Introduction: Since their discovery nearly five decades ago, liposomes have become the most well studied nanodelivery system. Researchers have exploited their amphipathic nature to sequester both lipophilic and hydrophilic compounds in order to make potent biodegradable and biocompatible delivery vehicles. However, despite their well-documented use, strategies for encapsulating therapeutics are limited and are often extremely specialized, hindering their widespread implementation. Several techniques, including passive and active loading strategies, have been proposed to formulate potent drug encapsulated liposomes. Passive loading has been recognized as a universal encapsulation technique, but often suffers from poor loading efficiencies. Conversely, active loading is extremely efficient, but is limited to weakly basic therapeutics, making it dependent on the chemical nature of the drug. Our work aims to build upon our SPIN (synthesis and purification of injectable nanocarriers) technology for liposome synthesis and develop an efficient and universal loading strategy. A novel equilibration technique is used as a one-step approach to co-load both lipophilic and hydrophilic molecules, simultaneously. We demonstrate the efficacy of co-loaded liposomes by encapsulating two front-line chemotherapeutics, Doxorubicin (DXR) and Paclitaxel (PTX), and measuring their cytotoxic effect on MDA-MB-231 metastatic breast cancer cells. To understand the atomic level interactions that occur between DXR, PTX, and the liposomal membrane during equilibration, we perform molecular dynamic simulations using NAMD (Nanoscale Molecular Dynamics). We believe that this scalable and robust technique for synthesizing potent co-loaded liposomes offers unparalleled advantages compared to contemporary encapsulation techniques, and will ultimately lead to the development of novel liposomal formulations for clinical intervention.
Materials and Methods: Liposomes composed of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC) and cholesterol at a 2:1 ratio are synthesized using our SPIN technique. The synthesized liposomes are equilibrated at the transition temperature (Tm) of DSPC (55 ºC) for up to 4 hours with DXR and PTX. The co-loaded liposomes are then filtered to remove non-encapsulated therapeutics and the drug to lipid ratio of the two molecules in the liposomes is calculated. Samples were incubated with MDA-MB-231 metastatic cancer cells and viability over time was analyzed using a WST-1 proliferation assay. All-atomic NAMD simulations were performed using the CHARMM force-field for both lipids and drugs. Simulations were conducted for 100 ns at temperatures of 37 OC, 55 OC, and 65 OC.
Results and Discussion: Liposomes were produced using our SPIN technique, which is an efficient and scalable synthesis approach. Using equilibration, we were able to achieve drug to lipid ratios of 0.04. Cells incubated with liposomal therapeutics showed a drastic decrease in viability over time as compared to controls. NAMD simulations to investigate the interactions between the drugs and the membrane show that electrostatic forces play a predominant role in the encapsulation of therapeutics.
