Nanocomposites loaded with PD-L1 siRNA and antitumor peptides, their preparation and application in anti-renal cancer

Abstract

This invention discloses a nanocomposite loaded with PD-L1 siRNA and an anti-tumor peptide, its preparation, and its application in treating renal cell carcinoma. The nanocomposite uses a dendritic macromolecule as its carrier, and the loaded peptide is Tat-HSPB1, whose amino acid sequence is shown in SEQ ID NO.1. The amino acid sequence of the loaded PD-L1 siRNA is shown in SEQ ID NO.2. The composite of this invention utilizes multiple interactions between phenylboronic acid, nucleic acids, and peptides to achieve stable co-assembly and acid-responsive intracellular release. While reducing the cytotoxicity of high-generation dendritic macromolecules, it effectively delivers siPD-L1 to specifically downregulate PD-L1 expression in renal tumor cells, thereby relieving tumor immunosuppression. Synergistically, with the anti-tumor activity of Tat-HSPB1 itself, this composite can significantly induce tumor cell death and inhibit its proliferation, providing a novel, highly efficient, and low-toxicity strategy for renal cell carcinoma immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to nanocomposites loaded with PD-L1 siRNA and antitumor peptides, their preparation and application in the treatment of renal cell carcinoma. Background Technology

[0002] Renal cell carcinoma (RCC) is a common malignant tumor of the urinary system. Metastatic RCC patients are insensitive to traditional radiotherapy and chemotherapy, and their 5-year survival rate is extremely low. In recent years, the combined use of targeted drugs (such as tyrosine kinase inhibitors TKIs) and PD-1 / PD-L1 immune checkpoint inhibitors has brought survival benefits to some patients. However, primary or acquired drug resistance is common in clinical practice, and TKIs are often accompanied by side effects such as hypertension, proteinuria, and hand-foot syndrome, which limits their long-term use. At the same time, immune checkpoint inhibitors themselves also have problems such as low target specificity, long half-life, high risk of autoimmune reactions, and limited response rate due to the tumor immunosuppressive microenvironment. Therefore, there is an urgent need to develop novel and efficient anti-tumor treatment strategies.

[0003] Silencing PD-L1 expression in tumor cells using small interfering RNA (siRNA) promises to achieve long-term immune checkpoint blockade and inhibit tumor immune escape. However, naked siRNA is easily degraded by nucleases under physiological conditions, and its negative charge makes it difficult to cross the cell membrane, necessitating the use of safe and efficient delivery carriers. While commonly used cationic polymers (such as polyethyleneimine derivatives) can achieve a certain degree of PD-L1 knockdown, they generally suffer from poor cell compatibility and uncontrollable structure. Furthermore, although nanoparticle drug delivery systems exhibit unique advantages in tumor targeting and controlled drug release, their industrial production is challenging, process control is complex, and costs are high. Additionally, some carriers (such as high-dose dendritic macromolecules) still exhibit significant cytotoxicity, hindering clinical translation.

[0004] Polyamide-amine dendrimers (PAMAMs), as a class of structurally well-defined and easily functionalized non-viral vectors, have attracted widespread attention in nucleic acid delivery. Phenylboronic acid (PBA) can simultaneously form reversible boron ester bonds with the cis-diol on the siRNA ribose ring and boron-nitrogen coordination bonds with the amino groups on the vector surface, thereby endowing the vector with self-assembly and acid-responsive dissociation properties, potentially enabling efficient and low-toxicity intracellular delivery. Meanwhile, functional peptides such as Tat-HSPB1 possess both membrane-penetrating and antitumor activities; co-assembly with the vector can further enhance the stability and therapeutic efficacy of the complex.

[0005] In summary, a PD-L1 siRNA delivery system that combines good cell compatibility, controllable structure, and synergistic antitumor activity is currently lacking. Those skilled in the art urgently need to construct a functionalized PAMAM nanocomplex for co-delivering PD-L1 siRNA and Tat-HSPB1 peptide to provide a novel strategy for tumor immunotherapy. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing nanocomposites loaded with PD-L1 siRNA and anti-tumor peptides, their preparation, and their application in treating renal cell carcinoma.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect is to provide a nanocomposite loaded with PD-L1 siRNA and an antitumor peptide, wherein the carrier of the nanocomposite is a dendritic macromolecule, the antitumor peptide is Tat-HSPB1, and its amino acid sequence is shown in SEQ ID NO.1: RKKRRQRRR-GPSWDPFRDWYPHSRLFDQAFGL; the amino acid sequence of the PD-L1 siRNA is shown in SEQ ID NO.2: CCUACUGGCAUUUGCUGA ACGCAUU.

[0008] Furthermore, the dendritic macromolecule is a fifth-generation polyamide-amine dendritic macromolecule G5.NH2 PAMAM, whose surface is modified with phenylboronic acid.

[0009] The second aspect is to provide a method for preparing the above-mentioned nanocomposite loaded with PD-L1 siRNA and anti-tumor peptides, including the following steps: Step 1: The fifth-generation polyamide-amine dendritic macromolecule G5.NH2 is reacted with 4-bromomethylphenylboronic acid to obtain the phenylboronic acid-modified functionalized dendritic macromolecule G5-PBA; Step 2: Mix the G5-PBA solution with the PD-L1 siRNA solution and incubate to obtain the intermediate complex G5-PBA / PD-L1 siRNA loaded with siRNA. Step 3: Mix the G5-PBA / PD-L1 siRNA solution with the peptide Tat-HSPB1 solution, incubate, and then perform ultrafiltration and centrifugation to obtain the final product G5-PBA / PD-L1 siRNA / Tat-HSPB1 nanocomposite loaded with siRNA and peptide.

[0010] Furthermore, in step one, the molar ratio of G5.NH2 to 4-bromomethylphenylboronic acid is 1:(30-50); the reaction solvent is dimethyl sulfoxide.

[0011] Furthermore, in step one, the reaction temperature is 65-70℃ and the reaction time is 20-26h; the reaction product is dialyzed in pure water for 2-3 days using a dialysis bag with a rejection capacity of 8000-14000Da, and then freeze-dried.

[0012] Furthermore, in step two, the mixing ratio of G5-PBA solution to PD-L1 siRNA solution is calculated as a nitrogen-to-phosphorus ratio (N / P), with N / P being (10-15):1.

[0013] Furthermore, in step two, the incubation time is 20-30 minutes; the incubation temperature is room temperature.

[0014] Furthermore, in step three, the mass ratio of G5-PBA / PD-L1 siRNA to Tat-HSPB1 is 1:(1-3).

[0015] Furthermore, in step three, the incubation time is 30-40 minutes; the incubation temperature is room temperature; and the parameters for ultrafiltration centrifugation are 4500g and centrifugation for 30-40 minutes.

[0016] The third aspect is the application of the aforementioned nanocomposite loaded with PD-L1 siRNA and anti-tumor peptides in the preparation of drugs for treating clear cell renal cell carcinoma.

[0017] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: This invention constructs a nanocomposite possessing both gene silencing and peptide antitumor activity by co-delivering PD-L1 siRNA and Tat-HSPB1 peptide through a phenylboronic acid-modified dendritic macromolecule. This composite utilizes the multiple interactions between phenylboronic acid, nucleic acids, and peptides to achieve stable co-assembly and acid-responsive intracellular release. While reducing the cytotoxicity of high-generation dendritic macromolecules, it effectively delivers siPD-L1 to specifically downregulate PD-L1 expression in renal tumor cells, thereby relieving tumor immunosuppression. Synergistically, the Tat-HSPB1 peptide itself exhibits antitumor activity, and this composite significantly induces tumor cell death and inhibits their proliferation. Its antitumor effect is significantly superior to that of the peptide alone, providing a novel, highly efficient, and low-toxicity strategy for renal cancer immunotherapy. Attached Figure Description

[0018] Figure 1 For G5-PBA 1 HNMR spectrum.

[0019] Figure 2 Agarose gel electrophoresis images of G5-PBA / PD-L1 siRNAs with different N / P ratios.

[0020] Figure 3In the figure, a represents the hydrodynamic diameter of G5-PBA / PD-L1 siRNA with different N / P ratios, and b represents the surface potential of G5-PBA / PD-L1 siRNA with different N / P ratios.

[0021] Figure 4 The flow cytometry results of siRNA phagocytosis after co-incubating the G5-PBA / Cy3-siPD-L1 complex with (a) Caki-1 and (b) A498 for 4 hours under different N / P conditions, and (c) fluorescence quantitative results.

[0022] Figure 5 The results of the CCK8 experiment after treating Caki-1 with different concentrations of G5-PBA / siPD-L1, Tat-HSPB1, and G5-PBA / siPD-L1 / Tat-HSPB1 are shown in the figure.

[0023] Figure 6 The image shows the results of WB experiments after treating Caki-1 with siPD-L1, G5-PBA / siPD-L1, and G5-PBA / siPD-L1 / Tat-HSPB1.

[0024] Figure 7 Statistical graphs of tumor volume (a) and body weight (b) in mice treated with PBS, Tat-HSPB1, G5-PBA / siPD-L1, and G5-PBA / siPD-L1 / Tat-HSPB1. Detailed Implementation

[0025] 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 invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0026] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available.

[0027] Example 1 This embodiment provides a nanocomposite loaded with PD-L1 siRNA and an anti-tumor peptide, the preparation method of which specifically includes: Step 1: Weigh 50 mg of G5.NH2 PAMAM (1.992 μmol) and dissolve it in 5 mL of DMSO. Weigh 23.24 mg of 4-bromomethylphenylboronic acid (Br-PBA, 96.09 μmol) and dissolve it in 5 mL of DMSO. Add the above Br-PBA solution to the vigorously stirred G5.NH2 PAMAM solution and react at 70 °C for 24 h. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyze it in pure water for 3 days. Finally, freeze-dry the product G5-PBA and store it in a sealed container at -20 °C.

[0028] Step 2: Dissolve 944 μg of G5-PBA in 0.472 mL of pure water to prepare a 2 mg / mL solution, and dissolve 56 μg of PD-L1 siRNA in 56 μL of pure water to prepare a 1 μg / μL PD-L1 siRNA solution. Mix the G5-PBA solution and the PD-L1 siRNA solution and incubate at room temperature for 30 min to obtain the intermediate complex G5-PBA / PD-L1 siRNA (which can also be referred to as G5-PBA / siPD-L1).

[0029] Step 3: Dissolve 3 mg of peptide Tat-HSPB1 in 1 mL of PBS, mix the Tat-HSPB1 solution with 1 mg of G5-PBA / PD-L1 siRNA, and incubate at room temperature for 30 min. After the reaction, ultrafilter and centrifuge (4500 g, 30 min) to obtain G5-PBA / PD-L1 siRNA / Tat-HSPB1 nanocomplex.

[0030] Example 2 Characterization 2.1 1H NMR spectrum of G5-PBA 6 mg of G5-PBA prepared in Example 1 was weighed and dissolved in 600 μL of deuterated water, and then the 1H NMR spectrum of the material was measured using a nuclear magnetic resonance spectroscopy instrument. The results are as follows: Figure 1 As shown, the proton peaks at chemical shifts of 2.2–3.5 ppm represent the methylene proton peaks of G5.NH2 PAMAM, while the characteristic proton peaks of PBA are at 7.0–8.0 ppm, indicating that PBA has been successfully modified onto the surface of G5.NH2 PAMAM. By integrating the proton peak regions of G5.NH2 PAMAM and PBA, it was calculated that each G5.NH2 PAMAM was modified with 39 PBA molecules.

[0031] 2.2 Compressibility of G5-PBA for PD-L1 siRNA The compressibility of G5-PBA on PD-L1 siRNA was determined using an agarose gel retardation assay. A series of G5-PBA / PD-L1 siRNA complexes were prepared with nitrogen-to-phosphorus ratios (N / P, i.e., the molar ratio of primary amino groups on the dendritic macromolecule surface to phosphate groups in the siRNA backbone) of 0.25, 0.5, 1, 2, 3, 4, and 5, with a fixed siRNA dosage of 1 μg / well. The complexes were incubated at room temperature for 20 min. A 1.0% (w / v) agarose gel containing nucleic acid dye (4S Green Plus) was prepared. The complexes were mixed with loading buffer and added to the gel wells, and electrophoresis was performed at 90 V for 30 min. After electrophoresis, the migration of PD-L1 siRNA was observed and recorded using a gel imaging system. The results are as follows: Figure 2 As shown, when the N / P ratio is 1, the siRNA in the G5-PBA / PD-L1 siRNA complex is completely blocked in the loading well, indicating that G5-PBA has a strong gene compression ability.

[0032] 2.3 Hydration kinetics diameter and potential characterization of the G5-PBA / siPD-L1 complex The G5-PBA prepared in Example 1 was dissolved in ultrapure water. With a fixed amount of PD-L1 siRNA (5 μg), a series of G5-PBA / PD-L1 siRNA complexes were prepared at nitrogen-to-phosphorus (N / P) ratios of 0.25, 0.5, 1, 2, 3, 4, and 5, and incubated at room temperature for 20 min. After incubation, ultrapure water was added to a final volume of 1 mL. The hydrodynamic particle size and surface zeta potential of the complexes were measured using a Malvern laser particle size analyzer (Malvern, UK) at a wavelength of 633 nm.

[0033] The results are as follows Figure 3 As shown, when the N / P ratio increased from 0.25 to 5, the surface Zeta potential of the G5-PBA / PD-L1 siRNA complex gradually increased with the increase of the ratio, and finally stabilized below 60 mV; the hydrated particle size of all complexes was around 600 nm. These results indicate that G5-PBA can effectively compress PD-L1 siRNA into nanocomplexes over a wide N / P range, and the complex surface is positively charged at high N / P ratios, demonstrating its potential as a gene delivery vector.

[0034] 2.4 Cell uptake experiment The phagocytic capacity of Caki-1 and A498 cells for the G5-PBA / Cy3-siPD-L1 complex was evaluated using these cell models. According to 1.5×10 5Cells were seeded at a density per well in 12-well cell culture plates using 1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS, and incubated at 37°C for 12 h at 5% CO2. The medium was then replaced with medium containing the G5-PBA / Cy3-siPD-L1 complex (N / P = 5, 10, 15, 20, 25, 50), and co-cultured with the cells at 37°C for 4 h at 5% CO2. The medium and complex in the wells were then discarded, and the cells were washed twice with PBS, digested with trypsin, collected by centrifugation, and their fluorescence intensity was analyzed by flow cytometry to characterize cellular uptake of the G5-PBA / Cy3-siPD-L1 complex.

[0035] The results are as follows Figure 4 As shown, the G5PBA / Cy3-siPD-L1 complex was successfully endocytosed by cells 4 hours after transfection, under different N / P ratios. With increasing N / P ratio, the fluorescence intensity initially increased and then decreased. The fluorescence intensity in Caki-1 cells was generally higher than that in A498 cells; therefore, Caki-1 cells were selected as the subsequent model for validation. Furthermore, the strongest fluorescence intensity was detected in Caki-1 cells at N / P = 15, indicating that the G5-PBA / Cy3-siPD-L1 complex was most easily taken up by Caki-1 cells at N / P = 15, exhibiting the highest endocytosis efficiency.

[0036] 2.5 Encapsulation efficiency of G5-PBA / siPD-L1 for Tat-HSPB1 To determine the encapsulation efficiency of Tat-HSPB1 by G5-PBA / siPD-L1, different masses of G5-PBA / siPD-L1 were weighed and mixed with FAM-labeled Tat-HSPB1, and incubated for 20 min. After incubation, unencapsulated Tat-HSPB1 was removed by centrifugation at 4500g for 30 min three times using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10000 Da. The solution in the outer tube was collected, and different concentrations of FITC-Tat-HSPB1 were prepared to construct a standard curve. Finally, the fluorescence intensity values ​​of the standard curve solution and the solution in the outer tube were measured using a multi-functional microplate reader. The encapsulation efficiency and drug loading were calculated using the formulas: Encapsulation efficiency = (Total amount of peptide added - Amount of unloaded peptide) / Total amount of peptide added × 100%, and Drug loading rate = Amount of loaded peptide / Total mass of complex × 100%. The results are shown in Table 1. When the mass ratio of G5-PBA / siPD-L1 to Tat-HSPB1 is 1:3, the encapsulation efficiency of Tat-HSPB1 by G5-PBA / siPD-L1 reaches 66.1%.

[0037] Table 1

[0038] 2.6 Characterization of hydration kinetic diameter, potential, and polydispersity index of G5-PBA / siPD-L1 and G5-PBA / siPD-L1 / Tat-HSPB1 1 mg each of G5-PBA / siPD-L1 and G5-PBA / siPD-L1 / Tat-HSPB1 prepared in Example 1 were dissolved in 1 mL of ultrapure water to determine their surface potential, hydration kinetic diameter, and polydispersity index. The results are shown in Table 2. The hydration kinetic diameter of G5-PBA / siPD-L1 was 101.2 nm, the surface potential was 47.9 mV, and the polydispersity index was 0.28. The hydration kinetic diameter of the G5-PBA / siPD-L1 / Tat-HSPB1 nanocomposite was 231.1 nm, the surface potential was 30 mV, and the polydispersity index was 0.256.

[0039] Table 2

[0040] Example 3 Cell Experiment 3.1 Cytotoxicity assay Caki-1 adherent cells in the logarithmic growth phase were digested with trypsin. Once most cells detached, culture medium was added to stop the digestion. The cells were collected and transferred to centrifuge tubes, centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in complete culture medium, counted, and the cell density adjusted to 5 x 10⁹ cells / mL. 4 Cells were seeded at a rate of 100 μL per well in 96-well plates and incubated overnight at 37°C with 5% CO2. The next day, different concentrations of G5-PBA / siPD-L1, Tat-HSPB1, and G5-PBA / siPD-L1 / Tat-HSPB1 were added, and the plates were returned to the incubator for another 22 hours. Then, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for another 2 hours. The absorbance at 450 nm was then measured using a microplate reader. The data were integrated, and the cell viability of each group was calculated.

[0041] The results are as follows Figure 5 As shown in (a): Within the concentration range of 0-16 μg / ml, the cell viability of G5-PBA / siPD-L1 was generally 80%-90%, with no significant cytotoxicity. However, significant cytotoxicity occurred when the concentration of G5-PBA / siPD-L1 exceeded 16 μg / ml. And as... Figure 5As shown in (bc), Tat-HSPB1 and G5-PBA / siPD-L1 / Tat-HSPB1 significantly inhibited human renal cancer cells Caki-1 in a gradient-dependent manner. At the same peptide concentration, G5-PBA / siPD-L1 / Tat-HSPB1 inhibited Caki-1 cells more strongly than Tat-HSPB1.

[0042] 3.2 Western Blot (Protein Immunoblotting) Experiment The silencing efficiency of G5-PBA / siPD-L1 on PD-L1 was evaluated using Caki-1 cells as a cell model. The cells were prepared at a density of 2 × 10⁶ cells per well. 5 Caki-1 cells were seeded in 6-well plates using 1640 medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. The cells were incubated at 37°C with 5% CO2 for 24 hours. The medium was then replaced with PBS, naked PD-L1 siRNA, G5-PBA / siPD-L1 complex (N / P = 15), and G5-PBA / siPD-L1 / Tat-HSPB1 complex (N / P = 15) and co-cultured with the cells for 4 hours. The medium was then replaced with 10% FBS and cultured for another 24 hours. Cells were digested with trypsin and collected, lysed on ice, and centrifuged at 12000 rpm for 5 minutes at 4°C. The supernatant protein was collected. SDS-PAGE electrophoresis, cell transfer, immunoassay, and ECL chemical contrast agent fixing were then performed sequentially, and the gel images were analyzed.

[0043] Experimental results are as follows Figure 6 As shown, naked siRNA and PBS showed almost no gene silencing, while G5-PBA / siPD-L1 and G5-PBA / siPD-L1 / Tat-HSPB1 exhibited significant gene silencing. This indicates that G5-PBA / siPD-L1 and G5-PBA / siPD-L1 / Tat-HSPB1 can effectively silence the expression of PD-L1 on the surface of Caki-1 cells.

[0044] Example 4 Animal Experiment All animal experiments were conducted strictly in accordance with animal protection association standards. 2-3 week old female BALB / c nude mice used in the experiments were purchased from Shanghai Jicui Pharmaceutical Co., Ltd. (Shanghai, China). 5 × 10 6 One Caki-1 cell was subcutaneously injected into a mouse, and the tumor volume was increased to approximately 35 mm. 3 At approximately 10:00 AM, the mice were randomly divided into 4 groups (5 mice in each group).

[0045] The mice were divided into the following groups: control group (PBS); G5-PBA / siPD-L1 group (N / P=15, siPD-L1=0.18mg / kg); Tat-HSPB1 group (Tat-HSPB1, 18mg / kg); and G5-PBA / siPD-L1 / Tat-HSPB1 group (N / P=15, siPD-L1=0.18mg / kg, Tat-HSPB1=18mg / kg). The materials for each group were injected into mice peritumorally. The day treatment began was designated as day 0. Treatment was administered once daily, and mouse weight and tumor size were recorded every 3 days.

[0046] from Figure 7 As shown in (a), compared to the PBS group, Tat-HSPB1, G5-PBA / siPD-L1, and G5-PBA / siPD-L1 / Tat-HSPB1 all exhibited a certain degree of tumor-inhibiting effect. Compared to the Tat-HSPB1 group, the tumor volume of G5-PBA / siPD-L1 / Tat-HSPB1 was smaller, possibly because the PD-L1 siRNA in the G5-PBA / siPD-L1 group endogenously inhibited PD-L1 expression on the surface of clear cell renal cell carcinomas, thus further reducing the binding of PD-1 and PD-L1, thereby restoring the immune response function of T cells and killing tumor cells. Figure 7 As can be seen in (b), the weight changes in each group were not significant, indicating that the material has good biocompatibility.

[0047] 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 content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanocomposite loaded with PD-L1 siRNA and an antitumor peptide, characterized in that, The carrier of the nanocomposite is a dendritic macromolecule, the antitumor polypeptide is Tat-HSPB1, and its amino acid sequence is shown in SEQ ID NO.1; the amino acid sequence of the PD-L1 siRNA is shown in SEQ ID NO.

2.

2. The nanocomposite loaded with PD-L1 siRNA and antitumor peptide according to claim 1, characterized in that, The dendritic macromolecule is a fifth-generation polyamide-amine dendritic macromolecule G5.NH2 PAMAM, whose surface is modified with phenylboronic acid.

3. A method for preparing a nanocomposite loaded with PD-L1 siRNA and an antitumor peptide as described in any one of claims 1-2, characterized in that, Includes the following steps: Step 1: The fifth-generation polyamide-amine dendritic macromolecule G5.NH2 is reacted with 4-bromomethylphenylboronic acid to obtain the phenylboronic acid-modified functionalized dendritic macromolecule G5-PBA; Step 2: Mix the G5-PBA solution with the PD-L1 siRNA solution and incubate to obtain the intermediate complex G5-PBA / PD-L1 siRNA loaded with siRNA. Step 3: Mix the G5-PBA / PD-L1 siRNA solution with the peptide Tat-HSPB1 solution, incubate, and then perform ultrafiltration and centrifugation to obtain the final product G5-PBA / PD-L1 siRNA / Tat-HSPB1 nanocomposite loaded with siRNA and peptide.

4. The preparation method according to claim 3, characterized in that, In step one, the molar ratio of G5.NH2 to 4-bromomethylphenylboronic acid is 1:(30-50); the reaction solvent is dimethyl sulfoxide.

5. The preparation method according to claim 3, characterized in that, In step one, the reaction temperature is 65-70℃ and the reaction time is 20-26h; the reaction product is dialyzed in pure water for 2-3 days using a dialysis bag with a rejection capacity of 8000-14000Da, and then freeze-dried.

6. The preparation method according to claim 3, characterized in that, In step two, the mixing ratio of G5-PBA solution and PD-L1siRNA solution is calculated as a nitrogen-to-phosphorus ratio (N / P), with N / P being (10-15):

1.

7. The preparation method according to claim 3, characterized in that, In step two, the incubation time is 20-30 minutes and the incubation temperature is room temperature.

8. The preparation method according to claim 3, characterized in that, In step three, the mass ratio of G5-PBA / PD-L1 siRNA to Tat-HSPB1 is 1:(1-3).

9. The preparation method according to claim 3, characterized in that, In step three, the incubation time is 30-40 minutes; the incubation temperature is room temperature; and the parameters for ultrafiltration centrifugation are 4500g and centrifugation for 30-40 minutes.

10. The use of the nanocomposite loaded with PD-L1 siRNA and antitumor peptide as described in any one of claims 1-2 in the preparation of a drug for treating clear cell renal cell carcinoma.

Images