ABSTRACT New treatment strategies are desperately needed to improve respiratory and airway protective functions in neuromuscular disorders that cause breathing impairment, ventilator-dependence and death, such as cervical spinal injury, ALS, encephalitis and neurotoxicity, among others. Targeted drug delivery to the respiratory neural network is a critical goal to effectively treat these neuromuscular disorders. Unfortunately, drug delivery to the central nervous system is restricted by the blood-brain barrier (BBB). Despite the critical need for effective treatment strategies to preserve/restore breathing ability, few options are currently available. The fundamental goal of this proposal is to test a promising and highly novel strategy of nanoparticle-based drug delivery to respiratory motor neurons and associated neural circuits. Motor neurons are unique since their axonal projections reach into the periphery, making it possible to bypass the BBB via retrograde axonal drug transport. However, it is not yet known if motor neuron axon transport mechanisms can be harnessed to carry therapeutic drugs to phrenic motor neurons and their associated pre- synaptic neural network. Motor neurons retrogradely transport certain substances to their cell bodies, including cholera toxin subunit beta (CtB), a non-toxic protein extensively used to label respiratory motor neurons. CtB- conjugated fluorophores are also retrogradely transported to phrenic motor neuron somata. However, CtB- conjugates do not directly affect neural/glial networks associated with targeted motor neurons. We propose to enable retrograde drug delivery via CtB and nanoparticles with unique properties that enable transport across motor neuron cell membranes, effectively delivering therapeutics to the relevant cellular network. Carbon quantum dots (CDots) are nanoparticles (<10 nm) with requisite characteristics for drug delivery, and can be utilized as nanoplatforms decorated with various molecules. Striking preliminary data show that, unlike direct CtB fluorophore conjugates, CtB conjugated to fluorophores via CDots label a subset of spinal interneurons beyond the phrenic motor neurons per se; this finding suggests that retrogradely transported CtB-CDot-conjugates do not remain confined within motor neurons, but distribute more broadly to pre-synaptic neurons in the neural network. We will test the hypotheses that intrapleurally injected CtB-CDot- bioconjugates: 1) distribute beyond phrenic motor neurons throughout spinal and brainstem respiratory neural circuitry (Aim 1); and 2) transport functional cargo to the phrenic motor circuit (Aim 2). This proposal fits the definition of high risk, high impact, appropriate for an R21 grant application since it will: 1) establish new methods of selective drug delivery across the blood brain barrier to the respiratory neural network; 2) yield fundamental knowledge concerning inter-neuronal transport mechanisms of CDot-conjugates; and 3) guide de...