2016 Enhanced Synthesis and Purification of PEGylated Liposomes for Targeted Drug Delivery Neil Parikh, Steven Roberts, and Nitin Agrawal nagrawa2@gmu.edu Abstract General Protocol Results Therapeutic intervention often relies on pharmaceuticals that are not targeted towards specific tissues within the body. This lack of specificity has been shown to cause issues with efficacy and cytotoxicity. Nanodelivery systems, such as liposomes, are promising vehicles for targeted theranostic intervention. The goal of this project is to achieve an enhanced synthesis and purification of PEGylated liposomes for targeted drug delivery. The synthesis process is characterized to determine the optimal concentrations of phospholipids and targeting moieties. A specialized phospholipid (DSPE-PEG-MAL) that covalently bonds ligands is integrated within the lipid shell, allowing for protein and cell binding. Here, two liposome production methods are explored, pre-insertion and post-insertion. In order to produce the pre-insertion liposomes, cholesterol, a phospholipid (DSPC), and a functionalized PEG-lipid (DSPE-PEG-MAL) are combined at varying molar ratios using a shaker, syringe pump and ultracentrifuge. The liposomes are created within a fluorescein and water solution, and incubated with metastatic cells for several time intervals. The fluorescence of the cells is quantified using ImageJ analysis. In order to further validate the data, dynamic light scattering (DLS) is employed. A second experiment is conducted to test the post-insertion protocol, which is the addition of DSPE-PEG-MAL after the formation of liposomes. DSPE-PEG-MAL is introduced to the liposome solution at varying volumes to match the predetermined molar concentration ratios and samples are shaken at distinct temperatures. The resulting liposome solutions are analyzed using DLS to determine similarity to first experiment. We have determined that the optimal conditions are a three hour interaction between cells and pre-insertion liposomes consisting of a 20:1 phospholipid to DSPE-PEG-MAL ratio. Pre-Insertion When comparing the three hour time interval, 20:1 molar ratio column of cells for volumes 1 uL, 5 uL and 10 uL of liposomes, we found that 10 uL produced the greatest fluorescence consistently across all five cells (Figure 1). When comparing across molar ratios, and keeping the time interval and volume constant, we found that the 20:1 ratio produced the greatest fluorescence compared to the baseline (Figure 2). Therefore, we determined that the optimal conditions for cell binding are a three hour interaction between cells and 10 uL of liposomes consisting of a 20:1 phospholipid to DSPE-PEG-MAL ratio. Post-Insertion After analyzing the data, we found that 50°C was the optimal temperature for post-insertion liposome production. Compared to the control solution, all PEGylated liposomes were larger, suggesting that the PEG was being incorporated into the outer part of lipid bilayer (Figure 3). We hypothesize that liposome synthesis should use a post-insertion protocol with molar concentration ratio of 20:1 and shaker temperature of 50°C. Final phospholipid and cholesterol molar concentrations are kept at 10 M and 5 M, respectively. Pre-insertion requires the addition of DSPE-PEG-MAL to the lipid solution prior to its injection while post-insertion requires DSPE-PEG-MAL to be added to the liposome colloid. Pre-Insertion Figure 1. Figure 3. The DSPC to DSPE-PEG-MAL molar concentration ratios varied as follows: 20:1, 40:1, 60:1, 80:1 and 100:1 Post-Insertion Objectives Enhance the liposome synthesis and purification process. Determine the optimal DSPC to DSPE-PEG-MAL ratio. Explore the distinction between pre-insertion and post-insertion protocols. Evaluate the efficiency of liposome to cell binding. Discover an enhanced drug delivery mechanism for potential cancer treatment. Figure 2. Obtain three Eppendorf tubes labeled DSPC, DSPE-PEG-MAL or cholesterol Measure out masses of each dependent on volume of liposome desired Dissolve in isopropanol based on solubility Shake at the following parameters: 450 speed, 50 °C and 15 minutes Cell 1 Cell 2 Pre-Insertion Protocol Cell 3 Cell 4 Add liposome solutions to 96-well cell plate Ultracentrifuge at the following parameters: 12,000 x g, 60 minutes and four degrees Celsius Measure and analyze fluorescence using fluorescent microscope and ImageJ program Syringe pump and 50 uL Beckman Syringe to add isopropanol solution to water and fluorescein solution Background Cell 5 Background Liposome: Spherical vesicle having at least one lipid bilayer. Core is surrounded by a hydrophobic membrane and hydrophilic solution dissolved within can undergo limited diffusion, allowing for controlled drug release. Fuse with another bilayer, such as the cell membrane, to deliver contents within the core to the cell. Used for the administration of nutrients and pharmaceuticals. Types of Liposomes: Unilamellar liposome is a spherical chamber or vesicle bounded by a single bilayer. Multilamellar liposome consists of concentric lipid bilayers. Additional Components: DSPE-PEG-MAL is a linear heterobifunctional PEGylation reagent and is used to prepare PEGylated liposomes. Fluorescein is a fluorescent chemical that allows us to determine if the liposome bound to the edge of the cell and is considered a model drug. Figure 4. Control PEG Cell Background Post-Insertion Protocol Fluorescein Conclusion In conclusion, we have determined the optimal parameters for liposome synthesis and purification, allowing for enhanced targeted drug delivery. We recommend that a post-insertion protocol be employed that incorporates a 20:1 DPSC to DSPE-PEG-MAL ratio and is shaken at 50°C for a sixty minute time period. In order to further test these results, we hope to use fluorescein as a model drug and analyze the fluorescence of the liposomes after they are added to metastatic cells for a three hour time interval. If abundant cell binding is found, we will further progress to test nanodrug delivery for cancer treatment. Syringe pump and 250 uL Beckman Syringe to add DSPC and cholesterol solution to water Add liposome solution to twenty-five Eppendorf tubes for molar concentration ratio and temperature combinations Place each set of six Eppendorf tubes into a shaker at the following parameters: 450 speed, 60 minutes and appropriate temperature (30°C, 40 °C, 50 °C and 60 °C) Use Dynamic Light Scattering (DLS) to analyze the resulting liposome solutions DSPE-PEG Liposome