We are combining nanotechnology and bioengineering to demonstrate that external and internal, genetically-encoded nanoparticles can be used in vivo to remotely regulate cellular activity. 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). In particular, we have demonstrated that nanoparticles conjugated to TRPV1 can be used to remotely activate it and mediate cellular activity, such as neuron action potentials, gene transcription, and protein production.
We have shown that the channel can be manipulated remotely to regulate gene expression in mice. This was achieved by decorating His6-tag modified TRPV1 channels (TRPV1His) with anti-His6 antibody-coated iron oxide nanoparticles (αHis6-IONPs) and subjecting them to an alternating magnetic field (AMF) (Figure 1) [Stanley et al. Science 336, 604-608 (2012)]. Upon exposure to an AMF, the IONPs activate the decorated channels and cause them to open, allowing calcium ion flux into the cytoplasm, which subsequently activates a gene under the control of a calcium-sensitive promoter. We tested the system in vitro in human embryonic kidney cells (HEK-293T) expressing TRPV1His and a bioengineered proinsulin gene under the control of a calcium promoter.Proinsulin levels were found to increase significantly when cells wereincubated with αHis6-IONPs and treated with an AMF (Figure 2) [Stanley et al. Science 336, 604-608 (2012)]. We also showed that exposure to an AMF stimulates insulin release from xenograft tumors thereby lowering blood glucose levels in diabetic mice. These studies established the efficacy of a novel platform for using nanotechnology to remotely control cellular response.
Recent reports have shown that intracellular, (super)paramagnetic ferritin nanoparticles can gate TRPV1, a non-selective cation channel, in a magnetic field. We have elucidated the effects of differing field strength and frequency as well as chemical inhibitors on channel gating using a Ca2+-sensitive promoter to express a secreted embryonic alkaline phosphatase (SEAP) reporter (Figure 3). Exposure of TRPV1-ferritin-expressing HEK-293T cells at 30°C to an alternating magnetic field of 501 kHz and 27.1 mT significantly increased SEAP secretion by ~82% relative to control cells, with lesser effects at other field strengths and frequencies. Between 30-32°C, SEAP production was strongly potentiated 3.3-fold by the addition of the TRPV1 agonist capsaicin (Figure 4). This potentiation was eliminated by the competitive antagonist AMG-21629, the NADPH oxidase assembly inhibitor apocynin, and the reactive oxygen species (ROS) scavenger N-acetylcysteine, suggesting that ROS contributes to magnetogenetic TRPV1 activation. These results provide a rational basis to address the heretofore unknown mechanism of magnetogenetics (Figure 5).
Finally, we have demonstrated that photostimulation of gold nanorods (AuNRs) using a tunable near-infrared (NIR) laser at specific longitudinal surface plasmon resonance (SPR) wavelengths can induce the selective and temporal internalization of calcium in HEK-293T cells via TRPV1 activation leading to gene expression [Sanchez-Rodriguez et al. Biotechnol. Bioeng. 113, 2228-2240(2016)]. Biotin-polyethylene glycol (PEG)-AuNRs coated with streptavidin Alexa Fluor-633 and biotinylated anti-His antibodies (Figure 6) were used to decorate cells genetically modified with His6-tagged TRPV1 temperature-sensitive ion channel (Figure 7) and AuNRs conjugated to biotinylated RGD peptide were used to decorate integrins in unmodified cells (Figure 7). Plasmonic activation can be stimulated at weak laser power (0.7-4.0 W·cm-2) without causing cell damage. Selective activation of TRPV1 channels could be controlled by laser power between 1.0-1.5 W·cm-2. Integrin targeting robustly stimulated calcium signaling due to a dense cellular distribution of nanoparticles (Figure 7) [Sanchez-Rodriguez et al. Biotechnol. Bioeng.2240 (2016)].
Such an approach represents a functional tool for combinatorial activation of cell signaling in heterogeneous cell populations. Our results suggest that it is possible to induce cell activation via NIR-induced AuNR heating through the selective targeting of membrane proteins in unmodified cells to produce calcium signaling and downstream expression of specific genes with significant relevance for both in vitro and therapeutic applications.
Jeffrey Friedman – Rockefeller University
Sarah Stanley – Icahn School of Medicine at Mount Sinai