Targeted and efficient activation of channelrhodopsins expressed in living cells via specifically-bound upconversion nanoparticles

Abstract

Optogenetics is an innovative technology now widely adopted by researchers in different fields of biological sciences. However, most light-sensitive proteins adopted in optogenetics are excited by ultraviolet or visible light which has a weak tissue penetration capability. Upconversion nanoparticles (UCNPs), which absorb near-infrared (NIR) light to emit shorter wavelength light, can help address this issue. In this report, we demonstrated the target selectivity by specifically conjugating the UCNPs with channelrhodopsin-2 (ChR2). We tagged the V5 epitope to the extracellular N-terminal of ChR2 (V5-ChR2m) and functionalized the surface of UCNPs with NeutrAvidin (NAv-UCNPs). After the binding of the biotinylated antibody against V5 onto the V5-ChR2m expressed in the plasma membrane of live HEK293T cells, our results showed that the NAv-UCNPs were specifically bound to the membrane of cells expressing V5-ChR2m. Without the V5 epitope or NAv modification, no binding of UCNPs onto the cell membrane was observed. For the cells expressing V5-ChR2m and bound with NAv-UCNPs, both 488 nm illumination and the upconverted blue emission from UCNPs by 980 nm excitation induced an inward current and elevated the intracellular Ca concentration. Our design reduces the distance between UCNPs and light-sensitive proteins to the molecular level, which not only minimizes the NIR energy required but also provides a way to guide the specific binding for optogenetics applications.

Study of near-infrared light-induced excitation of upconversion nanoparticles as a vector for non-viral DNA delivery

“…In this experiment, we used surface modication to modify hydrophobic UCNPs into hydrophilic UCNPs. 54,55 The mesoporous silica shell structure obtained by the sol-gel two-phase transfer strategy via surfactant modication can be obviously seen in Fig. 5.…”

Intracellular photoswitchable neuropharmacology driven by luminescence from upconverting nanoparticles

“…Here we show that these materials can also be co-loaded with a photoswitchable drug into living neurons via a patch pipette, and used to control ion channel conductivity. This approach is complementary to those proposed in other studies, with some encasing UCNPs in polymer films placed adjacent to opsin-transfected neurons [22][23][24] , linking UCNPs intimately to ion channels 25,26 , or simply injecting UCNPs into the extracellular space of living mice 27,29 . Our study relies on the physical confinement provided by the intracellular environment to bring the UCNPs into intimate proximity with an azobenzene (AB) photoswitch called AAQ 8 in CA1 neurons in living brain slices.…”

“…Subsequently, both spiropyran 19 and AB 20 photoswitches have been indirectly controlled using UCNP 21 . In parallel to these studies, biological photoswitches (light-gated ion channels [22][23][24][25][26][27][28][29] , and LOV domains 30 ) have been stimulated by the luminescence from UCNPs. Here we show that blue luminescence generated by 975 nm excitation of a NaYF 4 :TmYb nanoparticle can be used to stimulate cis to trans isomerization of the mono quaternary ammonium photoprobe AAQ.…”

Near‐Infrared Manipulation of Membrane Ion Channels via Upconversion Optogenetics

“…Other than the improvements of UNCP‐based photon‐transducers, localized stimulations could be additional approaches that contribute to more efficient and remote optogenetic manipulations. Recent study from Chen and co‐workers demonstrated that reducing the distance between UCNPs and light‐sensitive ion channel proteins (ChRs) to the molecular level could minimize the NIR energy and achieve highly specific optogenetic stimulation (Figure c) . The demonstrated study provides a generic approach on the basis of specific binding between NAv‐UCNPs and V5‐ChR2m on the cell membrane, which can be further duplicated for various optogenetic systems.…”

“…c) Targeted and efficient activation of ChRs expressed in living cells via specifically bound UCNPs. Reproduced with permission . Copyright 2017, Royal Society of Chemistry.…”