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Nanoparticle mediated delivery and small molecule triggered activation of proteins in the nucleus

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Version 2 2018-10-22, 10:49
Version 1 2018-09-15, 06:02
journal contribution
posted on 2018-10-22, 10:49 authored by Hsin-Yi Chiu, Jack A. Bates, Jonas Helma, Hanna Engelke, Hartmann Harz, Thomas Bein, Heinrich Leonhardt

Protein transfection is a versatile tool to study or manipulate cellular processes and also shows great therapeutic potential. However, the repertoire of cost effective techniques for efficient and minimally cytotoxic delivery remains limited. Mesoporous silica nanoparticles (MSNs) are multifunctional nanocarriers for cellular delivery of a wide range of molecules, they are simple and economical to synthesize and have shown great promise for protein delivery. In this work we present a general strategy to optimize the delivery of active protein to the nucleus. We generated a bimolecular Venus based optical sensor that exclusively detects active and bioavailable protein for the performance of multi-parameter optimization of protein delivery. In conjunction with cell viability tests we maximized MSN protein delivery and biocompatibility and achieved highly efficient protein transfection rates of 80%. Using the sensor to measure live-cell protein delivery kinetics, we observed heterogeneous timings within cell populations which could have a confounding effect on function studies. To address this problem we fused a split or dimerization dependent protein of interest to chemically induced dimerization (CID) components, permitting control over its activity following cellular delivery. Using the split Venus protein we directly show that addition of a small molecule dimerizer causes synchronous activation of the delivered protein across the entire cell population. This combination of cellular delivery and triggered activation provides a defined starting point for functional studies and could be applied to other protein transfection methods.

Funding

Financial support from the Deutsche Forschungsgemeinschaft (SFB 1032 and SPP1623/LE721/13), the Center for Nano Science (CeNS) and the Excellence Cluster Nanosystems Initiative Munich (NIM) is gratefully acknowledged.

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