A method for preparing a multifunctional nanocomposite delivery system CEL-TPP@siSurvivin / TDNP NPs and its application in breast cancer therapeutics.

Abstract

This invention discloses a method for preparing a multifunctional nanocomposite delivery system CEL-TPP@siSurvivin / TDNP NPs and its application in breast cancer treatment. The system is constructed by bridging triphenylphosphine-modified CEL with carboxyl-modified TPP, followed by electrostatic self-assembly of the product with negatively charged siSurvivin to form a nanocore, which is then encapsulated in turmeric-derived exosomes. This delivery system exhibits uniform spherical shape, a smooth surface, and good serum stability and cellular uptake capacity. TPP modification enables it to specifically target mitochondria, disrupt membrane potential, and synergistically knock down the survivin gene with siRNA, significantly promoting tumor cell apoptosis. This multifunctional nanocomposite delivery system overcomes the problem of poor water solubility of CEL and protects siRNA from nuclease degradation, achieving a highly efficient and synergistic anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine delivery technology, specifically relating to the preparation method of a multifunctional nanocomposite delivery system CEL-TPP@siSurvivin / TDNP NPs and its application in breast cancer treatment. Background Technology

[0002] Breast cancer (BC) is currently the leading cause of cancer death among women worldwide, and its incidence has been rising annually in recent years. Triple-negative breast cancer (TNBC) is a specific type characterized by insufficient expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor type 2 (HER2). Studies report that TNBC accounts for approximately 15%-20% of all breast cancer cases in the United States, exhibiting high malignancy and invasiveness, thus exacerbating the difficulty of treatment. Current clinical treatments include chemotherapy, immune checkpoint inhibitors, PARP inhibitors, and antibody-drug conjugates, with a gradual shift towards combination therapies.

[0003] Small interfering RNAs (siRNAs) selectively regulate gene expression through RNA interference mechanisms. Most siRNAs originate from exogenous or endogenous long double-stranded RNAs, which are cleaved into 21-23 nucleotide double strands in the cell and bind with other proteins to form the siRNA-induced silencing complex (RISC). Survivin, an apoptosis inhibitor, has biological mechanisms of action including: directly inhibiting caspase activity, blocking the execution pathway of apoptosis, and helping abnormal cells escape programmed cell death; as a regulator of mitosis, it participates in the regulation of centromere protein INCENP on chromosome centromeres, the maintenance of microtubule stability, and the function of spindle assembly checkpoints, directly promoting normal cell division and proliferation. In TNBC, overexpression of Survivin is closely related to apoptosis resistance and also significantly affects clinicopathological factors and prognosis. Therefore, knocking down Survivin gene expression with siRNA is a promising therapeutic strategy. The effective delivery of siRNA in vivo requires the assistance of an ideal delivery vector, including protection from degradation and renal clearance during systemic circulation, precise enrichment in target tissues, crossing cell membrane barriers to enter cells, and successful lysosomal escape to release siRNA into the cytoplasm. siRNA combats cancer through a strategy of precisely silencing specific pathogenic genes at the mRNA level, exhibiting high specificity. By designing the siRNA sequence, it can be targeted to genes with any known sequence, including targets lacking effective drugs, thereby achieving precise targeting and reducing off-target toxicity. siRNA-based gene therapy can specifically recognize and bind to target mRNA, inhibiting gene expression. Combining siRNA with other therapeutic modalities can exert a synergistic effect through multiple mechanisms, improving the success rate of solid tumor treatment and overcoming the limitations of single-therapy approaches.

[0004] Celastrol (CEL) is one of the main active ingredients of the traditional Chinese medicine Tripterygium wilfordii. It is a pentacyclic triterpenoid compound, appearing as red needle-like crystals or powder with poor water solubility. Its structural formula is as follows: Figure 1As shown, CEL exhibits good pharmacological activities in multiple aspects, including antitumor, anti-inflammatory, antioxidant, anti-obesity, neuroprotective, and hypoglycemic effects. Particularly in cancer treatment research, its multi-target and potent antitumor activity has shown inhibitory effects on various tumor cells, including lung cancer, liver cancer, breast cancer, colon cancer, leukemia, and glioma. However, as a highly lipophilic macromolecule, CEL has extremely poor water solubility, making it impossible to administer and maintain therapeutic concentrations via conventional routes. Secondly, the effective anti-therapeutic dose of CEL is very close to the dose that induces toxicity, potentially causing severe off-target toxicity. Finally, CEL has poor stability and may degrade under light or other environmental conditions, with some metabolites generated in vivo posing toxic risks. These limitations stem from the inherent physicochemical properties of CEL, restricting its clinical application. Currently, the development of nano-formulations, combination therapy strategies, molecular structural modification, and optimization of administration methods are gradually being used to improve the shortcomings of CEL.

[0005] Exosomes are membrane-like vesicles composed of various biomacromolecules, and their nanoscale diameter, low immunogenicity, and high biopermeability make them novel drug delivery components. Plant-derived exosomes exhibit greater safety and a lower risk of immunogenicity compared to other sources. Previous studies have shown that plant-derived exosomes possess anti-proliferative, pro-apoptotic, and anti-inflammatory activities, and have the potential to alter the tumor microenvironment and inhibit solid tumors. Experiments have shown that the good biocompatibility and stability of turmeric exosomes can improve the shortcomings of photosensitizers, such as easy aggregation and short half-life, making them a highly promising biodelivery carrier. Using turmeric-derived nanovesicles as a carrier to deliver survivin-targeting siRNA via intravenous injection demonstrated excellent tumor-suppressive effects while significantly improving the targeted silencing effect, and no significant change in mouse body weight during treatment, proving its advantages of both safety and high efficiency. Summary of the Invention

[0006] Objective of the Invention: Addressing the problems existing in the prior art, this invention provides a method for preparing a multifunctional nano-co-delivery system, CEL-TPP@siSurvivin / TDNP NPs, and its application in breast cancer treatment. The multifunctional nano-co-delivery system of this invention involves bridging CEL with a carboxyl-modified triphenylphosphine (TPP) molecule, electrostatically self-assembling it with siRNA (siSurvivin) targeting Survivin to form a nanocore, and then coating it with turmeric-derived plant exosomes to construct the multifunctional nano-co-delivery system CEL-TPP@siSurvivin / TDNP NPs. In vitro experimental results show that CEL-TPP@siSurvivin / TDNP NPs enhances tumor killing and significantly inhibits tumor growth.

[0007] Technical Solution: To achieve the above objectives, the present invention provides a method for preparing a multifunctional nanocomposite delivery system CEL-TPP@siSurvivin / TDNP NPs, comprising the following steps:

[0008] (1) Dissolve CEL, inorganic base and phosphorus-containing organic salt in organic solvent, react and purify;

[0009] (2) Turmeric vesicles were obtained by processing peeled turmeric;

[0010] (3) The product obtained in step (1) is mixed with siRNA (siSurvivin) targeting Survivin to obtain self-assembled nanoparticles; the self-assembled nanoparticles are then mixed with the turmeric vesicles obtained in step (2) at a mass ratio and processed to obtain the nano-co-delivery system.

[0011] Furthermore, in step (1), the molar ratio of triptolide CEL, inorganic alkali and phosphorus-containing organic salt is (0.5-2):(1-10):(1-5).

[0012] Preferably, in step (1), the molar ratio of triptolide CEL, inorganic alkali and phosphorus-containing organic salt is 1:4:2.

[0013] Further, the inorganic base in step (1) is selected from at least one of sodium bicarbonate, sodium carbonate, potassium bicarbonate, and ammonium bicarbonate; the phosphorus-containing organic salt is at least one of triphenylphosphine bromide (TPP-Br), tetraphenylphosphine bromide, tetraphenylphosphine iodide, and tetraphenylphosphine tetrafluoroborate; and the organic solvent is selected from at least one of N,N-dimethylacetamide, N-methyl-2-pyrrolidone, and dimethyl sulfoxide.

[0014] Preferably, the inorganic base in step (1) is sodium bicarbonate; the phosphorus-containing organic salt is triphenylphosphine bromide (TPP-Br); and the organic solvent is N,N-dimethylacetamide (DMF).

[0015] Further, in step (1), the reaction is a reflux reaction with a temperature of 50-100℃ and a reaction time of 15-20 h; the purification process includes extraction, filtration, rotary evaporation and column chromatography elution in sequence; wherein, the eluent for column chromatography elution is a mixed solvent of dichloromethane and methanol with a volume ratio of (15-25):(1-5).

[0016] Preferably, in step (1), the temperature of the reflux reaction is 60°C and the reaction time is 16 h; the purification process includes extraction, filtration, rotary evaporation and column chromatography elution in sequence; wherein, the eluent for column chromatography elution is a mixed solvent of dichloromethane and methanol, with a volume ratio of 20:1.

[0017] Further, the processing in step (2) includes grinding, differential centrifugation precipitation, ultracentrifugation and ultrasonic resuspension; wherein, the differential centrifugation precipitation includes: first centrifugation: room temperature, 2000-3000 × g, 15-25 min; second centrifugation: room temperature, 9000-10000 × g, 30-50 min; the conditions for ultracentrifugation are: 2-10℃, 130000-150000 × g, 1-3 h.

[0018] Preferably, the processing in step (2) includes grinding, differential centrifugation precipitation, ultracentrifugation and ultrasonic resuspension; wherein, the differential centrifugation precipitation includes: first centrifugation: room temperature, 3000 × g, 20 min; second centrifugation: room temperature, 10 000 × g, 40 min; the conditions for ultracentrifugation are: 4℃, 150 000 × g, 2 h.

[0019] Further, in step (3), the concentration of the product obtained in step (1) is 5-15 mg / mL; the concentration of the siRNA (siSurvivin) targeting Survivin is 15-25 μM; the two are diluted 10-15 times respectively, and the molar ratio of the two after dilution is (5-15):(0.5-3); the mass ratio of the self-assembled nanoparticles to the turmeric vesicles obtained in step (2) is (1-5):(0.5-2).

[0020] Preferably, in step (3), the concentration of the product obtained in step (1) is 10 mg / mL; the concentration of the siRNA (siSurvivin) targeting Survivin is 20 μM; the dilution factor is 10 times, and the molar ratio of the two after dilution is 10:1; the mass ratio of the self-assembled nanoparticles to the turmeric vesicles obtained in step (2) is 2:1.

[0021] Further, the mixing reaction process in step (3) includes vortexing and incubation; the treatment process is centrifugation; wherein the incubation conditions are 35-37℃ for 10-20 min; and the centrifugation conditions are 8000-12000 rpm for 10-20 min.

[0022] Preferably, the mixing reaction process in step (3) includes vortexing and incubation; the treatment process is centrifugation; wherein the incubation conditions are 37°C for 15 min; and the centrifugation conditions are 10,000 rpm for 15 min.

[0023] Preferably, the preparation method of the multifunctional nanocomposite delivery system CEL-TPP@siSurvivin / TDNP NPs of the present invention includes the following steps:

[0024] (1) Preparation of CEL-TPP: CEL (triptolide), NaHCO3 (sodium bicarbonate) and TPP-Br (triphenylphosphine bromide) were dissolved in DMF (N,N-dimethylformamide), heated, refluxed, and reacted for 16 h. CEL-TPP was obtained by extraction, filtration, rotary evaporation and purification.

[0025] (2) Extraction of turmeric vesicles: Peeled turmeric was ground, precipitated by differential centrifugation, precipitated by ultracentrifugation, and resuspended by ultrasonication to obtain turmeric vesicles.

[0026] (3) Synthesis of nanoparticles CEL-TPP@siSurvivin / TDNP NPs: CEL-TPP and siRNA targeting Survivin (siSurvivin) were diluted, mixed, vortexed and incubated to obtain self-assembled nanoparticles; then the self-assembled nanoparticles were mixed with turmeric vesicles, vortexed, incubated and centrifuged to obtain vesicle-coated nanoparticles.

[0027] The multifunctional nano-co-delivery system CEL-TPP@siSurvivin / TDNP NPs prepared by the method described in this invention.

[0028] The application of the non-multifunctional nanocomposite delivery system CEL-TPP@siSurvivin / TDNP NPs described in this invention in the preparation of drugs for treating breast cancer.

[0029] A medicament for treating breast cancer, characterized in that it comprises the multifunctional nano-co-delivery system CEL-TPP@siSurvivin / TDNP NPs as described in claim 8, and pharmaceutically acceptable excipients or carriers.

[0030] Furthermore, the pharmaceutical preparation is an injection, granules, tablets, capsules, pills, or oral liquid.

[0031] This invention modifies CEL with TPP to obtain CEL-TPP, which is then self-assembled with siSurvivin via electrostatic interaction and loaded onto TDNPs to obtain a multifunctional nano-co-delivery system CEL-TPP@siSurvivin / TDNP NPs.

[0032] This invention prepares a multifunctional nano-co-delivery system by combining TPP and CEL to obtain TPP-CEL. The chemical structure of TPP-CEL was verified by liquid chromatography-mass spectrometry (LC-MS) and 1H nuclear magnetic resonance spectroscopy (1H-NMR). It was then self-assembled with siSurvivin via electrostatic interaction and loaded onto TDNPs to form the multifunctional nano-co-delivery system CEL-TPP@siSurvivin / TDNP NPs. TEM observation showed that the nanoparticles were well-dispersed spherical structures. In vitro pharmacodynamic studies demonstrated that the system exhibits strong cell-inhibiting effects. 4T1 cell uptake studies showed that the system can be effectively internalized into 4T1 cells in a time-dependent manner. The system also allows lysosomal escape, effectively improving the bioavailability of therapeutic drugs and targeting cancer cells. These results demonstrate that this nanosystem can exert a good therapeutic effect on breast cancer and has promising application prospects.

[0033] This invention designs and synthesizes a multifunctional nanocomposite delivery system, CEL-TPP@siSurvivin / TDNP NPs, in which CEL-TPP and siSurvivin are self-assembled and loaded onto TDNPs. The NPs of this invention specifically target mitochondria, disrupting membrane potential and synergistically knocking down the survivin gene with siRNA, significantly enhancing tumor cell apoptosis. The CEL-TPP@siSurvivin / TDNP NPs constructed in this invention exhibit potent anti-tumor growth effects both in vivo and in vitro via lysosomal escape from tumor necrosomes (TNBC).

[0034] The multifunctional nano-co-delivery system constructed in this invention avoids the biotoxicity problems that carriers may cause compared to traditional delivery systems; it has better serum stability and cellular uptake capacity, and can better target tumor sites through lysosomal escape effects.

[0035] This invention utilizes the excellent biocompatibility of turmeric-derived plant exosomes to enhance the stability and tumor accumulation capacity of the system. Characterization shows that the prepared NPs are uniformly spherical with moderate particle size, good dispersibility, and excellent serum stability and drug sustained-release performance. In vitro studies show that the system can be efficiently taken up by 4T1 cells. Mitochondrial-targeted CEL-TPP and cytoplasm-released siSurvivin work synergistically to significantly increase the tumor cell apoptosis rate. In vivo experiments in a 4T1 tumor-bearing mouse model further demonstrate that CEL-TPP@siSurvivin / TDNP NPs have a significant ability to inhibit tumor growth.

[0036] This invention designs and constructs a multifunctional nanocomposite delivery system, CEL-TPP@siSurvivin / TDNP NPs, based on turmeric-derived plant exosomes: TPP-modified CELs and negatively charged siSurvivin self-assemble via electrostatic interactions to form a nanocore, which is then encapsulated within naturally derived turmeric exosomes. This nanoplatform exhibits excellent antitumor efficacy in both in vivo and in vitro TNBC models through the synergistic effect of chemotherapeutic drugs and siRNA.

[0037] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0038] (1) This invention prepared a multifunctional nano-co-delivery system, CEL-TPP@siSurvivin / TDNP NPs, which is self-assembled from CEL-TPP and siSurvivin and loaded onto TDNPs. The NPs exhibit a uniform spherical structure with good dispersibility. The particle size and PDI of the nanoparticles were monitored continuously for 7 days at 4°C, and no significant fluctuations were observed, confirming its good storage stability. This study utilizes the self-assembly of CEL-TPP and siSurvivin onto TDNPs to form a multifunctional nano-co-delivery system, which can target and deliver drugs to tumor sites, avoiding liver and kidney toxicity and improving therapeutic efficacy.

[0039] (2) The multifunctional nano-co-delivery system CEL-TPP@siSurvivin / TDNP NPs described in this invention has broad application prospects in the preparation of drugs for treating breast cancer; the system solves the problem of poor water solubility of CEL, protects siRNA from nuclease degradation, and achieves a highly efficient and synergistic anti-tumor effect.

[0040] (3) The preparation method of the multifunctional nano-co-delivery system CEL-TPP@siSurvivin / TDNP NPs described in this invention is simple, efficient and can be industrialized for production and application. Attached Figure Description

[0041] Figure 1 The structural formula of triptolide;

[0042] Figure 2 The synthetic route for CEL-TPP;

[0043] Figure 3 Transmission electron microscope image of CEL-TPP;

[0044] Figure 4 The images show the LC-MS spectrum of CEL-TPP (A) and the mass spectrum of CEL-TPP (B).

[0045] Figure 5The hydrogen NMR spectrum of CEL-TPP;

[0046] Figure 6 Transmission electron microscopy (TEM) image of CEL-TPP@siSurvivin / TDNP NPs;

[0047] Figure 7 Gel electrophoresis bands for storage stability testing (A) and serum stability testing of CEL-TPP@siSurvivin / TDNP NPs (B);

[0048] Figure 8 To detect the cytotoxic effects of different concentration groups on 4T1 cells using the CCK-8 assay;

[0049] Figure 9 Flow cytometry analysis of cell uptake efficiency and quantification after treatment of 4T1 cells with CEL-TPP@siSurvivin / TDNP NPs for 0, 0.5, 1, 2, 4, and 6 h (A), scale bar: 50 µm; CLSM image (B).

[0050] Figure 10 CLSM images and quantitative analysis of 4T1 cells treated with CEL-TPP@siSurvivin / TDNP NPs at 1, 2, 4, and 6 h, Lyso-Tracker Red staining, scale bar: 10µm. .

[0051] Figure 11 Representative images (A) of mouse tumors after tail vein injection of PBS, siSurvivin, CEL-TPP and NPs, respectively, and tumor weight (B) and tumor volume change curves after dissection (C) (n = 5). Detailed Implementation

[0052] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0054] Survivin-targeting siRNA sequence: sense strand: 5'-GCAAAGGAAACCAACAAUATT-3'; antisense strand: 5'-UAUUGUUGGUUUCCUUUGCTT-3', equimolar mixture;

[0055] Survivin-targeting siRNA with a 5-carboxyfluorescein (FAM) tag (FAM-siSurvivin), 5′ end modified with FAM: purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd., catalog number A03004;

[0056] Fetal bovine serum (Catalog No. BC-SE-FBS01), RPMI-1640 culture medium (Catalog No. BC-M-017), and trypsin-EDTA digestion solution (Catalog No. BC-CE-005) were all purchased from Jiangsu Nanjing Senbeijia Biotechnology Co., Ltd.

[0057] Example 1

[0058] Preparation and characterization of CEL-TPP

[0059] (1) Preparation of CEL-TPP

[0060] Synthetic routes such as Figure 2 As shown, CEL (triptolide, 1.0 eq, 50 mg, 0.11 mmol, structure as shown) was used. Figure 1 Add the solution (as shown) to a round-bottom flask containing 4 ml of DMF, then add sodium bicarbonate (NaHCO3, 4 eq, 37.33 mg, 0.44 mmol) and TPP-Br (triphenylphosphine bromide, 2 eq, 103.15 mg, 0.22 mmol), gradually raise the temperature to 60 °C, reflux, and react for 16 h (monitored by TLC to confirm complete reaction), then stop. Cool the reaction solution to room temperature, extract and filter, and rotary evaporate to obtain a dark brown liquid. Then perform column chromatography using dichloromethane:methanol = 20:1 (v / v) as eluent and silica gel as packing material to obtain orange powder CEL-TPP, which is stored at -20 °C. Its transmission electron micrograph is shown below. Figure 3 As shown in the figure, the average particle size of CEL-TPP is approximately 100 nm.

[0061] (2) Extraction of turmeric vesicles

[0062] Wash and peel 150g of turmeric, then grind it in a juicer to obtain juice. Centrifuge approximately 45ml of the juice at 3000×g for 20 min, then at 10000×g for 40 min to remove large ginger fibers. Divide the supernatant evenly into four 27.5ml ultracentrifuge tubes and ultracentrifuge at 150000×g (43937rpm) at 4℃ for 2 h. Resuspend the precipitate in 0.5ml of PBS in each tube by ultrasonic dispersion.

[0063] (3) Preparation of CEL-TPP@siSurvivin / TDNP NPs

[0064] Nanoparticles were prepared using a co-incubation method. A 10 mg / mL stock solution of CEL-TPP and siRNA targeting Survivin (siSurvivin, 20 μM) were dissolved in DMSO. These were diluted 10-fold with DEPC water and mixed at a molar ratio of 10:1. The mixture was vortexed for 5 min and incubated at 37°C for 15 min to obtain a suspension of self-assembled nanoparticles. This suspension was then mixed with a turmeric vesicle suspension at a mass ratio of 2:1, vortexed for 5 min, and incubated at 37°C for 15 min to obtain vesicle-coated nanoparticles. After incubation, the mixture was centrifuged at 10,000 rpm for 15 min to remove unbound free drug. The precipitate was collected to obtain the CEL-TPP@siSurvivin / TDNP nanoparticles. Transmission electron microscopy (TEM) images are shown below. Figure 6 As shown in the figure, CEL-TPP@siSurvivin / TDNP NPs are spherical particles with an average diameter of about 100nm, and they have a consistent shape and uniform size.

[0065] (3) Characterization of CEL-TPP

[0066] The prepared CEL-TPP was dissolved in methanol to prepare a 50 µg / mL solution. After filtration, the solution was placed in a sample vial and directly loaded onto an LC-MS sample. The molecular weight of CEL-TPP was verified using LC-MS. The results are as follows: Figure 4 As shown, at a detection wavelength of 400 nm, the target compound exhibits a single, sharp chromatographic peak at 8.727 minutes, indicating high product purity. Its corresponding mass spectrum is at m / z 754.3 [M]. + The quasi-molecular ion peak is displayed at [location], which is similar to that of CEL-TPP (C [other ion]). 46 H 53 O4P + The molecular weights of the compounds were highly consistent with their theoretical values, thus confirming the successful synthesis of the target conjugate. Furthermore, [the following was also employed]... 1 The structure of CEL-TPP was verified by H-NMR. Figure 5 As shown, the multiplets in the CEL-TPP chemical shift range of δ 7.64–7.82 ppm are attributed to protons on the benzene ring of the TPP group, while the signals in the range of δ 0.39–2.32 ppm correspond to various protons on the CEL backbone. These characteristic signals preliminarily confirm the existence of both CEL and TPP structural units. Furthermore, the doublet observed at δ 3.95–4.09 ppm can be attributed to methylene protons in the linking group (-CH2-CH2-CH2-), confirming that the three-carbon linking chain of TPP remains intact during the reaction and does not break.

[0067] (4) Characterization of CEL-TPP@siSurvivin / TDNP NPs

[0068] The prepared CEL-TPP@siSurvivin / TDNP nanoparticles were placed at 4°C. The storage stability of the nanoparticles was assessed by monitoring changes in particle size over time and the polydispersity index (PDI). Free siSurvivin and an equal volume of CEL-TPP@siSurvivin / TDNP nanoparticles were mixed with 10% FBS and incubated at room temperature for 0, 1, 2, 4, and 6 h, respectively. An equal volume of 0.2% SDS was then added to lyse the nanoparticles. Agarose gels were performed on samples at different time points under the conditions of 50 V for 10 min. The serum stability of the nanoparticles was assessed by comparing the intensity and integrity of the siSurvivin bands at different time points. The gel bands are shown below. Figure 7 As shown, this demonstrates that NPs have a significant protective effect against siRNA.

[0069] Example 2

[0070] In vitro antitumor activity of nanoparticles

[0071] Cell culture: The murine 4T1 breast cancer cell line was obtained from the National Center for Cell Science (NCCS) and cultured in RPMI-1640 medium containing 10% fetal bovine serum. Cells were grown at 37°C in 5% CO2, with the medium changed daily and passaged every other day at a 1:10 ratio.

[0072] Cell resuscitation procedure: Remove the cryovials from liquid nitrogen and quickly place them in a preheated 37°C water bath, gently shaking until the liquid is completely thawed. Then, in a laminar flow hood, transfer the cell suspension to a sterile centrifuge tube containing 10 mL of culture medium and centrifuge at 1000 rpm for 5 min. Discard the supernatant, resuspend the cells in 1 mL of culture medium, and then transfer the resuspended cells to a cell culture flask containing 10 mL of culture medium. After thorough mixing, incubate in an incubator.

[0073] Cell passage: When the cells cover 80%-90% of the culture flask, passage should be performed immediately. First, discard the original culture medium in the flask, add an appropriate amount of PBS for washing, and then discard the PBS. Next, add 2 mL of trypsin and place the flask in an incubator for 1 min of digestion. When the cells become round, quickly add double the volume of serum-containing culture medium to stop digestion. Afterward, gently pipette the cells to disperse them. Collect the cell suspension in a sterile centrifuge tube and centrifuge at 1000 rpm for 5 min. Discard the supernatant, resuspend the cells in culture medium, and aliquot them into new culture flasks for continued culture.

[0074] Cell cryopreservation: Prepare cryopreservation culture medium containing 7% DMSO. Select cells in the logarithmic growth phase, remove the old culture medium, wash with PBS, and then add trypsin to digest the cells into a single-cell state. After digestion, add serum-containing culture medium to terminate the digestion process. Next, gently mix the cell suspension and count the cells, adjusting the cell concentration to 5 × 10⁶ cells / year. 6 - 1×10 7 Cells / mL. The cell suspension was then aliquoted into cryovials, 1.0–1.5 mL per tube, and labeled with the cell name, cryopreservation date, and operator information. The cryovials were initially placed at -20°C for 2 hours, then transferred to -80°C overnight. The following day, the cryovials were transferred to liquid nitrogen for long-term storage.

[0075] Using 4T1 cells as a research model, the in vitro antitumor activity of CEL-TPP was determined using the CCK-8 assay. 4T1 cells were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density in 96-well plates. After the cells had expanded, the medium was changed by diluting different concentrations of CEL-TPP and CEL-TPP@siSurvivin / TDNP NPs with culture medium. The concentrations of CEL-TPP and CEL-TPP@siSurvivin / TDNP NPs in each group were 0, 0.5, 1.0, 1.5, and 2.0 μg / mL, respectively. After treatment, each group was incubated for 24 h, and then the medium was changed by adding 10 μL of fresh culture medium containing CCK-8 reagent to each well, and incubated for another 1 h. Finally, the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%.

[0076] The results are as follows Figure 8 As shown, compared with free CEL-TPP, CEL-TPP@siSurvivin / TDNP NPs exhibited a significantly enhanced tumor cell inhibitory effect in a concentration-dependent manner. This enhanced cytotoxicity can be attributed to two synergistic factors: first, the nanoparticles facilitated cellular endocytosis of the drug, leading to the intracellular release and exertion of more CEL-TPP; second, the successfully delivered siSurvivin effectively silenced the target gene, thereby synergistically promoting tumor cell apoptosis.

[0077] Example 3

[0078] Study on nanoparticle uptake by 4T1 cells

[0079] After nanoparticles are systematically delivered and enriched in target cells, their cellular uptake efficiency and lysosomal escape ability together constitute the key rate-limiting steps affecting the final therapeutic effect. FAM-siSurvivin was used as a tracer to quantitatively assess cellular uptake of nanoparticles.

[0080] The uptake of CEL-TPP@siSurvivin / TDNP NPs in 4T1 cells was observed by flow cytometry. Cells were loaded at a rate of 2 × 10⁶ cells / year. 5 Cells were seeded at a density of [insert density here] in six-well plates and incubated at 37°C for 24 h until complete adherence. Then, FAM-labeled CEL-TPP@siSurvivin / TDNP NPs were added for 0.5 h, 1 h, 2 h, 4 h, and 6 h, respectively. After incubation, cells were washed with PBS to remove residual drugs, digested with trypsin, centrifuged to remove the pellet, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / well. 5 Cells / ml. Finally, the fluorescence of cells was collected at different time points using flow cytometry.

[0081] The uptake of siSurvivin and CEL-TPP@siSurvivin / TDNP NPs in 4T1 cells was observed using CLSM. Cells were loaded at a concentration of 1.3 × 10⁶ cells / year. 5 Cells were seeded at a density suitable for laser confocal microscopy in small dishes and incubated at 37 °C for 24 h until complete adherence. Then, the respective drugs were added. After 6 h of incubation, cells were fixed with 4% paraformaldehyde for 15 min and gently washed three times with PBS. The nuclei were then stained with DAPI for 15 min and washed three times with PBS. Finally, cell uptake in each experimental group was observed using CLSM.

[0082] like Figure 9 Flow cytometry results showed that the proportion of FAM-positive cells increased significantly with prolonged incubation time, reaching a plateau after 2 hours. Notably, cellular uptake was relatively slow in the initial stage, but showed a sharp upward trend between 1 and 2 hours. This typical uptake kinetics is consistent with endocytosis, a time-dependent process regulated by multiple factors including cell type, endocytic pathway, microenvironment, and the physicochemical properties of the nanoparticles themselves. Furthermore, CLSM imaging, such as... Figure 9 B. A direct comparison was made between the cellular uptake of the nanoparticle formulation and free siRNA. Compared with the naked siRNA group, the nanoparticle group showed significantly higher fluorescence intensity, indicating enhanced cellular uptake. This suggests that the outer curcumin-derived vesicles, with their lipid bilayer structure, not only provide effective protection for the inner core, preventing degradation by nucleases, but also significantly improve uptake efficiency and actively promote the cellular internalization process through their inherent biocompatibility and affinity for the cell membrane.

[0083] Example 4

[0084] Lysosomal escape behavior of nanoparticles

[0085] After internalization into cells, most nanomaterials are captured by lysosomes and degraded due to the abundance of hydrolytic enzymes within them, thus failing to exert their therapeutic effect. Therefore, after nanoparticles are internalized into cells, most become trapped within lysosomes and degraded by the abundant hydrolytic enzymes, leading to therapeutic failure. Thus, achieving efficient lysosomal escape is a core challenge related to the efficiency of nucleic acid drug delivery. To evaluate the lysosomal escape of CEL-TPP@siSurvivin / TDNP NPs, 4T1 cells were loaded at 1.3 × 10⁻⁶ cells per cell line. 5 Cells were seeded at a density suitable for confocal microscopy in culture dishes for 24 h. Subsequently, cells were treated with 0.5 μg / mL free siSurvivin and 1.5 μg / mL CEL-TPP@siSurvivin / TDNP NPs, respectively, at time gradients of 1, 2, 3, 4, and 6 hours. Cells were washed three times with PBS, followed by incubation with the fluorescent lysosomal probe Lyso-Tracker RED for 30 min. After washing with the stain, Hochest staining was performed for 7 min. Finally, CLSM imaging was used to observe the co-localization of lysosomes and nanoparticles in the cells. Experimental results are shown below. Figure 10 The degree of co-localization for each group was quantified by calculating the Pearson correlation coefficient, which depends on the overlap between red and green fluorescence signals. Within 1-6 hours, the PCC value showed a trend of first increasing and then decreasing, indicating that a large number of nanoparticles entered the lysosomes at 2 hours, and most successfully escaped at 6 hours. This multifunctional nano-co-delivery system, CEL-TPP@siSurvivin / TDNP NPs, can avoid degradation, ensuring the drug reaches its target site and ultimately exerts its therapeutic effect.

[0086] Example 5

[0087] In vivo antitumor activity study of nanoparticles

[0088] Establishment of a mouse tumor model: Female BALB / c mice aged 7-8 weeks and weighing 18-20 g were used as research subjects. They were acclimatized for 7 days in a standard environment with a temperature range of 26-28℃ and a humidity level of 40%-60%. Subsequently, 4T1 cells in the logarithmic growth phase were harvested, digested with trypsin, washed twice with PBS, and the cell pellet was collected. The cells were resuspended in PBS, and the cell density was adjusted to 1×10⁻⁶. 7 Insulin was administered at a rate of 1 × 10⁶ mg / ml and kept in an ice bath until needed. The insulin was injected subcutaneously into the right pectoral pad of BALB / c mice using a syringe, with each mouse receiving 1 × 10⁶ mg / ml. 6A triple-negative breast cancer solid tumor model was established using [number] cells. Once the tumor volume reached 100 mm³, mice were randomly divided into four groups of five mice each. Each group received a different treatment: PBS, siSurvivin, CEL-TPP, or CEL-TPP@siSurvivin / TDNP NPs. Administration was via tail vein injection every other day. CEL was administered at a concentration of 2 mg / kg, corresponding to a siSurvivin concentration of 0.75 mg / kg. Body weight and tumor volume were recorded regularly during treatment. Tumor volume was calculated using the formula: Tumor volume = 0.5 × L × W 2 (L represents length, W represents width). Fourteen days after treatment, tumor tissue was removed, weighed, photographed, and a tumor growth curve was plotted. For example... Figure 11 As shown, the tumor growth rate was slowest in the CEL-TPP@siSurvivin / TDNP NPs treatment group. Based on tumor weight, the tumor growth inhibition rate of CEL-TPP@siSurvivin / TDNP NPs was 75.56%, which was 1.55 times that of the CEL-TPP group and 1.99 times that of the sisurvivin group. These results indicate that CEL-TPP@siSurvivin / TDNP NPs can co-deliver CEL-TPP and sisurvivin to the tumor site, inducing tumor cell apoptosis and exhibiting significant anti-tumor activity, which is significantly superior to single treatment.

Claims

1. A method for preparing a multifunctional nano-co-delivery system CEL-TPP@siSurvivin / TDNPNPs based on turmeric-derived plant exosomes, comprising the following steps: (1) Dissolve triptolide (CEL), inorganic base and phosphorus-containing organic salt in an organic solvent, react and purify; (2) Turmeric vesicles were obtained by processing peeled turmeric; (3) The product obtained in step (1) is mixed with siRNA (siSurvivin) targeting Survivin to obtain self-assembled nanoparticles; the self-assembled nanoparticles are then mixed with the turmeric vesicles obtained in step (2) at a mass ratio and processed to obtain the nano-co-delivery system.

2. The method for preparing the multifunctional nanocomposite delivery system CEL-TPP@siSurvivin / TDNP NPs according to claim 1, characterized in that, In step (1), the molar ratio of triptolide CEL, inorganic alkali and phosphorus-containing organic salt is (0.5-2):(1-10):(1-5).

3. The method for preparing the multifunctional nanocomposite delivery system CEL-TPP@siSurvivin / TDNP NPs according to claim 1, characterized in that, The inorganic base in step (1) is selected from at least one of sodium bicarbonate, sodium carbonate, potassium bicarbonate, and ammonium bicarbonate; the phosphorus-containing organic salt is selected from at least one of triphenylphosphine bromide (TPP-Br), tetraphenylphosphine chloride, tetraphenylphosphine bromide, tetraphenylphosphine iodide, and tetraphenylphosphine tetrafluoroborate; and the organic solvent is selected from at least one of N,N-dimethylacetamide, N-methyl-2-pyrrolidone, and dimethyl sulfoxide.

4. The method for preparing the multifunctional nanocomposite delivery system CEL-TPP@siSurvivin / TDNP NPs according to claim 1, characterized in that, In step (1), the reaction is a reflux reaction with a temperature of 50-100℃ and a reaction time of 15-20 h; the purification process includes extraction, filtration, rotary evaporation and column chromatography elution in sequence; wherein the eluent for column chromatography elution is a mixed solvent of dichloromethane and methanol with a volume ratio of (15-25):(1-5).

5. The method for preparing the multifunctional nanocomposite delivery system CEL-TPP@siSurvivin / TDNP NPs according to claim 1, characterized in that, The processing steps in step (2) include grinding, differential centrifugation precipitation, ultracentrifugation, and ultrasonic resuspension; wherein, the differential centrifugation precipitation includes: first centrifugation: room temperature, 2000-3000 × g, 15-25 min; second centrifugation: room temperature, 9000-10000 × g, 30-50 min; the conditions for ultracentrifugation are: 2-10℃, 130000-150000 × g, 1-3 h.

6. The method for preparing the multifunctional nanocomposite delivery system CEL-TPP@siSurvivin / TDNP NPs according to claim 1, characterized in that, The concentration of the product obtained in step (1) in step (3) is 5-15 mg / mL; the concentration of the siRNA (siSurvivin) targeting Survivin is 15-25 μM; the two are diluted 10-15 times respectively, and the molar ratio of the two after dilution is (5-15):(0.5-3); the mass ratio of the self-assembled nanoparticles to the turmeric vesicles obtained in step (2) is (1-5):(0.5-2).

7. The method for preparing the multifunctional nanocomposite delivery system CEL-TPP@siSurvivin / TDNP NPs according to claim 1, characterized in that, The mixing reaction process in step (3) includes vortexing and incubation; the treatment is centrifugation; wherein the incubation conditions are 35-37℃ for 10-20 min; and the centrifugation conditions are 8000-12000 rpm for 10-20 min.

8. The multifunctional nano-co-delivery system CEL-TPP@siSurvivin / TDNP NPs prepared by the method of claim 1.

9. The use of the multifunctional nanocomposite delivery system CEL-TPP@siSurvivin / TDNP NPs as described in claim 8 in the preparation of a medicament for treating breast cancer.

10. A drug for treating breast cancer, characterized in that, This includes the multifunctional nanocomposite delivery system CEL-TPP@siSurvivin / TDNP NPs as described in claim 8, as well as pharmaceutically acceptable excipients or carriers.

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