Developing Membrane Active Peptides for Endosomal Escape of Macromolecules

Membrane Active Peptides (MAPs) are an important class of short protein sequences that hold the potential to be used for a number of varied purposes such as antibacterial, antiviral, and drug delivery based therapies. Despite this great potential, the development of MAPs has been hindered by a lack of consensus about their mechanisms of activity. Herein we proposed that MAPs can be categorized into several categories differentiating how they interact with model cell membranes. In this dissertation we examined how these activities can be regulated by pH. We used this information to design and test MAPs with pH-dependant activity for drug delivery through both rational design and high-throughput screening. Finally, we tested our peptides both with model biophysical characterization techniques as well as cell based assays and made connections between the two. Our intention is that the work conducted in this dissertation will provide a framework for the development of effective and efficient Membrane Active Peptides

Photobuforin II, a fluorescent photoswitchable peptide

Antimicrobial peptide buforin II translocates across the cell membrane and binds to DNA. Its sequence is identical to a portion of core histone protein H2A making it a highly charged peptide. Buforin II has a proline residue in the middle of its sequence that creates a helix-hinge-helix motif which has been found to play a key role in its ability to translocate across the cell membrane. To explore the structure-function relationship of this proline residue this study has replaced P11 with a meta-substituted azobenzene amino acid (Z). The resultant peptide, photobuforin II, retained the secondary structure and membrane activity of the naturally occurring peptide while gaining new spectroscopic properties. Photobuforin II can be isomerized from its trans to cis isomer upon irradiation with ultra-violet (UV) light and from its cis to trans isomer upon irradiation with visible (VL). Photobuforin II is also fluorescent with an emission peak at 390 nm. The intrinsic fluorescence of the peptide was used to determine binding to the membrane and to DNA. VL-treated photobuforin II has a 2-fold lower binding constant compared to UV-treated photobuforin and causes 11-fold more membrane leakage in 3:1 POPC:POPG vesicles. Photobuforin II provides insights into the importance of structure function relationships in membrane active peptides while also demonstrating that azobenzene can be used in certain peptide sequences to produce intrinsic fluorescence

A Novel, Rapid, and Low-Volume Assay for Therapeutic Drug Monitoring of Posaconazole and Other Long-Chain Azole-Class Antifungal Drugs

This work describes an effective assay for TDM of long-chain azole-class antifungal drugs that can be used in diluted human serum samples. This assay will provide a quick, cost-effective method for monitoring concentrations of drugs such as posaconazole that exhibit well-documented pharmacokinetic variability. Our rGO-aptamer assay has the potential to improve health care for those struggling to treat fungal infections in rural or resource-limited setting.Clinicians need a better way to accurately monitor the concentration of antimicrobials in patient samples. In this report, we describe a novel, low-sample-volume method to monitor the azole-class antifungal drug posaconazole, as well as certain other long-chain azole-class antifungal drugs in human serum samples. Posaconazole represents an important target for therapeutic drug monitoring (TDM) due to its widespread use in treating invasive fungal infections and well-recognized variability of pharmacokinetics. The current “gold standard” requires trough and peak monitoring through high-pressure liquid chromatography (HPLC) or liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Other methods include bioassays that use highly susceptible strains of fungi in culture plates or 96-well formats to monitor concentrations. Currently, no method exists that is both highly accurate in detecting free drug concentrations and is also rapid. Herein, we describe a new method using reduced graphene oxide (rGO) and a fluorescently labeled aptamer, which can accurately assess clinically relevant concentrations of posaconazole and other long-chain azole-class drugs in little more than 1 h in a total volume of 100 µl

Alternative Antibiotics in Dentistry: Antimicrobial Peptides

The rise of antibiotic resistant bacteria due to overuse and misuse of antibiotics in medicine and dentistry is a growing concern. New approaches are needed to combat antibiotic resistant (AR) bacterial infections. There are a number of methods available and in development to address AR infections. Dentists conventionally use chemicals such as chlorohexidine and calcium hydroxide to kill oral bacteria, with many groups recently developing more biocompatible antimicrobial peptides (AMPs) for use in the oral cavity. AMPs are promising candidates in the treatment of (oral) infections. Also known as host defense peptides, AMPs have been isolated from animals across all kingdoms of life and play an integral role in the innate immunity of both prokaryotic and eukaryotic organisms by responding to pathogens. Despite progress over the last four decades, there are only a few AMPs approved for clinical use. This review summarizes an Introduction to Oral Microbiome and Oral Infections, Traditional Antibiotics and Alternatives & Antimicrobial Peptides. There is a focus on cationic AMP characteristics and mechanisms of actions, and an overview of animal-derived natural and synthetic AMPs, as well as observed microbial resistance

Faculty Research Showcase

At Seton Hall University, research is a range on multiple fields of knowledge, and this implies that it derives from different disciplines. As a result of this, the findings of this research are multi-dimensional. This research showcase exhibits how scientific knowledge is generated, and how critical that knowledge is in shaping decisions in our personal lives and in the public domain

Faculty Research Showcase

At Seton Hall University, research is a range on multiple fields of knowledge, and this implies that it derives from different disciplines. As a result of this, the findings of this research are multi-dimensional. This research showcase exhibits how scientific knowledge is generated, and how critical that knowledge is in shaping decisions in our personal lives and in the public domain