Joule heating and electroosmotic flow in cellular micro/nano electroporation.

Localized micro/nano-electroporation (MEP/NEP) shows tremendous potential in cell transfection with high cell viability, precise dose control, and good transfection efficacy. In MEP/NEP, micro or nanochannels are used to tailor the electric field distribution. Cells are positioned tightly by a micron or nanochannel, and the cargoes are delivered into the cell via the channel by electrophoresis (EP). Such confined geometries with micro and nanochannels are also widely used in sorting, isolation, and condensing of biomolecules and cells. Theoretical studies on the electrokinetic phenomena in these applications have been well established. However, for MEP/NEP applications, electrokinetic phenomena and their impact on the cell transfection efficiency and cell survival rate have not been studied comprehensively. In this work, we reveal the coupling between electric field, Joule heating, electroosmosis (EO), and EP in MEP/NEP at different channel sizes. A microfluidic biochip is used to investigate the electrokinetic phenomena in MEP/NEP on a single cell level. Bubble formation is observed at a threshold voltage due to Joule heating. The bubble is pushed to the cargo side due to EO and grows at the outlet of the nanochannel. As the voltage increases, the cargo transport efficiency decreases due to more intense EO, particularly for plasmid DNAs (3.5 kbp) with a low EP mobility. An 'electroporation zone' is defined for NEP/MEP systems with different channel sizes to avoid bubble formation and excessive EO velocity that may reduce the cargo delivery efficiency.

Extracellular Vesicular Analysis of Glypican 1 mRNA and Protein for Pancreatic Cancer Diagnosis and Prognosis.

Detecting pancreatic duct adenocarcinoma (PDAC) in its early stages and predicting late-stage patient prognosis undergoing chemotherapy is challenging. This work shows that the activation of specific oncogenes leads to elevated expression of mRNAs and their corresponding proteins in extracellular vesicles (EVs) circulating in blood. Utilizing an immune lipoplex nanoparticle (ILN) biochip assay, these findings demonstrate that glypican 1 (GPC1) mRNA expression in the exosomes-rich (Exo) EV subpopulation and GPC1 membrane protein (mProtein) expression in the microvesicles-rich (MV) EV subpopulation, particularly the tumor associated microvesicles (tMV), served as a viable biomarker for PDAC. A combined analysis effectively discriminated early-stage PDAC patients from benign pancreatic diseases and healthy donors in sizable clinical from multiple hospitals. Furthermore, among late-stage PDAC patients undergoing chemotherapy, lower GPC1 tMV-mProtein and Exo-mRNA expression before treatment correlated significantly with prolonged overall survival. These findings underscore the potential of vesicular GPC1 expression for early PDAC screenings and chemotherapy prognosis.

Engineering a tunable micropattern-array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ.

The molecular heterogeneity of extracellular vesicles (EVs) and the co-isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single-EV and particle (siEVP) protein and RNA assay (siEVP PRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The siEVP PRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single-particle resolution, the siEVP PRA outperformed the sensitivities of bulk-analysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross-validation. Despite the presence of single-EV-LP co-isolates in serum, the siEVP PRA detected GBM-associated vesicular RNA profiles in GBM patient siEVPs. The siEVP PRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.

Adaptive design of mRNA-loaded extracellular vesicles for targeted immunotherapy of cancer.

The recent success of mRNA therapeutics against pathogenic infections has increased interest in their use for other human diseases including cancer. However, the precise delivery of the genetic cargo to cells and tissues of interest remains challenging. Here, we show an adaptive strategy that enables the docking of different targeting ligands onto the surface of mRNA-loaded small extracellular vesicles (sEVs). This is achieved by using a microfluidic electroporation approach in which a combination of nano- and milli-second pulses produces large amounts of IFN-γ mRNA-loaded sEVs with CD64 overexpressed on their surface. The CD64 molecule serves as an adaptor to dock targeting ligands, such as anti-CD71 and anti-programmed cell death-ligand 1 (PD-L1) antibodies. The resulting immunogenic sEVs (imsEV) preferentially target glioblastoma cells and generate potent antitumour activities in vivo, including against tumours intrinsically resistant to immunotherapy. Together, these results provide an adaptive approach to engineering mRNA-loaded sEVs with targeting functionality and pave the way for their adoption in cancer immunotherapy applications.

A plasma miRNA-based classifier for small cell lung cancer diagnosis.

Introduction: Small cell lung cancer (SCLC) is characterized by poor prognosis and challenging diagnosis. Screening in high-risk smokers results in a reduction in lung cancer mortality, however, screening efforts are primarily focused on non-small cell lung cancer (NSCLC). SCLC diagnosis and surveillance remain significant challenges. The aberrant expression of circulating microRNAs (miRNAs/miRs) is reported in many tumors and can provide insights into the pathogenesis of tumor development and progression. Here, we conducted a comprehensive assessment of circulating miRNAs in SCLC with a goal of developing a miRNA-based classifier to assist in SCLC diagnoses. Methods: We profiled deregulated circulating cell-free miRNAs in the plasma of SCLC patients. We tested selected miRNAs on a training cohort and created a classifier by integrating miRNA expression and patients' clinical data. Finally, we applied the classifier on a validation dataset. Results: We determined that miR-375-3p can discriminate between SCLC and NSCLC patients, and between SCLC and Squamous Cell Carcinoma patients. Moreover, we found that a model comprising miR-375-3p, miR-320b, and miR-144-3p can be integrated with race and age to distinguish metastatic SCLC from a control group. Discussion: This study proposes a miRNA-based biomarker classifier for SCLC that considers clinical demographics with specific cut offs to inform SCLC diagnosis.

An immunogold single extracellular vesicular RNA and protein (Au SERP) biochip to predict responses to immunotherapy in non-small cell lung cancer patients.

Conventional PD-L1 immunohistochemical tissue biopsies only predict 20%-40% of non-small cell lung cancer (NSCLC) patients that will respond positively to anti-PD-1/PD-L1 immunotherapy. Herein, we present an immunogold biochip to quantify single extracellular vesicular RNA and protein (Au SERP) as a non-invasive alternative. With only 20 µl of purified serum, PD-1/PD-L1 proteins on the surface of extracellular vesicles (EVs) and EV PD-1/PD-L1 messenger RNA (mRNA) cargo were detected at a single-vesicle resolution and exceeded the sensitivities of their bulk-analysis conventional counterparts, ELISA and qRT-PCR, by 1000 times. By testing a cohort of 27 non-responding and 27 responding NSCLC patients, Au SERP indicated that the single-EV mRNA biomarkers surpass the single-EV protein biomarkers in predicting patient responses to immunotherapy. Dual single-EV PD-1/PD-L1 mRNA detection differentiated responders from non-responders with an accuracy of 72.2% and achieved an NSCLC diagnosis accuracy of 93.2%, suggesting the potential for Au SERP to provide enhanced immunotherapy predictions and cancer diagnoses within the clinical setting.

Phase 2 of extracellular RNA communication consortium charts next-generation approaches for extracellular RNA research.

The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013-2018), Phase 2 of the program (ERCC2, 2019-2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database. ERCC2 has established several Working Groups (Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, nomenclature, and Scientific Outreach) to promote collaboration between ERCC2 members and the broader scientific community. We expect that ERCC2's current and future achievements will significantly improve our understanding of exRNA biology and the development of accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays.

Microfluidic harvesting of breast cancer tumor spheroid-derived extracellular vesicles from immobilized microgels for single-vesicle analysis.

Investigating cellular and vesicular heterogeneity in breast cancer remains a challenge, which encourages the development of controllable in vitro systems that mimic the tumor microenvironment. Although three-dimensional cell culture better recapitulates the heterogeneity observed in tumor growth and extracellular vesicle (EV) biogenesis, the physiological relevance is often contrasted with the control offered by two-dimensional cell culture. Therefore, to challenge this misconception we developed a novel microfluidic system harboring highly tunable three-dimensional EV microbioreactors (EVµBRs) to model micrometastatic EV release in breast cancer while capitalizing on the convenient, low-volume, and sterile interface provided by microfluidics. The diameter and cellular occupancy of the EVµBRs could be precisely tailored to various configurations, supporting the formation of breast cancer tumor spheroids. To immobilize the EVµBRs within a microchannel and facilitate EV extraction, oxygen inhibition in free-radical polymerization was repurposed to rapidly generate two-layer hydrodynamic traps in situ using a digital-micromirror device (DMD)-based ultraviolet (UV) projection system. Breast cancer tumor spheroid-derived EVs were harvested with as little as 20 µL from the microfluidic system and quantified by single-EV immunofluorescence for CD63 and CD81. Despite the low-volume extraction, differences in biomarker expression and coexpression of the tetraspanins on single EVs were observed. Furthermore, the EVµBRs were capable of recapitulating heterogeneity at a cellular and vesicular degree, indicating the utility and robustness of the microfluidic system to investigate physiologically relevant EVs in breast cancer and other disease models.

Integrated therapy platform of exosomal system: hybrid inorganic/organic nanoparticles with exosomes for cancer treatment.

Recent studies have found that exosomes or extracellular vehicles (EVs) are associated with cancer metastasis, disease progression, diagnosis, and treatment, leading to a rapidly emerging area of exocrine vesicle research. Relying on the superior targeting function and bio-compatibility of exosomes, researchers have been able to deliver drugs to cancer stem cells deep within tumors in mouse models. Despite significant efforts made in this relatively new field of exosome research, progress has been held back by challenges such as inefficient separation methods, difficulties in characterization/tracking, and a lack of specific biomarkers. Therefore, current researches are devoted to combining nanomaterials with exosomes to improve these shortcomings. Adding inorganic/organic nanoparticles such as artificial liposomes and iron oxide can bring more drug options and various fluorescent or magnetic diagnostic possibilities to the exosome system. Moreover, the applications of exosomes need to be further evaluated under actual physiological conditions. This review article highlights the potential of exosome-biomimetic nanoparticles for their use as drug carriers to improve the efficacy of anticancer therapy.

Author Correction: Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation.

A Correction to this paper has been published: https://doi.org/10.1038/s41551-021-00725-w.

Immunomagnetic sequential ultrafiltration (iSUF) platform for enrichment and purification of extracellular vesicles from biofluids.

Extracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5-100 mL) can be significantly enriched (up to 1000 times), with up to 99% removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% ± 3.6%, 96.0% ± 2.0% and 94.0% ± 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads to target EV subpopulations. Serum from a cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).

On-chip multiplexed single-cell patterning and controllable intracellular delivery.

Conventional electroporation approaches show limitations in the delivery of macromolecules in vitro and in vivo. These limitations include low efficiency, noticeable cell damage and nonuniform delivery of cells. Here, we present a simple 3D electroporation platform that enables massively parallel single-cell manipulation and the intracellular delivery of macromolecules and small molecules. A pyramid pit micropore array chip was fabricated based on a silicon wet-etching method. A controllable vacuum system was adopted to trap a single cell on each micropore. Using this chip, safe single-cell electroporation was performed at low voltage. Cargoes of various sizes ranging from oligonucleotides (molecular beacons, 22 bp) to plasmid DNA (CRISPR-Cas9 expression vectors, >9 kb) were delivered into targeted cells with a significantly higher transfection efficiency than that of multiple benchmark methods (e.g., commercial electroporation devices and Lipofectamine). The delivered dose of the chemotherapeutic drug could be controlled by adjusting the applied voltage. By using CRISPR-Cas9 transfection with this system, the p62 gene and CXCR7 gene were knocked out in tumor cells, which effectively inhibited their cellular activity. Overall, this vacuum-assisted micropore array platform provides a simple, efficient, high-throughput intracellular delivery method that may facilitate on-chip cell manipulation, intracellular investigation and cancer therapy.

Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation.

Exosomes are attractive as nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and their ability to penetrate physiological barriers that are impermeable to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs, into cell-secreted exosomes leads to low yields. Here we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides. Compared with bulk electroporation and other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and a more than 103-fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic phosphatase and tensin homologue (PTEN)-deficient glioma mouse models, mRNA-containing exosomes restored tumour-suppressor function, enhanced inhibition of tumour growth and increased survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.

ROR1-targeted delivery of miR-29b induces cell cycle arrest and therapeutic benefit in vivo in a CLL mouse model.

Chronic lymphocytic leukemia (CLL) occurs in 2 major forms: aggressive and indolent. Low miR-29b expression in aggressive CLL is associated with poor prognosis. Indiscriminate miR-29b overexpression in the B-lineage of mice causes aberrance, thus warranting the need for selective introduction of miR-29b into B-CLL cells for therapeutic benefit. The oncofetal antigen receptor tyrosine kinase orphan receptor 1 (ROR1) is expressed on malignant B-CLL cells, but not normal B cells, encouraging us with ROR1-targeted delivery for therapeutic miRs. Here, we describe targeted delivery of miR-29b to ROR1+ CLL cells leading to downregulation of DNMT1 and DNMT3A, modulation of global DNA methylation, decreased SP1, and increased p21 expression in cell lines and primary CLL cells in vitro. Furthermore, using an Eµ-TCL1 mouse model expressing human ROR1, we report the therapeutic benefit of enhanced survival via cellular reprograming by downregulation of DNMT1 and DNMT3A in vivo. Gene expression profiling of engrafted murine leukemia identified reprogramming of cell cycle regulators with decreased SP1 and increased p21 expression after targeted miR-29b treatment. This finding was confirmed by protein modulation, leading to cell cycle arrest and survival benefit in vivo. Importantly, SP1 knockdown results in p21-dependent compensation of the miR-29b effect on cell cycle arrest. These studies form a basis for leukemic cell-targeted delivery of miR-29b as a promising therapeutic approach for CLL and other ROR1+ B-cell malignancies.

Phenotypic Plasticity of Invasive Edge Glioma Stem-like Cells in Response to Ionizing Radiation.

Unresectable glioblastoma (GBM) cells in the invading tumor edge can act as seeds for recurrence. The molecular and phenotypic properties of these cells remain elusive. Here, we report that the invading edge and tumor core have two distinct types of glioma stem-like cells (GSCs) that resemble proneural (PN) and mesenchymal (MES) subtypes, respectively. Upon exposure to ionizing radiation (IR), GSCs, initially enriched for a CD133+ PN signature, transition to a CD109+ MES subtype in a C/EBP-ß-dependent manner. Our gene expression analysis of paired cohorts of patients with primary and recurrent GBMs identified a CD133-to-CD109 shift in tumors with an MES recurrence. Patient-derived CD133-/CD109+ cells are highly enriched with clonogenic, tumor-initiating, and radiation-resistant properties, and silencing CD109 significantly inhibits these phenotypes. We also report a conserved regulation of YAP/TAZ pathways by CD109 that could be a therapeutic target in GBM.

Fabrication of Plano-Concave Plastic Lens by Novel Injection Molding Using Carbide-Bonded Graphene-Coated Silica Molds.

Injection molding of plastic optical lenses prevails over many other techniques in both efficiency and cost, however polymer shrinkage during cooling, high level of uneven residual stresses and refractive index variations have limited its potential use for high precision lenses fabrication. In this research, we adopted a newly-developed strong graphene network to both plain and convex fused silica mold surfaces and proposed a novel injection molding of plano-concave lenses with graphene coated fused silica molds. The unique combination of the graphene coating and fused silica substrate maximize the mechanical properties of the mold and coating materials, namely high hardness, low surface friction, and high heat preservation effect during cooling since fused silica has low thermal conductivity. This advanced injection molding process was implemented in molding of plano-concave lenses resulting in reduced polymer shrinkage. In addition, internal residual stresses, and refractive index variations were also analyzed and discussed in detail. Meanwhile, as a comparison of conventional injection mold material, aluminum mold inserts with the same shape and size were also diamond machined and then employed to mold the same plano-concave lenses. Finally, a simulation model using Moldex3D was utilized to interpret stress distributions of both graphene and aluminum molds and then validated by experiments. The comparison between graphene and aluminum molds reveals that the novel injection molding with carbide-bonded graphene coated fused silica mold inserts is capable of molding high quality optical lenses with much less shrinkage and residual stresses, but more uniform refractive index distribution.

PLAUR Confers Resistance to Gefitinib Through EGFR/P-AKT/Survivin Signaling Pathway.

BACKGROUND/AIMS: Tyrosine kinase inhibitor gefitinib significantly improves the survival of patients with non-small-cell lung cancer (NSCLC) by inhibiting epidermal growth factor receptor (EGFR) tyrosine kinase. However, patients eventually develop resistance to gefitinib through uncharacterized mechanisms. It is known that plasminogen activator urokinase receptor (PLAUR) plays an important role in cell proliferation, migration and apoptosis. However, the role of PLAUR, particularly exosomal PLAUR in gefitinib resistance in NSCLC has not been reported. The aim of this study is to determine the relationship between PLAUR and gefitinib resistance. METHODS: In this study, a tethered cationic lipoplex nanoparticle (TCLN) biochip containing molecular beacons was used as probes to detect PLAUR mRNA in plasma exosomes from patients with gefitinib-sensitive and -resistant NSCLC. In vitro, Real-time PCR was used to examine the expression of PLAUR mRNA and Western blot was applied to examine the expression of related proteins. The gene knockdown was achieved by Lentivirus based RNA silence technique. The cell counting kit-8 assay and EdU incorporation were used to examine cell proliferation. The flow cytometry was applied to determine cell apoptosis and cell cycle, while the mitochondrial membrane potential was measured by JC-1 dye assay. Signaling pathway affected by PLAUR knockdown was identified by cDNA Microarray. The effect of PLAUR knockdown on tumorigenesis was analyzed in vivo. RESULTS: We found that the exosomal PLAUR mRNA in the plasma of gefitinib-resistant NSCLC patients was significantly increased compared to that of gefitinib-sensitive NSCLC patients. The PLAUR mRNA and soluble PLAUR protein were also significantly increased in gefitinib-resistant human lung adenocarcinoma PC9R cells compared to gefitinib-sensitive PC9 cells. Silencing PLAUR in PC9R cells impaired mitochondrial membrane potential and increased cell apoptosis via EGFR/p-AKT/survivin signaling pathway. Furthermore, EGFR was upregulated in the geftinib-resistant PC9R cells, and knockdown of EGFR significantly increased cell apoptosis. CONCLUSIONS: Taken together, our results demonstrated that PLAUR induces geftinib-resistance through EGFR/p-AKT/survivin signaling pathway in gefitinib-resistant human lung adenocarcinoma cells. PLAUR could be a novel therapeutic target for gefitinib-resistant NSCLC patients.

Highly stretchable and ultrathin nanopaper composites for epidermal strain sensors.

Multifunctional electronics are attracting great interest with the increasing demand and fast development of wearable electronic devices. Here, we describe an epidermal strain sensor based on an all-carbon conductive network made from multi-walled carbon nanotubes (MWCNTs) impregnated with poly(dimethyl siloxane) (PDMS) matrix through a vacuum filtration process. An ultrasonication treatment was performed to complete the penetration of PDMS resin in seconds. The entangled and overlapped MWCNT network largely enhances the electrical conductivity (1430 S m-1), uniformity (remaining stable on different layers), reliable sensing range (up to 80% strain), and cyclic stability of the strain sensor. The homogeneous dispersion of MWCNTs within the PDMS matrix leads to a strong interaction between the two phases and greatly improves the mechanical stability (ca. 160% strain at fracture). The flexible, reversible and ultrathin (<100 µm) film can be directly attached on human skin as epidermal strain sensors for high accuracy and real-time human motion detection.

Extracellular mRNA detected by molecular beacons in tethered lipoplex nanoparticles for diagnosis of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains one of the major causes of cancer related deaths. Although ultrasonography (US), computed tomography (CT) and/or high-cost magnetic resonance imaging (MRI) have been shown to improve early detection of liver cancer and mortality rates in high-risk individuals, such imaging based methods are limited by high rates of false positivity leading to unnecessary patient anxiety and invasive procedures. Complementary blood biomarkers could increase the accuracy of early detection. Although Alpha-fetoprotein (AFP) in blood is widely used in HCC screening and diagnosis, the false-negative rate as high as 30% and 40% is found in advanced HCC and early stage HCC respectively. We detected AFP messenger RNA (mRNA) in extracellular vesicles (EVs) in patient plasma using designed molecular beacons and a novel tethered lipoplex nanoparticle (TLN) biochip. Together with glypican-3 (GPC-3) mRNA, another well-known HCC marker, we observed much improved performance of AFP protein-based HCC detection. Comparing normal donors (N = 38) and HCC patients (N = 40), our TLN biochip using EV AFP and GPC-3 mRNAs provided an AUC (area under the ROC curve) of 0.995, better than that of a single marker. This 2-mRNA combination also provided a perfect positive predictive value (PPV = 1) at a negative predictive value (NPV) of 0.95 and 20% prevalence, while the blood AFP protein or plasma EV GPC3 mRNA alone could only provide a PPV of 0.61 and 0.79 respectively at the same conditions. Thus, this facile new method may complement current models for risk stratification in liver cancer screening, therapeutic monitoring, and after-treatment surveillance. However, large scale validation will need to be conducted to confirm its clinical potential.