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In collaboration with Jeffrey Friedman’s group at The Rockefeller University, we are combining nanotechnology and bioengineering to demonstrate that nanoparticles can be used in vivo to remotely regulate protein expression. Calcium channels are of great therapeutic interest due to their numerous and varied functions throughout the body. One important channel, Transient Receptor Potential Vanilloid 1 (TRPV1), has gained great interest throughout the literature since its discovery. Understanding the channel’s role as a nociceptor has led to the development of treatments for a wide variety of diseases (e.g. pain caused by shingles).

We have shown that the channel can be manipulated remotely to regulate gene expression in mice [1]. This was achieved by decorating a His6 tag modified TRPV1 channel with antibody-coated iron oxide nanoparticles that are subjected to a low-frequency RF field. Upon activation, the channel opens, allowing a calcium ion flux into the cytoplasm, which then activates a calcium-promoted gene. Studying tumor xenografts expressing a bioengineered insulin gene, we showed that exposure to radio waves stimulates insulin release from the tumors and lowers blood glucose in mice. These approaches provide a platform for using nanotechnology to remotely activate cells to produce protein on demand. Future work includes the use of gold nanoparticles in a similar, light-based approach as well as exploring more fundamental questions such as the mechanism of channel opening.

 

fig1 protein delivery

Figure 1. TRPV1-Nanoparticle system. An anti-his tag antibody is covalently attached to an iron oxide nanoparticle which then binds to a his-tag on the channel. When subjected to RF, the energy is absorbed by the particle and causes the channel to open. This then causes a calcium ion flux, and subsequent calcium-promoted gene transcription.

 

fig2 protein delivery

Figure 2. Efficacy of system. Upon activation by RF waves, the particles cause the channel to open and the subsequent calcium flux promotes gene transcription. This effect is shut down by the channel antagonist Ruthenium red.

 


Nanoparticle-Mediated Cytoplasmic Delivery of Proteins to Target Cellular Machinery

Intracellular delivery of specific proteins and peptides may be used to influence signaling pathways and manipulate cell function, including stem cell fate. Despite recent advances in nanomaterial-based delivery systems, their applicability as carriers of cargo, especially proteins for targeting cellular components and manipulating cell function, is not well understood. We have demonstrated the ability of hydrophobic silica nanoparticles (SiNP) to deliver proteins, including enzymes and antibodies, to a diverse set of mammalian cells, including human breast cancer cell line (MCF-7), human embryonic kidney (HEK-293) and rat neural stem cells (NSCs), while preserving the activity of the biomolecule post-delivery. Using SiNP with a hydrophobic coating, we have explored the delivery and cytosolic activity of nanoparticle-protein conjugates and elucidated the mechanism of cytosolic transport.

Two such delivery cases have been studied. First, we designed a chimeric protein, GFP-FRATtide, where GFP acts as a biomarker for fluorescence detection, and FRATtide binds to and blocks the active site of glycogen synthase kinase-3β (GSK-3β), a protein kinase involved in Wnt signaling. The SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, resulting in an elevation of β-catenin levels due to GSK-3 β inhibition. Accumulation of β-catenin led to increased transcription of Wnt target genes, such as c-MYC, which instruct the cell to actively proliferate and remain in an undifferentiated state (Figure 1). Second, we have explored the cellular delivery of ribonuclease A (RNase A) and the antibody to phospho-Akt (pAkt) both of which resulted in the initiation of cell death due to either the degradation of mRNA or initiation of apoptosis. We, therefore, have shown that functional proteins can be delivered intracellularly in vitro using modified SiNP and used to target key signaling proteins and regulate cell signaling pathways for gain/loss of function and may also prove to be useful for in vivo delivery applications.

 

protein delivery

Figure 1. Schematic of SiNP-GFP-FRATtide preparation and its delivery into cells resulting in β -catenin accumulation by inhibition of GSK-3 β activity. SiNP-GFP-FRATtide conjugates are internalized into the cell cytosol, where FRATtide binds to GSK-3β and blocks the formation of the destruction complex.  Consequently, β -catenin ubiquitinylation and proteasomal degradation is prevented, causing cytosolic β -catenin to accumulate and enter the nucleus to promote target gene expression. APC, adenomatous polyposis coli; CK1a,casein kinase 1α; Fzd, Frizzled; GSK-3 β, glycogen synthase kinase-3β. Figure inspired from Barker and Clevers [2] and van Amerongen et al [3].

 

Reference

  1. S.A. Stanley, J.E. Gagner, S. Damanpour, M. Yoshida, J.S. Dordick, and J.M. Friedman (2012), “Radio-Wave Heating of Iron Oxide Nanoparticles Can Regulate Plasma Glucose in Mice” Science 336, 604-608.
  2. Barker N, Clevers H (2007), “Mining the Wnt pathway for cancer therapeutics”, Nature Reviews Drug Discovery 5, 997-1014.
  3. van Amerongen R, Nawijn M, Franca-Koh J, Zevenhoven J, van der Gulden H, Jonkers J, Berns A (2005), “Frat is dispensable for canonical Wnt signaling in mammals”, Genes & Development 19, 425-430.