Chemical imaging of nanocarriers in live cells and live animals

Hongtao Chen, Purdue University

Abstract

Nanomedicine deals with the development of nanoscale targeted drug delivery systems to improve the bioavailability of drugs. In order to deliver the nanocarriers and drugs to target cells in a tissue environment, it is critical to understand their targeting, cellular internalization and pharmacokinetics during systemic administration. My thesis work develops and employs advanced imaging techniques to provide useful information and deep understanding of the drug delivery via nanocarriers. The first part of my thesis focuses on the endocytosis of folate receptors (FRs) which are overexpressed in over 1/3 human cancers. Real-time fluorescence imaging and single particle tracking (SPT) analysis are used to study the intracellular trafficking of FR. FR endosomes migrating along microtubules is directly observed and bidirectional motions of FR endosomes are quantified by SPT. The role of microtubule-associated motor proteins, dynein and kinesin I, is demonstrated by microinjection with antibodies. Furthermore, our imaging tools allow the evaluation of the impact of cholesterol on intracellular transport of FR. It is found that cholesterol level regulates the FR trafficking by changing the endosomal distribution of Rab4 and Rab7 proteins which are adaptors for microtubule motors (Biophys. J., 2008, 94:1508-1520, front cover story). Using Förster resonance energy transfer (FRET) imaging, drug release from polymeric micelles, a superior nanocarrier for hydrophobic drugs, during cellular uptake and blood circulation is investigated. A FRET pair, DiIC 18(3) and DiOC18(3), is loaded into micelles to mimic hydrophobic drug molecules. By monitoring the FRET efficiency in live cells, the plasma membrane is demonstrated to be a temporal residence for micelle-released hydrophobic molecules before their delivery to target intracellular destinations (Proc. Natl. Acad. Sci. USA, 2008, 105: 6596-6601). By monitoring the FRET efficiency in blood stream, DiIC18 and DiOC18 are found to quickly escape from micelles. FRET spectroscopy studies demonstrate that α- and β-globulins are major factors for the observed fast release, whereas γ-globulins, albumin, and red blood cells play minor roles. This information can help the development of micelle-based nanocarriers which can be resistant to disassembly in circulation (Langmuir, 2008, 24:5213-17, front Cover Story). The same FRET technique is also used to study disulfide bond-reducing activity in endosomal compartments (Proc. Natl. Acad. Sci. USA, 2006, 103: 13872-13877). To monitor nanocarriers, such as novel metal nanoparticles and nanorods, in tissue environment and to study the interactions between extracellular matrix and stroma, a multimodal multiphoton microscopy is developed. Because different modalities can be used to image different biological structures and nanocarriers, herein an easy-to-operate approach is presented to perform Coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPF), second harmonic generation (SHG), and third harmonic generation (THG) imaging using a single laser source composed of an 80 MHz fs laser, an optical parametric oscillator (OPO), and a PPLN crystal for frequency doubling (Optics express, 2009, 17: 1282-1290). It allows simultaneous imaging of different biological structures, e.g. vibrationally resonant CARS imaging of CH-rich myelin sheath in fresh spinal tissues and lipid bodies in live cells, SHG imaging of collagen fibers in liver tissue, THG imaging of gold nanorods in cell, tissue and animals after administration. This platform can provide complementary and important information for bioscience research.

Degree

Ph.D.

Advisors

Cheng, Purdue University.

Subject Area

Analytical chemistry

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