Preparation method of nano delivery system H-C atC / siPD-L1 NPs and application of nano delivery system H-C atC / siPD-L1 NPs in treatment of triple negative breast cancer

Abstract

The invention discloses a preparation method of a nano delivery system H-C (at) C / siPD-L1NPs and application of the nano delivery system H-C (at) C / siPD-L1NPs in treatment of triple negative breast cancer, H4R6RGD and CM beta CD (carboxymethyl-beta-cyclodextrin) are grafted together to form a nano carrier H4R6RGD-CM beta CD, then H4R6RGD-CM beta CD (at) cellatrol / siPD-L1NPs (H-C (at) C / siPD-L1NPs) is prepared, the nano particle is of a spherical structure with good dispersity, and the nano particle has good biocompatibility and can be applied to treatment of triple negative breast cancer. The interaction force of co-assembly mainly comprises hydrophobic action and electrostatic adsorption action. The nano delivery system enhances cellular uptake, can effectively escape from lysosome and has good biocompatibility, and co-delivery of the tripterine and the siPD-L1 promotes tumor apoptosis, improves the immunosuppression microenvironment of tumors and shows the potential in the aspect of enhancing the curative effect of the triple negative breast cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-drug delivery, and specifically relates to a preparation method of a nano-delivery system HC@C / siPD-L1NPs and its application in the treatment of triple-negative breast cancer. Background Art

[0002] Currently, RNAi is a highly effective and specific gene-blocking technology that can rapidly analyze target gene function. Its rapid development has made it a powerful tool for functional genomics and reverse genetics research, and the technology has been widely used by researchers to study gene function, infectious diseases, and gene therapy for malignant tumors. However, several key factors limit its clinical use. First, its poor stability, sensitivity to endogenous nucleases, and easy clearance; second, RNA drugs, due to their negative charge and polymeric properties, have difficulty entering tumor cells to exert their effects; and most importantly, siRNA drugs are generally taken up by tumor cells through endocytosis and are easily engulfed by lysosomes within the cells. Lysosomal enzymes within lysosomes can degrade siRNA drugs, thus affecting their therapeutic effects.

[0003] Treatments using RNA interference technology offer many advantages over traditional treatments such as small molecules and protein-based drugs. First, compared to traditional chemotherapy drugs, siRNA treatment only has an antagonistic effect, while traditional treatments produce both agonist and antagonist effects. Second, the targets of siRNA treatment can exist anywhere, even "undruggable" targets, and siRNA is easier to synthesize than other types of drugs. Although siRNA therapy has more obvious advantages than chemotherapy, the combination of siRNA and small molecules can show better efficacy and improve prognosis for the same disease.

[0004] Cyclodextrin (CD) is a biocompatible cyclic oligosaccharide with a hydrophilic periphery and a well-defined hydrophobic cavity that can bind to a variety of organic molecules, including biologically relevant compounds. Cyclodextrin-based supramolecular nanocapsules have shown great potential in tumor therapy due to their good biocompatibility, low toxicity to healthy cells, and reliable targeted delivery and release of therapeutic drugs. However, single carboxymethyl-β-cyclodextrin lacks tumor targeting and penetration in vivo, so surface modification is required to improve its performance as a drug delivery material. If a cyclodextrin derivative can be designed to effectively deliver siRNA and chemotherapeutic drugs simultaneously, it will expand its application as a delivery material.

[0005] Cell-penetrating peptides (CPPs) are usually composed of thirty or fewer amino acids, and their ability to penetrate the membrane is related to the amino acid sequence. Since most bioactive substances have difficulty in targeting cells to penetrate the cell membrane to exert their therapeutic effects, for example, oligonucleotide drugs face the problem of low membrane penetration efficiency, and conventional delivery strategies such as liposomes, viral vectors, microinjection and electroporation may cause side effects such as high toxicity, poor specificity, immunogenicity and low delivery efficiency, there is an urgent need for a method to efficiently deliver large molecule drugs to cells. Due to the problems faced by conventional large molecule delivery strategies, CPPs have become increasingly popular in drug delivery and are widely used in the delivery of nucleic acids, proteins and chemotherapy drugs. Cell-penetrating peptides can be coupled to various drugs through covalent and non-covalent attachment to help drugs cross biological barriers. In addition, cell-penetrating peptides are also multifunctional carriers. When they are coupled with anti-cancer drugs to target various tumor markers, they can also be used for the specific delivery of tumor cells. CPPs have shown great potential in the efficient and specific delivery of anti-tumor drugs. When used in combination with tumor-targeting peptides, studies have shown that CPPs enhance the specific targeting of drugs to tumors and reduce the adverse reactions of anti-cancer drugs in the body. CPPs offer great potential for the efficient and specific delivery of anti-tumor chemotherapy drugs.

[0006] Celastrol (cela) is a quinone methylated pentacyclic triterpenoid compound, which is a natural active compound extracted from the root of Tripterygium wilfordii. Figure 1 As shown, it shows potential pharmacological activity in various diseases, including inflammation, obesity, cancer and bacterial diseases. Celastrol has potential anti-cancer effects. However, its poor water solubility, low bioavailability and high toxicity hinder its clinical application. Studies have shown that celastrol can exhibit cytotoxic, hepatotoxic and even neurotoxic effects at high concentrations or long-term exposure. In order to solve these problems, many drug delivery methods and technologies have been reported to improve the efficiency of celastrol and reduce its toxicity. In order to construct new celastrol delivery systems, some new formulation nanotechnologies such as liposomes, micelles, microemulsions and nanoparticles continue to emerge.

[0007] Programmed cell death protein 1 (PD-1) is a cell surface receptor initially discovered to be preferentially expressed in apoptotic cells. PD-1 was later identified as a key immune checkpoint regulating the threshold for T and B cell responses to antigens. As a key checkpoint for T cells, PD-1 plays a central role in regulating their cellular function. Programmed cell death ligand 1 (PD-L1), in addition to being expressed on the cell surfaces of T lymphocytes, B lymphocytes, dendritic cells (DCs), and macrophages, is also highly expressed on cancer cells. PD-L1 promotes tumor immune escape by inducing T cell exhaustion and immune tolerance. Therefore, abnormal upregulation of PD-L1 levels in cancer cells and some immune cells can lead to immune escape, inducing the formation of an immunosuppressive tumor microenvironment and promoting tumor growth. Blocking PD-1 / PD-L1 can alleviate T cell functional suppression, promote the release of proinflammatory cytokines, T cell proliferation, and CTL activation, restoring T cell cytotoxicity and inducing tumor regression, thereby resulting in better clinical outcomes. PD-1 / PD-L1 is an ideal immunotherapy target for restoring anti-tumor specific T cell effector function.

[0008] The tumor microenvironment (TME) is a highly structured ecosystem that includes a variety of immune cells, tumor-associated fibroblasts (CAFs), endothelial cells, and extracellular matrix (ECM). Targeting the TME has obvious advantages in treatment because the cancer cell genome is unstable and prone to drug resistance, while the non-tumor cells in the TME are genetically more stable and more fragile. On the other hand, treatment strategies targeting the tumor microenvironment (TME) need to be precisely targeted to specific phenotypic variations in non-tumor cells related to tumor growth, thereby preventing side effects that may be caused by targeting normal cells in other parts of the body. To achieve this goal, it is necessary to have a deep understanding of the differences between tumor-promoting host cells and normal host cells in the TME at the molecular and cellular levels. However, the broad spectrum of functional states of a large number of non-tumor host cells in the TME poses challenges to deciphering their mechanisms of action and identifying targets for intervention in the tumor-promoting TME. Summary of the Invention

[0009] Purpose of the invention: To address the problems existing in the prior art, the present invention designed a multifunctional transmembrane peptide, H4R6RGD, with the ability to target tumors, escape lysosomes, and promote cellular uptake. At the same time, a cyclodextrin derivative nanodelivery system, H4R6RGD-CMβCD@celastrol / siPD-L1 nanoparticles (HC@C / siPD-L1 NPs), modified with the multifunctional transmembrane peptide was developed to co-deliver Celastrol (cela) and siPD-L1. Under the acidic conditions of the tumor, histidine dissociates and protonates, the nanoparticles dissociate, and the lysosomal burst drug is successfully released into the cytoplasm. The co-loading of celastrol and siPD-L1 activates endoplasmic reticulum stress. This system not only has a good killing effect on tumor cells, but also improves the tumor immunosuppressive microenvironment and reduces toxic side effects.

[0010] Technical solution: To achieve the above objectives, the present invention provides a method for preparing a nano-delivery system HC@C / siPD-L1 NPs, comprising the following steps:

[0011] (1) Synthesis of H4R6RGD-CMβCD: Dissolve carboxymethyl-β-cyclodextrin in an organic solvent, adjust the solution pH, and stir. Mix and dissolve 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, add dropwise to the carboxymethyl-β-cyclodextrin solution, and stir to activate. Continue to add H4R6RGD, adjust the solution pH, and stir. Dialyze and freeze-dry to obtain H4R6RGD-CMβCD.

[0012] (2) Preparation of HC@cela: H4R6RGD-CMβCD obtained in step (1) was dissolved in water, and tripterygium wilfordii was dissolved in an organic solvent. The two were mixed, stirred, dialyzed, centrifuged, and the supernatant was freeze-dried to obtain HC@cela;

[0013] (3) Preparation of HC@C / siPD-L1 NPs: siPD-L1 was incubated with the HC@cela obtained in step (2), mixed, and vortexed to obtain HC@C / siPD-L1 NPs.

[0014] Wherein, in step (1), the mass ratio of carboxymethyl-β-cyclodextrin to H4R6RGD is 1-5:1-3, and the pH value of the solution is adjusted to 2-4 and then stirred at room temperature for 1-2 hours.

[0015] Wherein, in step (1), the molar ratio of the mixed dissolution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 0.5-2:0.5-1, and the stirring activation condition is 3-5 hours at room temperature.

[0016] Wherein, after adding H4R6RGD in step (1), the pH value of the solution is adjusted to 4-6 and stirred at room temperature for 10-12 hours.

[0017] Preferably, 40 mg of CMβCD was dissolved in anhydrous DMF, and then 0.5 mmol of EDCI and 0.5 mmol of NHS were dissolved in anhydrous DMF, and 30 mg of H4R6RGD was added.

[0018] Wherein, in step (2), the mass ratio of H4R6RGD-CMβCD to tripterygium wilfordii is preferably 1-3:1-4, the stirring conditions are 12-24h at room temperature, and the centrifugation conditions are 6000-8000rpm for 20-30min.

[0019] In step (3), the volume ratio of siPD-L1 to HC@cela is preferably 0.5-2:50-100, the incubation conditions are 50-60° C. water bath for 10-15 min, and the vortex conditions are 30-40 s.

[0020] The present invention relates to a nano-delivery system HC@C / siPD-L1 NPs prepared by the method for preparing the nano-delivery system HC@C / siPD-L1 NPs according to claim 1.

[0021] Furthermore, a nano-delivery system HC@C / siPD-L1 NPs is used in the preparation of drugs for treating breast cancer.

[0022] The drug for treating breast cancer described in the present invention comprises a nano drug delivery system HC@C / siPD-L1NPs and a pharmaceutically acceptable excipient or carrier.

[0023] Wherein, the pharmaceutical preparation is an injection, granule, tablet, capsule, pill or oral solution.

[0024] The structure of the nanocarrier (H4R6RGD-CMβCD) described in the present invention is as follows:

[0025]

[0026] The present invention uses an amide reaction to graft H4R6RGD with carboxymethyl-β-cyclodextrin to form the nanocarrier H4R6RGD-CMβCD. The chemical structure of H4R6RGD-CMβCD was verified by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance hydrogen spectrum (1H-NMR), and matrix-assisted laser desorption time-of-flight tandem mass spectrometry (MALDI-TOF-MS). It was stirred with a DMSO solution of celastrol overnight, dialyzed, centrifuged, and freeze-dried to obtain HC@cela. Subsequently, siPD-L1 was incubated with HC@cela and vortexed to obtain HC@C / siPD-L1 NPs. TEM observation showed that the nanoparticles had a smooth appearance, a uniform particle size of approximately 200 nm, and good dispersibility. In addition, the nanoparticles were formed mainly through hydrophobic interaction and electrostatic adsorption, and had good stability in serum. In vitro results demonstrated that the polymer H4R6RGD-CMβCD exhibited excellent biosafety, while the HC@cela complex exhibited superior antitumor efficacy compared to free tripterine. Furthermore, while the cytotoxicity of siPD-L1 alone was negligible, HC@C / siPD-L1 nanoparticles demonstrated robust antitumor activity. Cellular uptake and lysosomal escape are crucial for drug efficacy. Laser confocal microscopy revealed that HC@C / siRNA nanoparticles promoted drug uptake and lysosomal escape, primarily due to the presence of the H4R6RGD peptide on the nanoparticle surface, which enabled specific cellular accumulation and uptake. This demonstrates the targeting properties of the RGD peptide and the transmembrane activity of arginine within the material. These results suggest that the histidine in HC@C / siRNA protonates within lysosomes, generating a "proton sponge effect" that disrupts the lysosomal membrane and facilitates nanoparticle escape from lysosomes, thereby enhancing drug utilization and achieving better antitumor efficacy.

[0027] The present invention designed and synthesized a multifunctional peptide-modified cyclodextrin derivative nanosystem, HC@C / siRNA, to construct a platform for the co-delivery of tripterine and siRNA. The cyclodextrin derivative with a hydrophobic cavity and a positively charged peptide chain can effectively co-assemble with tripterine and siRNA to form spherical nanoparticles with uniform particle size. The HC@C / siPD-L1 nanoparticles exhibited efficient cellular uptake and were able to escape lysosomes, ensuring the effective intracellular delivery of siPD-L1. These results demonstrated that H4R6RGD-CMβCD can co-transport tripterine and siPD-L1, thereby exerting a synergistic anti-tumor effect. At the same time, these results provide experience for expanding the application range of cyclodextrin as a delivery agent. In summary, the multifunctional nano drug delivery system can promote tumor cell apoptosis, regulate the immunosuppressive microenvironment, enhance the anti-tumor effect, and provide a new strategy for improving the efficacy of tumor treatment.

[0028] Beneficial Effects: Compared with existing technologies, this invention offers the following significant advantages: It prepares a multifunctional peptide-modified cyclodextrin derivative nanosystem, HC@C / siRNA, which exhibits excellent stability in serum. The HC@C / siRNA nanoparticles promote cellular drug uptake and lysosomal escape, thereby enhancing drug utilization and enhancing anti-tumor efficacy. Furthermore, the polymer, H4R6RGD-CMβCD, exhibits excellent biosafety. While the cytotoxicity of siPD-L1 alone is negligible, the HC@C / siPD-L1 nanoparticles still exhibit significant anti-tumor effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the structural formula of tripterygium wilfordii;

[0030] Figure 2 The synthetic route of H4R6RGD-CMβCD;

[0031] Figure 3 Fourier transform infrared spectra of CMβCD (a), H4R6RGD (b), H4R6RGD-CMβCD (c), and H4R6RGD mixed CMβCD (d);

[0032] Figure 4 H NMR spectra of H4R6RGD-CMβCD, H4R6RGD, and CMβCD;

[0033] Figure 5 Matrix-assisted laser desorption / time-of-flight tandem mass spectra of CMβCD, H4R6RGD, and H4R6RGD-CMβCD (from top to bottom);

[0034] Figure 6Transmission electron microscopy images of HC@C / siRNANPs;

[0035] Figure 7 is the particle size distribution of HC@C / siRNANPs;

[0036] Figure 8 To detect the interaction force, the changes in UV absorbance of HC@cela and cela under the action of (a) DMSO, (b) NaCl, and (c) SDS, and (d) the changes in fluorescence signals of different groups of Cy5-siRNA were detected;

[0037] Figure 9 Serum stability test of HC@C / siRNANPs;

[0038] Figure 10 (A) Safety evaluation of H4R6RGD-CMβCD, (B) Cytotoxicity of cela and HC@cela on MDA-MB-231 cells after 24 h of incubation, (C) Cytotoxicity of siPD-L1 and HC@C / siPD-L1NPs in MDA-MB-231 cells after 24 h of incubation;

[0039] Figure 11 (A) CLSM analysis of siRNA distribution in MDA-MB-231 cells, DAPI staining of cell nuclei, scale bar, 20 μm, (B) Quantification of fluorescence intensity of cellular uptake of siRNA and HC@C / siRNA NPs;

[0040] Figure 12 Figure 3: CLSM observation of the damage of siRNA to lysosomes and intracellular distribution in MDA-MB-231 cells at different times. Lysosomes and cell nuclei were stained with Lyso-Tracker Red and DAPI, respectively. Scale bar, 20 μm. siRNA was labeled with FAM (FAM-siRNA). DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0042] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.

[0043] Example 1

[0044] Synthesis and characterization of H4R6RGD-CMβCD

[0045] H4R6RGD: The linear peptide HHHHRRRRRRRGD (H4R6RGD) was synthesized by Nanjing GenScript Biotechnology Co., Ltd. The structure is shown below:

[0046] HHHHRRRRRRRGD

[0047] CMβCD: Shanghai BiDe Pharmaceutical Technology Co., Ltd., product number: BD303994.

[0048] siPD-L1 (siRNA): (5′-CUGGGAGCCAUCUUAUUAUTT-3′, 5′-AUAAUAAGAUGGCUCCCAGTT-3′) Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0049] FAM was modified at the 5′ end of siPD-L1.

[0050] (1) Synthesis of H4R6RGD-CMβCD

[0051] Synthesis route such as Figure 2 As shown, 100 mg of CMβCD was dissolved in 3 mL of anhydrous DMF. An appropriate amount of 0.4 nM HCl was then added, and the solution was adjusted to pH 2 and stirred for 1 hour. 20.7 mg of EDCI and 12.4 mg of NHS were then dissolved in 1.5 mL of anhydrous DMF and added dropwise to the reaction solution. After stirring and activation for 3 hours, 40 mg of H4R6RGD was added to the reaction solution. Triethylamine solution was added to adjust the pH to 4 and stirred overnight. The reaction solution was dialyzed using a dialysis bag (MWCO: 2000 Da) and ultrapure water for 24 hours before lyophilization to obtain the nanocarrier H4R6RGD-CMβCD lyophilized powder, which was stored at -20°C until use.

[0052] (2) Characterization of H4R6RGD-CMβCD

[0053] Fourier transform infrared spectroscopy (FTIR): CMβCD, H4R6RGD, H4R6RGD-CMβCD and H4R6RGD mixed with CMβCD were sampled and analyzed by infrared spectroscopy using the potassium bromide pellet method. The wavelength range was 4000-400 cm -1 , with a resolution of 2cm -1 .

[0054] like Figure 3 As shown, CMβCD at 1602 cm -1 The stretching vibration peak of -COO- appears at 1672cm -1 The spectrum of the synthetic compound CMβCD-H4R6RGD shows a peak at 1658cm-1 The stretching vibration peak of amide C=O appeared at 1420cm -1 The stretching vibration peak of the amide CN bond appeared at , proving that the material CMβCD-H4R6RGD was synthesized.

[0055] Proton Nuclear Magnetic Resonance (1H-NMR): Appropriate amounts of CMβCD, H4R6RGD, and H4R6RGD-CMβCD were dissolved in deuterium oxide (D2O), respectively, and then placed into different nuclear magnetic resonance tubes, appropriately labeled, and then analyzed and detected using a proton nuclear magnetic resonance analyzer.

[0056] like Figure 4 As shown, specific proton peaks corresponding to the histidine imidazole ring are shown at 7.2 and 8.5 ppm. In addition, the H characteristic peak on the cyclodextrin ring is observed in the range of 3.3 to 4.3 ppm in the H NMR spectrum of H4R6RGD-CMβCD, indicating the presence of CMβCD. 1 The H NMR spectrum shows characteristic peaks for both H4R6RGD and CMβCD, demonstrating successful grafting of the H4R6RGD peptide onto the CMβCD surface. In the H4R6RGD-CMβCD spectrum, peaks characteristic of CMβCD are observed between δ = 5.0 and 6.5 ppm. Integration of the standard H NMR peaks revealed that the 7H residues obtained correspond to the seven glucopyranose units on 1 CMβCD. Integration of the CH2 chemical shift at the arginine guanidinium group in H4R6RGD between δ = 2.9 and 3.1 ppm yielded a 7.86H residue. 1 H-NMR integral analysis showed that the bonding ratio of H4R6RGD to CMβCD was 28%.

[0057] Matrix-assisted laser desorption / time of flight tandem mass spectrometry (MALDI-TOF-MS): First, appropriate amounts of CMβCD, H4R6RGD, and its derivative H4R6RGD-CMβCD were dissolved in deionized water and labeled. Deionized water was then used as a blank control, and the samples were analyzed and measured using a matrix-assisted laser desorption / mass spectrometer.

[0058] like Figure 5As shown, the relative molecular mass of CMβCD is 1541, and the relative molecular mass of CMβCD sodium salt is 1563. CMβCD exhibits a typical peak at a relative molecular mass of 1562.973, and H4R6RGD exhibits a typical peak at a relative molecular mass of 1826.286. The spectrum of H4R6RGD-CMβCD shows characteristic peaks of CMβCD and H4R6RGD, respectively, and a new H4R6RGD-CMβCD molecular weight peak appears at 3351.979, indicating that H4R6RGD has been successfully bound to cyclodextrin and that H4R6RGD-CMβCD has been successfully synthesized. Its structure is:

[0059]

[0060] Example 2

[0061] Preparation and characterization of HC@C / siPD-L1 NPs

[0062] (1) Preparation of HC@C / siPD-L1 NPs

[0063] The polymer H4R6RGD-CMβCD@Celastrol (HC@cela) was prepared using a saturated aqueous solution method. 39.8 mg of H4R6RGD-CMβCD prepared in Example 1 was dissolved in 1 mL of water. 1 mL of a DMSO solution containing 10 mg of tripterygium wilfordii was slowly added and stirred overnight. The mixture was then dialyzed against ultrapure water using a dialysis bag (MWCO: 2000 Da) for 24 hours to remove the organic solvent. The supernatant was centrifuged (6000 rpm, 30 minutes) and lyophilized to yield the product HC@cela. Subsequently, siPD-L1 (200 nM) and HC@cela aqueous solution (100 ng / ml) were incubated in a 50 °C water bath for 10 min, mixed at a ratio of 1:100 (volume ratio), and vortexed for 30 s to obtain nanoparticles HC@C / siPD-L1NPs (H4R6RGD-CMβCD@cela / siRNANPs). At the same time, FAM-modified siPD-L1 was used to prepare HC@C / FAM-siRNANPs.

[0064] (2) Characterization of HC@C / siPD-L1 NPs

[0065] Transmission electron microscopy (TEM) morphology and particle size analysis: An appropriate amount of H4R6RGD-CMβCD@cela / siRNA NPs solution was dripped onto a copper mesh and allowed to dry at room temperature. After the mesh was completely dry, the particle size and morphology of the nanoparticles were observed using a transmission electron microscope. The particle size of the nanoparticles was measured using a Zeta PALS high-resolution TEM and particle size analyzer.

[0066] like Figure 6Transmission electron microscopy results show that H4R6RGD-CMβCD@C / siPD-L1 NPs are spherical structures with a particle size of about 200nm. The experimental results show that the nanoparticles we prepared have good morphology and are evenly distributed spherical structures with a particle size of 200nm. The particle size of the nanoparticles was measured using Zeta PALS high-resolution potential and particle size analyzer. Figure 7 As shown, the particle size distribution of the nanoparticles is around 200 nm, the average particle size is 189.3 and the PDI is 0.132, which is consistent with the results shown by transmission electron microscopy. The potential of the nanoparticles is -1.097 mv.

[0067] Interaction force studies: HC@cela was incubated in 0.1 nM, 0.2 nM, 0.4 nM, and 0.8 nM NaCl solutions and 0.2% (w / v) SDS solution for 10 min. The absorbance at wavelengths of 300–550 nm was measured using a microplate reader. HC@C / siRNA NPs were synthesized using Cy5 fluorescently labeled siRNA. To 0.1 mL of HC@C / Cy5-siRNA solution containing 0.76 nmol / mL Cy5-siRNA, 60 mg / mL polyanion heparin sodium (5 μL) was added. After incubation for 5 min, the fluorescence signal of the Cy5-siRNA was tracked using a microplate reader (λex = 630 nm).

[0068] like Figure 8 As shown in the figure, the absorption peak of HC@cela did not change significantly in the presence of NaCl. However, the addition of sodium dodecyl sulfate (SDS) caused the absorption peak of HC@cela to disappear. This shift in absorption peaks indicates that the formation of HC@cela primarily utilizes the hydrophobic interaction between H4R6RGD-CMβCD and cela. Simultaneously, HC@C / Cy5-siRNA nanoparticles were prepared using Cy5-labeled siRNA, and the Cy5-siRNA was detected by fluorescence. Fluorescence detection revealed that the Cy5 fluorescence intensity in HC@C / Cy5-siRNA NPs was weaker than that of naked Cy5-siRNA, indicating that the Cy5-siRNA was effectively encapsulated in the nanoparticles. However, after the addition of sodium heparin to disrupt the electrostatic interaction, the fluorescence of the Cy5-siRNA was restored. This confirms that the electrostatic interaction between HC@cela and siRNA plays a key role in the coassembly of HC@C / siRNA NPs. These results confirmed our previous hypothesis that H4R6RGD-CMβCD can utilize the hydrophobic interaction between the cyclodextrin cavity and the drug and the electrostatic adsorption between the positively charged peptide chain and siRNA to promote the formation of co-assembled nanoparticles, thereby co-delivering cela and siPD-L1.

[0069] Serum stability assessment: Naked siRNA and an equal volume of nanoparticles were mixed with FBS and incubated at room temperature for 1, 3, 6, 12, and 24 hours. siRNA degradation was analyzed by electrophoresis on a 3% agarose gel at 80 V for 30 minutes. siRNA degradation was assessed by comparing the intensity and integrity of the siRNA bands at different time points.

[0070] like Figure 9 As shown in the figure, naked siRNA was completely degraded after 12 hours, while siRNA in HC@C / siRNANPs was still detectable even after 24 hours. This observation indicates that the nanoparticles effectively protected siRNA, reduced the effects of enzymes on siRNA, prolonged the half-life of siRNA, and improved the overall stability of the nanoparticles.

[0071] Example 3

[0072] Study on the safety of nanocarriers and in vitro activity of nanoparticles

[0073] Cell culture: MDA-MB-231 cell line was obtained from the National Center for Cell Science (NCCS) and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS).

[0074] Resuscitation: Gently shake the cryovial containing MDA-MB-231 cells removed from liquid nitrogen in a 37°C water bath until the liquid thaws. Then, aspirate the suspension in a clean bench and add it to a sterile centrifuge tube containing culture medium. Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add 1 mL of culture medium, gently pipette to resuspend the cells, and add them to a cell flask containing 10 mL of culture medium. Pipet and culture evenly. Incubate in an incubator at 37°C, 5% CO2.

[0075] Passaging: When the cells in the culture flask grow to 80%-90% of the bottom of the flask, the subculture operation should be carried out. The original culture medium should be poured out, and after washing with PBS, appropriate trypsin should be added. Digest in the incubator for about 1 minute. After the cells are observed to be round under the microscope, an equal volume of complete culture medium should be added to terminate the digestion. The cells should be blown, resuspended by centrifugation, and then added to the culture medium for further culturing.

[0076] Cryopreservation: Take cells in the logarithmic growth phase, remove the old culture medium, wash with PBS, add trypsin to digest into single cells, add an appropriate amount of serum-containing culture medium to terminate the digestion, pipette evenly, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cells in a dedicated serum-free cell freezing solution, and dilute the cells to 5×10 6 ~1×10 7Cells were then aliquoted into cryovials at 1-1.5 mL per tube. The cryovials were placed in a cryostat box cooled to room temperature and filled with isopropanol. The cryovials were stored at -80°C overnight. The next day, the cryovials were removed and transferred to a liquid nitrogen container.

[0077] Evaluation of nanocarrier safety and nanoparticle cytotoxicity: The CCK-8 method was used to detect the cytotoxicity of materials and nanoparticles. In vitro cytotoxicity tests were performed using the CCK-8 kit according to the manufacturer's instructions. MDA-MB-231 cells were placed in a 96-well plate at a concentration of 4,000 cells per well and incubated at 37°C and 5% CO2 for 24 hours. Different concentrations of cela and HC@cela were added to the wells. The concentrations of cela in the two were ensured to be 0.25, 0.5, 0.75, and 1 μg / mL, respectively. 24 hours after the addition of the drug, 10 μL of CCK-8 was added to each well and incubated for 1 hour. The absorbance value at 450 nm was measured using an enzyme reader. The formula for calculating cell viability is: Cell viability (%) = [(experiment-blank) / control-blank] × 100%. The results are shown in Figure 2. Figure 10 As shown in (A), the cell survival rate of the synthetic material is above 80%, and no obvious cell death or damage is observed even at high concentrations, indicating that the synthetic material has good biosafety.

[0078] Next, the inhibitory effect of H4R6RGD-CMβCD polymer loaded with tripterygium wilfordii (HC@cela) on MDA-MB-231 cells was evaluated ( Figure 10 B). Cells were incubated with different concentrations of tripterine (0.25, 0.5, 0.75, 1 μg / mL) for 24 hours, and the results showed that HC@cela had a significantly better killing effect on tumor cells than free cela drugs. In addition, the cytotoxicity of HC@cela was concentration-dependent. The higher the concentration of tripterine encapsulated, the better the killing effect on cells. This improved effect can be attributed to the enhanced cellular uptake of the polymer-encapsulated cells, thereby increasing the utilization of the drug in the cells and enhancing the anti-tumor activity. In addition, the cytotoxicity of naked siPD-L1 and HC@C / siPD-L1 NPs was evaluated ( Figure 10 C) Using the same method as above, siPD-L1 and HC@C / siPD-L1 NPs were used at concentrations of 100, 200, 300, and 400 nM siPD-L1. While siPD-L1 alone was minimally toxic to cells, the nanoparticle group exhibited a more pronounced cell-killing effect. This effect can be attributed to the co-delivery of cela and siPD-L1 by the nanoparticles.

[0079] These experiments demonstrated that the polymer H4R6RGD-CMβCD possesses excellent biosafety and that the HC@cela complex exhibits superior antitumor efficacy compared to free tripterine. Furthermore, HC@C / siPD-L1 nanoparticles exhibited excellent antitumor activity, while the cytotoxicity of siPD-L1 alone was negligible.

[0080] Evaluation of Nanoparticle Cellular Uptake: Confocal laser scanning microscopy (CLSM) was used to observe the uptake of HC@C / FAM-siRNA NPs in MDA-MB-231 cells. Cells were seeded in 20 mm glass-bottomed cell culture dishes and incubated at 37°C for 24 h until the cells were fully attached. They were then treated with siPD-L1 (naked siRNA group) and HC@C / siRNA (HC@C / siPD-L1 NPs) for 12 h. The final concentration of the HC@C / siPD-L1 NPs was 0.33 μg / mL of tripterine. The amount of naked FAM-siRNA was the same as the siRNA content in the HC@C / siPD-L1 NPs. The cells were then fixed with 4% paraformaldehyde for 15 min, and the nuclei were stained with DAPI for 15 min. Finally, the cells were washed three times with PBS for 5 min each time. The stained cells were then observed using a confocal laser scanning microscopy.

[0081] like Figure 11 As shown, the fluorescence intensity of the nanoparticle group was significantly higher than that of the naked siRNA group, indicating enhanced cellular uptake. This improved cellular uptake is primarily attributed to the presence of the H4R6RGD peptide on the nanoparticle surface, which facilitates targeting of MDA-MB-231 cells that overexpress αVβ3 integrin. The nanoparticles were able to specifically accumulate in these cells, and the positively charged polyarginine enhanced the nanoparticle's membrane penetration, indirectly demonstrating the targeting effect of the RGD peptide and the membrane-penetrating properties of arginine.

[0082] Study on the lysosomal escape behavior of nanoparticles: CLSM was used to detect the lysosomal escape behavior of HC@C / siRNA NPs. MDA-MB-231 cells were cultured at 2×10 5Cells were seeded at a density of 1000 cells / dish in laser confocal microscopy culture dishes and cultured for 24 hours until cell adhesion. They were then treated with naked FAM-siRNA and HC@C / FAM-siRNA NPs for 2, 6, and 8 hours, respectively. The final concentration of HC@C / FAM-siRNA NPs was 0.33 μg / mL of tripterine. The amount of naked FAM-siRNA was the same as the siRNA content in the HC@C / FAM-siRNA NPs. Lyso-Tracker Red (50 nM) was diluted with culture medium at a ratio of 1:1500 and added to the culture dish to stain lysosomes for 30 minutes. The cells were fixed with 4% paraformaldehyde for 15 minutes and stained with DAPI for 15 minutes. The cells were then washed with PBS three times for 5 minutes each to observe the lysosomal escape of the nanoparticles.

[0083] like Figure 12 As shown, the green fluorescence of the siRNA in the naked siRNA group almost completely overlapped with the red fluorescence of the lysosomes at all time points, and the overlap showed a strong yellow light, indicating that the naked siRNA failed to escape from the lysosomes. In contrast, although the green fluorescence of the siRNA in the HC@C / siRNANPs group overlapped with the red fluorescence of the lysosomes at 2 and 6 hours, the fluorescence intensity of the overlapping portion was significantly weaker than that of the naked siRNA group. At 8 hours, the red and green fluorescence began to separate, indicating that most of the nanoparticles had successfully escaped from the lysosomes. This escape behavior can be attributed to the presence of histidine in the polymer carrier used in this study. Within the lysosome, histidine is protonated, producing a "proton sponge effect." This action causes the lysosomal membrane to rupture, facilitating the escape of the nanoparticles from the lysosome.

Claims

1. A method for preparing a nano-delivery system HC@C / siPD-L1 NPs, characterized in that: The following steps are involved: (1) Synthesis of H4R6RGD-CMβCD: Carboxymethyl-β-cyclodextrin was dissolved in an organic solvent, and the pH of the solution was adjusted and stirred. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were mixed and dissolved, and then added dropwise to the carboxymethyl-β-cyclodextrin solution and stirred for activation. H4R6RGD was further added, and the pH of the solution was adjusted and stirred. The solution was dialyzed and freeze-dried to obtain the nanocarrier H4R6RGD-CMβCD. (2) Preparation of HC@cela: H4R6RGD-CMβCD obtained in step (1) was dissolved in water, and tripterygium wilfordii was dissolved in an organic solvent. The two were mixed, stirred, dialyzed, centrifuged, and the supernatant was freeze-dried to obtain HC@cela; (3) Alternatively, HC@C / siPD-L1 NPs may be further prepared by mixing siPD-L1 with the HC@cela obtained in step (2) and incubating the mixture to obtain HC@C / siPD-L1 NPs.

2. The method for preparing the nano-delivery system HC@C / siPD-L1 NPs according to claim 1, characterized in that: In step (1), the mass ratio of carboxymethyl-β-cyclodextrin to H4R6RGD is 1-5:1-3, and the pH value of the solution is adjusted to 2-4 and stirred at room temperature for 1-2 hours. The amino acid sequence of the H4R6RGD is HHHHRRRRRRRGD.

3. The method for preparing the nano-delivery system HC@C / siPD-L1 NPs according to claim 1, characterized in that: In step (1), the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to be dissolved is 0.5-2:0.5-1, and the stirring activation condition is 3-5 hours at room temperature.

4. The method for preparing the nano-delivery system HC@C / siPD-L1 NPs according to claim 1, characterized in that: After adding H4R6RGD in step (1), the pH value of the solution is adjusted to 4-6 and stirred at room temperature for 10-12 hours.

5. The method for preparing the nano-delivery system HC@C / siPD-L1 NPs according to claim 1, characterized in that: In step (2), the mass ratio of H4R6RGD-CMβCD to tripterygium wilfordii is preferably 1-3:1-4, the stirring conditions are 12-24 h at room temperature, and the centrifugation conditions are 6000-8000 rpm for 20-30 min.

6. The method for preparing the nano-delivery system HC@C / siPD-L1 NPs according to claim 1, characterized in that: In step (3), the volume ratio of siPD-L1 to HC@cela is preferably 0.5-2:50-100, and the incubation conditions are 50-60° C. water bath for 10-15 min and vortexing for 30-40 s.

7. A nanodelivery system HC@C / siPD-L1 NPs prepared by the method for preparing the nanodelivery system HC@C / siPD-L1 NPs according to claim 1.

8. Use of the nanodelivery system HC@C / siPD-L1 NPs according to claim 7 in the preparation of a drug for treating breast cancer.

9. A drug for treating breast cancer, characterized in that: The nano drug delivery system comprises the HC@C / siPD-L1 NPs according to claim 7 and a pharmaceutically acceptable excipient or carrier.

10. A nanocarrier H4R6RGD-CMβCD, characterized in that Its structure is as follows:

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