Construction of carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system and application of carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system in treatment of breast cancer

Abstract

The invention discloses construction of a carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system and application of the carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system in treatment of breast cancer, and belongs to the field of medicine.The novel multifunctional nanoparticle (TPP-Ce6 at-siPD-L1NPs) is developed on the basis of a carrier-free self-assembly strategy, amino-modified triphenylphosphine (TPP) and Ce6 are covalently linked to form an amphiphilic compound, and the amphiphilic compound is used for preparing the carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system. The TPP-Ce6-coated siPD-L1NPs is obtained by taking TPP-Ce6-coated siPD-L1NPs as a template and loading siPD-L1 by utilizing electrostatic interaction, the physicochemical properties of the TPP-Ce6-coated siPD-L1NPs are subjected to comprehensive characterization and in-vitro activity research, and the result shows that the nanoparticles can remarkably inhibit the proliferation of tumor cells and enhance the cytotoxicity of the TPP-Ce6-coated siPD-L1NPs through a mitochondrial targeting mechanism, the anti-tumor curative effect and safety of the nanoparticles are verified, obvious systematic toxicity is not caused, and the TPP-Ce6-coated good clinical application prospects are shown.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the construction of a carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system and its application in the treatment of breast cancer. Background Art

[0002] Triple-negative breast cancer (TNBC), as the most aggressive subtype of breast cancer, accounts for about 15% of all breast cancer cases. Its estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 are all negatively expressed. This molecular classification results in its insensitivity to endocrine therapy and HER2-targeted therapy. Clinically, surgical resection combined with platinum or taxane chemotherapy regimens has been used for a long time. However, problems such as the high systemic toxicity of chemotherapy drugs and the easy generation of drug resistance in tumors significantly limit the curative effect. The 5-year survival rate of patients is not high, and the risk of recurrence and metastasis is higher than that of other subtypes. In recent years, the introduction of immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies) has shown some efficacy in treating TNBC, but the single-drug response rate is low, and the immunosuppressive characteristics of the tumor microenvironment (TME) further weaken the treatment effect.

[0003] PDT is a targeted tumor treatment technology based on photochemical reactions, and its history can be traced back to the early 20th century. This therapy achieves precise killing of cancer cells through the ternary synergistic action of photosensitizers, specific wavelength light sources, and molecular oxygen. In recent years, PDT has become a research hotspot for TNBC treatment due to its local precise killing characteristics. The photosensitizer Ce6 induces apoptosis of tumor cells by generating ROS, but it has insufficient penetration into deep tumors, and there are limitations in penetration depth and spatial precision, oxygen dependence, phototoxicity, and long-term side effects. Moreover, single therapy is difficult to overcome the immunosuppression in the tumor microenvironment.

[0004] RNA interference therapy is a treatment technology based on the RNA interference mechanism. By designing small interfering RNA (siRNA) or short hairpin RNA (shRNA) to target the mRNA of specific pathogenic genes, inducing its degradation or inhibiting translation, gene expression can be precisely regulated. Silencing PD-L1 by siRNA in gene therapy can reverse tumor immune escape, but problems such as short half-life, easy off-target, easy degradation of siRNA, and low targeted delivery efficiency need to be solved urgently. Summary of the Invention

[0005] Objective of the Invention: Aiming at the problems existing in the prior art, the present invention provides a construction method of a carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system. The carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system constructed by the present invention utilizes the spatial confinement effect of the nanocarrier to achieve co-encapsulation, targeted delivery and synergistic release of the photosensitizer and the gene drug. The hydrophobic photosensitizer and negatively charged siRNA are co-loaded by electrostatic adsorption, which can not only protect siRNA from degradation, extend its half-life and reduce off-target effects, but also enrich in tumor sites through the enhanced permeability and retention effect (EPR), reduce the accumulation time in normal tissues, thereby reducing the phototoxicity and side effects of the photosensitizer. The two treatment methods synergistically fight breast cancer and enhance the anti-tumor effect.

[0006] The present invention also provides the carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system and its application.

[0007] Technical Solution: The construction method of a carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system according to the present invention includes the following steps:

[0008] (1) Synthesize TPP-NH2: Mix triphenylphosphine (TPP) and 3-bromo-1-aminopropane hydrobromide, add an organic solvent after fully mixing evenly, heat the obtained solution, filter after cooling to obtain a solid product, wash the solid product and then dissolve and precipitate it again, and dry the precipitate to obtain the product TPP-NH2;

[0009] (2) Synthesize TPP-Ce6: Add HOBT (1-hydroxybenzotriazole), DIPEA (N,N-diisopropylethylamine), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and TPP-NH2 to chlorin e6, stir the mixture in the dark under the protection of an inert gas until the reaction is complete, filter, extract the filtrate, purify the organic phase and then freeze-dry to obtain the required product TPP-Ce6;

[0010] (3) Synthesize nanoparticles TPP-Ce6@siPD-L1NPs: Take siPD-L1 and add it to the TPP-Ce6 solution, mix evenly, perform ultrasonic treatment, keep warm and stand still, and then dialyze to obtain the carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system TPP-Ce6@siPD-L1NPs.

[0011] Among them, in step (1), triphenylphosphine and 3-bromo-1-aminopropane hydrobromide are mixed in a mass ratio of 1:1 - 4:1, and then an acetonitrile solution is added.

[0012] Among them, in step (1), heat to 80 - 90 °C and reflux for 10 - 20 h, filter, wash the solid product, dissolve the solid product with isopropanol, precipitate again and dry to obtain the product.

[0013] Among them, in step (2), the mass ratio of Ce6 to TPP-NH2 is 1-2:2-3.

[0014] Preferably, in step (2), 100-200 mg of Ce6, 100-200 mg of HOBT, 400-500 mL of DIPEA, 100-200 mg of EDCI, and 200-300 mg of TPP-NH2 are taken.

[0015] Among them, the freeze-drying time in step (2) is 36-48 h.

[0016] Among them, in step (3), siPD-L1 is preheated at 50-60 °C for 3-5 min, and then TPP-Ce6 is added. The volume ratio of siPD-L1 to TPP-Ce6 is 1-3:48-144.

[0017] Among them, in step (3), ultrasonic treatment is performed at 400-500 W for 10-60 s, and the heat preservation and static settlement conditions are 1-2 h at 35-37 °C.

[0018] The carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system constructed by the method for constructing the carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system described in the present invention.

[0019] Application of the carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system described in the present invention in the preparation of a drug for treating breast cancer.

[0020] The drug for treating breast cancer described in the present invention is characterized by comprising the carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system described in claim 1 and a pharmaceutically acceptable excipient or carrier.

[0021] Among them, the pharmaceutical preparation is an injection, granule, tablet, capsule, pill or oral liquid.

[0022] Based on a carrier-free self-assembly strategy, the present invention develops a novel multifunctional nanoparticle (TPP-Ce6@siPD-L1 NPs): an amphiphilic complex is formed by covalently connecting aminomodified triphenylphosphine (TPP) with Ce6, and siPD-L1 is loaded by electrostatic interaction.

[0023] The present invention systematically explores the application potential of TPP-Ce6@siPD-L1 NPs in tumor treatment, covering the whole process from the preparation and characterization of the nanoparticles to the evaluation of in vivo efficacy and safety. First, TPP-Ce6@siPD-L1 NPs with mitochondrial targeting ability were successfully prepared, and their physicochemical properties were comprehensively characterized, including particle size, zeta potential, and morphology, etc., laying a foundation for subsequent experiments. Then, in vitro activity studies showed that the nanoparticles could significantly inhibit the proliferation of tumor cells and enhance their cytotoxicity through the mitochondrial targeting mechanism. Next, the anti-tumor mechanism of the nanoparticles was further explored, including changes in mitochondrial membrane potential and expression changes of apoptosis-related proteins. Finally, in vivo studies further verified the anti-tumor efficacy and safety of the nanoparticles. The results of animal experiments showed that the nanoparticles could significantly inhibit tumor growth without causing obvious systemic toxicity, showing good clinical application prospects.

[0024] The present invention has conducted systematic research on various aspects such as the preparation of nanoparticles, in vitro activity, mechanism of action, and in vivo efficacy, providing important experimental basis and theoretical support for the development of mitochondria-targeted nano-drugs.

[0025] The carrier-free nanoparticles prepared in the present invention co-deliver the Ce6 / siPD-L1 system without exogenous carriers, with both high drug loading and tumor mitochondrial targeting. They can induce apoptosis and activate the cGAS-STING pathway at the same time. Meanwhile, siPD-L1 silences the expression of PD-L1 in tumor cells, synergistically enhancing the immune effect. Further, the system of the present invention can generate ROS to induce apoptosis and activate the cGAS-STING immune pathway. At the same time, siPD-L1 relieves the inhibition of T cell function by silencing the expression of PD-L1 in tumor cells, forming an "immune positive cycle" and remodeling the immunosuppressive microenvironment.

[0026] The carrier-free nanoparticles prepared in the present invention can induce apoptosis of tumor cells, target gene silencing, and activate the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway immune pathway: when irradiated with near-infrared (NIR) light, the nanoparticles Ce6 / siPD-L1 can generate reactive oxygen species (ROS). The generated ROS will damage mitochondria. On the one hand, it induces apoptosis of tumor cells; on the other hand, the damaged mitochondria release mtDNA into the cytoplasm, which is recognized by cGAS and then activates the cGAS-STING pathway, thus promoting the activation of T cells and enhancing the immune response of the body. In addition, siPD-L1 targets and silences the expression of the PD-L1 gene, enhancing immune-mediated tumor cell destruction.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0028] The carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system constructed in the present invention is a self-assembled carrier-free nanoparticle integrating the chemotherapeutic drug Ce6 and siPD-L1, which has excellent therapeutic effects in breast cancer treatment.

[0029] The preparation method of TPP-Ce6@siPD-L1 NPs of the present invention is simple, the material cost is low, the prepared nanoparticles have the characteristics of spherical and smooth surfaces, show special cell uptake and lysosomal escape abilities, and at the same time these nanoparticles effectively deliver siPD-L1 to the tumor site, significantly down-regulating the expression of PD-L1 in tumor cells. In addition, the nanoparticles can target tumor mitochondria and induce apoptosis through reactive oxygen species generated by PDT. The infiltration of T cells can be enhanced by activating the cGAS-STING immune pathway, enhancing immunotherapy. Further, in vivo studies have shown that TPP-Ce6@siPD-L1 NPs significantly inhibit tumor growth and maintain good biosafety and low toxicity.

[0030] Generally speaking, the system using self-assembled carrier-free nanoparticles to co-deliver chemotherapeutic drugs and small interfering RNAs in the present invention provides a novel and promising strategy for improving the efficacy of tumor treatment. This material and method have great potential in future medical applications, especially in the treatment of breast cancer. Description of the Drawings

[0031] Figure 1 is the synthetic route diagram of TPP-Ce6;

[0032] Figure 2 is the stability of TPP-Ce6@siPD-L1 NPs in 10% FBS solution;

[0033] Figure 3 is the cell viability after treating 4T1 cells with different concentrations of drugs in each group under light (A); the cell viability after treating 4T1 cells with different concentrations of drugs in each group without light (B);

[0034] Figure 4 is the CLSM imaging analysis after co-incubating cells 4T1 with Ce6, TPP-Ce6 and TPP-Ce6@siPD-L1 NPs for 2, 4, 6 or 12 h respectively;

[0035] Figure 5 is the CLSM imaging analysis (A) after incubating cells 4T1 with Ce6, TPP-Ce6 and TPP-Ce6@siPD-L1 NPs respectively, and the fluorescence intensity analysis (B) after incubating cells 4T1 with Ce6, TPP-Ce6 and TPP-Ce6@siPD-L1 NPs respectively;

[0036] Figure 6Assessment of mitochondrial membrane potential stained with JC-1 (A), fluorescence intensity analysis of mitochondrial membrane potential stained with JC-1 (B);

[0037] Figure 7 For ex vivo tumor weight under different treatments (A); Curve of ex vivo tumor volume change (n = 5) (B).

[0038] Figure 8 Western blotting analysis of PD-L1, Caspase-3, BAX, and BCL-XL proteins in tumor tissues;

[0039] Western blotting analysis of cGAS, STING, TBK1, IRF3, and phosphorylated proteins in 94T1 cells;

[0040] Figure 10 For the relative levels of mRNA of CD8A, CD4, CD80, and CD86 in tumor tissues;

[0041] Figure 11 Transmission electron micrographs of solutions with different nitrogen-phosphorus ratios. Detailed implementation manners

[0042] The present invention will be further described below in conjunction with specific embodiments and the accompanying drawings.

[0043] 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.

[0044] The source of siPD-L1: Shanghai GenePharma Co., Ltd., which includes equimolar sequences 5'-3-: UGUUUGUCCAGAUUACCUCTT and 5'-3': GAGGUAAUCUGGACAAACATT.

[0045] Example 1

[0046] Preparation of multifunctional nanoparticles (TPP-Ce6@siPD-L1 NPs)

[0047] Weigh 1.0 g of TPP and 0.84 g of 3-bromoalanine hydrobromide and mix them well in a round-bottom flask. Then add 7 ml of acetonitrile solution. Under a reflux condenser, heat the resulting solution to 85 °C and reflux for 16 h. After the reaction mixture is cooled to room temperature, filter it through hexane to obtain a solid product. Subsequently, wash the solid with hexane for further purification. Dissolve the washed solid in 100 mL of isopropanol, and then precipitate a white powder by adding cold diethyl ether. Collect and dry the precipitate to obtain the product TPP-NH2; Weigh 100 mg of Ce6 into a round-bottom flask, and then sequentially add 115 mg of HOBT, 445 mL of DIPEA, 163 mg of EDCI, and 217.6 mg of TPP-NH2. Stir the mixture overnight under inert gas protection (protected from light) to ensure complete reaction. After the reaction is completed, filter the reaction solution to remove any insoluble impurities. Extract the filtrate with dichloromethane and water to separate the product, and then obtain the purified product by high-pressure preparative chromatography. Purification conditions: the product solvent is methanol, the mobile phase is acetonitrile. Set Flow rate: 10 ml / min, Detection: 254 nm, Monitor: 397 nm. Set the gradient pressure: first EQU, 5-5, 5 Min, then 5-5, 7 min, 5-95, 25 min, 95-95, 5 min. Finally, lyophilize the purified product for 36 - 48 h to obtain a black powder, which is the desired product TPP-Ce6; The synthesis route of TPP-Ce6 is as Figure 1 shown. First, modify TPP with an amino group, and then connect it with Ce6 through amide condensation to synthesize TPP-Ce6. Next, take 10 μL of siPD-L1 (200 nM) and place it in an enzyme-free centrifuge tube. Preheat it in a water bath at 60 °C for 3 - 5 min, add 480 μL of TPP-Ce6 (100 ng / ml), and make the components evenly distributed by vortex mixing. Treat it with ultrasound (500 W, 100%) for about 10 s to enhance the binding of TPP-Ce6 and siPD-L1. Place the mixed solution in a water bath at 37 °C for 1 h to promote the formation of TPP-Ce6@siPD-L1 NPs. After incubation, dialyze the solution containing TPP-Ce6@siPD-L1 NPs using a dialysis bag (MCOW: 8000 Da) for 48 h to remove any unreacted components or impurities, and obtain the multifunctional nanoparticles (TPP-Ce6@siPD-L1 NPs).

[0048] Example 2

[0049] Stability evaluation

[0050] To further evaluate the stability of the nanoparticles in serum, a gel electrophoresis experiment was conducted. 20 μl of naked siPD-L1 and an equal volume of nanoparticles (TPP-Ce6@siPD-L1 NPs prepared in Example 1), with siPD-L1 at 200 nM in both, were each mixed with 2 μl of 10% FBS and incubated at room temperature for 0, 6, 12, and 24 h respectively. Then, 20 μl of 0.2% SDS was added to dissociate the nanoparticles. Using a 3% agarose gel, under the conditions of a voltage of 50 V and electrophoresis for 40 min, the degradation of siPD-L1 was evaluated by comparing the intensity and integrity of the siPD-L1 bands at different time points. The results are as Figure 2 shown. Naked siPD-L1 began to be degraded after 6 h, while siPD-L1 in TPP-Ce6@siPD-L1 NPs could still be detected even after 24 h. This observation indicates that the nanoparticles effectively protected siPD-L1 from degradation, thus extending the half-life of siPD-L1.

[0051] Example 3

[0052] Safety assessment

[0053] The safety and cytotoxicity of TPP-Ce6@siPD-L1 NPs were studied by the CCK8 method. The CCK8 kit was used for in vitro cytotoxicity tests according to the manufacturer's instructions. 4T1 cells were seeded in 96-well plates at a cell density of 8×10 3 per well and incubated for 24 h. Then, different concentrations of Ce6, TPP-Ce6, and TPP-Ce6@siPD-L1 NPs (NPs) prepared in Example 1 were added to the wells. After incubation for 6 h, the light irradiation group was subjected to light irradiation treatment (660 nm, 15 mW / cm 2 , 10 min). The non-irradiation group was set under the same experimental conditions. The concentrations of Ce6 in each group were 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 μg / mL respectively. After incubation for 12 h after each group's treatment, 10 μL of CCK8 was added to each well and incubated for another 1 h. Finally, the absorbance value at 450 nm was measured by an enzyme-linked immunosorbent assay (ELISA) reader. As Figure 3 shown, under non-irradiation conditions, the cell viability of all tested concentrations of the nanoparticles remained above 95%, indicating that the synthetic materials have good biosafety. However, under light irradiation conditions (660 nm, 15 mW / cm 2 , 10 min), TPP-Ce6@siPD-L1 NPs showed significantly better tumor cell killing effects than Ce6 and TPP-Ce6. Specifically, at a concentration of 0.6 μg / mL, the cell mortality rate was close to 95%, showing a significant killing effect.

[0054] Example 4

[0055] Uptake ability evaluation

[0056] CLSM was used to observe the uptake of Ce6, TPP-Ce6, and TPP-Ce6@siPD-L1 NPs prepared in Example 1 by 4T1 cells. The cells were seeded in a confocal dish at a density of 1.3×10 5 , incubated at 37 °C for 24 h until the cells were completely adherent, and then treated with drugs in each group for 2 h, 4 h, 6 h, and 12 h, respectively. The final concentration of the drugs in each group was ensured to be 0.3 mg / kg. After incubation, the cells were fixed with 4% paraformaldehyde for 20 min and rinsed 3 times with PBS. Subsequently, the cell nuclei were stained with DAPI for 15 min, and finally washed 3 times with PBS. Finally, the uptake of cells in each experimental group was observed by CLSM. As Figure 4 shown, the cell nuclei were stained with DAPI, and the red fluorescence of Ce6 was visible under near-infrared light. With the extension of the incubation time, the red fluorescence intensity of Ce6 increased in a time-dependent manner. Within just 2 h, TPP-Ce6@siPD-L1 NPs showed significant fluorescence signals, indicating their rapid internalization by cells. Subsequently, after 4 h of incubation, TPP-Ce6 began to internalize. After 6 h of incubation, the tumor cells began to internalize Ce6. TPP-Ce6@siPD-L1 NPs demonstrated high tumor cell uptake ability and were significantly superior to Ce6 and TPP-Ce6, reducing the accumulation time in other tissues and lowering the risk of phototoxicity.

[0057] Example 5

[0058] Cellular ROS content evaluation

[0059] ROS plays a key role in the interaction with intracellular biomacromolecules and often leads to damage of important organelles such as mitochondria and lysosomes. ROS generation was detected by CLSM imaging. 4T1 cells were cultured in a confocal dish at a density of 1.3×10 5 cells per dish for 24 h, and then treated with Ce6, TPP-Ce6, and TPP-Ce6@siPD-L1 NPs prepared in Example 1 for 12 h, respectively. The experiment was divided into a light irradiation group and a non-light irradiation group. After 6 h of drug treatment, the final concentration of the drugs in each group was ensured to be 0.3 mg / kg, irradiated with 660 nm light for 5 min, incubated for another 2 h, then incubated with DCFH-DA for 30 min, and finally rinsed 3 times with PBS. Finally, the generation of green fluorescence of intracellular ROS was observed by CLSM. As Figure 5 shown in A, the cells treated with TPP-Ce6@siPD-L1 NPs and 660 nm light irradiation showed strong fluorescence compared with the control group without light irradiation, as Figure 5As shown in Figure B, compared with Ce6 and TPP-Ce6 after light irradiation, its fluorescence intensity is also more obvious. This strong fluorescence indicates a significant increase in the production of intracellular ROS. ROS plays a crucial role in the interaction with intracellular biomacromolecules and often causes damage to important organelles such as mitochondria and lysosomes. The good ROS production ability of the nanoparticles lays the foundation for subsequent escape from lysosomes and damage to mitochondria.

[0060] Example 6

[0061] Evaluation of cell MMP content

[0062] A decrease in mitochondrial membrane potential (MMP) is usually regarded as a marker of early apoptotic events in cells. In healthy cells, the MMP is higher, and the JC-1 dye forms aggregates and emits red fluorescence. In contrast, in cells with reduced MMP, JC-1 transforms into a monomeric form and emits green fluorescence. Red fluorescence indicates JC-1 aggregates. To detect the effect of the nanoparticles on mitochondrial membrane potential, a JC-1 detection kit was used according to the instructions. 4T1 cells were seeded in a confocal microscope culture dish at a density of 1.3×10 5 and cultured for 24 h. Then, Ce6, TPP-C6, and TPP-Ce6@siPD-L1NPs prepared in Example 1 were added respectively and incubated for 6 h. The final drug concentration in each group was ensured to be 0.3 mg / kg. After treatment, the cells were washed 3 times with PBS and then incubated with JC-1 reagent at 37 °C for 30 min. Finally, the damage of mitochondrial membrane potential in each group of cells was observed by CLSM imaging. As Figure 6 shown, when detecting different light treatment groups, an increase in green fluorescence and a decrease in red fluorescence were observed. This change in fluorescence indicates a decrease in MMP, which is a known indicator of early apoptosis. In these groups, the TPP-Ce6@siPD-L1NPs group had the strongest green fluorescence and was significantly superior to Ce6 and TPP-Ce6, indicating the most significant destructive effect on MMP. As Figure 6 shown in Figure B, the results indicate that TPP-Ce6@siPD-L1NPs can effectively damage the mitochondrial membrane potential under light conditions, thereby causing mitochondrial damage in tumor cells. Example 7

[0063] Evaluation of tumor growth rate

[0064] Establish a triple-negative breast cancer model: The mice used in the experiment were BALB / c white mice (7 - 8 weeks old, 18 - 20 g, female). After purchase, they were allowed to adapt to the environment for 2 - 3 days. Take 4T1 cells in the logarithmic growth phase, digest them with trypsin, wash them with PBS and collect the cells. Centrifuge at 1000 rpm for 5 min and repeat 2 times to obtain cell pellets. Finally, prepare them into 1×10 7Cell suspension at a concentration of [X] cells / mL was placed in an ice-water mixture for later use. Each mouse was inoculated with [X] cells, which were subcutaneously injected into the right chest pad of BALB / c mice using an insulin syringe to establish a triple-negative breast cancer solid tumor model. 6

[0065] In vivo anti-tumor research: When the tumor volume grew to about 100 mm 3 , the mice were randomly divided into 4 groups of 5 mice each. Each group received different treatments: Ce6+L, TPP-Ce6+L, TPP-Ce6@siPD-L1NPs+L, and a PBS group was set as a control. The administration method was tail vein injection, which was carried out on the 1st and 4th days. The dose of Ce6 in all administration groups was 2 mg / kg. For the groups that required light treatment, 6 hours after tail vein injection, the tumor site of the mice was irradiated with 660 nm near-infrared light for 10 min. During the treatment period, the body weight changes and tumor volume of the mice were regularly recorded. The formula for calculating the tumor volume was: Tumor volume = 0.5 × L × W2 (L is the length and W is the width). After 14 days of treatment, the tumor tissues were removed for weighing and photographing, and a tumor growth curve was plotted.

[0066] As Figure 7 shown, the tumor growth rate of the experimental group treated with TPP-Ce6@siPD-L1NPs was the slowest among all experimental groups. Specifically, compared with the experimental group treated with only Ce6 light irradiation and the experimental group treated with TPP-Ce6 light irradiation, the TPP-Ce6@siPD-L1NPs light irradiation group achieved a very significant tumor suppression effect. TPP-Ce6@siPD-L1NPs could efficiently deliver Ce6 and siPD-L1 to the tumor site, induce apoptosis of tumor cells, and exhibit excellent anti-tumor activity. While siPD-L1 alone as a nucleic acid could not accumulate in the tumor and would not produce an effect. At the same time, the tumor suppression effect of TPP-Ce6@siPD-L1NPs in this invention, especially in terms of tumor mass, significantly exceeded that of Ce6 and TPP-Ce6, showing an obvious synergistic effect.

[0067] Example 8

[0068] 4T1 cells in the logarithmic growth phase were taken and seeded at a density of 1×10 7 It should be noted that the specific cell concentration values in and are missing in the original text and are represented by [X] and [Y] respectively in the translation. You need to fill in the correct values according to the actual situation.Inoculate at a density in a petri dish with a diameter of 5 cm and culture for 24 h. Subsequently, treat the cells with PBS, Ce6+L, TPP-Ce6+L, siPD-L1, TPP-Ce6@siPD-L1 NPs, and TPP-Ce6@siPD-L1 NPs+L prepared in Example 1 for 12 h (where "L" represents light irradiation), and ensure that the Ce6 concentration in each group is 0.3 mg / kg. For the light irradiation group, after incubating the drug with the cells for 6 h, irradiate with 660 nm light for 5 min and then continue to incubate for 2 h. After the treatment, collect the cells and centrifuge to obtain cell pellets (1000 rpm, 5 min). Subsequently, add 100 μL of RIPA lysis buffer containing a protease inhibitor (Phosphatase Inhibitor Cocktail (100×), Selleck, B15001) and a phosphatase inhibitor (Protease Inhibitior Cocktail (100×), Selleck, B14001). Place the cells in an ultrasonic crusher and break them for 10 s, then centrifuge at 12000 rpm for 20 min at 4 °C, and collect the supernatant containing cell proteins. Use a BCA protein concentration assay kit to detect the protein concentration in the supernatant. After denaturing the protein samples in a boiling water bath for 5 min, store them in a -80 °C refrigerator for later use.

[0069] Separate the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the conditions of a constant voltage of 170 V for 50 min. Subsequently, transfer the separated proteins onto a PVDF membrane under the conditions of a constant current of 300 mA for 50 min. Wash the membrane 4 times with TBST buffer, 5 min each time. To block non-specific binding sites, block the PVDF membrane with 5% bovine serum albumin (BSA) at room temperature for 1 h. After blocking, wash the membrane with TBST buffer 4 times again, 5 min each time. Next, incubate the membrane with specific primary antibodies overnight at 4 °C. The primary antibodies used include BCL-Xl, BAX, Caspase-3, β-actin, PD-L1, cGAS, STING, P-STING, IRF3, P-IRF3, TBK1, P-TBK1. After incubating with the primary antibodies, wash the membrane 4 times with TBST buffer, 5 min each time. Subsequently, incubate the membrane with the secondary antibody at room temperature for 1 h. Use an ECL luminescence solution to develop the protein bands on the membrane, and perform exposure and photography in a bio-imaging system. Finally, quantitatively analyze the gray values of the protein bands with the aid of Image J software. The results are as Figure 8As shown, in terms of protein expression in tumor tissues, the level of the pro-apoptotic protein BAX in the tumor tissues of the TPP-Ce6@siPD-L1 NPs group was significantly increased, while the expressions of the anti-apoptotic proteins total Caspase-3 and BCL-XL were significantly decreased. At the same time, it showed the strongest inhibitory effect on the expression of the PD-L1 gene. As Figure 9 shown, the phosphorylation of related proteins indicated the successful activation of this pathway, suggesting that TPP-Ce6@siPD-L1 NPs had a good effect on inducing apoptosis of tumor cells. Among them, the phosphorylation of cGAS-STING related proteins indicated the successful activation of this pathway.

[0070] Example 9

[0071] 4T1 cells were seeded on a culture dish with a diameter of 5 cm at a density of 1×10 7 , cultured for 24 h, and then the cells were treated with the PBS prepared in Example 1, Ce6 + light, TPP-Ce6 + light, siPD-L1, TPP-Ce6@siPD-L1 NPs, and TPP-Ce6@siPD-L1 NPs + light for 12 h. Light irradiation group: After the drug was incubated with the cells for 6 h, the cells were irradiated with 660 nm light for 5 min and then incubated for another 2 h. Then, the cell pellet was collected by centrifugation (4°C, 1000 rpm, 5 min).

[0072] First, mRNA was extracted using a cell / tissue total RNA isolation kit (FreeZol Reagent). Then, RT-qPCR was performed according to the following procedure: 1. RNA reverse transcription; 2. Q-PCR detection.

[0073] 1. RNA reverse transcription: cDNA was obtained using HiScript III RT SuperMix for qPCR. The system for removing genomic DNA is shown in Table 1, and the reaction conditions were 42°C for 2 min. The reverse transcription system is shown in Table 2, and the reaction conditions were 37°C for 15 min; 85°C for 5 s.

[0074] Table 1. Removal of genomic DNA (16.0 μL system)

[0075]

[0076] Table 2. Reverse transcription system

[0077]

[0078] 2. q-PCR detection: Detect the concentration of the reverse-transcribed DNA. Aliquot the samples and store them at -80 °C for later use. Use Tap ProUniversal SYBR qPCR Master Mix to detect the mRNA concentration. The Real Time PCR reaction system (20.0 μL system) is shown in Table 3 below:

[0079] Table 3. q-PCR detection reaction system

[0080]

[0081] Perform the PCR reaction according to the conditions shown in Table 4:

[0082] Table 4. PCR reaction

[0083]

[0084] 3. Application of q-PCR detection results 2 -ΔΔCt Calculate and statistically analyze using method 2. The results are as Figure 10 shown. The mRNA levels of immune markers CD86, CD80, CD4, and CD8A were quantitatively analyzed using RT-qPCR technology. The results showed that the expression levels of these genes in the TPP-Ce6@siPD-L1NPs group were higher compared to the other two groups, indicating that TPP-Ce6@siPD-L1NPs enhanced the immune ability of T cells and could reshape the immunosuppressive microenvironment.

[0085] Example 10

[0086] Prepare TPP-Ce6@siPD-L1NPs according to the method of Example 1, with the difference that the nitrogen-phosphorus ratios in TPP-Ce6 and siPD-L1 are adjusted to 20:1, 10:1, 1:1, and 1:10 respectively. TPP-Ce6 provides ammonium ions and siPD-L1 provides phosphorus. The nitrogen-phosphorus ratios in TPP-Ce6 and siPD-L1 in Example 1 were 10:1. By adjusting the addition amounts of the raw materials, the nitrogen-phosphorus ratio was adjusted. Characterized by transmission electron microscopy, the results are as Figure 11 shown. Nanoparticles could not be prepared with other ratios, and the result prepared with 10:1 was the best.

Claims

1. A method for constructing a carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system, characterized in that, The following steps are involved: (1) Synthesis of TPP-NH2: Triphenylphosphine and 3-bromopropylamine hydrobromide are mixed, and an organic solvent is added after thorough mixing. The resulting solution is heated, cooled, and filtered to obtain a solid product. The solid product is washed, dissolved again, and precipitated. The precipitate is dried to obtain the product TPP-NH2; (2) Synthesis of TPP-Ce6: HOBT, DIPEA, EDCI, and TPP-NH2 were added to dihydrochlorin e6, and the mixture was stirred in the dark under inert gas protection until the reaction was complete. The filtrate was filtered and extracted, and the organic phase was purified and freeze-dried to obtain the desired product TPP-Ce6; (3) Synthesis of nanoparticles TPP-Ce6@siPD-L1 NPs: siPD-L1 was added to TPP-Ce6 solution, mixed evenly, and then ultrasonicated. After keeping warm and standing, the mixture was dialyzed to obtain the carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system TPP-Ce6@siPD-L1NPs.

2. The construction method according to claim 1, characterized in that, In step (1), triphenylphosphine and 3-bromopropylamine hydrobromide are preferably mixed in a mass ratio of 1:1-4:1, and then the acetonitrile solution is added.

3. The construction method according to claim 1, characterized in that In step (1), the reaction mixture is heated to 80-90° C. and refluxed for 10-20 h. After filtering, the solid product is washed and dissolved in isopropanol, and the solid product is precipitated and dried again to obtain the product.

4. The construction method according to claim 1, characterized in that In step (2), the mass ratio of Ce6 and TPP-NH2 is 1-2:2-3.

5. The construction method according to claim 1, characterized in that In step (3), siPD-L1 is preheated at 50-60°C for 3-5 minutes, and then TPP-Ce6 is added. The volume ratio of siPD-L1 to TPP-Ce6 is 1-3:48-144.

6. The construction method according to claim 1, wherein, In step (3), ultrasonic treatment is performed at 400-500W for 10-60s, and the temperature is kept at 35-37°C for 1-2h.

7. A carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system constructed by the construction method of the carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system according to claim 1.

8. Use of the carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system 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 comprises the carrier-free nanoparticle co-delivery Ce6 / siPD-L1 system according to claim 1 and a pharmaceutically acceptable excipient or carrier.

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

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