Remotely Tuning Cell Functions With Near-infrared Upconversion Mediated Optogenetics

During the past decade,exciting advances in optogenetics have emerged in varieties of research fields including cell signaling transduction,neuroscience,tumor therapy and microbiological engineering.However,the optogenetic proteins currently available are mostly responsive to the short wavelength photons of ultraviolet,blue or green light,which greatly limits the application of optogenetics in vivo due to their poor tissue penetration ability.In the experiments using the traditional optogenetic techniques,the optical fibers must be implanted in vivo through invasive surgeries can bring great pains to the host and elevate the risk of unexpected animal behaviors.Upconversion nanoparticles are composed of lanthanide rare earth elements,enabling the conversion of near-infrared light into ultraviolet-visible light.Thanks to the low phototoxicity and deep tissue penetration featured by the near-infrared light,the integration of near-infrared upconversion nanoparticles and optogenetics promises addressing the limitations of current optogenetic proteins.In my dissertation,I have developed a versatile platform based on near-infrared upconversion mediated optogenetic(UMO),and explored its performance in a series of biomedical applications,including the development of non-invasively ameliorating blood glucose levels in type 2 diabetes,demonstration of reversing tumor chemotherapy resistance,and promotion of peripheral nerve injury repair.The main contents of my dissertation include the following sections.Chapter 1:A brief overview of the developments,applications,and limitations of optogenetic techniques,as well as introduction of upconversion nanomaterials.The rationales of justifying the projects,and a brief description of my research contents.Chapter 2:Type 2 diabetes,lowly responsive to insulin injection,is a difficult chronic disease which requires therapeutic breakthroughs.Herein,we integrate upconversion nanoparticles with the optogenetic technique to selectively activate the PI3K/AKT signaling pathways,in an insulin-independent manner for type 2 diabetic treatments.After induction of type 2 diabetic mice model,we have delivered the optogenetic plasmids to the mouse liver,the main site of glucose metabolism,by high-pressure hydrodynamic transfection.The upconversion nanoparticles have been modified with polyethylene glycol for the improvement in dispersibility and biocompatibility,and further modified with glycyrrhetinic acid to specifically target hepatocytes surface receptors for the enhanced uptake of nanoparticles.When the liver is exposed to the near-infrared light,the optogenetic proteins expressed in the liver cells can respond to the blue light transduced by upconversion nanomaterials.It activates the signaling pathways that promote glycogen synthesis and inhibit gluconeogenesis,thus lowering the blood glucose to normal levels.Our approach features fast responsiveness(in seconds)to the near-infrared light,deep tissue penetration(up to centimeters),and tunable dosage by adjusting laser irradiation.As a novel optogenetic tool,it offers the opportunity of developing new alternative strategies to meet the clinical challenges in the treatment of type 2 diabetes.Chapter 3:Tumor drug resistance is a severe challenge to endanger cancer treatment.In this project,I have designed a new system of near-infrared light triggered optogenetics in order to reverse tumor drug resistance for enhanced chemotherapy.Redesigning the optogenetic plasmids allows me to precisely regulating the intracellular localization of the tumor suppressor gene phosphatase and tensin homolog deleted on chromosome ten(PTEN),thereby inhibiting the highly activated phosphoinositide 3-kinase/AKT(PI3K/AKT)pathway in association with tumor growth and proliferation.Simultaneously,the expression of multidrug transporter protein P-glycoprotein(P-gp)can be suppressed,rending the drugresistant cells vulnerable to chemotherapies again.The polyethyleneimine-modified upconversion nanoparticles have been tested as the vehiccles to deliver optogenetic plasmids,after modifiication with hyaluronic acid on the surface for targeting enhancement.In the in vitro and in vivo experiments,I have demonstrated gene transfection in a high efficiency with my approach.The NIR laser can selectively activate the optogenetic proteins expressed in the tumor cells at the specific tumor site.Consequently,it enables accumulation of chemotherapeutic drug molecules in the tumor cells,leading to apoptosis of initially drugresistant tumors.The features of our approach,including the targeted delivery of nanomaterials,the selective activation of near-infrared light,and the reversal of tumor drug resistance,synergistically allows for safe antitumor treatment at low doses of chemotherapeutics.Chapter 4:The clinical treatment of severe peripheral nerve injuries mainly relies on neurosurgeries.In this project,I have developed a novel strategy to precisely guide and promote peripheral nerve injury repair by UMO.It relies on adeno-associated virus infection to specifically express the blue light-sensitive excitatory ion channel protein channelrhodopsin 2(ChR2)in the motor nerve of mice.Then,I have constructed a tibial nerve complete transection model by surgical injury,and injected acetylcholine-modified upconversion nanoparticles that specifically target cholinergic neuron near the injured site.Under the guidance of near-infrared light,the ChR2 protein in the specific neuronal cells can be activated,switching on the ion channels for a cascade of signaling events.This method facilitates precisely guiding and promoting the repair and reconnection of injured peripheral nerves by enhancing stimulation of nerve loops,and restoring innervation to neuromuscular junctions that were denervated due to nerve injuries.This new concept of recovering peripheral nerve injuries in a non-invasive and precisely controlled manner has great potential for clinical translation.Chapter 5:A summary of the projects in my doctoral studies.Discussion of current limitation of my approaches and new potential solutions,and outlook on the future research proposals.