Purpose: High grade glioma is highly malignant, as the tumor cells reproduce quickly and infiltrate rapidly. Brain being as a least permeable organ, which is protected by endothelial cell layer, called blood brain barrier (BBB), which restricts the entry of chemotherapeutic agents. In this study, we developed a dual functionalized liposomal delivery system by modifying the surface with transferrin (Tf) for receptor targeting and a cell penetrating peptide, PFVYLI (PFV) to increase translocation of doxorubicin (Dox) and Erlotinib (Erlo) across the BBB into glioblastoma (U87) tumor cells. The objective of this study was to determine the efficacy of co-delivery of doxorubicin and erlotinib encapsulated dual functionalized liposomes on tumor regression using an in vitro brain tumor model.
Methods: Tf and PFV were coupled to the activated terminal end of NHS-PEG(2000)-DSPE via nucleophilic substitution reaction individually. PFV-PEG(2000)-DSPE and erlotinib along with other phospholipids were used to formulate PFV-liposomes by thin film hydration. Post–insertion technique was used to prepare dual functionalized liposomes by incorporating Tf-micelles into PFV-liposomes. pH gradient method was used to entrap doxorubicin into liposomes. The entrapment of Dox and Erlo was quantified by HPLC. The cellular uptake of dual functionalized liposomes into different cells including U87, brain endothelial (bEnd.3), and glial cells was determined by HPLC. Hemolysis study was performed to determine the potential interaction between erythrocytes and PFV. The culture inserts carrying brain endothelial cells on luminal side and glial cells on abluminal side formed a tight barrier which was placed on glioblastoma grown PLGA-chitosan scaffold to form an in vitro brain tumor model. The gradual tumor growth inside scaffold was monitored using hematoxylin-eosin staining. The integrity of constructed barrier was determined by measuring transendothelial electrical resistance (TEER) using EVOM2 and the flux of sodium fluorescein (Na-F) across the endothelial co-culture barrier layer. The transport of liposomal nanoparticles was measured across in vitro brain tumor model. Finally, the anti-tumor efficacy of anti-cancer chemotherapeutics encapsulated liposomes were evaluated quantitatively by determining the tumor cell viability inside scaffold by MTT assay as well as qualitatively by live and dead cell fluorescence imaging.
Results: The particle size and zeta potential of liposomes were found to be < 200 nm and ~8 mV, respectively. The entrapment efficiency of Dox and Erlo was found to be ~65% and ~53%, respectively. The quantitative estimation of Dox and Erlo cellular uptake into different cells was ~66% and ~65%, respectively which further confirmed qualitatively by fluorescence images. The integrity of barrier demonstrated the TEER value of 178.4 ± 10 Ω cm2 and endothelial permeability coefficient, Pe of 2.17 x 10-6 cm/s, respectively which confirmed the tightness of the endothelial co-culture barrier. Hemolysis study revealed that Tf-PFV liposomes showed no significant increase in the hemoglobin release up to 600 nmoles of phospholipid concentration and considered non-toxic for in vivo administration. The percent liposomal transport was increased from 8.05 % and 8.7% for Tf and PFV liposomes to 12.67% for Tf-PFV over the period of 24 h. The percent tumor cell viability was observed to be decreased from 77.84 ± 1.57% for Dox-Erlo encapsulated plain liposomes to 47.85 ± 1.06% for Dox-Erlo encapsulated Tf-PFV liposomes for 24 h treatment which was further confirmed by fluorescence images of the scaffold sections.
Conclusion: The dual-functionalized liposomes were successfully designed to improve the transport of anti-cancer chemotherapeutics across a co-cultured endothelial barrier into a glioblastoma tumor grown scaffold. Incorporation of PFV to Tf-liposomes showed better biocompatibility, high cellular uptake, and efficient transport of Dox and Erlo across brain endothelial barrier and into the tumor present inside the scaffold, which resulted in regression of the glioblastoma tumor in the scaffold. Therefore, demonstrates the efficient antitumor efficacy of dual functionalized liposomes.