Neural Interface Based on Nanomaterials for Transfecting and Stimulating Neuron Cells

基於神經與納米材料交互開發的細胞轉染和細胞刺激方法研究

Student thesis: Doctoral Thesis

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Award date25 Jan 2018

Abstract

The physical and chemical properties of nanomaterials enabled the reaction of many biology interfaces that can be used to manipulate and control biological system at fundamental, molecular levels with a high degree of specificity. In neuroscience research, the applications of nanomaterials for interfacing with neuron have been widely used in different applications, such as drug delivery, imaging, photo activation, electrical stimulation and signal recording and so on. In this thesis, we exploited different nanomaterials to develop novel engineering interface that addresses two major challenges in traditional neuroscience research.

We firstly, describe a novel platform utilizing diamond nanoneedles arrays to facilitated efficient, reliable and vector-free cytosolic delivery. The controlled delivery of foreign molecules and materials into living cells are of great value in genetic manipulation of cells. In the intracellular delivery process, a significant barrier is to cross the cell membrane. This problem is particularly challenging for neuron cells. There is no available commercial tool which is vector-free can solve this issue, and many advanced techniques do not work very well in the transfecting neuron, even though they could get effectively results in other cell systems. Neuron interface based on the nanostructure of needles which could disrupt the membranes can potentially provide a better solution for this issue. By adoption of new this technique, the cellar membrane is deformed by an array of cylindrical nanoneedles with the controlled force around a few nano newtons. We show that our technique has the capability to deliver the broad range of molecules and materials into the cytoplasm of primary neuron in adherent culture. In particular, for delivering nuclear acids into neurons, our technique produces at least eightfold improvement (~45% versus ~1–5%) in transfection efficiency with a dramatically shorter experimental protocol, when compared with the commonly used lipofection approach. Our diamond-nanoneedle provides a powerful tool for non-viral genetic manipulation in primary neuron cells.

In many cases, electrophysiology is used for characterizing the functional phenotype resulted from genetic manipulation, often involving electrical or optical modulation of neural activity. For these applications, nanomaterials based neural interface also provide great opportunities.

We secondly focus on developing a novel neural stimulation technique by using upconversion technology. Upconversion nanoparticles (UCNPs) are the materials that could convert two or more low-energy pump photons from the near-infrared (NIR) spectral region to a higher-energy output photon with a shorter wavelength in the visible spectrum. This property renders UCNPs the potential to be used for stimulating Channelrhodopsin proteins, which are mostly activated by visible light. In this way, tissue penetrating NIR can be directly used for regular optogenetic experiments, bypassing the huge spectral gap between NIR and visible spectrum (VIS). In this study, UCNPs were used as a transducer element to convert remotely applied NIR irradiation to visible light that reliably evoked neuronal spiking activity. In addition to electrophysiology characterization by patch-clamping in culture cells, we further demonstrate the application of the upconversion based neural stimulation strategy in freely moving animals, and successfully conditioned their behavioral activities by using visual cortical and ventral tegmental area (VTA) activation by remotely applied NIR illumination. The specific flexible optrode based on upconversion nanoparticles (UCNPs) was also developed for neural activity manipulation by epidural spinal cord stimulation. We demonstrate that the NIR light stimulation on spinal cord which expresses ChR2 and implanted with the optrode could evoke the corresponding muscle activities, and these locomotion activities could be characterized with the electromyography (EMG) recording. In combination with a robotic laser projection system, UCNPs-optrode could modulate the behavioral activation of the freely moving mice in an open field area by setting NIR light on the target spine. Compared to traditional optogenetic methods requiring fiber optics for light delivery, these results demonstrate the feasibility of a tetherless NIR-optogenetic strategy based on UCNPs.

The application of nanomaterials to neuroscience research has significantly expanded the toolbox for manipulating the complex nervous system at different levels, providing innovative neural interfaces that can achieve unprecedented temporal and spatial control over the system. In this thesis, we particularly focused on developing novel nanostructure-based tools for chemical, genetic and electrical regulation of neural activity, solving the long-haunting problems that have technically hurdle many experiments. We believe our methods based on nanomaterials can open up the new possibility and flexibility for both basic and translational neuroscience research. We also believe that continuous development of new technology of neuron interface based on nanomaterials will make significant contributions to clinical care and prevention in general and neuroscience in the foreseeable future.

    Research areas

  • Neural interface, Nanomaterials , Neural transfection , Neural stimulation