Nanocomposite for cancer photothermal therapy and preparation method and application thereof

Abstract

The invention belongs to the technical field of nano-composites, and particularly relates to a nano-composite for cancer photothermal therapy and a preparation method and application thereof. The nano-polymer is composed of hollow mesoporous copper sulfide nano-particles located on the inner layer and a GSH responsive polymer located on the outer layer, can achieve a broad-spectrum and targeted killing effect on tumors by means of the photo-thermal regulation effect and the GSH consumption effect, and has the advantages of being small in side effect, high in targeting performance and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanocomposites, and in particular relates to a nanocomposite for cancer photothermal therapy, a preparation method and an application thereof. Background Art

[0002] Cancer is one of the major diseases that threaten human life. According to the World Health Organization, cancer causes more than 9 million deaths each year, and its incidence and mortality rates continue to rise. The occurrence of cancer is closely related to multiple factors such as cell gene mutations, immune escape, and changes in the tumor microenvironment. It is highly heterogeneous and complex, and different types of cancer have different biological characteristics and clinical manifestations. Traditional cancer treatments, such as surgical resection, radiotherapy, and chemotherapy, although they have made significant breakthroughs in the past few decades, are often accompanied by some side effects, such as normal tissue damage and the development of drug resistance. In addition, the heterogeneity and drug resistance mechanisms of tumor cells lead to uncertainty in treatment effects, which seriously limits the application of traditional treatments in cancer treatment. Therefore, there is an urgent need to develop new cancer treatments that are more precise, efficient, and have fewer side effects.

[0003] In recent years, with the advancement of nanotechnology, the application of nanocarriers in cancer therapy has attracted widespread attention. Compared with traditional drug formulations, nanocarriers offer advantages such as controllability and reduced toxic side effects. New nanocarriers, created by encapsulating specific drugs within nanocarriers or chemically coupling them to polymer backbones and nanoparticle surfaces, not only prolong drug circulation in the body but also promote their accumulation at tumor sites through enhanced permeability and retention, enabling passive targeting. Furthermore, active targeting can be effectively achieved by conjugating nanocarriers with ligands that specifically target receptors overexpressed on tumor cells. Furthermore, nanomaterials can respond to external stimuli (such as magnetic fields, temperature, light, and ultrasound) or tumor microenvironmental characteristics (such as weakly acidic pH, high glutathione (GSH) concentrations, and hypoxia), leading to controlled release of drugs in a spatiotemporally specific manner. Near-infrared (NIR) light-triggered photothermal therapy, in particular, exhibits promising application prospects by activating nanocarriers to generate localized hyperthermia, effectively killing tumor cells.

[0004] CN114873629A provides a hollow mesoporous copper sulfide nanomedicine loaded with adriamycin, which has good liver cancer targeting, but this targeting limits the scope of use of nanomedicines, that is, reduces the applicability to other cancers. CN110898221A provides a hollow mesoporous copper sulfide prepared using hyaluronic acid, copper salts and sulfides, which can load adriamycin and other drugs, and has a higher photothermal conversion efficiency. However, it still utilizes the photothermal effect of hollow mesoporous copper sulfide and the cytotoxicity of chemotherapy drugs, that is, it is limited to the anti-tumor spectrum of chemotherapy drugs themselves. And how to improve the broad spectrum applicability of hollow mesoporous copper sulfide nanomedicines while improving the anti-tumor effect of its photothermal therapy, there is still a lack of mature technology. Summary of the Invention

[0005] In response to the above technical problems, the purpose of the present invention is to provide a dual-effect nanocomposite with photothermal effect and GSH (glutathione) consumption. This dual-response nanocomposite can not only achieve efficient local photothermal conversion by triggering near-infrared light, thereby utilizing the photothermal effect to kill tumor cells locally, but can also respond to the high concentration of GSH (glutathione) in the tumor microenvironment and consume the high concentration of GSH in the tumor microenvironment, thereby enhancing the therapeutic effect and reducing damage to normal tissues. As a result, this nanocomposite has the potential to be precise, effective, and have fewer side effects as a new cancer treatment strategy. The specific scheme of the present invention is as follows:

[0006] A nanocomposite for cancer photothermal therapy, the nanocomposite comprising hollow mesoporous copper sulfide nanoparticles located in an inner layer and a GSH-responsive polymer located in an outer layer. The structure of the GSH-responsive polymer is shown in Formula I, wherein x is a natural number of 40 to 200, m is a natural number of 2 to 20, and n is a natural number of 2 to 20:

[0007]

[0008] Preferably, in the structure of the GSH-responsive polymer, x is 114, m is 6, and n is 6.

[0009] The method for preparing the aforementioned nanocomposite comprises the following steps:

[0010] S1: Preparation of hollow mesoporous copper sulfide nanoparticles;

[0011] S2: Preparation of GSH-responsive polymers:

[0012] S21: Synthesis of polymer monomer: 4-hydroxybenzyl alcohol and p-nitrophenyl chloroformate are reacted to prepare intermediate A, intermediate A is reacted with acryloyl chloride to prepare intermediate B, and intermediate B is reacted with cystamine dihydrochloride to prepare polymer monomer C;

[0013] S22: Synthesis of a GSH-responsive polymer: preparing an intermediate D using a polymer monomer compound C, 1,3-di(4-piperidinyl)propane, and a polyethylene glycol derivative having an amino group, and reacting the intermediate D with 1,3-diaminopropane to prepare the GSH-responsive polymer;

[0014] S3: Preparation of nanocomposites: dissolving hollow mesoporous copper sulfide nanoparticles in deionized water, adding polyacrylic acid, stirring the reaction, centrifuging, washing with water, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stirring the reaction, adding a dimethyl sulfoxide solution of a GSH-responsive polymer, stirring the reaction, washing with water, and centrifuging to obtain the nanocomposite.

[0015] Preferably, the method for preparing the hollow mesoporous copper sulfide nanoparticles comprises the following steps:

[0016] A mixed solution containing PVP-K30 and CuCl2 was prepared, and N2H4·H2O solution and NaOH solution were added to the mixed solution to prepare Cu2O nanospheres. After the reaction, Na2S solution was added to react in an oil bath to prepare hollow mesoporous copper sulfide nanoparticles.

[0017] Preferably, in step S21, the molar ratio of 4-hydroxybenzyl alcohol to p-nitrophenyl chloroformate is (0.5-2):1.

[0018] Preferably, in step S21, the molar ratio of intermediate A to acryloyl chloride is (0.5-2):1.

[0019] Preferably, in step S21, the molar ratio of intermediate B to cystamine dihydrochloride is (0.5-3):1.

[0020] Preferably, in step S22, the molar ratio of the polymer monomer compound C to 1,3-di(4-piperidinyl)propane and NH2-polyethylene glycol is (10-20):10:1.

[0021] Preferably, in the step S22, the feeding amount of 1,3-diaminopropane is calculated as (0.2-1):1 based on the molar ratio of polymer monomer compound C:1,3-diaminopropane.

[0022] Preferably, the polyethylene glycol derivative with an amino group in step S22 is a methoxy polyethylene glycol amino derivative. Further preferably, the methoxy polyethylene glycol amino derivative is mPEG5000-NH2.

[0023] The present invention further provides use of the aforementioned nanocomposite in preparing a nanocomposite for cancer photothermal therapy.

[0024] Beneficial effects:

[0025] (1) The nanocomplex of the present invention has the dual tumor inhibitory effects of local photothermal conversion and consumption of excessive GSH in the tumor microenvironment, without the use of cytotoxic anti-tumor drugs, and has the multiple advantages of targeting and low side effects;

[0026] (2) In the nanocomplex of the present invention, the GSH-responsive polymer in the outer layer has a good effect of consuming GSH in the tumor microenvironment, and when combined with hollow mesoporous copper sulfide nanoparticles, it unexpectedly significantly improves the anti-tumor effect of photothermal therapy.

[0027] (3) The nanocomposite of the present invention exhibits excellent anti-tumor effects even under conventional near-infrared laser (808 nm laser) irradiation. Although the nanocomposite preparation method of the present invention is simple and does not employ complex techniques such as coupled receptor targeting molecules, it still overcomes the low photothermal conversion efficiency of conventional hollow mesoporous copper sulfide nanoparticles and achieves excellent anti-tumor effects without side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the synthetic route of polymer monomer C in the preparation of the nanocomposite of the present invention;

[0029] Figure 2 For polymer monomer C 1 H NMR spectrum;

[0030] Figure 3 The present invention provides a synthetic route for the GSH-responsive polymer used in the preparation of the nanocomposite.

[0031] Figure 4 The GSH-responsive polymer of the present invention is 1 H NMR spectrum;

[0032] Figure 5 The particle size characterization results of the hollow mesoporous copper sulfide nanoparticles prepared in the nanocomposite of the present invention;

[0033] Figure 6 is a transmission electron microscope photograph of the nanocomposite of the present invention;

[0034] Figure 7 is the particle size characterization result of the nanocomposite of the present invention;

[0035] Figure 8 is the ultraviolet absorption spectrum of the nanocomposite and hollow mesoporous copper sulfide of the present invention;

[0036] Figure 9 is the photothermal curve of the nanocomposite of the present invention;

[0037] Figure 10 The results of the tumor volume changes in mice treated with the nanocomplex of the present invention are as follows;

[0038] Figure 11 The weight change results of mice treated with tumors treated with the nanocomplex of the present invention;

[0039] Figure 12 The results of the investigation on the GSH consumption capacity of different polymers are shown in Figure 2. DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments are only used to explain the present invention and are not used to limit the scope of protection of the present invention.

[0041] The abbreviations and full names in Chinese and English used in the following examples are shown in Table 1.

[0042] Table 1 Abbreviations

[0043]

[0044]

[0045] Example 1 Preparation and characterization of nanocomposites

[0046] Synthesis of polymer monomers: see Figure 1 The synthetic route is as follows.

[0047] 4-Hydroxybenzyl alcohol (8.06 mmol) and triethylamine (8.06 mmol) were dissolved in anhydrous tetrahydrofuran (20 mL). Under nitrogen, a solution of p-nitrochlorophenylformate in tetrahydrofuran (8.87 mmol, 10 mL) was slowly added. After reacting in an ice bath for 3 h, 150 mL of ethyl acetate was added to the mixture. The organic phase was washed with water and saturated brine, dried over anhydrous magnesium sulfate, concentrated in vacuo, and purified by silica gel column chromatography (dichloromethane:ethyl acetate = 20:1) to obtain Intermediate A.

[0048] Intermediate A (3.46 mmol) was dissolved in a mixture of 20 mL of tetrahydrofuran and 10 mL of dichloromethane. The mixture was placed on ice and, under nitrogen, triethylamine (6.92 mmol) and a dichloromethane solution of acryloyl chloride (3.46 mmol, 10 mL) were added. After 1 h of reaction, the mixture was concentrated in vacuo, and 100 mL of dichloromethane was added. The organic phase was washed with water and saturated brine, dried over anhydrous magnesium sulfate, concentrated in vacuo, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 6:1) to obtain Intermediate B.

[0049] Finally, intermediate B (2.91 mmol) was dissolved in tetrahydrofuran solution (20 mL), and triethylamine (5.82 mmol) and cystamine dihydrochloride (1.46 mmol, 10 mL) in dimethyl sulfoxide solution were added respectively. The mixture was reacted at room temperature for 30 min. 150 mL of ethyl acetate was added to the mixture. The organic phase was washed with water and saturated brine respectively and dried over anhydrous magnesium sulfate. After concentration in vacuo, it was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain polymer monomer C (yield 58.71%). The NMR data of polymer monomer C are as follows (NMR spectrum see Figure 2 ): 1 H-NMR (400MHz, CDCl3): δ7.36(4H), 7.17-7.08(4H), 6.44(2H), 6.15(2H), 5.85(2H), 5.17(4H), 3.61(4H), 2.91(4H).

[0050] (2) Synthesis of GSH-responsive polymers: see Figure 3 The synthetic route is as follows.

[0051] Polymer monomer C (1.2 mmol), 1,3-di(4-piperidinyl)propane (1.0 mmol), and mPEG5000-NH2 (molecular weight ~5000 Da, 0.1 mmol, commercially available product, trade name PS1-N-5K) were dissolved in 3 mL of chloroform. The mixture was stirred at 60°C under nitrogen for 48 hours to prepare intermediate D. 1,3-Diaminopropane (2.2 mmol) was then added to the reaction system and stirred at 60°C for 2 hours. The reaction was then terminated by cooling to room temperature. The product was precipitated in anhydrous ether and collected by centrifugation (2000 rpm, 5 minutes) to obtain a GSH-responsive polymer (yield 64.57%).

[0052] The NMR data of the GSH-responsive polymer are as follows (NMR spectrum see Figure 4 ): 1 H NMR(400MHz,DMSO-d6):1H NMR(400MHz,DMSO-d6)δ7.18-6.93(25H),6.68(27H),4.04-3.60(68H),3.3 9(456H),2.90(14H),2.75(52H),2.24(43H),1.80(11H),1.67-1.15(106H).

[0053] In the structure of this polymer, x is 114, m is 6, and n is 6.

[0054] (3) Preparation of HMCuS: PVP-K30 (polyvinyl pyrrolidone K30, 1.2 g) and CuCl2 solution (0.5 mol·L -1 , 800 μL) was added to 75 mL of deionized water and stirred at room temperature for 5 min. Then, NaOH solution (0.01 mol·L -1 , 75 mL) and N2H4·H2O (40 μL) to form Cu2O nanospheres. After reacting at room temperature for 5 min, Na2S solution (320 mg·mL -1 , 1 mL) and reacted in an oil bath at 80°C for 2 h. After the reaction, the mixture was washed three times with deionized water and anhydrous ethanol, respectively, and HMCuS was obtained by centrifugation (11000 rpm, 10 min).

[0055] The particle size characterization results of the HMCuS are as follows Figure 5 As shown, the average particle size is about 120 nm.

[0056] (4) Preparation of nanocomposites: HMCuS (10 mg) was dissolved in 10 mL of deionized water, and polyacrylic acid (PAA) (100 mg, calculated by adding amount, the concentration of PAA added to the reaction solution was 10 mg mL -1 ) was stirred at room temperature for 12 h, the mixed solution was centrifuged, the solid was washed three times with water, 10 ml of water was added, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) (10 mg) and N-hydroxysuccinimide (NHS) (7 mg) were added to the mixture and stirred at room temperature for 30 min. Then, the GSH-responsive polymer prepared in the above step (2) (50 mg, calculated by the amount added, the concentration of the GSH-responsive polymer in the reaction solution after addition was 25 mg mL) was added to the above solution. -1 ) in dimethyl sulfoxide solution and continued stirring for 24 h. After the reaction, it was washed three times with deionized water and centrifuged (11000 rpm, 10 min) to obtain a nanocomposite.

[0057] The transmission electron microscope photos, particle size characterization results, UV absorption spectrum and photothermal curve of the nanocomposite are shown in Figure 6 、 7 , 8, 9. Figure 7 As shown in FIG, the average particle size of the nanocomposite is about 195 nm; Figure 8 As shown, the nanocomposite exhibits strong near-infrared absorption at 800-1000 nm due to the presence of HMCuS, indicating that it has a large photothermal potential.

[0058] Figure 9 The conditions and methods for the photothermal curve determination are as follows: different concentrations of nanocomposite solutions (0-200 μg mL -1) at 808nm(1.0W / cm 2 ) laser irradiation for 600s, and the temperature was recorded in real time by an infrared thermal imager (FOTRIC323Pro+). The results showed that the photothermal effect of the nanocomposite was dose-dependent and enhanced with increasing concentration and irradiation time. After 10 minutes of irradiation, the temperature increased steadily. -1 The nanocomposite solution was irradiated under near-infrared light for 10 minutes, and the temperature could reach above 45°C, which is sufficient for photothermal therapy.

[0059] Example 2 Investigation of the Effect of Photothermal Therapy on Nanocomposites

[0060] According to the method of Example 1, HMCuS, GSH responsive polymer (hereinafter referred to as polymer) and nanocomplex were prepared respectively for in vivo effect investigation.

[0061] PBS suspension of CT26 colon cancer cells (cell density: 2×10 7 cells / mL) were injected into the right hind leg of six-week-old female Balb / c nude mice (18-20 g) subcutaneously, with 100 μL injected into each nude mouse. 3 The mice were divided into six groups and given the following treatments: saline, saline + laser, HMCuS, HMCuS + laser, polymer, and nanocomposite + laser. 200 μL (2.5 mg / kg for all groups except saline) was injected into the tail vein every two days for 14 days (i.e., 7 injections per animal). 8 hours after each injection, the tumor site of the mouse was irradiated with 808 nm laser (10 min, 1 W / cm 2 The tumor volume and body weight of mice were recorded every day.

[0062] The formula for calculating tumor volume is:

[0063] The results are as follows Figure 10 、 11 As shown by Figure 10 It can be seen that the tumor volume of the nanocomposite + laser group was significantly lower than that of the other groups, including the HMCuS + laser group. From the 4th to the 14th day, the difference between the tumor volume of the nanocomposite + laser group and the tumor volume of the saline + laser group was also significantly greater than the sum of the difference between the tumor volume of the polymer group and the saline + laser group, and the difference between the tumor volume of the HMCuS + laser group and the saline + laser group. This shows that the HMCuS and polymer in the nanocomposite have an unexpected synergistic effect in photothermal therapy. Figure 11 It can be seen that there was no significant difference in body weight between the nanocomplex + laser group and the normal saline group, or between the normal saline + laser group, indicating that the nanocomplex has good safety.

[0064] Example 3 Results of GSH consumption capacity study of different polymers

[0065] A GSH-responsive polymer was prepared according to the method of Example 1. PEG-SS-PLGA was prepared according to the literature (Zhang Z, Cheng W, Pan Y, Jia L. An anticancer agent-loaded PLGA nanomedicine with glutathione-response and targeted delivery for the treatment of lung cancer. J Mater Chem B. 2020; 8(4): 655-665.) and the GSH consumption capacity was tested.

[0066] 0.5 mL of GSH-responsive polymer and PEG-SS-PLGA solutions at different concentrations (0, 0.02 mM, 0.04 mM, 0.08 mM, 0.12 mM, 0.16 mM, and 0.2 mM) were added to 0.5 mL of GSH solution (0.2 mM) and reacted at 37°C for 4 h. After the reaction, 0.5 mL of the mixed solution was added to 2.5 mL of DNTB analysis solution (0.1 mM) and reacted in the dark for 10 min. The absorbance was immediately measured at 412 nm.

[0067] The calculation formula of GSH is: y = 2.5916x - 0.0029R 2 =0.999, where x is the absorbance and y is the GSH concentration.

[0068] See the results Figure 12 After reacting PEG-SS-PLGA and the GSH-responsive polymer with GSH at 37°C for 4 hours, their in vitro GSH-depletion abilities were determined. Compared to PEG-SS-PLGA, the GSH-responsive polymers of the present invention exhibited significant GSH-depletion effects, depleting GSH in a dose-dependent manner. These results confirm that the GSH-responsive polymers possess superior GSH-depletion abilities.

Claims

1. A nanocomposite for cancer photothermal therapy, characterized in that The nanocomposite is composed of hollow mesoporous copper sulfide nanoparticles located in the inner layer and a GSH-responsive polymer located in the outer layer. The structure of the GSH-responsive polymer is shown in Formula I, wherein x is a natural number of 40 to 200, m is a natural number of 2 to 20, and n is a natural number of 2 to 20:

2. The nanocomposite for cancer photothermal therapy according to claim 1, characterized in that: In the structure of the GSH-responsive polymer of formula I, x is 114, m is 6, and n is 6.

3. The method for preparing the nanocomposite for cancer photothermal therapy according to claim 1, characterized in that: The process includes the following steps: S1: Preparation of hollow mesoporous copper sulfide nanoparticles; S2: Preparation of GSH-responsive polymers: S21: Synthesis of polymer monomer: 4-hydroxybenzyl alcohol and p-nitrophenyl chloroformate are reacted to prepare intermediate A, intermediate A is reacted with acryloyl chloride to prepare intermediate B, and intermediate B is reacted with cystamine dihydrochloride to prepare polymer monomer C; S22: Synthesis of a GSH-responsive polymer: preparing an intermediate D using a polymer monomer compound C, 1,3-di(4-piperidinyl)propane, and a polyethylene glycol derivative having an amino group, and reacting the intermediate D with 1,3-diaminopropane to prepare the GSH-responsive polymer; S3: Preparation of nanocomposites: dissolving hollow mesoporous copper sulfide nanoparticles in deionized water, adding polyacrylic acid, stirring the reaction, centrifuging, washing with water, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stirring the reaction, adding a dimethyl sulfoxide solution of a GSH-responsive polymer, stirring the reaction, washing with water, and centrifuging to obtain the nanocomposite.

4. The method for preparing a nanocomposite for cancer photothermal therapy according to claim 3, characterized in that: The preparation method of the hollow mesoporous copper sulfide nanoparticles comprises the following steps: A mixed solution containing PVP-K30 and CuCl2 was prepared, and N2H4·H2O solution and NaOH solution were added to the mixed solution to prepare Cu2O nanospheres. After the reaction, Na2S solution was added to react in an oil bath to prepare hollow mesoporous copper sulfide nanoparticles.

5. The method for preparing a nanocomposite for cancer photothermal therapy according to claim 3, characterized in that: In the step S21, the molar ratio of 4-hydroxybenzyl alcohol to p-nitrophenyl chloroformate is (0.5-2):1; in the step S21, the molar ratio of intermediate A to acryloyl chloride is (0.5-2):1; in the step S21, the molar ratio of intermediate B to cystamine dihydrochloride is (0.5-3):

1.

6. The method for preparing a nanocomposite for cancer photothermal therapy according to claim 3, characterized in that: In the step S22, the molar ratio of the polymer monomer compound C, 1,3-di(4-piperidinyl)propane and NH2-polyethylene glycol is (10-20):10:

1.

7. The method for preparing a nanocomposite for cancer photothermal therapy according to claim 3, characterized in that: In the step S22, the feeding amount of 1,3-diaminopropane is calculated as (0.2-1):1 based on the molar ratio of polymer monomer compound C:1,3-diaminopropane.

8. The method for preparing a nanocomposite for cancer photothermal therapy according to claim 3, characterized in that: The polyethylene glycol derivative with an amino group in step S22 is a methoxy polyethylene glycol amino derivative.

9. The method for preparing a nanocomposite for cancer photothermal therapy according to claim 8, characterized in that: The methoxy polyethylene glycol amino derivative is mPEG5000-NH2.

10. Use of the nanocomposite for cancer photothermal therapy according to claim 1 or the nanocomposite for cancer photothermal therapy obtained by the preparation method according to any one of claims 3 to 9 in preparing a nanocomposite for cancer photothermal therapy.

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