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Core–Shell–Shell Upconversion Nanoparticles with Enhanced Emission for Wireless Optogenetic Inhibition Xudong Lin, Xian Chen, Wenchong Zhang, Tianying Sun, Peilin Fang, Qinghai Liao, Xi Chen, Jufang He, Ming Liu, Feng Wang, and Peng SHI Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b04339 • Publication Date (Web): 26 Dec 2017 Downloaded from http://pubs.acs.org on December 26, 2017
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Core–Shell–Shell Upconversion Nanoparticles with Enhanced Emission for Wireless Optogenetic Inhibition Xudong Lin†,#, Xian Chen‡, ∇, #, Wenchong Zhang†, Tianying Sun‡, Peilin Fang†, Qinghai Liao⊥, Xi Chen∥, Jufang He∥, Ming Liu⊥, Feng Wang‡,§,*, Peng Shi†,§,* †
Department of Mechanical and Biomedical Engineering City University of Hong Kong Kowloon, Hong Kong SAR, China 999077 ‡
Department of Materials Science and Engineering City University of Hong Kong Kowloon, Hong Kong SAR, China 999077 ∇College
of Materials Science and Engineering Shenzhen University Shenzhen, China 518060 ⊥
Department of Electrical and Computer Engineering Hong Kong University of Science and Technology Kowloon, Hong Kong SAR, China 999077
∥ Department
of Biomedical Science City University of Hong Kong Kowloon, Hong Kong SAR, China 999077
§
Shenzhen Research Institute City University of Hong Kong Shenzhen, China 518000
#
These authors contributed equally to this work.
*
Correspondence should be addressed to Dr. Shi Peng,
[email protected] Dr. Wang Feng,
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ABSTRACT Recent advances in upconversion technology have enabled optogenetic neural stimulation using remotely applied optical signals, but limited success has been demonstrated for neural inhibition by using this method, primarily due to the much higher optical power and more redshifted excitation spectrum that are required to work with the appropriate inhibitory opsin proteins. To overcome these limitations, core–shell–shell upconversion nanoparticles (UCNPs) with a hexagonal phase are synthesized to optimize the doping contents of ytterbium ions (Yb3+) and to mitigate Yb-associated concentration quenching. Such UCNP’s emission contains an almost 3fold enhanced peak around 540-570 nm, matching the excitation spectrum of a commonly used inhibitory opsin protein, halorhodopsin. The enhanced UCNPs are utilized as optical transducers to develop a fully implantable upconversion-based device for in vivo tetherless optogenetic inhibition, which is actuated by near-infrared (NIR) light irradiation without any electronics. When the device is implanted into targeted sites deep in the rat brain, the electrical activity of the neurons is reliably inhibited with NIR irradiation and restores to normal level upon switching off the NIR light. The system is further used to perform tetherless unilateral inhibition of the secondary motor cortex in behaving mice, achieving control their motor functions. This study provides an important and useful supplement to the upconversion-based optogenetic toolset, which is beneficial for both basic and translational neuroscience investigations.
KEYWORDS: neural inhibition, upconversion, near infrared light, halorhodopsin, wireless optogenetics, lanthanide-doped nanoparticles
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INTRODUCTION Selective modulation, including stimulation and inhibition, of neurons is essential for the dissection of neural circuits and the understanding of brain functions
1, 2
. Recently, functional
nanomaterials have attracted considerable attention in the field of neural modulation because of their unique physicochemical properties
3-7
. Many studies have demonstrated that, when coupled
with tissue-penetrating near-infrared (NIR) light or ultrasound, functional nanomaterials possess the possibility to enable wireless physiological manipulation in biological organisms. For example, gold nanoparticles and gold nanorods have been utilized to stimulate neurons via photo-thermal effects 4, 5
; piezoelectric nanoparticles have also been demonstrated to enable wireless neural stimulation
during ultrasound signals actuation 8. However, most of the applications have focused on the in vitro demonstration of neuronal stimulation; there have been limited in vivo practices, and neuronal inhibition is largely neglected. As one special type of nanomaterials, lanthanide-doped nanoparticles can sequentially absorb multiple discrete lower-energy photons and emit higher-energy photons. The upconversion capability provides the potential to use NIR for remote regulation of various physiological function in living organisms
9-11
. Especially, upconversion technique has recently been combined with
optogenetics for more flexible neural modulation
12-14
. In this framework, the upconversion
nanoparticles (UCNPs) were used as optical transducers to convert NIR irradiation to visible lights for optogenetic stimulation of neuronal cells that are genetically modified to express light-sensitive ion channel proteins on their membrane12-15. Ideally, the upconversion-based optogenetic strategy not only provides a solution to remotely deliver excitation signal into deep tissues by taking advantage of NIR’s penetrating capability, but also renders the possibility to adjust UCNPs’ emission by tuning their physical, structural and chemical compositions to accommodate different
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opsin proteins
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13, 14
. However, there has been limited progress of applying upconversion technique
in mammalian animals to suppress their neural function, primarily due to the unsuitable emission and the low upconversion efficiency from UCNPs. The ability to manipulate the optical output of the UCNPs with an enhanced emission is important for their bio-applications, especially for the neuronal inhibition. Compared to neural stimulation, the optogenetic tools for neuronal inhibition typically requires much higher optical power to excite 16. Various approaches have been used to tune the spectrum of the emission 17-19. Among these approaches, a highly enhanced emission has potential to be achieved by precise control different combinations of lanthanide dopants and dopant concentration 19. Great efforts have also been made towards enhancing the intensity of visible light emission from UCNPs 20-22. In some cases, the thermal effect constrains relevant bio-applications 23. As a more practical scheme, the UCNP absorption efficiency could be enhanced via material selection, structural design, and precise scale control 19, 22.
NaYF4 is the most efficient host
material for upconversion luminescence, because Yb3+ ions have much larger absorption crosssection than other lanthanide ions at 980-nm wavelength and can transfer the excitation energy efficiently to activator ions. In principle, a high Yb content can enhance upconversion process due to a capacity of absorption and sustain excitation energy. However, the excitation energy can migrate a long distance through Yb sublattice especially at high Yb content, which lead to a depletion of excitation energy (also known as concentration quenching). By imposing a spatial confinement of Yb, the long distance energy migration can be suppressed and the upconversion processes can be enhanced
24
. Although strategies for preparing high Yb3+ doped cubic NaYF4
particles with a small size have been demonstrated 25-27, small UCNPs with a hexagonal phase are preferred for much higher upconverison efficiency compared to the nanoparticles with a cubic phase 28, 29
. However, the paradox between high Yb-content doping and the size increase of the hexagonal
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NaYF4 limits its utilities. In this study, we fabricated hexagonal-phase NaYF4-based UCNPs with a specially designed core–shell–shell structure. β-NaYF4 core particles were used as a template to control the growth of a β-NaYF4:Yb/Er lattice, and an another shell of β-NaYF4 was further introduced to provide a spatial confinement to the Yb lattice to mitigate the high Yb-content associated concentration quenching. In this way, we can optimize the doping ratio of Yb3+ to obtain UNCPs with substantially enhanced upconversion emission, especially at the wavelength around 550 nm. This emission specifically matches the working spectrum of enhanced natronomonas halorhodopsin (eNpHR), a commonly used opsin protein for optogenetic inhibition 30, 31. We then embedded the UCNPs into a glass micropipette to form an ultra-small, fully implantable upconversion device for in vivo applications. When the enhanced UCNP device was placed close to targeted brain circuits, the electrical activities of the eNpHR–expressing neurons could be reliably inhibited upon remote application of NIR illumination and immediately recovered to normal level by switching off the NIR signal, which is not accessible by using the regular UCNPs that we previously reported. By combining the novel material design and our instrumentation development, we successfully achieved an upconversion-based wireless optogenetic inhibition in the motor cortex of behaving mice, and modulated the locomotion behavior of the animals in an open field. In addition, the novel design concept of confining energy migration among different layers of lattice structures can be a general and versatile strategy to enhance upconversion emission in other types of nanoparticles. RESULTS Synthesis and characterization of the core-shell-shell UCNPs
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To prepare the NIR light-powered device with an emission spectrum matching the excitation wavelength of eNpHR (Figure 1a), we first fabricated core–shell–shell UCNPs composed of lanthanide-doped NaYF4, which is extensively used as an UCNP host material because of its low phonon energy and high optical transparence
32
. As demonstrated in our
previous work, the depletion of the excitation energy in upconversion nanocrystals with a high Yb3+ content can be mitigated by suppressing the long-distance energy migration through the spatial confinement of the excitation energy
24
. However, the hexagonal NaYF4 nanoparticles
doped with a high Yb content usually have larger and uneven sizes (Figure S1). To address this problem, we used β-NaYF4 core nanoparticles as a template to precisely control the growth of a β-NaYF4:Yb/Er
lattice.
A
core–shell–shell
structure
[NaYF4@NaYF4:Yb/Er
(58/2
mol%)@NaYF4] was shown to suppress the concentration quenching, when compared to the commonly used core-shell UCNPs [NaYF4:Yb/Er (58/2 mol%)@NaYF4] (Figure S2). In this regard, a series of NaYF4@NaYF4:Yb/Er (x/2 mol%)@NaYF4 core–shell–shell nanocrystals with different Yb3+ contents confined in the middle layer were fabricated via a layer-by-layer epitaxial growth process (Figure 1a). Transmission electron microscopy (TEM) images revealed the uniform morphology of the nanoparticles, which had an average size of 40 nm (Figure 1b). High-resolution TEM revealed the single-crystalline nature of the core–shell–shell nanocrystals, which was evidenced by the clear lattice fringes spaced at 0.518 nm (Figure 1c). This agrees well with the lattice spacing in the (100) planes of hexagonal-phase NaYF4. With the application of an NIR laser (980-nm), the core–shell–shell UCNPs comprising Er3+ dopants with different Yb3+ contents emitted the light with peaks around 540 nm, as indicated by the photoluminescence spectra, which exhibited characteristic emission peaks assigned to the 4G11/2 and 2H9/2→ 4I15/2 (380 and 410 nm), 2H11/2 and 4S3/2 → 4I15/2 (528 and 543 nm) transitions of Er3+
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(Figure 1d, e). Notably, both the emissions around 410 nm and 540-570 nm were enhanced when the Yb3+ content increased from 18 to 78 mol%. When the Yb3+ content increased to 98 mol%, the 410 nm emission further increased, whereas the 540-570 nm emission remained almost unchanged. The maximum emission intensities of 540-570 nm range were obtained at 78 mol%. In theory, the 540-570 nm emission is a two-photon process and becomes saturated earlier than the three-photon generated 410 nm emission, as we increased the Yb3+ content. Using this method, we significantly improved the upconversion efficiency, particularly for the 540-570 nm emission. These enhanced UCNPs can well address the need for use with inhibitory opsin protein, epNHR. Biocompatible package of the core–shell–shell UCNPs To make the UCNPs biocompatible for in vivo experiments, they were loaded and sealed in a glass micro-pipette to form a micro-device (Figure 2a), allowing us to place the UNCPs in close proximity to neurons in a densely packed manner. This package method has been shown to render the UCNP device extremely good biocompatibility with brain tissues 13. We exploited this dense design to create an electronics-free, fully implantable, ultra-small (
