Nanoparticles formulated from poly(D,L-lactide-co-glycolide) (PLGA) and poly(lactide) (PLA) are being extensively investigated for different therapeutic applications such as for sustained drug, vaccine, and gene delivery. For many of these applications, it is necessary to study the intracellular distribution as well as the tissue uptake of nanoparticles to optimize the efficacy of the encapsulated therapeutic agent. Fluorescence and electron microscopic techniques are usually used for the above purposes. Colloidal gold particles and fluorescent polystyrene, which are generally used as model particles for electron and fluorescence microscopy, respectively, may not be suitable alternatives to PLGA/PLA nanoparticles for these studies mainly because of the differences in their physical properties and also because they do not contain any therapeutic agent. The aim of the present study was to develop and characterize PLGA nanoparticle formulations that would be suitable for confocal/fluorescence and transmission electron microscopic (TEM) studies. Towards this objective, PLGA nanoparticles containing 6-coumarin as a fluorescent marker and osmium tetroxide as an electron microscopic marker with bovine serum albumin (BSA) as a model protein were formulated. Different physical properties of marker-loaded nanoparticles such as particle size, zeta potential, residual PVA content and protein-loading were compared with those of unloaded nanoparticles and were found to be not significantly different. Furthermore, marker-loaded nanoparticle formulations were non-toxic to the cells as unloaded nanoparticles. Nanoparticles loaded with 6-coumarin were found to be useful for studying intracellular nanoparticle uptake and distribution using confocal microscopy while osmium tetroxide-loaded nanoparticles were found to be useful for studying nanoparticle uptake and distribution in cells and tissue using TEM. It was concluded that 6-coumarin and osmium tetroxide could serve as useful fluorescence and electron microscopy probes, respectively, for incorporation into nanoparticles to study their cellular and tissue distribution.
Bibliographical noteFunding Information:
Grant support from the National Institutes of Health (HL 57234) and the Nebraska Research Initiative, Gene Therapy Program. J.P. and S.S. are supported by pre-doctoral and post-doctoral fellowships from the American Heart Association. Predoctoral fellowship to S.P. (DAMD-17-02-1-0506) from Department of Army, the U.S. Army Medical Research Association Activity, 820 Chandler Street, Fort Detrick, MD 21702-5014. We would like to thank Janice Taylor, confocal laser microscopy core facility at UNMC, for her assistance with the microscopic studies and Ms. Elaine Payne for providing administrative assistance. We would like to thank the core electron microscopy facility, Department of Genetics, Cell Biology and Anatomy, UNMC, for assistance with TEM studies.
- Biodegradable polymers
- Confocal microscopy
- Drug delivery
- Transmission electron microscopy