CF7). The polymer CF7 of the present invention can be self-assembled to form nanoparticles, which can be used as a nano-diagnosis and treatment agent for tumors. The folic acid molecules coupled to its skeleton can target and recognize tumor cells with high expression of folic acid receptors, and the Cy7 molecule can be used for near-infrared fluorescence imaging. and photodynamic therapy.

Therefore the object of the present invention is to provide a kind of polymer CF7 and preparation method thereof. Another object of the present invention is to provide nanoparticles formed from the polymer and its preparation method and use.

The invention provides a kind of polymer CF7, and its structural formula is:

Wherein n is the number of chitosan derivative repeating units.

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

Wherein n is the number of chitosan derivative repeating units.

In the reaction formula, 1 is chitosan; 2 is N-phthalimide-based chitosan; 3 is 6-bromo-6-deoxy-N-phthalimide-chitosan; 4 is 6-azido-6-deoxy-N-phthalimido-chitosan; 5 is polymer CF7.

The synthetic method of CF7 of the present invention comprises the following steps:

Step a: Weighing chitosan 1, reacting with phthalic anhydride to replace the amino group, to obtain N-phthalimine chitosan 2;

Step b: carry out bromine substitution reaction on the 6-position hydroxyl on the product 2 to obtain the product 6-bromo-6-deoxy-N-phthalimido-chitosan 3;

Step c: Substituting the 6-position bromine on the product 3 with an azido group to obtain 6-azido-6-deoxy-N-phthalimide-chitosan 4;

Step d: Perform Click reaction of product 4 with ALK-FA and ALK-Cy7 under the catalysis of anhydrous copper sulfate and vitamin C sodium salt to obtain product 5; wherein ALK-FA is obtained by reacting folic acid and propargylamine ( Guo, Z.; Zhang, P.;Song, M.; Wu, X.; Liu, C.; Zhao, Z.; Lu, J.; Zhang, X., Synthesis and preliminary evaluation of novel Tc-99m-labeled folate derivative via clickreaction for SPECT imaging. Applied Radiation And Isotopes 2014, 91, 24-30), ALK-Cy7 is obtained by a series of reactions between phenylhydrazine and 3-methyl-2-butanone (Yang, Z.; Lee , JH; Jeon,HM; Han, JH; Park, N.; He, Y.; Lee, H.; Hong, KS; Kang, C.; Kim, JS, Folate-Based Near-Infrared Fluorescent Theranostic Gemcitabine Delivery. J Am Chem Soc 2013, 135, (31), 11657-11662).

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

The specific reaction steps of polymer CF7 of the present invention are as follows:

Step a: Weigh chitosan 1, dissolve in anhydrous DMF, add phthalic anhydride, nitrogen protection, stir and heat in an oil bath at 120°C. At the end of the reaction, the reaction solution was poured into ice water, and a yellow-white precipitate was precipitated. Suction filtration, the solid was washed with ether and acetone, and dried to obtain N-phthalimine chitosan 2;

Step b: Weigh the product 2, dissolve it in N-methylpyrrolidone (NMP), add N-bromosuccinimide (NBS) and triphenylphosphine (TPP). Under the protection of nitrogen, the reaction was carried out at 80° C. for two hours. After the reaction was completed, the reaction solution was poured into ethanol to precipitate a solid. After centrifugation, the product was collected, washed three times with ethanol and acetone and dried to obtain a brownish red solid 3;

Step c: Weigh 3 and dissolve it in N-methylpyrrolidone. Sodium azide (NaN ₃ ) was added, under nitrogen protection, and reacted at 80°C for 4 hours. After the reaction was completed, the reaction solution was poured into ethanol to precipitate a solid. Centrifuge to collect the product, and wash the product three times successively with ethanol, secondary water and acetone. Drying afforded 4 as a brown solid;

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 the beaker . Reaction at 80°C for 72 hours. After the reaction, the reaction solution was added to a dialysis bag, dialyzed with pure water for 72 hours, and freeze-dried to obtain the product 5 (CF7). Among them, ALK-FA was obtained by the reaction of folic acid and propargylamine, and ALK-Cy7 was obtained by the reaction of phenylhydrazine And 3-methyl-2-butanone obtained through a series of reactions.

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

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

The polymers described in the present invention can form nanoparticles and methods for their preparation. The method is to dissolve polymer CF7 in dimethyl sulfoxide, then slowly add it dropwise into a beaker filled with pure water with a syringe, stir and mix, and stand at room temperature. The polymer forms nanoparticles by self-assembly. The specific steps are: mix the polymer described in the present invention with dimethyl sulfoxide into a 0.1-1 mg/ml solution, then use a syringe to draw 1 ml, and slowly drop it into a solution containing 20-50 ml of pure water in a beaker, stirred, and allowed to stand at room temperature for 0.5-1 hour, the polymer forms nanoparticles through self-assembly.

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

The action principle of the present invention is as follows: 1, improving the solubility of chitosan; 2, targeting and delivering Cy7 to cancer cells, and using it for near-infrared fluorescence imaging and photodynamic therapy.

The beneficial effects of the present invention are:

1. The polymers of the present invention are self-assembled to form nanoparticles with a particle size of less than 300 nanometers, which can be delivered intravenously and targeted to tumor sites.

2. The polymer of the present invention and the nanoparticles formed thereof overcome the defect of poor solubility of chitosan, and at the same time utilize the active targeting effect of folic acid on the surface of the nanocarrier and the folic acid receptor on the surface of tumor cells to selectively concentrate on Tumor cells, and Cy7 molecules in nanoparticles can also be used for near-infrared fluorescence imaging and photodynamic therapy of tumor cells.

Description of drawings

Fig. 1 Infrared spectra of Cs-N3, C7 and CF7 prepared in Example 1 and Example 2.

Fig. 2 embodiment 1, the ultraviolet spectrum of Cs-N3 prepared in embodiment 3, CF7 and CF.

Fig. 3 is the fluorescence spectrum of Cs-N3, CF7 and C7 prepared in Example 1 and Example 2.

Fig. 4 is a confocal imaging diagram of nanoparticles CF7Ns and C7Ns prepared in Example 4 and Example 5.

Fig. 5 In vitro cytotoxicity of nanoparticles CF7Ns and C7Ns prepared in Example 4 and Example 5.

detailed description

Below, the present invention will be further described through examples, but the invention is not limited to these examples, and various changes or equivalent replacements can be made within the scope of the claims of the present invention.

Chitosan 1 was purchased from Shanghai Boao Biotechnology Co., Ltd., with a molecular weight of 60 kilodaltons and a deacetylation degree of 90%.

Example 1

Synthesis of Polymer CF7:

Step a: Weigh 800 mg of chitosan and dissolve in 60 mL of anhydrous DMF, then add 1.6 g of phthalic anhydride, under nitrogen protection, stir and heat in an oil bath at 120°C. When the reaction solution became clear, the reaction was terminated. The reaction solution was poured into an appropriate amount of ice water, and a white precipitate was precipitated. After suction filtration, the solid was washed three times with ether and acetone respectively to remove excess phthalic anhydride, and dried to obtain product 2.

Step b: Weigh 100 mg of product 2, add 10 mL of N-methylpyrrolidone (NMP), heat and stir to dissolve. When the solution was cooled and placed in ice water, 616 mg N-bromosuccinimide (NBS) and 902 mg triphenylphosphine (TPP) were added. React at 80°C for two hours under nitrogen protection. After the reaction was over, the mixture was poured into 100 mL of ethanol, and a solid was precipitated. The product was collected by centrifugation (5000 r/min), and washed three times with ethanol and acetone. After drying, 3 was obtained as a brown solid.

Step c: Weigh 50 mg of product 3 and dissolve in 5 mL of N-methylpyrrolidone, add 50 mg of sodium azide (NaN ₃ ), under nitrogen protection, stir and heat at 80°C for 4 hours. After the reaction, the reaction solution was poured into 50 mL of ethanol, and a solid was precipitated. The product was collected by centrifugation (8000 r/min), and the product was washed three times with ethanol, secondary water and acetone. After drying a brown solid 4 (Cs-N ₃ ) was obtained. According to infrared spectrogram analysis, the bromine at the 6-position of the product 3 was replaced by an azido group. According to the infrared spectrum analysis, as shown in Figure 1, the product 4 (Cs-N ₃ ) has an infrared absorption peak at 2100 cm ^-1 , indicating that the azido group has successfully replaced the bromine at the 6-position.

Step d: Weigh 20 mg of product 4, dissolve in 5 mL dimethyl sulfoxide, add 10 mg ALK-FA, 10 mg ALK-Cy7. The flask was sealed with a rubber stopper and protected with nitrogen after evacuation. First add 2.5 mg copper sulfate pentahydrate (dissolved in 100 μL secondary water) dropwise to the flask with a 1 mL syringe, and then add 2 mg sodium ascorbate (dissolved in 100 μL secondary water) dropwise. The reactants were reacted at 50 °C for 72 h in the dark. After the reaction, the reaction solution was dialyzed for 72 hours with a 14000 dialysis bag. After dialysis, the solids in the dialysis bags were lyophilized. According to infrared spectrogram analysis, the azido group at position 6 in product 4 reacted with the alkynyl group in ALK-FA and ALK-Cy7 to form a triazole ring. According to the infrared spectrum analysis, as shown in Figure 1, CF7 has no infrared absorption peak at 2100 cm ^-1 , indicating that the azido group has successfully reacted with the alkyne group to form a triazole ring. And there are two absorption peaks at 1531 cm ^-1 and 1638 cm ^-1 , indicating that folic acid is successfully connected to the chitosan backbone. The product was dissolved in dimethyl sulfoxide to measure the ultraviolet absorption, as shown in Figure 2, CF7 has ultraviolet absorption at 280nm, indicating that folic acid has been successfully connected to the chitosan skeleton. The product was dissolved in dimethyl sulfoxide, the excitation wavelength was 633nm, and the fluorescence intensity was measured. As shown in Figure 3, CF7 has the characteristic peak of ALK-Cy7 at 801 nm, indicating that ALK-Cy7 has also been successfully connected to the chitosan backbone.

Example 2

Synthesis of Chitosan-Cy7 Polymer (C7):

Weigh 10 mg of the product 4 of Example 1, dissolve it in 5 mL of dimethyl sulfoxide, and add 10 mg of ALK-Cy7. The flask was sealed with a rubber stopper and protected with nitrogen after evacuation. First add 2.5 mg copper sulfate pentahydrate (dissolved in 100 μL secondary water) dropwise to the flask with a 1 mL syringe, and then add 2 mg sodium ascorbate (dissolved in 100 μL secondary water) dropwise. The reactants were reacted at 50 °C for 72 h in the dark. After the reaction, the reaction solution was dialyzed for 72 hours with a 14000 dialysis bag. After dialysis, the product was lyophilized. According to infrared spectrogram analysis, the 6-position azido group in product 4 reacted with the alkynyl group in ALK-Cy7 to form a triazole ring. As shown in Figure 1, chitosan-Cy7 polymer (C7) has no infrared absorption peak at 2100 cm ^-1 , indicating that the azide group has successfully reacted with the alkyne group in ALK-Cy7 to form a triazole ring. The product was dissolved in dimethyl sulfoxide, the excitation wavelength was 633nm, and its fluorescence spectrum was measured. As shown in Figure 3, CF7 has the characteristic peak of ALK-Cy7 at 801 nm, indicating that ALK-Cy7 has also been successfully connected to the chitosan backbone.

Example 3

Synthesis of chitosan-FA polymer (CF):

Weigh 10 mg of the product 4 of Example 1, dissolve it in 5 mL dimethyl sulfoxide, and add 10 mg ALK-FA. The flask was sealed with a rubber stopper and protected with nitrogen after evacuation. First add 2.5 mg copper sulfate pentahydrate (dissolved in 100 μL secondary water) dropwise to the flask with a 1 mL syringe, and then add 2 mg sodium ascorbate (dissolved in 100 μL secondary water) dropwise. The reactants were reacted at 50 °C for 72 h in the dark. After the reaction, the reaction solution was dialyzed for 72 hours with a 14000 dialysis bag. After dialysis, the product was lyophilized. According to infrared spectrogram analysis, the azido group at position 6 in product 4 reacted with the alkynyl group in ALK-FA to form a triazole ring. As shown in Figure 1, chitosan-FA polymer (CF) has no infrared absorption peak at 2100 cm ^-1 , indicating that the azido group has successfully reacted with the alkyne group in ALK-FA to form a triazole ring. And there were two absorption peaks at 1531 cm ^-1 and 1638 cm ^-1 , which indicated that ALK-FA was successfully linked to the chitosan backbone. The product was dissolved in dimethyl sulfoxide to measure the UV absorption, as shown in Figure 2, CF7 has UV absorption at 280nm, indicating that ALK-FA has been successfully connected to the chitosan skeleton.

Example 4

Polymer CF7 is used for the preparation method of drug nanoparticle:

The CF7 prepared in Example 1 was dissolved in dimethyl sulfoxide, and then slowly added dropwise into a beaker filled with pure water with a syringe, stirred and mixed, and allowed to stand at room temperature. The polymer forms nanoparticles by self-assembly. The specific steps are: mix CF7 with dimethyl sulfoxide to make a 0.1-1 mg/ml solution, then draw 1 ml with a syringe, slowly add it dropwise to a beaker filled with 20-50 ml of pure water, stir, and keep at room temperature After standing still for 0.5~1 hour, the polymer formed nanoparticles (CF7Ns) by self-assembly.

Example 5

Chitosan-Cy7 polymer (C7) is used for the preparation method of drug nanoparticle:

Dissolve C7 in dimethyl sulfoxide, then slowly drop it into a beaker filled with pure water with a syringe, stir and mix, and let stand at room temperature. The polymer forms nanoparticles by self-assembly. The specific steps are: mix C7 with dimethyl sulfoxide to make a 0.1-1 mg/ml solution, then draw 1 ml with a syringe, slowly add it dropwise to a beaker filled with 20-50 ml of pure water, stir, and keep at room temperature After standing still for 0.5~1 hour, the polymer formed nanoparticles (C7Ns) by self-assembly.

Example 6

The human cervical cancer cell line Hela cells (folate receptor overexpression cells) and human liver cancer cell line HepG2 cells (folate receptor low expression) were used as test cell lines (the cells were purchased from the Cell Resource Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences).

Cell culture method: Take out the Hela cell culture tube from the liquid nitrogen tank, quickly melt and thaw it in a 37°C water bath, then centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and take 1 mL of DMEM complete culture solution to blow the cell pellet Evenly, transfer to a culture bottle so that the medium in the bottle is 4 mL, and place it in a 37°C, 5% (v/v) CO ₂ incubator for culture. Take out HepG2 cells frozen in liquid nitrogen, thaw in water at 37°C, transfer the cell suspension into a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL RPMI 1640 complete culture solution, and gently Pipette evenly, transfer the cell suspension to a culture bottle, add 3 mL RPMI 1640 complete culture solution, and place the culture bottle in a 5% (v/v) CO ₂ , 37°C incubator for cultivation.

Confocal imaging experiment: Hela cells and HepG2 cells were digested and placed in a 24-well plate overnight. After the cells were completely adhered to the wall, they were washed twice with PBS and mixed with 20 μg/mL of CF7Ns and C7Ns of Example 4 and Example 5 respectively. Incubate at 37°C for 4 h. Then wash twice with PBS, incubate and fix with 4wt% paraformaldehyde for 10 min, wash twice with PBS, then add DAPI, incubate at room temperature in the dark for 10 min, and then wash twice with PBS. Laser confocal imaging.

The results of confocal imaging of nanoparticles are shown in Figure 4. It can be seen from Figure 4 that in Hela cells, the fluorescence intensity of CF7Ns is stronger than that of C7Ns, while in HepG2 cells, the fluorescence intensity of CF7Ns is comparable to that of C7Ns, indicating that the modification of folic acid can increase the folate receptor overexpression tumor cell line Uptake of nanomedicines. Moreover, CF7Ns can be targeted and delivered to cell lines with high expression of folate receptors for imaging, providing an effective means for tumor treatment.

Example 7

The human cervical cancer cell line Hela cells (folate receptor overexpression cells) and human liver cancer cell line HepG2 cells (folate receptor low expression) were used as test cell lines (the cells were purchased from the Cell Resource Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences).

Cell culture method: Take out the Hela cell culture tube from the liquid nitrogen tank, quickly melt and thaw it in a 37°C water bath, then centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and take 1 mL of DMEM complete culture solution to blow the cell pellet Evenly, transfer to a culture bottle so that the medium in the bottle is 4 mL, and place it in a 37°C, 5% (v/v) CO ₂ incubator for culture. Take out HepG2 cells frozen in liquid nitrogen, thaw in water at 37°C, transfer the cell suspension into a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL RPMI 1640 complete culture solution, and gently Pipette evenly, transfer the cell suspension to a culture bottle, add 3 mL RPMI 1640 complete culture solution, and place the culture bottle in a 5% (v/v) CO ₂ , 37°C incubator for cultivation.

Cytotoxicity test: Select Hela or HepG2 cells in logarithmic phase and in good condition, digest with trypsin to make the cell concentration 0.5-1×10 ⁵ cells/mL, and prepare a cell suspension. The amount of 100 µL cell suspension per well was inoculated into a 96-well plate, and after 24 hours of culture, 40 μg/mL Cs-N ₃ in Example 1, 40 μg/mL of CF7Ns and CFNs in Example 4 and Example 5 were added , a solvent control group and a blank control group were also set up. In order to investigate the effect of photodynamic therapy, the present invention divides the experimental group adding CF7Ns and CFNs into four groups, two groups are irradiated with infrared light (NIR), and the other two groups do not use infrared light. light exposure. After incubation for 24 hours, discard the old medium, wash with PBS 3 times, add 90 µL of serum-free, phenol red-free 1640 medium and 10 µL of MTT solution to each well, continue to incubate for 4 hours, and carefully discard the supernatant solution, add 150 µL dimethyl sulfoxide to each well, and shake in the dark for 10 minutes to dissolve all the blue-purple crystals, measure the absorbance of each well at a wavelength of 570 nm with a multifunctional microplate reader, and calculate according to the following formula cell viability. Survival rate (%) = (absorption value of experimental group - absorption value of solvent control group) / (absorption value of blank group - absorption value of solvent control group).

The cytotoxicity results of nanoparticles are shown in Fig. 5 . It can be seen from Figure 5 that Cs-N ₃ is less toxic to the two types of cells, and both CF7Ns and C7Ns can kill cells to varying degrees. In Hela cells, the toxicity of CF7Ns is stronger than that of C7Ns. When the cells are exposed to infrared light, the toxicity of CF7Ns+NIR (infrared light irradiation) is stronger than that of CF7Ns, and the toxicity of C7Ns+NIR is also stronger than that of C7Ns; while In HepG2 cells, the toxicity of C7Ns was comparable to that of CF7Ns, and the toxicity of CF7Ns+NIR was comparable to that of C7Ns+NIR. This suggests that the modification of folic acid can increase the toxicity of nanomedicines to folate receptor-overexpressing tumor cells, thereby improving the antitumor selectivity. It also shows that photodynamic therapy can enhance the antitumor effect.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (7)

1. a folic acid-chitosan-Cy7 polymer with tumor targeting, is characterized in that: structural formula is as follows: ; Where n is the number of repeating units of chitosan derivatives. 2. The folic acid-chitosan-Cy7 polymer according to claim 1, characterized in that: the molecular weight of the folic acid-chitosan-Cy7 polymer is 100-1000 kilodaltons. 3. a method for preparing folic acid-chitosan-Cy7 polymer as claimed in claim 1, is characterized in that: reaction formula is as follows: ; Wherein n is the number of chitosan derivative repeating units; The specific steps are: Step a: Weighing chitosan 1, reacting with phthalic anhydride to replace the amino group, to obtain N-phthalimine chitosan 2; Step b: performing a bromine substitution reaction on the 6-hydroxyl group on product 2 to obtain product 3; Step c: Substituting the 6-position bromine on product 3 with an azido group to obtain product 4; Step d: performing a Click reaction on product 4 with alkynyl-modified folic acid ALK-FA and alkynyl-modified heptamethine cyanine dye ALK-Cy7 under the catalysis of anhydrous copper sulfate and vitamin C sodium salt to obtain product 5; Among them, ALK-FA is obtained by the reaction of folic acid and propynylamine, and ALK-Cy7 is obtained by a series of reactions of phenylhydrazine and 3-methyl-2-butanone. 4. the preparation method of folic acid-chitosan-Cy7 polymer according to claim 3 is characterized in that: the weight average molecular weight of described chitosan 1 is 10-1000 kilodalton. 5. the preparation method of folic acid-chitosan-Cy7 polymer according to claim 3 is characterized in that: the mass ratio of product 4 and ALK-FA and ALK-Cy7 is 2: 1: 1. 6. a kind of medicine nanoparticle that folic acid-chitosan-Cy7 polymer as claimed in claim 1 is made. 7. The drug nanoparticle according to claim 6, characterized in that: its preparation method comprises the following steps: making the folic acid-chitosan-Cy7 polymer into 0.1-1 mg/kg with dimethyl sulfoxide milliliter solution, then draw 1 milliliter with a syringe, drop it into a beaker filled with 20-50 milliliters of pure water at a rate of one drop per second, stir, and let stand at room temperature for 0.5-1 hour, the polymer forms nanoparticles through self-assembly .