Engineered exosome targeting bladder cancer cells as well as preparation method and application of engineered exosome

Abstract

The invention relates to an engineered exosome for targeting bladder cancer cells as well as a preparation method and application of the engineered exosome, and the engineered exosome is specific siRNA (small interfering Ribonucleic Acid) which is subjected to surface modification with concanavalin A and loaded with CASC11, and is marked as ConA-EXO-siCASC11. According to the invention, the engineered exosome is modified by ConA, so that the CASC11-siRNA is effectively delivered to the BLCA cells, the therapeutic effect of the CASC11-siRNA on the BLCA cis-platinum resistance is explored, an emerging therapeutic scheme is provided for the cis-platinum resistance BLCA, and the development of the field of precise treatment based on the engineered exosome is promoted.

Description

Technical Field

[0001] This invention belongs to the field of bladder cancer treatment technology, and specifically relates to an engineered exosome that targets bladder cancer cells, its preparation method, and its application. Background Technology

[0002] Bladder cancer (BLCA) is one of the most common malignant tumors of the genitourinary system, characterized by aggressive progression and widespread treatment resistance. First-line treatment for advanced BLCA is cisplatin-based chemotherapy, which induces tumor cell necrosis and apoptosis by disrupting DNA structure through the formation of Pt / DNA adducts. However, only 35% of patients with metastatic BLCA initially respond to cisplatin chemotherapy, and most of these initially responsive patients eventually develop resistance. Overcoming cisplatin resistance in BLCA is a significant challenge, necessitating the identification of key molecules and the exploration of effective treatment strategies.

[0003] Exosomes are nanoscale extracellular vesicles ranging from 30 to 150 nm, stably residing within the tumor microenvironment (TME). Exosomes play a crucial role at various stages of tumor development by exchanging functional substances between homologous and heterologous cells. Furthermore, exosomes can encapsulate a variety of molecules, including RNA, peptides, and lipids, enabling them to specifically target recipient cells within the TME, offering a promising avenue for enhancing cancer treatment efficacy through precise drug delivery to tumor cells. However, most unmodified injectable exosomes remain in the liver or spleen rather than at the tumor site. Emerging engineered exosome technologies have demonstrated the potential to improve tumor retention rates by modifying exosomes with tumor-targeting ligands. Exosomes modified with the LAMP2B / Bla-1 fusion protein can selectively target sialic acid-Tn antigen on the surface of bladder tumor cells, thereby promoting highly efficient drug delivery to BLCA cells. Concanavalin A (ConA) is a highly efficient tumor-targeting ligand with a high affinity for mannose, which is highly expressed on the surface of BLCA cells. Fan et al. created a ConA-EPI conjugate, which utilizes mannose-bound ConA to specifically deliver epirubicin (EPI) to BLCA cells, thereby improving antitumor efficacy. However, little is known about engineered exosomes involved in the effective treatment of BLCA, and it remains unknown whether ConA-modified exosomes can promote their distribution at BLCA sites. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide an engineered exosome targeting bladder cancer cells, its preparation method and application. The engineered exosomes are modified with ConA to effectively deliver CASC11-siRNA to BLCA cells and explore its therapeutic effect on cisplatin-resistant BLCA. This provides a new treatment option for cisplatin-resistant BLCA and promotes the development of precision medicine based on engineered exosomes.

[0005] This invention provides an engineered exosome that targets bladder cancer cells. The engineered exosome is a surface-modified concanavalin A-containing siRNA loaded with CASC11, denoted as ConA-EXO-siCASC11.

[0006] This invention also provides a method for preparing engineered exosomes targeting bladder cancer cells, comprising the following steps:

[0007] BMSC-derived exosomes loaded with siCASC11 were isolated; the isolated BMSC exosomes were biotinylated by binding DSPE-PEG-biotin; the biotinylated exosomes were bound to streptavidin to form streptavidin-coupled biotinylated exosomes; and then incubated with biotin-ConA to obtain engineered exosomes targeting bladder cancer cells.

[0008] Preferably, the concentration of the DSPE-PEG-biotin is 0.01-10 μmol / L.

[0009] Preferably, the mass ratio of the biotin-modified exosomes to streptavidin is 1:1 to 3:1.

[0010] The present invention also provides the use of engineered exosomes targeting bladder cancer cells in combination with cisplatin and / or immune checkpoint inhibitors.

[0011] This invention also provides the application of engineered exosomes targeting bladder cancer cells in the preparation of drugs for treating bladder cancer.

[0012] Beneficial effects

[0013] This invention uses ConA-modified engineered exosomes to effectively deliver CASC11-siRNA to BLCA cells and explores its therapeutic effect on cisplatin-resistant BLCA, providing a novel treatment option for cisplatin-resistant BLCA and promoting the development of precision medicine based on engineered exosomes. Attached Figure Description

[0014] Figure 1 This is a schematic diagram illustrating the synthesis of the engineered exosomes of this invention.

[0015] Figure 2 Representative transmission electron microscopy images of bone marrow mesenchymal stem cell exosomes containing siCASC11 or siNC.

[0016] Figure 3 Tracking analysis and size determination of exosome nanoparticles containing siCASC11 or siNC from bone marrow mesenchymal stem cells.

[0017] Figure 4This is a schematic diagram of cisplatin treatment in tumor-bearing mice.

[0018] Figure 5 This is a schematic diagram illustrating the treatment of tumor-bearing mice with exosomes.

[0019] Figure 6 This is an in vivo imaging study of the distribution of various exosomes in tumor-bearing mice. Detailed Implementation

[0020] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0021] Example 1

[0022] I. Materials and Methods

[0023] 1. Cell Culture and Processing

[0024] Human bladder urothelial carcinoma cell line T24 was cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin. Human bone marrow mesenchymal stromal cells (BMSCs) were cultured in DMEM (low glucose, Gibco, USA) medium containing 15% FBS, 1% penicillin-streptomycin, and 5 ng / ml bFGF. All cells were maintained in a humidified incubator at 37°C and 5% CO2, routinely identified by short tandem repeat analysis, and periodically treated with mycoplasma scavenging agents.

[0025] 2. Small interfering RNA (siRNAs) and plasmid transfection

[0026] CASC11-targeting siRNA and control siRNA were purchased from Shanghai Gemma Gene Co., Ltd. The CASC11-targeting siRNA was pre-incubated in transfection medium (Opti-MEM, Gibco, USA) and then transiently transfected using Lipofectamine 3000 reagent (Invitrogen, USA).

[0027] 3. Engineered exosomes labeled with Bio-ConA

[0028] Biotin-modified BMSC exosomes were generated using 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine-poly(DSPE-PEG). First, BMSC exosome membranes were incubated with DSPE-PEG-biotin (Sigma-Aldrich, USA) at specified concentrations (0, 0.01, 0.1, 1, and 10 μmol / L) at 37°C for 30 min. Excess free DSPE-PEG-biotin was removed by ultracentrifugation at 100,000 g for 75 min. The DSPE-PEG-biotin-labeled BMSC exosomes were then incubated with streptavidin-PE antibody (1:200, ThermoFisher Scientific, USA). Flow cytometry was then used to determine the proportion of DSPE-PEG-biotin-labeled cells. Fluorescence microscopy (Nikon, Japan) was also used to evaluate the DSPE-PEG-biotin-labeled cells. Streptavidin-PE was added as a secondary antibody for DSPE-PEG-biotin-modified cells.

[0029] Subsequently, streptavidin was added to biotinylated exosomes to obtain the streptavidin-Bio-EXO complex. Streptavidin-Bio-EXO was then combined with biotinylated ConA (Sigma-Aldrich, USA) to form a Bio-ConA@Bio-EXO hydrogel. 50 μL of PBS containing streptavidin was mixed with 2 × 10⁻⁶ ppm of PBS. 10 One non-biotin-grafted BMSC exosome or 2 × 10 10 Bio-EXO (1:1, 2:1, and 3:1) conjugated with streptavidin was mixed with 50 μL of 20% (wt / vol) biotinylated ConA dissolved in 0.5% (wt / vol) lithium acylphosphonate photoinitiator solution to obtain engineered exosome ConA-EXO-siCASC11.

[0030] 4. Animal experiments

[0031] T24 cells (1×10) 6 Twenty 4-week-old female BALB / c nude mice were subcutaneously injected with exosomes (100 μg each) into the right axilla. Seven days later, the mice were intravenously injected daily with equal volumes of PBS, parental exosomes (EXO-NC, 100 μg), conventional engineered exosomes (EXO-siCASC11, 100 μg), and targeted engineered exosomes (ConA-EXO-siCASC11, 100 μg) for 15 days. Cisplatin (10 mg / kg) was administered intraperitoneally every 3 days. Tumor volume was measured every three days during treatment. At the end of the 15-day exosome treatment period, the mice were euthanized with CO2, and tumor tissue was harvested for subsequent histological analysis.

[0032] II. Experimental Results

[0033] 1. Synthesis and characterization of engineered exosome ConA-EXO-siCASC11 targeting BLCA cells

[0034] The schematic diagram illustrates that the synthesis of engineered exosomes is achieved through a series of carefully crafted steps: a) isolating BMSC-derived exosomes loaded with siNC or siCASC11. b) conjugating the isolated BMSC exosomes with DSPE-PEG-biotin to introduce a biotin moiety onto their surface. c) conjugating the biotin-modified exosomes with streptavidin to form streptavidin-coupled biotinylated exosomes. d) subsequently incubating with biotin-ConA to achieve ConA-coupled biotinylated exosomes, thereby enhancing their targeting specificity. Figure 1 ).

[0035] First, the self-assembly of DSPE-PEG-Biotin into the membrane of BMSCs was analyzed. BMSCs were incubated with gradient concentrations of DSPE-PEG-Biotin (0, 0.01, 0.1, 1, and 10 μmol / L). Flow cytometry analysis showed a significant increase in the biotinylated exosome population, with approximately 75% biotinylated cells generated after incubation with 10 μmol / L DSPE-PEG-Biotin. These results indicate that the lipid tail of DSPE-PEG-Biotin self-assembles into the membrane of BSMCs, providing a basis for the construction of engineered exosomes. Subsequently, the biotinylated BMSC exosomes were incubated with streptavidin, followed by incubation with ConA-Biotin to obtain appropriately engineered exosomes, named ConA-EXO-siCASC11. TEM analysis revealed nanoscale spherical vesicles with diameters ranging from 50 to 200 nm. Furthermore, after modifying their surfaces with ConA-coupled biotin, the morphologies of ConA-EXO-siCASC11 and ConA-EXO-siNC remained intact. Figure 2 NTA analysis shows that the peak diameters of ConA-EXO-siCASC11 and ConA-EXO-siNC are 164.8 nm and 175.3 nm, respectively. Figure 3 The experimental results collectively demonstrate the successful and efficient engineering of ConA-modified BMSC exosomes with minimal impact on their biophysical properties.

[0036] 2. ConA-EXO-siCASC11 enhances cisplatin sensitivity.

[0037] To evaluate the in vivo antitumor effects of targeted engineered exosomes combined with cisplatin, tumor-bearing mice in each group were administered PBS, EXO-NC, EXO-siCASC11, or ConA-EXO-siCASC11 via tail vein injection once daily. Cisplatin (10 mg / kg) was administered intraperitoneally every 3 days. Figure 4 , 5Compared with cisplatin alone, tumors from the EXO-siCASC11 and ConA-EXO-siCASC11 groups showed reduced tumor weight and volume when CASC11 was targeted (P<0.0001). Figure 6 Furthermore, compared with the EXO-siCASC11 group, the ConA-EXO-siCASC11 group showed a more significant anti-tumor effect on tumor size (P<0.01), indicating that engineered exosomes designed to simultaneously target BLCA cells and deliver siRNA targeting CASC11 can significantly enhance the anti-tumor efficacy of cisplatin.

Claims

1. An engineered exosome targeting bladder cancer cells, characterized in that: The engineered exosomes are surface-modified concanavalin A-specific siRNAs loaded with CASC11, denoted as ConA-EXO-siCASC11.

2. A method for preparing engineered exosomes targeting bladder cancer cells, comprising the following steps: BMSC-derived exosomes loaded with siCASC11 were isolated; the isolated BMSC exosomes were biotinylated by binding DSPE-PEG-biotin; the biotinylated exosomes were bound to streptavidin to form streptavidin-coupled biotinylated exosomes; and then incubated with biotin-ConA to obtain engineered exosomes targeting bladder cancer cells.

3. The preparation method according to claim 2, characterized in that: The concentration of the DSPE-PEG-Biotin is 0.01-10 μmol / L.

4. The preparation method according to claim 2, characterized in that: The mass ratio of the biotin-modified exosomes to streptavidin is 1:1 to 3:

1.

5. The use of an engineered exosome targeting bladder cancer cells as described in claim 1 in combination with cisplatin and / or an immune checkpoint inhibitor.

6. The use of an engineered exosome targeting bladder cancer cells as described in claim 1 in the preparation of a medicament for treating bladder cancer.

Images