The use of small RNAs (miRNAs or siRNAs) for cancer therapy is expanding, but delivery challenges persist. Current nanocarriers often suffer from low bioavailability, insufficient loading capacity, poor transport across biological barriers, and limited delivery to the tumor microenvironment (TME). Extracellular vesicles (EVs), particularly those derived from mammalian cells, have emerged as potential drug delivery vehicles. However, their clinical translation is hampered by concerns such as immunogenicity, low loading efficiency, low yield, and high production costs. To address these issues, this research explores the use of plant-derived vesicles (PDVs) as an alternative delivery system for small RNAs in cancer therapy. PDVs are membrane-coated vesicles released from plants, possessing biocompatibility, the ability to be taken up by mammalian cells, and stability in the human body. Their intrinsic therapeutic properties, including anti-inflammatory and antioxidant effects, further enhance their appeal. This study aims to develop a novel drug delivery system leveraging the advantages of PDVs while overcoming their existing limitations, focusing specifically on efficient delivery of small RNAs to the TME for effective cancer treatment.

The existing literature extensively covers the potential of small RNAs like microRNAs (miRNAs) and small interfering RNAs (siRNAs) as therapeutic agents for various diseases, including cancer. However, challenges in targeted delivery to the tumor microenvironment (TME) and overcoming systemic clearance rates limit their effectiveness. Several studies have investigated the use of nanocarriers, including liposomes and polymeric nanoparticles, for RNA delivery. While some success has been achieved in neurological and metabolic disorders, the application in cancer therapy remains challenging due to complexities of the TME. Mammalian-derived extracellular vesicles (EVs) are another promising delivery system under intense investigation; however, concerns over immunogenicity, low loading capacity, scalability, and cost-effectiveness hamper their widespread use. A growing body of research highlights the potential of plant-derived vesicles (PDVs) as biocompatible and readily scalable alternatives. Studies have demonstrated PDV uptake by mammalian cells and their intrinsic therapeutic properties. This study builds upon this existing knowledge, specifically focusing on engineering PDVs for efficient miRNA delivery in cancer.

This study employed a systematic approach to develop a novel drug delivery system. First, various edible plants were screened based on criteria including low allergenic potential, cost-effectiveness, high PDV yield, and cell uptake. Watermelon was selected as the optimal source based on superior PDV yield and cell uptake in several cell types representative of the ovarian cancer microenvironment (ID8 ovarian cancer cells, MOECs, CAFs, and THP-1 monocytes). Watermelon PDVs were characterized by nanotrack analysis and transmission electron microscopy (TEM). In vivo biodistribution studies in immunocompetent mice bearing ID8 ovarian tumors demonstrated efficient PDV delivery to the TME. Next, a hybrid exosomal polymeric (HEXPO) nanoparticle system was engineered. Generation 3 PAMAM dendrimers were chosen for their high branching structure and ability to complex with miRNAs. The dendrimers, complexed with miRNA (miR146a mimic or control miRctrl), were combined with watermelon PDVs to create the HEXPO nanoparticles, which were characterized for size, charge, and loading efficiency using various techniques including nanotracking analysis, TEM, electrophoresis retardation assay, spectrophotometry, and small-particle flow cytometry. miR146a was selected as the therapeutic miRNA candidate based on its association with improved overall survival in ovarian cancer patients and its involvement in pathways related to angiogenesis and inflammation/immunity. The therapeutic efficacy of HEXPO-miR146a was evaluated in three in vivo ovarian cancer mouse models (ID8, A2780, and OVCAR8) by assessing tumor growth, angiogenesis parameters (microvessel density), and mouse body weight. In vitro experiments were conducted to validate the anti-angiogenic effects of miR146a. RF24 endothelial cells were transfected with miR146a or miRctrl, and tube formation assays were performed using both direct transfection and conditioned medium from transfected A2780 and OVCAR8 cells. In vivo matrigel plug assays were also used to assess angiogenesis. An angiogenesis array analyzed the levels of secreted factors from transfected cancer cells. RNA sequencing was performed on transfected RF24, A2780, and OVCAR8 cells to identify the genes regulated by miR146a and related pathways. Further analysis investigated IRAK1 and SERPINE1 expression using quantitative real-time PCR and Western blotting, and SERPINE1 levels were measured in cell supernatants by ELISA. In silico target prediction analysis was used to determine if SERPINE1 was directly regulated by miR146a.

This study successfully developed a novel HEXPO nanoparticle system for efficient delivery of therapeutic miRNAs to the TME. Watermelon was identified as an optimal source of PDVs due to its high yield and efficient cellular uptake. HEXPO nanoparticles showed a size range of 100–300 nm and a near-neutral charge. Flow cytometry revealed high loading efficiency of miRNA into the HEXPO system (45-71% into dendrimers, 96-98% into PDVs). In vivo studies demonstrated robust therapeutic efficacy of HEXPO-miR146a in three ovarian cancer models, resulting in a significant reduction in tumor weight (67% in A2780, 55% in ID8, 69% in OVCAR8). A marked reduction in angiogenesis was observed in the TME (51% in OVCAR8, 33% in ID8). In vitro studies confirmed the dual anti-angiogenic effect of miR146a, demonstrating a decrease in tube formation in endothelial cells both directly after transfection and indirectly via conditioned medium from cancer cells. RNA sequencing revealed miR146a's impact on genes involved in angiogenesis and other pathways. SERPINE1 was identified as a key downstream target of miR146a, likely indirectly regulated via IRAK1 downregulation. Additionally, increased CD8+ lymphocyte density was observed in the TME of ID8 tumors treated with HEXPO-miR146a, suggesting a potential role for miR146a in anti-tumor immunity.

This research successfully addresses the challenge of efficient small RNA delivery for cancer therapy by developing a novel HEXPO nanoparticle platform utilizing watermelon-derived PDVs and PAMAM dendrimers. The results demonstrate significant therapeutic efficacy in vivo and provide mechanistic insight into the anti-angiogenic role of miR146a. The high loading efficiency and favorable biodistribution of HEXPO nanoparticles suggest its potential as a superior delivery system compared to existing methods. The observed anti-angiogenic effects of miR146a, both direct and indirect, highlight its therapeutic potential in cancer treatment. The identification of SERPINE1 and IRAK1 as key regulatory molecules involved in the anti-angiogenic mechanisms provides valuable insights into the molecular mechanisms underlying miR146a's effects. The observed increased CD8+ lymphocyte density in the TME suggests the potential for a synergistic effect between miR146a-mediated anti-angiogenesis and immune cell infiltration. Future research could focus on exploring combination therapies with immune checkpoint inhibitors.

This study successfully developed and characterized HEXPO, a novel, cost-effective, and biocompatible drug delivery system for therapeutic small RNAs, demonstrating potent anti-tumor activity in vivo. The findings highlight the previously unknown anti-angiogenic role of miR146a and its potential for enhancing anti-tumor immunity. Future work could focus on exploring combination therapies, optimizing the HEXPO formulation, and translating these findings into clinical trials.

The study primarily focused on ovarian cancer models. Further investigation is needed to confirm the efficacy and generalizability of the HEXPO system across diverse cancer types. While the in vivo studies demonstrated a favorable safety profile, more extensive toxicological studies are warranted before clinical translation. The mechanism of SERPINE1 regulation by miR146a requires further elucidation.