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

Today, antibodies are considered to be the most powerful tools for specific visualization of cellular compartments at the molecular level aimed at the study of cellular processes. They are indispensable for proteomic analyses, protein localization and detection of post-translational modifications. However, the application of full-length antibodies is restricted to fixed cells, meaning dead cells, since the massive sizes (~150 kD) and complex folding structures, including intermolecular disulphide bridges, limit their use in living cells via the transient expression approach or via direct delivery. As a result, the idea of engineering recombinant small antibodies for real time dynamic protein tracing in living cells has received much attention. A variety of recombinant small antibodies including immunoglobulin (Ig) derived Fab (~50 kD) and scFv (~25 kD), as well as non-Ig derived monobody (~10 kD) and affibody (~6.5 kD) protein scaffolds have been generated in the last decades for this purpose1. Nanobodies (~14 kD) are the single-domain antigen-binding fragments derived from camelid’s single-chain IgG2. They have a binding affinity and specificity similar to conventional antibodies, but are much smaller in size and exhibit higher stability. When conjugated with fluorescent proteins or organic dyes, the fluorescent nanobodies, named chromobodies, become molecular probes that can trace the dynamics of endogenous cellular structures in living cells. Chromobodies have successfully shown their antigen detection efficacy on cytoskeleton, histone protein and DNA replication complexes, and have revealed the spatio-temporal protein changes during cell cycles3. In our previous report4, HIV-specific chromobodies have been generated and used for real time visualization of HIV assembly in living cells. These studies demonstrate that chromobodies are promising protein reporters for the study of cellular processes in living cells. However, to date the application of chromobodies for live cell imaging was limited due to the need to introduce them genetically, followed by subsequent cytosolic expression. To broaden the flexibility and use of chromobodies in biomedical applications (e.g., manipulation of cell function for disease treatment), direct intracellular delivery of the molecular probes would be highly desirable. However, intracellular protein delivery is challenging firstly because the large size of proteins leads to difficulties with passive diffusion through the cell membrane or with endocytosis. The following endosomal trapping of internalized proteins further limits the protein functions in cells. A few studies of non-carrier intracellular protein delivery aimed to enhance the cellular uptake efficiency in combination with endosomolytic agents to increase the protein delivery efficacy5,6. For example, Erazo-Oliveras et al. co-incubated dimerized cell-penetrating peptide TAT (dfTAT) with proteins (EGFP and Cre recombinase, etc.) in culture media, the results show that proteins were successfully crossing the cell membrane with the help of dfTAT and presenting their functions in cytosols5; D’Astolfo et al. used hyperosmolality NaCl buffer to induce the highly efficient micropinocytosis of proteins. In combination with a transduction compound (a propanebetaine), intracellular protein release was achieved6. Alternatively, using carriers such as cationic polyplexes with high cellular uptake efficiency for proteins may improve the generally low efficiency of protein uptake. However, the complex structures and wide variety of surface charges of proteins make it difficult to design a general carrier for universal protein delivery7. The second challenge of protein delivery is that proteins are susceptible to proteolytic cleavage. Proteins must maintain their tertiary structure to preserve their functionality during the delivery process. To address these bottlenecks, we surmised that mesoporous silica nanoparticles (MSNs) could be promising candidates for serving as efficient and versatile protein delivery vehicles. For example, 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. In the past years, the development of MSNs for biomedical applications has greatly increased. The achievements include delivery of chemotherapeutic agents for cancer therapy8, intracellular protein delivery for manipulation of cell function9,10, and oligonucleotide delivery for gene therapy11,12. 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. Importantly, MSNs were found to be biocompatible within certain concentration ranges13. In this study, we synthesize multifunctional large-pore MSNs for intracellular chromobody delivery. Metal chelate complexes are covalently attached on the internal silica surface and used for pH-responsive coordination binding of His6-tagged chromobodies. The binding affinity and pH-stimulated release of various metal ions (Fe2+, Co2+, Ni2+, Cu2+, Zn2+ and Ca2+) with His6-tagged chromobodies are examined by colorimetric measurements in vitro. Mouse embryonic fibroblasts (MEFs) expressing EGFP fused to LMNA (an inner nuclear membrane protein) (MEF-G-LMNA) are generated for the detection of GFP-specific chromobody release and function in intracellular delivery experiments. 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.