Profiling DNA Cargos in Single Extracellular Vesicles via Hydrogel-Based Droplet Digital Multiple Displacement Amplification.

Due to the substantial heterogeneity among extracellular vesicle (EV) subpopulations, single-EV analysis has the potential to elucidate the mechanisms behind EV biogenesis and shed light on the myriad functions, leading to the development of novel diagnostics and therapeutics. While many studies have been devoted to reveal between-EV variations in surface proteins and RNAs, DNA cargos (EV-DNA) have received little attention. Here, we report a hydrogel-based droplet digital multiple displacement amplification approach for the comprehensive analysis of EV-DNA at the single-EV level. Single EVs are dispersed in thousands of hydrogel droplets and lysed for DNA amplification and identification. The droplet microfluidics strategy empowers the assay with single-molecule sensitivity and capability for absolute quantification of DNA-containing EVs. In particular, our findings indicate that 5-40% EVs are associated with DNA, depending on the cell of origin. Large EVs exhibit a higher proportion of DNA-containing EVs and a more substantial presence of intraluminal DNA, compared to small EVs. These DNA-containing EVs carry multiple DNA fragments on average. Furthermore, both double-stranded DNA and single-stranded DNA were able to be detected at the single-EV level. Utilizing this method, the abundance, distribution, and biophysical properties of EV-DNA in various EV populations are evaluated. The DNA level within EVs provides insight into the status of the originating cells and offers valuable information on the outcomes of anticancer treatments. The utilization of single-EV analysis for EV-DNA holds significant promise for early cancer detection and treatment response monitoring.

Extruded small extracellular vesicles: splinters of circulating tumour cells may promote cancer metastasis?

We speculate ruptured circulating tumour cells (CTC) in capillaries could release a large number of small extracellular vesicle-like vesicles, namely mechanically extruded sEV (sEVme), which can encapsulate chromosomal DNA fragments. These sEVme have similar physicochemical properties compared to small extracellular vesicles spontaneously secreted by living cells (sEVss), and thus sEVme and sEVss cannot be effectively distinguished based on their size or membrane protein markers. Meanwhile, these sEVme derived from CTC inherit oncogenic payloads, deliver cargo through the bloodstream to recipient cells, and thus may promote cancer metastasis. The validation of this speculation could facilitate our understanding of EV biogenesis and cancer pathology. The potential finding will also provide a theoretical foundation for burgeoning liquid biopsy using DNA fragments derived from harvested sEV.

Self-Assembly of Extracellular Vesicle-like Metal-Organic Framework Nanoparticles for Protection and Intracellular Delivery of Biofunctional Proteins.

The intracellular delivery of biofunctional enzymes or therapeutic proteins through systemic administration is of great importance in therapeutic intervention of various diseases. However, current strategies face substantial challenges owing to various biological barriers, including susceptibility to protein degradation and denaturation, poor cellular uptake, and low transduction efficiency into the cytosol. Here, we developed a biomimetic nanoparticle platform for systemic and intracellular delivery of proteins. Through a biocompatible strategy, guest proteins are caged in the matrix of metal-organic frameworks (MOFs) with high efficiency (up to ∼94%) and high loading content up to ∼50 times those achieved by surface conjunction, and the nanoparticles were further decorated with the extracellular vesicle (EV) membrane with an efficiency as high as ∼97%. In vitro and in vivo study manifests that the EV-like nanoparticles can not only protect proteins against protease digestion and evade the immune system clearance but also selectively target homotypic tumor sites and promote tumor cell uptake and autonomous release of the guest protein after internalization. Assisted by biomimetic nanoparticles, intracellular delivery of the bioactive therapeutic protein gelonin significantly inhibits the tumor growth in vivo and increased 14-fold the therapeutic efficacy. Together, our work not only proposes a new concept to construct a biomimetic nanoplatform but also provides a new solution for systemic and intracellular delivery of protein.

Nucleus of Circulating Tumor Cell Determines Its Translocation Through Biomimetic Microconstrictions and Its Physical Enrichment by Microfiltration.

The mechanism of cells passing through microconstrictions, such as capillaries and endothelial junctions, influences metastasis of circulating tumor cells (CTCs) in vivo, as well as size-based enrichment of CTCs in vitro. However, very few studies observe such translocation of microconstrictions in real time, and thus the inherent biophysical mechanism is poorly understood. In this study, a multiplexed microfluidic device is fabricated for real-time tracking of cell translocation under physiological pressure and recording deformation of the whole cell and nucleus, respectively. It is found that the deformability and size of the nucleus instead of the whole cell dominate cellular translocation through microconstrictions under a normal physiological pressure range. More specifically, cells with a large and stiff nucleus are prone to be blocked by relatively small constrictions. The same phenomenon is also observed in the size-based enrichment of CTCs from peripheral blood of metastatic cancer patients. These findings are different from a popular viewpoint that the size and deformability of a whole cell mainly determine cell translation through microconstrictions, and thus may elucidate interactions between CTCs and capillaries from a new perspective and guide the rational design of size-based microfilters for rare cell enrichment.

A Spontaneous 3D Bone-On-a-Chip for Bone Metastasis Study of Breast Cancer Cells.

Bone metastasis occurs at ≈70% frequency in metastatic breast cancer. The mechanisms used by tumors to hijack the skeleton, promote bone metastases, and confer therapeutic resistance are poorly understood. This has led to the development of various bone models to investigate the interactions between cancer cells and host bone marrow cells and related physiological changes. However, it is challenging to perform bone studies due to the difficulty in periodic sampling. Herein, a bone-on-a-chip (BC) is reported for spontaneous growth of a 3D, mineralized, collagenous bone tissue. Mature osteoblastic tissue of up to 85 µm thickness containing heavily mineralized collagen fibers naturally formed in 720 h without the aid of differentiation agents. Moreover, co-culture of metastatic breast cancer cells is examined with osteoblastic tissues. The new bone-on-a-chip design not only increases experimental throughput by miniaturization, but also maximizes the chances of cancer cell interaction with bone matrix of a concentrated surface area and facilitates easy, frequent observation. As a result, unique hallmarks of breast cancer bone colonization, previously confirmed only in vivo, are observed. The spontaneous 3D BC keeps the promise as a physiologically relevant model for the in vitro study of breast cancer bone metastasis.

Label-Free Virus Capture and Release by a Microfluidic Device Integrated with Porous Silicon Nanowire Forest.

Viral diseases are perpetual threats to human and animal health. Detection and characterization of viral pathogens require accurate, sensitive, and rapid diagnostic assays. For field and clinical samples, the sample preparation procedures limit the ultimate performance and utility of the overall virus diagnostic protocols. This study presents the development of a microfluidic device embedded with porous silicon nanowire (pSiNW) forest for label-free size-based point-of-care virus capture in a continuous curved flow design. The pSiNW forests with specific interwire spacing are synthesized in situ on both bottom and sidewalls of the microchannels in a batch process. With the enhancement effect of Dean flow, this study demonstrates that about 50% H5N2 avian influenza viruses are physically trapped without device clogging. A unique feature of the device is that captured viruses can be released by inducing self-degradation of the pSiNWs in physiological aqueous environment. About 60% of captured viruses can be released within 24 h for virus culture, subsequent molecular diagnosis, and other virus characterization and analyses. This device performs viable, unbiased, and label-free virus isolation and release. It has great potentials for virus discovery, virus isolation and culture, functional studies of virus pathogenicity, transmission, drug screening, and vaccine development.

A Nanostructured Microfluidic Immunoassay Platform for Highly Sensitive Infectious Pathogen Detection.

Rapid and simultaneous detection of multiple potential pathogens by portable devices can facilitate early diagnosis of infectious diseases, and allow for rapid and effective implementation of disease prevention and treatment measures. The development of a ZnO nanorod integrated microdevice as a multiplex immunofluorescence platform for highly sensitive and selective detection of avian influenza virus (AIV) is described. The 3D morphology and unique optical property of the ZnO nanorods boost the detection limit of the H5N2 AIV to as low as 3.6 × 103 EID50 mL-1 (EID50 : 50% embryo infectious dose), which is ≈22 times more sensitive than conventional enzyme-linked immunosorbent assay. The entire virus capture and detection process could be completed within 1.5 h with excellent selectivity. Moreover, this microfluidic biosensor is capable of detecting multiple viruses simultaneously by spatial encoding of capture antibodies. One prominent feature of the device is that the captured H5N2 AIV can be released by simply dissolving ZnO nanorods under slightly acidic environment for subsequent off-chip analyses. As a whole, this platform provides a powerful tool for rapid detection of multiple pathogens, which may extent to the other fields for low-cost and convenient biomarker detection.

On-demand one-step synthesis of monodisperse functional polymeric microspheres with droplet microfluidics.

A simple and robust method for one-step synthesis of monodisperse functional polymeric microspheres was established by generation of reversed microemulsion droplets in aqueous phase inside microfluidic chips and controlled evaporation of the organic solvent. Using this method, water-soluble nanomaterials can be easily encapsulated into biodegradable Poly(D,L-lactic-co-glycolic acid) (PLGA) to form functional microspheres. By controlling the flow rate of microemulsion phase, PLGA polymeric microspheres with narrow size distribution and diameters in the range of ∼50-100 µm were obtained. As a demonstration of the versatility of the approach, high-quality fluorescent CdTe:Zn(2+) quantum dots (QDs) of various emission spectra, superparamagnetic Fe3O4 nanoparticles, and water-soluble carbon nanotubes (CNTs) were used to synthesize fluorescent PLGA@QDs, magnetic PLGA@Fe3O4, and PLGA@CNTs polymeric microspheres, respectively. In order to show specific applications, the PLGA@Fe3O4 were modified with polydopamine (PDA), and then the silver nanoparticles grew on the surfaces of the PLGA@Fe3O4@PDA polymeric microspheres by reducting the Ag(+) to Ag(0). The as-prepared PLGA@Fe3O4@PDA-Ag microspheres showed a highly efficient catalytic reduction of the 4-nitrophenol, a highly toxic substance. The monodisperse uniform functional PLGA polymeric microspheres can potentially be critically important for multiple biomedical applications.

Flexible micro spring array device for high-throughput enrichment of viable circulating tumor cells.

BACKGROUND: The dissemination of circulating tumor cells (CTCs) that cause metastases in distant organs accounts for the majority of cancer-related deaths. CTCs have been established as a cancer biomarker of known prognostic value. The enrichment of viable CTCs for ex vivo analysis could further improve cancer diagnosis and guide treatment selection. We designed a new flexible micro spring array (FMSA) device for the enrichment of viable CTCs independent of antigen expression. METHODS: Unlike previous microfiltration devices, flexible structures at the micro scale minimize cell damage to preserve viability, while maximizing throughput to allow rapid enrichment directly from whole blood with no need for sample preprocessing. Device performance with respect to capture efficiency, enrichment against leukocytes, viability, and proliferability was characterized. CTCs and CTC microclusters were enriched from clinical samples obtained from breast, lung, and colorectal cancer patients. RESULTS: The FMSA device enriched tumor cells with 90% capture efficiency, higher than 10(4) enrichment, and better than 80% viability from 7.5-mL whole blood samples in <10 min on a 0.5-cm(2) device. The FMSA detected at least 1 CTC in 16 out of 21 clinical samples (approximately 76%) compared to 4 out of 18 (approximately 22%) detected with the commercial CellSearch® system. There was no incidence of clogging in over 100 tested fresh whole blood samples. CONCLUSIONS: The FMSA device provides a versatile platform capable of viable enrichment and analysis of CTCs from clinically relevant volumes of whole blood.

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.

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 reported 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 MCAo mice support the continued use of mBEC-EVs to optimize the therapeutic potential of mitochondria-containing EVs in preclinical mouse models.

Nanoscale coordination polymers enabling antioxidants inhibition for enhanced chemodynamic therapy.

Reactive oxygen species (ROS) generation to induce cell death is an effective strategy for cancer therapy. In particular, chemodynamic therapy (CDT), using Fenton-type reactions to generate highly cytotoxic hydroxyl radical (•OH), is a promising treatment modality. However, the therapeutic efficacy of ROS-based cancer treatment is still limited by some critical challenges, such as overexpression of enzymatic and non-enzymatic antioxidants by tumor cells, as well as the low tumor targeting efficiency of therapeutic agents. To address those problems, biomimetic CuZn protoporphyrin IX nanoscale coordination polymers have been developed, which significantly amplify oxidative stress against tumors by simultaneously inhibiting enzymatic and non-enzymatic antioxidants and initiating the CDT. In this design, cancer cell membrane camouflaged nanoparticle exhibits an excellent homotypic targeting effect. After being endocytosed into tumor cells, the nanoparticles induce depletion of the main non-enzymatic antioxidant glutathione (GSH) by undergoing a redox reaction with GSH. Afterward, the redox reaction generated cuprous ion (Cu+) works as a CDT agent for •OH generation. Furthermore, the released Zn protoporphyrin IX strongly inhibits the activity of the typical enzymatic antioxidant heme oxygenase-1. This tetra-modal synergistic strategy endows the biomimetic nanoparticles with great capability for anticancer therapy, which has been demonstrated in both in vitro and in vivo studies.

An implantable Fabry-Pérot pressure sensor fabricated on left ventricular assist device for heart failure.

Continuous flow left ventricular assist devices (LVADs) are commonly used as bridge-to-transplantation or destination therapy for heart failure patients. However, non-optimal pumping speeds can reduce the efficacy of circulatory support or cause dangerous ventricular arrhythmias. Optimal flow control for continuous flow LVADs has not been defined and calls for an implantable pressure sensor integrated with the LVAD for real-time feedback control of pump speed based on ventricular pressure. A MEMS pressure sensor prototype is designed, fabricated and seamlessly integrated with LVAD to enable real-time control, optimize its performance and reduce its risks. The pressure sensing mechanism is based on Fabry-Pérot interferometer principle. A biocompatible parylene diaphragm with a silicon mirror at the center is fabricated directly on the inlet shell of the LVAD to sense pressure changes. The sensitivity, range and response time of the pressure sensor are measured and validated to meet the requirements of LVAD pressure sensing.

Affinity-Based Enrichment of Extracellular Vesicles with Lipid Nanoprobes.

Extracellular vesicles (EVs) are lipid-bilayer-enclosed vesicles with sub-micrometer size that are released by various cells. EVs contain a tissue-specific signature wherein a variety of proteins and nucleic acids are selectively packaged. Growing evidence has shown important biological roles and clinical relevance of EVs in diseases. For EV-related studies to thrive, rapid efficient isolation of pure EVs is a prerequisite. However, lengthy procedure, low yield, low throughput, and high contaminants stemmed from existing isolation approaches hamper both basic research and large-scale clinical implementation. We have shown that lipid nanoprobes (LNP) enable spontaneous labeling and rapid isolation of EVs by coupling with magnetic enrichment. Recently, we further developed a one-step EV isolation platform that utilizes EV size-matched silica nanostructures and surface-conjugated LNPs with an integrated microfluidic mixer. EVs, derived from up to 2-ml clinical plasma, can be processed with this point-of-care device using optimized flow rate. Subsequently, contents of isolated EVs can be extracted on-chip and eluted from the device for downstream molecular analyses. The LNP-functionalized microfluidic device combined with state-of-the-art analysis platforms could have great potential in promoting EV-centered research and clinical use in the future.

Vertically Aligned Carbon Nanotubes as a Unique Material for Biomedical Applications.

Vertically aligned carbon nanotubes (VACNTs), a unique classification of CNT, highly oriented and normal to the respective substrate, have been heavily researched over the last two decades. Unlike randomly oriented CNT, VACNTs have demonstrated numerous advantages making it an extremely desirable nanomaterial for many biomedical applications. These advantages include better spatial uniformity, increased surface area, greater susceptibility to functionalization, improved electrocatalytic activity, faster electron transfer, higher resolution in sensing, and more. This Review discusses VACNT and its utilization in biomedical applications particularly for sensing, biomolecule filtration systems, cell stimulation, regenerative medicine, drug delivery, and bacteria inhibition. Furthermore, comparisons are made between VACNT and its traditionally nonaligned, randomly oriented counterpart. Thus, we aim to provide a better understanding of VACNT and its potential applications within the community and encourage its utilization in the future.

An Ultrafast One-Step Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Detection of SARS-CoV-2.

We developed an ultrafast one-step RT-qPCR assay for SARS-CoV-2 detection, which can be completed in only 30 min on benchtop Bio-Rad CFX96. The assay significantly reduces the running time of conventional RT-qPCR: reduced RT step from 10 to 1 min, and reduced the PCR cycle of denaturation from 10 to 1 s and extension from 30 to 1 s. A cohort of 60 nasopharyngeal swab samples testing showed that the assay had a clinical sensitivity of 100% and a clinical specificity of 100%.

The Role of Extracellular Vesicles in the Pathogenesis and Treatment of Autoimmune Disorders.

Extracellular vesicles (EVs) are important players in autoimmune diseases, both in disease pathogenesis and as potential treatments. EVs can transport autoimmune triggers throughout the body, facilitating the process of antigen presentation. Understanding the link between cellular stress and EV biogenesis and intercellular trafficking will advance our understanding of autoimmune diseases. In addition, EVs can also be effective treatments for autoimmune diseases. The diversity of cell types that produce EVs leads to a wide range of molecules to be present in EVs, and thus EVs have a wide range of physiological effects. EVs derived from dendritic cells or mesenchymal stem cells have been shown to reduce inflammation. Since many autoimmune treatments are focused only on symptom management, EVs present a promising avenue for potential treatments. This review looks at the different roles EVs can play in autoimmune diseases, from disease pathology to diagnosis and treatment. We also overview various methodologies in isolating or generating EVs and look to the future for possible applications of EVs in autoimmune diseases.

Microfluidics in Single-Cell Virology: Technologies and Applications.

Microfluidics has proven to be a powerful tool for probing biology at the single-cell level. However, it is only in the past 5 years that single-cell microfluidics has been used in the field of virology. An array of strategies based on microwells, microvalves, and droplets is now available for tracking viral infection dynamics, identifying cell subpopulations with particular phenotypes, as well as high-throughput screening. The insights into the virus-host interactions gained at the single-cell level are unprecedented and usually inaccessible by population-based experiments. Therefore, single-cell microfluidics, which opens new avenues for mechanism elucidation and development of antiviral therapeutics, would be a valuable tool for the study of viral pathogenesis.

Magnetically Driven Nanotransporter-Assisted Intracellular Delivery and Autonomous Release of Proteins.

Biofunctional proteins such as active enzymes and therapeutic proteins show tremendous promise in disease treatment. However, intracellular delivery of proteins is facing substantial challenges owing to their vulnerability to degradation and denaturation and the presence of various biological barriers such as their low membrane transport efficiency. Herein, we report a magnetically driven and redox-responsive nanotransporter (MRNT) for highly efficient intracellular delivery of biofunctional proteins. The MRNT has remarkably high cargo capacity, compared with that without nanoscale cargo compartments. We have demonstrated the directional and dynamic motion of the MRNT using both nanoparticle tracking analysis and magnetic driving evaluation. Moreover, the active MRNT can translocate into the cytosol and sense the reducing cytosolic environment to discharge protein cargoes autonomously. The internalization mechanism of the MRNT has been studied using endocytosis inhibitors. Under the magnetic drive, the MRNT can promote a protein transduction efficiency of over 95%, and the intracellular protein delivery by the active MRNT shows significantly higher (∼4 times) enzymatic activity and therapeutic efficiency than those achieved by the static ones. Our proof-of-concept study provides a valuable tool for intracellular protein transduction and contributes to biotechnology and protein therapeutics.

Extracellular Vesicles as Potential Biomarkers for Early Detection and Diagnosis of Pancreatic Cancer.

Pancreatic carcinoma (PC) is highly metastatic, and it tends to be detected at advanced stages. Identifying and developing biomarkers for early detection of PC is crucial for a potentially curative treatment. Extracellular vesicles (EVs) are bilayer lipid membrane-structured nanovesicles found in various human bodily fluids, and they play important roles in tumor biogenesis and metastasis. Cancer-derived EVs are enriched with DNA, RNA, protein, and lipid, and they have emerged as attractive diagnostic biomarkers for early detection of PC. In this article, we provided an overview of the cell biology of EVs and their isolation and analysis, and their roles in cancer pathogenesis and progression. Multiplatform analyses of plasma-based exosomes for genomic DNA, micro RNA, mRNA, circular RNA, and protein for diagnosis of PC were critically reviewed. Numerous lines of evidence demonstrate that liquid biopsy with analysis of EV-based biomarkers has variable performance for diagnosis of PC. Future investigation is indicated to optimize the methodology for isolating and analyzing EVs and to identify the combination of EV-based biomarkers and other clinical datasets, with the goal of improving the predictive value, sensitivity, and specificity of screening tests for early detection and diagnosis of PC.