Biology subjects

Manickam, D. S.

Publications and source records attributed to Manickam, D. S..

5 recordsLinked to original sources

bioRxiv2025

Purinergic receptor activation rectifies autism-associated endothelial dysfunction

Early cerebrovascular alterations affect brain maturation by impacting trophic support and energy supply. Recent evidence in a 16p11.2 deletion mouse model of autism spectrum disorder (ASD) revealed brain endothelial abnormalities postnatally. Yet, the endothelial alterations eliciting these changes remain unknown. Isolation of brain endothelial cells (ECs) from 14-day old male 16p11.2-deficient and wild-type mice revealed that 16p11.2 deletion-induced endothelial dysfunction is linked to a bioenergetic failure, with reduced intracellular ATP. Intra- or extra-cellular ATP supplementation rescued the function of 16p11.2-deficient ECs in vitro via P2 purinergic receptor activation, specifically P2Y2 receptors. Activating P2Y2 receptors restored cerebrovascular reactivity in 16p11.2-deficient parenchymal arterioles ex vivo and rescued 16p11.2 deletion-associated mouse behaviors. Taken together, this study demonstrates that metabolic reprogramming of brain ECs via purinergic receptor engagement represents a possible therapeutic avenue for ASD.

cell biology↗

bioRxiv2025

Ionic liquid-coated lipid nanoparticles promote enhanced red blood cell hitchhiking

Lipid nanoparticles (LNPs) have transformed the delivery of nucleic acid therapeutics; however, their natural tropism favors the liver resulting in clearance by the reticuloendothelial system, with less than 1% of the injected dose reaching challenging targets such as brain. Biocompatible ionic liquids (ILs) are tunable materials that can modulate nanoparticle interactions with blood components. Choline trans-2-hexenoate (C2HA) is an IL known to facilitate red blood cell (RBC) hitchhiking of PLGA polymeric nanoparticles and reduces hepatic uptake and therefore enabling transport to distant non-hepatic organs. We wanted to determine if C2HA coatings can show similar RBC hitchhiking effects with LNPs. We previously demonstrated that IL-coated LNPs reduce serum protein binding to LNPs--a key contributor to rapid clearance via the liver. While LNPs coated with choline trans-2-hexenoate at 1:1 and 1:2 cation: anion ratios decreased mouse serum protein binding and improved cellular uptake into brain endothelial cells (BECs) and motor neurons, they did not show hitchhiking behavior. To identify IL formulations capable of this behavior, we screened higher IL cation: anion ratios (1:3 and 1:5) for LNP coating and optimized IL volumes that allowed stable particle diameters. The resulting IL-coated LNPs successfully hitchhiked on both mouse and human RBCs and significantly enhanced uptake in b.End3 mouse BECs, and NSC-34 neuroblastoma cells compared to uncoated LNPs. 1:3 IL-coated LNPs demonstrated the most pronounced improvement in RBC binding. Whole blood pharmacokinetic and biodistribution analyses demonstrated that IL-coating significantly extends the circulation time of LNPs and results in reduced hepatic uptake alongside increased routing to the brain, compared to standard LNPs. These findings reveal that ILs can be leveraged to re-engineer clinically approved LNP platforms to promote RBC hitchhiking behavior and further be developed for drug delivery to challenging extra-hepatic targets such as the brain. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/649225v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@3fadceorg.highwire.dtl.DTLVardef@abc821org.highwire.dtl.DTLVardef@1e00156org.highwire.dtl.DTLVardef@177ba2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Mitochondria-containing extracellular vesicles from mouse vs. human brain endothelial cells for ischemic stroke therapy

Ischemic stroke-induced mitochondrial dysfunction in the blood-brain barrier-forming brain endothelial cells (BECs) results in long-term neurological dysfunction post-stroke. We previously data from a pilot study where intravenous administration of human BEC (hBEC)-derived mitochondria-containing extracellular vesicles (EVs) showed a potential efficacy signal in a mouse middle cerebral artery occlusion (MCAo) model of stroke. We hypothesized that EVs harvested from donor species homologous to the recipient species (e.g., mouse) may improve therapeutic efficacy, and therefore, use of mouse BEC (mBEC)-derived EVs may improve post-stroke outcomes in MCAo mice. We investigated potential differences in the mitochondria transfer of EVs derived from the same species as the recipient cell (mBEC-EVs and recipient mBECs or hBECs-EVs and recipient hBECs) vs. cross-species EVs and recipient cells (mBEC-EVs and recipient hBECs or vice versa). Our results showed that while both hBEC- and mBEC-EVs transferred EV mitochondria, mBEC-EVs outperformed hBEC-EVs in increasing ATP levels and improved recipient mBEC mitochondrial function via increasing oxygen consumption rates. mBEC-EVs significantly reduced brain infarct volume and neurological deficit scores compared to vehicle-injected MCAo mice. The superior therapeutic efficacy of mBEC-EVs in a mouse MCAo stroke support the continued use of mBEC-EVs to optimize the therapeutic potential of mitochondria-containing EVs in preclinical mouse models. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/575903v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d1deb4org.highwire.dtl.DTLVardef@74157corg.highwire.dtl.DTLVardef@12eb991org.highwire.dtl.DTLVardef@990a47_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Effect of homotypic vs. heterotypic interactions on the cellular uptake of extracellular vesicles

Extracellular vehicles (EVs) are an emerging class of drug carriers and are primarily reported to be internalized into recipient cells via a combination of endocytic routes such as clathrin-mediated, caveolae-mediated and macropinocytosis pathways. In this work, (1) we investigated potential effects of homotypic vs. heterotypic interactions by studying the cellular uptake of homologous EVs (EV donor cells and recipient cells of the same type) vs. heterologous EVs (EV donor cells and recipient cells of different types) and (2) determined the route of EV internalization into low pinocytic/hard-to-deliver cell models such as brain endothelial cells (BECs). We used BECs and macrophages as low-pinocytic and phagocytic cell models, respectively, to study the effect of homotypic vs. heterotypic interactions on EV uptake in the recipient cells. Homotypic interactions led to a greater extent of uptake into the recipient BECs compared to heterotypic interactions. However, we did not see a complete reduction in EV uptake into recipient BECs when endocytic pathways were blocked using pharmacological inhibitors. Our results suggest that EVs primarily use membrane fusion to enter low-pinocytic recipient BECs instead of relying on endocytosis. Lipophilic PKH67 dye-labeled EVs but not intravesicular esterase-activated calcein ester-labeled EVs severely reduced particle uptake into BECs while phagocytic macrophages internalized both types of EV-labeled particles to comparable extents. Our results also highlight the importance of carefully choosing labeling dye chemistry to study EV uptake, especially in the case of low pinocytic cells such as BECs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=171 HEIGHT=200 SRC="FIGDIR/small/563628v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@d5cb50org.highwire.dtl.DTLVardef@172d1dorg.highwire.dtl.DTLVardef@192caf6org.highwire.dtl.DTLVardef@4b16ba_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

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 utility in either applications in 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/454207v2_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@1ac52acorg.highwire.dtl.DTLVardef@67dbb0org.highwire.dtl.DTLVardef@880526org.highwire.dtl.DTLVardef@a1693_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

bioengineering↗