Live Cell Imaging with Fluorescent Polymer Bioconjugates

DESCRIPTION (provided by applicant): The primary goal of this research is to develop nanometer-sized, stable, highly fluorescent, nontoxic, and biocompatible conjugated polymer bioconjugates (CPB) for live cell imaging. The most commonly used fluorophores in cell imaging, cell tracking, and in vivo imaging are organic molecules or green fluorescent proteins (GFPs). They have proven valuable in optical imaging and been applied to the study of single molecules. However, poor photostability causes difficulties in long-term monitoring of cellular events with high sensitivity and resolution. Quantum dots (QDs) are a rapidly developing fluorescent material because of their excellent optical properties. However, the inherent toxicity of QDs (mainly from a heavy metal core such as divalent cadmium ion) causes concerns in long-term monitoring of cellular events, despite demonstrations of low toxicity in living organisms. Here, we propose to develop a new class of fluorescent material that has superior optical properties and no long-term toxicity. Nomadics has developed and applied amplifying fluorescent polymers (AFPs) for the trace detection of biomolecules such as oligonucleotides, proteins, and pathogens. AFP is a class of conjugated polymers with a characteristic pentiptycene unit that preserves superior optical properties even in solid state. The optical characteristics of AFPs are high molar extinction coefficient, high quantum yield, and high photostability. In this proposed work, the AFP will be synthesized with biocompatible monomers, such as poly(ethylene glycol) (PEG) containing monomers, and used as a foundation for CPBs. An all-organic CPB is expected to be nontoxic or less toxic to living cells. In the proposed research, we will focus on the fabrication of multi-functioned CPBs and the demonstration of cell permeability and biocompatibility. Three specific aims are proposed: 1) Fabrication and isolation of CPBs with a target size of less than 20 nm. CPBs will be forms of either collapsed particles or pseudo-micelles. With well-established polymerization techniques, we can control both size and functionality. 2) Attachment of cell-penetrating conjugates (mainly peptides), purification, and quantification. Specific amino sequences for target proteins in cells will be coupled into CPB for the specificity study. 3) Introduction into cells. Permeability, distribution, nonspecific binding, specificity, and toxicity of CPBs will be examined as functions of size, nature of conjugates, surface functionalities, and types of cell lines. Knowledge of endocytosis from the permeability screening of CPBs will be very useful. Successful development of CPB will provide novel platforms for understanding signaling pathways, which will strongly impact the understanding of complex cellular process.