Functionalized lipid nanocarriers (LNCs) for targeting intracellular delivery of siRNA

Gene therapy through RNA interference (RNAi) is rapidly emerging as a personalized approach to cancer treatment. Small interfering RNAs (siRNAs) play a pivotal role in this strategy by selectively silencing target genes, thereby enabling the inhibition of disease-associated proteins. However, the success of siRNA-based therapies depends heavily on the development of efficient delivery systems capable of crossing the cell membrane, protecting siRNA from degradation, and releasing it into the cytoplasm.1 To address this challenge, we developed lipid nanocarriers (LNCs) using the main component of the eukaryotic cell membrane (POPC) to enhance the biocompatibility of the entire system, an ionizable lipid (DODMA) to ensure siRNA encapsulation, cholesterol to improve the stability of the lipid bilayer, and PEGylated lipids (DOPE-PEG2000-COOH and DOPE-PEG2000-maleimide) to help shield LNCs from recognition and clearance by the immune system. LNCs were characterized using chemico-physical techniques. DLS and SANS allowed us to obtain the hydrodynamic radius (75 ± 1 nm) of the designed LNCs and the bilayer thickness (0.44 ± 0.02 nm); ζ-potential measurements suggested a positive surface charge (48.3 ± 0.7 mV) at acidic pH values, typical of endosomal and tumoral environments; EPR spectra provided insights into the lipid bilayer microstructure. To enhance targeted delivery, we functionalized LNCs with CGSG-T7 peptide, selected for its affinity with the transferrin receptor, which is overexpressed on the blood-brain barrier and the blood–tumor barrier in glioma.2 This strategy aims to increase the accumulation of the therapeutic payload at the tumor site, thereby improving therapeutic efficacy. siRNA pGL3 was successfully encapsulated into the functionalized LNCs (CGSG-T7@LNCs), as demonstrated by agarose gel electrophoresis. Additionally, the interaction between CGSG-T7@LNCs and Large Unilamellar Vesicles (LUVs) mimicking the tumor cell membrane was demonstrated using SANS and acquiring TEM and CLSM images. Preliminary flow cytometry experiments were performed to assess the internalization efficiency of LNCs in different cell populations, including cells overexpressing the hTfR receptor targeted by the CGSG-T7 peptide.