Polyelectrolyte-based Nanoparticles for Gene and Protein Delivery

Polyelectrolytes have emerged as a versatile, simple and promising tool to deliver therapeutic payloads. Their ability to form complexes with oppositely charged polymers or biomacromolecules has led to various applications in the pharmaceutical and biotechnology industries. In this thesis, three different polyelectrolyte delivery systems have been developed and explored for potential uses in gene and protein delivery. Polyvinylamine (PVAm) nanogels with different amounts of surface charge and degradability were used to systematically inspect gene transfection efficiency and cytotoxicity. Transfection efficiency of non-degradable nanogels increased with increasing amounts of positive charge. Intriguingly, acid-labile nanogels bearing low charge showed sustained gene transfection and low cytotoxicity. An intricate balance between transfection efficiency and cytotoxicity is crucial for gene vectors. These results led to an exploration of less toxic, small polycations. Historically, polyplexes using small polycationic peptides such as TAT have shown relatively poor gene transfection, however, previous studies in our group showed that the transfection efficiency could be enhanced by condensing these large polyplexes using calcium. In this thesis, the LABL peptide targeting intercellular cell-adhesion molecule-1 (ICAM-1) was conjugated to TAT peptide using a polyethylene glycol (PEG) spacer. Though the transfection efficiency of TAT polyplexes was reduced by PEGylation, TAT complexes targeting ICAM-1 were able to restore high levels of gene transfection. Thus, targeted TAT polyplexes offer promise for gene delivery to sites of injury or inflammation. Next, other PEGylation strategies using polyelectrolytes were explored. Repifermin, a truncated version of fibroblast growth factor-10 (FGF-10) also known as keratinocyte growth factor-2 (KGF-2), is a heparin-binding protein with potent regenerative properties. The protein unfolds and aggregates at relatively low temperature (~37 oC). The thermal stability of several FGFs was enhanced by electrostatic interactions with polyanions. PEG was grafted to the polyanions pentosan polysulfate (PPS) and dextran sulfate (DS). The potential uses of polyanion conjugates were explored using a variety of spectroscopic and calorimetric methods, and dynamic light scattering. PPS-PEG and DS-PEG conjugates were able to stabilize KGF-2 by increasing the melting temperature of the protein complexes. Though there are several parameters that could be optimized to improve the protein structure upon binding, polyanion-PEG conjugates, however, are encouraging reagents that can improve the thermal stability of heparin-binding proteins via electrostatic PEGylation

Targeting Peptides Derived from Phage Display for Clinical Imaging

Phage display is a high-throughput technology used to identify peptides or proteins with high and specific binding affinities to a target, which is usually a protein biomarker or therapeutic receptor. In general, this technique allows peptides with a particular sequence to be presented on a phage particle. Peptides derived from phage display play an important role in drug discovery, drug delivery, cancer imaging, and treatment. Phage peptides themselves can act as sole therapeutics, for example, drugs, gene therapeutic, and immunotherapeutic agents that are comprehensively described elsewhere. In this chapter, we discuss phage selection and screening procedures in detail including some modifications to reduce nonspecific binding. In addition, the rationale for discovery and utilization of phage peptides as molecular imaging probes is focused upon. Molecular imaging is a new paradigm that uses advanced imaging instruments integrated with specific molecular imaging probes. Applications include monitoring of metabolic and molecular functions, therapeutic response, and drug efficacy, as well as early cancer detection, personalized medicine, and image-guided therapy

Preparation and Characterization of phenytoin Sodium-Controlled Release Solid Dosage Forms

This research aimed to prepare and characterize phenytoin sodium-controlled release solid dosage forms. Phenytoin sodium and hydroxypropyl methylcellulose (HPMC) were mixed at the ratio of 1:0 - 1:3 using physical mixing (PM) and solid dispersion (SD) methods. Compared with the Fourier transform infrared (FTIR) spectrum of phenytoin sodium and HPMC, the spectrum of the obtained mixture using the PM method demonstrated that there were no significant interactions between phenytoin and HPMC. However, the presence of interactions between phenytoin sodium and HPMC was detected when using the SD method. Disintegration time (DT) of all prepared capsules using various ratios of HPMC was less than 15 min. For the in vitro release of capsules, phenytoin released from phenytoin incorporated with HPMC was more than that from non-incorporated phenytoin. Phenytoin released from the formulated capsule with HPMC was increased with increasing of the amount of HPMC. Two pre-formulations (phenytoin sodium and HPMC= 1:2 and 1:3) were used to formulate tablets. Both tablet formulations met the requirement criteria for thickness, hardness, and weight variation (USP41). For the in vitro release of tablets, phenytoin released from the formulated tablet was lower than 5%. This was due to the formulated tablet remaining a viscous white gel in the dissolution basket at the end of the experiment. In conclusion, incorporating phenytoin with HPMC might be suitable for sustained phenytoin release in oral administered tablets. However, DT will be increased and the appropriate ratio of phenytoin sodium and HPMC will be investigated in further studies

Calcium-crosslinked LABL-TAT complexes effectively target gene delivery to ICAM-1 expressing cells

Targeted gene delivery using non-viral vectors is a highly touted scheme to reduce the potential for toxic or immunological side effects by reducing dose. In previous reports, TAT polyplexes with DNA have shown relatively poor gene delivery. The transfection efficiency has been enhanced by condensing TAT/DNA complexes to a small particle size using calcium. To explore the targetability of these condensed TAT complexes, LABL peptide targeting intercellular cell-adhesion molecule-1 (ICAM-1) was conjugated to TAT peptide using a polyethylene glycol (PEG) spacer. PEGylation reduced the transfection efficiency of TAT, but TAT complexes targeting ICAM-1 expressing cells regained much of the lost transfection efficiency. Targeted block peptides properly formulated with calcium offer promise for gene delivery to ICAM-1 expressing cells at sites of injury or inflammation

Progress in Molecular Imaging in Endoscopy and Endomicroscopy for Cancer Imaging

Imaging is an essential tool for effective cancer management. Endoscopes are important medical instruments for performing in vivo imaging in hollow organs. Early detection of cancer can be achieved with surveillance using endoscopy, and has been shown to reduce mortality and to improve outcomes. Recently, great advancements have been made in endoscopic instruments, including new developments in optical designs, light sources, optical fibers, miniature scanners, and multimodal systems, allowing for improved resolution, greater tissue penetration, and multispectral imaging. In addition, progress has been made in the development of highly-specific optical probes, allowing for improved specificity for molecular targets. Integration of these new endoscopic instruments with molecular probes provides a unique opportunity for significantly improving patient outcomes and has potential to further improve early detection, image guided therapy, targeted therapy, and personalized medicine. This work summarizes current and evolving endoscopic technologies, and provides an overview of various promising optical molecular probes