Co-delivery of small molecules and siRNA via Janus Based Nanopieces for Solid Tumor Treatment

Introduction: Despite advancements in drug delivery technologies, there remains a significant need for vehicles that enhance targeting and biodistribution in hard-to-penetrate tissues. Solid tumors, for instance, pose significant challenges due to their dense extracellular matrix (ECM). Furthermore, multidrug resistance (MDR) is a major obstacle to effective chemotherapy, reducing drug potency and efficacy. Small molecule drugs, commonly used in chemotherapeutics, can induce apoptosis in cancer cells but often fail alone due to drug resistance. Combining small molecules with other therapies is essential for enhancing therapeutic success. Small interfering RNAs (siRNAs) involved in RNA interference play a crucial role in gene silencing and hold significant therapeutic potential. However, cancer cells frequently form spheroids with a dense ECM, impeding drug penetration. To address these challenges, we formulated a novel family of rod-shaped delivery vehicles named Janus base nanopieces (JBNps). These JBNp nanorods consist of bundles of DNA-inspired Janus base nanotubes (JBNts) with intercalated therapeutic cargoes, leading to improved penetration into the dense ECM of solid tumors. Then, we utilized the rod-shaped JBNp platform to co-deliver doxorubicin (DOX) and MDR siRNA, enabling the silencing of MDR genes and inducing effective apoptosis in cancer cells and spheroids. Our findings highlight the potential of JBNps to overcome drug resistance, improve drug penetration in cancer spheroids, and enhance the overall efficacy of cancer treatments. This study successfully developed a rod-shaped delivery vehicle for improved tissue penetration, offering significant insights and advancements in cancer therapy.

Materials and

Methods: The JBNt with intercalated doxorubicin (JBNt-DOX) was fully self-assembled into a nanotubular structure, after which siRNAs were successfully incorporated into the JBNt-DOX by sonicated with a sonicator. The morphology of JBNp-DOX-siRNA was observed via transmission electron microscopy (TEM). SKOV-3 cells were transfected with JBNp-DOX with fluorescently labeled siRNA for 3 days. The intracellular co-delivery ability of JBNp was demonstrated via confocal laser-scanning microscopy (CLSM) images. For endosomal escape study, lysotracker was used before fixing. The colocalization of siRNA and endosome flurescence was quantified based upon Pearson’s R values. Female 8- week-old NU/J mice were inoculated subcutaneously with 5 × 106 SKOV-3 cells supplemented with matrigel and allowed to grow for 4 weeks until the tumor size was 200 mm3. Mice were separated randomly into two treatment groups: Saline and JBNp-DOX-siRNA (n=6). All the treatments were administered intravenously every six days for 21 days. Mice were treated with Doxorubicin dose of 0.86 mg/kg. Tumor growth curves were plotted based on the calculation of volume = ½ (length × width2). Mice were sacrificed 3 days after the last dose for histological analysis and tumor statistics. The tumor weight was measured after scarification. Tissues were collected, and cryosections were prepared for immunofluorescence. The anti-active caspase antibody and Click-iT Plus TUNEL Assay were used to identify caspase-3 and TUNEL activity, respectively. Cryosectioned organs were stained with H&E for histological analysis.

Results, Conclusions, and Discussions: (Figure 1A) The self-assembled JBNt-DOX was sonicated with siRNA to prepare JBNp-DOX-siRNA. (Figure 1B) TEM image of nanopieces loaded with DOX and siRNA (JBNp-DOX-siRNA). (Figure 1C) The CLSM image of cell delivery of JBNp shows the JBNp successfully delivered DOX (red) and siRNA (green) into cells. (Figure 1D and 1E) The JBNp showed better endosomal escape ability compared to Lipofectamine 2000. (Figure 2A) After the tumor volume in the SKOV-3 tumor-bearing nude mice model reached approximately 200 mm3, mice were intravenously injected with saline and JBNp-DOX-siRNA every 6 days for 4 times. (Figure 2B) After 21 days, tumors were excised and weighed. Compared to the saline group, mice treated with JBNp-DOX-siRNA showed significantly decreased tumor weight. (Figure 2C) Tumor growth of the JBNp-DOX-siRNA was inhibited during 21-day treatment compared with the saline group. (Figure 2D) Tumors treated with JBNp-DOX-siRNA showed high expression of cleaved caspase-3 and TUNEL, indicating early and late apoptosis respectively. (Figure 2E) Histopathological analysis of major organs, including heart, lung, liver, spleen, and kidney, showed no difference was observed between saline and JBNp-DOX-siRNA groups.

In summary, we have developed a novel family of rod-shaped delivery vehicles, JBNp, designed to co-load small molecules and siRNAs. As a proof-of-concept, the JBNp encapsulated DOX and siRNA, successfully co-delivering them both in vitro and in vivo. When applied to an ovarian cancer model, the JBNps significantly enhanced the EPR effect, indicating the potential for combinational therapy.

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