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Magnetogenetics and Near Infrared for Remote Control of Cell Function

 

In collaboration with Jeffrey Friedman at the Rockefeller University and Sarah Stanley at the Icahn School of Medicine at Mount Sinai, 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 MG-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 MG-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.

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Figure MG-1. TRPV1-IONP system [Stanley et al. Science 336, 604-608 (2012)]. The IONP is covalently coated in anti-His6 antibodies allowing it to bind to the His6-tag on the channel. When subjected to alternating magnetic fields, the nanoparticle activates the channel and causes it to open. This results in calcium ion flux into the cell and the subsequent expression of a calcium-promoted gene.

 

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Figure MG-2. Efficacy of TRPV1-nanoparticle system in HEK-293T cells [Stanley et al. Science 336, 604-608 (2012)]. Upon activation by alternating magnetic fields, 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.

 

Temporally regulating gene expression and cellular activity are invaluable for elucidating underlying physiological processes and could have therapeutic implications. Building upon our TRPV1-IONP system, we developed a genetically encoded system for remote regulation of gene expression by either AMF stimulation or exposure to a magnetic field. Intracellularly, ferritin binds, converts, and stores excess iron ions as paramagnetic ferrihydrite nanoparticles[Stanley et al. Nat. Medicine 21, 92-98 (2015)]. We thus introduced and constitutively produced green fluorescent protein (GFP)-tagged ferritin light and heavy chain dimer fusion proteins, which integrate with endogenous ferritin light and heavy chain monomers to form chimeric GFP-tagged ferritin 24-mers (GFP-ferritin). The GFP-ferritin nanoparticles associate with camelid anti-GFP nanobody TRPV1 fusion proteins (αGFP-TRPV1), allowing for transduction of noninvasive AMF or permanent magnetic fields into channel activation.  This, in turn, initiates calcium-dependent transgene expression(Figure MG-3) [Stanley et al. Nature 531, 647-650 (2016)]. In mice with viral expression of these genetically encoded components, remote stimulation of insulin transgene expression by AMF or static magnetic field exposure lowers blood glucose. This robust, repeatable method for remote regulation in vivo may ultimately have applications in basic science, technology, and therapeutics, as we demonstrated in altering the feeding behavior of mice using the magnetogenetics system (Figure MG-4) [Stanley et al. Nature 531, 647-650 (2016)].

 

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Figure MG-3. TRPV1-ferritin system [Stanley et al. Nature 531, 647-650 (2016)]. An anti-GFP camelid nanobody is expressed on the N-terminus of the TRPV1 and binds to a GFP-tag chimerically integrated into the ferritin nanoparticle (GFP-ferritin). When subjected to alternating magnetic fields, the nanoparticle activates the channel and causes it to open. This results in calcium ion flux into the cell and the subsequent expression of a calcium-promoted gene.

 

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Figure MG-4. Activation and inhibition of glucose-sensing neurons in the ventromedial hypothalamus of mice [Stanley et al. Nature 531, 647-650 (2016)]. AMF treatment of GK-Cre (Gck) and wild-type (WT) mice expressing the TRPV1-ferritin system showed significant (i) reduction in insulin levels and upregulation in (ii) glucagon levels and (iii) glucose-6-phosphatase expression (* p < 0.05, *** p < 0.005). Data shown as Mean ± S.E.M.

 

These approaches provide a platform for using nanotechnology to remotely control cellular response through cell signaling, gene transcription, and protein expression. We are now broadening the capabilities of this platform, exploring the range of potential nanoparticle interactions available to remotely regulate cellular activity. To this end, 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 MG-5i) were used to decorate cells genetically modified with His6-tagged TRPV1 temperature-sensitive ion channel (Figure MG-5ii) and AuNRs conjugated to biotinylated RGD peptide were used to decorate integrins in unmodified cells (Figure MG-5iii). 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 MG-6) [Sanchez-Rodriguez et al. Biotechnol. Bioeng.2240 (2016)]. 

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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.

 

Figure MG-5. TRPV1-AuNR and integrins-AuNR systems [Sanchez-Rodriguez et al. Biotechnol. Bioeng. 113, 2228-2240 (2016)]. (i) Antibody functionalization of gold nanorods using biotin-PEG, streptavidin, and biotinylated anti-His6 antibody. For RGD functionalized AuNR, biotinylated RGD peptide was used in place of biotinylated antibody. (ii) The anti-His6 antibody functionalized AuNR is conjugated to the His6-tag on the TRPV1 channel. When subjected to NIR light, the nanoparticle activates the channel and causes it to open. This results in calcium ion flux into the cell and the subsequent fluorescence of Fluo-4 AM. (iii) The RGD peptide functionalized AuNR is conjugated to integrins on the cell surface. When subjected to NIR light, the nanoparticle likely polarizes the cell membrane, resulting in calcium ion flux into the cell and the subsequent expression of a calcium-promoted gene.

    

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Figure MG-6. Response to NIR light stimulation of functionalized AuNRs on HEK-293T cells. Comparison of the Fluo-4 AM signal after NIR treatment of NR720 functionalized with either anti-His6 or RGD peptide at 0.8, 1.5, and 4.0 W·cm-2. Anti-His6 antibody AuNRs produced fluorescence in HEK-293T cells above a threshold of ~ 1.0 W·cm-2 while RGD peptide AuNRs showed substantial fluorescence even at 0.8 W·cm-2 [Sanchez-Rodriguez et al. Biotechnol. Bioeng. 113, 2228-2240(2016)].

Collaborators:

Jeffrey Friedman - Rockefeller University

Sarah Stanley - Icahn School of Medicine at Mount Sinai