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.

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.

Vesicular Antibodies: Shedding Light on Antibody Therapeutics with Cell Membrane Nanotechnology.

The high stability of antibodies and their ability to precisely bind to antigens and endogenous immune receptors, as well as their susceptibility to protein engineering, enable antibody-based therapeutics to be widely applied in cancer, inflammation, infection, and other disorders. Nevertheless, the application of traditional antibody-based therapeutics has certain limitations, such as high price, limited permeability, and protein engineering complexity. Recent breakthroughs in cell membrane nanotechnology have deepened the understanding of the critical role of membrane protein receptors in disease treatment, enabling vesicular-antibody-based therapeutics. Here, the concept of vesicular antibodies that are obtained by modifying target antibodies onto cell membranes for biomedical applications is proposed. Given that an antibody is basically a protein, as an extension of this concept, vesicles or membrane-coated nanoparticles that use surface antibodies and protein receptors on cell membranes for biomedical applications as vesicular antibodies are defined. Furthermore, several engineering strategies for vesicular antibodies are summarized and how vesicular antibodies can be used in a variety of situations is highlighted. In addition, current challenges and future prospects of vesicular antibodies are also discussed. It is anticipated this perspective will provide new insights on the development of next-generation antibodies for enhanced therapeutics.

Neutrophil membrane-coated immunomagnetic nanoparticles for efficient isolation and analysis of circulating tumor cells.

The isolation and analysis of scarce circulating tumor cells (CTCs) with immunomagnetic nanoparticles (IMNs) have shown promising outcomes in noninvasive cancer diagnosis. However, the IMNs adsorb nonspecific proteins after entering into biofluids and the formed protein coronas cover surface targeting ligands, limiting the detection efficiency of IMNs. In addition, the interaction between surface targeting ligands and white blood cells (WBCs) significantly limits the purity of CTCs isolated by IMNs. Furthermore, the interfacial collision of nanoparticles and cells has negative effects on the viability of isolated CTCs. All of these limitations synthetically restrict the isolation and analysis of rare CTCs for early diagnosis and precision medicine. Here, we proposed that surface functionalization of IMNs with neutrophil membranes can simultaneously reduce nonspecific protein adsorption, enhance the interaction with CTCs, reduce the distraction from WBCs, and improve the viability of isolated CTCs. In spiked blood samples, our neutrophil membrane-coated IMNs (Neu-IMNs) exhibited a superior separation efficiency from 41.36% to 96.82% and an improved purity from 40.25% to 90.68% when compared to bare IMNs. Additionally, we successfully isolated CTCs in 19 out of total 20 blood samples from breast cancer patients using Neu-IMNs and further confirmed the feasibility of the isolated CTCs for downstream cell sequencing. Our work provides a new perspective on engineered IMNs for efficient isolation and analysis of CTCs, paving the way for early noninvasive diagnosis of cancer.

Noninvasive Optical Isolation and Identification of Circulating Tumor Cells Engineered by Fluorescent Microspheres.

Circulating tumor cells (CTCs) are rare, meaning that current isolation strategies can hardly satisfy efficiency and cell biocompatibility requirements, which hinders clinical applications. In addition, the selected cells require immunofluorescence identification, which is a time-consuming and expensive process. Here, we developed a method to simultaneously separate and identify CTCs by the integration of optical force and fluorescent microspheres. Our method achieved high-purity separation of CTCs without damage through light manipulation and avoided additional immunofluorescence staining procedures, thus achieving rapid identification of sorted cells. White blood cells (WBCs) and CTCs are similar in size and density, which creates difficulties in distinguishing them optically. Therefore, fluorescent PS microspheres with high refractive index (RI) are designed here to capture the CTCs (PS-CTCs) and increase the average index of refraction of PS-CTCs. In optofluidic chips, PS-CTCs were propelled to the collection channel from the sample mixture, under the radiation of light force. Cells from the collection outlet were easily identified under a fluorescence microscope due to the fluorescence signals of PS microspheres. This method provides an approach for the sorting and identification of CTCs, which holds great potential for clinical applications in early diagnosis of disease.

A light-induced hydrogel responsive platform to capture and selectively isolate single circulating tumor cells.

Isolation of circulating tumor cells (CTCs) from patients is a challenge due to the rarity of CTCs. Recently, various platforms to capture and release CTCs for downstream analysis have been developed. However, most of the reported release methods provide external stimuli to release all captured cells, which lead to lack of specificity in the pool of collected cells, and the external stimuli may affect the activity of the released cells. Here, we presented a simple method for single-cell recovery to overcome the shortcomings, which combined the nanostructures with a photocurable hydrogel, chondroitin sulfate methacryloyl (CSMA). In brief, we synthesized gelatin nanoparticles (Gnps) and modified them on flat glass (Gnp substrate) for the specific capture of CTCs. A 405 nm laser was projected onto the selected cells, and then CSMA was cured to encapsulate the selected CTCs. Unselected cells were removed with MMP-9 enzyme solution, and selected CTCs were recovered using a microcapillary. Finally, the photocurable hydrogel-encapsulated cells were analyzed by nucleic acid detection. In addition, the results suggested that the isolation platform showed good biocompatibility and successfully achieved the isolation of selected cells. In summary, our light-induced hydrogel responsive platform holds certain potential for clinical applications.

Emerging Microfluidic Technologies for the Detection of Circulating Tumor Cells and Fetal Nucleated Red Blood Cells.

Blood tests have been a powerful tool for the clinical analysis of many diseases. With the advances in microfluidic technology, two more specific indicators from the circulation system, namely, emerging "liquid biopsy" of circulating tumor cells (CTCs) and fetal nucleated red blood cells (fNRBCs), can be screened and analyzed as a simple blood test for the noninvasive diagnosis of cancers as well as fetal disorders. The unique feature of precisely manipulating a trace of fluid endows microfluidic devices with the ability to isolate CTCs or fNRBCs from numerous blood cells with high performance, which undoubtedly facilitates biomedical applications of these two kinds of rare cells. In this review, advanced developments in microfluidic technologies focusing on the detection and sorting of rare CTCs and fNRBCs from peripheral blood are summarized. The development of microfluidic devices incorporated with various multifunctional microstructures and nanomaterials for enhancing the sensitivity, purity, and viability of CTC or fNRBC detection enables CTC molecular analysis and fNRBC-based noninvasive prenatal diagnosis (NIPD). These microfluidics-based approaches provide great potential opportunities in noninvasive cancer diagnosis or NIPD applications.

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.

ZnO nanowire-integrated bio-microchips for specific capture and non-destructive release of circulating tumor cells.

Circulating tumor cells (CTCs) are one type of significant biomarker in cancer patients' blood that have been attracting attention from researchers for decades, and their efficient and viable isolation is of vital importance in cancer prevention and treatment. However, the development of efficient and low-cost bio-microchips still faces significant challenges. In this paper, we construct a novel three-dimensional micro-nano bio-microchip that has dual functions of specifically capturing and non-destructively releasing cancer cells. ZnO nanowire arrays were vertically grown on the surface of a polydimethylsiloxane (PDMS) pillar substrate with a gear structure (ZnO-coated G-PDMS pillar microchips). The gear structure provides more binding sites for antibodies and target cancer cells, while ZnO nanowires provide a rough surface for CTC attachment and size-specific effects for retaining CTCs. For subsequent culture and bioanalysis, the captured CTCs can be non-destructively released with high efficiency and good viability using a mild acidic solution treatment. Furthermore, the manufacturing process of the G-PDMS pillar microchips is convenient and low-cost, and the preparation approach of the ZnO nanowire is mature and simple to operate. In particular, the bio-microchips showed high capture efficiency (91.11% ± 5.53%) and excellent cell viability (96%) using a spiked cell sample. Moreover, we successfully achieved the specific fluorescent labeling of CTCs in 9 clinical breast cancer patients' samples. The ZnO-coated G-PDMS pillar microchips not only have great potential for new target drug development for cancer stem cells but also open up new opportunities for individualized treatment.

An Acoustic Droplet-Induced Enzyme Responsive Platform for the Capture and On-Demand Release of Single Circulating Tumor Cells.

The recovery of rare single circulating tumor cells (CTCs) from patients has great potential to facilitate the study of cell heterogeneity and cancer metastasis, which may promote the development of individualized cancer immunotherapy. Herein, a versatile single-cell recovery approach that utilizes an acoustic droplet-induced enzyme responsive platform for the capture and on-demand release of single CTCs is proposed. The platform combines a multifunctional enzyme-responsive gelatin nanoparticle (GNP)-decorated substrate (GNP-chip) for specific capture with an acoustic droplet positioning technique to realize on-demand release of single CTCs. The acoustic droplet dispenser is employed to generate oxidized alginate microdroplets containing the MMP-9 enzyme (OA-MMP-9) with controllable size and precise positioning upon the cell-attached GNP-chip, allowing controlled cell-surface biodegradation under enzymatic reactions followed by calcium chloride (CaCl2) solution treatment to form single-cell encapsulated calcium alginate hydrogels. Benefitting from the existence of hydrogels, the released cells could be efficiently recovered by microcapillary. Results demonstrate that the encapsulated cells maintain good cell morphology in the hydrogels, which allow further single-cell nucleic acid analysis. As a proof-of-concept platform, this approach enables reliable and efficient retrieval of single CTCs and holds the potential for versatility in single-cell analysis systems.

Biomimetic Immunomagnetic Nanoparticles with Minimal Nonspecific Biomolecule Adsorption for Enhanced Isolation of Circulating Tumor Cells.

Immunomagnetic micro/nanoparticles (IMNs) have been widely used to isolate rare circulating tumor cells (CTCs) from blood samples for early diagnosis of cancers. However, when entering into biofluids, IMNs nonspecifically adsorb biomolecules and the in situ formed biomolecule corona covers IMN surface ligands and weakens the targeting capabilities of IMNs. In this work, we demonstrated that by surface coating of IMNs with red blood cell (RBC)-derived vesicles, the obtained biomimetic particles (RBC-IMNs) basically adsorb no biomolecules and maintain the CTC targeting ability when exposed to plasma. Compared to IMNs, RBC-IMNs exhibited an excellent cell isolation efficiency in spiked blood samples, which was improved to 95.71% from 60.22%. Furthermore, by using RBC-IMNs, we successfully isolated CTCs in 28 out of 30 prostate cancer patient blood samples and further showed the robustness of RBC-IMNs in downstream cell sequencing. The work presented here provides a new insight into developing targeted nanomaterials for biological and medical applications.

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.

Capture and "self-release" of circulating tumor cells using metal-organic framework materials.

Capturing circulating tumor cells (CTCs) from peripheral blood for subsequent analyses has shown potential in precision medicine for cancer patients. Broad as the prospect is, there are still some challenges that hamper its clinical applications. One of the challenges is to maintain the viability of the captured cells during the capturing and releasing processes. Herein, we have described a composite material that could encapsulate a magnetic Fe3O4 core in a MIL-100 shell (MMs), which could respond to pH changes and modify the anti-EpCAM antibody (anti-EpCAM-MMs) on the surface of MIL-100. After the anti-EpCAM-MMs captured the cells, there was no need for additional conditions but with the acidic environment during the cell culture process, MIL-100 could realize automatic degradation, leading to cell self-release. This self-release model could not only improve the cell viability, but could also reduce the steps of the release process and save human and material resources simultaneously. In addition, we combined clinical patients' case diagnosis with the DNA sequencing and next generation of RNA sequencing technologies in the hope of precision medicine for patients in the future.

A microfluidic platform utilizing anchored water-in-oil-in-water double emulsions to create a niche for analyzing single non-adherent cells.

Non-adherent cells play key roles in various biological processes. Studies on this type of cell, especially at single-cell resolution, help reveal molecular mechanisms underlying many biological and pathological processes. The emerging microfluidics technology has developed effective methods for analyzing cells. However, it remains challenging to treat and monitor single live non-adherent cells in an in situ, long-term, and real-time manner. Herein, a microfluidic platform was set up to generate and anchor cell-laden water-in-oil-in-water (W/O/W) double emulsions (DEs) to investigate these cells. Within the device, W/O/W DEs encapsulating non-adherent cells were generated through two adjacent flow-focusing structures and subsequently anchored in an array of microchambers. These droplets maintained the W/O/W structure and the anchorage status in the continuous perfusion fluid for at least one week. The mass transfer of different molecules with suitable molecular weights and partition coefficients between the interior and exterior of W/O/W DEs could be regulated by perfusion fluid flow rates. These features endow this platform with potential to continuously supply encapsulated non-adherent cells with nutrients or small-molecule stimuli/drugs through fluid perfusion. Meanwhile, the confinement of cells in the anchored DEs favored long-term monitoring of cellular dynamic behaviors and responses. As a proof of concept, fluorescein diacetate (FDA) was employed to visualize the cellular uptake and biochemical metabolism of TF-1 human erythroleukemia cells. We believe that this W/O/W DE anchorage and perfusion platform would benefit single-cell-level studies as well as small-molecule drug discovery requiring live non-adherent cells.

Engineered red blood cells for capturing circulating tumor cells with high performance.

Filtration of circulating tumor cells (CTCs) in peripheral blood is of proven importance for early cancer diagnosis, treatment monitoring, metastasis diagnosis, and prognostic evaluation. However, currently available strategies for enriching CTCs, such as magnetic activated cell sorting (MACS), face serious problems with purity due to nonspecific interactions between beads and leukocytes in the process of capturing. In the present study, the tumor-targeting molecule folic acid (FA) and magnetic nanoparticles (MNPs) were coated on the surface of red blood cells (RBCs) by hydrophobic interaction and chemical conjugation, respectively. The resulting engineered RBCs rapidly adhered to CTCs and the obtained CTC-RBC conjugates were isolated in a magnetic field. After treatment with RBC lysis buffer and centrifugation, CTCs were released and captured. The duration of the entire process was less than three hours. Cell counting showed that the capture efficiency was above 90% and the purity of the obtained CTCs was higher than 75%. The performance of the proposed method exceeded that of MACS® beads (80% for capture efficiency and 20% for purity) under the same conditions. The obtained CTCs could be successfully re-cultured and proliferated in vitro. Our engineered RBCs have provided a novel method for enriching rare cells in the physiological environment.

Gelatin Nanoparticle-Coated Silicon Beads for Density-Selective Capture and Release of Heterogeneous Circulating Tumor Cells with High Purity.

Background: Circulating tumor cells (CTCs) are a burgeoning topic in cancer biomarker discovery research with minimal invasive blood draws. CTCs can be used as potential biomarkers for disease prognosis, early cancer diagnosis and pharmacodynamics. However, the extremely low abundance of CTCs limits their clinical utility because of technical challenges such as the isolation and subsequent detailed molecular and functional characterization of rare CTCs from patient blood samples. Methods: In this study, we present a novel density gradient centrifugation method employing biodegradable gelatin nanoparticles coated on silicon beads for the isolation, release, and downstream analysis of CTCs from colorectal and breast cancer patients. Results: Using clinical patient/spiked samples, we demonstrate that this method has significant CTC-capture efficiency (>80%) and purity (>85%), high CTC release efficiency (94%) and viability (92.5%). We also demonstrate the unparalleled robustness of our method in downstream CTC analyses such as the detection of PIK3CA mutations. Conclusion: The efficiency and versatility of the multifunctional density microbeads approach provides new opportunities for personalized cancer diagnostics and treatments.

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.

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.

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.

Microfluidic Electroporation-Facilitated Synthesis of Erythrocyte Membrane-Coated Magnetic Nanoparticles for Enhanced Imaging-Guided Cancer Therapy.

Biomimetic cell membrane-coated nanoparticles (CM-NPs) with superior biochemical properties have been broadly utilized for various biomedical applications. Currently, researchers primarily focus on using ultrasonic treatment and mechanical extrusion to improve the synthesis of CM-NPs. In this work, we demonstrate that microfluidic electroporation can effectively facilitate the synthesis of CM-NPs. To test it, Fe3O4 magnetic nanoparticles (MNs) and red blood cell membrane-derived vesicles (RBC-vesicles) are infused into a microfluidic device. When the mixture of MNs and RBC-vesicles flow through the electroporation zone, the electric pulses can effectively promote the entry of MNs into RBC-vesicles. After that, the resulting RBC membrane-capped MNs (RBC-MNs) are collected from the chip and injected into experimental animals to test the in vivo performance. Owing to the superior magnetic and photothermal properties of the MN cores and the long blood circulation characteristic of the RBC membrane shells, core-shell RBC-MNs were used for enhanced tumor magnetic resonance imaging (MRI) and photothermal therapy (PTT). Due to the completer cell membrane coating, RBC-MNs prepared by microfluidic electroporation strategy exhibit significantly better treatment effect than the one fabricated by conventional extrusion. We believe the combination of microfluidic electroporation and CM-NPs provides an insight into the synthesis of bioinpired nanoparticles to improve cancer diagnosis and therapy.