Composite drug-loaded exosome for overcoming cancer trail resistance and preparation method thereof

Abstract

The application discloses a compound drug-loaded exosome for overcoming cancer TRAIL drug resistance and a preparation method thereof, relates to the technical field of compound nano-drug preparation, and comprises an engineered exosome and an interfering RNA targeting a natriuretic peptide receptor A gene, wherein the engineered exosome is derived from a cell capable of expressing a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and carries the TRAIL protein; the interfering RNA is loaded in the engineered exosome; and the engineered exosome and the interfering RNA synergistically act to silence the NPRA gene through the interfering RNA to overcome the drug resistance of cancer cells to TRAIL. The application can silence the NPRA gene and remodel the apoptosis sensitivity of tumor cells through multiple channels, thereby efficiently overcoming the TRAIL drug resistance.

Description

Technical Field

[0001] This invention relates to the field of composite nanomedicine preparation technology, and in particular to a composite drug-loaded exosome that overcomes cancer TRAIL resistance and its preparation method. Background Technology

[0002] Tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) is considered a highly promising anticancer drug due to its selective induction of tumor cell apoptosis. To overcome its limitations as a recombinant protein, researchers have developed a delivery platform based on engineered exosomes (EVs). By genetically engineering cells (such as mesenchymal stem cells) to secrete exosomes carrying the TRAIL protein (EV-T), this strategy has significantly improved the stability and targeted delivery efficiency of TRAIL, representing an important direction in this field.

[0003] However, this technological approach faces a fundamental challenge: the inherent or acquired resistance of tumor cells to TRAIL. Existing solutions primarily focus on optimizing the carrier itself (e.g., increasing yield, enhancing targeting) or combining it with broad-spectrum chemotherapy drugs. These methods do not directly intervene in the intrinsic molecular pathways leading to resistance, thus having limited effectiveness in restoring TRAIL sensitivity and potentially increasing non-specific toxicity. The core bottleneck of current technology lies in the lack of a synergistic therapeutic strategy capable of actively and precisely reversing the TRAIL resistance mechanisms within tumor cells. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides a method for preparing composite drug-loaded exosomes to overcome cancer TRAIL resistance, solving the technical problem in the prior art of lacking a synergistic therapeutic strategy that can precisely reverse tumor cell TRAIL resistance by actively intervening in intrinsic molecular pathways.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a composite drug-loaded exosome that overcomes cancer TRAIL resistance and a method for preparing the same, comprising, Engineered exosomes derived from cells expressing tumor necrosis factor-associated apoptosis-inducing ligands, such that the exosomes carry the TRAIL protein; and interfering RNA targeting the natriuretic peptide receptor A gene, wherein the interfering RNA is encapsulated within the engineered exosomes; The engineered exosomes work synergistically with the interfering RNA to silence the NPRA gene, thereby overcoming cancer cells' resistance to TRAIL.

[0007] As a preferred embodiment of the composite drug-loaded exosomes for overcoming TRAIL resistance in cancer according to the present invention, wherein: the cells capable of expressing TRAIL are mesenchymal stem cells that have been genetically engineered to overexpress TRAIL.

[0008] Furthermore, the selection of genetically engineered mesenchymal stem cells (MSCs) overexpressing TRAIL as the cell source for engineered exosome production is based on multiple advantages. MSCs are adult stem cells with self-renewal and multi-lineage differentiation potential, exhibiting low immunogenicity and natural tumor tropism, which endows their secreted exosomes with potential targeted delivery advantages. By genetically engineering MSCs to stably and at high levels express TRAIL, it is ensured that the exosomes continuously secreted by these cells (i.e., EV-Ts) are enriched with TRAIL protein on their membrane surface. TRAIL protein bands were clearly detected in the lysates of EV-Ts, directly confirming that engineered MSCs successfully produced exosomes carrying the therapeutic protein TRAIL. This strategy avoids complex post-processing chemical modifications or physical loading of exosomes that could affect their biological activity, directly obtaining naturally occurring therapeutic exosomes carrying functional proteins.

[0009] As a preferred embodiment of the composite drug-loaded exosomes for overcoming TRAIL resistance in cancer according to the present invention, wherein the engineered exosomes are exosomes isolated and purified from the culture supernatant of mesenchymal stem cells overexpressing TRAIL by ultracentrifugation.

[0010] Furthermore, low-speed centrifugation removes cells and cell debris, followed by higher-speed centrifugation to remove impurities such as apoptotic bodies and large vesicles. Finally, ultracentrifugation (e.g., above 100,000 × g) precipitates EV-Ts with sizes ranging from tens to over one hundred nanometers. The resulting EV-Ts exhibit a typical cup-shaped or biconcave disc-shaped morphology, a classic characteristic of exosomes. Nanoflow cytometry results further confirmed that the obtained EV-Ts had a concentrated particle size distribution of approximately 70 nm (median) and an appropriate concentration, consistent with the typical size distribution range of exosomes. In addition, Western blot verification of the purified product using exosome markers (such as TSG101) is a crucial quality control step to confirm that the obtained particles are exosomes rather than other cell vesicles or protein aggregates. Ultracentrifugation effectively preserves the native structure and biological integrity of exosomes, providing an ideal carrier for subsequent siRNA loading.

[0011] As a preferred embodiment of the composite drug-loaded exosomes for overcoming cancer TRAIL resistance as described in this invention, wherein the interfering RNA targeting the natriuretic peptide receptor A gene is a small interfering RNA.

[0012] Furthermore, small interfering RNA (siRNA) is used as a molecular tool to target and silence the natriuretic peptide receptor A gene. siRNA is a double-stranded RNA molecule approximately 21-23 nucleotides long. Through RNA interference, it can efficiently and specifically degrade target mRNA within cells, thereby inhibiting the expression of the target protein at the translational level. Whether transfected via liposomes or delivered via the EV-T described in this invention, siRNA targeting NPRA effectively knocks down the protein expression level of NPRA in A549 cells in a dose-dependent manner. This highly efficient gene silencing effect is one of the core mechanisms by which this invention overcomes TRAIL resistance. Compared to other gene silencing tools (such as shRNA), siRNA, as a mature synthetic molecule, offers flexible sequence design, stable synthesis processes, and avoids potential risks associated with viral vectors. Encapsulating it within EV-T cells using physical methods protects it from degradation by nucleases in vivo. Utilizing the delivery function of exosomes, siRNA is efficiently introduced into target cells, achieving specific silencing of the NPRA gene, thereby reversing the resistance of tumor cells to the TRAIL protein carried by EV-T.

[0013] Secondly, the present invention provides a method for preparing composite drug-loaded exosomes that overcome TRAIL resistance in cancer, comprising: Step 1, obtaining engineered exosomes: culturing engineered cells that overexpress TRAIL, and separating and purifying engineered exosomes carrying TRAIL protein from their culture supernatant; Step 2: Loading interfering RNA: Interfering RNA targeting the natriuretic peptide receptor A gene is loaded into the engineered exosomes obtained in Step 1 using a physical loading method to form composite drug-loaded exosomes. As a preferred embodiment of the method for preparing composite drug-loaded exosomes to overcome TRAIL resistance in cancer according to the present invention, wherein: the engineered cells in step one are mesenchymal stem cells overexpressing TRAIL; and the separation and purification method includes at least one of ultracentrifugation, size exclusion chromatography, or polymer precipitation.

[0014] Furthermore, when isolating and purifying TRAIL-carrying exosomes (EV-Ts) from engineered cell culture supernatants, ultracentrifugation, size exclusion chromatography (SEC), and polymer precipitation are explicitly listed as optional separation and purification methods, demonstrating the flexibility and scalability of this invention. For example, polymer precipitation (using polyethylene glycol) is simple to operate and has high throughput, making it suitable for rapid acquisition of exosomes; size exclusion chromatography can achieve high-purity separation based on differences in molecular size. These methods can effectively enrich nanovesicles with particle sizes matching the characteristics of exosomes while maintaining their structural integrity and biological activity. The claims list multiple methods, providing flexible technical pathways for different application scenarios (such as laboratory research and future large-scale production), thus broadening the scope of protection beyond a single separation method.

[0015] As a preferred embodiment of the method for preparing composite drug-loaded exosomes to overcome TRAIL resistance in cancer according to the present invention, wherein the physical loading method in step two is ultrasonic treatment.

[0016] Furthermore, by utilizing the cavitation effect, microjets, and mechanical shear forces generated by ultrasound, micropores are instantaneously formed on the exosome membrane or its structure is temporarily loosened, allowing siRNA molecules in the external solution to passively diffuse into the vesicles. Through ultrasound treatment, siRNA was successfully loaded into EV-Ts, forming a functional complex. Compared with methods such as chemical transfection or electroporation, the ultrasound loading method has advantages such as simple operation, relatively mild conditions, no need to introduce additional chemical reagents, and avoidance of potential damage to the vesicle structure caused by high voltage. This method can effectively achieve siRNA encapsulation. As a preferred embodiment of the method for preparing composite drug-loaded exosomes to overcome TRAIL resistance in cancer according to the present invention, wherein: chloroquine, an endosome / lysosome inhibitor, is used concurrently or before administering the composite drug-loaded exosomes to target cells or subjects; the chloroquine is used to inhibit autophagy and block the lysosomal degradation pathway of exosomes to improve the delivery efficiency of interfering RNA and the silencing effect of target genes.

[0017] Furthermore, chloroquine, an endosomal / lysosomal inhibitor, is used concurrently or prior to administering the compound drug-loaded exosomes to the target cells or subjects. Through chloroquine's specific inhibitory effect on autophagy, the fusion process of the compound drug-loaded exosomes with lysosomes after endocytosis is effectively blocked. This prevents the interfering RNA loaded on the exosomes from being degraded by acidic hydrolases, ensuring that the interfering RNA can be released into the cytoplasm in its intact and active form, thereby significantly improving its silencing efficiency and therapeutic effect on the target gene.

[0018] In a preferred embodiment of the method for preparing composite drug-loaded exosomes to overcome TRAIL resistance in cancer according to the present invention, the lysosome-mediated autophagy inhibitor is chloroquine or a pharmaceutically acceptable salt thereof.

[0019] Furthermore, by neutralizing the acidic environment within the endosome / lysosome, it inhibits hydrolytic enzyme activity and, due to its "proton sponge effect," causes endosome membrane rupture, thereby promoting the escape of its contents into the cytoplasm. In all key in vitro functional experiments, 50 µM chloroquine was explicitly added to the experimental system. This consistent experimental design irrefutably demonstrates the necessity of chloroquine as a lysosome-mediated autophagy inhibitor for the synergistic therapeutic effect of the complex of this invention.

[0020] As a preferred embodiment of the method for preparing composite drug-loaded exosomes to overcome TRAIL resistance in cancer according to the present invention, wherein: the composite drug-loaded exosomes prepared by the method are capable of: (a) Effectively downregulates the expression of NPRA protein in target cancer cells; (b) Upregulates the expression of the death receptor DR5 in target cancer cells; (c) Downregulate the expression of one or more anti-apoptotic proteins selected from cFLIP, MCL-1, BCL-2, XIAP and cIAP1 in target cancer cells; (d) Inhibit the activation of the NF-κB signaling pathway in target cancer cells.

[0021] Furthermore, (a) effectively downregulates the expression of NPRA protein in target cancer cells: This is the most direct and upstream effect of the present invention. After treating A549 cells with siNPR1@EV-T prepared in this invention, the intracellular NPRA protein level can be significantly reduced in a dose-dependent manner, proving that it has successfully achieved the function of targeted gene silencing.

[0022] (b) Upregulation of the expression of the death receptor DR5 in target cancer cells and (c) downregulation of the expression of one or more anti-apoptotic proteins: these two points reveal the pivotal regulatory role of the present invention in reversing drug resistance and initiating apoptosis. Figure 5 (a) Western blot results showed that siNPR1@EV-T treatment remodeled intracellular apoptosis signaling pathways: on the one hand, the expression of the death receptor DR5 (one of the receptors for TRAIL) was upregulated, essentially "opening the door to apoptosis signaling"; on the other hand, the expression levels of several key anti-apoptotic factors, including cFLIP, MCL-1, BCL-2, XIAP, and cIAP1, were significantly inhibited, essentially "removing multiple brakes from the cell." This dual effect greatly enhanced the cell's sensitivity to TRAIL-induced apoptosis.

[0023] (d) Inhibition of NF-κB signaling pathway activation in target cancer cells: This is another key aspect of the invention. NF-κB is a classic pro-survival and anti-apoptotic signaling pathway. siNPR1@EV-T treatment significantly reduced the phosphorylation level of p65 protein (P-p65), i.e., inhibited NF-κB pathway activation. This further shuts down cellular survival signals, synergistically with the effects of points (b) and (c) above, jointly pushing cells towards irreversible apoptosis.

[0024] The beneficial effects of this invention are as follows: engineered exosomes (EV-T) are isolated and purified from the culture supernatant of engineered cells (such as mesenchymal stem cells) overexpressing TRAIL. Then, siRNA is loaded into the EV-T via physical methods such as sonication. This EV-T can be selectively combined with lysosomal autophagy inhibitors such as chloroquine. This complex can synergistically deliver apoptosis-inducing signals and drug resistance reversal elements, and by silencing the NPRA gene, it remodels the apoptosis sensitivity of tumor cells through multiple pathways, thereby effectively overcoming TRAIL resistance. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Figure showing the results of characterizing and identifying TRAIL-expressing exosomes derived from engineered MSCs.

[0027] Figure 2 The image shows the results of detecting NPRA expression in various cancer cells and efficiently knocking down NPRA expression in cancer cells using siNPR1@EV-T complex exosomes encapsulated with siRNA.

[0028] Figure 3 Figure showing the results of siNPR1@EV-T efficiently and specifically overcoming the resistance of lung cancer cell lines to TRAIL.

[0029] Figure 4 Figure showing apoptosis in A549 cells that have been strongly induced to tolerate TRAIL by siNPR1@EV-T.

[0030] Figure 5 Immunoblot analysis of components of the TRAIL and NF-κB signaling pathways.

[0031] Figure 6 This image shows the complete regression of subcutaneous A549 xenograft tumors induced by siNPR1@EV-T treatment in an animal tumor model.

[0032] Figure 7 A schematic diagram illustrating the mechanism of siNPR1@EV-T in overcoming TRAIL tolerance and effectively treating tumors. Detailed Implementation

[0033] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0034] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0035] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0036] Reference Figures 1-7 This is one embodiment of the present invention, which provides a composite drug-loaded exosome for overcoming TRAIL resistance in cancer and a method for preparing the same, comprising the following steps: Example 1: Preparation, characterization, and silencing effect of the siNPR1@EV-T complex on the NPRA gene in A549 cells. 1. Purpose This embodiment aims to illustrate the specific preparation process of the composite drug-loaded exosome (siNPR1@EV-T) of the present invention, and to perform basic characterization of the prepared engineered exosome (EV-T) and the final complex. Simultaneously, it verifies the ability of this complex to deliver siRNA in vitro and efficiently silence the target gene NPRA.

[0037] 2. Materials and Methods Preparation of engineered exosomes (EV-T): Mesenchymal stem cells overexpressing human TRAIL protein were used. Cell culture supernatant was collected and purified by differential ultracentrifugation (sequential centrifugation at 300×g, 2,000×g, and 10,000×g to remove cells and debris, followed by final ultracentrifugation at 100,000×g to precipitate exosomes) to obtain EV-T. Morphology was observed using transmission electron microscopy, particle size and concentration were analyzed by nanoflow cytometry, and the TRAIL protein and exosome marker TSG101 carried by EV-T were detected by Western blot.

[0038] Preparation of siNPR1@EV-T: The purified EV-T was mixed with small interfering RNA targeting the human NPRA gene, and the siRNA was loaded into EV-T by sonication (sonication power, sonication for several seconds, ice bath interval) to form the siNPR1@EV-T complex.

[0039] Cellular gene silencing assay: Human non-small cell lung cancer A549 cells were used as a model. Cells were divided into the following groups: PBS control group, EV-T group, groups treated with different concentrations (0, 100, 200, 400, 600 nM) of free siNPR1 (transfected with Lipofectamine 2000), and groups treated with different concentrations (0, 62, 103, 180, 260 nM) of siNPR1@EV-T. The siNPR1@EV-T treatment groups received an additional 50 µM chloroquine in the culture medium. Cells were collected after 48 hours of treatment.

[0040] Detection method: The expression level of NPRA protein in each group of cells was detected by Western blot. The protein bands were semi-quantitatively analyzed by optical density scanning.

[0041] 3. Results EV-T characterization: The prepared EV-Ts exhibited typical cup-shaped or biconcave disc-shaped vesicle structures under TEM; the particle size distribution was concentrated, with a median particle size of approximately 70.2 nm; Western blot confirmed that they highly expressed TRAIL protein and the exosome marker TSG101.

[0042] Gene silencing effect: In A549 cells treated with siNPR1@EV-T, the expression level of NPRA protein showed a significant dose-dependent decrease. The knockdown effect of NPRA was most pronounced under 260 nM siNPR1@EV-T treatment.

[0043] This effect depends on the presence of chloroquine; the silencing effect is weakened in groups without chloroquine.

[0044] The silencing efficiency of siNPR1@EV-T is comparable to or even better than that of an equal dose of free siNPR1 delivered via commercial transfection reagents.

[0045] 4. Conclusion In this embodiment, engineered exosomes (EV-T) carrying TRAIL were successfully prepared, and siRNA targeting NPRA was efficiently loaded using an ultrasonic method. The obtained siNPR1@EV-T complex can effectively deliver functional siRNA into A549 cells and specifically, in a dose-dependent manner, silence NPRA gene expression, demonstrating its feasibility as a gene delivery system.

[0046] Example 2: Study on the efficacy and mechanism of siNPR1@EV-T complex in overcoming TRAIL resistance and inducing apoptosis in vitro. 1. Purpose This embodiment aims to verify the effect of the siNPR1@EV-T complex on reversing the resistance of tumor cells to TRAIL and inducing apoptosis in vitro, and to preliminarily explore its molecular mechanism of action.

[0047] 2. Materials and Methods Cytotoxicity assays: Tumor cell lines with different TRAIL sensitivities, A549, NCI-H23, and H727, as well as normal mesenchymal stem cells (UC-MSCs), were selected. Cells were treated with EV-T, EV-T loaded with disordered siRNA (scEV-T), and siNPR1@EV-T for 24 hours, respectively. All treatments included 50 µM chloroquine. Cell viability was assessed using a CCK-8 assay kit.

[0048] Apoptosis detection: Flow cytometry: A549 cells were treated with PBS (Ctrl), EV-T, scEV-T or siNPR1@EV-T for 24 hours, and the apoptosis rate was quantitatively analyzed by flow cytometry using FITC-Annexin V / PI double staining.

[0049] Immunoblotting: The expression of apoptosis-initiating protein cleavage caspase-3 and initiating protein cleavage caspase-9 in the treated A549 cells was detected by Western blot.

[0050] Mitochondrial membrane potential detection: The JC-1 fluorescent probe was used for staining, and the changes in mitochondrial membrane potential (ΔΨm) were observed and quantitatively analyzed by fluorescence intensity using confocal microscopy.

[0051] Mechanistic protein detection: After A549 cells were treated with the above groups for 24 hours, the expression changes of the following proteins were detected by Western blot: death receptors DR4 and DR5; anti-apoptotic proteins cFLIP, MCL-1, BCL-2, XIAP, and cIAP1; total p65 and its phosphorylated form P-p65 of the NF-κB pathway.

[0052] 3. Results Cytotoxicity: As shown in Fig. 3, siNPR1@EV-T significantly reduced the viability of three types of tumor cells: A549, H23, and H727, while exhibiting minimal cytotoxicity against normal UC-MSC cells. EV-T or scEV-T treatment alone showed limited cytotoxicity against A549 cells, indicating that silencing NPRA is key to restoring TRAIL sensitivity.

[0053] Induction of apoptosis: Flow cytometry results showed that siNPR1@EV-T treatment significantly increased the proportion of early and late apoptosis in A549 cells.

[0054] Western blot analysis showed that the levels of cleavage caspase-3 and caspase-9 were significantly upregulated in the siNPR1@EV-T group.

[0055] JC-1 staining showed that siNPR1@EV-T treatment led to a significant decrease in mitochondrial membrane potential (reduced red / green fluorescence ratio), indicating that mitochondrial apoptosis was triggered.

[0056] Molecular mechanism: Compared with the control group, siNPR1@EV-T treatment can: Significantly upregulated the expression of the death receptor DR5.

[0057] The expression of multiple anti-apoptotic proteins (cFLIP, MCL-1, BCL-2, XIAP, cIAP1) was significantly downregulated.

[0058] It inhibits the activation of the NF-κB pathway (manifested as a decrease in phosphorylated p65 levels).

[0059] 4. Conclusion This embodiment demonstrates that the siNPR1@EV-T complex can effectively reverse the resistance of various tumor cell lines to TRAIL through synergistic action, specifically inducing tumor cell apoptosis while having minimal impact on normal cells. Its mechanism of action is closely related to multi-target regulation involving upregulation of DR5, downregulation of key anti-apoptotic proteins, and inhibition of the NF-κB survival-promoting pathway.

[0060] Example 3: Evaluation of the in vivo antitumor efficacy of the siNPR1@EV-T complex 1. Purpose This embodiment aims to evaluate the in vivo therapeutic efficacy and safety of the siNPR1@EV-T complex in tumor-bearing animal models.

[0061] 2. Materials and Methods Animal model establishment: BALB / c nude mice were subcutaneously inoculated with A549 cells until the tumor volume grew to approximately 200 mm. 3 At that time, the mice were randomly divided into 4 groups (n=6).

[0062] Dosage regimen: Intratumoral injection therapy for 10 consecutive days. Groups are as follows: Control group (Ctrl): injected with normal saline.

[0063] EV-T group: 117.3 μg EV-Ts (equivalent to 4 ng TRAIL) were injected.

[0064] scEV-T group: 117.3 μg EV-Ts loaded with 100 μg of disordered siRNA were injected.

[0065] siNPR1@EV-T group: 117.3 μg EV-Ts injected with 100 μg siNPR1.

[0066] All injectable formulations contain 50 μM chloroquine.

[0067] Observation indicators: Tumor volume and mouse weight were measured every 2-3 days to plot growth curves.

[0068] After treatment, the mice were euthanized, the tumors were removed, and the mice were weighed.

[0069] Histological analysis: Twelve hours after the third dose, three mice from each group were sacrificed to harvest tumors. Tumor tissue was processed as follows: H&E staining was used to observe tissue morphology.

[0070] Immunohistochemical staining was used to detect the expression of the proliferation marker Ki67 and the apoptosis marker cleaved caspase-3 (C-Casp-3), and a semi-quantitative score (H-SCORE) was performed.

[0071] 3. Results Tumor suppression effect: The siNPR1@EV-T treatment group showed a very strong tumor suppression effect, with tumor growth almost completely inhibited. At the experimental endpoint, most mice showed complete tumor regression, and the final tumor weight was significantly lower than that of other groups. The EV-T group and scEV-T group showed only mild tumor suppression effects.

[0072] Safety: During the treatment, the body weight of mice in all groups remained stable, with no significant decrease or abnormality, indicating that the treatment was well tolerated.

[0073] Histological analysis: In the tumor tissue of the siNPR1@EV-T group, Ki67 positive cells were significantly reduced, while caspase-3 cleavage positive cells were significantly increased, indicating that tumor cell proliferation was inhibited and apoptosis was strongly activated.

[0074] The interfering RNA can effectively downregulate the expression of NPRA protein in target cancer cells. Specifically, in Western blotting experiments, compared with the untreated control group (Ctrl), the gray value of the band corresponding to NPRA protein in cancer cells treated with the interfering RNA (siNPR1) was significantly reduced. Moreover, this treatment had no significant effect on the expression level of death receptor DR4. At the same time, it significantly downregulated the expression levels of cFLIP, MCL-1, XIAP and cIAP-1 proteins and inhibited the phosphorylation of p65 protein.

[0075] This embodiment demonstrates that the siNPR1@EV-T complex prepared in this invention can significantly inhibit tumor growth and even completely regress tumors in an A549 lung cancer xenograft model via intratumoral administration, with efficacy far superior to single TRAIL delivery (EV-T) or non-targeted drug-loaded exosomes (scEV-T). No significant systemic toxicity was observed at effective doses, exhibiting good therapeutic potential and safety.

[0076] In summary, this invention isolates and purifies engineered exosomes (EV-Ts) from the culture supernatant of engineered cells (such as mesenchymal stem cells) overexpressing TRAIL. Then, siRNA is loaded into the EV-Ts using physical methods such as sonication. This EV-T can be selectively combined with lysosomal autophagy inhibitors such as chloroquine. This complex synergistically delivers apoptosis-inducing signals and drug resistance reversal elements, and by silencing the NPRA gene, it remodels the apoptosis sensitivity of tumor cells through multiple pathways, thereby effectively overcoming TRAIL resistance.

[0077] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A composite drug-loaded exosome for overcoming TRAIL resistance in cancer, characterized in that: include, Engineered exosomes derived from cells expressing the tumor necrosis factor-associated apoptosis-inducing ligand TRAIL, such that the exosomes carry the TRAIL protein; and interfering RNA targeting the natriuretic peptide receptor A gene, wherein the interfering RNA is encapsulated within the engineered exosomes; The engineered exosomes work synergistically with the interfering RNA to silence the NPRA gene, thereby overcoming the resistance of cancer cells to TRAIL. Western blotting results show that the interfering RNA can effectively downregulate the expression of NPRA protein in target cancer cells.

2. The composite drug-loaded exosome for overcoming cancer TRAIL resistance as described in claim 1, characterized in that: The cells that can express TRAIL are mesenchymal stem cells that have been genetically engineered to overexpress TRAIL.

3. The composite drug-loaded exosome for overcoming cancer TRAIL resistance as described in claim 2, characterized in that: The engineered exosomes are exosomes isolated and purified from the culture supernatant of mesenchymal stem cells overexpressing TRAIL by ultracentrifugation.

4. The composite drug-loaded exosome for overcoming cancer TRAIL resistance as described in claim 3, characterized in that: The interfering RNA targeting the natriuretic peptide receptor A gene is a small interfering RNA.

5. A method for preparing a composite drug-loaded exosome to overcome cancer TRAIL resistance, based on the composite drug-loaded exosome to overcome cancer TRAIL resistance as described in any one of claims 1 to 4, characterized in that: include, Step 1: Obtaining engineered exosomes: Culture engineered cells overexpressing TRAIL, and isolate and purify engineered exosomes carrying the TRAIL protein from their culture supernatant; Step 2, Loading interfering RNA: Interfering RNA targeting the natriuretic peptide receptor A gene is loaded into the engineered exosomes obtained in Step 1 using a physical loading method to form a composite drug-loaded exosome.

6. The method for preparing composite drug-loaded exosomes to overcome cancer TRAIL resistance as described in claim 5, characterized in that: The engineered cells in step one are mesenchymal stem cells overexpressing TRAIL; the separation and purification methods include at least one of ultracentrifugation, size exclusion chromatography, or polymer precipitation.

7. The method for preparing composite drug-loaded exosomes to overcome cancer TRAIL resistance as described in claim 5, characterized in that: The physical loading method in step two is ultrasonic treatment.

8. The method for preparing composite drug-loaded exosomes to overcome cancer TRAIL resistance as described in claim 5, characterized in that: Chloroquine, an endosome / lysosome inhibitor, is used concurrently or prior to administration of the compound drug-loaded exosomes to target cells or subjects. Chloroquine is used to inhibit autophagy and block the lysosomal degradation pathway of exosomes to improve the delivery efficiency of interfering RNA and the silencing effect of target genes.

9. The method for preparing composite drug-loaded exosomes to overcome cancer TRAIL resistance as described in claim 5, characterized in that: The lysosome-mediated autophagy inhibitor is chloroquine or a pharmaceutically acceptable salt thereof.

10. The method for preparing composite drug-loaded exosomes to overcome cancer TRAIL resistance as described in claim 5, characterized in that: The composite drug-loaded exosomes prepared by the method can: (a) Effectively downregulates the expression of NPRA protein in target cancer cells; (b) Upregulates the expression of the death receptor DR5 in target cancer cells; (c) Downregulate the expression of one or more anti-apoptotic proteins selected from cFLIP, MCL-1, BCL-2, XIAP and cIAP1 in target cancer cells; (d) Inhibit the activation of the NF-κB signaling pathway in target cancer cells.

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