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Population-based analysis of cell-penetrating peptide uptake using a microfluidic droplet trapping array.

ABSTRACT: Cell-penetrating peptides (CPPs) have garnered significant attention as a method to introduce reporters and therapeutics into intact cells. While numerous studies have been performed identifying new CPP sequences, relatively little is known about their uptake efficiency at the single-cell level. Here, a droplet microfluidic trapping array was used to characterize CPP uptake across a population of single intact cells. The microfluidic device allowed for facile and rapid isolation and analysis of single-cell fluorescence in a 787-member overhead trapping array with >?99% droplet trapping efficiency. The permeability efficiencies of four different CPPs were studied and compared in HeLa cells. Population analysis was performed using linkage hierarchical cluster analysis by R programming to bin cells into subpopulations expressing very low to very high peptide uptake efficiencies. CPP uptake was observed to be heterogeneous across the population of cells with peptide concentration and sequence both playing important roles in the diversity of CPP uptake, the overall peptide uptake efficiency, and the intracellular homogeneity of peptide distribution. This microfluidic-based analytical approach finds application in personalized medicine and provides new insight in the heterogeneity of CPP uptake which has the potential to affect both biosensor and drug internalization in intact cells. Graphical abstract .

SUBMITTER: Safa N

PROVIDER: S-EPMC6472966 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Project description:The use of high-throughput multiplexed screening platforms has attracted significant interest in the field of on-site disease detection and diagnostics for their capability to simultaneously interrogate single-cell responses across different populations. However, many of the current approaches are limited by the spectral overlap between tracking materials (e.g., organic dyes) and commonly used fluorophores/biochemical stains, thus restraining their applications in multiplexed studies. This work demonstrates that the downconversion emission spectra offered by rare earth (RE)-doped β-hexagonal NaYF4 nanoparticles (NPs) can be exploited to address this spectral overlap issue. Compared to organic dyes and other tracking materials where the excitation and emission is separated by tens of nanometers, RE elements have a large gap between excitation and emission which results in their spectral independence from the organic dyes. As a proof of concept, two differently doped NaYF4 NPs (europium: Eu3+, and terbium: Tb3+) were employed on a fluorescent microscopy-based droplet microfluidic trapping array to test their feasibility as spectrally independent droplet trackers. The luminescence tracking properties of Eu3+-doped (red emission) and Tb3+-doped (green emission) NPs were successfully characterized by co-encapsulating with genetically modified cancer cell lines expressing green or red fluorescent proteins (GFP and RFP) in addition to a mixed population of live and dead cells stained with ethidium homodimer. Detailed quantification of the luminescent and fluorescent signals was performed to confirm no overlap between each of the NPs and between NPs and cells. Thus, the spectral independence of Eu3+-doped and Tb3+-doped NPs with each other and with common fluorophores highlights the potential application of this novel technique in multiplexed systems, where many such luminescent NPs (other doped and co-doped NPs) can be used to simultaneously track different input conditions on the same platform. Graphical abstract ᅟ.

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Population-based analysis of cell-penetrating peptide uptake using a microfluidic droplet trapping array.

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