Biology subjects

Soundara Manickam, D.

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

6 recordsLinked to original sources

bioRxiv2026

Mitochondrial and bioenergetic processes are enriched in large-but are reciprocally depleted in small extracellular vesicles

Extracellular vesicles (EVs) are a promising class of drug delivery carriers for a broad range of therapeutic applications. We have previously demonstrated that brain endothelial cell (BEC)-derived large EVs (>200 nm in particle diameter) increased mitochondrial function in recipient BECs and improved therapeutic outcomes in a mouse model of ischemic stroke. In the current study, we demonstrate using size-resolved quantitative proteomics and gene-set enrichment analysis that mitochondrial and bioenergetic processes are enriched in large- but are reciprocally depleted in small EVs (exosomes, <200 nm in particle diameter). These results are striking and reveal the selective packaging of mitochondrial cargo in large but not small EVs. These strong mechanistic data anchor the rationale for the continued development of large EV mitochondria as a potential approach for the treatment of multiple diseases associated with mitochondrial dysfunction.

bioengineering↗

bioRxiv2026

Bioreactor cultures of brain endothelial cells produce extracellular vesicles with comparable characteristics as that of batch cultures

Our prior works have demonstrated the therapeutic potential extracellular vesicles (EVs) derived from batch cultures of brain endothelial cells (BECs) owing to their ability to transfer mitochondria to injured recipient cells. However, conventional batch/flask cultures limit scalable and reproducible production of EVs. Here, we evaluated a CELLine AD1000 bioreactor as a platform for sustained production of EVs from mouse b.End3 BECs and compared bioreactor-derived (BR) EVs with EVs obtained conventional batch cultures (BT). Large EVs (lEVs) and small (sEVs) were isolated using a sequential centrifugation protocol and were characterized using dynamic light scattering, nanoparticle tracking analysis, microBCA assay, western blotting, proteomics analysis, and their ability to rescue bioenergetics in oxygen-glucose deprived (OGD) BECs. Bioreactor cultures maintained high cell viabilities (~90%) for up to eight weeks and produced approximately three- to four-fold greater EV protein yields than batch cultures. BR EVs showed reproducible particle characteristics consistent with EV subtypes derived from BT cultures. CD63, CD9 and Arf6 were detected in EV fractions, whereas the mitochondrial outer-membrane protein TOMM20 was enriched in lEVs. Proteomic analyses revealed that bioreactor-derived lEVs retained the mitochondrial cargo signature observed in batch-derived lEVs. Both bioreactor and batch EVs restored relative ATP levels in ODG-exposed BECs. These findings suggest that bioreactor BEC cultures are a scalable approach for generating functionally active BEC-EVs for downstream characterization and therapeutic development.

bioengineering↗

bioRxiv2026

Sequential- vs. density gradient- centrifugation for the isolation of mitochondria-containing extracellular vesicles

A subset of extracellular vehicles (EVs) with particle diameters >200 nm, large vesicles (lEVs) contain mitochondria that increase recipient cell bioenergetics. To date, sequential centrifugation (SC) is the most reported protocol to separate lEVs from the smaller EVs (<200 nm)/exosomes. We have previously demonstrated that lEVs derived from brain endothelial cells (BECs) using the standard SC method transferred their innate mitochondria to recipient BECs, increased recipient BEC bioenergetics, reduced brain infarct volume, and improved behavioral outcomes in a mouse model of transient ischemic stroke. Despite their promising therapeutic activity, SC-isolated lEVs are likely a mixture of mitochondria-containing lEVs and non-mitochondria-containing lEVs. We hypothesized that subsequent purification of SC-isolated lEVs using density-gradient centrifugation (DGC) may yield a purer sample of mitochondria-containing lEVs. We established a DGC protocol to purify lEVs. In this pilot study, lEVs isolated using SC and DGC protocols were compared to determine their physicochemical characteristics and their effects on recipient BEC bioenergetics. SC-lEVs and DGC-lEVs both significantly restored ATP levels in OGD-injured BECs with no difference between groups. However, a Seahorse mitochondrial function assay revealed distinct functional effects: SC-lEVs did not significantly alter respiration, whereas DGC-lEVs induced a dose-dependent increase in oxygen consumption rate, indicating enhanced oxidative phosphorylation. These findings demonstrate that DGC purification yields a more mitochondria-enriched and functionally potent lEV preparation with an enhanced capacity to restore oxidative phosphorylation in ischemic BECs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/732469v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@102b68borg.highwire.dtl.DTLVardef@11e7df1org.highwire.dtl.DTLVardef@de7161org.highwire.dtl.DTLVardef@1e5d64c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Mitochondria-containing large extracellular vesicles target mouse motor neurons upon intramuscular injection

Amyotrophic Lateral Sclerosis (ALS) is a neurological disorder that causes progressive degeneration of motor neurons. Mitochondrial dysfunction accelerates neurodegeneration aggravating the severity of ALS. We hypothesized that increasing the mitochondrial function of motor neurons may promote neuronal survival. Therefore, we investigated the potential of neuron-derived mitochondria containing extracellular vehicles (EVs) as a novel therapeutic approach for ALS using differentiated NSC-34 cells as a surrogate for neurons. Neuron derived-large EVs (lEVs) but not small EVs (sEVs) contained mitochondria. However, we observed increased cell viability and oxygen consumption rates in heat-stressed neurons treated with both sEVs and lEVs suggesting improved mitochondrial function in recipient neurons. The increased oxygen consumption rates in sEV-treated heat-stressed neurons was accompanied by a greater proton leak compared to lEV treatment. The greater proton leak observed with sEVs likely suggests a lower efficiency of oxidative phosphorylation compared to that achieved by cells treated with mitochondria-containing lEVs. These findings suggest that mitochondrial components present in sEVs, such as proteins and mitochondrial DNA, may too contribute to improving cellular respiration. Furthermore, we have demonstrated that lEV mitochondria are transported into the lumbar spinal cord motor neurons following intramuscular injection in C57BL/6 mice in a EV dose-dependent manner. Collectively, for the first time, we have demonstrated the therapeutic effects of neuronal EVs in recipient heat-stressed neurons and the delivery of lEV mitochondria to spinal cord motor neurons in vivo without any EV surface modifications for neuronal targeting. Further studies will determine the therapeutic efficacy of mitochondria-containing EVs in the SOD1G93A transgenic mouse model of ALS. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/675842v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@af590aorg.highwire.dtl.DTLVardef@1fed11dorg.highwire.dtl.DTLVardef@52d339org.highwire.dtl.DTLVardef@1a97182_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Lipidoid nanoparticles increase ATP uptake into hypoxic brain endothelial cells

Lipidoid nanoparticles (LNPs) are clinically successful carriers for nucleic acid delivery to liver and muscle targets. Their ability to load and deliver small molecule drugs has not been reported yet. We propose that the delivery of adenosine triphosphate (ATP) to brain endothelial cells (BECs) lining the blood-brain barrier may increase cellular energetics of the injured BECs. We formulated and studied the physicochemical characteristics of ATP-loaded LNPs using the C12-200 ionizable cationic lipid and other helper lipids. Polyethylene glycol-dimyristoyl glycerol (PEG-DMG), one of the helper lipids, played a crucial role in maintaining colloidal stability of LNPs over time whereas the inclusion of both ATP and PEG-DMG maintained the colloidal stability of LNPs in the presence of serum proteins. ATP-LNPs formulated with PEG-DMG resulted in a 7.7- and 6.6-fold increased uptake of ATP into normoxic and hypoxic BECs, respectively. Altogether, our results demonstrate the potential of LNPs as a novel carrier for the delivery of small molecular mass actives to BECs--a CNS target. HighlightsO_LILNPs were formulated with ATP, a small molecule drug C_LIO_LIPEG-DMG plays a critical role in maintaining particle stability over tim C_LIO_LIATP and PEG-DMG play a critical role in maintaining particle stability in 10% serum C_LIO_LIATP-LNPs were internalized by normoxic and hypoxic brain endothelial cells (BECs) C_LIO_LILNP delivery to BECs broadens its applicability to CNS targets C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/487513v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1a315eaorg.highwire.dtl.DTLVardef@1f3d649org.highwire.dtl.DTLVardef@17989fdorg.highwire.dtl.DTLVardef@526ce2_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Extracellular Vesicles Deliver Mitochondria and HSP27 Protein to Protect the Blood-Brain Barrier

Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of endothelial cell-derived extracellular vesicles (EVs) and utilized mixtures of EV/exogenous heat shock protein 27 (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, which subsequently colocalized with the mitochondrial network of the recipient primary human BECs. BECs treated with m/lEVs increased relative ATP levels and displayed superior mitochondrial function. Importantly, m/lEVs isolated from oligomycin (mitochondrial complex V inhibitor) or rotenone (mitochondrial complex I inhibitor)-exposed BECs (RTN-m/lEVs or OGM-m/lEVs) did not increase BECs ATP levels compared to naive m/lEVs. In contrast, RTN-sEV and OGM-sEV functionality in increasing cellular ATP levels was minimally impacted in comparison to naive sEVs. Intravenously administered m/lEVs showed a reduction in brain infarct sizes compared to vehicle-injected mice in a mouse middle cerebral artery occlusion model of ischemic stroke. We formulated binary mixtures of human recombinant HSP27 protein with EVs: EV/HSP27 and ternary mixtures of HSP27 and EV with cationic polymer poly (ethylene glycol)-b-poly (diethyltriamine): (PEG-DET/HSP27)/EV. (PEG-DET/HSP27)/EV and EV/HSP27 mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two-punch approach to increase BEC metabolic function and tight junction integrity is a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/466491v5_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@dc5493org.highwire.dtl.DTLVardef@134ad7forg.highwire.dtl.DTLVardef@16a8929org.highwire.dtl.DTLVardef@15315bc_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMedium-to-large extracellular vesicles (m/lEVs), not small EVs contain mitochondria C_LIO_LIm/lEVs increased ATP and mitochondrial function in brain endothelial cells (BECs) C_LIO_LIm/lEVs from oligomycin-exposed BECs did not increase recipient BEC ATP levels C_LIO_LIIntravenously injected m/lEVs reduced brain infarct sizes in a mouse stroke model C_LIO_LIEV/HSP27 mixtures reduced small and large dextran molecule permeability across BECs C_LI

bioengineering↗