NIR-II light-driven multifunctional nano-enzyme for treating melanoma and preparation method of NIR-II light-driven multifunctional nano-enzyme

Abstract

The invention discloses an NIR-II light-driven multifunctional nano enzyme for melanoma treatment and a preparation method thereof, and belongs to the technical field of nano biomedical material preparation, and the preparation method comprises the following steps: accurately weighing pure element substances platinum and selenium according to a ratio, then inserting into a quartz tube, heating to 700-800 DEG C, and reacting to obtain a compound; the compound is naturally cooled to the environment temperature, then the quartz tube is broken, and Pt-Se powder is obtained through collection; the method comprises the following steps: grinding Pt-Se powder into fine powder, then putting the fine powder into a chitosan solvent for ultrasonic treatment, centrifuging after ultrasonic treatment, removing supernate after centrifuging, removing redundant chitosan solvent, centrifuging again, and obtaining nano-enzyme from the supernate, namely PS-CS. The prepared nano-enzyme can effectively convert H2O2 into ROS under laser irradiation, so that the anti-tumor efficiency is improved, and a novel and comprehensive treatment strategy is provided for treatment of 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 huge challenge to traditional therapeutic approaches. In these hypoxic microenvironments, tumor cells exhibit higher levels of H2O2, which provides opportunities for innovative therapeutic interventions. Taking advantage of this unique phenomenon, researchers have begun to explore 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 to cancer cells. The appeal of nanozymes lies not only in their ability to mimic the catalytic power 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 via 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 susceptible to infection, complicating treatment and increasing costs. In this study, we developed platinum- and selenium-based nanozymes, Pt-Se@Chitosan (PS@CS), which have multienzyme activity and strong photothermal conversion efficiency, and can convert H2O2 into reactive oxygen species, thereby inducing cell death. In this study, we found that PS@CS has excellent antitumor effects both in vitro and in vivo, providing an innovative integrated therapeutic approach for melanoma treatment. Summary of the invention

[0004] In view of the above problems, the present invention provides a NIR-II light-driven multifunctional nanozyme for the treatment of melanoma and a preparation method thereof. The prepared nanozyme has good biocompatibility, multiple enzyme mimetic activities, including POD, CAT and OXD, and excellent photothermal conversion ability. Under laser irradiation, PS@CS can effectively convert H2O2 into ROS, thereby improving the anti-tumor efficiency, providing a novel and comprehensive treatment 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, heated to 700-800°C for reaction for 80-120 hours to obtain a compound;

[0008] S2: The compound obtained by 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 powder was centrifuged at 8000-12000 rpm for 30-60 min. The supernatant was removed after centrifugation, and the excess chitosan solvent was removed. The powder 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 implementation, the pressure in the quartz tube in step S1 drops 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 implementation, 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 nanozyme has a thickness of 16-18 nm and a particle size of 80-120 nm. 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 with antitumor application 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 method to prepare Pt-Se powder, and obtains the ultrathin structure of PS@CS nanozyme by ultrasonic treatment. The research shows that Pt-Pt bond does not exist inside the nanozyme structure, thus 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 activities;

[0020] (3) The nanozyme prepared by the present invention has good biocompatibility, revealing the safety of nanozymes. 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, multiple enzyme mimetic activities including POD, CAT and OXD, and excellent photothermal conversion ability. 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. 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 The 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 result 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 The absorbance curves of PS@CS aqueous solution at different concentrations;

[0028] Figure 7The 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] Fig. 9 The temperature curve of three on / off cycles of PS@CS aqueous solution under 1064 nm laser irradiation;

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

[0032] Fig.11 ESR spectra confirming H2O2 consumption by PS@CS nanozyme under laser irradiation;

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

[0034] Fig.13 ESR spectra of PS@CS nanozyme in OXD enzyme activity;

[0035] Fig.14 The hemolysis rate of PS@CS nanozymes with different concentrations;

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

[0037] Fig.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] Fig.17 Representative images of live / dead cells of B16F10 cells after PS@CS+laser treatment and curves of the control group;

[0039] Fig.18 ROS detection diagram of B16F10 cells in the control group and PS@CS+laser treatment group;

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

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

[0042] Fig.21 The changes in tumor volume of mice in the three groups on different days of treatment;

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

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

[0045] Fig.24 TUNEL and Ki67 staining analysis of tumors in the three treatment groups;

[0046] Fig.25 H&E staining pathological analysis 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] Embodiment 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 are accurately weighed in a molar ratio of 1:2, and then inserted into a quartz tube. The pressure of the quartz tube is reduced to below 0.001Pa, and heated to 700℃ for reaction at a heating rate of 5℃ / min for 100h to obtain a compound;

[0051] S2: The compound obtained by 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 centrifuged at 1500 rpm for 60 minutes to obtain nanozyme from the supernatant, which was recorded as PS@CS.

[0053] Embodiment 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 are accurately weighed in a molar ratio of 1:1, and then inserted into a quartz tube. The pressure of the quartz tube is reduced to below 0.001 Pa, and heated to 800°C for reaction at a heating rate of 8°C / min for 80 hours to obtain a compound;

[0056] S2: The compound obtained by 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 centrifuged at 2000 rpm for 45 minutes to obtain nanozyme from the supernatant, which was recorded as PS@CS.

[0058] Embodiment 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 are accurately weighed in a molar ratio of 1:3, and then inserted into a quartz tube. The pressure of the quartz tube is reduced to below 0.001 Pa, and heated to 720°C for reaction at a heating rate of 6°C / min for 120 hours to obtain a compound;

[0061] S2: The compound obtained by 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 a 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 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 the ultrathin structure of PS@CS nanozyme

[0064] The PS@CS nanozyme prepared in Example 1 was evaluated for its morphology and size distribution by scanning transmission electron microscopy (STEM), showing a sheet-like 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] To verify the atomically dispersed Pt, the extended X-ray absorption fine structure (EXAFS) method was used to gain insight into the specific features of the local coordination environment of Pt inside the PS@CS nanozyme. The characteristic peaks appearing in the Fourier transform EXAFS spectrum of the R-space PS@CS are likely attributed to the presence of Pt-Se bonds, and no obvious characteristic peaks belonging to Pt-Pt bonds were detected (e.g. Figure 4 A- Figure 4 In addition, the maximum 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, such as 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 Fig. 9 The results showed that no significant degradation occurred during the recycling process. The photothermal conversion efficiency of PS@CS nanozyme was 34.1% ( Fig.10 According to these research results, PS@CS nanozyme can be regarded as a photothermal agent, which concentrates 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. Fig.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. Fig.12 In addition, under laser irradiation, the OXD enzyme activity of PS@CS can induce ·O2 - Generation (such as Fig.13 As shown in Figure 2, and with the increase of O2, O2 - The generation of is also enhanced, because PS@CS nanozymes can produce more O2 by catalyzing H2O2 in the CAT enzyme reaction. These results show that the enzyme-mimicking catalytic activity of PS@CS is improved and enhanced under laser irradiation.

[0069] 3.PS@CS has good biocompatibility

[0070] The biocompatibility of the product prepared in Example 1 was tested by hemolysis test. 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 without 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 Fig.16 AM / PI staining was used to image live 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 in Figure 2A). Fig.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 in Figure 2A). Fig.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 (e.g. Fig.19 As shown in Fig. 20 and Fig.21 As shown in the figure, PS@CS+laser treatment has a better anti-tumor effect than 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 (e.g. Fig. 22 The tumor inhibition effect was further confirmed by H&E, and 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). Fig.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, and the number 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 2A). Fig.24 In addition, histological analysis of major organs by H&E staining showed no pathological changes after various therapeutic interventions (e.g. Fig.25 Overall, 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 variants are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. Specific examples are used in this article to illustrate the principle and implementation 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 implementation of the present invention. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, for ordinary technicians in this technical field, without departing from the principle of the present invention, several improvements, modifications or changes can be made, and the above technical features can also 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 protection scope 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, heated to 700-800°C for reaction for 80-120 hours to obtain a compound; S2: The compound obtained by 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 powder was centrifuged at 8000-12000 rpm for 30-60 min. The supernatant was removed after centrifugation, and the excess chitosan solvent was removed. The powder 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 drops 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 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.

9. The NIR-II light-driven multifunctional nanozyme according to claim 7 has anti-tumor application under laser irradiation.

Images