Development and characterization of hybrid liposome-exosome nanoparticles for targeted drug delivery in glioblastoma

Glioblastoma (GBM) is the most aggressive brain malignancy in adults [1]. The complex microenvironment of GBM presents significant obstacles to traditional drug delivery methods, prompting the exploration of nanoparticles (NPs). The fusion of liposomes (LPs) and exosomes (EXOs) is considered a promising strategy to enhance drug carrier biocompatibility, targeting properties, and cellular uptake [2]. The goal of our research was to synthesize and characterize hybrid NPs for the delivery of doxorubicin (DOX) to GBM cells. Two LP formulations, F1 and F2, were prepared via the thin layer evaporation method, each with distinct lipid molar ratios: 6:3:1 for DPPC:CHOL:DSPEmPEG2000 in F1 and 7:4:6:1 for DPPC:DPPS:CHOL:DSPEmPEG2000 in F2. Lipid films were hydrated with either a PBS solution (10 mM, pH 7.4), ammonium sulphate solution (250 mM, pH 5.5), or M0 macrophage-derived EXOs in PBS, following extrusion through membrane filters. For loaded LPs, DOX was entrapped using a pH gradient and remote loading procedure, with entrapment efficiency (EE%) determined by fluorescence. Size, polydispersity index (PdI), and zeta potential (ZP) were assessed using dynamic light scattering. Long-term stability of all NPs at 4°C was evaluated weekly for a month. The short-term stability of empty LPs and the drug release of loaded LPs in DMEM with 10% FBS at different pH levels were assessed under stirring at 37°C for 72 hours. The average size of F1, F2, M0-F1 and M0-F2 NPs was below 152 nm, while loaded LPs increased in diameter by up to 20 nm. The PdI of all NPs was < 0.2. ZP of empty NPs was –13.2 ± 1.2 mV (F1), –23.4 ± 1.9 mV (F2), -36.4 ± 3.9 mV (M0-F1), and -24.6 ± 2.4 mV (M0-F2), while DOX loading altered ZP from 6 to 13 mV. F1, F2, DOX-F2, M0-F1 and M0-F2 remained stable at 4°C for a month. However, the PdI of DOX-F1 slightly increased in the third week. Empty LPs remained stable in stressful conditions for 72 hours. The EE% of F2 LPs was 40% higher compared to F1. Finally, DOX was released more rapidly from F2 NPs at pH 5.5, increasing selectivity for the tumor microenvironment. Overall, the F2 liposomal formulation showed promising characteristics for developing hybrid nanocarriers, making M0-F2 NPs a strong candidate for surface modifications and further in vitro studies.