Characterization of the SIM-A9 cell line as a model of activated microglia in the context of neuropathic pain.

Resident microglia of the central nervous system are being increasingly recognized as key players in diseases such as neuropathic pain. Biochemical and behavioral studies in neuropathic pain rodent models have documented compelling evidence of the critical role of ATP mediated-P2X4R-brain-derived neurotrophic factor (BDNF) signaling pathway in the initiation and maintenance of pain hypersensitivity, a feature driving neuropathic pain-related behavior. The goal of this study was to develop and characterize an in vitro cell line model of activated microglia that can be subsequently utilized for screening neuropathic pain therapeutics. In the present study, we characterized the SIM-A9 microglia cell line for key molecules in the P2X4R-BDNF signaling axis using a combination of biochemical techniques and developed an ATP-activated SIM-A9 microglia model. We present three novel findings: first, SIM-A9 cells expressed P2X4R and BDNF proteins, second, ATP, but not LPS, was cytocompatible with SIM-A9 cells and third, exposure of cells to optimized ATP concentrations for defined periods increased intracellular expression of Iba1 and BDNF proteins. Increased Iba1 levels confirmed microglia activation and increased BDNF expression confirmed ATP-mediated stimulation of the P2X4R signaling pathway. We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown

Extracellular Vesicles Derived from a Human Brain Endothelial Cell Line Increase Cellular ATP Levels

Engineered cell-derived extracellular vesicles (EVs) such as exosomes and microvesicles hold immense potential as safe and efficient drug carriers due to their lower immunogenicity and inherent homing capabilities to target cells. In addition to innate vesicular cargo such as lipids, proteins, and nucleic acids, EVs are also known to contain functional mitochondria/mitochondrial DNA that can be transferred to recipient cells to increase cellular bioenergetics. In this proof-of-concept study, we isolated naïve EVs and engineered EVs loaded with an exogenous plasmid DNA encoding for brain-derived neurotrophic factor (BDNF-EVs) from hCMEC/D3, a human brain endothelial cell line, and RAW 264.7 macrophages. We tested whether mitochondrial components in naïve or engineered EVs can increase ATP levels in the recipient brain endothelial cells. EVs (e.g., exosomes and microvesicles; EXOs and MVs) were isolated from the conditioned medium of either untreated (naïve) or pDNA-transfected (Luc-DNA or BDNF-DNA) cells using a differential centrifugation method. RAW 264.7 cell line-derived EVs showed a significantly higher DNA loading and increased luciferase expression in the recipient hCMEC/D3 cells at 72 h compared with hCMEC/D3 cell line-derived EVs. Naïve EVs from hCMEC/D3 cells and BDNF-EVs from RAW 264.7 cells showed a small, but a significantly greater increase in the ATP levels of recipient hCMEC/D3 cells at 24 and 48 h post-exposure. In summary, we have demonstrated (1) differences in exogenous pDNA loading into EVs as a function of cell type using brain endothelial and macrophage cell lines and (2) EV-mediated increases in the intracellular ATP levels in the recipient hCMEC/D3 monolayers

DNA Polyplexes of a Phosphorylcholine-Based Zwitterionic Polymer for Gene Delivery

Purpose: We tested polyplexes of a diblock polymer containing a pH-responsive, endosomolytic core (dimethylaminoethyl methacrylate and butyl methacrylate; DB) and a zwitterionic Poly (methacryloyloxyethyl phosphorylcholine) (PMPC) corona for the delivery of plasmid DNA (pDNA) to glioblastoma cells. Methods: We studied the physicochemical characteristics of the DNA polyplexes such as particle hydrodynamic diameter and surface potential. Cytocompatibility of free PMPC-DB polymer and pDNA polyplexes with U-87MG and U-138MG glioma cell lines were evaluated using the ATP assay. The transfection activity of luciferase pDNA polyplexes was measured using a standard luciferase assay. Anti-proliferative, apoptotic, and cell migration inhibitory activities of PMPC-DB/Interferon-beta (IFN-β1) pDNA polyplexes were examined using ATP assay, flow cytometry, and wound closure assay, respectively. Results: PMPC-DB copolymer condensed pDNA into nanosized polyplexes. DNA polyplexes showed particle diameters ranging from ca. 100–150 nm with narrow polydispersity indices and near electroneutral zeta potential values. PMPC-DB/Luciferase pDNA polyplexes were safe and showed an 18-fold increase in luciferase expression compared to the gold standard PEI polyplexes in U-87MG cells. PMPC-DB/IFN-β1 polyplexes induced apoptosis, demonstrated anti-proliferative effects, and retarded cell migration in glioblastoma cells. Conclusion: The results described herein should guide the future optimization of PMPC-DB/DNA delivery systems for in vivo studies

Development of Lipidoid Nanoparticles for siRNA Delivery to Neural Cells

Lipidoid nanoparticles (LNPs) are the delivery platform in Onpattro, the first FDA-approved siRNA drug. LNPs are also the carriers in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While these applications have demonstrated that LNPs effectively deliver nucleic acids to hepatic and muscle cells, it is unclear if LNPs could be used for delivery of siRNA to neural cells, which are notoriously challenging delivery targets. Therefore, the purpose of this study was to determine if LNPs could efficiently deliver siRNA to neurons. Because of their potential delivery utility in either applications for the central nervous system and the peripheral nervous system, we used both cortical neurons and sensory neurons. We prepared siRNA-LNPs using C12-200, a benchmark ionizable cationic lipidoid along with helper lipids. We demonstrated using dynamic light scattering that the inclusion of both siRNA and PEG-lipid provided a stabilizing effect to the LNP particle diameters and polydispersity indices by minimizing aggregation. We found that siRNA-LNPs were safely tolerated by primary dorsal root ganglion neurons. Flow cytometry analysis revealed that Cy5 siRNA delivered via LNPs into rat primary cortical neurons showed uptake levels similar to Lipofectamine RNAiMAX-the gold standard commercial transfection agent. However, LNPs demonstrated a superior safety profile, whereas the Lipofectamine-mediated uptake was concomitant with significant toxicity. Fluorescence microscopy demonstrated a time-dependent increase in the uptake of LNP-delivered Cy5 siRNA in a human cortical neuron cell line. Overall, our results suggest that LNPs are a viable platform that can be optimized for delivery of therapeutic siRNAs to neural cells

Engineering Extracellular Vesicles to Modulate Their Innate Mitochondrial Load

Introduction: Extracellular vesicles (EVs) are promising carriers for the delivery of biotherapeutic cargo such as RNA and proteins. We have previously demonstrated that the innate EV mitochondria in microvesicles (MVs), but not exosomes (EXOs) can be transferred to recipient BECs and mouse brain slice neurons. Here, we sought to determine if the innate EV mitochondrial load can be further increased via increasing mitochondrial biogenesis in the donor cells. We hypothesized that mitochondria-enriched EVs (“mito-EVs”) may increase the recipient BEC ATP levels to a greater extent than naïve MVs. Methods: We treated NIH/3T3, a fibroblast cell line and hCMEC/D3, a human brain endothelial cell (BEC) line using resveratrol to activate peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), the central mediator of mitochondrial biogenesis. Naïve EVs and mito-EVs isolated from the non-activated and activated donor cells were characterized using transmission electron microscopy, dynamic light scattering and nanoparticle tracking analysis. The effect of mito-EVs on resulting ATP levels in the recipient BECs were determined using Cell Titer Glo ATP assay. The uptake of Mitotracker Red-stained EVs into recipient BECs and their colocalization with recipient BEC mitochondria were studied using flow cytometry and fluorescence microscopy. Results: Resveratrol treatment increased PGC-1α expression in the donor cells. Mito-MVs but not mito-EXOs showed increased expression of mitochondrial markers ATP5A and TOMM20 compared to naïve MVs. TEM images showed that a greater number of mito-MVs contained mitochondria compared to naïve MVs. Mito-MVs but not mito-EXOs showed a larger particle diameter compared to their naïve EV counterparts from the non-activated cells suggesting increased mitochondria incorporation. Mito-EVs were generated at higher particle concentrations compared to naïve EVs from non-activated cells. Mito-EVs increased the cellular ATP levels and transferred their mitochondrial load into the recipient BECs. Mito-MV mitochondria also colocalized with recipient BEC mitochondria. Conclusions: Our results suggest that the pharmacological modulation of mitochondrial biogenesis in the donor cells can change the mitochondrial load in the secreted MVs. Outcomes of physicochemical characterization studies and biological assays confirmed the superior effects of mito-MVs compared to naïve MVs—suggesting their potential to improve mitochondrial function in neurovascular and neurodegenerative diseases

Microvesicles transfer mitochondria and increase mitochondrial function in brain endothelial cells

We have demonstrated, for the first time that microvesicles, a sub-type of extracellular vesicles (EVs) derived from hCMEC/D3: a human brain endothelial cell (BEC) line transfer polarized mitochondria to recipient BECs in culture and to neurons in mice acute brain cortical and hippocampal slices. This mitochondrial transfer increased ATP levels by 100 to 200-fold (relative to untreated cells) in the recipient BECs exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. We have also demonstrated that transfer of microvesicles, the larger EV fraction, but not exosomes resulted in increased mitochondrial function in hypoxic endothelial cultures. Gene ontology and pathway enrichment analysis of EVs revealed a very high association to glycolysis-related processes. In comparison to heterotypic macrophage-derived EVs, BEC-derived EVs demonstrated a greater selectivity to transfer mitochondria and increase endothelial cell survival under ischemic conditions