Glutamate-Leucine Block Copolypeptides for Drug Delivery
Chemotherapy treatments involving the delivery of naked drugs to the body must overcome many complications such as poor solubility, enzymatic degradation, and clearance from the body, all of which can result in the drug having a short circulation half-life, low efficacy, and undesirable side effects. One solution to overcome these problems is to encapsulate the drug within a nano-sized drug delivery vehicle. Nano-sized drug delivery vehicles are advantageous since they can protect the drug from degradation during its circulation in the body, release the drug in a controlled manner, and provide passive targeting to the tumor tissue.
Many materials for drug delivery vehicles have been investigated. Liposomes, which are vesicles composed of natural or synthetic phospholipids, have been thoroughly investigated, and many liposomal formulations have been successful in the market. However, one limitation of liposomes is that they are less stable due to being comprised of relatively smaller molecules that exhibit weaker attractive interactions in a self-assembled vesicle. This disadvantage has motivated the development of other materials for drug delivery such as synthetic polymers, which are longer molecules that can exhibit stronger attractive interactions in a self-assembled vesicle. Synthetic control of the polymers also allows for fine tuning of the hydrophilic and hydrophobic chain lengths.
Another material that has been recently gaining popularity for use in drug delivery is the polypeptide. These amino acid-based building blocks provide further advantages. Similar to polymers, monodisperse polypeptides can be synthesized with precise control due to recent advances in polymerization techniques, and the longer chains provide stability for the self-assembled vesicles. They are also naturally occurring and have the potential to be biocompatible. These polypeptides can also adopt secondary structures to further improve the stability of the vesicles. Our laboratory previously investigated the novel poly(L-glutamate)60-b-poly(L-leucine)20 (E60L20) block copolypeptide synthesized by the laboratory of Timothy Deming. In this polypeptide, the hydrophilic glutamate segment assumes a random coil while the hydrophobic leucine segment forms an alpha helix. The E60L20 block copolypeptide therefore has a truncated cone shape that favors self-assembly into vesicles (EL vesicles). The size of the EL vesicles could be controlled by serial extrusion, and the EL vesicles were not cytotoxic to cells. Additionally, when previously conjugated with transferrin (Tf), a widely used cancer targeting ligand, the EL vesicles exhibited increased cellular uptake. These characteristics suggested that the EL vesicle could be used as a potential drug delivery vehicle.
This thesis extended our investigation of these vesicles, specifically, the thesis focused on the ability of the EL vesicle to encapsulate and deliver doxorubicin (DOX), a commonly used chemotherapeutic. Polyethylene glycol (PEG) was conjugated to the EL vesicles to maintain vesicle stability during the drug loading process and for providing stability in the future for in vivo applications. DOX was successfully encapsulated while maintaining stable EL vesicles using a modified pH-ammonium sulfate gradient method. Tf was then conjugated to the drug-loaded EL vesicles to create a stable, targeted drug delivery vehicle. A mathematical model was developed to predict drug release from the targeted, drug-loaded EL vesicles by considering the transient diffusion of DOX across the vesicle bilayer and the time-dependent mass balances on DOX in the interior core and exterior aqueous solution. Release profiles were predicted by applying the method of lines approach with an ordinary differential equation solver in MATLAB. In vitro release experiments were performed to confirm the predicted release profiles, and the DOX diffusion coefficient in the vesicle bilayer was estimated. An in vitro cytotoxicity assay was subsequently performed with a cancer cell line with both the targeted and non-targeted, drug-loaded EL vesicles. The targeted, drug-loaded EL vesicles demonstrated an improved drug delivery efficacy compared to the non-targeted, drug-loaded EL vesicles.