OBJECTIVE: Construct a population of uniformly sized anionic nanoparticles (NPs) with consistent size, composition, and charge that can be loaded with traditional water-soluble synthetic drugs and, alternatively, protein therapeutics in the lumen and on the surface of the vesicles. DESCRIPTION: Current medical treatments for casualties are heavily reliant upon diffusion of water-soluble synthetic drugs to reach their site of action to protect critical cells and tissues. Typically, synthetic drugs need to be administered at levels such that non-target sites can saturate prior to reaching physiologically relevant concentrations. To minimize drug dosage and maximize the time to effective dose, nanoparticle carriers can be tailored to focus drug delivery to those critical cells and tissues and shift the time tables towards better protection and, ultimately, faster recovery for patients. NPs are increasingly used in applications for drug delivery and other biomedical technologies. Their small size range (1-100 nm diameter) affords them unique properties that can be significantly leveraged to improve systemic delivery and cell-mediated absorption. Their high surface-to-volume ratios enable them to carry significant quantities of synthetic small molecule drugs, as well as protein drugs. The intrinsic properties of the NP's constituents can also be exploited to intricately specify which cells are targeted by the NPs and their payload. Most somatic cells in the human body have an anionic (negative) membrane potential, including neurons. The membrane potential is consistently maintained by gated ion channels, assisting the separation of cytoplasm from extracellular fluids. Neurons have the unique characteristic of switching their membrane potential from negative to positive, albeit transiently, during action potentials. It has been shown that negatively charged NPs are attracted to electrically active neurons, regardless of their size, shape, or composition (Dante et al., 2017).This complimentary charge-charge attraction between NPs and neurons is the cornerstone for this SBIR initiative. PHASE I: The main goal of Phase I is to formulate and construct non-toxic NP constituents which will consistently produce uniformly sized NPs with a negative surface charge. The NPs must have the capacity to be easily loaded with water-soluble synthetic drugs. The NPs must also have the capacity to lumen load protein drugs with encapsulation efficiencies greater than 25% with no decrement to enzyme activity. Developing an efficient NP loading strategy will be paramount to completing Phase I. As a separate milestone, protein drugs must also be functionalized to adhere to the surface of the NPs by any means, as long as there is no interference with enzyme catalytic efficiency. The NPs must be capable of retaining these drugs in a closed vesicle of any shape. NPs can take various shapes during production, including spherical vesicles or rod-shaped hexasomes (Angelova et al., 2017; Eygeris et al., 2020). NP size distributions will be measured using quasi-electric light scattering (QELS) or a similar method. Diffusion coefficients are used to develop population characteristics in terms of hydrodynamic radius (Rh) by quantifying dynamic fluctuations in scattered light. After extrusion, NPs should have a narrow Rh histogram. Some variation in NP sizes will exist within any singular population, which should be captured using QELS and monitored across three (3) distinct production batches. Stability of the nanoparticles can be evaluated by incubating NP samples at elevated temperatures (e.g., 37 C) in buffer and measuring population shifts in Rh. PHASE II: The main goal of Phase II is to evaluate cytotoxicity of the primary NP product from Phase I. A colorimetric thiazolyl blue tetrazolium bromide (MTT) assay for assessing cell metabolic activity or a similar assay measuring cytotoxicity effects may be used. Cytotoxicity can also be assessed in cultured neurons using fluorescent dyes (SYTO 13 and Hoechst 33342) to monitor membrane fluidity and neuron viability (Hubbard et al., 2012). Attraction to neurons in vitro may also assist in developing confidence in the NP net charge (e.g., anionic). Primary neuronal cell cultures can potentially be used to assist in determining cytotoxicity and neuronal attraction simultaneously. A stability study of loaded NPs spiked into animal plasma will need to be completed at room temperature and at 37 C. This will help evaluate the stability of the NP in an ex vivo milieu. NP size and size distribution changes can be monitored using QELS to determine if osmotic shifts will impact loading buffer ionic strength or command the use of loading adjuvants. PHASE III DUAL USE APPLICATIONS: The main goal of Phase III is to show stability and efficacy of the engineered NP with full payloads of both synthetic small molecule drugs and protein drugs in either the lumen or adorned to the surface in a small animal study comparing safety and efficacy results to a non-NP approach using the same drugs. Stability will be measure by injecting loaded NPs into an animal and evaluating their ability to be cleared from the bloodstream by following protein drug pharmacokinetics. This will be a follow on experiment to the study in Phase II where loaded NPs are spiked into animal plasma to evaluate stability ex vivo. Ultimately, this phase of the SBIR will involve direct contact between key DoD laboratories involved in neurological and surgical research, allowing for collaborative assessment using advanced injury models. To evaluate the ability of the NPs to confer neuroprotective capabilities, animals shall be challenged with paraoxon, an organophosphorous compound that is known to inhibit acetylcholinesterase, a key enzyme involved in the transmission of nerve signals at the neuromuscular junction. The proposed animal study will incorporate two therapeutic approaches by examining protective efficacies against 2 x LD50 challenges of paraoxon using conventional chemotherapeutics (atropine and 2-PAM) compared to anionic NPs loaded with these drugs or with a protein-based drug designed to hydrolyze organophosphorus threat agents or loaded with a combination of small molecule and protein drugs. Mice would be an ideal choice because they maintain a body temperature similar to humans at 37 C, putting the experimental NPs under conditions that they will encounter when transitioned to clinical trials. These animals also have a small blood volume which will minimize the use of the experimental NP drugs. As an advanced application of the NPs, they may be transitioned to the administration of pain relieving drugs for both kinetic and thermal traumatic wounds. The neurons responsible for transmitting pain from the source undergo excessive depolarization to send the pain signal back to the central nervous system. Time is the most critical factor in treating any traumatic wound of any kind. In drug development, drug onset of action is critical to success of the product. The short-term effect of these new therapeutic vesicles is that they will provide an improvement in targeting neurons and delivering drugs faster once administered. These NPs will also help extend limited supplies of pharmacological drugs needed in a crisis by using a smaller drug quantity per person, thereby helping more people. Additionally, as a transition product that could also have additional use in the Chemical-Biological Defense Program, a future direction that would be beneficial is in the area of kinetic or thermal traumatic wounds that are exacerbated by the presence of chemical agents. This area is lacking because, currently, a patient's skin can be decontaminated, but there is not a decontamination product appropriate for use in wounds. Without proper decontamination of wounds, the chemical warfare agent continues to be absorbed into the patient's bloodstream. As an adjunct to conventional wound treatment, the anionic nanoparticles could be applied, potentially in the form of a bandage-based treatment solution, to detoxify chemical agents sustainably, immediately, and locally, while traditional administration of therapeutics would attempt to treat the whole body. Ultimately, this new drug design will open the possibility to reformulate the way we present therapeutic drugs to patients, with an emphasis on treating combined injuries involving traumatic wounds contaminated with chemical warfare agents. REFERENCES: 1. Angelova, A., Garamus, V., Angelova, B., Tian, Z., Li, Y., and Zou, A. Advances in structural design of lipid-based nanoparticle carriers for delivery of macromolecular drugs, phytochemicals and anti-tumor agents. 2017. Advances in Colloidal and Interface Science 249: 331-345. 2. Dante, S., Petrelli, A., Petrini, E., Marotte, R., Maccione, A., Alabastri, A., Quarta, A., DeDenato, F., Ravasenga, T., Sathya, A., Cingolani, R., Zaccaria, R., Berdondini, L., Barberis, A., and Pellegrino, T. Selective targeting of neurons with inorganic nanoparticles: revealing the crucial role of nanoparticle surface charge. 2017. ACS Nano 11: 6630-6640. 3. Eygeris, Y., Patel, S. Jozic, A., and Sahay, G. Deconvoluting lipid nanoparticle structure for messenger RNA delivery. 2020. NANO Letters 20: 4543-4549. 4. Hubbard, K., Gut, I., Scheeler, S., Lyman, M. and McNutt, P. Compatibility of SYTO 13 and Hoechst 33342 for longitudinal imaging of neuron viability and cell death. 2012. BMC Research Notes 5: 437-442. KEYWORDS: neurons, nanoparticles, anionic, drug delivery, pain management, and chemical agents
