A NIR-II light-driven multifunctional nanozyme for melanoma treatment and its preparation method

Abstract

The present invention discloses a NIR-II light-driven multifunctional nanozyme for melanoma treatment and a preparation method thereof, belonging to the field of nanobiomedical material preparation technology. The method comprises the following steps: accurately weighing pure elemental substances platinum and selenium in proportion, inserting them into a quartz tube, and heating them to 700-800°C for reaction to obtain a compound; naturally cooling the compound to ambient temperature, then breaking the quartz tube to collect Pt-Se powder; grinding the Pt-Se powder into a fine powder, and then ultrasonically treating it in a chitosan solvent. After ultrasonication, the mixture is centrifuged, the supernatant is removed after centrifugation, the excess chitosan solvent is removed, and the nanozyme is obtained from the supernatant, which is denoted as PS@CS. Under laser irradiation, the prepared nanozyme PS@CS can effectively convert H2O2 into ROS, thereby improving anti-tumor efficiency and providing a novel and comprehensive treatment strategy for melanoma.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-biomedical material preparation, and specifically relates to a NIR-II light-driven multifunctional nanozyme for melanoma treatment and a preparation method thereof. Background Art

[0002] The rapid growth of tumors often outstrips the development of adequate vascular networks, leading to hypoxia, which poses a significant challenge to traditional therapeutic approaches. In these hypoxic microenvironments, tumor cells exhibit elevated levels of H2O2, providing opportunities for innovative therapeutic interventions. Exploiting this unique phenomenon, researchers have begun exploring the potential of nanozymes to catalyze the conversion of H2O2 into O2, superoxide radicals (·O2 - ) and hydroxyl radicals (·OH), thereby resolving oxygen deficiency and enhancing cytotoxicity against cancer cells. The appeal of nanozymes lies not only in their ability to mimic the catalytic capabilities of natural enzymes, but also in their rational design and multifunctional integration. Incorporating photothermal properties into these nanozymes further expands their therapeutic scope, enabling precise and localized cancer ablation by converting light energy into heat through photothermal therapy (PTT).

[0003] Melanoma is the most common type of skin cancer and is primarily treated surgically. However, complete removal of tumor cells is challenging, and surgical wounds are prone to infection, complicating treatment and increasing costs. In this study, we developed platinum- and selenium-based nanozymes, Pt-Se@Chitosan (PS@CS), which possess multienzymatic activity and strong photothermal conversion efficiency, converting H2O2 into reactive oxygen species, thereby inducing cell death. In this study, we found that PS@CS exhibited excellent antitumor effects both in vitro and in vivo, providing an innovative combined therapeutic approach for melanoma treatment. Summary of the Invention

[0004] To address the above problems, the present invention provides a NIR-II light-driven multifunctional nanozyme for melanoma treatment and a preparation method thereof. The prepared nanozyme has good biocompatibility and multiple enzyme mimetic activities, including POD, CAT and OXD, as well as excellent photothermal conversion capabilities. Under laser irradiation, PS@CS can effectively convert H2O2 into ROS, thereby improving the anti-tumor efficiency, providing a novel and comprehensive therapeutic strategy for the treatment of melanoma.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for preparing a NIR-II light-driven multifunctional nanozyme for melanoma treatment comprises the following steps:

[0007] S1: Pure elemental substances platinum and selenium are accurately weighed in proportion, then inserted into a quartz tube and heated to 700-800°C for reaction for 80-120 hours to obtain a compound;

[0008] S2: The compound obtained after the complete reaction in step S1 is naturally cooled to ambient temperature, and then the quartz tube is broken to collect the Pt-Se powder;

[0009] S3: The Pt-Se powder obtained in step S2 was ground into fine powder, and then placed in a chitosan solvent for ultrasonic treatment. After ultrasonication, the mixture was centrifuged at 8000-12000 rpm for 30-60 min. The supernatant was removed after centrifugation, and the excess chitosan solvent was removed. The mixture was centrifuged at 1500-3000 rpm for 30-60 min to obtain nanozyme from the supernatant, which was recorded as PS@CS.

[0010] In a preferred embodiment, the molar ratio of pure elemental substances platinum and selenium in step S1 is 1:1-3.

[0011] In a preferred embodiment, the pressure in the quartz tube in step S1 is reduced to below 0.001 Pa.

[0012] In a preferred embodiment, the heating rate in step S1 is 5-8°C / min.

[0013] In a preferred embodiment, the concentration of the chitosan solvent in step S3 is 0.8-1 mg / mL, and the solid-liquid ratio of the fine powder to the chitosan solvent is 5-20 mg:1 mL.

[0014] In a preferred embodiment, the ultrasonic treatment time in step S3 is 18-48 hours.

[0015] NIR-II light-driven multifunctional nanozyme prepared by a method.

[0016] In a preferred embodiment, the thickness of the nanozyme is 16-18 nm, the particle size is 80-120 nm, and its structure does not contain Pt-Pt metal bonds but contains Pt-Se bonds, so it is a platinum metal single-atom nanozyme.

[0017] A NIR-II light-driven multifunctional nanozyme has anti-tumor applications under laser irradiation.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention adopts chemical vapor transport to prepare Pt-Se powder, and obtains the ultrathin structure of PS@CS nanozyme by ultrasonic treatment. The research shows that the Pt-Pt bond does not exist inside the nanozyme structure, thereby verifying the existence of atomically dispersed Pt in PS@CS nanozyme;

[0019] (2) The nanozyme prepared by the present invention can be regarded as a photothermal agent, which concentrates light energy into heat energy. Under near-infrared II (NIR-II) light irradiation, its photothermal conversion efficiency is high and its photothermal stability is good. Under laser irradiation, the enzyme-mimicking catalytic activity of PS@CS is improved and enhanced, and it has the catalytic activity of multiple enzyme-mimicking enzymes.

[0020] (3) The nanozyme prepared by the present invention has good biocompatibility, revealing the safety of the nanozyme. Moreover, under laser irradiation, PS@CS has significant potential for effective tumor treatment at the cellular and animal levels without causing adverse reactions to major organs.

[0021] (4) The PS@CS nanozyme prepared in the present invention has good biocompatibility and possesses multiple enzyme mimetic activities, including POD, CAT, and OXD, as well as excellent photothermal conversion ability. Under laser irradiation, PS@CS can effectively convert H2O2 into ROS, thereby improving the anti-tumor efficiency and providing a novel and comprehensive therapeutic strategy for the treatment of melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the STEM image of PS@CS nanozyme, where Figure 1 A is a STEM image with a magnified size of 100 nm. Figure 1 B is a STEM image with a magnified size of 50 nm;

[0023] Figure 2 STEM-DF image and corresponding elemental mapping of PS@CS nanozyme;

[0024] Figure 3 AFM image of PS@CS nanozyme;

[0025] Figure 4 The EXAFS spectrum and fitting results of PS@CS nanozyme are shown in Figure 2. Figure 4 A is the EXAFS spectrum and fitting result of PS@CS nanozyme in R space, Figure 4 B is the EXAFS spectrum and fitting results of PS@CS nanozyme in k-space;

[0026] Figure 5 is the EXAFS wavelet transform diagram of PS@CS nanozyme, where Figure 5 A is the wavelet transform diagram of PS@CS, Figure 5 B is the wavelet transform image of platinum foil;

[0027] Figure 6 is the absorbance curve of PS@CS aqueous solution at different concentrations;

[0028] Figure 7is the temperature curve of PS@CS aqueous solution at different concentrations;

[0029] Figure 8 The temperature curve of PS@CS aqueous solution under different laser power densities;

[0030] Figure 9 The temperature curve of PS@CS aqueous solution under 1064 nm laser irradiation for three on / off cycles;

[0031] Figure 10 This is the photothermal conversion efficiency diagram of PS@CS aqueous solution;

[0032] Figure 11 ESR spectra of PS@CS nanozyme confirming H2O2 consumption under laser irradiation;

[0033] Figure 12 This is the ESR spectrum of PS@CS nanozyme in POD enzyme activity;

[0034] Figure 13 ESR spectrum of PS@CS nanozyme in OXD enzyme activity;

[0035] Figure 14 The hemolysis rate of PS@CS nanozymes at different concentrations;

[0036] Figure 15 The morphology of red blood cells incubated with different concentrations of PS@CS;

[0037] Figure 16 The bar graph shows the survival rate of B16F10 cells after treatment with different concentrations of PS@CS under laser irradiation or without irradiation;

[0038] Figure 17 Representative images of live / dead cells of B16F10 cells after PS@CS+laser treatment and curves of the control group;

[0039] Figure 18 Figure 2 shows ROS detection of B16F10 cells in the control group and PS@CS+laser treatment group;

[0040] Figure 19 For light irradiation (1064nm, 1.0W / cm 2 In situ thermal imaging of tumor-bearing mice after ).

[0041] Figure 20 These are photos of different tumors under the three treatment groups;

[0042] Figure 21 The changes in tumor volume of mice in the three groups at different days of treatment;

[0043] Figure 22The weight changes of mice in the three groups on different days under treatment;

[0044] Figure 23 H&E tumor sections of the three treatment groups;

[0045] Figure 24 Figure 2 is the TUNEL and Ki67 staining analysis of tumors in the three treatment groups;

[0046] Figure 25 These are H&E staining pathological analysis images of the main organs (heart, liver, spleen, lung, and kidney) of mice in the three treatment groups. DETAILED DESCRIPTION

[0047] In order to enable those skilled in the art to better understand the technical solution, the present invention is described in detail below in conjunction with embodiments. The description in this section is only exemplary and explanatory and should not have any limiting effect on the scope of protection of the present invention.

[0048] Example 1:

[0049] A method for preparing a NIR-II light-driven multifunctional nanozyme for melanoma treatment comprises the following steps:

[0050] S1: Pure elemental substances platinum and selenium were accurately weighed in a molar ratio of 1:2, and then inserted into a quartz tube. The pressure of the quartz tube was reduced to below 0.001 Pa, and the reaction was heated to 700°C at a heating rate of 5°C / min for 100 hours to obtain the compound;

[0051] S2: The compound obtained after the complete reaction in step S1 is naturally cooled to ambient temperature, and then the quartz tube is broken to collect the Pt-Se powder;

[0052] S3: The Pt-Se powder obtained in step S2 was ground into fine powder, and then placed in a chitosan solvent for ultrasonic treatment. The concentration of the chitosan solvent was 1 mg / mL, and the solid-liquid ratio of the fine powder to the chitosan solvent was 10 mg:1 mL. After ultrasonic treatment for 24 hours, the mixture was centrifuged at 12000 rpm for 30 minutes. The supernatant was removed after centrifugation, and the excess chitosan solvent was removed. The mixture was then centrifuged at 1500 rpm for 60 minutes to obtain nanozyme from the supernatant, which was recorded as PS@CS.

[0053] Example 2:

[0054] A method for preparing a NIR-II light-driven multifunctional nanozyme for melanoma treatment comprises the following steps:

[0055] S1: Pure elemental substances platinum and selenium were accurately weighed in a molar ratio of 1:1, and then inserted into a quartz tube. The pressure of the quartz tube was reduced to below 0.001 Pa, and the reaction was heated to 800°C at a heating rate of 8°C / min for 80 hours to obtain the compound;

[0056] S2: The compound obtained after the complete reaction in step S1 is naturally cooled to ambient temperature, and then the quartz tube is broken to collect the Pt-Se powder;

[0057] S3: The Pt-Se powder obtained in step S2 was ground into fine powder, and then placed in a chitosan solvent for ultrasonic treatment. The concentration of the chitosan solvent was 0.8 mg / mL, and the solid-liquid ratio of the fine powder to the chitosan solvent was 5 mg:1 mL. After ultrasonic treatment for 30 hours, the mixture was centrifuged at 8000 rpm for 60 minutes. The supernatant was removed after centrifugation, and the excess chitosan solvent was removed. The mixture was then centrifuged at 2000 rpm for 45 minutes to obtain nanozyme from the supernatant, which was recorded as PS@CS.

[0058] Example 3:

[0059] A method for preparing a NIR-II light-driven multifunctional nanozyme for melanoma treatment comprises the following steps:

[0060] S1: Pure elemental substances platinum and selenium were accurately weighed in a molar ratio of 1:3, and then inserted into a quartz tube. The pressure of the quartz tube was reduced to below 0.001 Pa, and the reaction was heated to 720°C at a heating rate of 6°C / min for 120 h to obtain the compound;

[0061] S2: The compound obtained after the complete reaction in step S1 is naturally cooled to ambient temperature, and then the quartz tube is broken to collect the Pt-Se powder;

[0062] S3: The Pt-Se powder obtained in step S2 was ground into fine powder, and then placed in chitosan solvent for ultrasonic treatment. The concentration of the chitosan solvent was 0.9 mg / mL, and the solid-liquid ratio of the fine powder to the chitosan solvent was 20 mg:1 mL. After ultrasonic treatment for 20 hours, the mixture was centrifuged at 10,000 rpm for 45 minutes. The supernatant was removed after centrifugation, and the excess chitosan solvent was removed. The mixture was then centrifuged at 3,000 rpm for 30 minutes to obtain nanozyme from the supernatant, which was recorded as PS@CS.

[0063] 1. Data Analysis of PS@CS Nanozyme Ultrathin Structure

[0064] The PS@CS nanozyme prepared in Example 1 was evaluated for its morphology and size distribution by scanning transmission electron microscopy (STEM), which showed a flake structure with good size distribution (e.g. Figure 1 A- Figure 1In addition, energy dispersive X-ray spectroscopy (EDX) elemental mapping was performed (as shown in Figure 2 The thickness of the nanozyme was verified by atomic force microscopy (AFM), and the ultra-thin thickness of the nanozyme was determined to be 16-18nm. Figure 3 shown.

[0065] In order to verify the atomically dispersed Pt, the extended X-ray absorption fine structure (EXAFS) method was used to gain in-depth insights into the specific characteristics of the local coordination environment of Pt within the PS@CS nanozyme. The characteristic peaks appearing in the Fourier transform EXAFS spectrum of PS@CS in R space are likely attributed to the presence of Pt-Se bonds, and no obvious characteristic peaks belonging to Pt-Pt bonds were detected (such as Figure 4 A- Figure 4 In addition, the maximum value of the wavelet transform (WT) of PS@CS is attributed to the Pt-Se coordination (as shown in Figure 5 A), which is consistent with Figure 5 This is different from the Pt foil shown in B. The results of this study clearly show that Pt-Pt bonds are not present inside the structure, thus verifying the presence of atomically dispersed Pt in the PS@CS nanozyme.

[0066] 2. Catalytic activity of various enzyme mimics under near-infrared II (NIR-Ⅱ) light irradiation

[0067] The PS@CS nanozyme prepared in Example 1 was made into PS@CS aqueous solutions of different concentrations, and the absorbance spectrum showed a single broad absorption band from the visible light (vis) to the near infrared (NIR) region (e.g. Figure 6 In order to explore the photothermal conversion ability of PS@CS nanozymes, the photothermal heating curve of PS@CS aqueous solution was monitored and captured, as shown in Figure 7 As shown. When laser irradiated (1064nm, 1.0W / cm2), the temperature of the PS@CS aqueous solution increased in a dose-dependent manner with increasing concentration. On the other hand, no significant change in the temperature of pure water was observed, indicating that the addition of PS@CS nanozymes effectively promoted the conversion of near-infrared-II light into thermal energy. Similarly, it was found that the photothermal conversion efficiency of the PS@CS aqueous solution depends on the power density, as shown in Figure 2. Figure 8 The photothermal stability of PS@CS was evaluated by allowing the PS@CS solution to undergo multiple cyclic temperature changes, as shown in Figure 9 It shows that no significant degradation occurs during the recycling process. The photothermal conversion efficiency of PS@CS nanozyme is 34.1% (e.g. Figure 10 These results indicate that PS@CS nanozyme can be considered as a photothermal agent, which can concentrate light energy into heat energy.

[0068] Based on the above research, the catalytic activity of various enzyme mimics under near-infrared-Ⅱ irradiation was further studied using electron spin resonance (ESR) spectroscopy. Figure 11 As shown in Figure 2, under NIR-Ⅱ laser irradiation, PS@CS has a stronger ability to consume H2O2. In addition, in this enzymatic reaction, PS@CS exhibits better POD enzyme activity under NIR-Ⅱ irradiation (e.g. Figure 12 In addition, under laser irradiation, the OXD enzyme activity of PS@CS can induce O2 - Generation (such as Figure 13 As shown), and with the increase of O2, O2 - The generation of O2 is also enhanced, which is because PS@CS nanozymes can produce more O2 by catalyzing H2O2 in the CAT enzyme reaction. These results show that the catalytic activity of the enzyme-mimicking PS@CS is improved and enhanced under laser irradiation.

[0069] 3. PS@CS has good biocompatibility

[0070] The biocompatibility test of the product prepared in Example 1 was carried out by hemolysis test, and the experimental results are as follows: Figure 14-15 As shown, it is proved that PS@CS nanozyme has good biocompatibility and reveals the safety of nanozyme.

[0071] 4. Study the potential of PS@CS for tumor therapy at the cellular and animal levels

[0072] The study investigated the anti-tumor effect of PS@CS by treating B16F10 cells with different concentrations of PS@CS under laser irradiation or without irradiation. The study found that only high concentrations of PS@CS showed moderate tumor cell killing ability in the absence of laser irradiation. Under laser irradiation, PS@CS showed significant anti-tumor effects in vitro, especially at high concentrations, killing almost all tumor cells (such as Figure 16 AM / PI staining was used to image the living and dead cells after PS@CS+ laser treatment, and it was found that almost all PS@CS+ laser-treated cells died, while the control group survived (as shown). Figure 17 Subsequently, DCFH-DA and DHE probes were used to detect intracellular ROS generation, confirming that laser irradiation promoted significant ROS generation in tumor cells, leading to cell death (as shown). Figure 18 shown).

[0073] The therapeutic effect of PS@CS on mice with melanoma was further studied. The mice were divided into three groups: PBS, PS@CS, and PS@CS+laser. First, the photothermal performance of PS@CS after intratumoral injection was tested. The results showed that under a 1064nm laser with a power density of 1.0W / cm2, the temperature of the tumor site quickly reached and stabilized at 45°C (such as Figure 19 As shown). Figure 20 and Figure 21 As shown in the results, PS@CS+laser treatment had a better anti-tumor effect than the other groups. This indicates that PS@CS has the highest effect in inhibiting tumor growth under near-infrared laser. The weight loss in the PS@CS+laser group was negligible, confirming the good biosafety in the short term (such as Figure 22 The tumor inhibition effect was further confirmed by H&E. The results showed that the control group showed obvious nuclear polymorphism and fission, while the PS@CS+laser group showed varying degrees of cell swelling and necrosis (as shown in Figure 2). Figure 23 Finally, TUNEL and Ki67 staining were used to observe tumor proliferation and apoptosis. The results showed that the abundance of TUNEL-positive cells in the PS@CS+laser group was high, while the abundance of Ki67-positive cells was low, while the opposite was true in the PBS group, indicating that PS@CS+laser had a strong anti-tumor effect on B16F10 tumor-bearing mice (as shown in Figure 2). Figure 24 In addition, histological analysis of major organs by H&E staining showed no pathological changes after various therapeutic interventions (e.g. Figure 25 Collectively, these findings suggest that PS@CS has significant potential for effective tumor treatment under laser irradiation without causing adverse effects on major organs.

[0074] It should be noted that, in this article, the terms: include, contain and any other variations are intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article or device. Specific examples are used herein to illustrate the principles and implementation methods of the technical solution of the present invention. The above examples are only used to help understand the method of the present invention and its core idea. The above is only a preferred embodiment of the present invention. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of the present invention, several improvements, modifications or changes can be made, and the above technical features can be combined in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the present invention to other occasions without improvement, should be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a NIR-II light-driven multifunctional nanozyme for melanoma treatment, characterized in that: The following steps are involved: S1: Pure elemental substances platinum and selenium are accurately weighed in proportion, then inserted into a quartz tube and heated to 700-800°C for reaction for 80-120 hours to obtain a compound; S2: The compound obtained after the complete reaction in step S1 is naturally cooled to ambient temperature, and then the quartz tube is broken to collect the Pt-Se powder; S3: The Pt-Se powder obtained in step S2 was ground into fine powder, and then placed in a chitosan solvent for ultrasonic treatment. After ultrasonication, the mixture was centrifuged at 8000-12000 rpm for 30-60 min. The supernatant was removed after centrifugation, and the excess chitosan solvent was removed. The mixture was centrifuged at 1500-3000 rpm for 30-60 min to obtain nanozyme from the supernatant, which was recorded as PS@CS.

2. The method for preparing a NIR-II light-driven multifunctional nanozyme for melanoma treatment according to claim 1, characterized in that: In step S1, the molar ratio of pure elemental substances platinum and selenium is 1:1-3.

3. The method for preparing a NIR-II light-driven multifunctional nanozyme for melanoma treatment according to claim 1, characterized in that: In step S1, the pressure in the quartz tube is reduced to below 0.001 Pa.

4. The method for preparing a NIR-II light-driven multifunctional nanozyme for melanoma treatment according to claim 1, characterized in that: The heating rate in step S1 is 5-8°C / min.

5. The method for preparing a NIR-II light-driven multifunctional nanozyme for melanoma treatment according to claim 1, characterized in that: The concentration of the chitosan solvent in step S3 is 0.8-1 mg / mL, and the solid-liquid ratio of the fine powder to the chitosan solvent is 5-20 mg:1 mL.

6. The method for preparing a NIR-II light-driven multifunctional nanozyme for melanoma treatment according to claim 1, characterized in that: The ultrasonic treatment time in step S3 is 18-48 hours.

7. A NIR-II light-driven multifunctional nanozyme prepared by the method according to any one of claims 1 to 6.

8. The NIR-II light-driven multifunctional nanozyme for melanoma treatment according to claim 7, characterized in that: The nanozyme has a thickness of 16-18 nm and a particle size of 80-120 nm. Its structure does not contain a Pt-Pt metal bond but contains a Pt-Se bond.

9. Use of the NIR-II light-driven multifunctional nanozyme according to claim 7 in the preparation of anti-tumor drugs.

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