Purpose: Glioblastoma multiforme (GBM) is a WHO grade IV primary central nervous system tumor. Drug delivery to brain has been a major hurdle for treatment of central nervous system disorders, due to impermeability and selectivity of the blood brain barrier (BBB), which separates the blood from the cerebral parenchyma and limits the uptake of most chemotherapeutics into brain. Therefore, there is a need to develop a delivery system to deliver chemotherapeutics to brain. The objective of this research was to design and characterize dual-functionalized liposomes surface modified with transferrin (Tf) for receptor targeting and cell penetrating peptides (CPPs, - HIV-1 TAT, Penetratin, and QLPVM) for enhanced cell penetration for efficient co-delivery of doxorubicin (Dox) and erlotinib (Erlo) to brain for treating glioblastoma.
Methods: CPPs and transferrin were coupled with terminal NHS-activated DSPE-PEG-2000 via nucleophilic substitution reaction separately. CPP-liposomes were prepared by using erlotinib and CPP-PEG-DSPE with other phospholipids through thin film hydration method. Dual-functionalized liposomes were prepared by incorporating Tf-micelles into CPP-liposomes using post-insertion technique. Doxorubicin was incorporated into liposomes by pH gradient method. The entrapment efficiencies of Dox and Erlo were quantified by HPLC. The cellular uptake was studied by determining the amount of Dox and Erlo into different cells including glioblastoma (U87), brain endothelial (bEnd.3), and glial cells by HPLC. A tight co-culture barrier was formed by seeding bEnd.3 and glial cells on upper and bottom side of culture insert, respectively. This tightly formed co-culture barrier was placed on the glioblastoma grown PLGA-chitosan scaffold to form in vitro brain tumor model. The efficacy of dual functionalized liposomes was evaluated quantitatively by determining glioblastoma tumor regression by MTT assay as well as qualitatively by live and dead cell fluorescence imaging using in vitro brain tumor model. The in vivo biodistribution of Dox and Erlo encapsulated dual functionalized liposomes was studied in nude mice at 24 h time point.
Results: The particle size of liposomes were found to be < 200 nm. The entrapment efficiency of doxorubicin and erlotinib were found to be ~63% and ~48%, respectively. Quantitative uptake of Dox and Erlo encapsulated dual-functionalized liposomes showed more than 75% and 70%, respectively in all the three cell lines compared to ~22% and ~21%, respectively for plain liposomes, which confirmed the efficacy of dual-functionalized liposomes over conventional liposomes without any functionalization. The endothelial permeability coefficient, Pe of 2.65 x 10-6 cm/s which shows the tightness of the endothelial co-culture barrier. Hemolysis study demonstrated no significant hemolytic activity of dual functionalized liposomes upto 600 nM phospholipid concentration. The transport study showed more than 13% transport of liposomes across endothelial co-culture barrier. The microscopic images of scaffolds containing glioblastoma tumor after 24h treatment with dual-functionalized liposomes showed tumor regression by 55%. Dual functionalized liposomes showed more than 5% of Dox and Erlo translocation across BBB into mice brain.
Conclusion: Dual-functionalized liposomes showed biocompatibility, high cellular uptake, and efficient translocation of drug molecules across brain endothelial barrier, in vitro and in vivo.