Determination of the fate of administered nanoparticles in a biological system, or nanoparticle biodistribution, is critical in evaluating a nanoparticle formulation for nanomedical purposes. Unlike their small-molecule drug antecedents, nanoparticles pose unique challenges in the design of appropriate biodistribution studies due to their nanoscale size and subsequent detection signal. Current, “gold standard” methods to determine nanoparticle biodistribution in vitro and ex vivo fall into one of two categories: bulk- cell analysis, in which large numbers of cells or whole tissues are scanned for the presence of nanoparticles, or single-cell analysis, in which individual cells are probed for positive nanoparticle uptake. However, these methods are greatly limited due to their trade-offs in detection thresholds. Bulk-cell analysis methods, such as those that rely on fluorescence labeling or magnetic nanoparticle core materials, can rapidly detect nanoparticles over large areas of sample but only when they are present in micron-sized aggregates. On the other hand, single-cell analysis methods, such as electron microscopy and atomic force microscopy, can detect small numbers of nanoparticles inside single cells but only scanning sections of a single cell at a time. These trade-offs in nanoparticle detection dem- onstrate an overwhelming need for a sensitive and efficient imaging-based method that can (1) detect small numbers of (or even single) nanoparticles, (2) associate nanoparticle uptake with cell type, and (3) allow for rapid detection over large areas of in vitro and ex vivo samples. We propose a novel method for single nanoparticle detection that is based on bio-sensing principles, coined “nano- barcoding.” Nanobarcoding utilizes a non-endogenous oligonucleotide, or “nanobarcode,” conjugated to the nanoparticle surface to amplify the detection signal from a single nanoparticle via in situ PCR. In situ PCR combines the extreme sensitivity of PCR and the cell-localizing ability of in situ hybridization and is traditionally utilized for rapid detection of low-copy DNA, which is analogous to small numbers of nanoparticles, inside fixed cells of a cell monolayer or histological tissue section. Nanobarcoding has the potential to facilitate rapid and accurate detection of single nanoparticles inside cells for whole-body biodistribution studies. Moreover, nanobar- coding can be applied to the detection of more than one nanoparticle type to study the effects of physicochemical properties, targeting mechanisms, and route of entry on nanoparticle biodistribution. Results from such nanoparticle biodistribution studies can lead to a better understanding of nanoparticle-cell interactions, guide the design of improved theragnostic nanoparticle systems, and direct future investigative paths in nanomedicine.
Burton Morgan 121, Purdue University, West Lafayette, IN