The effectiveness of a drug candidate depends on its ability to distribute to its target site of action after administration. Thus, a primary concern for drug delivery labs like the Pun lab is preventing drugs from being cleared from the bloodstream by the body's renal system before they are able to accumulate to therapeutic levels at their site of action. In short, one important goal in drug delivery research is to find ways to extend a drug's blood circulation half-life. Conjugating drugs to the large molecular weight molecule polyethylene glycol (PEG) to slow their clearance kinetics is the current gold-standard method, but a crucial drawback is that PEG's large size leads to its potentially toxic buildup in tissues like the liver. To get around this problem, my project aims to develop a drug delivery platform that will allow small molecule drugs to reversibly bind, or in other words "hitchhike" onto human serum albumin (HSA), an abundant protein in blood plasma with an extraordinarily long half-life. At this point in my project, I have successfully synthesized a novel fatty acid monomer with a methacrylate functionality that can be used to copolymerize the monomer with therapeutic small-molecules or peptides to improve their circulation half-life. The next steps will be to copolymerize the fatty acid monomer with pGmMA, a water-soluble polymer, and use biolayer interferometry to test the fatty acid monomer's ability to coordinate to albumin, which will confirm its efficacy as a drug delivery platform. If successful, this project has the potential to provide a generalizable improvement to the pharmacokinetics of various kinds of small-molecule drugs or peptides, enhancing their potency and overall ease of treatment. 

My project intends to discover a DNA aptamer, a single stranded DNA oligonucleotide, that binds selectively to the protein Interleukin-6 (IL-6). IL-6 has an important role in the immune system response and in excess it is known to cause inflammation. Aptamers exhibit binding affinities like that of antibodies but are ~50 times cheaper to produce. The method of aptamer discovery is through SELEX (Systematic Evolution of Ligands by Exponential Enrichment) which involves the selection from an aptamer library that contains 52N random nucleotide region and constant 5’ and 3’ 18 base pair regions for PCR amplification. Positive and negative selection are completed by incubating aptamer libraries with IL-6 or random protein immobilized on magnetic beads respectively. After each round selected aptamer sequences are amplified with a polymerase chain reaction (PCR) with primers that anneal to the constant regions. Reverse primer has biotin on 5’ end that is used later for strand separation with streptavidin agarose. After each round aptamer pool is going to be sequenced using nanopore sequencing platform till the enrichment of IL-6 specific sequences is observed. Binding will be tested through an enzyme-linked immunosorbent assay (ELISA) using the fam on 3’ end. The end goal of this project is to design a cost-effective method of IL-6 depletion from patients blood, allowing for cost-effective method of treatment for overactive immune system inflammation in sepsis patients.

VLA-4 is an integrin expressed on immune cells that plays an important role in their extravasation into tissues during an immune response. In the autoimmune disease multiple sclerosis (MS), pathogenic T cells extravasate and attack nerve cells by using VLA-4 to bind to VCAM-1, a cell adhesion molecule on endothelial cells that line blood vessels. Current treatments for MS rely on antibodies to bind VLA-4 and block its interaction with VCAM-1, thus preventing a pathogenic immune response. However, antibodies are expensive to manufacture, and their binding cannot be easily regulated to control drug-induced side effects. Aptamers are single-stranded DNA or RNA molecules that fold into sequence-defined structures capable of binding to their targets with affinities and specificities comparable to antibodies. Being chemically synthesized, they are much cheaper to manufacture and offer minimal batch-to-batch differences. Unlike antibodies, their binding in vivo can be rapidly reversed using a reversal agent, which could alleviate the side effects of disease treatments. However, aptamers have limitations in vivo: degradation by nucleases in blood serum, and rapid clearance into urine through the glomerular filtration barrier. This project focuses on the development of a VLA-4 aptamer for treating MS. We found that the VLA-4 aptamer prevents soluble VCAM-1 from binding to VLA-4-expressing leukocytes by flow cytometry. We then showed that the aptamer blocks VLA-4/VCAM-1 mediated leukocyte adhesion in vitro. We are currently assessing aptamer blockade of leukocyte transendothelial migration. We are also designing modifications to improve the stability of the aptamer for in vivo uses. Successful development of the aptamer will lead to an alternative treatment modality for MS with a potentially improved safety profile.