Photo-thermal-enzyme catalysis nano-particles as well as preparation method and application thereof

Abstract

The invention relates to the technical field of nano biological medicines, and discloses a photo-thermal-enzyme catalysis nano-particle as well as a preparation method and application thereof. The photo-thermal-enzyme catalysis nano-particles are constructed by crosslinking water-soluble self-doped polyaniline nano-particles, human serum albumin, lactate oxidase and collagenase through a thioacetal linker capable of being split by active oxygen. Collagenase and lactate oxidase (LOx) are integrated into the same nanoparticle system through a thioacetal (TK) linker, the TK linker is split under the action of tumor microenvironment active oxygen (ROS) and H2O2 generated by catalysis of the LOx, the activity of the two enzymes is synchronously activated, a collagen matrix (physical barrier) is degraded, lactic acid (chemical barrier) is removed, the tumor microenvironment is efficiently remodeled, and the tumor microenvironment can be effectively inhibited. The problems that the regulation effect of a single barrier is limited, and the tumor microenvironment is difficult to improve are solved.

Description

Technical Field

[0001] This invention relates to the field of nanobiomedicine technology, and in particular to a photothermal-enzyme catalytic nanoparticle, its preparation method, and its application. Background Technology

[0002] The rise of nanomedicine has provided new strategies for precision oncology treatment. Compared to traditional small-molecule drugs, nanomedicines, with their unique physicochemical properties, have shown significant advantages in improving pharmacokinetics, increasing bioavailability, achieving passive targeted accumulation of tumors, and reducing systemic toxicity. Furthermore, the multifunctionality of nanoplatforms allows them to integrate imaging, therapy, and monitoring, laying the foundation for building an integrated diagnostic and therapeutic system. However, despite their promising prospects, the clinical translation of nanomedicines has been extremely slow, with only a handful of nano-formulations approved globally for the treatment of solid tumors.

[0003] For a long time, the design of nanomedicines in the treatment of solid tumors has been mainly based on the enhanced permeability-retention effect (EPR). This theory posits that the poor vascular integrity, increased permeability, and impaired lymphatic drainage in tumor tissues allow nanoparticles of specific sizes to selectively accumulate at the tumor site. However, with further research, the clinical effectiveness of the EPR is facing serious challenges. Quantitative analysis shows that, on average, less than 1% (approximately 0.7%) of injectable nanoparticles can be successfully delivered to the tumor lesion through this effect. This extremely low delivery efficiency has become a fundamental bottleneck restricting the efficacy of nanomedicines and hindering their clinical translation.

[0004] One of the core reasons for the low efficiency of nanomedicine delivery lies in the multiple physiological barriers present in the tumor microenvironment (TME). Among these, the dense extracellular matrix (ECM), composed of cross-linked collagen fibers and hyaluronic acid, constitutes the primary physical barrier. This abnormal matrix structure not only significantly increases interstitial pressure, severely hindering the penetration and uniform distribution of large nanoparticles deep within the tumor, but also repels the infiltration of immune effector cells such as cytotoxic T lymphocytes through physical shielding, leading to the formation of an immunosuppressive microenvironment. To address this physical barrier, researchers have attempted to integrate matrix-degrading enzymes such as collagenase into nanodelivery systems, aiming to improve the perfusion of nanomedicines and immune effector cells by in-situ enzymatically hydrolyzing the ECM and remodeling tumor vasculature and interstitial pressure.

[0005] In addition to physical barriers, the unique metabolic characteristics of tumors create another significant chemical barrier. Driven by the Warburg effect, tumor cells tend to engage in highly efficient glycolysis even under aerobic conditions, leading to a large accumulation of lactic acid in the tumor microenvironment. This excessive accumulation of lactic acid is not only a product of tumor metabolic reprogramming but also a potent immunosuppressive signal. Studies have shown that a high-concentration lactic acid microenvironment can induce tumor-associated macrophages (TAMs) to polarize towards the pro-tumor M2 type, while directly inhibiting the proliferation and cytotoxic activity of effector T cells, leading to their functional exhaustion. Furthermore, lactic acid can act as a signaling molecule to activate cancer-associated fibroblasts (CAFs), enhancing their ability to synthesize and secrete collagen, thereby exacerbating ECM deposition and creating a vicious cycle where physical and chemical barriers reinforce each other. To eliminate lactate, researchers introduced lactate oxidase (LOx), an enzyme that specifically catalyzes the conversion of lactate into pyruvate and hydrogen peroxide (H2O2), thereby remodeling the immunosuppressive microenvironment by consuming lactate. However, directly using exogenous enzymes in vivo faces challenges such as uncontrollable activity, easy inactivation, potential immunogenicity, and systemic side effects caused by non-specific catalysis, severely limiting their therapeutic efficacy.

[0006] Therefore, achieving synergistic regulation of physical and chemical barriers in the tumor microenvironment while ensuring the precise and controllable release of therapeutic factors has become a critical technical challenge that urgently needs to be addressed. Developing an intelligent nanoplatform capable of responding to specific signals in the tumor microenvironment and achieving spatiotemporally controllable delivery of biomolecules such as enzymes is of significant research and application value for overcoming multiple delivery barriers and improving anti-tumor efficacy. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide a photothermal-enzyme catalytic nanoparticle, its preparation method and application, which aims to solve the problem that existing nanomedicines are difficult to synergistically break through the dual barriers of the tumor microenvironment.

[0008] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a photothermal-enzyme catalytic nanoparticle, wherein the photothermal-enzyme catalytic nanoparticle comprises water-soluble self-doped polyaniline nanoparticles, human serum albumin, lactate oxidase and collagenase cross-linked by a reactive oxygen species-cleavable thioacetal linker.

[0009] To verify the successful preparation of the nanoparticles, dynamic light scattering (DLS), zeta potential (Z-Potential), and transmission electron microscopy (TEM) were performed. The results showed that compared to the self-doped polyaniline nanoparticles and the enzyme components, the cross-linked nanoparticles had a 50-fold increase in particle size, reaching 197.6 nm. Z-Potential results also indicated that compared to the high electronegativity of -24.1 mV on the surface of the self-doped polyaniline nanoparticles, the surface potential of the cross-linked nanoparticles was significantly reduced to -3.5 mV, demonstrating successful cross-linking between the self-doped polyaniline nanoparticles and the enzyme. Finally, TEM observation revealed the morphology of the nanoparticles, showing spherical particles around 200 nm in size. Furthermore, the addition of hydrogen peroxide to the nanoparticle solution resulted in the nanoparticles being fragmented into even smaller particles, with the DLS measurement showing a particle size of 4.5 nm. In summary, these findings demonstrate the successful cross-linking of the nanoparticles and their ROS-responsiveness.

[0010] Optionally, the photothermal-enzyme catalytic nanoparticles have a hydrodynamic diameter of 197.6 nm and a surface potential of -3.5 mV.

[0011] Optionally, in the photothermal-enzyme catalytic nanoparticles, the mass ratio of water-soluble self-doped polyaniline nanoparticles, human serum albumin, lactate oxidase, collagenase, and reactive oxygen species cleavable thioacetal linkers is 20:5:1:5:10.

[0012] Based on previous research and experimentation with different reactant ratios, the proportions of each reactant component were adjusted according to the DLS results of the nanoparticles and the catalytic effect of the enzymes in the nanoparticles. The results showed that the reactant ratio could form nanoparticles with suitable and uniform particle size, and the nanoparticles could be effectively cleaved in the presence of hydrogen peroxide, releasing the enzymes and carrying out catalytic action.

[0013] Optionally, the water-soluble self-doped polyaniline nanoparticles have a hydrodynamic diameter of 3.9 nm and a surface potential of -24.1 mV.

[0014] Optionally, the thioacetal linker is used to break under the action of reactive oxygen species in the tumor microenvironment to achieve responsive release of water-soluble self-doped polyaniline nanoparticles, lactate oxidase, and collagenase.

[0015] A second aspect of the present invention provides a method for preparing the above-described photothermal-enzyme catalytic nanoparticles, the method comprising the following steps: Water-soluble self-doped polyaniline nanoparticles, human serum albumin, lactate oxidase and collagenase were mixed, and a thioacetal linker that can be cleaved by reactive oxygen species was added to obtain a mixture. The mixture was subjected to a cross-linking reaction to obtain the photothermal-enzyme catalytic nanoparticles.

[0016] Optionally, the crosslinking reaction is performed under the following conditions: the mixture is stirred at room temperature and 450 rpm for 15 min. After the reaction, the product is ultrafiltered three times using a 50 kDa ultrafiltration centrifuge tube to remove unreacted reactants, and then filtered through a 0.2 μm needle filter membrane to obtain the photothermal-enzyme catalytic nanoparticles.

[0017] Optionally, the preparation of the water-soluble self-doped polyaniline nanoparticles includes the following steps: mixing aniline-N-propanesulfonic acid with aniline and performing a copolymerization reaction to obtain the water-soluble self-doped polyaniline nanoparticles.

[0018] Optionally, the mass ratio of aniline-N-propanesulfonic acid to aniline is 100:1.

[0019] Optionally, the copolymerization reaction conditions are as follows: aniline-N-propanesulfonic acid to aniline are fed at a mass ratio of 100:1, and ammonium persulfate catalyst is added. Then, the mixture is stirred at 450 rpm for 3 hours at room temperature. The reaction is stopped when the solution turns emerald green. The reactants are washed with acetone to precipitate the reactants. The precipitate is collected and vacuum dried to finally obtain a dark green solid, which is the water-soluble self-doped polyaniline nanoparticle.

[0020] A third aspect of the present invention provides the application of the above-described photothermal-enzyme catalytic nanoparticles in the preparation of tumor diagnostic and therapeutic agents.

[0021] Optionally, the tumor diagnostic and therapeutic agents can synergistically reshape the tumor microenvironment through photothermal therapy and enzyme catalysis, thereby achieving precision tumor treatment.

[0022] Optionally, the tumor is a solid tumor.

[0023] Optionally, the tumor is breast cancer in situ.

[0024] Compared with existing technologies, this invention has the following beneficial effects: 1. Achieving synergistic remodeling of the tumor microenvironment through dual barriers: This invention integrates collagenase and lactate oxidase (LOx) into the same nanoparticle system through a thioacetal (TK) linker. Under the action of reactive oxygen species (ROS) and H2O2 catalyzed by LOx in the tumor microenvironment, the TK linker cleaves, simultaneously activating the activity of the two enzymes, degrading the collagen matrix (physical barrier) and clearing lactate (chemical barrier) respectively, efficiently remodeling the tumor microenvironment, and solving the problem of limited regulation effect of single barriers and difficulty in improving the tumor microenvironment; 2. Enzyme activity Spatiotemporal controllability reduces systemic side effects: The cross-linked structure can temporarily inhibit enzyme activity during blood circulation, avoiding systemic side effects caused by premature activation of exogenous enzymes; only at the tumor site, after ROS triggers the cleavage of TK linkers, enzyme activity is restored, achieving precise localized activation of enzyme catalysis. Combined with the targeted nature of photothermal therapy, this further enhances treatment safety; 3. Photothermal-enzyme synergistic effect enhances anti-tumor efficacy: The water-soluble self-doped polyaniline nanoparticles (PSPA) in the nanoparticles possess pH-responsive NIR-II photothermal properties, activating in the acidic tumor microenvironment. This technology achieves gentle photothermal ablation of tumor cells while inducing immunogenic cell death (ICD). Combined with the immunosuppressive relief resulting from the remodeling of the tumor microenvironment via enzymatic catalysis, it can effectively activate anti-tumor immunity, achieving a synergistic anti-tumor effect of "tumor microenvironment remodeling - photothermal therapy - immune activation," and can achieve near-complete tumor growth inhibition in in situ breast cancer. 4. Excellent biocompatibility and high safety: The nanoparticles use human serum albumin (HSA) and water-soluble polyaniline as carriers, exhibiting good biocompatibility. In vivo experiments show that these nanoparticles do not cause significant changes in mouse body weight and have no adverse effects on the heart or liver. 5. No significant damage to major organs such as spleen, kidneys, and lungs, and no abnormalities in liver and kidney function. It can also alleviate tumor-induced systemic inflammation and restore immune homeostasis, providing a safety guarantee for clinical application. 6. Strong tumor penetration and uniform remodeling effect: The ultra-small PSPA (3.9nm) released after the nanoparticles are disassembled can achieve deep penetration of tumors. At the same time, the released enzymes can be evenly distributed in tumor tissue, realizing the comprehensive remodeling of the tumor microenvironment. This further improves the uniformity of the distribution of therapeutic drugs in tumor tissue and solves the problem that traditional nanoparticles are difficult to penetrate dense tumor tissue and the microenvironment remodeling is incomplete. Attached Figure Description

[0025] Figure 1 a) is a graph showing the surface potential detection results of the nanoparticles prepared in Examples 1 and 2 of the present invention; b) is a graph showing the hydrodynamic diameter of the nanoparticles prepared in Examples 1 and 2 of the present invention; c) is a TEM image of the PCLN nanoparticles in Example 2 of the present invention.

[0026] Figure 2This is a schematic diagram illustrating the preparation of PCLN nanoparticles and the functions of each component in Example 2 of the present invention.

[0027] Figure 3 a is a hydrodynamic diameter diagram of PCLN after H2O2 treatment in Example 3 of the present invention; b is a TEM image of PCLN after H2O2 treatment in Example 3 of the present invention.

[0028] Figure 4 a) is the pH response absorption spectrum in Example 4 of the present invention; b) is the pH response photothermal heating curve in Example 4 of the present invention; c) is the pH response thermal imaging diagram in Example 4 of the present invention; d) is the photothermal cycling curve in Example 4 of the present invention.

[0029] Figure 5 a) is a graph showing the collagen degradation results in Example 5 of the present invention; b) is a graph showing the lactic acid degradation results in Example 5 of the present invention.

[0030] Figure 6 a) is a graph showing the cell-killing effect under different lactic acid conditions in Example 6 of the present invention; b) is a graph showing the cell-killing effect of different groups in Example 6 of the present invention.

[0031] Figure 7 a) is an in vivo photothermal imaging image of different groups of mice in Example 7 of the present invention; b) is a temperature rise curve of different groups of mice in Example 7 of the present invention.

[0032] Figure 8 a) Tumor growth curves of different groups of mice in Example 8 of the present invention; b) Tumor images of different groups of mice after the experiment in Example 8 of the present invention; c) Comparison of spleen weight of different groups of mice in Example 8 of the present invention; d) Comparison of spleen images of different groups of mice in Example 8 of the present invention.

[0033] Figure 9 :a represents the CD8 concentration in the spleens of different groups of mice in Example 9 of this invention. + The results of cytotoxic T lymphocyte detection; b is the CD107a concentration in the spleen of different groups of mice in Example 9 of this invention. + The positive rate; c is the CD62L concentration in the spleen of different groups of mice in Example 9 of this invention. + Cell ratio.

[0034] Figure 10a) shows the weight changes of mice in different groups in Example 10; b) shows the H&E staining results of major organs of mice in different groups in Example 10 of the present invention; c) shows the serum aspartate aminotransferase (AST) biochemical index results of mice in different groups in Example 10 of the present invention (n=5); d) shows the serum alanine aminotransferase (ALT) biochemical index results of mice in different groups in Example 10 of the present invention (n=5); f) shows the serum urea (UREA) biochemical index results of mice in different groups in Example 10 of the present invention (n=5); g) shows the serum creatinine (CREA) biochemical index results of mice in different groups in Example 10 of the present invention (n=5); h) shows the serum uric acid (UA) biochemical index results of mice in different groups in Example 10 of the present invention (n=5). Detailed Implementation

[0035] This invention provides photothermal-enzyme catalytic nanoparticles, their preparation method, and applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them, and are intended to illustrate the present invention only and not to limit it in any way. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1: Synthesis of water-soluble self-doped polyaniline nanoparticles (PSPA) (1) Preparation of SPA In the synthesis of SPA, 5.5 mL of aniline was first taken, followed by 1.2 mL of propanesulfonate lactone, which was then slowly added dropwise to the aniline in a warm water bath. The mixture was then stirred at 450 rpm for 3 hours at room temperature until solidification. Afterwards, an organic solvent precipitation method was used, followed by thorough washing with acetone and filtration until the solid turned white. Finally, the white solid was collected and dried overnight under vacuum to remove residual acetone.

[0038] (2) Preparation of PSPA 100 mg of SPA was added to 2 mL of water and stirred at 450 rpm. Simultaneously, 160 mg of APS was dissolved in 0.3 mL of water and sonicated to ensure complete dissolution. Separately, 0.1 mL of aniline and 0.1 mL of concentrated hydrochloric acid were added to 0.8 mL of water, mixed thoroughly, and diluted 10-fold. 0.035 mL of this diluted solution was added to the SPA solution (SPA:aniline = 100:1). Then, the APS solution was added dropwise to the mixture containing SPA and aniline, and the mixture was stirred at room temperature for 3 hours. After the reaction was complete, an organic solvent precipitation method was used. The mixture was thoroughly washed with acetone, filtered, and the blackish-green solid was collected. The solid was dried overnight under vacuum to remove residual acetone. The performance of PSPA was detected using dynamic light scattering (DLS) and a Zeta potentiometer. Figure 1 a and Figure 1 As shown in Figure b, PSPA has a hydrodynamic diameter of 3.9 nm and a surface potential of -24.1 mV. It exhibits good water solubility and dispersibility, and can be used as a photothermal reagent for efficient loading into nanomedicine systems.

[0039] Example 2: Preparation of photothermal-enzyme catalyzed nanoparticles (PCLN) 2 mg of the PSPA solid prepared in Example 1 was dissolved in 1 mL of distilled water and filtered through a 0.2 μm needle filter. The pH of the PSPA solution was adjusted to between 7.5 and 8.0, followed by the addition of 1 mg of the ROS-degradable thioacetal (TK) crosslinking agent NHS-TK-NHS, then 0.5 mg of HSA, 0.1 mg of LOx, and 0.5 mg of collagenase. The mixture was stirred at 450 rpm for 20 minutes at room temperature. After the reaction, the mixture was ultrafiltered three times through a 50 kDa ultrafiltration tube to remove unreacted impurities and concentrated to obtain PCLN photothermal-enzyme catalytic nanomedicine. The preparation of PCLN and the functions of its components are as follows: Figure 2 As shown.

[0040] Using the same method, without adding LOx, PCN was prepared; using the EDC / NHS chemical method (without using TK linkers), PSPA was crosslinked with HSA, LOx, and collagenase to prepare the non-responsive nanoconjugate PCLN0.

[0041] The properties of each nanoparticle were detected using DLS and Zeta potentiometer. For example... Figure 1 a and Figure 1 As shown in Figure b, the hydrodynamic diameter of PCN is 229.8 nm and its surface potential is -7.6 mV; the hydrodynamic diameter of PCLN is 197.6 nm and its surface potential is -3.5 mV; the hydrodynamic diameter of PCLN0 is 197.6 nm and its surface potential is -3.3 mV, indicating that PSPA has successfully cross-linked with protein and enzyme components to form a photothermal-enzyme catalytic nanomedicine system. Figure 1 a and Figure 1 (b) TEM image shows ( Figure 1 (c) PCLN consists of irregularly shaped spherical nanoparticles of uniform size.

[0042] Example 3: Detection of ROS-responsive disassembly performance of PCLN photothermal-enzyme catalyzed nanoparticles The PCLN prepared in Example 2 was dispersed in PBS buffer containing H2O2 (100 μM, simulating the ROS level of the tumor microenvironment). After incubation at 37°C for different times, the hydrodynamic diameter change was detected by DLS, and the morphological changes were observed by TEM to verify the disassembly and assembly ability of the nanomedicine, providing support for the photothermal-enzyme synergistic effect. DLS results showed that the hydrodynamic diameter of PCLN significantly decreased from 197.6 nm to 4.5 nm under the action of H2O2, which is close to the size of PSPA (polystyrene- ... Figure 3 (a). TEM image shows ( Figure 3 (b) After incubation, the particles disassemble and release small-sized PSPA particles, indicating that the TK linker can be cleaved by ROS (H2O2), realizing the programmed disassembly and assembly of PCLN photothermal-enzyme catalytic nanomedicine, and ensuring the precise release of enzymes and photothermal components at the tumor site.

[0043] Example 4: Detection of pH-responsive photothermal properties of PCLN photothermal-enzyme catalyzed nanoparticles 50 μg / mL PSPA and PCLN (calculated based on the concentration including PSPA) were dispersed in PBS buffer at different pH values ​​(6.0 (simulating the acidic microenvironment of tumors) and 7.5 (simulating the normal tissue environment)). The absorption spectra were detected using a UV-Vis-NIR spectrophotometer. Subsequently, PCLN solutions under different pH conditions (pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5) were irradiated with a 1064 nm laser (power 0.4 W / cm²). The temperature changes of the solutions were monitored using an infrared camera to evaluate and verify the photothermal performance.

[0044] Absorption data show that the near-infrared absorption intensity of PCLN in acidic environments is significantly higher than that in neutral environments. Figure 4 (a). Photothermal experiments showed that the temperature rise of the PCLN solution under acidic conditions was significantly greater than that under neutral conditions, and the higher the PCLN concentration, the more significant the temperature rise. This indicates that the PCLN photothermal-enzyme catalyzed nanomedicine has good pH-responsive NIR-II photothermal properties and can precisely exert photothermal therapeutic effects at tumor sites. Figure 4 b and Figure 4 (c) Furthermore, no significant differences were observed after five cycles of heating and cooling, demonstrating that PCLN exhibits good photothermal stability. Figure 4(d).

[0045] Example 5: Detection of ROS-activated enzyme activity in PCLN photothermal-enzyme catalyzed nanoparticles (1) Collagenase activity detection Using FITC-labeled gelatin as a collagen mimic substrate, PCLN, PCLNO, and free collagenase were incubated with FITC-gelatin, divided into H2O2-added and non-H2O2-added groups. After incubation at 37℃ for 24 h, the supernatant was collected by centrifugation, and the fluorescence intensity of the supernatant was detected using a fluorescence spectrophotometer (excitation wavelength 488 nm, emission wavelength 520 nm). Higher fluorescence intensity indicated stronger collagenase activity and more complete gelatin degradation, verifying the physical barrier remodeling ability of nanoparticles. Figure 5 As shown in Figure a, the fluorescence intensity of the supernatant of the PCLN group increased significantly after the addition of H2O2, indicating that ROS can activate the collagenase activity in PCLN. The collagen degradation rate of PCLN is lower than that of free collagenase, but significantly higher than that of PCLN0 (non-responsive type), indicating that the collagenase activity is partially inhibited in the cross-linked nanomedicine system and is only restored after ROS triggers the cleavage of TK linkers, thus achieving targeted activation of collagenase and efficient degradation of the tumor physical barrier.

[0046] (2) Detection of lactate oxidase (LOx) activity Using a lactic acid assay kit, PCLN was dispersed in a lactic acid-containing solution and incubated at 37°C. Samples were taken at 0, 15, 30, 60, 90, and 120 minutes to detect changes in lactic acid concentration, assess the catalytic activity of LOx, and verify the chemical barrier scavenging ability of the nanoparticles. Figure 5 As shown in Figure b, after the addition of PCLN, the lactate concentration in the solution continued to decrease with the extension of incubation time, and the lactate concentration dropped to the lowest level at 120 minutes. This indicates that LOx in PCLN can effectively catalyze the degradation of lactate after ROS activation, thereby clearing the tumor chemical barrier and synergistically completing the remodeling of the tumor microenvironment with collagenase.

[0047] Example 6: In vitro synergistic cytotoxicity detection of PCLN photothermal-enzyme catalyzed nanoparticles First, the cytotoxicity of PCLN and PCLN0 was compared under lactate and lactate-free conditions. Breast cancer cells (4T1 cells) were used as model cells, and the cell viability of different groups was detected using the CCK8 assay. Figure 6 As shown in Figure a, compared to the case without lactate, both PCLN and PCLN0 were significantly reduced in the presence of lactate. Furthermore, the cytotoxicity of the PCLN group was significantly higher than that of the PCLN0 group. This demonstrates that LOx catalyzing lactate to produce H2O2 not only has a certain cytotoxic effect but also further breaks the TK linker, restoring enzyme activity.

[0048] Furthermore, the in vitro synergistic antitumor effect of PCLN photothermal-enzyme catalyzed nanoparticles was evaluated. Breast cancer cells (4T1 cells) were used as model cells and divided into the following groups: PBS group, PSPA group, PCN group, PCLN group, PBS+L group, PSPA+L group, PCN+L group, and PCLN+L group; the laser irradiation conditions were 1064 nm and 1.5 W / cm². 2 The irradiation time was 10 minutes. Cell viability in different groups was detected using the CCK8 assay. Figure 6 As shown in Figure b, the cell survival rate of the PCLN+L group was the lowest, significantly lower than that of the other groups. The results of cell viability staining showed that the PCLN+L group had the highest proportion of dead cells, indicating that the combination of PCLN photothermal-enzyme catalytic nanoparticles and NIR-II photothermal therapy has significant synergistic cytotoxicity and can effectively kill tumor cells, demonstrating the synergistic effect of photothermal-enzyme catalysis.

[0049] Example 7: Detection of the in vivo photothermal effect of PCLN photothermal-enzyme catalyzed nanoparticles 4T1 orthotopic breast cancer-bearing mice were randomly divided into 4 groups: PBS group, PSPA group, PCN group, and PCLN group, with 5 mice in each group. The corresponding preparation (dose 15 mg / kg) was injected into the tail vein. 4 h after injection, the tumor site was irradiated with a 1064 nm laser (0.4 W / cm²). The temperature change of the tumor site was monitored by an infrared thermal imager for 10 minutes to verify the in vivo photothermal properties of the nanoparticles.

[0050] like Figure 7 a and Figure 7 As shown in Figure b, mice treated with the PCLN nanoparticles described in this invention exhibited significantly better temperature rise at the tumor site after light irradiation compared to other control groups (PSPA and PCN). Specifically, the PCLN group reached the ideal thermotherapy temperature range of 42.0℃ (42-45℃), while the PSPA and PCN groups reached 40.2℃ and 40.9℃, respectively. This significant difference in temperature rise demonstrates that PCLN nanomedicines possess higher bioavailability and tumor-specific accumulation capacity in complex in vivo environments, overcoming the shortcomings of traditional photothermal agents such as low enrichment rate and rapid heat dissipation, thus achieving precise photothermal intervention at the tumor site.

[0051] Example 8: In vivo therapeutic effect detection of PCLN photothermal-enzyme catalyzed nanoparticles 4T1 orthotopic breast cancer-bearing mice were randomly divided into 5 groups of 5 mice each: PBS group, PCLN group, PSPA+L group, PCN+L group, and PCLN+L group. The treatment regimen was as follows: a single injection of the preparation (dose 15 mg / kg) via tail vein, followed by laser irradiation (1064 nm, 0.4 W / cm², 10 minutes / session) 4 hours after injection. During the treatment period, the weight and tumor volume of the mice were measured every 2 days, and tumor growth curves were plotted. After 18 days of treatment, the mice were sacrificed, the tumors were removed and photographed, the tumor weight and spleen weight were measured, liver and kidney function were tested, and H&E staining of major organs was performed to observe organ damage. The therapeutic efficacy, safety, and tumor microenvironment remodeling effect of the nanomedicine were comprehensively evaluated.

[0052] Tumor growth curves and tumor images indicate ( Figure 8 a and Figure 8 In group b), compared to the PBS group, the tumor-suppressing effects of photothermal therapy alone (PSPA+L) or enzyme regulation alone (PCLN without irradiation) were not ideal. The combination of photothermal therapy and a single enzyme (PCN+L), while enhancing the therapeutic effect, did not completely inhibit tumor growth. Only the PCLN+L group achieved near-complete tumor growth inhibition, with significantly smaller tumor volume compared to the other groups. Furthermore, spleen weight measurement and spleen imaging showed ( Figure 8 c and Figure 8 In the PBS group (d), mice showed significant splenomegaly (a marker of tumor-induced systemic inflammation and myeloid-derived suppressor cell expansion), while the spleen size and weight of mice in the PCLN+L group were significantly reduced. This indicates that synergistic treatment can effectively alleviate systemic immunosuppression and restore immune homeostasis, supporting the effect of tumor microenvironment remodeling.

[0053] Example 9: In vivo antitumor immune activation of PCLN photothermal-enzyme catalyzed nanoparticles Eighteen days after treatment, mice in each group were sacrificed and their spleen tissue was collected. Single-cell suspensions were prepared, and flow cytometry was used to detect immune cell subsets in the spleen to evaluate the effect of the PCLN photothermal-enzyme catalytic nanoparticles on the body's anti-tumor immune response.

[0054] Flow cytometry analysis showed that after PCLN+L treatment, CD8 levels in mice decreased. + Cytotoxic T lymphocytes ( Figure 9 a) and its secretory degranulation marker CD107a + The positive rate was significantly increased ( Figure 9 (b) CD62L, which also represents immune memory capacity. + The cell ratio was also increased. Figure 9(c). The above experimental data show that the PCLN nanoparticles described in this invention, under near-infrared light irradiation, can directionally reshape the immunosuppressive microenvironment through the synergistic intervention of photothermal therapy and enzyme catalytic reaction, thereby inducing a highly efficient anti-tumor immune response. Its therapeutic effect is significantly better than that of single photothermal or single drug groups, demonstrating an excellent synergistic effect.

[0055] Example 10: In vivo biosafety assay of PCLN photothermal-enzyme catalyzed nanoparticles Throughout the treatment process, the mice's body weight was monitored, and 18 days after treatment, major organs such as the heart, liver, spleen, kidneys, and lungs of each group of mice were collected for H&E staining. Body weight monitoring ( Figure 10 (a) and organ H&E staining ( Figure 10 (b) The results showed that there was no significant change in body weight in all groups of mice, and no significant lesions were observed in the major organs such as the heart, liver, spleen, kidneys, and lungs of the PCLN+L group mice; liver and kidney function tests ( Figure 10 The results showed that there was no significant difference in liver and kidney function indicators between the PCLN group and the PBS group, indicating that the PCLN photothermal-enzyme catalyzed nanomedicine has good biocompatibility and low systemic toxicity.

[0056] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A photothermal-enzyme catalyzed nanoparticle, characterized in that, The photothermal-enzyme catalytic nanoparticles are constructed by cross-linking water-soluble self-doped polyaniline nanoparticles, human serum albumin, lactate oxidase, and collagenase through reactive oxygen species-cleavable thioacetal linkers.

2. The photothermal-enzyme catalytic nanoparticles according to claim 1, characterized in that, The photothermal-enzyme catalytic nanoparticles have a hydrodynamic diameter of 197.6 nm and a surface potential of -3.5 mV.

3. The photothermal-enzyme catalytic nanoparticles according to claim 1, characterized in that, In the photothermal-enzyme catalytic nanoparticles, the mass ratio of water-soluble self-doped polyaniline nanoparticles, human serum albumin, lactate oxidase, collagenase, and reactive oxygen species cleavable thioacetal linkers is 20:5:1:5:

10.

4. The photothermal-enzyme catalytic nanoparticles according to claim 1, characterized in that, The water-soluble self-doped polyaniline nanoparticles have a hydrodynamic diameter of 3.9 nm and a surface potential of -24.1 mV.

5. A method for preparing photothermal-enzyme catalyzed nanoparticles according to any one of claims 1-4, characterized in that, The preparation method includes the following steps: Water-soluble self-doped polyaniline nanoparticles, human serum albumin, lactate oxidase and collagenase were mixed, and a thioacetal linker that can be cleaved by reactive oxygen species was added to obtain a mixture. The mixture was subjected to a cross-linking reaction to obtain the photothermal-enzyme catalytic nanoparticles.

6. The method for preparing photothermal-enzyme catalyzed nanoparticles according to claim 5, characterized in that, The conditions for the crosslinking reaction are as follows: the mixture is stirred at room temperature and 450 rpm for 15 min.

7. The method for preparing photothermal-enzyme catalyzed nanoparticles according to claim 5, characterized in that, The preparation of the water-soluble self-doped polyaniline nanoparticles includes the following steps: mixing aniline-N-propanesulfonic acid with aniline and performing a copolymerization reaction to obtain the water-soluble self-doped polyaniline nanoparticles.

8. The use of the photothermal-enzyme catalytic nanoparticles according to any one of claims 1-4 in the preparation of tumor diagnostic and therapeutic agents.

9. The application according to claim 8, characterized in that, The tumor diagnostic and therapeutic agents synergistically reshape the tumor microenvironment through photothermal therapy and enzyme catalysis, thereby achieving precision tumor treatment.

10. The application according to claim 8, characterized in that, The tumor is a solid tumor.

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