Neurobehavioral Characterization of Perinatal Oxycodone-Exposed Offspring in Early Adolescence.

The opioid epidemic has received considerable attention, but the impact on perinatal opioid-exposed (POE) offspring remains underexplored. This study addresses the emerging public health challenge of understanding and treating POE children. We examined two scenarios using preclinical models: offspring exposed to oxycodone (OXY) in utero (IUO) and acute postnatal OXY (PNO). We hypothesized exposure to OXY during pregnancy primes offspring for neurodevelopmental deficits and severity of deficits is dependent on timing of exposure. Notable findings include reduced head size and brain weight in offspring. Molecular analyses revealed significantly lower levels of inflammasome-specific genes in the prefrontal cortex (PFC). Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) highlighted the enrichment of genes associated with mitochondrial and synapse dysfunction in POE offspring. Western blot analysis validated IPA predictions of mitochondrial dysfunction in PFC-derived synaptosomes. Behavioral studies identified significant social deficits in POE offspring. This study presents the first comparative analysis of acute PNO- and IUO-offspring during early adolescence finding acute PNO-offspring have considerably greater deficits. The striking difference in deficit severity in acute PNO-offspring suggests that exposure to opioids in late pregnancy pose the greatest risk for offspring well-being.

Role of the Gut Bacteria-Derived Metabolite Phenylacetylglutamine in Health and Diseases.

Over the past few decades, it has been well established that gut microbiota-derived metabolites can disrupt gut function, thus resulting in an array of diseases. Notably, phenylacetylglutamine (PAGln), a bacterial derived metabolite, has recently gained attention due to its role in the initiation and progression of cardiovascular and cerebrovascular diseases. This meta-organismal metabolite PAGln is a byproduct of amino acid acetylation of its precursor phenylacetic acid (PAA) from a range of dietary sources like egg, meat, dairy products, etc. The microbiota-dependent metabolism of phenylalanine produces PAA, which is a crucial intermediate that is catalyzed by diverse microbial catalytic pathways. PAA conjugates with glutamine and glycine in the liver and kidney to predominantly form phenylacetylglutamine in humans and phenylacetylglycine in rodents. PAGln is associated with thrombosis as it enhances platelet activation mediated through the GPCRs receptors α2A, α2B, and ß2 ADRs, thereby aggravating the pathological conditions. Clinical evidence suggests that elevated levels of PAGln are associated with pathology of cardiovascular, cerebrovascular, and neurological diseases. This Review further consolidates the microbial/biochemical synthesis of PAGln and discusses its role in the above pathophysiologies.

Novel RNA-Seq Signatures Post-Methamphetamine and Oxycodone Use in a Model of HIV-Associated Neurocognitive Disorders.

In the 21st century, the effects of HIV-associated neurocognitive disorders (HAND) have been significantly reduced in individuals due to the development of antiretroviral therapies (ARTs). However, the growing epidemic of polysubstance use (PSU) has led to concern for the effects of PSU on HIV-seropositive individuals. To effectively treat individuals affected by HAND, it is critical to understand the biological mechanisms affected by PSU, including the identification of novel markers. To fill this important knowledge gap, we used an in vivo HIV-1 Transgenic (HIV-1 Tg) animal model to investigate the effects of the combined use of chronic methamphetamine (METH) and oxycodone (oxy). A RNA-Seq analysis on the striatum-a brain region that is primarily targeted by both HIV and drugs of abuse-identified key differentially expressed markers post-METH and oxy exposure. Furthermore, ClueGO analysis and Ingenuity Pathway Analysis (IPA) revealed crucial molecular and biological functions associated with ATP-activated adenosine receptors, neuropeptide hormone activity, and the oxytocin signaling pathway to be altered between the different treatment groups. The current study further reveals the harmful effects of chronic PSU and HIV infection that can subsequently impact neurological outcomes in polysubstance users with HAND.

In utero and post-natal opioid exposure followed by mild traumatic brain injury contributes to cortical neuroinflammation, mitochondrial dysfunction, and behavioral deficits in juvenile rats.

Maternal opioid use poses a significant health concern not just to the expectant mother but also to the fetus. Notably, increasing numbers of children born suffering from neonatal opioid withdrawal syndrome (NOWS) further compounds the crisis. While epidemiological research has shown the heightened risk factors associated with NOWS, little research has investigated what molecular mechanisms underly the vulnerabilities these children carry throughout development and into later life. To understand the implications of in utero and post-natal opioid exposure on the developing brain, we sought to assess the response to one of the most common pediatric injuries: minor traumatic brain injury (mTBI). Using a rat model of in utero and post-natal oxycodone (IUO) exposure and a low force weight drop model of mTBI, we show that not only neonatal opioid exposure significantly affects neuroinflammation, brain metabolites, synaptic proteome, mitochondrial function, and altered behavior in juvenile rats, but also, in conjunction with mTBI these aberrations are further exacerbated. Specifically, we observed long term metabolic dysregulation, neuroinflammation, alterations in synaptic mitochondria, and impaired behavior were impacted severely by mTBI. Our research highlights the specific vulnerability caused by IUO exposure to a secondary stressor such as later life brain injury. In summary, we present a comprehensive study to highlight the damaging effects of prenatal opioid abuse in conjunction with mild brain injury on the developing brain.

Impact of Adolescent Nicotine Exposure in Pre- and Post-natal Oxycodone Exposed Offspring.

Perinatal exposure to prescription opioids pose a critical public health risk. Notably, research has found significant neurodevelopmental and behavioral deficits between in utero (IUO) and postnatal (PNO) oxycodone-exposed offspring but there is a notable gap in knowledge regarding the interaction of these groups to other drug exposure, particularly nicotine exposure. Nicotine's widespread use represents a ubiquitous clinical interaction that current research does not address. Children often experiment with drugs and risky behavior; therefore, adolescence is a key timepoint to characterize. This study employed an integrated systems approach to investigate escalating nicotine exposure in adolescence and subsequent nicotine withdrawal in the IUO- and PNO-offspring. Western blot analysis found synaptic protein alterations, especially upregulation of synaptophysin in IUO-withdrawal animals. RT-qPCR further validated immune dysfunction in the central nervous system (CNS). Peripheral nicotine metabolism was consistent with increased catabolism of nicotine concerning IUO animals. Lastly, behavioral assays found subtle deficits to withdrawal in nociception and anxiety-like behavior. This study showed, for the first time, the vulnerabilities of PNO- and IUO-exposed groups concerning nicotine use during early adolescence and withdrawal. Graphical Abstract.

An Integrated Systems Approach to Decode the Impact of Adolescent Nicotine Exposure in Utero and Postnatally Oxycodone Exposed Offspring.

Perinatal exposure to prescription opioids pose a critical public health risk. Notably, research has found significant neurodevelopmental and behavioral deficits between in utero (IUO) and postnatal (PNO) oxycodone-exposed offspring but there is a notable gap in knowledge regarding the interaction of these groups to other drug exposure, particularly nicotine exposure. Nicotine's widespread use represents a ubiquitous clinical interaction that current research does not address. Children often experiment with drugs and risky behavior; therefore, adolescence is a key timepoint to characterize. This study employed an integrated systems approach to investigate escalating nicotine exposure in adolescence and subsequent nicotine withdrawal in the IUO- and PNO-offspring. Western blot analysis found alterations of the blood-brain barrier (B.B.B.) and synaptic proteins. RT-qPCR further validated immune dysfunction in the central nervous system (CNS) consistent with compromised B.B.B. Peripheral nicotine metabolism was consistent with increased catabolism of nicotine concerning PNO & IUO, a predictor of greater addiction risk. Lastly, behavioral assays found subtle deficits to withdrawal in nociception and anxiety-like behavior. This study showed, for the first time, the vulnerabilities of PNO- and IUO-exposed groups concerning nicotine use during early adolescence and withdrawal.

Identification of YWHAH as a Novel Brain-Derived Extracellular Vesicle Marker Post Long-Term Midazolam Exposure during Early Development.

Recently, the long-term use of sedative agents in the neonatal intensive care unit (NICU) has raised concerns about neurodevelopmental outcomes in exposed neonates. Midazolam (MDZ), a common neonatal sedative in the NICU, has been suggested to increase learning disturbances and cognitive impairment in children. However, molecular mechanisms contributing to such outcomes with long-term MDZ use during the early stages of life remain unclear. In this study, we for the first time elucidate the role of brain-derived extracellular vesicles (BDEVs), including mining the BDEV proteome post long-term MDZ exposure during early development. Employing our previously established rodent model system that mimics the exposure of MDZ in the NICU using an increasing dosage regimen, we isolated BDEVs from postnatal 21-days-old control and MDZ groups using a differential sucrose density gradient. BDEVs from the control and MDZ groups were then characterized using a ZetaView nanoparticle tracking analyzer and transmission electron microscopy analysis. Next, using RT-qPCR, we examined the expression of key ESCRT-related genes involved in EV biogenesis. Lastly, using quantitative mass spectrometry-based proteomics, we mined the BDEV protein cargo that revealed key differentially expressed proteins and associated molecular pathways to be altered post long-term MDZ exposure. Our study characterized the proteome in BDEV cargo from long-term MDZ exposure at early development. Importantly, we identified and validated the expression of YWHAH as a potential target for further characterization of its downstream mechanism and a potential biomarker for the early onset of neurodevelopment and neurodegenerative diseases. Overall, the present study demonstrated long-term exposure to MDZ at early development stages could influence BDEV protein cargo, which potentially impact neural functions and behavior at later stages of development.

Effect of Combined Methamphetamine and Oxycodone Use on the Synaptic Proteome in an In Vitro Model of Polysubstance Use.

Polysubstance use (PSU) generally involves the simultaneous use of an opioid along with a stimulant. In recent years, this problem has escalated into a nationwide epidemic. Understanding the mechanisms and effects underlying the interaction between these drugs is essential for the development of treatments for those suffering from addiction. Currently, the effect of PSU on synapses-critical points of contact between neurons-remains poorly understood. Using an in vitro model of primary neurons, we examined the combined effects of the psychostimulant methamphetamine (METH) and the prescription opioid oxycodone (oxy) on the synaptic proteome using quantitative mass-spectrometry-based proteomics. A further ClueGO analysis and Ingenuity Pathway Analysis (IPA) indicated the dysregulation of several molecular functions, biological processes, and pathways associated with neural plasticity and structural development. We identified one key synaptic protein, Striatin-1, which plays a vital role in many of these processes and functions, to be downregulated following METH+oxy treatment. This downregulation of Striatin-1 was further validated by Western blot. Overall, the present study indicates several damaging effects of the combined use of METH and oxy on neural function and warrants further detailed investigation into mechanisms contributing to synaptic dysfunction.

Distinct Synaptic Vesicle Proteomic Signatures Associated with Pre- and Post-Natal Oxycodone-Exposure.

The current opioid crisis, which has ravaged all segments of society, continues to pose a rising public health concern. Importantly, dependency on prescription opioids such as oxycodone (oxy) during and after pregnancy can significantly impact the overall brain development of the exposed offspring, especially at the synapse. A significant knowledge gap that remains is identifying distinct synaptic signatures associated with these exposed offspring. Accordingly, the overall goal of this current study was to identify distinct synaptic vesicle (SV) proteins as signatures for offspring exposed to oxy in utero (IUO) and postnatally (PNO). Using a preclinical animal model that imitates oxycodone exposure in utero (IUO) and postnatally (PNO), we used a quantitative mass spectrometry-based proteomics platform to examine changes in the synaptic vesicle proteome on post-natal day 14 (P14) IUO and PNO offspring. We identified MEGF8, associated with carpenter syndrome, to be downregulated in the IUO offspring while LAMTOR4, associated with the regulator complex involved in lysosomal signaling and trafficking, was found to be upregulated in the PNO groups, respectively. Their respective differential expression was further validated by Western blot. In summary, our current study shows exposure to oxy in utero and postnatally can impact the SV proteome in the exposed offspring and the identification of these distinct SV signatures could further pave the way to further elucidate their downstream mechanisms including developing them as potential therapeutic targets.

Methamphetamine Induces the Release of Proadhesive Extracellular Vesicles and Promotes Syncytia Formation: A Potential Role in HIV-1 Neuropathogenesis.

Despite the success of combinational antiretroviral therapy (cART), the high pervasiveness of human immunodeficiency virus-1 (HIV)-associated neurocognitive disorders (HAND) poses a significant challenge for society. Methamphetamine (meth) and related amphetamine compounds, which are potent psychostimulants, are among the most commonly used illicit drugs. Intriguingly, HIV-infected individuals who are meth users have a comparatively higher rate of neuropsychological impairment and exhibit a higher viral load in the brain than infected individuals who do not abuse meth. Effectively, all cell types secrete nano-sized lipid membrane vesicles, referred to as extracellular vesicles (EVs) that can function as intercellular communication to modulate the physiology and pathology of the cells. This study shows that meth treatments on chronically HIV-infected promonocytic U1 cells induce the release of EVs that promote cellular clustering and syncytia formation, a phenomenon that facilitates HIV pathogenesis. Our analysis also revealed that meth exposure increased intercellular adhesion molecule-1 (ICAM-1) and HIV-Nef protein expression in both large (10 K) and small (100 K) EVs. Further, when meth EVs are applied to uninfected naïve monocyte-derived macrophages (MDMs), we saw a significant increase in cell clustering and syncytia formation. Furthermore, treatment of MDMs with antibodies against ICAM-1 and its receptor, lymphocyte function-associated antigen 1 (LFA1), substantially blocked syncytia formation, and consequently reduced the number of multinucleated cells. In summary, our findings reveal that meth exacerbates HIV pathogenesis in the brain through release of proadhesive EVs, promoting syncytia formation and thereby aiding in the progression of HIV infection in uninfected cells.

A comprehensive study to delineate the role of an extracellular vesicle-associated microRNA-29a in chronic methamphetamine use disorder.

Extracellular vesicles (EVs), which express a repertoire of cargo molecules (cf. proteins, microRNA, lipids, etc.), have been garnering a prominent role in the modulation of several cellular processes. Here, using both non-human primate and rodent model systems, we provide evidence that brain-derived EV (BDE) miRNA, miR-29a-3p (mir-29a), is significantly increased during chronic methamphetamine (MA) exposure. Further, miR-29a levels show significant increase both with drug-seeking and reinstatement in a rat MA self-administration model. We also show that EV-associated miR-29a is enriched in EV pool comprising of small EVs and exomeres and further plays a critical role in MA-induced inflammation and synaptodendritic damage. Furthermore, treatment with the anti-inflammatory drug ibudilast (AV411), which is known to reduce MA relapse, decreased the expression of miR-29a and subsequently attenuated inflammation and rescued synaptodendritic injury. Finally, using plasma from MUD subjects, we provide translational evidence that EV-miR29a could potentially serve as a biomarker to detect neuronal damage in humans diagnosed with MA use disorder (MUD). In summary, our work suggests that EV-associated miR-29a-3p plays a crucial role in MUD and might be used as a potential blood-based biomarker for detecting chronic inflammation and synaptic damage.

Integrated Systems Analysis of Mixed Neuroglial Cultures Proteome Post Oxycodone Exposure.

Opioid abuse has become a major public health crisis that affects millions of individuals across the globe. This widespread abuse of prescription opioids and dramatic increase in the availability of illicit opioids have created what is known as the opioid epidemic. Pregnant women are a particularly vulnerable group since they are prescribed for opioids such as morphine, buprenorphine, and methadone, all of which have been shown to cross the placenta and potentially impact the developing fetus. Limited information exists regarding the effect of oxycodone (oxy) on synaptic alterations. To fill this knowledge gap, we employed an integrated system approach to identify proteomic signatures and pathways impacted on mixed neuroglial cultures treated with oxy for 24 h. Differentially expressed proteins were mapped onto global canonical pathways using ingenuity pathway analysis (IPA), identifying enriched pathways associated with ephrin signaling, semaphorin signaling, synaptic long-term depression, endocannabinoid signaling, and opioid signaling. Further analysis by ClueGO identified that the dominant category of differentially expressed protein functions was associated with GDP binding. Since opioid receptors are G-protein coupled receptors (GPCRs), these data indicate that oxy exposure perturbs key pathways associated with synaptic function.

Society on NeuroImmune Pharmacology COVID-19 Virtual Workshop.

This brief report collects the program and abstracts of the Society on NeuroImmune Pharmacology (SNIP) COVID-19 Virtual Workshop held on April 9, 2021. The workshop consisted of four symposia: Symposium 1: Molecular approaches to COVID-19 pathogenesis and underlying mechanisms; Symposium 2: Therapeutic and vaccine approaches to COVID-19; Symposium 3: Early Career Investigator talks; and Symposium 4: Diversity and Inclusion SNIP Committee (DISC) program: Well-being and reflections. The workshop also featured four special talks on COVID-19 and funding opportunities from the National Institute on Alcohol Abuse and Alcoholism (NIAAA); COVID-19 and funding opportunities from the National Institute on Drug Abuse (NIDA); opportunities from NIH for early career investigator (ECI) fellows; and neurologic and psychiatric complications of SARS-CoV-2 infection. Presenters included NIH officials, SNIP members, and non-member scientists whose abstracts were submitted and accepted for inclusion in the virtual event hosted by the University of Nebraska Medical Center via Zoom webinar. A special theme issue of SNIP's official journal, the Journal of Neuroimmune Pharmacology (JNIP), will collect select papers from the workshop along with other related manuscripts in a special theme issue titled "Neuroimmune Pharmacology of SARS-CoV-2."

Extracellular Vesicles from Infected Cells Are Released Prior to Virion Release.

Here, we have attempted to address the timing of EV and virion release from virally infected cells. Uninfected (CEM), HIV-1-infected (J1.1), and human T cell leukemia virus-1 (HTLV-1)-infected (HUT102) cells were synchronized in G0. Viral latency was reversed by increasing gene expression with the addition of serum-rich media and inducers. Supernatants and cell pellets were collected post-induction at different timepoints and assayed for extracellular vesicle (EV) and autophagy markers; and for viral proteins and RNAs. Tetraspanins and autophagy-related proteins were found to be differentially secreted in HIV-1- and HTLV-1-infected cells when compared with uninfected controls. HIV-1 proteins were present at 6 h and their production increased up to 24 h. HTLV-1 proteins peaked at 6 h and plateaued. HIV-1 and HTLV-1 RNA production correlated with viral protein expression. Nanoparticle tracking analysis (NTA) showed increase of EV concentration over time in both uninfected and infected samples. Finally, the HIV-1 supernatant from the 6-h samples was found not to be infectious; however, the virus from the 24-h samples was successfully rescued and infectious. Overall, our data indicate that EV release may occur prior to viral release from infected cells, thereby implicating a potentially significant effect of EVs on uninfected recipient cells prior to subsequent viral infection and spread.

Generational Effects of Opioid Exposure.

The inheritance of substance abuse, including opioid abuse, may be influenced by genetic and non-genetic factors related to the environment, such as stress and socioeconomic status. These non-genetic influences on the heritability of a trait can be attributed to epigenetics. Epigenetic inheritance can result from modifications passed down from the mother, father, or both, resulting in either maternal, paternal, or parental epigenetic inheritance, respectively. These epigenetic modifications can be passed to the offspring to result in multigenerational, intergenerational, or transgenerational inheritance. Human and animal models of opioid exposure have shown generational effects that result in molecular, developmental, and behavioral alterations in future generations.

Role of microRNAs in the pathophysiology of addiction.

Addiction is a chronic and relapsing brain disorder characterized by compulsive seeking despite adverse consequences. There are both heritable and epigenetic mechanisms underlying drug addiction. Emerging evidence suggests that non-coding RNAs (ncRNAs) such as microRNAs (miRNAs), long non-coding RNAs, and circular RNAs regulate synaptic plasticity and related behaviors caused by substances of abuse. These ncRNAs modify gene expression and may contribute to the behavioral phenotypes of addiction. Among the ncRNAs, the most widely researched and impactful are miRNAs. The goal in this systematic review is to provide a detailed account of recent research involving the role of miRNAs in addiction. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions RNA in Disease and Development > RNA in Disease.

Characterization of the intergenerational impact of in utero and postnatal oxycodone exposure.

Prescription opioid abuse during and after pregnancy is a rising public health concern. While earlier studies have documented that offspring exposed to opioids in utero have impaired neurodevelopment, a significant knowledge gap remains in comparing the overall development between offspring exposed in utero and postnatally. Adding a layer of complexity is the role of heredity in the overall development of these exposed offspring. To fill in these important knowledge gaps, the current study uses a preclinical rat model mimicking oxycodone (oxy) exposure in utero (IUO) and postnatally (PNO) to investigate comparative and intergenerational effects in the two different treatment groups. While significant phenotypic attributes were observed with the two treatments and across the two generations, RNA sequencing revealed alterations in the expression of key synaptic genes in the two exposed groups in both generations. RNA sequencing and post validation of genes using RT-PCR highlighted the differential expression of several neuropeptides associated with the hypocretin system, a system recently implicated in addiction. Further, behavior studies revealed anxiety-like behaviors and social deficits that persisted even in the subsequent generations in the two treatment groups. To summarize, our study for the first time reveals a new line of investigation on the potential risks associated with oxy use during and after pregnancy, specifically the disruption of neurodevelopment and intergenerational impact on behavior.

Role of Extracellular Vesicles in Substance Abuse and HIV-Related Neurological Pathologies.

Extracellular vesicles (EVs) are a broad, heterogeneous class of membranous lipid-bilayer vesicles that facilitate intercellular communication throughout the body. As important carriers of various types of cargo, including proteins, lipids, DNA fragments, and a variety of small noncoding RNAs, including miRNAs, mRNAs, and siRNAs, EVs may play an important role in the development of addiction and other neurological pathologies, particularly those related to HIV. In this review, we summarize the findings of EV studies in the context of methamphetamine (METH), cocaine, nicotine, opioid, and alcohol use disorders, highlighting important EV cargoes that may contribute to addiction. Additionally, as HIV and substance abuse are often comorbid, we discuss the potential role of EVs in the intersection of substance abuse and HIV. Taken together, the studies presented in this comprehensive review shed light on the potential role of EVs in the exacerbation of substance use and HIV. As a subject of growing interest, EVs may continue to provide information about mechanisms and pathogenesis in substance use disorders and CNS pathologies, perhaps allowing for exploration into potential therapeutic options.

Role of Brain Derived Extracellular Vesicles in Decoding Sex Differences Associated with Nicotine Self-Administration.

Smoking remains a significant health and economic concern in the United States. Furthermore, the emerging pattern of nicotine intake between sexes further adds a layer of complexity. Nicotine is a potent psychostimulant with a high addiction liability that can significantly alter brain function. However, the neurobiological mechanisms underlying nicotine's impact on brain function and behavior remain unclear. Elucidation of these mechanisms is of high clinical importance and may lead to improved therapeutics for smoking cessation. To fill in this critical knowledge gap, our current study focused on identifying sex-specific brain-derived extracellular vesicles (BDEV) signatures in male and female rats post nicotine self-administration. Extracellular vesicles (EVs) are comprised of phospholipid nanovesicles such as apoptotic bodies, microvesicles (MVs), and exosomes based on their origin or size. EVs are garnering significant attention as molecules involved in cell-cell communication and thus regulating the pathophysiology of several diseases. Interestingly, females post nicotine self-administration, showed larger BDEV sizes, along with impaired EV biogenesis compared to males. Next, using quantitative mass spectrometry-based proteomics, we identified BDEV signatures, including distinct molecular pathways, impacted between males and females. In summary, this study has identified sex-specific changes in BDEV biogenesis, protein cargo signatures, and molecular pathways associated with long-term nicotine self-administration.

Mesenchymal Stem Cell-Derived Extracellular Vesicles: Challenges in Clinical Applications.

Stem cell therapy has garnered much attention and application in the past decades for the treatment of diseases and injuries. Mesenchymal stem cells (MSCs) are studied most extensively for their therapeutic roles, which appear to be derived from their paracrine activity. Recent studies suggest a critical therapeutic role for extracellular vesicles (EV) secreted by MSCs. EV are nano-sized membrane-bound vesicles that shuttle important biomolecules between cells to maintain physiological homeostasis. Studies show that EV from MSCs (MSC-EV) have regenerative and anti-inflammatory properties. The use of MSC-EV, as an alternative to MSCs, confers several advantages, such as higher safety profile, lower immunogenicity, and the ability to cross biological barriers, and avoids complications that arise from stem cell-induced ectopic tumor formation, entrapment in lung microvasculature, and immune rejection. These advantages and the growing body of evidence suggesting that MSC-EV display therapeutic roles contribute to the strong rationale for developing EV as an alternative therapeutic option. Despite the success in preclinical studies, use of MSC-EV in clinical settings will require careful consideration; specifically, several critical issues such as (i) production methods, (ii) quantification and characterization, (iii) pharmacokinetics, targeting and transfer to the target sites, and (iv) safety profile assessments need to be resolved. Keeping these issues in mind, the aim of this mini-review is to shed light on the challenges faced in MSC-EV research in translating successful preclinical studies to clinical platforms.