Responsive controlled-release nitric oxide photothermal gel and its preparation method and application

Abstract

The present invention discloses a responsive controlled-release nitric oxide photothermal hydrogel and a preparation method and application thereof. The hydrogel is prepared by a polycarbonate macromolecular nitric oxide donor (PNTC) and chitosan (CI) modified by a cyanine photosensitizer through a Schiff base crosslinking reaction. The injectable self-healing multifunctional hydrogel of the present invention responsively controls NO release through the photothermal effect of the photosensitizer, synergistically removes pathogenic drug-resistant bacterial biofilms, kills tumor cells, and improves the immune microenvironment. The hydrogel has a simple preparation method, good biocompatibility, excellent performance, and good research and application prospects.

Description

Technical Field

[0001] The invention relates to polymer materials and biomedical materials, preparation methods and uses, and in particular to responsive controlled-release nitric oxide photothermal water gel and a preparation method and use thereof. Background Art

[0002] The hydrogel system has a unique 3D porous network structure with excellent swelling properties, mechanical properties, permeability and biocompatibility. The local administration method can avoid systemic toxicity and play a protective barrier role. However, traditional dressing-type hydrogels have certain limitations in practical applications, such as large-area invasive implantation surgery, non-adhesion to tissue surfaces, multiple traumas and increased risk of infection. At the same time, they are easily damaged by external factors during processing and use, resulting in gel cracks, thereby shortening the service life and weakening the use effect. In recent years, the introduction of injectable and self-healing functions into hydrogel materials is conducive to innovating the use of hydrogels, extending the service life of materials, and improving the therapeutic effect and safety of use. The injectable self-healing properties of hydrogels are based on dynamic covalent bonds or non-covalent bonds, which enable them to have both the reversibility of non-covalent bonds and the stability of covalent bonds. At present, injectable self-healing hydrogels based on phenylboronic acid esters, disulfide bonds, hydrazides, and imines have been reported.

[0003] Among them, dynamic smart hydrogels based on Schiff bases have attracted widespread attention as injectable self-healing materials. Schiff base reactions can occur through the reversible condensation of amines and aldehydes / ketones. Under physiological conditions, no additional reagents or catalysts are required. They can be formed by in situ self-crosslinking under mild reaction conditions, with adjustable gel kinetics and good biocompatibility. The coupling, dynamic uncoupling and recoupling properties of reversible bonds give Schiff base hydrogels shear-thinning and self-healing properties, which can maximize the conformation to irregular wound surfaces and reduce irritation during drug administration. Therefore, injectable self-healing hydrogels based on Schiff bases have broad potential for biomedical applications.

[0004] Chitosan is the only known alkaline polysaccharide with a positive charge. It is obtained by removing the acetyl group of chitin in hot alkaline solution. Compared with other natural polysaccharides, chitosan has the advantages of good biocompatibility and low cytotoxicity, and has developed rapidly in the biomedical field. Its inherent reactive amino groups make it easy to realize Schiff base hydrogels based on chitosan, and at the same time, it is also conducive to further functional modification of it.

[0005] Nitric oxide (NO) can be used as a special therapeutic agent, playing an important role in eliminating tumors, killing bacteria, removing bacterial biofilms, and improving the immunosuppressive microenvironment. To this end, numerous NO delivery systems have been developed. Among them, light irradiation is a non-invasive means of regulating NO release, which has the advantages of time and space controllability and adjustable wavelength / intensity. Near-infrared (NIR) light has become one of the widely used light sources due to its effective deep tissue penetration and weak absorption of water and tissue fluid. Among them, photothermal therapy (PTT), which converts near-infrared light (NIR) energy into thermal energy through photothermal transducers (PTA), is a model of precision treatment for cancer, bacterial biofilm infection, and immunosuppression widely used in clinical and scientific research. At the same time, it is expected to achieve a controllable synergistic therapeutic effect of nitric oxide gas therapy and phototherapy.

[0006] Therefore, how to find an injectable self-healing multifunctional hydrogel with both photothermal effect and nitric oxide release function deserves further study. Summary of the invention

[0007] Purpose of the invention: The present invention provides an injectable self-healing multifunctional hydrogel having a photothermal effect and capable of inducing the release of nitric oxide (NO).

[0008] Another object of the present invention is to provide a preparation method and use of the hydrogel.

[0009] The multifunctional hydrogel material obtained by the present invention controls NO release responsively through the photothermal effect of the photosensitizer, and the photothermal therapy and NO gas therapy synergistically kill pathogenic drug-resistant bacteria, remove pathogenic drug-resistant bacterial biofilms, damage tumor cells, and promote the repolarization of M2 macrophages to M1. The present invention combines NO gas therapy with photothermal therapy, and is applied to break the interaction between tumor development, bacterial biofilm infection, and immunosuppression, thereby exerting a better therapeutic effect, and providing ideas for the study of the interaction between tumors, bacterial biofilms, and immunity.

[0010] Technical solution: The responsive controlled-release nitric oxide photothermal gel is prepared by a polycarbonate macromolecular nitric oxide donor (PNTC) and chitosan (CI) modified by a cyanine photosensitizer through a Schiff base cross-linking reaction.

[0011] The preparation method of the responsive controlled-release nitric oxide photothermal water gel,

[0012] (1) The preparation method of PNTC is as follows: a nitrate cyclocarbonate (NTC) monomer is subjected to ring-opening polymerization with an initiator to obtain a copolymer with a terminal hydroxyl group, and then the terminal hydroxyl group is aldehyde-formylated to obtain the copolymer;

[0013] (2) CI is prepared by reacting a cyanine photosensitizer with 6-aminocaproic acid (EACA) to obtain a carboxylated cyanine photosensitizer (I-COOH), which is then amidated with chitosan pretreated with dilute acid to obtain CI;

[0014] (3) Dissolving the obtained PNTC and CI separately to obtain two hydrogel precursor solutions, mixing the two precursor solutions uniformly, and allowing them to stand to form gel.

[0015] Further, the molecular weight of the NTC block is 1000Da-15000Da; the initiator block is selected from polyethylene glycol (HO-PEG-OH) or multi-arm polyethylene glycol (n-arm-PEG-OH) (n≥3) with a main chain non-functionalized or functionalized with a molecular weight of 1000Da-100000Da. The polyethylene glycol or multi-arm polyethylene glycol (n≥3) is one or more of a straight chain or a multi-chain, presenting one or more of a linear, star-shaped, tree-shaped or forked structure; the polyethylene glycol or multi-arm polyethylene glycol (n≥3) is one or more of a main chain non-functionalized, single or multi-functionalized.

[0016] Furthermore, the cyanine photosensitizer includes IR780, IR783, IR808, IR820 and their respective iodides; the molecular weight of the chitosan is 10kDa-1000kDa; and in the chitosan modified with the cyanine photosensitizer, the modification rate of the cyanine photosensitizer is 5%-50%.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0018] 1. An injectable self-healing multifunctional hydrogel with photothermal effect and NO-inducing ability was constructed, and on-demand NO-responsive photothermal release was achieved in a high GSH-reducing environment through light control.

[0019] 2. The biocompatible injectable self-healing Schiff base hydrogel prepared by the present invention combines photothermal therapy with gas therapy, avoiding the use of chemotherapy drugs and secondary debridement treatment, which is beneficial to avoid conventional drug resistance means of tumors and bacterial biofilms and achieves good therapeutic effects.

[0020] 3. The multifunctional hydrogel prepared by the present invention can play a "three-in-one" therapeutic role of clearing pathogenic resistant bacterial biofilms, killing tumor cells, and promoting the repolarization of M2 macrophages to M1 macrophages, and is expected to break the malignant promoting relationship among bacterial biofilms, tumors, and immunosuppression.

[0021] 4. The multifunctional hydrogel has a simple preparation method, good biocompatibility, excellent therapeutic effect, and has good research and development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : The H-NMR spectrum of the polycarbonate macromolecular nitric oxide donor (PNTC) in Example 1;

[0023] Figure 2 : UV spectrum of photosensitizer-modified chitosan (CI) in Example 2;

[0024] Figure 3 : Gel photo of hydrogel PNCI in Example 3;

[0025] Figure 4 : Characterization of the injectable self-healing properties of the hydrogel PNCI in Example 4;

[0026] Figure 5 : Temperature rise curves of the hydrogel PNCI under different light irradiation powers in Example 5;

[0027] Figure 6 : Experimental results of nitric oxide release in Example 6;

[0028] Figure 7 : In vitro antibacterial test results in Example 7;

[0029] Figure 8 : The results of the in vitro anti-biofilm experiment in Example 8;

[0030] Fig. 9 : The in vitro anti-tumor experimental results in Example 9;

[0031] Fig.10 : The experimental results of promoting repolarization of M1 macrophages in vivo in Example 10. DETAILED DESCRIPTION

[0032] Example 1: Preparation of polycarbonate macromolecular nitric oxide donor (PNTC)

[0033] The synthesis, synthesis route and process of PNTC are as follows:

[0034]

[0035] Cyclic carbonate monomer (NTC), four-arm polyethylene glycol (4-arm-PEG-OH) and polymerization catalyst are co-dissolved in anhydrous dichloromethane (DCM) and reacted under nitrogen protection. The terminator triethylamine (TEA) is added to terminate the reaction. The reaction solution is dripped into icy ether and vacuum dried to obtain a terminal hydroxyl polymer (4-arm-PEG-PNTC-OH). It is reacted with p-formylbenzoic acid (4-FABH) at room temperature. After the reaction is completed, icy ether is added dropwise and vacuum dried to obtain a polycarbonate macromolecular nitric oxide donor (PNTC).

[0036] Example 2: Preparation of photosensitizer-modified chitosan (CI):

[0037] The synthesis route and process of CI are as follows:

[0038]

[0039] IR-780 iodide and 6-aminocaproic acid (EACA) were dissolved in anhydrous N, N-dimethylformamide (DMF), and triethylamine (TEA) was slowly added dropwise. The mixture was protected from light and nitrogen and heated for reaction. After the reaction, the solvent was removed by vacuum distillation to obtain a crude product, which was purified by column chromatography to obtain the product carboxylated cyanine photosensitizer (I-COOH).

[0040] Chitosan was completely dissolved in a 3% acetic acid aqueous solution, dialyzed, and freeze-dried to obtain pretreated chitosan (CS), which was dissolved in deionized water.

[0041] Dissolve carboxylated cyanine photosensitizer (I-COOH), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) in N,N-dimethylformamide (DMF) and stir at room temperature. Transfer to the deionized water solution of pretreated chitosan (CS) and react at room temperature in the dark. After the reaction, dialyze and freeze-dry to obtain photosensitizer-modified chitosan (CI).

[0042] Example 3: Preparation of functional hydrogel with photothermal effect and capable of inducing NO release

[0043] Weigh two gel components and dissolve them in sterile PBS respectively to obtain two hydrogel precursor solutions.

[0044] The hydrogel precursor solutions of PNTC and CI were mixed at a volume ratio of 2:1 to obtain the functional hydrogel PNCI with photothermal effect and the ability to induce NO release. Figure 3 , The inverted gelation of PNTC and CI at a volume ratio of 2:1. Both hydrogel precursor solutions are in a flowing state. When mixed together, they gel immediately. When placed inverted, they do not flow and are in a gel state.

[0045] Example 4: Characterization of injectable self-healing properties of functional hydrogels with photothermal effect and NO release

[0046] The injectable self-healing behavior of PNCI hydrogel was evaluated using a rotational rheometer. The test sample was gelled in a mold, placed on the lower plate of the rheometer, and subjected to alternating cyclic strain tests of low shear strain (0.1%) and high shear strain (200%). Figure 4As shown in the figure, under low strain, the storage modulus (G') of PNCI hydrogel is greater than the loss modulus (G"), and under high strain, the loss modulus (G") of PNCI hydrogel is greater than the storage modulus (G'), indicating that the structure of the hydrogel is destroyed under high strain and a stable and rapid sol-gel transition occurs during the cycle, indicating that the hydrogel has excellent injectable self-healing properties.

[0047] Example 5: Photothermal effect of functional hydrogel with photothermal effect and inducing NO release

[0048] PNCI hydrogel was prepared in the mold according to the above ratio and process, and then irradiated with a 660nm near-infrared laser at different powers. The infrared thermal imager recorded the temperature changes in real time during the process to study the photothermal effect of the hydrogel. Figure 4 As shown, the prepared hydrogel has an adjustable photothermal effect and excellent photothermal conversion performance, with obvious and controllable temperature rise, and has the potential to meet different treatment needs.

[0049] Example 6: In vitro NO release experiment of functional hydrogel with photothermal effect and NO release induction

[0050] The amount of NO released by the hydrogel in vitro was determined by the Griess reagent detection method. The gel was prepared according to the corresponding conditions in Example 3 and Example 4, and the experiment was carried out under the conditions of simulating the high glutathione reduction environment (8mM GSH) of the bacterial biofilm in vitro. After each group of samples was processed, a certain amount of the leaching liquid was taken out according to the instructions and mixed with the Griess reagent, and the microplate reader was used to detect at UV = 540nm. The results are shown in FIG. Figure 5 As shown, the prepared hydrogel has the characteristics of GSH responsive release, and in the presence of photothermal effect, the release amount and release rate of NO are significantly increased, indicating that the functional hydrogel with photothermal effect and capable of inducing NO release disclosed in the present invention has excellent near-infrared radiation control and high GSH reduction environment response NO release performance, and has good practical application potential.

[0051] Example 7: In vitro antibacterial effect of functional hydrogels with photothermal effect and NO release induction

[0052] The gel in vitro antibacterial experiment was conducted using multiple-resistant Staphylococcus aureus (MRSA) as a model bacterium. The bacteria were cultured in Luria Bertani medium until the logarithmic growth phase and the density was adjusted to 10 7 CFU / ml. After each group was treated as required, the plate coating method was used to analyze the in vitro antibacterial effect of each group of hydrogels. Figure 7As shown, under in vitro conditions, photothermal therapy combined with NO gas therapy has a significant bacterial killing effect.

[0053] Example 8: In vitro anti-biofilm effect of functional hydrogels with photothermal effect and NO release induction

[0054] The anti-biofilm effect of the gel in vitro was evaluated using multiple-resistant Staphylococcus aureus (MRSA) as a model bacterium. The resistant Staphylococcus aureus was placed in Luria Bertani medium and cultured in a 96-well plate in a 37°C incubator according to the operating procedures to obtain bacterial biofilm. After treatment in each group, the in vitro anti-biofilm effect of each group of hydrogels was analyzed by crystal violet staining. The quantitative results are shown in Figure 8 As shown, it is shown that the functional hydrogel with photothermal effect and capable of inducing NO release disclosed in the present invention has obvious anti-bacterial biofilm effect. After treatment, the survival rate of mature biofilm of drug-resistant Staphylococcus aureus formed in vitro is only 28.4%.

[0055] Example 9: In vitro antitumor effect of functional hydrogel with photothermal effect and NO release induction

[0056] B16-F10 melanoma tumor cells were used as model cells to evaluate the in vitro anti-tumor effect of the gel. B16-F10 melanoma tumor cells were placed in 1640 culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin dual antibody solution and cultured in a 48-well plate at 37°C and 5% CO2 incubator according to the operating procedures. After hydrogel treatment, the in vitro anti-tumor effect of each group of hydrogels was analyzed by the MTT method. The quantitative results are shown in Fig. 9 As shown, the functional hydrogel with photothermal effect and capable of inducing NO release disclosed in the present invention has obvious anti-tumor effect, and the cell survival rate is only 8.7%, showing excellent tumor cell killing ability.

[0057] Example 10: In vivo macrophage repolarization effect of functional hydrogels with photothermal effect and NO release induction

[0058] To study the repolarization effect of hydrogel on macrophages in mice, 5×10 6 The cell suspension (100ul) of B16-F10 cells was implanted subcutaneously on the back of C57BL / 6 (female, 6-7 weeks old). The tumor size was about 150mm 3 The mice were randomly divided into 5 groups, and about 99% of the tumor was removed, leaving 1% to simulate the micro-residue of the tumor after surgery. Then 50ul of MRSA liquid (10 8CFU / ml) were dripped onto the surgical bed and randomly divided into five groups after complete absorption. After the bacterial biofilm matured, the corresponding treatments were given. After three days, the whole tissue of the affected area was removed and tested by flow cytometry. Fig.10 As shown, compared with the control group, the number of M1 macrophages was significantly increased under the synergistic effect of NO gas therapy and photothermal therapy.

Claims

1. A responsive controlled-release nitric oxide photothermal gel, characterized in that: It is prepared by the cross-linking reaction of chitosan modified by polycarbonate macromolecular nitric oxide donor and cyanine photosensitizer. Among them, the polycarbonate macromolecular nitric oxide donor is obtained by ring-opening polymerization of nitrate cyclocarbonate monomers with initiators to obtain a copolymer with a terminal hydroxyl group, and then the terminal hydroxyl group is aldehyde-formylated, and the initiator is polyethylene glycol or multi-arm polyethylene glycol with three arms or more; The chitosan modified with cyanine photosensitizer is obtained by reacting cyanine photosensitizer with 6-aminocaproic acid to obtain carboxylated cyanine photosensitizer, which is then subjected to amidation reaction with chitosan pretreated with dilute acid.

2. The method for preparing the responsive controlled-release nitric oxide photothermal gel according to claim 1, characterized in that: The steps include: (1) dissolving a polycarbonate macromolecular nitric oxide donor and chitosan modified with a cyanine photosensitizer respectively to obtain two hydrogel precursor solutions; (2) The two solutions in the previous step are mixed thoroughly and allowed to stand to form a gel.

3. The responsive controlled-release nitric oxide photothermal gel according to claim 1, characterized in that: In the polycarbonate macromolecular nitric oxide donor, the molecular weight of the nitrate cyclocarbonate block is 1000Da-15000Da, and the initiator block is selected from a main chain non-functionalized or functionalized polyethylene glycol or a multi-arm polyethylene glycol with three arms or more and a molecular weight of 1000Da-100000Da.

4. The method for preparing the responsive controlled-release nitric oxide photothermal gel according to claim 2, characterized in that: The cyanine photosensitizers include IR780, IR783, IR808, IR820 and their respective iodides; the molecular weight of the chitosan is 10kDa-1000kDa; in the chitosan modified with the cyanine photosensitizers, the modification rate of the cyanine photosensitizers is 5%-50%.

5. The method for preparing the responsive controlled-release nitric oxide photothermal gel according to claim 2, characterized in that: The mass concentrations of the polycarbonate macromolecular nitric oxide donor and the chitosan modified by the cyanine photosensitizer in the two hydrogel precursor solutions are both 1%-25%.

6. The method for preparing the responsive controlled-release nitric oxide photothermal gel according to claim 2, characterized in that: The volume ratio of the polycarbonate macromolecular nitric oxide donor hydrogel precursor solution to the cyanine photosensitizer-modified chitosan hydrogel precursor solution is 1:1-7:

1.

7. Use of the responsive controlled-release nitric oxide photothermal gel according to claim 1 in the preparation of drugs for removing bacterial biofilms, drugs for treating tumors, and drugs for promoting the repolarization of M2 macrophages to M1.

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