Tumor-Targeted Delivery of IL-2 by Fusing with a pH Low Insertion Peptide for Antitumor Immunotherapy.

As a pleiotropic cytokine, interleukin-2 (IL-2) can effectively regulate lymphocyte proliferation, survival, and active antitumor immune responses in tumor microenvironments. Although the ability of IL-2 to boost immune responses was reported in cancer patients, its short circulating half-life and high toxicity hinder its broad and continual clinical application. Herein, we developed a novel tumor target agent by fusing pH low insertion peptides (pHLIP) with IL-2, forming the fusion protein pHLIP-IL2. Based on the low pH insertion property of pHLIP, the pHLIP-IL2 fusion protein could be selectively delivered to the acidic tumor microenvironments and then promote the proliferation of killer immune cells to elicit tumor regression. We found that pHLIP-IL2 fusion proteins can be significantly enriched in tumor tissues and can effectively reduce tumor size in diverse tumor models, including breast cancer and melanoma, without apparent adverse effects. These data suggest that the pHLIP-IL2 fusion protein may be a promising solution for the continual and extensive application of IL-2, and pHLIP-IL2 is a potential and valuable therapeutic drug for cancer patients with antitumor immunotherapy.

Study on the Properties and Synergistic Antioxidant Effects of Novel Bifunctional Fusion Proteins Expressed Using the UTuT6 System.

Important antioxidant enzymes, glutathione peroxidase (GPx) and superoxide dismutase (SOD), are involved in maintaining redox balance. They can protect each other and result in more efficiently removing excessive reactive oxygen species (ROS), protecting cells against injury, and maintaining the normal metabolism of ROS. In this study, human cytosolic GPx (hGPx1) and human phospholipid hydroperoxide GPx (hGPx4) genes were integrated into the same open reading frame with human extracellular SOD active site (SOD3-72P) genes, respectively, and several novel fusion proteins were obtained by using the UTuT6 expression system for the first time. Among them, Se-hGPx1UAG-L4-SOD3-72P is the bifunctional fusion protein with the highest GPx activity and the best anti-hydrogen peroxide inactivation ability thus far. The Se-hGPx4UAG-L3-SOD3-72P fusion protein exhibits the strongest alkali and high temperature resistance and a greater protective effect against lipoprotein peroxidation damage. Se-hGPx1UAG-L4-SOD3-72P and Se-hGPx4UAG-L3-SOD3-72P fusion proteins both have good synergistic and antioxidant abilities in H2O2-induced RBCs and liver damage models. We believe that this research will help with the development of novel bifunctional fusion proteins and the investigation of the synergistic and catalytic mechanisms of GPx and SOD, which are important in creating novel protein therapeutics.

Nanoparticle-Based Combination Therapy Enhances Fulvestrant Efficacy and Overcomes Tumor Resistance in ER-Positive Breast Cancer.

Nanoparticles (NP) spanning diverse materials and properties have the potential to encapsulate and to protect a wide range of therapeutic cargos to increase bioavailability, to prevent undesired degradation, and to mitigate toxicity. Fulvestrant, a selective estrogen receptor degrader, is commonly used for treating patients with estrogen receptor (ER)-positive breast cancer, but its broad and continual application is limited by poor solubility, invasive muscle administration, and drug resistance. Here, we developed an active targeting motif-modified, intravenously injectable, hydrophilic NP that encapsulates fulvestrant to facilitate its delivery via the bloodstream to tumors, improving bioavailability and systemic tolerability. In addition, the NP was coloaded with abemaciclib, an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), to prevent the development of drug resistance associated with long-term fulvestrant treatment. Targeting peptide modifications on the NP surface assisted in the site-specific release of the drugs to ensure specific toxicity in the tumor tissues and to spare normal tissue. The NP formulation (PPFA-cRGD) exhibited efficient tumor cell killing in both in vitro organoid models and in vivo orthotopic ER-positive breast cancer models without apparent adverse effects, as verified in mouse and Bama miniature pig models. This NP-based therapeutic provides an opportunity for continual and extensive clinical application of fulvestrant, thus indicating its promise as a treatment option for patients with ER-positive breast cancer. SIGNIFICANCE: A smart nanomedicine encapsulating fulvestrant to improve its half-life, bioavailability, and tumor-targeting and coloaded with CDK4/6 inhibitor abemaciclib to block resistance is a safe and effective therapy for ER-positive breast cancer.

Regulation of metabolism in pancreatic ductal adenocarcinoma via nanotechnology-enabled strategies.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal malignancy with insidious onset and early distal metastasis. Metabolic reprogramming, the autonomous changes in cellular bioenergetics driven by aberrant genetic events and crosstalk between cancer and non-cancer cells in the desmoplastic microenvironment, is pivotal for the rapid progression of PDAC. As an attractive therapeutic target, nucleoside metabolism is regulated by various anti-metabolic drugs for the clinical treatment of PDAC. Despite various challenges, such as poor drug delivery efficiency and off-target side effects, metabolic modification and intervention are emerging as promising strategies for PDAC therapy, enabled by the rapid development of nanotechnology-based drug delivery strategies. In this review, we discuss the metabolic characteristics of PDAC and highlight how the development of nanomedicine has boosted the development of new therapeutics for PDAC by modulating critical targets in metabolic reprogramming.

Reactive Human Plasma Glutathione Peroxidase Mutant with Diselenide Bond Succeeds in Tetramer Formation.

Plasma glutathione peroxidase (GPx3) belongs to the GPx superfamily, and it is the only known secreted selenocysteine (Sec)-containing GPx in humans. It exists as a glycosylated homotetramer and catalyzes the reduction of hydrogen peroxide and lipid peroxides, depending on the Sec in its active center. In this study, a previously reported chimeric tRNAUTuT6 was used for the incorporation of Sec at the UAG amber codon, and the mature form of human GPx3 (hGPx3) without the signal peptide was expressed in amber-less E. coli C321.ΔA.exp. Reactive Sec-hGPx3, able to reduce H2O2 and tert-butyl hydroperoxide (t-BuOOH), was produced with high purity and yield. Study of the quaternary structure suggested that the recombinant Sec-hGPx3 contained an intra-molecular disulfide bridge but failed to form tetramer. Mutational and structural analysis of the mutants with three Cys residues, individually or jointly replaced with Ser, indicated that the formation of intra-molecular disulfide bridges involved structure conformational changes. The secondary structure containing Cys77 and Cys132 was flexible and could form a disulfide bond, or form a sulfhydryl-selenyl bond with Sec49 in relative mutants. Mutation of Cys8 and Cys132 to Sec8 and Sec132 could fix the oligomerization loop through the formation of diselenide bond, which, in turn, facilitated tetramer formation and noticeably improved the GPx activity. This research provides an important foundation for the further catalysis and functional study of hGPx3.

CXC Chemokine Receptor 2 Accelerates Tubular Cell Senescence and Renal Fibrosis via ß-Catenin-Induced Mitochondrial Dysfunction.

Renal fibrosis is a common feature of various chronic kidney diseases (CKD). However, its underlying mechanism has not been totally clarified. C-X-C motif chemokine receptor (CXCR) family plays a role in renal fibrosis, however, detailed mechanisms have not been elucidated. Here, we report that CXCR2 has a potential role in tubular cell senescence and renal fibrosis, and is associated with ß-catenin-activated mitochondrial dysfunction. CXCR2 is one of most increased members among CXCR family in unilateral ureteral obstruction (UUO) mice. CXCR2 was expressed primarily in tubules and co-localized with p16INK4A, a cellular senescence marker, and ß-catenin. Administration of SB225002, a selective CXCR2 antagonist, significantly inhibited the activation of ß-catenin signaling, restored mitochondrial function, protected against tubular cell senescence and renal fibrosis in unilateral ureteral obstruction (UUO) mice. In unilateral ischemia-reperfusion injury (UIRI) mice, treatment with interlukin-8 (IL-8), the ligand of CXCR2, further aggravated ß-catenin activation, mitochondrial dysfunction, tubular cell senescence and renal fibrosis, whereas knockdown of p16INK4A inhibited IL-8-induced these effects. In vitro, SB225002 inhibited mitochondrial dysfunction and tubular cell senescence. Furthermore, ICG-001, a ß-catenin signaling blocker, significantly retarded CXCR2-induced cellular senescence and fibrotic changes. These results suggest that CXCR2 promotes tubular cell senescence and renal fibrosis through inducing ß-catenin-activated mitochondrial dysfunction.

Reducing Postoperative Recurrence of Early-Stage Hepatocellular Carcinoma by a Wound-Targeted Nanodrug.

New strategies to decrease risk of relapse after surgery are needed for improving 5-year survival rate of hepatocellular carcinoma (HCC). To address this need, a wound-targeted nanodrug is developed, that contains an immune checkpoint inhibitor (anti-PD-L1)and an angiogenesis inhibitor (sorafenib)). These nanoparticles consist of highly biocompatible mesoporous silica (MSNP) that is surface-coated with platelet membrane (PM) to achieve surgical site targeting in a self-amplified accumulation manner. Sorafenib is introduced into the MSNP pores while covalently attaching anti-PD-L1 antibody on the PM surface. The resulting nano-formulation, abbreviated as a-PM-S-MSNP, can effectively target the surgical margin when intraperitoneally (IP) administered into an immune competent murine orthotopic HCC model. Multiple administrations of a-PM-S-MSNP generate potent anti-HCC effect and significantly prolong overall mice survival. Immunophenotyping and immunochemistry staining reveal the signatures of favorable anti-HCC immunity and anti-angiogenesis effect at tumor sites. More importantly, microscopic inspection of a-PM-S-MSNP treated mice shows that 2 out 6 are histologically tumor-free, which is in sharp contrast to the control mice where tumor foci can be easily identified. The data suggest that a-PM-S-MSNP can efficiently inhibit post-surgical HCC relapse without obvious side effects and holds considerable promise for clinical translation as a novel nanodrug.

PEGylation and antioxidant effects of a human glutathione peroxidase 1 mutant.

Human glutathione peroxidase1 (hGPx1) is a good antioxidant and potential drug, but the limited availability and poor stability of hGPx1 have affected its development and application. To solve this problem, we prepared a hGPx1 mutant (GPx1M) with high activity in an Escherichia coli BL21(DE3)cys auxotrophic strain using a single protein production (SPP) system. In this study, the GPx1M was conjugated with methoxypolyethylene glycol-succinimidyl succinate (SS-mPEG, Mw = 5 kDa) chains to enhance its stability. SS-mPEG-GPx1M and GPx1M exhibited similar enzymatic activity and stability toward pH and temperature change, and in a few cases, SS-mPEG-GPx1M was discovered to widen the range of pH stability and increase the temperature stability. Lys 38 was confirmed as PEGylated site by liquid-mass spectrometry. H9c2 cardiomyoblast cells and Sprague-Dawley (SD) rats were used to evaluate the effects of GPx1M and SS-mPEG-GPx1M on preventing or alleviating adriamycin (ADR)-mediated cardiotoxicity, respectively. The results indicated that GPx1M and SS-mPEG-GPx1M had good antioxidant effects in vitro and in vivo, and the effect of SS-mPEG-GPx1M is more prominent than GPx1M in vivo. Thus, PEGylation might be a promising method for the application of GPx1M as an important antioxidant and potential drug.

Improvement of stability and in vivo antioxidant effect of human glutathione peroxidase mutant by PEGylation.

Human glutathione peroxidase (GPx), as an important kind of antioxidant enzyme, is often used for the removal of reactive oxygen species. Unfortunately, the application has been hindered by its limited source and poor stability. To solve these problems, human glutathione peroxidase mutant (GPxM) with high activity and yield was obtained using Escherichia coli BL21(DE3) cys auxotrophic strain and the single-protein production system in our previous work. However, the antioxidant effect of this novel recombinant protein drug in animals has not been demonstrated, and its immunogenicity and short biological half-life as a biological macromolecule may have seriously hindered its clinical application. Therefore, it is important to find an effective strategy to address the above issues. In this work, PEGylated GPxM was prepared by conjugating the corresponding mutant with monomethoxy polyethylene glycol succinimidyl succinate (SS-mPEG). We researched the structure, stability, pharmacokinetic properties, antioxidant effect in vivo and protective mechanism against adriamycin (ADR)-mediated cardiotoxicity of modified products, and compared with the above properties of GPxM. The results revealed that GPxM had an excellent antioxidant effect in vivo, and PEGylation can enhance the stability, half-life and antioxidant effect of GPxM while reducing immunogenicity. In addition, the above improvement of PEGylated GPx1M was stronger than that of monoPEGylated GPx4M. Hence, PEGylation might be an effective method to broaden the applications of GPxM as the important antioxidant drug, especially the PEGylated GPx1M with high antioxidant effect.

Protective Effects and Mechanisms of Recombinant Human Glutathione Peroxidase 4 on Isoproterenol-Induced Myocardial Ischemia Injury.

Ischemic heart disease (IHD) is a cardiovascular disease with high fatality rate, and its pathogenesis is closely related to oxidative stress. Reactive oxygen species (ROS) in oxidative stress can lead to myocardial ischemia (MI) injury in many ways. Therefore, the application of antioxidants may be an effective way to prevent IHD. In recent years, glutathione peroxidase 4 (GPx4) has received increasing attention due to its antioxidant effect. In a previous study, we used the new chimeric tRNAUTuT6 to express highly active recombinant human GPx4 (rhGPx4) in amber-less Escherichia coli. In this study, we established an isoproterenol- (ISO-) induced MI injury model in rats and an in vitro model to research the protective effect and mechanism of rhGPx4 on MI injury. The results showed that rhGPx4 could reduce the area of myocardial infarction and ameliorate the pathological injury of heart tissue, significantly reduce ISO-induced abnormalities on electrocardiogram (ECG) and cardiac serum biomarkers, protect mitochondrial function, and attenuate cardiac oxidative stress injury. In an in vitro model, the results also confirmed that rhGPx4 could inhibit ISO-induced oxidative stress injury and cardiomyocyte apoptosis. The mechanism of action of rhGPx4 involves not only the inhibition of lipid peroxidation by eliminating ROS but also keeping a normal level of endogenous antioxidant enzymes by eliminating ROS, thereby preventing oxidative stress injury in cardiomyocytes. Additionally, rhGPx4 could inhibit cardiomyocyte apoptosis through a mitochondria-dependent pathway. In short, rhGPx4, a recombinant antioxidant enzyme, can play an important role in the prevention of IHD and may have great potential for application.

Molecularly engineered truncated tissue factor with therapeutic aptamers for tumor-targeted delivery and vascular infarction.

Selective occlusion of tumor vasculature has proven to be an effective strategy for cancer therapy. Among vascular coagulation agents, the extracellular domain of coagulation-inducing protein tissue factor, truncated tissue factor (tTF), is the most widely used. Since the truncated protein exhibits no coagulation activity and is rapidly cleared in the circulation, free tTF cannot be used for cancer treatment on its own but must be combined with other moieties. We here developed a novel, tumor-specific tTF delivery system through coupling tTF with the DNA aptamer, AS1411, which selectively binds to nucleolin receptors overexpressing on the surface of tumor vascular endothelial cells and is specifically cytotoxic to target cells. Systemic administration of the tTF-AS1411 conjugates into tumor-bearing animals induced intravascular thrombosis solely in tumors, thus reducing tumor blood supply and inducing tumor necrosis without apparent side effects. This conjugate represents a uniquely attractive candidate for the clinical translation of vessel occlusion agent for cancer therapy.

Platelet-Membrane-Coated Nanoparticles Enable Vascular Disrupting Agent Combining Anti-Angiogenic Drug for Improved Tumor Vessel Impairment.

Compared with traditional chemotherapeutics, vascular disruption agents (VDAs) have the advantages of rapidly blocking the supply of nutrients and starving tumors to death. Although the VDAs are effective under certain scenarios, this treatment triggers angiogenesis in the later stage of therapy that frequently leads to tumor recurrence and treatment failure. Additionally, the nonspecific tumor targeting and considerable side effects also impede the clinical applications of VDAs. Here we develop a customized strategy that combines a VDA with an anti-angiogenic drug (AAD) using mesoporous silica nanoparticles (MSNs) coated with platelet membrane for the self-assembled tumor targeting accumulation. The tailor-made nanoparticles accumulate in tumor tissues through the targeted adhesion of platelet membrane surface to damaged vessel sites, resulting in significant vascular disruption and efficient anti-angiogenesis in animal models. This study demonstrates the promising potential of combining VDA and AAD in a single nanoplatform for tumor eradication.

Study on antioxidant effect of recombinant glutathione peroxidase 1.

Glutathione peroxidase 1 (GPx1) is an important antioxidant selenium enzyme and has a good prospect for drug development. However, the expression of GPx1 requires a complex expression mechanism, which makes the drug development of recombinant GPx1 (rGPx1) difficult. In the previous study, we expressed highly active rhGPx1 in amber-less Escherichia coli by using a novel chimeric tRNAUTuT6. However, the antioxidant effect of rhGPx1 at the cellular and animal levels has not been verified. In this study, we established isoproterenol (ISO)-induced oxidative stress injury models to study the antioxidant effect of rhGPx1 at the cellular and animal levels. Meanwhile, in order to more accurately reflect the antioxidant effect of rGPx1 in mice, we used the same method to express recombinant mouse GPx1 (rmGPx1) as a control for rhGPx1. The results of a study showed that rhGPx1 has a good antioxidant effect at the cellular and animal levels. However, due to species differences, rhGPx1 had immunogenicity in mice and antibodies of rhGPx1 could inhibit its antioxidant activity, so the antioxidant effect of rhGPx1 was not as good as rmGPx1 in mice. Nevertheless, this study provides a reliable theoretical basis for the development of rhGPx1 as an antioxidant drug.

Engineering Biomimetic Platesomes for pH-Responsive Drug Delivery and Enhanced Antitumor Activity.

Biomimetic camouflage, i.e., using natural cell membranes for drug delivery, has demonstrated advantages over synthetic materials in both pharmacokinetics and biocompatibility, and so represents a promising solution for the development of safe nanomedicine. However, only limited efforts have been dedicated to engineering such camouflage to endow it with optimized or additional properties, in particular properties critical to a "smart" drug delivery system, such as stimuli-responsive drug release. A pH-responsive biomimetic "platesome" for specific drug delivery to tumors and tumor-triggered drug release is described. This platesome nanovehicle is constructed by merging platelet membranes with functionalized synthetic liposomes and exhibits enhanced tumor affinity, due to its platelet membrane-based camouflage, and selectively releases its cargo in response to the acidic microenvironment of lysosomal compartments. In mouse cancer models, it shows significantly better antitumor efficacy than nanoformulations based on a platesome without pH responsiveness or those based on traditional pH-sensitive liposomes. A convenient way to incorporate stimuli-responsive features into biomimetic nanoparticles is described, demonstrating the potential of engineered cell membranes as biomimetic camouflages for a new generation of biocompatible and efficient nanocarriers.

Quantitative determination of monotropein in rat plasma and tissue by LC-MS/MS and its application to pharmacokinetic and tissue distribution studies

Abstract A selective and sensitive liquid chromatography tandem with mass spectrometry was developed and validated for accurate determination of monotropein in rat plasma and tissues. All biological samples were prepared by simple protein precipitation method using catalpol as an internal standard. The analyte and internal standard were separated on a C18 analytical column with 2 min of run time, at flow rate of 0.5 ml/min. The detection was performed on a triple-quadrupole tandem mass spectrometer equipped with negative-ion electrospray ionization by selected-reaction monitoring of the transitions at m/z 389→147 for monotropein and m/z 361→169 for the internal standard. The calibration curves for plasma and tissue samples were linear over the concentration range of 4-2000 ng/ml, with a lower limit of quantification of 4 ng/ml. The method was successfully applied to a pharmacokinetic and tissue distribution study of monotropein in rats.

Platelet membrane-based and tumor-associated platelettargeted drug delivery systems for cancer therapy.

Platelets have long been known to play critical roles in hemostasis by clumping and clotting blood vessel injuries. Recent experimental evidence strongly indicates that platelets can also interact with tumor cells by direct binding or secreting cytokines. For example, platelets have been shown to protect circulating cancer cells in blood circulation and to promote tumor metastasis. In-depth understanding of the role of platelets in cancer progression and metastasis provides promising approaches for platelet biomimetic drug delivery systems and functional platelet-targeting strategies for effective cancer treatment. This review highlights recent progresses in platelet membrane-based drug delivery and unique strategies that target tumor-associated platelets for cancer therapy. The paper also discusses future development opportunities and challenges encountered for clinical translation.

A Sensitive LC-MS-MS Method for Quantification of 1,6-O,O-Diacetylbritannilactone in Rat Plasma and its Application in a Pharmacokinetic Study.

1,6-O,O-Diacetylbritannilactone is a natural sesquiterpene lactone isolated from Inula britannica that has displayed cytotoxic effects against several human cancer cell lines. In this study, a selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed for determination of 1,6-O,O-diacetylbritannilactone in rat plasma. Chromatographic separation was achieved on an Agilent C18 column (4.6 mm × 75 mm, 3.5 µm) with methanol and water (80:20, v/v) as the mobile phase. An ESI source was applied and operated in positive ion mode; selected-reaction monitoring was used for quantification using target fragment ions m/z 373.2→312.9 for 1,6-O,O-diacetylbritannilactone and m/z 331.2→144.1 for the IS. Calibration plots were linear in the range of 1.5-1350 ng/mL for 1,6-O,O-diacetylbritannilactone in rat plasma. Intra- and inter-day precisions were <8.5%, and the accuracy ranged from -2.7 to 12.8%. The LC-MS-MS method was successfully applied in a pharmacokinetic study of 1,6-O,O-diacetylbritannilactone in rats.

Efficient Expression of Glutathione Peroxidase with Chimeric tRNA in Amber-less Escherichia coli.

The active center of selenium-containing glutathione peroxidase (GPx) is selenocysteine (Sec), which is is biosynthesized on its tRNA in organisms. The decoding of Sec depends on a specific elongation factor and a Sec Insertion Sequence (SECIS) to suppress the UGA codon. The expression of mammalian GPx is extremely difficult with traditional recombinant DNA technology. Recently, a chimeric tRNA (tRNAUTu) that is compatible with elongation factor Tu (EF-Tu) has made selenoprotein expression easier. In this study, human glutathione peroxidase (hGPx) was expressed in amber-less Escherichia coli C321.ΔA.exp using tRNAUTu and seven chimeric tRNAs that were constructed on the basis of tRNAUTu. We found that chimeric tRNAUTu2, which substitutes the acceptor stem and T-stem of tRNAUTu with those from tRNASec, enabled the expression of reactive hGPx with high yields. We also found that chimeric tRNAUTuT6, which has a single base change (A59C) compared to tRNAUTu, mediated the highest reactive expression of hGPx1. The hGPx1 expressed exists as a tetramer and reacts with positive cooperativity. The SDS-PAGE analysis of hGPx2 produced by tRNAUTuT6 with or without sodium selenite supplementation showed that the incorporation of Sec is nearly 90%. Our approach enables efficient selenoprotein expression in amber-less Escherichia coli and should enable further characterization of selenoproteins in vitro.

Suppression of Tumor Energy Supply by Liposomal Nanoparticle-Mediated Inhibition of Aerobic Glycolysis.

Aerobic glycolysis enables cancer cells to rapidly take up nutrients (e.g., nucleotides, amino acids, and lipids) and incorporate them into the biomass needed to produce a new cell. In contrast to existing chemotherapy/radiotherapy strategies, inhibiting aerobic glycolysis to limit the adenosine 5'-triphosphate (ATP) yield is a highly efficient approach for suppressing tumor cell proliferation. However, most, if not all, current inhibitors of aerobic glycolysis cause significant adverse effects because of their nonspecific delivery and distribution to nondiseased organs, low bioavailability, and a narrow therapeutic window. New strategies to enhance the biosafety and efficacy of these inhibitors are needed for moving them into clinical applications. To address this need, we developed a liposomal nanocarrier functionalized with a well-validated tumor-targeting peptide to specifically deliver the aerobic glycolysis inhibitor 3-bromopyruvate (3-BP) into the tumor tissue. The nanoparticles effectively targeted tumors after systemic administration into tumor-bearing mice and suppressed tumor growth by locally releasing 3-BP to inhibit the ATP production of the tumor cells. No overt side effects were observed in the major organs. This report demonstrates the potential utility of the nanoparticle-enabled delivery of an aerobic glycolysis inhibitor as an anticancer therapeutic agent.

Field-Free Isolation of Exosomes from Extracellular Vesicles by Microfluidic Viscoelastic Flows.

Exosomes, molecular cargos secreted by almost all mammalian cells, are considered as promising biomarkers to identify many diseases including cancers. However, the small size of exosomes (30-200 nm) poses serious challenges in their isolation from complex media containing a variety of extracellular vesicles (EVs) of different sizes, especially in small sample volumes. Here we present a viscoelasticity-based microfluidic system to directly separate exosomes from cell culture media or serum in a continuous, size-dependent, and label-free manner. Using a small amount of biocompatible polymer as the additive in the media to control the viscoelastic forces exerted on EVs, we are able to achieve a high separation purity (>90%) and recovery (>80%) of exosomes. The proposed technique may serve as a versatile platform to facilitate exosome analyses in diverse biochemical applications.