Part I : Peptide functionalized materials for gram-negative bacteria targeted PDT and LPS sensing. Part II : Small peptide and membrane lipid based self-assembled nanostructures for controlled drug/gene delivery

• Main Author: Liu, Fang
• Other Authors: Xing Bengang
• Format: Theses and Dissertations
• Language: English
• Published: 2014
• Subjects: DRNTU::Science::Medicine::Biosensors
• Online Access: http://hdl.handle.net/10356/61686
• Institution: Nanyang Technological University

Part I: Peptide Functionalized Materials for Gram-negative Bacteria Targeted PDT and LPS Sensing Combating infections caused by Gram-negative bacteria is of great importance and remains a big challenge in antimicrobial studies. Generally, Gram-negative bacterial pathogens have additional outer membranes mainly composed of highly conserved and unique lipopolysaccharide (LPS) molecules, which serve as permeability barriers to prevent the cell bodies from toxic antimicrobial molecules1-2 including many photosensitizers, which have been successfully used in photoinactivation of Gram-positive strains.3-4 Furthermore, LPS, a potent inducer to human innate immune systems, hence called endotoxin, is now primarily responsible for sepsis associated with the infections of Gram-negative pethogens.5-6 Therefore, the most promising strategy is use of LPS neutralizing peptide as the binding ligand to deliver antibacterial agents meanwhile minimize the side effects.7 The first objective of the research in part I is the design and synthesis of photosensitizer-peptide conjugates for multivalent targeted cellular imaging and photodynamic inactivation (PDI) against Gram-negative bacterial strains. This simple and specific strategy was based on the conjugation of protophophyrin IX (PpIX), a photosensitizer (PS), with lipopolysaccharide (LPS) binding peptides through a divalent manner. The fluorescent imaging and photodynamic antimicrobial chemotherapy (PACT) studies indicated that the divalent PpIX-peptide conjugate could really serve as a fluorescence probe to image the bacterial cells. Furthermore, those synthetic porphyrin-peptide conjugates indicated promising activities against Gram-negative pathogens even for those with antibiotics resistance upon the exposure to light irradiation. In part I, the second objective is the development of dual-functional nanosystems for both detection and clearance of bacterial lipopolysaccharide (LPS or endotoxin). The dual-functional nanoparticles were synthesized by integration of core-shell Fe3O4@SiO2 magnetic nanoparticles with perylene-diimide (PDI) conjugated LPS-recognizing peptides. The detection mechanism of our rational design is mainly based on the fact that the specific interactions between LPS and neutralizing peptide could break up the π-π stacking conformation of neighbouring PDI molecules on Fe3O4@SiO2 nanoparticle surfaces, and thus efficiently turn on the self-quenched fluorescence. More importantly, the stable and easily controlled LPS sensing nano-platform can also rapidly remove the toxic LPS from bacterial cell lysates and human serum without obvious loss of total proteins. Using the detoxified serum supplemented medium as macrophage culture medium could greatly decrease the nitric oxide generation of macrophages that was based on the inflammatory stimulation by LPS. This was the first example of a simple and novel magnetic nano-platform to address the challenging requirement for both effective detection and removal of LPS in biological samples. Part II: Small Peptide and Membrane Lipid Based Self-assembled Nanostructures for Controlled Drug/gene Delivery The usage of nanoscale materials may greatly facilitate the development of novel therapeutic methods for controllable release of bioactive agents at location of diseases with desired dosage, which has been considered to be of clinical significance to guarantee the curative effect while minimizing the drug resistance and other side effects.8-10 Among the available nanocarriers, the self-assembled nanostructures from highly biocompatible materials (e.g. oligo-peptide, membrane lipids, etc) have been extensively studied due to their unique features of high water content, high drug/gene loading efficiency and promising therapeutic effect.11-13 More importantly, they are naturally biocompatible and biodegradable thus ensuring desirable bio-safety, whereas in terms of inorganic nanoparticle-based delivery systems, additional surface modifications were often required to achieve a good biocompatibility. In part II, my first project is the development of novel drug-loaded nanogels for pH sensitive drug release, which will be described in chapter 3. In our research, the most commonly used model drug doxorubicin (Dox) was conjugated to an Fmoc group protected short peptide through an acid-labile hydrazone-bond to form a peptide-Dox prodrug. Cellular imaging studies displayed that the peptide nanogels could self-deliver drugs into cells through endocytosis and therefore provided effective antitumor activities toward resistant tumor cells. Moreover, our results also unequivocally indicated that ROS could serve as one non-negligible factor to overcome the endo/lysosomal barriers and to facilitate the fast translocation of caged drugs into nucleus. This study provided a simple strategy to investigate the details regarding the rapid intracellular drug redistribution by overcoming the different biological barriers in resistant cancer cells, which could help to better understand the mechanisms of drug delivery, and it may also facilitate the rational design of new carrier systems towards the enhanced drug activity in cancer therapy. The second project of the part is the rational design of novel clickable membrane vesicles (MVs) for tumor-targeted drug/gene delivery. The cell derived MVs are of important self-assembled membrane structures, which are found to play critical roles in antigen presentation, cell to cell communication, cell migration and so on.14-16 In our research, a novel chemical engineered membrane vesicle was developed by introducing an amphiphilic benzophenone derivative into membranes. Meanwhile upon Ca2+ stimulation, a greatly increased production yield of MV from HEK 293 cells was achieved in our research. Our preliminary results indicated that these MVs could be functionalized with additional azido tagged ligands (e.g. Cy5 molecules, oligo His6, etc) through click chemistry. The subsequent study using such clickable MVs as controlled drug/gene delivery vehicles is still in progress.