Authors: Connor Purcell, Kadhambari Rajendran, Wafik S. El-Deiry, Theresa M. Raimondo
Published: 2025-04-21
DOI: 10.1158/1538-7445.am2025-1865
Source: Full article
The design of delivery vehicles to protect microRNAs (miRNAs) from degradation, enable tissue-specific targeting, and avoid potential off-target effects is a central challenge for their therapeutic application. miRNA encapsulation by lipid nanoparticles (LNPs) has been used to extend in vivo half-life, and LNPs may also be conjugated to an antigen targeting moiety to guide delivery to tumors. In vitro studies have identified miR-6883 as a promising therapeutic agent which targets CDK4/6, induces apoptosis, and reduces HIF1α levels in colorectal and breast cancer. In the present study, we employ a partial factorial design of experiments (DOE) to determine the optimal molar ratios of LNP components for on-target tumor potency, and use the miRNA-LNPs to further explore the potential therapeutic application of miR-6883. Methods: LNPs were formulated with MC3 or Lipid 331, DOTAP, DSPC, cholesterol, and DMG-PEG2k. Aqueous and lipid phases were combined a 3:1 ratio, followed by rapid pipetting and overnight dialysis. Encapsulation and RNA concentration were determined by Ribogreen assay. Polydispersity index (PDI) and diameter were determined by dynamic light scattering. Cancer cell lines were reverse transfected with 50nM of LNP-encapsulated RNA (miR-6883 or control). Viability was determined via CellTiter-Glo assay and protein levels by western blot. Fluorescent imaging and flow cytometry were performed to evaluate uptake of fluorescently-labeled LNPs. Partial factorial DOE was used to survey a design space consisting of a 4-part mixture (ionizable lipid, phospholipid, cholesterol, and PEG molar ratios) and a binary categorical variable to include 5% cationic lipid (JMP software). Results: Initial dosing with fluorescently labeled LNPs showed a dose-dependent increase in fluorescence, at lipid doses as low as 1µg/mL. Following formation of 12 LNP formulations based on the DOE screening design, LNPs had a maximum RNA encapsulation of 94.32%, PDIs from 0.07-.279 and diameters from 98.85-240.3nm. Multiple formulations showed potent anticancer activity, with a maximal 93.52% reduction in viability observed in HCT116 after 72h of 50nM miR-6883 compared to an untreated control. Protein level modulation of CDK4 and CDK6 was observed in all LNPs tested, as well as a reduction of HIF1α after 4 days. Notably, multiple samples exhibited an increase in CDK6, warranting further investigation. Conclusions: Our DOE identified candidate miR-6883-LNP formulations for further optimization. Future work will assess the optimized miR-LNP therapeutic in murine models of colorectal and breast cancer. Currently, we are generating antibody-conjugated LNPs, with the aim of increasing potency and mitigating off target toxicity, and are continuing LNP optimization for in vivo studies.