Intracellular chromobody delivery by mesoporous silica nanoparticles for antigen targeting and visualization in real time

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.

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

“…Endosomal rupture is triggered using chloroquine[19] and FRB-VC is released to the cytosol. FRB-VC diffuses to the nucleus and interacts with FKBP-VN, which is primed for dimerization with pre-bound rapamycin.…”

“…Large-pore MSNs were surface-functionalized with NTA-Ni complexes (MSN-Ni) through a series of modifications (Figure 2(a)) to accommodate pH dependent binding and release of the His-tagged FRB-VC[10,11,19]. STEM and SEM images (Figure 2(b)) indicated that the resulting nitrilotriacetic acid-modified MSNs (MSN-NTAs) exhibit uniform particle size and coral surface morphology with irregular pore shape (pore size ranging from 10 – 40 nm) consistent with the structure of the un-functionalized form (Figure S1).…”

“…MSNs can be generated in large volumes at low financial and labor expenditure [18]. Having previously demonstrated the delivery of bioactive proteins using our large pore MSN variant [19] we sought to produce an optimized delivery protocol that maximizes efficiency whilst retaining good biocompatibility, i.e. the integrity and viability of cells.…”

Nanobodys: Strategien zur chemischen Funktionalisierung und intrazelluläre Anwendungen

“…Dies kann entweder durch Destabilisierung der endosomalen Membran mittels Lipiden oder osmotischen Drucks oder aber durch die Translokation des Biomoleküls durch Transmembranporen erreicht werden . 2016 entwickelten Chiu und Mitarbeiter großporige mesoporöse Siliciumdioxid‐Nanopartikel (MSNs), die mit Nitrilotriessigsäure(NTA)‐Gruppen an der inneren Oberfläche der MSNs funktionalisiert sind (Abbildung d) . Die Aktivierung des Komplexes mit verschiedenen Metallionen ermöglichte die kovalente Bindung eines His 6 ‐markierten GFP‐Chromobody.…”

A Pathogen-Specific Cargo Delivery Platform Based on Mesoporous Silica Nanoparticles

“…achieved is through antibody-antigen interactions, and indeed, MSN-antibody conjugates have been reported that were used for analyte detection, 14-17 theranostics/imaging, [17][18][19][20][21][22] or cell targeting. [23][24][25][26][27][28] However, these examples demonstrate a specific recognition of an antigen by the MSN-antibody conjugate, but the recognition event itself does not stimulate a signal or killing response, i.e., there is no antigen-responsive gatekeeping mechanism that would selectively control cargo release only in the presence of a specific antigen or pathogen.…”