Esophageal adenocarcinoma (EAC) is a type of cancer with the most rapidly increasing incidence rate in developed nations. EAC occurs as a result of the metaplasia-dysplasia-adenocarcinoma sequence initiated by Barrett’s esophagus. In this sequence of events, patients develop Barrett’s esophagus, wherein the acidic damage of gastroesophageal reflux disease (GERD) causes the esophageal epithelium to take on an intestinal epithelium phenotype. This metaplasia can then progress to Barrett’s esophagus with either low-grade or high-grade dysplasia. Patients that develop high-grade dysplasia (HGD) are at great risk for esophageal adenocarcinoma, and hence there is a need for higher specificity in the labeling of pre-cancerous tissues for endoscopic detection. Current imaging of dysplastic lesions is done through traditional endoscopy with the use of contrast agents for differential staining. However, current contrast agents have a low signal-to-noise ratio (SNR), and dysplastic lesions are often missed. Therefore, for early detection of esophageal adenocarcinoma, we are creating a contrast agent that increases SNR via fluorophore-labeled peptides that bind specifically to tissues with high-grade dysplasia. Peptides are ideal as targeting ligands as they can bind specifically to surface proteins on dysplastic cells and increase the likelihood of clinicians obtaining biopsies of pre-cancerous tissue. In the past year, we have isolated potential peptide sequences that bind immortalized cells lines generated from dysplastic tissues. A bacteriophage library was subjected to subtractive whole cell panning for isolation of phage expressing HGD-binding peptide sequences. The phages were added in vitro to cultured cell lines of normal esophageal tissue and non-dysplastic Barrett’s esophageal tissue to eliminate those that were not selective for dysplasia. After subtractive whole cell panning, left over phage were sequenced to determine the peptide sequences on them. Future work includes binding studies using flow cytometry to demonstrate that fluorophore-labeled peptides bind preferentially to dysplastic cell lines independent of the bacteriophage.

As our understanding of the genetic basis for the pathophysiology of many diseases has broadened, gene therapy, which is the delivery of genetic material into cells in order to supplement or alter defective genes, is being explored as a potential treatment option. Despite its promise, no gene therapies have been approved for clinical use due to the lack of safe and effective vectors. There are many obstacles that must be overcome in order to successfully transfect a cell with exogenous DNA. First, the vector-DNA complex must navigate through the extracellular environment and be uptaken into the target cell. Then, the genetic material must achieve endosomal escape, be released from the complex, and then translocate into the nucleus where it can be transcribed and translated into functional or therapeutic proteins. In the Pun Lab, we previously synthesized a block-statistical copolymer comprised of different hydrophilic and hydrophobic segments aimed at providing a plethora of functionalities to the formed polyplexes. Due to this polymer’s unique architecture, it exhibits transfection efficiencies higher than branched polyethyleneimine, the gold standard for nonviral gene delivery, but still fails to reach the same level of transfection efficiency as seen with viral vectors. In this project, two additional modifications will be made to the copolymer and a library of well-defined polyplexes with varying formulations and structures will be synthesized and evaluated in vitro and in vivo. These modifications, guanidinylation and conjugation of a targeting peptide, aim to increase uptake into cells through electrostatic interactions and increase localization to the target cell type. From these studies, the ideal method to incorporate both modifications into one system to improve transfection efficiency and the structure-function effects on transfection efficiency will be evaluated.

Macrophages of our immune system can be broadly classified as pro-inflammatory M1 or anti-inflammatory M2 macrophages. Imbalance in the ratio of these two phenotypes has been associated with various chronic diseases, suggesting that targeting drugs to specific populations of macrophages may achieve therapeutic benefit. Phage display is one method of identifying peptides that can be used for targeting cell populations. In whole-cell peptide phage display, a library of bacteriophage, in which individual phage display a unique peptide sequence on a coat protein, is applied to target cells. Phages that preferentially bind target cells are isolated, and their peptide sequences can then be obtained by DNA sequencing. Targeting M1 macrophages has possible applications in treating arthritis, diabetes, and atherosclerosis. Recently, we identified a novel phage-bound peptide sequence (hM1pep), using phage display, that exhibited selective binding to M1 macrophages. I synthesized the peptide form of hM1pep and evaluated its selectivity of M1 macrophage binding in vitro using flow cytometry; however hM1pep showed insignificant binding. Tumor-associated macrophages (TAMs), a type of M2 macrophage, have been shown to facilitate tumor growth and are associated with decreased survival of cancer patients. Previously, we identified a peptide that preferentially bound to TAMs in mice. We demonstrated that peptide-mediated TAM elimination improved survival in mice, suggesting that an analogous strategy in humans could be a potential cancer therapy adjuvant. In this work, I attempted to identify human M1 and M2 macrophage binding peptides by panning against human M1 and M2 macrophages, while using M2 and M1 macrophages as a subtractive panning step, respectively. Targeting M2 macrophages has potential uses in treating diseases associated with increased tissue levels of M2 macrophages, including cancer, fibrosis, and asthma.

Thermosensitive hydrogels have been actively investigated as a potential approach to preventing the recurrence of tumors after surgery. However, current hydrogels remain limited by their low drug retention rate and non-degradability, preventing their use in neural applications. Using a trifunctional dibromomaleimide-alkyne based cross linker, this project aims to synthesize a thermosensitive, biodegradable polyethylene glycol (PEG) hydrogel for the delivery of anti-tumor drugs. The crosslinker is synthesized through condensation of dibromomaleic anhydride with propargylamine and substitution of bromine groups with cysteamine. Our trifunctional crosslinker will provide the following properties: (a) crosslinking of two thermosensitive, PEG based polymers, (b) reducible to allow for triggered degradation and (c) site for biologic conjugation by click chemistry. With the site for biologic conjugation, we will be able to physically attach the drug to the gel and make drug release dependent on the more sustained gel degradation rather than passive diffusion. The liquid, thermosensitive gel will be able to fully fill the neural cavity after tumor removal and release a drug over a sustained period of time. Preliminary synthesis of crosslinker using cysteamine shows the fluorescent color indicative of substituted maleimide in addition to active amine reactivity. Initial crosslinking of thermosensitive polymers indicate an increase in hydrophillicity and swelling with increased temperature. Future experiments will include increasing length of crosslinker arms with PEG spacers for more effective linking and attaching a biomolecule to the alkyne group of the crosslinker. The toxicity and release profile of the crosslinked polymers will be characterized to retune the polymers to create a hydrogel with high localization and minimal toxicity.