Ferroptosis induces membrane blebbing in placental trophoblasts.

Ferroptosis is a regulated, non-apoptotic form of cell death, characterized by hydroxy-peroxidation of discrete phospholipid hydroperoxides, particularly hydroperoxyl (Hp) forms of arachidonoyl- and adrenoyl-phosphatidylethanolamine, with a downstream cascade of oxidative damage to membrane lipids, proteins and DNA, culminating in cell death. We recently showed that human trophoblasts are particularly sensitive to ferroptosis caused by depletion or inhibition of glutathione peroxidase 4 (GPX4) or the lipase PLA2G6. Here, we show that trophoblastic ferroptosis is accompanied by a dramatic change in the trophoblast plasma membrane, with macro-blebbing and vesiculation. Immunofluorescence revealed that ferroptotic cell-derived blebs stained positive for F-actin, but negative for cytoplasmic organelle markers. Transfer of conditioned medium that contained detached macrovesicles or co-culture of wild-type target cells with blebbing cells did not stimulate ferroptosis in target cells. Molecular modeling showed that the presence of Hp-phosphatidylethanolamine in the cell membrane promoted its cell ability to be stretched. Together, our data establish that membrane macro-blebbing is characteristic of trophoblast ferroptosis and can serve as a useful marker of this process. Whether or not these blebs are physiologically functional remains to be established.

Integrated unbiased multiomics defines disease-independent placental clusters in common obstetrical syndromes.

BACKGROUND: Placental dysfunction, a root cause of common syndromes affecting human pregnancy, such as preeclampsia (PE), fetal growth restriction (FGR), and spontaneous preterm delivery (sPTD), remains poorly defined. These common, yet clinically disparate obstetrical syndromes share similar placental histopathologic patterns, while individuals within each syndrome present distinct molecular changes, challenging our understanding and hindering our ability to prevent and treat these syndromes. METHODS: Using our extensive biobank, we identified women with severe PE (n = 75), FGR (n = 40), FGR with a hypertensive disorder (FGR + HDP; n = 33), sPTD (n = 72), and two uncomplicated control groups, term (n = 113), and preterm without PE, FGR, or sPTD (n = 16). We used placental biopsies for transcriptomics, proteomics, metabolomics data, and histological evaluation. After conventional pairwise comparison, we deployed an unbiased, AI-based similarity network fusion (SNF) to integrate the datatypes and identify omics-defined placental clusters. We used Bayesian model selection to compare the association between the histopathological features and disease conditions vs SNF clusters. RESULTS: Pairwise, disease-based comparisons exhibited relatively few differences, likely reflecting the heterogeneity of the clinical syndromes. Therefore, we deployed the unbiased, omics-based SNF method. Our analysis resulted in four distinct clusters, which were mostly dominated by a specific syndrome. Notably, the cluster dominated by early-onset PE exhibited strong placental dysfunction patterns, with weaker injury patterns in the cluster dominated by sPTD. The SNF-defined clusters exhibited better correlation with the histopathology than the predefined disease groups. CONCLUSIONS: Our results demonstrate that integrated omics-based SNF distinctively reclassifies placental dysfunction patterns underlying the common obstetrical syndromes, improves our understanding of the pathological processes, and could promote a search for more personalized interventions.

Unc-13 homolog D mediates an antiviral effect of the chromosome 19 microRNA cluster miR-517a.

The function of microRNAs (miRNAs) can be cell autonomous or communicated to other cell types and has been implicated in diverse biological processes. We previously demonstrated that miR-517a-3p (miR-517a), a highly expressed member of the chromosome 19 miRNA cluster (C19MC) that is transcribed almost exclusively in human trophoblasts, attenuates viral replication via induction of autophagy in non-trophoblastic recipient cells. However, the molecular mechanisms underlying these effects remain unknown. Here, we identified unc-13 homolog D (UNC13D) as a direct, autophagy-related gene target of miR-517a, leading to repression of UNC13D. In line with the antiviral activity of miR-517a, silencing UNC13D suppressed replication of vesicular stomatitis virus (VSV), whereas overexpression of UNC13D increased VSV levels, suggesting a role for UNC13D silencing in the antiviral activity of miR-517a. We also found that miR-517a activated NF-κB signaling in HEK-293XL cells expressing TLR8, but the effect was not specific to C19MC miRNA. Taken together, our results define mechanistic pathways that link C19MC miRNA with inhibition of viral replication.

Isolation of exosomes from whole blood by integrating acoustics and microfluidics.

Exosomes are nanoscale extracellular vesicles that play an important role in many biological processes, including intercellular communications, antigen presentation, and the transport of proteins, RNA, and other molecules. Recently there has been significant interest in exosome-related fundamental research, seeking new exosome-based biomarkers for health monitoring and disease diagnoses. Here, we report a separation method based on acoustofluidics (i.e., the integration of acoustics and microfluidics) to isolate exosomes directly from whole blood in a label-free and contact-free manner. This acoustofluidic platform consists of two modules: a microscale cell-removal module that first removes larger blood components, followed by extracellular vesicle subgroup separation in the exosome-isolation module. In the cell-removal module, we demonstrate the isolation of 110-nm particles from a mixture of micro- and nanosized particles with a yield greater than 99%. In the exosome-isolation module, we isolate exosomes from an extracellular vesicle mixture with a purity of 98.4%. Integrating the two acoustofluidic modules onto a single chip, we isolated exosomes from whole blood with a blood cell removal rate of over 99.999%. With its ability to perform rapid, biocompatible, label-free, contact-free, and continuous-flow exosome isolation, the integrated acoustofluidic device offers a unique approach to investigate the role of exosomes in the onset and progression of human diseases with potential applications in health monitoring, medical diagnosis, targeted drug delivery, and personalized medicine.

Human placental trophoblasts confer viral resistance to recipient cells.

Placental trophoblasts form the interface between the fetal and maternal environments and serve to limit the maternal-fetal spread of viruses. Here we show that cultured primary human placental trophoblasts are highly resistant to infection by a number of viruses and, importantly, confer this resistance to nonplacental recipient cells by exosome-mediated delivery of specific microRNAs (miRNAs). We show that miRNA members of the chromosome 19 miRNA cluster, which are almost exclusively expressed in the human placenta, are packaged within trophoblast-derived exosomes and attenuate viral replication in recipient cells by the induction of autophagy. Together, our findings identify an unprecedented paracrine and/or systemic function of placental trophoblasts that uses exosome-mediated transfer of a unique set of placental-specific effector miRNAs to directly communicate with placental or maternal target cells and regulate their immunity to viral infections.

MicroRNAs in placental health and disease.

MicroRNAs (miRNAs) constitute a large family of small noncoding RNAs that are encoded by the genomes of most organisms. They regulate gene expression through posttranscriptional mechanisms to attenuate protein output in various genetic networks. The discovery of miRNAs has transformed our understanding of gene regulation and sparked intense efforts intended to harness their potential as diagnostic markers and therapeutic tools. Over the last decade, a flurry of studies has shed light on placental miRNAs but has also raised many questions regarding the scope of their biologic action. Moreover, the recognition that miRNAs of placental origin are released continually in the maternal circulation throughout pregnancy suggested that circulating miRNAs might serve as biomarkers for placental function during pregnancy. Although this generated much enthusiasm, recently recognized challenges have delayed the application of miRNA-based biomarkers and therapeutics in clinical practice. In this review, we summarize key findings in the field and discuss current knowledge related to miRNAs in the context of placental biology.

Dronc caspase exerts a non-apoptotic function to restrain phospho-Numb-induced ectopic neuroblast formation in Drosophila.

Drosophila neuroblasts have served as a model to understand how the balance of stem cell self-renewal versus differentiation is achieved. Drosophila Numb protein regulates this process through its preferential segregation into the differentiating daughter cell. How Numb restricts the proliferation and self-renewal potentials of the recipient cell remains enigmatic. Here, we show that phosphorylation at conserved sites regulates the tumor suppressor activity of Numb. Enforced expression of a phospho-mimetic form of Numb (Numb-TS4D) or genetic manipulation that boosts phospho-Numb levels, attenuates endogenous Numb activity and causes ectopic neuroblast formation (ENF). This effect on neuroblast homeostasis occurs only in the type II neuroblast lineage. We identify Dronc caspase as a novel binding partner of Numb, and demonstrate that overexpression of Dronc suppresses the effects of Numb-TS4D in a non-apoptotic and possibly non-catalytic manner. Reduction of Dronc activity facilitates ENF induced by phospho-Numb. Our findings uncover a molecular mechanism that regulates Numb activity and suggest a novel role for Dronc caspase in regulating neural stem cell homeostasis.

High-yield and rapid isolation of extracellular vesicles by flocculation via orbital acoustic trapping: FLOAT.

Extracellular vesicles (EVs) have been identified as promising biomarkers for the noninvasive diagnosis of various diseases. However, challenges in separating EVs from soluble proteins have resulted in variable EV recovery rates and low purities. Here, we report a high-yield ( > 90%) and rapid ( < 10 min) EV isolation method called FLocculation via Orbital Acoustic Trapping (FLOAT). The FLOAT approach utilizes an acoustofluidic droplet centrifuge to rotate and controllably heat liquid droplets. By adding a thermoresponsive polymer flocculant, nanoparticles as small as 20 nm can be rapidly and selectively concentrated at the center of the droplet. We demonstrate the ability of FLOAT to separate urinary EVs from the highly abundant Tamm-Horsfall protein, addressing a significant obstacle in the development of EV-based liquid biopsies. Due to its high-yield nature, FLOAT reduces biofluid starting volume requirements by a factor of 100 (from 20 mL to 200 µL), demonstrating its promising potential in point-of-care diagnostics.

Polo inhibits progenitor self-renewal and regulates Numb asymmetry by phosphorylating Pon.

Self-renewal and differentiation are cardinal features of stem cells. Asymmetric cell division provides one fundamental mechanism by which stem cell self-renewal and differentiation are balanced. A failure of this balance could lead to diseases such as cancer. During asymmetric division of stem cells, factors controlling their self-renewal and differentiation are unequally segregated between daughter cells. Numb is one such factor that is segregated to the differentiating daughter cell during the stem-cell-like neuroblast divisions in Drosophila melanogaster, where it inhibits self-renewal. The localization and function of Numb is cell-cycle-dependent. Here we show that Polo (ref. 13), a key cell cycle regulator, the mammalian counterparts of which have been implicated as oncogenes as well as tumour suppressors, acts as a tumour suppressor in the larval brain. Supernumerary neuroblasts are produced at the expense of neurons in polo mutants. Polo directly phosphorylates Partner of Numb (Pon, ref. 16), an adaptor protein for Numb, and this phosphorylation event is important for Pon to localize Numb. In polo mutants, the asymmetric localization of Pon, Numb and atypical protein kinase C are disrupted, whereas other polarity markers are largely unaffected. Overexpression of Numb suppresses neuroblast overproliferation caused by polo mutations, suggesting that Numb has a major role in mediating this effect of Polo. Our results reveal a biochemical link between the cell cycle and the asymmetric protein localization machinery, and indicate that Polo can inhibit progenitor self-renewal by regulating the localization and function of Numb.

The impact of opioids on the transcriptional landscape of human villous trophoblasts.

INTRODUCTION: Opioid use disorder (OUD) is implicated in major obstetrical diseases such as fetal growth restriction. Whether or not opioids directly impact placental trophoblast development and function remains unclear. We sought to examine the expression of opioid receptors (OPRs) in villous trophoblasts and the effect of opioids on placental transcriptomics. METHODS: Trophoblast stem (TS) cells and primary human trophoblast (PHT) cells from healthy term placentas were used to assess OPR expression in conditions that enhance trophoblast stemness vs differentiation. Placental RNAseq was conducted using our retrospective cohorts of pregnant people with OUD vs controls, both without major obstetrical complications. RT-qPCR was used to determine the effect of fentanyl on the expression of putative opioid targets and stemness or differentiation-associated genes in TS and PHT cells. RESULTS: Three main OPRs, including OPRM1, OPRD1, and OPRK1 were expressed in term PHT cells cultured in the stemness medium, whereas only OPRD1 and OPRK1 were expressed in TS cells. Interestingly, upon induction of differentiation, the expressed OPR mRNAs in TS or in PHT cells were downregulated. We found 286 differentially expressed long RNAs in placentas from the OUD participants vs controls. While three putative opioid targets differed their expression in stemness vs differentiation states of trophoblasts, fentanyl had no effect on their expression or the expression of major stemness or differentiation-relevant genes in TS and PHT cells. DISCUSSION: Trophoblastic expression of OPRs and opioid RNA targets is impacted by cell differentiation, suggesting differential susceptibility of villous trophoblasts to the effect of opioids.

Defining trophoblast injury patterns in the transcriptomes of dysfunctional placentas.

Trophoblast injury is central to clinically relevant placenta dysfunction. We hypothesized that the mRNA of primary human trophoblasts, exposed to distinct injuries in vitro, capture transcriptome patterns of placental biopsies obtained from common obstetrical syndromes. We deployed a CIBERSORTx deconvolution method to correlate trophoblastic RNAseq-based expression matrices with the transcriptome of omics-defined placental dysfunction patterns in vivo. We found distinct trophoblast injury patterns in placental biopsies from women with fetal growth restriction and a hypertensive disorder, or in biopsies clustered by their omics analysis. Our RNAseq data are useful for defining the contribution of trophoblast injuries to placental dysfunction syndromes.

The lipase cofactor CGI58 controls placental lipolysis.

In eutherians, the placenta plays a critical role in the uptake, storage, and metabolism of lipids. These processes govern the availability of fatty acids to the developing fetus, where inadequate supply has been associated with substandard fetal growth. Whereas lipid droplets are essential for the storage of neutral lipids in the placenta and many other tissues, the processes that regulate placental lipid droplet lipolysis remain largely unknown. To assess the role of triglyceride lipases and their cofactors in determining placental lipid droplet and lipid accumulation, we assessed the role of patatin like phospholipase domain containing 2 (PNPLA2) and comparative gene identification-58 (CGI58) in lipid droplet dynamics in the human and mouse placenta. While both proteins are expressed in the placenta, the absence of CGI58, not PNPLA2, markedly increased placental lipid and lipid droplet accumulation. These changes were reversed upon restoration of CGI58 levels selectively in the CGI58-deficient mouse placenta. Using co-immunoprecipitation, we found that, in addition to PNPLA2, PNPLA9 interacts with CGI58. PNPLA9 was dispensable for lipolysis in the mouse placenta yet contributed to lipolysis in human placental trophoblasts. Our findings establish a crucial role for CGI58 in placental lipid droplet dynamics and, by extension, in nutrient supply to the developing fetus.

Assessing hypoxic damage to placental trophoblasts by measuring membrane viscosity of extracellular vesicles.

INTRODUCTION: As highly sophisticated intercellular communication vehicles in biological systems, extracellular vesicles (EVs) have been investigated as both promising liquid biopsy-based disease biomarkers and drug delivery carriers. Despite tremendous progress in understanding their biological and physiological functions, mechanical characterization of these nanoscale entities remains challenging due to the limited availability of proper techniques. Especially, whether damage to parental cells can be reflected by the mechanical properties of their EVs remains unknown. METHODS: In this study, we characterized membrane viscosities of different types of EVs collected from primary human trophoblasts (PHTs), including apoptotic bodies, microvesicles and small extracellular vesicles, using fluorescence lifetime imaging microscopy (FLIM). The biochemical origin of EV membrane viscosity was examined by analyzing their phospholipid composition, using mass spectrometry. RESULTS: We found that different EV types derived from the same cell type exhibit different membrane viscosities. The measured membrane viscosity values are well supported by the lipidomic analysis of the phospholipid compositions. We further demonstrate that the membrane viscosity of microvesicles can faithfully reveal hypoxic injury of the human trophoblasts. More specifically, the membrane of PHT microvesicles released under hypoxic condition is less viscous than its counterpart under standard culture condition, which is supported by the reduction in the phosphatidylethanolamine-to-phosphatidylcholine ratio in PHT microvesicles. DISCUSSION: Our study suggests that biophysical properties of released trophoblastic microvesicles can reflect cell health. Characterizing EV's membrane viscosity may pave the way for the development of new EV-based clinical applications.

Small extracellular vesicles from plasma of women with preeclampsia increase myogenic tone and decrease endothelium-dependent relaxation of mouse mesenteric arteries.

Preeclampsia (PE) is a common syndrome of pregnancy, characterized by new-onset hypertension and proteinuria after gestational week 20, or new onset of hypertension and significant end-organ dysfunction. In the worst cases, it can threaten the survival of both mother and baby. Extracellular vesicles (EVs) are lipid-bilayer nanoparticles released from cells. They are involved in cell-cell communication and transport of diverse cargo molecules. Small extracellular vesicles (sEVs, exosomes) are defined by their size and biogenesis within the endocytic compartment of the cell or reverse budding of the plasma membrane. The function of circulating gestational EVs, released from maternal organs or the placenta, remains to be explored. Here, we focused on sEVs that circulate in the maternal blood in the third trimester of human pregnancy and hypothesized that sEVs from pregnant women with PE play a role in regulation of vessel tone. When compared to sEVs from women with uncomplicated pregnancies, ex vivo exposure of isolated mouse mesenteric arteries to sEVs purified from the plasma of pregnant women with PE led to constriction in response to intraluminal pressure. This effect was not observed using microvesicles from the plasma of women with PE or using PE plasma that was depleted of EVs. Blood vessels exposed to sEVs from women with PE were also more resistant to methacholine-stimulated relaxation. Immunofluorescence microscopy confirmed the presence of sEVs within the vessel wall. Together, these data support the notion that circulating sEVs from pregnant women play a role in the regulation of arterial tone.

Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery.

Mitochondria form dynamic tubular networks that undergo frequent morphological changes through fission and fusion, the imbalance of which can affect cell survival in general and impact synaptic transmission and plasticity in neurons in particular. Some core components of the mitochondrial fission/fusion machinery, including the dynamin-like GTPases Drp1, Mitofusin, Opa1, and the Drp1-interacting protein Fis1, have been identified. How the fission and fusion processes are regulated under normal conditions and the extent to which defects in mitochondrial fission/fusion are involved in various disease conditions are poorly understood. Mitochondrial malfunction tends to cause diseases with brain and skeletal muscle manifestations and has been implicated in neurodegenerative diseases such as Parkinson's disease (PD). Whether abnormal mitochondrial fission or fusion plays a role in PD pathogenesis has not been shown. Here, we show that Pink1, a mitochondria-targeted Ser/Thr kinase linked to familial PD, genetically interacts with the mitochondrial fission/fusion machinery and modulates mitochondrial dynamics. Genetic manipulations that promote mitochondrial fission suppress Drosophila Pink1 mutant phenotypes in indirect flight muscle and dopamine neurons, whereas decreased fission has opposite effects. In Drosophila and mammalian cells, overexpression of Pink1 promotes mitochondrial fission, whereas inhibition of Pink1 leads to excessive fusion. Our genetic interaction results suggest that Fis1 may act in-between Pink1 and Drp1 in controlling mitochondrial fission. These results reveal a cell biological role for Pink1 and establish mitochondrial fission/fusion as a paradigm for PD research. Compounds that modulate mitochondrial fission/fusion could have therapeutic value in PD intervention.

Trophoblastic extracellular vesicles and viruses: Friends or foes?

Cells produce cytoplasmic vesicles to facilitate the processing and transport of RNAs, proteins, and other signaling molecules among intracellular organelles. Moreover, most cells release a range of extracellular vesicles (EVs) that mediate intercellular communication in both physiological and pathological settings. In addition to a better understanding of their biological functions, the diagnostic and therapeutic prospects of EVs, particularly the nano-sized small EVs (sEVs, exosomes), are currently being rigorously pursued. While EVs and viruses such as retroviruses might have evolved independently, they share a number of similar characteristics, including biogenesis pathways, size distribution, cargo, and cell-targeting mechanisms. The interplay of EVs with viruses has profound effects on viral replication and infectivity. Our research indicates that sEVs, produced by primary human trophoblasts, can endow other non-placental cell types with antiviral response. Better insights into the interaction of EVs with viruses may illuminate new ways to attenuate viral infections during pregnancy, and perhaps develop new antiviral therapeutics to protect the feto-placental unit during critical times of human development.

RNA Network Interactions During Differentiation of Human Trophoblasts.

In the human placenta, two trophoblast cell layers separate the maternal blood from the villous basement membrane and fetal capillary endothelial cells. The inner layer, which is complete early in pregnancy and later becomes discontinuous, comprises the proliferative mononuclear cytotrophoblasts, which fuse together and differentiate to form the outer layer of multinucleated syncytiotrophoblasts. Because the syncytiotrophoblasts are responsible for key maternal-fetal exchange functions, tight regulation of this differentiation process is critical for the proper development and the functional role of the placenta. The molecular mechanisms regulating the fusion and differentiation of trophoblasts during human pregnancy remain poorly understood. To decipher the interactions of non-coding RNAs (ncRNAs) in this process, we exposed cultured primary human trophoblasts to standard in vitro differentiation conditions or to conditions known to hinder this differentiation process, namely exposure to hypoxia (O2 < 1%) or to the addition of dimethyl sulfoxide (DMSO, 1.5%) to the culture medium. Using next generation sequencing technology, we analyzed the differential expression of trophoblastic lncRNAs, miRNAs, and mRNAs that are concordantly modulated by both hypoxia and DMSO. Additionally, we developed a model to construct a lncRNA-miRNA-mRNA co-expression network and inferred the functions of lncRNAs and miRNAs via indirect gene ontology analysis. This study improves our knowledge of the interactions between ncRNAs and mRNAs during trophoblast differentiation and identifies key biological processes that may be impaired in common gestational diseases, such as fetal growth restriction or preeclampsia.

Extracellular vesicles promote transkingdom nutrient transfer during viral-bacterial co-infection.

Extracellular vesicles (EVs) are increasingly appreciated as a mechanism of communication among cells that contribute to many physiological processes. Although EVs can promote either antiviral or proviral effects during viral infections, the role of EVs in virus-associated polymicrobial infections remains poorly defined. We report that EVs secreted from airway epithelial cells during respiratory viral infection promote secondary bacterial growth, including biofilm biogenesis, by Pseudomonas aeruginosa. Respiratory syncytial virus (RSV) increases the release of the host iron-binding protein transferrin on the extravesicular face of EVs, which interact with P. aeruginosa biofilms to transfer the nutrient iron and promote bacterial biofilm growth. Vesicular delivery of iron by transferrin more efficiently promotes P. aeruginosa biofilm growth than soluble holo-transferrin delivered alone. Our findings indicate that EVs are a nutrient source for secondary bacterial infections in the airways during viral infection and offer evidence of transkingdom communication in the setting of polymicrobial infections.

Term Human Placental Trophoblasts Express SARS-CoV-2 Entry Factors ACE2, TMPRSS2, and Furin.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has had a massive impact on human lives worldwide. While the airborne SARS-CoV-2 primarily affects the lungs, viremia is not uncommon. As placental trophoblasts are directly bathed in maternal blood, they are vulnerable to SARS-CoV-2. Intriguingly, the human fetus is largely spared from SARS-CoV-2 infection. We tested whether the human placenta expresses the main SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and furin and showed that ACE2 and TMPRSS2 are expressed in the trophoblast rather than in other placental villous cells. While furin is expressed in the main placental villous cell types, we surveyed, trophoblasts exhibit the highest expression. In line with the expression of these entry factors, we demonstrated that a SARS-CoV-2 pseudovirus could enter primary human trophoblasts. Mechanisms underlying placental defense against SARS-CoV-2 infection likely involve postentry processing, which may be germane for mitigating interventions against SARS-CoV-2.IMPORTANCE Pregnant women worldwide have been affected by COVID-19. As the virus is commonly spread to various organs via the bloodstream and because human placental trophoblasts are directly bathed in maternal blood, feto-placental infection by SARS-CoV-2 seems likely. However, despite the heightened risk to pregnant women, thus far the transmission risk of COVID-19 to the feto-placental unit seems extremely low. This has been recently attributed to a negligible expression of SARS-CoV-2 entry factors in the human placenta. We therefore sought to explore the expression of the entry factors ACE2 and TMPRSS2 in the different cell types of human placental villi. Using a combination of transcriptome sequencing (RNA-seq), real-time quantitative PCR (RT-qPCR), in situ hybridization, and immunofluorescence, we found that trophoblasts, but not the other main villous cell types, express ACE2 and TMPRSS2, with a broad expression of furin. Correspondingly, we also showed that primary human trophoblasts are permissive to entry of SARS-CoV-2 pseudovirus particles.

Acoustofluidic centrifuge for nanoparticle enrichment and separation.

Liquid droplets have been studied for decades and have recently experienced renewed attention as a simplified model for numerous fascinating physical phenomena occurring on size scales from the cell nucleus to stellar black holes. Here, we present an acoustofluidic centrifugation technique that leverages an entanglement of acoustic wave actuation and the spin of a fluidic droplet to enable nanoparticle enrichment and separation. By combining acoustic streaming and droplet spinning, rapid (<1 min) nanoparticle concentration and size-based separation are achieved with a resolution sufficient to identify and isolate exosome subpopulations. The underlying physical mechanisms have been characterized both numerically and experimentally, and the ability to process biological samples (including DNA segments and exosome subpopulations) has been successfully demonstrated. Together, this acoustofluidic centrifuge overcomes existing limitations in the manipulation of nanoscale (<100 nm) bioparticles and can be valuable for various applications in the fields of biology, chemistry, engineering, material science, and medicine.