UCLA

Delivery of naked drugs in the body faces many challenges, such as poor solubility in blood and interstitial fluids, enzymatic and proteolytic degradation in the liver, clearance by the kidneys, recognition by the immune system, inability to cross the cell membrane, and nonspecific toxicity to normal cells. Over the years, drug encapsulation and delivery through nanocarriers have been sought as a potential approach to overcome many of these obstacles. Traditionally, research in this area has been dominated by vesicles assembled from lipids. These lipid based vesicles, also known as liposomes, are currently the most widely used vesicle for drug delivery with commercial formulations (DOXIL® and MyocetTM) in the market. However, liposomes are intrinsically unstable in vivo unless they are coated with polymers such as polyethylene glycol (PEG), which decreases their clearance from the body. Even with the addition of PEG, there are limitations as the added PEG chain increases the hydrophilic head group of the lipids, increasing their tendency to form micelles instead of bilayers. Due to these limitations, researchers have investigated alternative systems, such as polymer-based vesicles or polymersomes. The disproportionality that exists between the hydrophilic PEG head group and the hydrophobic tail of a lipid can be overcome by synthesizing synthetic PEG amphiphilic polymers with longer hydrophobic segments. In addition, by synthesizing building blocks that are larger than lipids, membrane stability can be improved along with slower leakage of drugs. However, some polymersomes suffer from lacking the ability to incorporate biofunctionality without impairing their ability to self-assemble into vesicles.

In order to contribute to this exciting drug delivery field and find a material that could potentially improve upon current lipids and synthetic polymers, our lab investigated the use of novel amino acid-based polymers as the building blocks for forming vesicles. The use of amino acids offers many advantages. They have an intrinsic biocompatibility, which implies great potential for low immunogenicity and toxicity. The variety of amino acids and their many possible sequence combinations can be used to custom design polypeptides with different chemical properties and biofunctionalities. Moreover, recent advances in polymerization techniques allow the synthesis of long chains of monodisperse polypeptides. Vesicles formed from polypeptides can offer increased stability over conventional liposomes by providing thicker membranes that contribute to increased attractive interactions between the building blocks. We have investigated vesicles comprised of polypeptides that have been synthesized by Dr. Timothy Deming's group (UCLA), which employs a transition metal-mediated α-amino acid N-carboxyanhydride (NCA) polymerization technique to synthesize the amphiphilic block copolypeptides. The vesicles formed from these polypeptides can be prepared in different sizes in bulk quantities.

This thesis focused on investigating the potential for using these polypeptide vesicles as drug delivery vehicles. Initially, vesicles comprised of positively charged polypeptides were studied, specifically the lysine-leucine (K60L20) and arginine-leucine (R60L20) block copolypeptides. Due to their positive charge, these vesicles have a disadvantage of being toxic to cells through interactions with the net-negatively charged plasma membranes. Therefore, in order to identify a design criterion for producing positively charged vesicles with low cytotoxicity, we focused on optimizing the hydrophilic/hydrophobic ratio of the polypeptide by varying the length of the hydrophobic block while maintaining a constant length for the hydrophilic block. The K60L20 polypeptides were used for this study due to their relative ease of preparation compared to the R60L20 polypeptides. It was found that varying the hydrophilic/hydrophobic ratio in the lysine-leucine block copolypeptide affects its ability to form vesicles, where polypeptides with long hydrophobic segments formed less toxic vesicles with appropriate sizes for drug delivery. Among the copolypeptides investigated, the K60L20 copolypeptide composition showed the most potential for drug delivery applications, as this copolypeptide was able to form monodisperse nanoscale vesicles with the least amount of micelles and small aggregates that were more toxic than the vesicles themselves.

The optimized block copolypeptide composition was then applied to the arginine-leucine block copolypeptide, and the ability of these arginine-based vesicles to transfect mammalian cells was systematically investigated. The arginine-leucine R60L20 block copolypeptide was studied, since the arginine residues had previously been shown to enhance the delivery of cargo into cells. Plasmid DNA was used as our model therapeutic, since only low concentrations of plasmid DNA are required for an effect. This was an important point, since high concentrations of R60L20 vesicles will lead to significant cytotoxicity. Our transfection results with our R60L20 vesicles demonstrated that there is potential for using this novel material as a transfection agent, as they were able to achieve transfection with low cytotoxicity and immunogenicity.

In contrast to plasmid DNA, high concentrations of the vesicles are required when delivering small molecule chemotherapeutics due to their IC50 values being in the micromolar range. Since the R60L20 vesicles themselves become cytotoxic at high concentrations, we also investigated the negatively charged vesicle formed from the glutamate-leucine (E60L20) block copolypeptide. A main advantage of these vesicles, over the positively charged vesicles, is that they are less toxic to cells. However, this is also a disadvantage, since the electrostatic repulsive interactions between the vesicles and the net-negatively charged cell membranes can inhibit the vesicles from entering cells. We were able to overcome this limitation by conjugating the vesicle surface with transferrin (Tf), a ligand that has been commonly used for targeting many types of cancer cells. We investigated the intracellular trafficking behavior of these Tf-conjugated EL vesicles, and they were shown to recycle back to the cell surface once they were internalized. This behavior was similar to what has been observed for other nanoparticle systems conjugated with Tf.

Even with this recycling behavior, other Tf-conjugated nanoparticle systems were found to exhibit improved drug delivery efficacy. Accordingly, we investigated the ability of our Tf-E60L20 vesicles to deliver the chemotherapeutic doxorubicin (DOX). Before performing the in vitro efficacy study, the vesicle surface was conjugated with poly(ethylene glycol) (PEG) to improve its stability for future in vivo applications. The targeting Tf ligand was then conjugated to the resulting vesicle, and DOX was then encapsulated in the vesicle interior. A previously developed mathematical model was applied to our system to predict trends that helped guide experiments and allowed for reduced experimentation and faster optimization. Once the carrier was complete, in vitro cytotoxicity studies were performed to demonstrate proof-of-concept that the newly developed Tf-conjugated vesicles exhibit a significant improvement in drug delivery efficacy over the non-targeted version.