Small interfering RNA (siRNA) therapy in acute myeloid leukemia (AML) is a promising strategy as the siRNA molecule can specifically target proteins involved in abnormal cell proliferation. The development of a clinically applicable method for delivering siRNA molecules is imperative due to the challenges involved in effectively delivering the siRNA into cells. We investigated the delivery of siRNA to AML MOLM-13 cells with the use of two lipid-substituted polyethyleneimines (PEIs), a commercially available reagent (Prime-Fect) and a recently reported reagent with improved lipid substitution (PEI1.2k-PHPA-Lin9). The siRNAs utilized in this study were targeting the oncogenes FLT3 and KMT2A::MLLT3. Both lipopolymers gave similar-size siRNA complexes (210–220 nm) with positive ζ-potentials (+17 to +25 mV). While the binding efficiency of both lipopolymers to siRNA were similar, PEI1.2k-PHPA-Lin9 complexes were more resistant to heparin-induced dissociation. The quantitative analysis of gene silencing performed by qPCR as well as immunostaining/flow cytometry indicated significant reduction in both FLT3 expression and FLT3 protein after specific siRNA delivery. The desired inhibition of cell growth was attained with both FLT3 and KMT2A::MLLT3 siRNAs, and the combination provided more potent effects in both cell growth and colony formation assays. Induction of apoptosis was confirmed after specific siRNA treatments using the Annexin V assay. Using Luc(+) MOLM-13 cells, the growth of the xenografted cells was shown to be retarded with Prime-Fect-delivered FLT3 siRNA, unlike the siRNA delivered with PEI1.2k-PHPA-Lin9. These results demonstrate the potential of designed lipopolymers in implementing RNAi (via delivery of siRNA) for inhibition of leukemia growth and provide evidence for the feasibility of targeting different oncogenes using siRNA-mediated therapy.

Background/Objectives: Messenger RNA (mRNA) delivery to circulating, suspension-growing cells remains challenging due to their inherent barriers to mRNA uptake. This study aimed to develop a novel lipopolymer-based nanoparticle (NP) platform to improve mRNA delivery and gene expression in suspension-growing cells and investigate the influence of NP physicochemical properties on delivery efficiency. Methods: Polyethyleneimine (PEI) was functionalized with lipidic moieties through degradable urethane linkages to generate lipopolymers with varying lipid modifications. mRNA delivery and gene expression were evaluated in suspension-growing myeloid leukemia cells in vitro and following systemic administration in mice. The effects of incorporating the anionic additive Trans-Booster (TB) into binary and ternary NPs were investigated. Machine learning analysis using SHapley Additive exPlanations (SHAP) was performed across 125 formulations to identify key physicochemical determinants of delivery performance. Results: Geraniol-conjugated PEI demonstrated the highest gene expression among the lipid modifications investigated. Incorporation of TB further enhanced transfection efficiency by modulating NP surface charge. In mice, binary NPs preferentially enhanced mRNA delivery to the spleen and lungs, whereas ternary NPs promoted gene expression primarily in the liver and intestine. SHAP analysis identified TB–charge interactions, TB presence, steric hindrance, lipid double bonds, and effective hydrophobicity as key determinants of mRNA delivery efficacy. Conclusions: The developed lipopolymer-based NP platform demonstrates potential for improving mRNA delivery to suspension-growing cells. Lipid functionalization and TB incorporation influenced transfection efficiency and tissue distribution, while SHAP analysis provided insights into the physicochemical factors governing delivery performance. These findings support further development of engineered lipopolymer NPs for mRNA-based applications targeting circulating cells.