Drug-loaded nanoparticles for regulating tumor microenvironment and preparation method and application thereof

Abstract

This invention relates to a method for preparing drug-loaded nanoparticles that regulate the tumor microenvironment. The steps include: S1, dispersing CaCO3 nanoparticles in a first solvent, adding dopamine hydrochloride solution, lysine solution, and ammonia, reacting for a first time, terminating the reaction, and separating the solid and liquid to obtain the first nanoparticles; S2, dispersing the first nanoparticles in a second solvent, adding alendronate sodium solution, adjusting the pH to neutral, reacting for a second time to obtain the second nanoparticles; S3, dispersing the second nanoparticles in a second solvent, adding dimethyl sulfoxide, shikonin solution, and dihydroporphyrin E6 solution, reacting for a third time, and separating the solid and liquid to obtain the drug-loaded nanoparticles. The drug-loaded nanoparticles of this invention can be used for multi-target therapy, targeting tumor cells. In addition to directly killing tumor cells with ROS generated by PDT and SHK, they induce ICD in cancer cells, reverse the acidic tumor microenvironment, and increase CD4+. + T cell infiltration and a reduction in Treg cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a drug-loaded nanoparticle for regulating the tumor microenvironment, its preparation method, and its application. Background Technology

[0002] Osteosarcoma (OS) is the most common primary malignant bone tumor in adolescents, exhibiting highly aggressive clinical manifestations. Due to its high heterogeneity and high rate of lung metastasis, treatment is extremely challenging. For the past 30 years, neoadjuvant chemotherapy has been the gold standard for treating this disease, aiming to maximize tumor resection while preventing recurrence. However, early-stage, occult tumor cells mean that cancer can recur at any time in the future, and the 5-year survival rate for some metastatic patients is less than 20%. Therefore, immunotherapy has emerged as one of the most promising treatment options for bone tumors.

[0003] Currently, existing immunotherapies for osteosarcoma have poor efficacy, primarily due to the fact that osteosarcoma is an immunosuppressive "cold" tumor and its complex and variable tumor microenvironment (TME). Clinical samples of osteosarcoma show a high proportion of myeloid cells (especially TAM2 type) and a low proportion of effector T cells. Therefore, simply administering immune activators, relying solely on the limited number of T cells, is insufficient to elicit a significant immune response to kill tumor cells. Given the high heterogeneity of osteosarcoma and the lack of targeted therapeutic targets, reshaping the TME of osteosarcoma and relieving the immunosuppressive microenvironment is a crucial treatment approach for prolonging patient survival.

[0004] Acidity, as one of the most prominent characteristics of the tumor microenvironment, is primarily caused by abnormal tumor metabolism, hypoxia, and vascular dysfunction. Existing research has shown that the acidic tumor microenvironment is a significant contributor to the immunosuppressive microenvironment. For example, it can inhibit the function of T cells and NK cells, promoting the recruitment of immunosuppressive cells (MDSCs, Treg cells). Furthermore, it can regulate macrophage function, promoting the survival of M2-type macrophages and creating an environment unaffected by the immune system for cancer cells. Therefore, reversing the acidic microenvironment to promote macrophage polarization towards the M1 type can alleviate the immunosuppressive microenvironment holistically.

[0005] Photodynamic therapy (PDT) has been shown to induce immunogenic cell death (ICD) by generating reactive oxygen species (ROS) that trigger oxidative stress in the endoplasmic reticulum (ER), leading to the transport of large amounts of ER calreticulin (CRT) to the cell membrane surface and the release of other related damage-associated molecular patterns (DAMPs), ultimately resulting in the immunogenic death of tumor cells. Secondly, the type I mechanism of PDT shows that it can achieve an anti-tumor response by recruiting macrophages in the tumor microenvironment (TME) and polarizing them to the M1 type. However, the ROS generated in PDT have a very short half-life, and their intracellular diffusion depth is also greatly limited, which makes it difficult to induce effective ER oxidative stress. Therefore, PDT alone is difficult to achieve strong and durable adaptive anti-tumor efficacy in cancer immunotherapy and often needs to be combined with other methods to enhance the ICD effect on tumors.

[0006] Given the characteristics of the osteosarcoma microenvironment and the limitations of single treatment methods, there is an urgent need for drug-loaded nanoparticles that can regulate the tumor microenvironment, as well as their preparation methods and applications. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing drug-loaded nanoparticles for regulating the tumor microenvironment, their preparation method, and their applications.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of this invention is to provide a method for preparing drug-loaded nanoparticles that regulate the tumor microenvironment, comprising the steps of:

[0010] S1. CaCO3 nanoparticles are dispersed in a first solvent, and hydrochloric acid dopamine solution, lysine solution and ammonia are added. After the reaction time is first, the reaction is terminated, and the first nanoparticles are obtained after solid-liquid separation.

[0011] S2. The first nanoparticles are dispersed in the second solvent, alendronate sodium (ALN) solution is added, the pH is adjusted to neutral, and after a second reaction time, the second nanoparticles are obtained.

[0012] S3. The second nanoparticles are dispersed in a second solvent, and dimethyl sulfoxide (DMSO), shikonin (SHK) solution and dihydroporphyrin e6 (Ce6) solution are added. After a third reaction time, solid-liquid separation is performed to obtain the drug-loaded nanoparticles.

[0013] Preferably, the steps further include:

[0014] S0. Preparation of the CaCO3 nanoparticles: CaCl2 solution is added dropwise to Na2CO3 solution. After the fourth reaction time, the reaction is terminated. After solid-liquid separation, the CaCO3 nanoparticles are obtained.

[0015] Preferably, in step S0,

[0016] The reaction conditions include: an ice bath and uniform stirring;

[0017] The termination reaction includes adding 10 times the volume of deionized water;

[0018] The solid-liquid separation includes: filtration, high-speed centrifugation, discarding the supernatant, washing multiple times, and vacuum drying.

[0019] Preferably, in step S1,

[0020] The first solvent includes: ethanol and deionized water;

[0021] The reaction conditions include: room temperature;

[0022] The termination reaction includes: adding 5 times the volume of deionized water;

[0023] The solid-liquid separation includes high-speed centrifugation and multiple washing processes.

[0024] Preferably, in step S2,

[0025] The second solution is deionized water;

[0026] The pH adjustment includes adding Tris-HCl buffer;

[0027] The reaction conditions include: room temperature and stirring.

[0028] Preferably, in step S3,

[0029] The dimethyl sulfoxide is added before the shikonin solution and / or the dihydroporphyrin E6 solution;

[0030] The reaction conditions include: protection from light, room temperature, and uniform stirring.

[0031] The solid-liquid separation includes: high-speed centrifugation, multiple washing processes, and vacuum drying.

[0032] A second aspect of the present invention is to provide drug-loaded nanoparticles prepared by the preparation method described above.

[0033] A third aspect of the present invention is to provide the use of drug-loaded nanoparticles as described above in the preparation of drugs for treating osteosarcoma.

[0034] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0035] The drug-loaded nanoparticles of the present invention are based on polydopamine (PDA), which has a surface that can be modified with multiple functions. ALN with bone-targeting ability is modified on its surface, and the photosensitizer Ce6 and SHK are co-adsorbed onto the surface of CaCO3 by utilizing its excellent adsorption properties to prepare "pomegranate" shaped drug-loaded nanoparticles.

[0036] The drug-loaded nanoparticles of this invention can be used for multi-target therapy, targeting tumor cells. The ROS generated by PDT and SHK can directly kill tumor cells, induce ICD in cancer cells, reverse the acidic tumor microenvironment, regulate the transformation of tumor-associated macrophages TAM2 into anti-tumor TAM1, and increase CD4. + T cell infiltration and reduction of Treg cells achieve a long-lasting immune effect. Attached Figure Description

[0037] Figure 1 A shows scanning electron microscope images of each nanoparticle;

[0038] Figure 1 B is a particle size characterization diagram of each nanoparticle;

[0039] Figure 2 This is a graph showing the drug release rate of drug-loaded nanoparticles in vitro.

[0040] Figure 3 This is the ultraviolet absorption spectrum of the drug-loaded nanoparticles;

[0041] Figure 4 In vitro cytotoxicity diagrams of various nanoparticles;

[0042] Figure 5 Figures showing the cell apoptosis and necrosis induced by various nanoparticles;

[0043] Figure 6 ROS generation capability diagrams for each nanoparticle;

[0044] Figure 7 The in vitro ICD-inducing capacity of each nanoparticle is shown in the graph.

[0045] Figure 8 Diagrams showing macrophage polarization induced by various nanoparticles;

[0046] Figure 9 In vivo targeting capability diagram of each nanoparticle;

[0047] Figure 10 The in vivo anti-tumor effects of various nanoparticles are shown in the diagram. Detailed Implementation

[0048] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0049] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0050] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0051] Material

[0052] Shikonin, dihydroporphyrin e6, and alendronate sodium were purchased from Aladdin; LPS and recombinant mouse interleukin-4 (IL-4) were provided by PeproTech (Rocky Hill, NJ, USA). Primary antibodies against CD206, iNOS, cleaved-caspase-3, CRT, and GAPDH were purchased from Cell Signaling Technology (Boston, USA). CCK-8 assay kit, Annexin V-FITC / PI apoptosis kit, DCFH-DA probe, Mito-Tracker Green, Lyso-Tracker Green, and DAPI probe were purchased from Beyotime Biotechnology. FITC anti-mouse / human CD11b, PerCP-Cy5.5 anti-mouse F4 / 80, Pacific Blue™ anti-mouse CD86, PE / Cy7 anti-mouse CD206, FITC anti-mouse CD45, APC-Fire™ 750 anti-mouse CD3, PE-Cy7 anti-mouse CD4, PerCP-Cy5.5 anti-mouse CD8α, PE anti-mouse CD25, Alexa Fluor 647 anti-mouse Foxp3, and AF700 anti-mouse granzyme B were purchased from BioLegend (San Diego, CA, USA). Mouse IFN-γ and TNF-α ELISA kits were sourced from Linko Biotechnology Co., Ltd. (Hangzhou, China). Other chemicals were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0053] Example 1

[0054] This embodiment provides a drug-loaded nanoparticle for regulating the tumor microenvironment and its preparation method. The preparation steps include:

[0055] S0. Under ice-water bath and uniform stirring at 1200 r / min, 5 mL of 2 mol / L CaCl2 solution was slowly added dropwise to 10 mL of 1 mol / L Na2CO3 solution. After stirring continuously for 10 min, 10 times the volume of deionized water was added to terminate the reaction. The solution was slowly filtered through a 220 nm pore size filter, centrifuged at 10000 r / min for 10 min and the supernatant was discarded. After washing with deionized water, the solution was centrifuged again and repeated 3 times. After vacuum drying, CaCO3 nanoparticles were obtained.

[0056] S1. Disperse 50 mg of CaCO3 nanoparticles in 50 mL of the first solvent (ethanol and deionized water in a volume ratio of 1:1). After mixing thoroughly, add 1 mL of 0.2 mg / mL dopamine hydrochloride solution, 48 mL of 10 mmol / L lysine solution, and 1 mL of 30% ammonia solution. After reacting at room temperature for a period of time, add 5 times the amount of deionized water to terminate the reaction. Centrifuge at 10000 r / min for 10 min and discard the supernatant. Wash with anhydrous ethanol and deionized water alternately, repeating 3 times to obtain the first nanoparticles (NPs).

[0057] S2. Disperse 50 mg of the first nanoparticles in deionized water, add 10 mL of 1 mol / L alendronate sodium (ALN) solution, add Tris-HCl buffer to adjust the pH to 8, stir the reaction at room temperature for 12 hours to obtain the second nanoparticles (A-NPs).

[0058] S3. Disperse 50 mg of the second nanoparticles in deionized water, add 50 mL of dimethyl sulfoxide (DMSO), stir thoroughly, and then add shikonin (SHK) solution and dihydroporphyrin e6 (Ce6) solution in no particular order. The molar ratio of shikonin to dihydroporphyrin e6 is 0.6:1. Stir at a constant speed for 24 hours at room temperature in the dark, centrifuge at 10000 r / min for 10 min, wash with deionized water, repeat 3 times, and then vacuum dry to obtain the drug-loaded nanoparticles (A-NPs@(SHK+Ce6)).

[0059] In summary, the drug-loaded nanoparticles of the present invention are based on polydopamine (PDA), which has a multifunctional surface modification capability. ALN with bone-targeting ability is modified on its surface, and its excellent adsorption properties are utilized to co-adsorb the photosensitizer Ce6 and SHK onto the surface of CaCO3 to prepare "pomegranate"-shaped drug-loaded nanoparticles.

[0060] Example 2

[0061] The drug synergistic index was determined using a CCK-8 assay. Based on the KI index, drug loading was determined. The chemical structure of the drug-loaded nanoparticles was detected using infrared spectroscopy. The particle size, zeta potential, and polydispersity index (PDI) of the nanoparticles were measured using a Malvern (UK) nanoparticle size analyzer. The morphology of the nanoparticles was observed using a field-emitting scanning electron microscope (SEM) (Hitachi, S4800). The encapsulation efficiency and drug loading of the ALN-modified nanoparticles were determined using fluorescence spectrophotometry and ultraviolet spectrophotometry, and calculated using the following formula:

[0062] Encapsulation efficiency (%) = (M 包封药物 ) / (M 添加药物 )×100%;

[0063] Drug loading (%) = (M 包封药物 ) / (M 纳米颗粒 )×100%.

[0064] Stability and in vitro drug release of drug-loaded nanoparticles: The stability of the nanoparticles was assessed by dispersing them in PBS containing 10% FBS. Particle size changes were examined at different time points. The in vitro drug release rate of the drug-loaded nanoparticles was measured using dialysis. ALN-modified drug-loaded nanoparticles were placed in dialysis bags (MWCO, 8kDa-14kDa) and suspended in PBS containing 0.05% sodium dodecyl sulfate at different pH values ​​(7.4, 6.8, and 5.2), and gently shaken at 100 rpm at 37°C. At specified time points, 1 mL of release medium was taken, and the concentrations of dihydroporphyrin E6 and shikonin were measured using a UV spectrophotometer, with equal volumes of fresh medium added.

[0065] Cell culture: Mouse osteosarcoma cell line K7M2, mouse macrophage cell line RAW264.7, and mouse fibroblasts L929 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. K7M2 and RAW264.7 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. Mouse fibroblasts L929 were cultured in ECM containing 5% FBS, 1% ECG, and 1% penicillin / streptomycin. All cell lines were stored in an incubator at 37°C containing 5% CO2 and 95% air.

[0066] In vitro cytotoxicity: K7M2 cells in logarithmic growth phase were seeded at a density of 5000 cells / well in 96-well plates and cultured for 24 hours. Cell morphology was then observed. Cells were treated with free Ce6, SHK, SHK+Ce6, NPs@(SHK+Ce6), and A-NPs@(SHK+Ce6), respectively. After 24 hours of culture, the Ce6, SHK+Ce6, NPs@(SHK+Ce6), and A-NPs@(SHK+Ce6) groups were subjected to PDT treatment. All groups were cultured for another 24 hours, and cell viability was assessed using the CCK-8 assay kit according to the manufacturer's instructions.

[0067] Cell damage site study: K7M2 cells in logarithmic growth phase were seeded at a density of 5000 cells / well in 35 mm confocal dishes. Cells were treated with A-NPs@(SHK+Ce6) for 24 hours. Mitochondrial and lysosomal nuclear probes were used for staining, and the colocalization of drugs with subcellular organelles was observed by laser confocal microscopy.

[0068] Flow cytometry analysis: (1) To quantitatively analyze the apoptosis rate of K7M2 cells before and after different treatment groups, PDT treatment was performed after 24 hours of co-culture. Cells and supernatant were collected after another 24 hours of culture and stained with V-FITC / PI apoptosis detection kit. All samples were detected by flow cytometry. (2) To detect the ability of cells to generate reactive oxygen species, K7M2 cells before and after different treatment groups were stained with DCFH-DA kit and the ability of cells to generate ROS was analyzed by flow cytometry. (3) To detect the ICD induced by PDT and shikonin, calreticulin CRT staining was performed and the ability of cells and tumor tissues to induce ICD was analyzed by flow cytometry. (4) To evaluate the in vivo polarization ability of drug-loaded nanoparticles, tumor tissues from different treatment groups were digested with hyaluronidase and DNase. Red blood cells were then separated using red blood cell lysis buffer, and cells were extracted using lymphocyte separation buffer for T cell sorting. The cells were then incubated with specific biomarkers for immune cells, such as CD86, CD206, Foxp3, and granzyme B. The cell suspension was filtered, and flow cytometry was used to analyze the content of myeloid cells and T cells in the tumor tissue. Quantitative analysis was performed on the proportion of M1 and M2 macrophages and CD4+. + and CD8 + The proportion of T cells.

[0069] Western blotting: After treatment, cells or tumor tissues were collected from each treatment group and lysed on ice for 30 minutes using pre-prepared RIPA lysis buffer. Following lysis, the cells were centrifuged at 12000g for 30 minutes at 4°C. The supernatant was collected and BCA quantified. Samples were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). The membrane was incubated at room temperature for 1 hour (with TBST containing 5% skim milk powder), followed by overnight incubation with primary antibody at 4°C. The membrane was washed with TBST, incubated with secondary antibody for 2 hours, washed with TBST again, and developed. GAPDH or β-action was used as a control.

[0070] qRT-PCR and ELISA: After treatment, cells and tumor tissues were collected. Total RNA was extracted from macrophages using TRIzol reagent (Thermo, USA) and reverse transcribed into cDNA using the TaKaRa PrimeScript RT kit (TaKaRa, Japan) according to the manufacturer's instructions. Real-time quantitative PCR was then performed using SYBR Premixed Dimer™ (TaKaRa, Japan) according to the manufacturer's instructions. β-action was used as a control to normalize mRNA levels. Cytokine levels (IFN-γ and TNF-α) in culture medium or tumor tissues were detected using an ELISA kit according to the manufacturer's instructions.

[0071] In vivo biodistribution: Using the K7M2 tibial orthotopic model, when the tumor volume reached 150 mm... 3 -200mm 3 Balb / c mice were randomly divided into three groups: SHK+Ce6, Nps@(SHK+Ce6), and A-Nps@(SHK+Ce6). The intravenous injection dose was 5 mg / kg, and real-time fluorescence detection was performed on the mice at different time points (0h-24h) using a small animal in vivo fluorescence imaging system. Images were processed using Living Image 4.5.1 software. To further investigate the distribution of nanoparticles in different tissues of mice, mice were anesthetized and sacrificed at 6h and 12h after intravenous injection. The heart, liver, spleen, lung, kidney, brain, and tumors were then isolated and placed in culture dishes. The fluorescence intensity was detected using an in vivo fluorescence imaging system with an excitation wavelength of 410 nm and an emission wavelength of 660 nm.

[0072] In vivo efficacy experiment: Using the K7M2 tibial orthotopic model, when the tumor volume reached 250mm... 3 -300mm 3Balb / c mice were randomly divided into groups of five. The treatment groups were: control (no treatment), SHK (5 mg / kg), Ce6-P (5 mg / kg + light), (SHK+Ce6)-P (5 mg / kg + light), NPs@(SHK+Ce6) (5 mg / kg), NPs@(SHK+Ce6)-P (5 mg / kg + light), and A-NPs@(SHK+Ce6) (5 mg / kg + light). All treatment groups received the medication for three consecutive days. The photodynamic therapy (PDT) group received PDT six hours after the third day of medication. Mouse body weight was monitored at days 1, 3, 5, 7, 10, 12, and 14 post-PDT, and tumor volume was measured with calipers. Mice were sacrificed after 14 days, tumors were removed, and weighed. Tumor growth curves were plotted, and tumor inhibition rates were calculated. One day after photodynamic therapy, three mice were selected from each group, sacrificed, and their tumors were removed. The tumor tissue was placed in 4% paraformaldehyde, prepared into sections, and then subjected to HE and TUNEL examinations.

[0073] Tumor volume calculation formula: V(mm) 3 = (length × width) 2 )÷2.

[0074] Tumor inhibition rate (%) = (1-W) treat ÷W control )×100%

[0075] Statistical analysis: All data were analyzed using Graphpad Prism 8.0 and IBM SPSS 18. Data are presented as mean ± standard deviation, and each experiment was repeated three times. Adjusted t-tests were used to compare differences between groups. Statistically significant differences were described as *P < 0.05, **P < 0.01, and ***P < 0.001.

[0076] Experimental results: SEM showed that the drug-loaded nanoparticles exhibited a "pomegranate" shape. Figure 1 A); The CaCO3 nanoparticles have a particle size of approximately 160 nm, the first nanoparticles (NPs) have a particle size of approximately 185 nm, the average particle size of the drug-loaded first nanoparticles (Nps@(SHK+Ce6)) is approximately 190 nm, and the drug-loaded nanoparticles (A-NPs@(SHK+Ce6)) have a particle size of approximately 200 nm. Nanoparticles of this diameter can enter the bloodstream and exert their effects. Figure 1 B). Figure 2The drug-loaded nanoparticles (A-NPs@(SHK+Ce6)) exhibited rapid release under acidic conditions (pH=5.2); the encapsulation efficiencies of the drug-loaded nanoparticles (A-NPs@(SHK+Ce6)) for SHK and Ce6 were 61.59±4.13% and 56.74±4.09%, respectively, with drug loadings of 10.24±1.69% and 9.87±1.75%, respectively. The drug-loaded nanoparticles (A-NPs@(SHK+Ce6)) were confirmed, and the UV absorption spectrum showed that the maximum absorption wavelength of the drug-loaded nanoparticles (A-NPs@(SHK+Ce6)) was at 665 nm. Figure 3 ).

[0077] The CCK-8 assay evaluated the antitumor activity of drug-loaded nanoparticles. Results showed that compared to the single use of free Ce6-P or SHK, the combination group (SHK+Ce6)-P effectively inhibited the proliferation of osteosarcoma cells. The IC50 of NPs@(SHK+Ce6)-P was significantly higher than that of (SHK+Ce6)-P. 50 The value decreased significantly, showing a statistically significant difference. There was no statistically significant difference in the in vitro therapeutic effect of drug-loaded nanoparticles before and after ALN modification. Figure 4 Previous studies have shown that PDT induces tumor cell death through both apoptosis and necrosis. Annexin FITC / PI assays showed that NPs@(SHK+Ce6)-P and (SHK+Ce6)-P significantly increased apoptosis and necrosis rates compared to the free drug group alone. Figure 5 This may be related to the ROS generated.

[0078] PDT-induced ICD triggers the ER stress response through the production of ROS, leading to the transport of large amounts of calreticulin (CRT) to the cell membrane surface and the release of other related DAMPs, ultimately resulting in immunogenic death of tumor cells. In this patent, using the ROS probe DCFH-DA staining, all experimental groups undergoing PDT showed significantly increased ROS production compared to the unexposed groups. Furthermore, the combined drug group (SHK+Ce6)-P produced more ROS than the drug-only groups (Ce6-P and SHK), indicating that both PDT and SHK can generate ROS to kill tumor cells, and that their combination enhances the effect. Figure 6 Simultaneously, after SHK or Ce6-P treatment, CRT exposure on K7M2 tumor cells significantly increased. Figure 7 ).

[0079] TAMs exhibit high plasticity, polarizing between TAM1 and TAM2 types. Multiple studies have shown that TAM2 type is associated with immunosuppression and poor prognosis. Since macrophages constitute a large proportion of osteosarcoma samples, regulating macrophage transformation to TAM1 type and remodeling TAMs to eliminate immunosuppressive barriers is an effective means to improve anti-tumor efficacy. In TransWell experiments, after co-culturing TAM / K7M2, different experimental groups were treated with SHK, Ce6-P, SHK+Ce6, (SHK+Ce6)-P, A-Nps@(SHK+Ce6), and A-Nps@(SHK+Ce6)-P, respectively. The PDT group received 2J of light for 1 min. Immunofluorescence results showed that the SHK and Ce6-P groups reflected downregulation of the M2 type marker (CD206) and upregulation of the M1 type marker (TNF-α). In the (SHK+Ce6)-P group, after combining the two drugs with PDT, the M1 type polarization trend was obvious. Compared to the combined free drug group, the ALN-modified nanoparticles exhibited more pronounced M1-type polarization. This also indicates that the addition of CaCO3 can modulate the acidic microenvironment and reverse the TAM2-type polarization. Figure 8 ).

[0080] Photosensitizers emit light in the near-infrared (NIR) region and can be used as fluorophores for in vivo imaging, helping to adjust parameters during PDT treatment. Simultaneously, the fluorescence of photosensitizers can distinguish between normal tissue and lesion areas, serving as an image-guided tool for optical biopsy, etc. In this patent, bone-targeting ALN-modified nanoparticles are used to enhance targeting and improve the efficacy of PDT, allowing ROS generated during PDT to exert maximum efficacy within their half-life, thus improving therapeutic effects. To investigate the biodistribution of nanoparticles in vivo and determine the drug administration and illumination interval, in vivo fluorescence imaging was performed at different time points after intravenous injection of 5 mg / kg nanoparticles in a K7M2 tibial orthotopic mouse model. The results showed that both Nps@(SHK+Ce6) and A-Nps@(SHK+Ce6) were effectively distributed to the tumor site, but A-Nps@(SHK+Ce6) showed higher tumor accumulation. At 12 hours, the mice were euthanized and dissected; in vitro imaging results also showed that the tumor accumulation of both nanoparticles before and after ALN modification was significantly higher than that of the free drug. Figure 9 ).

[0081] The antitumor effect of A-Nps@(SHK+Ce6) photodynamic therapy was evaluated using an immunologically active K7M2 tibial orthotopic tumor mouse model. The results showed that both ALN-modified and unmodified nanoparticles effectively inhibited tumor growth after photodynamic therapy, demonstrating the benefits of multi-target combination regulation of the microenvironment and the definite therapeutic effect of SHK-enhanced photodynamic therapy. Figure 10Tunel examination of tumor sections showed that A-Nps@(SHK+Ce6)-P induced extensive apoptosis in the tissues, and there was no significant decrease in animal body weight during treatment. H&E results also showed no significant toxic side effects on normal organs.

[0082] In summary, the drug-loaded nanoparticles of this invention can serve as multi-target therapy, targeting tumor cells. The ROS generated by PDT and SHK can directly kill tumor cells, induce ICD in cancer cells, reverse the acidic tumor microenvironment, regulate the transformation of tumor-associated macrophages TAM2 into anti-tumor TAM1, and increase CD4+. + T cell infiltration and reduction of Treg cells achieve a long-lasting immune effect.

[0083] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing drug-loaded nanoparticles for treating osteosarcoma, characterized in that the steps include... include: S1. CaCO3 nanoparticles are dispersed in a first solvent, and hydrochloric acid dopamine solution, lysine solution and ammonia are added. After the reaction time is first, the reaction is terminated, and the first nanoparticles are obtained after solid-liquid separation. The first solvent includes ethanol and deionized water; S2. Disperse the first nanoparticles in the second solvent, add alendronate sodium solution, adjust the pH to neutral, and after a second reaction time, obtain the second nanoparticles. The second solvent is deionized water; S3. The second nanoparticles are dispersed in a second solvent, and dimethyl sulfoxide, shikonin solution and dihydroporphyrin E6 solution are added. After a third reaction time, solid-liquid separation is performed to obtain the drug-loaded nanoparticles.

2. The preparation method according to claim 1, characterized in that, The steps also include: S0. Preparation of the CaCO3 nanoparticles: CaCl2 solution is added dropwise to Na2CO3 solution. After the fourth reaction time, the reaction is terminated. After solid-liquid separation, the CaCO3 nanoparticles are obtained.

3. The preparation method according to claim 2, characterized in that, In step S0, The reaction conditions include: an ice bath and constant stirring. The termination reaction includes adding 10 times the volume of deionized water; The solid-liquid separation includes: filtration, high-speed centrifugation, discarding the supernatant, washing multiple times, and vacuum drying.

4. The preparation method according to claim 1, characterized in that, In step S1, The reaction conditions include: room temperature; The termination reaction includes: adding 5 times the volume of deionized water; The solid-liquid separation includes high-speed centrifugation and multiple washing processes.

5. The preparation method according to claim 1, characterized in that, In step S2, The pH adjustment includes adding Tris-HCl buffer; The reaction conditions include: room temperature and stirring.

6. The preparation method according to claim 1, characterized in that, In step S3, The dimethyl sulfoxide is added before the shikonin solution and / or the dihydroporphyrin E6 solution; The reaction conditions include: protection from light, room temperature, and uniform stirring. The solid-liquid separation includes: high-speed centrifugation, multiple washing processes, and vacuum drying.

7. A drug-loaded nanoparticle prepared by the preparation method according to any one of claims 1-6.

8. The use of the drug-loaded nanoparticles as described in claim 7 in the preparation of a drug for treating osteosarcoma.

Images