Construction of ROS-responsive self-delivery carrier-free nanoparticles and application of ROS-responsive self-delivery carrier-free nanoparticles in breast cancer treatment

Abstract

The invention discloses construction of ROS response type self-delivery carrier-free nanoparticles and application of the ROS response type self-delivery carrier-free nanoparticles in breast cancer treatment, firstly, a TLR agonist R848 synthesizes a dimer TKdR through an ROS sensitive bond in one step, then the dimer TKdR and a free photosensitizer Ce6 are co-assembled to form a nano self-assembly body (psTKdR NAs), and an R848 dimer-based photoactivatable supramolecular self-assembly multi-drug delivery system is constructed, pDT can effectively induce ICD to stimulate an autoimmune system, meanwhile, Ce6 can generate ROS under activation of specific light and further induce breakage of TK to release drugs, and R848 can drive cellular immune response and cause polarization of M1 macrophages and is used for tumor immunotherapy. The ROS-responsive intelligent prodrug is designed and introduced into the construction of a nano system, so that the stability of the drug and the sensitivity of a disease site can be improved, and the synergistic curative effect of an intelligent drug delivery system or a traditional drug combination therapy is expected to be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of photodynamic immunotherapy for tumors, and particularly relates to the construction of ROS-responsive self-delivery carrier-free nanoparticles and their application in the treatment of breast cancer. Background Art

[0002] Triple-negative breast cancer has high invasion and high migration abilities. Inhibiting the metastasis and development of triple-negative breast cancer and inducing the death of tumor cells have attracted increasing attention. Currently, most anti-cancer drugs have poor water solubility, anti-proliferative effects, and problems such as poor selectivity and severe side effects. To solve these serious problems, many novel methods based on nanotechnology have been applied in biomedicine, such as liposomes, inorganic nanoparticles (NPs), or polymer micelles, etc. Although significant progress has been made, the use of carrier-based nanotechnology for anti-cancer drug delivery still lacks loading efficiency in terms of pharmacokinetics and biodistribution, and its long-term toxicity remains unknown. To further improve the drug loading capacity of the nanosystem and reduce the use of polymer materials, a simple and "green" carrier-free nanosystem has gradually gained the favor of researchers. Carrier-free nano self-assemblies with higher safety have shown great potential in the diagnosis and treatment of tumors. Carrier-free nano self-assemblies refer to nano drug delivery systems formed by the self-assembly of active drugs without the use or with very little use of excipients.

[0003] As a specific and selective cancer treatment method, photodynamic therapy (PDT) has received extensive attention in recent years. However, the effect of using photodynamic therapy alone is still limited. Especially in the deep part of tumor tissues, the weak light penetration ability limits the treatment range. Therefore, combination therapy has gradually become an effective new means of treating cancer, which can improve the efficacy of photodynamic therapy and overcome its limitations. Among many drug self-assembly systems, the photosensitizer Ce6, as a common photosensitizer, can generate singlet oxygen 1 O2 under the irradiation of near-infrared light, and has a strong anti-tumor effect.

[0004] Tumor-associated macrophages (TAMs) are the most abundant immunosuppressive cells in most solid tumors. They are closely associated with tumor cell invasion and migration, as well as poor prognosis. Tumor-associated macrophages (TAMs) can be divided into M1 and M2 subtypes, which have opposing immune functions. M1-like macrophages enhance anti-tumor immunity by activating T cells and phagocytosing tumor cells, while M2-like macrophages promote tumor growth by suppressing T cell activity. The phenotypes of these two subtypes can be mutually transformed under certain stimuli, and their plasticity provides an effective strategy for tumor immunotherapy. Therefore, macrophage polarization may enhance the immunotherapeutic efficacy of PDT. A mature platform is urgently needed for PDT-initiated immunotherapy. Toll-like receptors are the most important pattern recognition receptors in innate immunity and are widely expressed on various tumor cells, dendritic cells, B cells, macrophages, and other cells. Studies have shown that reximod (R848) is a potent agonist of Toll-like receptors 7 and 8 (TLR7 / TLR8), which can drive humoral and cellular immune responses, leading to M1 macrophage polarization and potential applications in tumor immunotherapy. However, the use of R848 alone can easily lead to problems such as excessive immune activation, poor specificity, and different individual reactions. Summary of the invention

[0005] Purpose of the invention: In response to the problems existing in the prior art, the present invention constructs a carrier-free nanoparticle that is a ROS-responsive self-delivering photoimmunomodulator. The carrier-free nanoparticle constructed by the present invention not only reduces the use of polymer materials, but also further improves the drug loading capacity of the nanosystem; the present invention designs a ROS-sensitive responsive prodrug (TKdR) to enable rapid and controllable release after ROS exposure, which is crucial to reducing premature leakage or release of therapeutic drugs before reaching the tumor site.

[0006] In addition, the nanoparticles prepared by the present invention can also provide applications for effectively inducing ICD to stimulate the autoimmune system through PDT and combining with the R848 immunomodulator (reximod) to exert synergistic anti-tumor efficacy.

[0007] Technical solution: The method for constructing carrier-free nanoparticles for ROS-responsive self-delivery of photoimmunomodulators of the present invention comprises the following steps:

[0008] (1) Resimod and thioketal were weighed in an organic solvent, and 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added respectively;

[0009] (2) stirring the reaction mixture obtained in step (1), washing and drying the organic layer after extraction, purifying and separating to obtain a product liquid, pre-freezing the product liquid and then vacuum freeze-drying it to obtain the product TKdR;

[0010] (3) Dissolve the product TKdR and free Ce6 obtained in step (2) in dimethyl sulfoxide, add it to double-distilled water under an ultrasonic water bath to prepare a nano-solution, and obtain the nanoparticles psTKdRNps through dialysis, which are carrier-free nanoparticles of a ROS-responsive self-delivery photoimmunomodulator.

[0011] Among them, the mass ratio of resimotil, thioacetal, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in step (1) is 4-6:1-3:3-5:5-7.

[0012] Preferably, weigh 400-600 mg of resimotil and 100-300 mg of thioacetal in 10-20 mL of anhydrous dichloromethane, and then add 300-500 mg of 4-dimethylaminopyridine and 500-700 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride respectively.

[0013] Among them, in step (2), the reaction mixture is stirred at 15-25 °C for 8-14 h, the obtained product solution is pre-frozen at -80-100 °C for 8-12 h, and the storage temperature of the product TKdR is 0-4 °C.

[0014] Preferably, the extractant in step (2) is any one of DCM, chloroform, ethyl acetate, and ether.

[0015] Preferably, in step (2), the organic layer is washed with any one or several of citric acid, saturated NaHCO3, dilute hydrochloric acid, dilute sulfuric acid, saturated sodium carbonate, and dilute sodium hydroxide solution, and the organic layer is dried with any one or several of anhydrous Na2SO4, anhydrous magnesium sulfate, and anhydrous calcium chloride.

[0016] Among them, in step (3), TKdR and free Ce6 are dissolved in dimethyl sulfoxide at a molar ratio of 1:0.8-1:1.2, then double-distilled water is added, and the dialysis time is 22-26 h.

[0017] Furthermore, a carrier-free nanoparticle of a self-delivery photoimmunomodulator prepared by the method for constructing a carrier-free nanoparticle of a ROS-responsive self-delivery photoimmunomodulator.

[0018] Among them, the application of the carrier-free nanoparticle of the self-delivery photoimmunomodulator in the preparation of a drug for treating breast cancer.

[0019] A drug for treating breast cancer, comprising the carrier-free nanoparticle psTKdRNps of the self-delivery photoimmunomodulator described in claim 1 and a pharmaceutically acceptable excipient or carrier.

[0020] Among them, the pharmaceutical preparation is an injection, granule, tablet, capsule, pill, or oral liquid.

[0021] The present invention constructs a carrier-free nanoparticle of a ROS-responsive self-delivery photoimmunomodulator for the combined immuno-photodynamic therapy of tumors. First, in this self-assembly system, the TLR agonist R848 is synthesized into a dimer TKdR through a ROS-sensitive bond to achieve controllable release of ROS. Subsequently, it is co-assembled with the free photosensitizer Ce6 to form a nano self-assembly (psTKdRNAs). The carrier-free nanoparticle is constructed by combining photodynamic and immunotherapy without the need to use an additional carrier. In the nanoparticle constructed by the present invention, PDT can effectively induce ICD to stimulate the body's own immune system. The combination of PDT and immunotherapy can provide a mutually reinforcing strategy for the spatio-temporal treatment of superficial and metastatic tumors. At the same time, Ce6 will generate ROS under specific light activation, which will further induce the cleavage of TK to release the drug. R848 can drive the cellular immune response, leading to the polarization of M1 macrophages for tumor immunotherapy. Designing a ROS-responsive intelligent prodrug using the ROS generated by PDT and introducing it into the construction of the nano system can improve the stability of the drug in the in vivo circulation and the sensitivity at the disease site, and is expected to achieve a synergistic effect of the intelligent drug delivery system superior to single therapy or traditional drug combination therapy. The present invention combines R848 and CE6. CE6 can be used as a photosensitizer to mediate photodynamic therapy, accurately target the lesion site, and reduce the systemic immune stimulation intensity of R848. Their synergistic effect can also enhance the local immune activation efficiency, reduce non-specific reactions, improve the treatment effect and reduce the risk of adverse reactions. Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0022] In the present invention, R848 (synthesizing dimer TKdR) and the photosensitizer Ce6 are self-assembled into stable carrier-free nanoparticles without adding additional excipients, etc., establishing a green, safe and efficient platform for nano-therapy; the PDT effect of Ce6 induces immunogenic cell death, and at the same time triggers the cleavage of the ROS-sensitive bond to achieve the precise spatio-temporal delivery of R848 to achieve a synergistic anti-cancer effect, showing a more powerful tumor inhibitory effect compared with single drugs; activating toll-like receptor signaling and cytokine interaction activates T cells and lymphocytes, significantly enhancing the immune-mediated tumor suppression effect. Description of the Drawings

[0023] Figure 1 1H NMR spectra of R848 (A) and TKdR (B);

[0024] Figure 2 Peak positions (A) and corresponding molecular weights (B) of TKdR;

[0025] Figure 3Preparation and characterization of psTKdR Nps: TEM images (A) and particle size distribution (B) of the self-assembled nanodrug with TKdR and Ce6 at a molar ratio of 1:1, and changes in the hydrodynamic diameter and polydispersity index (PDI) of the nanodrug over 7 days at different feeding ratios (C);

[0026] Figure 4 CCK8 assay for cell viability of 4T1 cells treated with Ce6, Ce6+, psTKdRNps, and psTKdRNps+: without light illumination (A), 660 nm near-infrared light illumination (B);

[0027] Figure 5 Schematic diagram of the administration cycle by tail vein injection in a mouse model;

[0028] Figure 6 Curves of tumor volume changes after intravenous injection of the control group, Ce6, and psTKdRNps;

[0029] Figure 7 H&E staining and TUNEL staining images of tumor tissues;

[0030] Figure 8 In vivo macrophage polarization and anti-tumor immune activation;

[0031] Figure 9 Analysis of the degradation of psTKdR nanoparticles (Nps) incubated in different concentrations of hydrogen peroxide (H2O2) by high performance liquid chromatography (HPLC). Detailed implementation manners

[0032] The present invention will be further described below in conjunction with specific embodiments and the accompanying drawings.

[0033] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all purchased from conventional biochemical reagent companies unless otherwise specified.

[0034] Example 1

[0035] Synthesis of the prodrug TKdR

[0036] Weigh 500 mg of resiquimod (R848, 2.2 eq) and 182.45 mg of thioacetal ketal (TK, 1.0 eq), add them to 15 mL of anhydrous dichloromethane (DCM), then successively add 388.6 mg of 4-dimethylaminopyridine (DMAP, 4.4 eq) and 609.75 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 4.4 eq). Stir for 12 hours at room temperature (20 °C), then extract with DCM. Wash the organic layer with 5% citric acid and saturated NaHCO₃, and dry with anhydrous Na₂SO₄. Dissolve the dried product in methanol. Set acetonitrile as the mobile phase, set Flow rate: 10 ml / min, Detection: 254 nm, Monitor: 397 nm, and set the gradient pressure: first EQU, 5 - 5, 5 Min, then 5 - 5, 7 min, 5 - 95, 25 min, 95 - 95, 5 min. Obtain the chromatographically purified product solution through high-pressure purification and separation. Pre-freeze the obtained product solution at -80 °C overnight, and then obtain the product through vacuum freeze-drying. Store it at 4 °C for later use, and perform NMR characterization on the obtained product.

[0037] As Figure 1 shown, from the characterization data of the nuclear magnetic resonance hydrogen spectrum, the hydroxyl peak at δ = 4.49 ppm has disappeared, and this peak corresponds to the hydroxyl group in the raw material R848. At the same time, new peaks appear in the ¹H NMR spectrum of the product. Two triplet peaks appear at δ = 2.58 ppm and δ = 2.83 ppm, indicating that there are two coupled methylene groups near the thioacetal bond. In addition, the singlet at δ = 1.58 ppm corresponds to the methyl group attached to the thioacetal bond. These chemical shifts are attributed to the reaction between the hydrogen atom of the hydroxyl group on R848 and the carboxyl group of the thioacetal bond. As Figure 2 shown, liquid chromatography-mass spectrometry analysis shows that the product TKdR elutes at 6.1 min, and the molecular weight corresponding to this time in the mass spectrum is 845, where 423 is the molecular weight of the dimer depolymerized into monomers in the mobile phase. The above results indicate the successful synthesis of the TKdR prodrug.

[0038] Example 2

[0039] Characterization results of the physicochemical properties of nanoparticles psTKdR Nps

[0040] The nanoparticles psTKdR Nps constructed from TKdR and Ce6 were prepared by the nanoprecipitation method. TKdR (5 mg) and free Ce6 (3.53 mg) were dissolved in 1 mL of dimethyl sulfoxide (DMSO) at a molar ratio of 1:1, and then slowly added to 9 mL of double-distilled water under ultrasonic water bath at room temperature and ultrasonicated for 10 min to prepare a nanoparticle solution. Subsequently, the nanoparticle solution was dialyzed in a dialysis bag with a molecular weight of 8000 Da for 24 h to obtain the psTKdR Nps solution.

[0041] The self-assembled nanoparticles (psTKdR Nps) prepared in Example 2 were characterized for their morphology and particle size by transmission electron microscopy (TEM) and dynamic light scattering (DLS), respectively, as Figure 3 shown. The nanoparticles exhibited a uniform circular structure with good dispersibility. The average particle size of psTKdR Nps was 122.3 nm, and the polydispersity index (PDI) was 0.097. psTKdR NPS was negatively charged with a potential of -34 mV. To further evaluate the stability of the nanoparticles, the changes in hydrodynamic size and PDI were monitored over a period of 7 days. The data results showed no significant fluctuations in particle size, confirming the good stability of psTKdR nanoparticles.

[0042] Example 3

[0043] In vitro antitumor results of the nanoparticles psTKdR Nps

[0044] The CCK8 method was used to determine cell viability. 4T1 cells in the logarithmic growth phase were selected and seeded into 96-well plates at a density of 4×10 3 cells / well and cultured in an incubator for 24 h until the cells adhered. Then, the cells were treated with Ce6, Ce6 + light, psTKdR Nps, and psTKdR Nps + light, respectively. The concentration of Ce6 was kept constant at 0.4 μg / mL in each group. After the drug was incubated with the cells for 20 h, light was irradiated with 660 nm near-infrared light for 5 min, and each group was incubated for an additional 24 h. Finally, 10 μL of CCK8 solution was added to each well in the 96-well plate and incubated for 1 h. The absorbance value at 450 nm was measured using a microplate reader, and the cell viability was calculated. The cell viability calculation formula: Cell viability (%) = (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group) × 100%

[0045] The cytotoxic effect of psTKdR Nps was evaluated in vitro by the CCK-8 cell viability assay, as Figure 4As shown, in the absence of light and in a normoxic environment, Ce6 and psTKdR Nps exhibited extremely low cytotoxicity at all treatment concentrations. Conversely, after excitation by a specific light (660 nm near-infrared light), Ce6 and psTKdR Nps showed concentration-dependent photocytotoxic effects on 4T1 cells. The results demonstrated the light-regulatory advantage of photodynamic therapy (PDT) in inhibiting tumor growth. Notably, compared with the Ce6+ group, the cell viability of the psTKdR Nps+ group was significantly reduced to the lowest level.

[0046] Example 4

[0047] In vivo antitumor activity study of nanoparticle psTKdR Nps

[0048] Balb / c female mice (4 - 6 weeks old, 18 - 20 g) were acclimatized for one week. 4T1 cells in the logarithmic growth phase were digested with trypsin, rinsed 3 times with PBS after digestion, and then the cells were collected. After centrifugation (1000 rpm, 30 min), cell pellets were obtained and resuspended in PBS to prepare a cell suspension of 7×10 6 cells / mL. The cell suspension was inoculated subcutaneously into the left mammary fat pad of BALB / c mice at a dose of 100 μL per mouse to establish a triple-negative breast cancer solid tumor model.

[0049] When the tumor volume reached approximately 80 mm 3 ³, the mice were randomly divided into 3 groups: control group, Ce6+ group, and psTKdR Nps+ group prepared in Example 1. The “+” indicates that the tumors received specific light irradiation (660 nm laser). On days 1, 3, 5, 7, 9, 10, 11, and 13, the mice received intravenous injection of different experimental drugs, ensuring that the dose of R848 in the psTKdR Nps+ group was 2.5 mg / kg and the dose of Ce6 was 2.5 mg / kg, and ensuring that the dose of Ce6 in the Ce6+ group was 2.5 mg / kg. Two hours after injection, the tumor sites were irradiated with 660 nm laser for 10 minutes. The tumor volume was evaluated every two days, and the tumor volume was calculated as: volume = 0.5 × L × W² (L: length, W: width). On day 15, the mice were euthanized, and multiple tissues such as heart, liver, spleen, lung, kidney, and tumor tissues were collected. The excised tissues were weighed and photographed, and the tumor growth curve was plotted for further analysis. The dissected tumor tissues were fixed with 4% paraformaldehyde, and H&E and TUNEL staining were used to detect apoptotic cells in the tumor tissues.

[0050] The established triple-negative breast cancer solid tumor mouse model is as Figure 5As shown, different experimental drugs were intravenously injected into three groups of tumor-bearing mice. Two hours after each treatment, the tumor sites were irradiated with 660 nm laser light to activate the photodynamic therapy (PDT) effect mediated by psTKdR Nps. During the entire treatment period, the tumor volume was measured every two days to construct a tumor growth curve. As Figure 6 shown, compared with the control group, the tumor growth in the Ce6+ treatment group slowed down to a certain extent. Compared with the other two treatment groups, the psTKdR Nps+ group had a significantly stronger inhibitory effect on tumor growth, with the smallest tumor volume and the best anti-tumor effect. As Figure 7 shown, hematoxylin-eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining further confirmed these findings. Histopathological analysis of tumor tissues by H&E staining showed that compared with the control group, the cell density in the Ce6+ treatment group decreased, and the cell density in the psTKdR Nps+ group was the lowest, with incomplete cell nuclei, indicating that psTKdR Nps caused extensive damage to tumor tissues. In TUNEL staining, there were very few apoptotic cells (red fluorescence) in the control group. Red fluorescence appeared in both the Ce6+ group and the psTKdR Nps+ group, and the red fluorescence in the psTKdR Nps+ group was significantly enhanced, indicating that psTKdR Nps could significantly induce tumor cell apoptosis.

[0051] Example 5

[0052] Detection of immune-related mRNA levels in tumor tissues

[0053] Weigh 0.1 g of the tumor tissue treated in Example 4, add 1 mL of PBS, and grind it with an electric homogenizer until there are no obvious tissue blocks. Centrifuge the grinding solution at 2000 rpm for 5 min, take out the precipitate after washing three times with PBS, extract total RNA using FreeZol reagent Vazyme#R711, perform RNA reverse transcription using the reverse transcription kit Vazyme#MR101, and use the Vazyme#Q331-02 real-time fluorescence quantitative PCR kit for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to analyze specific cytokines and immune-related factors in cells and tumor-infiltrating M1 / M2 type tumor-associated macrophages (TAMs). Among them, CD4 and CD8 are important surface markers of T cells, which play a key role in the immune response, can specifically recognize and target and destroy tumor cells, and contribute to anti-tumor immunity.

[0054] Comprehensive analysis of the expression of immune-related genes in tumor samples was performed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). As Figure 8As shown in Figures 8A and 8B, compared with the control group, the expression levels of CD4 and CD8 in the psTKdR Nps+ group were significantly increased, and this finding indicates the effectiveness of psTKdR nanoparticles in activating cytotoxic T lymphocytes (CTLs) for immunotherapy; DCs are crucial in initiating the adaptive immune response and are usually induced by highly immunogenic tumor cells. To evaluate the maturation of DCs triggered by immunogenic cell death associated with photodynamic therapy, the expression levels of CD80 and CD86 were measured; as Figure 8 shown in Figures 8C and 8D, compared with the non-irradiated group, both the Ce6 group and the psTKdR Nps+ group showed increased expression of CD80 and CD86, indicating that PDT-induced ICD of tumor cells triggered an immune response. The expression of CD80 and CD86 in the psTKdR Nps+ group was higher than that in the Ce6 group, which may be due to the synergistic effect between R848 and PDT-induced ICD; as Figure 8 shown in Figure 8E, the expression of Foxp3, a regulatory T cell marker that inhibits the immune response, was significantly decreased, indicating that the psTKdR Nps+ group could improve the immunosuppressive microenvironment in vivo; as Figure 8 shown in Figures 8F, 8G, and 8H, in the psTKdR Nps+ group, the expression of CD206, a key marker of M2 macrophages, was significantly reduced, and the levels of pro-inflammatory markers iNOS and TNFα related to M1 macrophages were significantly increased. Compared with the Ce6+ group, a significant polarization from the M2 to the M1 macrophage phenotype was observed in the psTKdR Nps+ group, indicating that macrophages in tumor tissues were effectively polarized during immunotherapy, thus exerting a strong immune response.

[0055] Example 6

[0056] Determination of Reactive Oxygen Species-Sensitive Drug Release

[0057] To evaluate the responsiveness of the TK bond to reactive oxygen species (ROS), hydrogen peroxide (H2O2) was used to simulate the ROS environment. In the presence of 50 mM hydrogen peroxide (H2O2), the release of R848 from psTKdR nanoparticles (Nps) was monitored by high performance liquid chromatography (HPLC). A 5 mL suspension of the nanoparticle psTKdR Nps solution prepared in Example 1 was transferred to a dialysis bag (8000 Da) and immersed in 20 mL of phosphate buffer (PBS) containing 50 mM H2O2 at a temperature maintained at 37 °C. At time intervals of every 20 min, the dialysis fluid was collected, and the concentration of R848 was quantitatively analyzed by high performance liquid chromatography.

[0058] Given that psTKdR nanoparticles have a strong ability to generate reactive oxygen species, hydrogen peroxide (H2O2) was used to simulate the reactive oxygen environment to study the reactive oxygen-sensitive drug release characteristics of psTKdR nanoparticles. The sensitivity of TK under the reactive oxygen environment can be used to evaluate the hydrolysis behavior of prodrugs upon activation by reactive oxygen species. As Figure 9 shown, the dialysis fluid was irradiated for 5 min under the activation of 660 nm light, and the reactive oxygen species generated by Ce6 in the nanoparticles effectively cleaved TK (thioketal), triggering the release of the active drug R848. The results showed that in the presence of 50 mM H2O2, the hydrolysis rate of the prodrug was accelerated, and approximately 50% of R848 was released within 90 min, with a release rate of 80% after 3 h. In contrast, in the absence of H2O2, the degradation of the prodrug was negligible. These findings indicate that introducing a reactive oxygen-sensitive linker in prodrug design enables responsive drug release. This rapid and controllable release upon exposure to reactive oxygen species is crucial for minimizing premature leakage or release of therapeutic drugs before reaching the tumor site.

Claims

1. A method for constructing a carrier-free nanoparticle of a ROS-responsive self-delivery photoimmunomodulator, characterized in that, It includes the following steps: (1) Weigh resimot and thioacetal in an organic solvent, and add 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride respectively; (2) Stir the reaction mixture obtained in step (1), extract and then wash and dry the organic layer, purify and separate to obtain a product solution, pre-freeze the product solution and then freeze-dry it under vacuum to obtain the product TKdR; (3) Dissolve the product TKdR and chlorin e6 obtained in step (2) in an organic solvent, add it to double-distilled water under an ultrasonic water bath to prepare a nano-solution, and obtain the carrier-free nanoparticles psTKdRNps of the ROS-responsive self-delivery photoimmunomodulator through dialysis.

2. The construction method according to claim 1, wherein In step (1), the mass ratio of resimot, thioacetal, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is preferably 4-6:1-3:3-5:5-7.

3. The construction method according to claim 1, characterized in that, In step (2), stir the reaction mixture at 15-25 °C for 8-14 h, and pre-freeze the obtained product solution at -80-100 °C for 8-12 h.

4. The construction method according to claim 1, characterized in that In step (2), the extractant is any one or more of DCM, chloroform, ethyl acetate and ether.

5. The construction method according to claim 1, characterized in that, In step (2), wash the organic layer with any one or several of citric acid, saturated NaHCO3, dilute hydrochloric acid, dilute sulfuric acid, saturated sodium carbonate and dilute sodium hydroxide solution, and dry the organic layer with any one or several of anhydrous Na2SO4, anhydrous magnesium sulfate and anhydrous calcium chloride.

6. The construction method according to claim 1, characterized in that In step (3), dissolve TKdR and Ce6 in a molar ratio of 1:0.8-1:1.2 in dimethyl sulfoxide, then add double-distilled water, and the dialysis time is 22-26 h.

7. A carrier-free nanoparticle of a self-delivery photoimmunomodulator prepared by the method for constructing a carrier-free nanoparticle of the ROS-responsive self-delivery photoimmunomodulator according to claim 1.

8. Use of the carrier-free nanoparticle of the ROS-responsive self-delivery photoimmunomodulator according to claim 7 in the preparation of a drug for treating breast cancer.

9. A drug for treating breast cancer, characterized in that, It includes the carrier-free nanoparticle psTKdRNps of the self-delivery photoimmunomodulator according to claim 1 and a pharmaceutically acceptable excipient or carrier.

10. The application according to claim 8, characterized in that, The pharmaceutical preparation is an injection, granule, tablet, capsule, pill or oral liquid.

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