Hybrid Cellular Nanovesicles Block PD-L1 Signal and Repolarize M2 Macrophages for Cancer Immunotherapy.

The PD1/PD-L1 immune checkpoint blocking is a promising therapy, while immunosuppressive tumor microenvironment (TME) and poor tumor penetration of therapeutic antibodies limit its efficacy. Repolarization of tumor-associated macrophages (TAMs) offers a potential method to ameliorate immunosuppression of TME and further boost T cell antitumor immunity. Herein, hybrid cell membrane biomimetic nanovesicles (hNVs) are developed by fusing M1 macrophage-derived nanovesicles (M1-NVs) and PD1-overexpressed tumor cell-derived nanovesicles (PD1-NVs) to improve cancer immunotherapy. The M1-NVs promote the transformation of M2-like TAMs to M1-like phenotype and further increase the release of pro-inflammatory cytokines, resulting in improved immunosuppressive TME. Concurrently, the PD1-NVs block PD1/PD-L1 pathway, which boosts cancer immunotherapy when combined with M1-NVs. In a breast cancer mouse model, the hNVs efficiently accumulate at the tumor site after intravenous injection and significantly inhibit the tumor growth. Mechanically, the M1 macrophages and CD8+ T lymphocytes in TME increase by twofold after the treatment, indicating effective immune activation. These results suggest the hNVs as a promising strategy to integrate TME improvement with PD1/PD-L1 blockade for cancer immunotherapy.

Microfluidics-Assisted Fluorescence Mapping of DNA Phosphorothioation.

As the key player of a new restriction modification system, DNA phosphorothioate (PT) modification, which swaps oxygen for sulfur on the DNA backbone, protects the bacterial host from foreign DNA invasion. The identification of PT sites helps us understand its physiological defense mechanisms, but accurately quantifying this dynamic modification remains a challenge. Herein, we report a simple quantitative analysis method for optical mapping of PT sites in the single bacterial genome. DNA molecules are fully stretched and immobilized in a microfluidic chip by capillary flow and electrostatic interactions, improving the labeling efficiency by maximizing exposure of PT sites on DNA while avoiding DNA loss and damage. After screening 116 candidates, we identified a bifunctional chemical compound, iodoacetyl-polyethylene glycol-biotin, that can noninvasively and selectively biotinylate PT sites, enabling further labeling with streptavidin fluorescent nanoprobes. With this method, PT sites in PT+ DNA can be easily detected by fluorescence, while almost no detectable ones were found in PT- DNA, achieving real-time visualization of PT sites on a single DNA molecule. Collectively, this facile genome-wide PT site detection method directly characterizes the distribution and frequency of DNA modification, facilitating a better understanding of its modification mechanism that can be potentially extended to label DNAs in different species.

Noninvasive prenatal diagnosis targeting fetal nucleated red blood cells.

Noninvasive prenatal diagnosis (NIPD) aims to detect fetal-related genetic disorders before birth by detecting markers in the peripheral blood of pregnant women, holding the potential in reducing the risk of fetal birth defects. Fetal-nucleated red blood cells (fNRBCs) can be used as biomarkers for NIPD, given their remarkable nature of carrying the entire genetic information of the fetus. Here, we review recent advances in NIPD technologies based on the isolation and analysis of fNRBCs. Conventional cell separation methods rely primarily on physical properties and surface antigens of fNRBCs, such as density gradient centrifugation, fluorescence-activated cell sorting, and magnetic-activated cell sorting. Due to the limitations of sensitivity and purity in Conventional methods, separation techniques based on micro-/nanomaterials have been developed as novel methods for isolating and enriching fNRBCs. We also discuss emerging methods based on microfluidic chips and nanostructured substrates for static and dynamic isolation of fNRBCs. Additionally, we introduce the identification techniques of fNRBCs and address the potential clinical diagnostic values of fNRBCs. Finally, we highlight the challenges and the future directions of fNRBCs as treatment guidelines in NIPD.

Enhanced Isolation of Fetal Nucleated Red Blood Cells by Enythrocyte-Leukocyte Hybrid Membrane-Coated Magnetic Nanoparticles for Noninvasive Pregnant Diagnostics.

Fetal nucleated red blood cells (fNRBCs) in maternal peripheral blood containing the whole genetic information of the fetus may serve for noninvasive pregnant diagnostics (NIPD). However, the fetal cell-based NIPD is seriously limited by the poor purity of the isolated fNRBCs. Recently, the biomimetic cell membrane-camouflaged nanoparticles containing outstanding features have been widely used to detect and isolate rare cells from the peripheral blood samples. In this work, enythrocyte (RBC) and leukocyte (WBC) membranes are fused and coated onto magnet nanoparticles and then modified with anti-CD147 to isolate fNRBCs from the maternal peripheral blood with significant efficiency (∼90%) and purity (∼87%) in simulated spiked blood samples. Further, fNRBCs were isolated and identified from a series of maternal peripheral blood samples coming from pregnant women of 11-13 gestational weeks, and different chromosomal aneuploidies were diagnosed using fNRBCs isolated from maternal blood in early pregnancy. Our strategy may offer additional opportunity to overcome the limitations of current cell-based NIPD platforms.

High-throughput isolation of fetal nucleated red blood cells by multifunctional microsphere-assisted inertial microfluidics.

Being easy, safe and reliable, non-invasive prenatal diagnosis (NIPD) has been greatly pursued in recent years. Holding the complete genetic information of the fetus, fetal nucleated red blood cells (fNRBCs) are viewed as a suitable target for NIPD application. However, effective separating fNRBCs from maternal peripheral blood for clinic use still faces great challenges, given that fNRBCs are extremely rare in maternal blood circulation. Here, by combining the high-throughput inertial microfluidic chip with multifunctional microspheres as size amplification, we develop a novel method to isolate fNRBCs with high performance. To enlarge the size difference between fNRBCs and normal blood cells, we use the gelatin coated microspheres to capture fNRBCs with anti-CD147 as specific recognizer at first. The size difference between fNRBCs captured by the microspheres and normal blood cells makes it easy to purify the captured fNRBCs through the spiral microfluidic chip. Finally, the purified fNRBCs are mildly released from the microspheres by enzymatically degrading the gelatin coating. Cell capture efficiency about 81%, high purity of 83%, as well as cell release viability over 80% were achieved using spiked samples by this approach. Additionally, fNRBCs were successfully detected from peripheral blood of pregnant women with an average of 24 fNRBCs per mL, suggesting the great potential of this method for clinical non-invasive prenatal diagnosis.

Electrospun degradable Zn-Mn oxide hierarchical nanofibers for specific capture and efficient release of circulating tumor cells.

Constructing biological affinity devices is considered as an effective strategy for isolating circulating tumor cells (CTCs), and electrospun nanofibers (ESNFs) have recently received attention. However, the current research focuses on polymer fibers, and fabricating stimuli-responsive inorganic nanofibers for cancer diagnosis and analysis is still challenging. In this work, Zn-Mn oxide nanofibers (ZnMnNFs) are used to capture and purify cancer cells after modification with specific antibodies. Then, the hierarchical nanofibers are degraded by reductive weak acid to release the captured cells efficiently without residues. Fusion of Zn and Mn, two transition metals, enhances the surface activity of oxides so that ZnMnNFs are easier to be degraded and modified. By using MCF-7 cancer cells, the cell capture efficiency of ZnMnNFs is up to 88.2%. Furthermore, by using citric acid, it is discovered that, by comparison with Mn oxide nanofibers, the cell release efficiency of ZnMnNFs is improved to 95.1% from 15.4%. In addition, the viability of released cells exceeds 90%. Lastly, the robustness of ZnMnNFs substrates is tested in peripheral blood from breast cancer patients (BCP) and colorectal cancer patients (CCP). Combined with fluorescence labeling, CTCs are confirmed to be isolated from all the clinical samples. This is the first trial of using ternary inorganic ESNFs for cancer cell capture. It is anticipated that the degradable ESNFs will provide biocompatible theranostic platforms and overcome the current limitations of cell release for high-precision gene analysis.

A Biomimetic Nanodecoy Traps Zika Virus To Prevent Viral Infection and Fetal Microcephaly Development.

Zika virus (ZIKV) has emerged as a global health threat due to its unexpected causal link to devastating neurological disorders such as fetal microcephaly; however, to date, no approved vaccine or specific treatment is available for ZIKV infection. Here we develop a biomimetic nanodecoy (ND) that can trap ZIKV, divert ZIKV away from its intended targets, and inhibit ZIKV infection. The ND, which is composed of a gelatin nanoparticle core camouflaged by mosquito medium host cell membranes, effectively adsorbs ZIKV and inhibits ZIKV replication in ZIKV-susceptible cells. Using a mouse model, we demonstrate that NDs significantly attenuate the ZIKV-induced inflammatory responses and degenerative changes and thus improve the survival rate of ZIKV-challenged mice. Moreover, by trapping ZIKV, NDs successfully prevent ZIKV from passing through physiologic barriers into the fetal brain and thereby mitigate ZIKV-induced fetal microcephaly in pregnant mice. We anticipate that this study will provide new insights into the development of safe and effective protection against ZIKV and various other viruses that threaten public health.

Mechanical Distension Induces Serotonin Release from Intestine as Revealed by Stretchable Electrochemical Sensing.

The role of endogenous serotonin (5-HT) in gastrointestinal motility is still highly controversial. Although electrochemical techniques allow for direct and real-time recording of biomolecules, the dynamic monitoring of 5-HT release from elastic and tubular intestine during motor reflexes remains a great challenge because of the specific peristalsis patterns and inevitable passivation of the sensing interface. A stretchable sensor with antifouling and decontamination properties was assembled from gold nanotubes, titanium dioxide nanoparticles, and carbon nanotubes. The sandwich-like structure endowed the sensor with satisfying mechanical stability and electrochemical performance, high resistance against physical adsorption, and superior efficiency in the photodegradation of biofouling molecules. Insertion of the sensor into the lumen of rat ileum (the last section of the small intestine) successfully mimics intestinal peristalsis, and simultaneous real-time monitoring of distension-evoked 5-HT release was possible for the first time. Our results unambiguously reveal that mechanical distension of the intestine induces endogenous 5-HT overflow, and 5-HT level is closely associated with the physiological or pathological states of the intestine.

TiO2 nanopillar arrays coated with gelatin film for efficient capture and undamaged release of circulating tumor cells.

Circulating tumor cells (CTCs) are important for the detection and treatment of cancer. Nevertheless, a low density of circulating tumor cells makes the capture and release of CTCs an obstacle. In this work, TiO2 nanopillar arrays coated with gelatin film were synthesized for efficient capture and undamaged release of circulating tumor cells. The scanning electron microscope and atomic force microscope images demonstrate that the substrate has a certain roughness. The interaction between the cell membrane and the nanostructure substrate contributes to the efficient capture of CTC (capture efficiency up to 94.98%). The gelatin layer has excellent biocompatibility and can be rapidly digested by matrix metalloproteinase (MMP9), which realizes the non-destructive release of CTCs (0.1 mg ml-1, 5 min, nearly 100% release efficiency, activity 100%). Therefore, by our strategy, the CTCs can be efficiently captured and released undamaged, which is important for subsequent analysis.

Macrophage membrane-coated iron oxide nanoparticles for enhanced photothermal tumor therapy.

Nanotechnology possesses the potential to revolutionize the diagnosis and treatment of tumors. The ideal nanoparticles used for in vivo cancer therapy should have long blood circulation times and active cancer targeting. Additionally, they should be harmless and invisible to the immune system. Here, we developed a biomimetic nanoplatform with the above properties for cancer therapy. Macrophage membranes were reconstructed into vesicles and then coated onto magnetic iron oxide nanoparticles (Fe3O4 NPs). Inherited from the Fe3O4 core and the macrophage membrane shell, the resulting Fe3O4@MM NPs exhibited good biocompatibility, immune evasion, cancer targeting and light-to-heat conversion capabilities. Due to the favorable in vitro and in vivo properties, biomimetic Fe3O4@MM NPs were further used for highly effective photothermal therapy of breast cancer in nude mice. Surface modification of synthetic nanomaterials with biomimetic cell membranes exemplifies a novel strategy for designing an ideal nanoplatform for translational medicine.

Highly sensitive and rapid isolation of fetal nucleated red blood cells with microbead-based selective sedimentation for non-invasive prenatal diagnostics.

Non-invasive prenatal diagnostics (NIPD) has been an emerging field for prenatal diagnosis research. Carrying the whole genome coding of the fetus, fetal nucleated red blood cells (FNRBCs) have been pursued as a surrogate biomarker traveling around in maternal blood. Here, by combining a unique microbead-based centrifugal separation and enzymatic release, we demonstrated a novel method for FNRBC isolation from the blood samples. First, the gelatin-coated silica microbeads were modified with FNRBC-specific antibody (anti-CD147) to capture the target cells in the blood samples. Then, the density difference between microbead-bound FNRBCs and normal blood cells enables the purification of FNRBCs via an improved high-density percoll-based separation. The non-invasive release of FNRBCs can then be achieved by enzymatically degrading the gelatin film on the surface of the microbeads, allowing a gentle release of the captured target cells with as high as 84% efficiency and ∼80% purity. We further applied it to isolate fetal cells from maternal peripheral blood. The released cells were analyzed by real-time polymerase chain reaction to verify their fetal origin and fluorescent in situ hybridization to detect fetal chromosome disorders. This straightforward and reliable alternative platform for FNRBC detection may have the potential for realizing facile NIPD.

Erythrocyte membrane-coated gold nanocages for targeted photothermal and chemical cancer therapy.

Recently, red blood cell (RBC) membrane-coated nanoparticles have attracted much attention because of their excellent immune escapability; meanwhile, gold nanocages (AuNs) have been extensively used for cancer therapy due to their photothermal effect and drug delivery capability. The combination of the RBC membrane coating and AuNs may provide an effective approach for targeted cancer therapy. However, few reports have shown the utilization of combining these two technologies. Here, we design erythrocyte membrane-coated gold nanocages for targeted photothermal and chemical cancer therapy. First, anti-EpCam antibodies were used to modify the RBC membranes to target 4T1 cancer cells. Second, the antitumor drug paclitaxel (PTX) was encapsulated into AuNs. Then, the AuNs were coated with the modified RBC membranes. These new nanoparticles were termed EpCam-RPAuNs. We characterized the capability of the EpCam-RPAuNs for selective tumor targeting via exposure to near-infrared irradiation. The experimental results demonstrate that EpCam-RPAuNs can effectively generate hyperthermia and precisely deliver the antitumor drug PTX to targeted cells. We also validated the biocompatibility of the EpCam-RAuNs in vitro. By combining the molecularly modified targeting RBC membrane and AuNs, our approach provides a new way to design biomimetic nanoparticles to enhance the surface functionality of nanoparticles. We believe that EpCam-RPAuNs can be potentially applied for cancer diagnoses and therapies.

Platelet-Facilitated Photothermal Therapy of Head and Neck Squamous Cell Carcinoma.

Here, we present a platelet-facilitated photothermal tumor therapy (PLT-PTT) strategy, in which PLTs act as carriers for targeted delivery of photothermal agents to tumor tissues and enhance the PTT effect. Gold nanorods (AuNRs) were first loaded into PLTs by electroporation and the resulting AuNR-loaded PLTs (PLT-AuNRs) inherited long blood circulation and cancer targeting characteristics from PLTs and good photothermal property from AuNRs. Using a gene-knockout mouse model, we demonstrate that the administration of PLT-AuNRs and localizing laser irradiation could effectively inhibit the growth of head and neck squamous cell carcinoma (HNSCC). In addition, we found that the PTT treatment augmented PLT-AuNRs targeting to the tumor sites and in turn, improved the PTT effects in a feedback manner, demonstrating the unique self-reinforcing characteristic of PLT-PTT in cancer therapy.

Synthetic nanoparticles camouflaged with biomimetic erythrocyte membranes for reduced reticuloendothelial system uptake.

Suppression of the reticuloendothelial system (RES) uptake is one of the most challenging tasks in nanomedicine. Coating stratagems using polymers, such as poly(ethylene glycol) (PEG), have led to great success in this respect. Nevertheless, recent observations of immunological response toward these synthetic polymers have triggered a search for better alternatives. In this work, natural red blood cell (RBC) membranes are camouflaged on the surface of Fe3O4 nanoparticles for reducing the RES uptake. In vitro macrophage uptake, in vivo biodistribution and pharmacokinetic studies demonstrate that the RBC membrane is a superior alternative to the current gold standard PEG for nanoparticle 'stealth'. Furthermore, we systematically investigate the in vivo potential toxicity of RBC membrane-coated nanoparticles by blood biochemistry, whole blood panel examination and histology analysis based on animal models. The combination of synthetic nanoparticles and natural cell membranes embodies a novel and biomimetic nanomaterial design strategy and presents a compelling property of functional materials for a broad range of biomedical applications.

Red Blood Cell Membrane as a Biomimetic Nanocoating for Prolonged Circulation Time and Reduced Accelerated Blood Clearance.

For decades, poly(ethylene glycol) (PEG) has been widely incorporated into nanoparticles for evading immune clearance and improving the systematic circulation time. However, recent studies have reported a phenomenon known as "accelerated blood clearance (ABC)" where a second dose of PEGylated nanomaterials is rapidly cleared when given several days after the first dose. Herein, we demonstrate that natural red blood cell (RBC) membrane is a superior alternative to PEG. Biomimetic RBC membrane-coated Fe(3)O(4) nanoparticles (Fe(3)O(4) @RBC NPs) rely on CD47, which is a "don't eat me" marker on the RBC surface, to escape immune clearance through interactions with the signal regulatory protein-alpha (SIRP-α) receptor. Fe(3)O(4) @RBC NPs exhibit extended circulation time and show little change between the first and second doses, with no ABC suffered. In addition, the administration of Fe(3)O(4) @RBC NPs does not elicit immune responses on neither the cellular level (myeloid-derived suppressor cells (MDSCs)) nor the humoral level (immunoglobulin M and G (IgM and IgG)). Finally, the in vivo toxicity of these cell membrane-camouflaged nanoparticles is systematically investigated by blood biochemistry, hematology testing, and histology analysis. These findings are significant advancements toward solving the long-existing clinical challenges of developing biomaterials that are able to resist both immune response and rapid clearance.

Genetically Engineered Cytomembrane Nanovaccines for Cancer Immunotherapy.

Cancer nanovaccines have attracted widespread attention by inducing potent cytotoxic T cell responses to improve immune checkpoint blockade (ICB) therapy, while the lack of co-stimulatory molecules limits their clinical applications. Here, a genetically engineered cancer cytomembrane nanovaccine is reported that simultaneously overexpresses co-stimulatory molecule CD40L and immune checkpoint inhibitor PD1 to elicit robust antitumor immunity for cancer immunotherapy. The CD40L and tumor antigens inherited from cancer cytomembranes effectively stimulate dendritic cell (DC)-mediated immune activation of cytotoxic T cells, while the PD1 on cancer cytomembranes significantly blocks PD1/PD-L1 signaling pathway, synergistically stimulating antitumor immune responses. Benefiting from the targeting ability of cancer cytomembranes, this nanovaccines formula shows an enhanced lymph node trafficking and retention. Compared with original cancer cytomembranes, this genetically engineered nanovaccine induces twofold DC maturation and shows satisfactory precaution efficacy in a breast tumor mouse model. This genetically engineered cytomembrane nanovaccine offers a simple, safe, and robust strategy by incorporating cytomembrane components and co-stimulatory molecules for enhanced cancer immunotherapy.

Magneto-controllable capture and release of cancer cells by using a micropillar device decorated with graphite oxide-coated magnetic nanoparticles.

Aiming to highly efficient capture and analysis of circulating tumor cells, a micropillar device decorated with graphite oxide-coated magnetic nanoparticles is developed for magneto-controllable capture and release of cancer cells. Graphite oxide-coated, Fe3 O4 magnetic nanoparticles (MNPs) are synthesized by solution mixing and functionalized with a specific antibody, following by the immobilization of such modified MNPs on our designed micropillar device. For the proof-of-concept study, a HCT116 colorectal cancer cell line is employed to exam the capture efficiency. Under magnetic field manipulation, the high density packing of antibody-modified MNPs on the micropillars increases the local concentration of antibody, as well as the topographic interactions between cancer cells and micropillar surfaces. The flow rate and the micropillar geometry are optimized by studying their effects on capture efficiency. Then, a different number of HCT116 cells spiked in two kinds of cell suspension are investigated, yielding capture efficiency >70% in culture medium and >40% in blood sample, respectively. Moreover, the captured HCT116 cells are able to be released from the micropillars with a saturated efficiency of 92.9% upon the removal of applied magnetic field and it is found that 78% of the released cancer cells are viable, making them suitable for subsequent biological analysis.

Biocompatible TiO2 nanoparticle-based cell immunoassay for circulating tumor cells capture and identification from cancer patients.

We demonstrate the isolation of circulating tumor cells (CTCs) with a biocompatible nano-film composed of TiO2 nanoparticles. Due to the enhanced topographic interaction between nano-film and cancer cell surface, cancer cells (HCT116) spiked into PBS and healthy blood can be recovered from the suspension, whose efficiencies were respectively 80 % and 50 %. Benifit from the biocompatibility of this nano-film, in-situ culture of the captured cancer cells is also available, which provides an alternative selection when the capture cell number was inadequate or the sample cannot be analyzed immediately. For the proof-of-concept study, we use this nano-film to separate the circulating tumor cells from the colorectal and gastric cancer patient peripheral blood samples and the captured CTCs are identified by a three-colored immunocytochemistry method. We investigated the cancer cells capture strength at the nano-bio interface through exposing the cells to fluid shear stress in microfluidic device, which can be utilized to increase the purity of CTCs. The result indicated that 50 % of the captured cells can be detached from the substrate when the fluid shear stress was 180 dyn cm(-2). By integration of this CTCs capture nano-film with other single cell analysis device, we expected to further explore their applications in genome sequencing based on the captured CTCs.

Solution-gated graphene field effect transistors integrated in microfluidic systems and used for flow velocity detection.

Solution-gated graphene field effect transistors (SGGT) were integrated in microfluidic systems. The transfer characteristics of a SGGT with an Ag/AgCl gate electrode shifted horizontally with the change of the ionic concentration of KCl solution in the microchannel and the relationship can be fitted with the Nernst equation, which was attributed to the change of the potential drop at the Ag/AgCl electrode. Therefore the gate electrode is one important factor for the ion sensitive property of the SGGT. Then SGGTs were used as flow velocity sensors, which were based on measuring the streaming potentials in microfluidic channels. A linear relationship between the shift of the transfer curve of the SGGT and the flow velocity was obtained, indicating that the SGGT is a promising transducer for measuring flow velocity in a microchip. Since the streaming potential is influenced by the three physical quantities, including the flow velocity, the ionic strength of the fluid and the zeta potential of the substrate, the device can be used for sensing any one of the three quantities when the other two were known. It is noteworthy that SGGTs have been used for various types of chemical and biological sensors. Array of the devices integrated in multichannel microchips are expected to find many important applications in the lab-on-a-chip systems in the future.