Drug-loaded nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a drug-loaded nanoparticle as well as a preparation method and application thereof. The drug-loaded nanoparticles comprise active components and a carrier, wherein the active components comprise a hydrophobic small-molecule inhibitor and PAEMA; the carrier comprises an amphiphilic polymer bonded with a photosensitizer; the amphiphilic polymer bonded with the photosensitizer is coated with a hydrophobic small-molecule inhibitor and PAEMA (Polyacrylamide); wherein the amphiphilic polymer bonded with the photosensitizer comprises the photosensitizer, a hydrophilic chain segment and a hydrophobic chain segment. Precise drug delivery is achieved through characteristic transformation of the nano-carrier driven by tumor acidity, M2 type macrophages can be removed, dendritic cells can be induced to be mature, tumor cells can be killed, and therefore the synergistic anti-tumor effect of photodynamic therapy and immunotherapy is enhanced. Therefore, various drugs can be accurately delivered to target cells with different spatial distributions, so that the cell killing level and the antigen presenting cell mediated immunocompetence are enhanced at the same time.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanomedicine, in particular to a drug-loaded nanoparticle, a preparation method and application thereof. BACKGROUND

[0002] The treatment of malignant tumors still faces great challenges worldwide. For malignant proliferative tumors, although traditional treatment methods can directly eliminate cancer cells, it is still difficult to completely avoid tumor recurrence and metastasis. With the development of immunology, the tumor immune escape mechanism is considered to be one of the main reasons for the deterioration of tumors. Emerging immunotherapy focuses on activating the function of the anti-tumor immune system to recognize and kill cancer cells, and has achieved remarkable clinical results. For example, immune checkpoint antibody anti-PD1 is used to activate T cells to kill tumor cells. However, single immunotherapy has poor effect on most solid tumors, with low patient response rate and limited survival benefit. Today, researchers have proposed a strategy of synchronously eliminating tumor cells and activating the immune system to improve anti-tumor efficacy.

[0003] The development of nanomedicine opens up a new way for targeted tumor tissue combined therapy. Most co-delivery nanocarriers aim to deliver therapeutic drugs to tumor cells, while some nanomedicines can activate immune cells by inducing tumor immunogenic cell death (ICD) when killing tumor cells. However, the tumor microenvironment (TME) can limit its therapeutic effect and weaken the immune activation effect. On the one hand, the dense extracellular matrix of tumors significantly hinders the penetration and distribution of nanomedicines. On the other hand, antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages (TAMs) in the tumor microenvironment usually exhibit immature and immunosuppressive phenotypes, which severely weaken their ability to present tumor antigens and activate T cells. Therefore, during most stages of treatment, there are still problems of insufficient anti-tumor activity and poor infiltration of effector T cells.

[0004] Studies have shown that effective immunotherapy mainly depends on two key immune steps: activation of tumor antigen presentation by APCs in the tumor or immune organs (such as lymph nodes and spleen), and activation of T cells. Therefore, nanocarriers that can simultaneously deliver anticancer drugs and immunotherapeutic drugs to tumors have been proven to be more effective. However, as a key target cell for immunotherapy, tumor-associated macrophages (M2-TAM) account for about 50% of immune cells in tumors and are mainly distributed in the perivascular area. This unique localization feature provides new challenges and opportunities for precisely delivering different drugs to tumor cells and APCs.

[0005] Therefore, it is urgent to develop a drug-loaded nanoparticle that can effectively enrich in the tumor site and co-deliver two different drugs to achieve in situ killing of tumor cells and activation of APCs. SUMMARY

[0006] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the first aspect of the present application provides a drug-loaded nanoparticle, which can activate APC-mediated immune response under the condition of tumor acidity, promote small-sized nanocarrier to deliver photosensitizer to the deep tumor, and then kill tumor cells and cause immunogenic cell death (ICD) under the condition of light, thereby further activating DCs, and achieving the effect of enhancing the combination of photodynamic therapy and immunotherapy.

[0007] The second aspect of the present application also provides a preparation method of the drug-loaded nanoparticle.

[0008] The third aspect of the present application also provides an application of the drug-loaded nanoparticle.

[0009] According to the first aspect of the present application, a drug-loaded nanoparticle is provided, comprising an active ingredient and a carrier: The active ingredient comprises a hydrophobic small molecule inhibitor and poly-2-(azepan-1-yl) ethyl methacrylate (PAEMA); The carrier comprises an amphiphilic polymer bonded with a photosensitizer; The hydrophobic small molecule inhibitor and poly-2-(azepan-1-yl) ethyl methacrylate (PAEMA) are coated in the amphiphilic polymer bonded with the photosensitizer; The poly-2-(azepan-1-yl) ethyl methacrylate (PAEMA) can realize a characteristic transition from hydrophobic to hydrophilic under the condition of acidity; and the amphiphilic polymer bonded with the photosensitizer comprises the photosensitizer, a hydrophilic segment and a hydrophobic segment.

[0010] According to a preferred embodiment of the present application, the average particle size of the drug-loaded nanoparticle is 20 nm ~ 70 nm.

[0011] According to a preferred embodiment of the present application, the hydrophobic small molecule inhibitor accounts for 1%-5% of the total mass of the drug-loaded nanoparticle.

[0012] According to a preferred embodiment of the present application, the photosensitizer is selected from at least one of chlorin e6 (Ce6), demethylated chlorophyllin a or verteporfin.

[0013] According to a preferred embodiment of the present application, the number average molecular weight of the hydrophilic segment is 2000 ~ 5000 Da. For example, it includes 2000 Da, 2500 Da, 3000 Da, 3500 Da, 4000 Da, 4500 Da, 5000 Da or a sub-range composed of any two of the above values.

[0014] According to a preferred embodiment of the present application, the hydrophilic segment is selected from polyethylene glycol.

[0015] According to a preferred embodiment of the present application, the number average molecular weight of the hydrophobic segment is 1000-5500 Da. For example, 1000 Da, 1500 Da, 2000 Da, 3000 Da, 3500 Da, 4000 Da, 4500 Da, 5000 Da, 5500 Da or a sub-range consisting of any two of the above values.

[0016] According to a preferred embodiment of the present application, the hydrophobic segment is selected from at least one of polylactic acid, polylactic acid, polycaprolactone or polyphosphoester.

[0017] According to a preferred embodiment of the present application, the hydrophobic small molecule inhibitor is selected from at least one of Sunitinib, Pacritinib, Ertugliflozin or Vimsertinib.

[0018] According to a preferred embodiment of the present application, the photosensitizer and the amphiphilic polymer are connected by a chemical bond.

[0019] The drug-loaded nanoparticles according to the embodiments of the present application have at least the following beneficial effects: The present application uses an amphiphilic polymer bonded with a photosensitizer to encapsulate a hydrophobic small molecule inhibitor and PAEMA, poly-2-(azepan-1-yl)ethyl methacrylate (PAEMA) can realize the characteristic transformation from hydrophobic to hydrophilic under acidic conditions; finally, an acid-responsive drug-loaded nanoparticle is obtained, which effectively encapsulates the hydrophobic small molecule inhibitor and PAEMA by the hydrophobic interaction between the hydrophobic segment of the amphiphilic polymer and the hydrophobic small molecule inhibitor and PAEMA, and self-assembles to form an acid-responsive drug-loaded nanosystem. The acid-responsive drug-loaded nanoparticle can effectively enrich at the tumor site and co-deliver two different drugs, achieving in situ killing of tumor cells and activation of APCs. At the tumor site, the released hydrophobic small molecule inhibitor eliminates M2-TAM, and the protonated PAEMA activates DCs, inducing DC maturation under tumor pH conditions. The delivery photosensitizer shrinks in size to reach the deep tumor, and then kills tumor cells and causes tumor immunogenic death (ICD) through photodynamic therapy under light conditions, thereby further activating DCs, ultimately activating CD8 +T cells, enhance the effect of photodynamic immunotherapy combined treatment. Finally, the present application realizes precise drug delivery through the characteristic transformation of tumor acidity driven nano-carriers, which can not only eliminate M2-TAM and induce DC maturation, but also kill tumor cells, thereby enhancing the synergistic anti-tumor effect of PDT and immunotherapy. Furthermore, the present application can precisely deliver various drugs to target cells with different spatial distribution, thereby simultaneously enhancing the cell killing ability and APC-mediated immune activity, providing a new breakthrough point for anti-tumor drug delivery strategies.

[0020] According to a second aspect of the present application, a method for preparing the drug-loaded nanoparticles as described in the first aspect of the present application is provided, comprising the following steps: S1, mixing the amphiphilic polymer of bonded photosensitizer, PAEMA and hydrophobic small molecule inhibitor respectively dissolved in the first solvent, dropping into the second solvent under stirring, and stirring to form drug-loaded nanogel by self-assembly; S2, removing the first solvent and centrifuging to remove free hydrophobic small molecule inhibitor to obtain drug-loaded nanoparticles.

[0021] According to a preferred embodiment of the present application, the mass ratio of the amphiphilic polymer of bonded photosensitizer and the hydrophobic small molecule inhibitor is 5-10:1. For example, it includes 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or a sub-range consisting of any two of the above ratios.

[0022] According to a preferred embodiment of the present application, the mass ratio of the PAEMA and the amphiphilic polymer of bonded photosensitizer is 0.1-1:1. For example, it includes 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.7:1, 0.8:1, 0.9:1, 1:1 or a sub-range consisting of any two of the above ratios.

[0023] According to a preferred embodiment of the present application, the concentration of the amphiphilic polymer of bonded photosensitizer after being dissolved in the first solvent is 5 mg / mL-20 mg / mL. For example, the concentration includes 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 12 mg / mL, 14 mg / mL, 15 mg / mL, 20 mg / mL or a sub-range consisting of any two of the above values.

[0024] According to a preferred embodiment of the present application, the concentration of the hydrophobic small molecule inhibitor after being dissolved in the first solvent is 10 mg / mL-20 mg / mL. For example, the concentration includes 10 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL or a sub-range consisting of any two of the above values.

[0025] According to a preferred embodiment of the present application, the concentration of the PAEMA after being dissolved in the first solvent is 2.5-5 mg / mL.

[0026] According to a preferred embodiment of the present application, the first solvent comprises tetrahydrofuran.

[0027] According to a preferred embodiment of the present application, the second solvent comprises water.

[0028] According to a preferred embodiment of the present application, the volume ratio of the first solvent and the second solvent is 1:5-10.

[0029] According to a preferred embodiment of the present application, in step S2, the centrifugal speed is 3000-5000 rpm, and the centrifugal time is 5-10 min.

[0030] The third aspect of the present application provides a use of the drug-loaded nanoparticle of the first aspect of the present application in the preparation of an anti-tumor drug.

[0031] According to a preferred embodiment of the present application, the tumor comprises at least one of breast cancer, colorectal cancer, and melanoma.

[0032] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent from the description, or can be learned by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0033] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the accompanying drawings, in which: Figure 1 For Example 1 Ce6 Synthesis of SNP / B and application schematic diagram; Figure 2 For Example 1 Ce6 Particle size diagram and transmission electron microscope diagram of SNP / B; Figure 3 For Example 1 Ce6 Acid-responsive particle size change and drug release diagram of SNP / B; Figure 4 For Example 1 Ce6 M2-TAM clearance effect diagram of SNP / B; Figure 5 For Example 1 Ce6 DC activation effect diagram of SNP / B; Figure 6 For Example 1 Ce6 Acid-responsive penetration effect diagram of SNP / B; Figure 7 Figure 1 shows the in vivo anti-tumor effect of SNP / B for Example 1 Ce6 Figure 2 shows the in vivo anti-tumor immune effect of SNP / B for Example 1. Figure 8 Figure 1 shows the in vivo anti-tumor effect of SNP / B for Example 1 Ce6 Figure 2 shows the in vivo anti-tumor immune effect of SNP / B for Example 1. DETAILED DESCRIPTION

[0034] The following are specific embodiments of the present application, and the technical solutions of the present application are further described in conjunction with the embodiments, but the present application is not limited to these embodiments.

[0035] The reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0036] Some of the raw materials used in the following examples are as follows: Tetrahydrofuran (THF) was purchased from Shanghai Chemical Reagent Co., Ltd., Sorafenib (BLZ-945) was purchased from Shanghai Hanxiang Biological Technology Co., Ltd., Ce6 was purchased from Shanghai Maikelin Biotechnology Co., Ltd., polyethylene glycol-poly-lactic acid (PEG-PLA) was purchased from Xi'an Ruixi Biological Technology Co., Ltd., 4-cyano-4-(thiobenzoyl) valeric acid (CPDB), 2-(hexamethylene imine) ethyl methacrylate (AEMA) and cyclohexyl methacrylate (CHMA) were purchased from Aldrich Industrial Co.

[0037] The amphiphilic polymer (PEG-PLA-Ce6) bonded with Ce6 was synthesized according to the reported method (Preeti Kumari, Sri Vishnu Kiran, Rompicharla, Himanshu Bhatt, Balaram Ghosh, Swati Biswas, Development of Chlorin E6-Conjugated Poly(Ethylene Glycol)-Poly(D,L-Lactide) Nanoparticles for Photodynamic Therapy, Nanomedicine 2019, 14, 819).

[0038] Poly 2-(azepan-1-yl)ethyl methacrylate (PAEMA) and non-acid responsive polymer (PCHMA) were synthesized according to the reported method (Weijia Xu, Junxia Wang, Liangjie Jin, Yueqiang Zhu, Xianzhu Yang, A tumor acidity-driven transformable polymeric nanoassembly with deep tumor penetration and membrane-anchoring capability for targeted photodynamic therapy, Biomaterials 2021, 276, 121024).

[0039] Other biochemical reagents are domestic conventional analytical reagents.

[0040] Reagent preparation: 20 mg / mL BLZ-945 solution: 10 mg hydrophobic BLZ-945 was weighed and dissolved in THF to 20 mg / mL.

[0041] 20 mg / mL PEG-PLA-Ce6 solution: 10 mg PEG-PLA-Ce6 was weighed and dissolved in THF to 20 mg / mL.

[0042] 5 mg / mL PAEMA solution: 10 mg PAEMA was weighed and dissolved in THF to 5 mg / mL.

[0043] 5 mg / mL PCHMA solution: 10 mg PCHMA was weighed and dissolved in THF to 5 mg / mL.

[0044] Example 1 Example 1 provides an acid-responsive drug-loaded nanoparticle (named as: Ce6 SNP / B), the synthesis route thereof is shown in Figure 1 The SNP / B is prepared by the following steps: S1, 0.35 mL PEG-PLA-Ce6 (20 mg / mL) solution, 0.6 mL PAEMA (5 mg / mL) solution and 0.05 mL BLZ-945 (20 mg / mL) solution were vortexed, and then were added dropwise into a 25 mL flask containing 5 mL ultrapure water under stirring. After stirring for 1 h, the solution was taken out, and THF was removed by rotary evaporation to obtain about 5 mL Ce6 SNP / B.

[0045] S2, the above results Ce6 Remove unsealed BLZ-945 by SNP / B centrifugation at 5000 rpm for 5 min, and collect the supernatant. Ce6 SNP / B.

[0046] Comparative Example 1 Comparative Example 1 provides a drug-loaded nanoparticle (named: Ce6 The preparation steps for INP / B are as follows: S1. Take 0.35 mL of PEG-PLA-Ce6 (20 mg / mL) solution, 0.6 mL of PCHMA (5 mg / mL) solution, and 0.05 mL of BLZ-945 (20 mg / mL) solution, vortex, and add dropwise to a 25 mL flask containing 5 mL of ultrapure water under stirring. After stirring for 1 h, remove the solution, remove THF by rotary evaporation, and obtain approximately 5 mL of... Ce6 INP / B.

[0047] S2, the above results Ce6 Remove unsealed BLZ-945 by INP / B centrifugation at 5000 rpm for 5 min, and collect the supernatant. Ce6 INP / B.

[0048] Performance testing The samples prepared in Example 1 and Comparative Example 1 respectively Ce6 SNP / B and Ce6 The physicochemical properties of INP / B were identified.

[0049] Will Ce6 SNP / B (approximately 1 mg) was suspended in ultrapure water, and the particle size of TMPD NPs was measured using a Zetasizer Nano ZSE (Malvern, UK) dynamic light scattering instrument; Ce6 SNP / B suspension droplets were placed on a copper grid with a film, dried for 10 min, and their morphology was observed using a field emission transmission electron microscope (Talos F200X). Figure 2 As shown in A, its particle size distribution Ce6 SNP / B particles have a diameter of approximately 70 nm and exhibit a uniform spherical shape. Adjustment Ce6 The acidity of the SNP / B solution was observed using the same method. Ce6 SNP / B shrinks to spherical particles of approximately 20 nm under pH 6.5 conditions. However, Ce6 INP / B maintains unchanged particle size and morphology under different pH conditions (e.g. Figure 2 (As shown in B in the diagram).

[0050] After freeze-drying the nanoparticles, the particles were dissolved and disrupted using THF. The BLZ-945 content in the nanoparticles was determined at a wavelength of 280 nm using a UV-Vis spectrophotometer. The encapsulation efficiency (EE) of BLZ-945 was calculated as follows: EE (%) = (mass of BLZ-945 in nanoparticles / total mass of BLZ-945) × 100%; The encapsulation efficiency of BLZ-945 was measured to be approximately 30%, and BLZ-945 accounted for 3% of the drug-loaded nanoparticles.

[0051] Measurement using a dynamic light scattering instrument Ce6 SNP / B and Ce6 The particle size variation of INP / B under different pH conditions revealed... Ce6 The particle size of SNP / B begins to decrease sharply around pH 6.8, but... Ce6 The particle size of INP / B remains basically unchanged (e.g. Figure 3 (As shown in A). Monitoring was performed using an ultraviolet spectrophotometer. Ce6 SNP / B and Ce6 Drug release of INP / B under different pH conditions was found. Ce6 SNP / B can release approximately 60% of BLZ-945 after incubation at pH 6.8 for 5 minutes (e.g., ...). Figure 3 (As shown in B). These results demonstrate that acid-responsive drug-loaded nanoparticles ( Ce6 SNP / B can achieve acid-responsive scaling and drug release.

[0052] Experimental Example 1: Acid-responsive drug-loaded nanoparticles of the present invention ( Ce6 Verification of the effect of SNP / B on in vitro activation of APC: 1. Ce6 SNP / B's effect on clearing M2-TAM Under pH 7.4 or pH 6.5 conditions, M2-TAM obtained from the differentiation of RAW264.7 was combined with... Ce6 SNP / B or Ce6 INP / B co-incubation. After 24 hours, cells were collected and stained with fluorescently labeled flow cytometry antibodies. The ratio of M1-TAM to M2-TAM was analyzed by flow cytometry to assess the loading of cells. Ce6 The scavenging effect of SNP / B on M2-TAM under specific pH conditions. The specific experimental procedure is as follows: RAW264.7 cells were seeded into 24-well plates (1*10⁻⁶ cells per well). 5 (cells / well), after induction with IL-4 (20 ng / mL) for 24 hours, were replaced with cells containing Ce6 SNP / B or Ce6INP / B (BLZ-945, 2.0 µg / mL) was incubated in 1640 medium (pH 7.4 or pH 6.5) for 24 h. The untreated medium served as the PBS group, with three replicates per group. Cells were collected and stained with fluorescently labeled flow cytometry antibodies (anti-F4 / 80-Per CP / cyamine 5.5, anti-CD80-FITC, anti-CD206-PE). F4 / 80 levels were analyzed by flow cytometry. + CD80 + M1-TAM and F4 / 80 + CD206 + M2-TAM ratio. Results are as follows: Figure 4 As shown in A and 4B, Ce6 In the SNP / B (pH 6.5) group, M2-TAM had the lowest proportion, while M1-TAM had the highest proportion, indicating that... Ce6 Acid-responsive drug release from SNP / B is beneficial for clearing M2-TAM and exerting the anti-tumor effect of M1-TAM.

[0053] 2. Ce6 The effect of SNP / B activating DC DC2.4 cells were mixed with... under pH 7.4 or pH 6.5 conditions. Ce6 SNP / B or Ce6 INP / B co-incubation. After 24 hours, cells were collected and stained with fluorescently labeled flow cytometry antibodies. DC maturation was analyzed by flow cytometry. The specific experimental procedure is as follows: DC2.4 cells were seeded into 24-well plates (1*10⁻⁶ cells per well). 5 (cells / well), and containing Ce6 SNP / B or Ce6 INP / B (BLZ-945, 2.0 µg / mL) was incubated in 1640 medium (pH 7.4 or pH 6.5) for 24 h. The untreated medium served as the PBS group, with three replicates per group. Cells were collected and stained with fluorescently labeled flow cytometry antibodies (anti-MCHCII-AF700, anti-CD80-FITC, anti-CD86-PE). Markers of DC maturation were analyzed by flow cytometry. Results are as follows: Figure 5 As shown in A, B, and C, Ce6 The expression of MHCII, CD80, and CD86 in the SNP / B (pH 6.5) group was higher than that in other groups, indicating that... Ce6 Acid-responsive protonation of SNP / B promotes DC maturation.

[0054] Experimental Example 2: Acid-responsive drug-loaded nanoparticles of the present invention ( Ce6Verification of deep penetration effect of SNP / B: The 3D cell spheroids were incubated with SNP / B or INP / B at pH 7.4 or pH 6.5 for 2 hours, and then the distribution of nanoparticles in the 3D cell spheroids was observed by laser confocal. The specific test process is as follows: Ce6 The 3D cell spheroids were incubated with SNP / B or INP / B at pH 7.4 or pH 6.5 for 2 hours, and then the distribution of nanoparticles in the 3D cell spheroids was observed by laser confocal. The specific test process is as follows: Ce6 The 3D cell spheroids were incubated with SNP / B or INP / B at pH 7.4 or pH 6.5 for 2 hours, and then the distribution of nanoparticles in the 3D cell spheroids was observed by laser confocal. The specific test process is as follows: 4T1 cells were inoculated into low-adhesion 24-well plates (0.5*10 5 cells / well), and 3D cell spheroids were obtained after about one week of culture. The 3D cell spheroids were incubated with SNP / B or INP / B at pH 7.4 or pH 6.5. After 2 hours, the fresh culture medium was replaced, and the distribution of nanoparticles in the 3D cell spheroids was observed by laser confocal. The results are shown in Ce6 The 3D cell spheroids were incubated with SNP / B or INP / B at pH 7.4 or pH 6.5 for 2 hours, and then the distribution of nanoparticles in the 3D cell spheroids was observed by laser confocal. The specific test process is as follows: Ce6 The 3D cell spheroids were incubated with SNP / B or INP / B at pH 7.4 or pH 6.5 for 2 hours, and then the distribution of nanoparticles in the 3D cell spheroids was observed by laser confocal. The specific test process is as follows: Figure 6 The 3D cell spheroids were incubated with SNP / B or INP / B at pH 7.4 or pH 6.5 for 2 hours, and then the distribution of nanoparticles in the 3D cell spheroids was observed by laser confocal. The specific test process is as follows: Ce6 The 3D cell spheroids were incubated with SNP / B or INP / B at pH 7.4 or pH 6.5 for 2 hours, and then the distribution of nanoparticles in the 3D cell spheroids was observed by laser confocal. The specific test process is as follows: Ce6 The 3D cell spheroids were incubated with SNP / B or INP / B at pH 7.4 or pH 6.5 for 2 hours, and then the distribution of nanoparticles in the 3D cell spheroids was observed by laser confocal. The specific test process is as follows:

[0055] Verification of enhanced anti-tumor effect and immune activation effect of acid-responsive drug-loaded nanoparticles (SNP / B) in vivo Ce6 Verification of enhanced anti-tumor effect and immune activation effect of acid-responsive drug-loaded nanoparticles (SNP / B) in vivo 1. Therapeutic effect of acid-responsive drug-loaded nanoparticles (SNP / B) at the animal level Ce6 Therapeutic effect of acid-responsive drug-loaded nanoparticles (SNP / B) at the animal level 1х10 6 BALB / c mice were injected with 1х10 3 BALB / c mice were injected with 1х10 Ce6 BALB / c mice were injected with 1х10 Ce6 BALB / c mice were injected with 1х10 Ce6 BALB / c mice were injected with 1х10 Ce6 BALB / c mice were injected with 1х10 Ce6 BALB / c mice were injected with 1х10 Ce6 BALB / c mice were injected with 1х10 Ce6The INP / B (L+) group was treated with 660 nm light at an intensity of 0.5 W / cm for 5 min. Throughout the treatment, the tumor volume was measured every two days using calipers. The formula for calculating the tumor volume is as follows: Volume (mm) 3 = 0.5 × length × width 2 Experimental results are as follows Figure 7 As shown, the tumors in the PBS group grew the fastest. Ce6 INP / B (L-) Ce6 INP / B (L+) and Ce6 The SNP / B(L-) group showed a certain inhibitory effect on tumor growth. Ce6 The SNP / B (L+) group exhibited the most significant inhibitory effect on tumor growth, which may be due to the acid-responsive drug-loaded nanoparticles of this invention. Ce6 SNP / B can simultaneously enhance tumor killing and anti-tumor immune effects under light conditions.

[0056] 2. Acid-responsive drug-loaded nanoparticles ( Ce6 SNP / B) immune enhancement effect at the animal level Tumor tissue was isolated and minced, and digested with a digestive solution (containing collagenase IV 1 mg / ml + DNase 100 µg / ml + hyaluronidase 100 µg / ml) for 45 min. After filtering, the tissue was centrifuged (450 g, 5 min), and the supernatant was discarded. Each tube was resuspended in 6 mL of 40% Percoll solution, centrifuged (800 g, 20 min, 6°C, 2°C), and the supernatant was discarded. Then, each tube was lysed with 2 mL of erythrocyte lysis buffer for 3 min, capped with PBS, centrifuged (450 g, 5 min), and the supernatant was discarded. After flow cytometry counting, 1 million cells were taken per tube, and 30 µL of CD16 / 32 antibody solution was added. The cells were blocked for 15 min, followed by 30 µL of fluorescently labeled antibody mixture. The cells were incubated at 4 ℃ in the dark for 45 min. Each tube was capped with PBS solution, centrifuged (3000 g, 2 min), resuspended, filtered, and the M2-TAM ratio was detected by flow cytometry.

[0057] To verify the activation of the immune system at the tumor site, mice were euthanized, tumor tissue was isolated, and lymph nodes and spleen were drained. After grinding the lymph nodes, the cell suspension was transferred to centrifuge tubes via a filter and centrifuged (450 g, 5 min), discarding the supernatant. CD16 / 32 antibody solution was added to each tube of cells, and the cells were blocked at 4 ℃ for 15 min. Then, 30 µL of fluorescently labeled antibody mixture was added, and the cells were incubated at 4 ℃ in the dark for 45 min. After incubation, each tube was filled with PBS solution and centrifuged (3000 g, 2 min) to remove excess antibody. The cells were then resuspended in 200 μL of PBS, filtered into flow cytometry tubes, and the maturity of dendritic cells (DCs) was detected by flow cytometry.

[0058] After grinding the spleen, the cell suspension was transferred to centrifuge tubes through a filter and centrifuged (450 g, 5 min). 1 mL of erythrocyte lysis buffer was added to each tube, and lysis was performed at room temperature for 3 min. PBS was added to stop lysis, and the tubes were centrifuged again (450 g, 5 min), discarding the supernatant. CD16 / 32 antibody solution was added to each tube, and the cells were blocked at 4 ℃ for 15 min. Then, 30 µL of fluorescently labeled antibody mixture was added, and the tubes were incubated at 4 ℃ in the dark for 45 min. After incubation, each tube was filled with PBS and centrifuged (3000 g, 2 min) to remove excess antibody. 200 μL of PBS was added to resuspend the cells, and the mixture was filtered into flow cytometry tubes for loading. Effector T cell activity was detected by flow cytometry. All antibodies were obtained from Biolegend.

[0059] The results are as follows Figure 8 As shown in A, B, and C, Ce6 The SNP / B group had the lowest M2-TAM, which was 50% of that in the PBS group. Ce6 The SNP / B group had the highest proportion of mature DC cells, which was 1.5 times that of the PBS group; Ce6 The SNP / B group had the highest proportion of effector T cells, twice that of the PBS group. These results indicate that... Ce6 SNP / B, under acidic conditions in tumors, can respond by releasing BLZ-945 and shrinking in size, simultaneously achieving BLZ-945's elimination of tumor cells and Ce6's deep penetration, thereby effectively killing tumor cells and inducing DC maturation under light irradiation. Therefore, the acid-responsive drug-loaded nanoparticles of this invention ( Ce6 SNP / B can enhance the effect of photodynamic therapy and induce a strong immune response through multiple pathways, thereby significantly inhibiting tumors.

[0060] The present invention has been described in detail above with reference to the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. Drug-loaded nanoparticles, characterized in that, The active ingredient includes a hydrophobic small molecule inhibitor and poly 2-(azepan-1-yl) ethyl methacrylate; The active ingredient includes a hydrophobic small molecule inhibitor and poly 2-(azepan-1-yl) ethyl methacrylate; The carrier includes a photosensitizer-bonded amphiphilic polymer; The carrier includes a photosensitizer-bonded amphiphilic polymer; The photosensitizer-bonded amphiphilic polymer includes a photosensitizer, a hydrophilic segment and a hydrophobic segment.

2. The drug-loaded nanoparticle of claim 1, wherein, The average particle size of the drug-loaded nanoparticles is 20 nm to 70 nm.

3. The drug-loaded nanoparticle of claim 1, wherein, The hydrophobic small molecule inhibitor accounts for 1% to 5% of the total mass of the drug-loaded nanoparticles.

4. The drug-loaded nanoparticle of claim 1, wherein, The photosensitizer is selected from at least one of chlorin e6, de-magnesium chlorophyllin a or verteporfin; And / or; the hydrophilic segment is selected from polyethylene glycol; And / or; the hydrophobic segment is selected from at least one of polylactic acid, polycaprolactone or polyphosphate.

5. The drug-loaded nanoparticle of claim 1, wherein, The hydrophobic small molecule inhibitor is selected from at least one of sunitinib, pesdecatinib, edotecarin or vimsertinib.

6. A method of preparing the drug-loaded nanoparticle according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: S1, mixing the photosensitizer-bonded amphiphilic polymer, poly 2-(azepan-1-yl) ethyl methacrylate and hydrophobic small molecule inhibitor dissolved in the first solvent respectively, and dropping into the second solvent under stirring, and stirring to form drug-loaded nanogel by self-assembly; S2, remove the first solvent, centrifuge to remove free hydrophobic small molecule inhibitor, and obtain drug-loaded nanoparticles.

7. The production method according to claim 6, wherein In step S1, the mass ratio of the photosensitizer-bonded amphiphilic polymer and the hydrophobic small molecule inhibitor is 5 to 10:1; and / or; The mass ratio of the poly 2-(azepan-1-yl) ethyl methacrylate and the photosensitizer-bonded amphiphilic polymer is 0.1 to 1:

1.

8. The preparation method according to claim 6, characterized in that, The concentration of the photosensitizer-bonded amphiphilic polymer after being dissolved in the first solvent is 5 mg / mL to 20 mg / mL; And / or; the concentration of the hydrophobic small molecule inhibitor after being dissolved in the first solvent is 10 mg / mL to 20 mg / mL.

9. The drug-loaded nanoparticles of any one of claims 1 to 5 for use in the preparation of an antitumor drug.

10. Use according to claim 9, characterized in that, The tumor includes at least one of breast cancer, colorectal cancer and melanoma.

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