Abstract
Chromobodies have recently drawn great attention as bioimaging nanotools. They offer high antigen binding specificity and affinity comparable to conventional antibodies, but much smaller size and higher stability. Chromobodies can be used in live cell imaging for specific spatio-temporal visualization of cellular processes. To date, functional application of chromobodies requires lengthy genetic manipulation of the target cell. Here, we develop multifunctional large-pore mesoporous silica nanoparticles (MSNs) as nanocarriers to directly transport chromobodies into living cells for antigen-visualization in real time. The multifunctional large-pore MSNs feature high loading capacity for chromobodies, and are efficiently taken up by cells. By functionalizing the internal MSN surface with nitrilotriacetic acid-metal ion complexes, we can control the release of His6-tagged chromobodies from MSNs in acidified endosomes and observe successful chromobody-antigen binding in the cytosol. Hence, by combining the two nanotools, chromobodies and MSNs, we establish a new powerful approach for chromobody applications in living cells.
Highlights
With passive diffusion through the cell membrane or with endocytosis
Mouse embryonic fibroblasts (MEFs) expressing EGFP fused to LMNA (MEF-G-LMNA) are generated for the detection of GFP-specific chromobody release and function in intracellular delivery experiments
We successfully developed NTA-M2+-complex-modified mesoporous silica nanoparticles (MSNs) as carriers for intracellular chromobody delivery
Summary
With passive diffusion through the cell membrane or with endocytosis. The following endosomal trapping of internalized proteins further limits the protein functions in cells. We surmised that mesoporous silica nanoparticles (MSNs) could be promising candidates for serving as efficient and versatile protein delivery vehicles Their surface can be modified with different functionalities and charges to accommodate different proteins; their pore sizes are tunable to fit different cargo sizes; their framework is stable and can effectively protect cargos from environmental degradation, and they can be efficiently taken up by cells. The achievements include delivery of chemotherapeutic agents for cancer therapy[8], intracellular protein delivery for manipulation of cell function[9,10], and oligonucleotide delivery for gene therapy[11,12] These studies demonstrate that MSNs can efficiently control the release of cargos in the target tissue/cells as well as effectively protect cargos from degradation. The successful chromobody delivery, release from the endosomes and binding to the target structures can be confirmed by the fluorescence co-localization signals of EGFP and chromobodies on the LMNA structure
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