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.

In Situ Microreaction Platform Based on Acoustic Droplet Manipulation for Ultra-High-Precision Multiplex Bioassay.

Liquid droplets rectors have been used in clinical diagnosis, high throughput screening and bioassay. However, it is challenging for droplet reactors to be used in practical applications due to the difficulty of uniformly mixing ultrasmall volumes of samples and the lack of rapid and high-precision detection protocols. Here, we have developed an acoustic droplet system for rapid and efficient biological detection and chemical screening. By employing acoustic wave devices, rapid and nondestructive uniform mixing of ∼nL-µL droplets can be achieved. By the acoustophoretic force, the perturbation of the droplets can quickly concentrate the sample and increase the detection limit by five times. Through the color reaction and the coordinated detection of photodiodes, we have developed a biomarker detection protocol with short reaction time and high accuracy. As a proof-of-concept application, we demonstrated that this system can detect ultrasmall or low-abundance samples faster and more accurately, highlighting its wide application in analytical chemistry, basic research, and clinical medicine.

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.

On-chip rapid drug screening of leukemia cells by acoustic streaming.

Rapid and personalized single-cell drug screening testing plays an essential role in acute myeloid leukemia drug combination chemotherapy. Conventional chemotherapeutic drug screening is a time-consuming process because of the natural resistance of cell membranes to drugs, and there are still great challenges related to using technologies that change membrane permeability such as sonoporation in high-throughput and precise single-cell drug screening with minimal damage. In this study, we proposed an acoustic streaming-based non-invasive single-cell drug screening acceleration method, using high-frequency acoustic waves (>10 MHz) in a concentration gradient microfluidic device. High-frequency acoustics leads to increased difficulties in inducing cavitation and generates acoustic streaming around each single cell. Therefore, single-cell membrane permeability is non-invasively increased by the acoustic pressure and acoustic streaming-induced shear force, which significantly improves the drug uptake process. In the experiment, single human myeloid leukemia mononuclear (THP-1) cells were trapped by triangle cell traps in concentration gradient chips with different cytarabine (Ara-C) drug concentrations. Due to this dual acoustic effect, the drugs affect cell viability in less than 30 min, which is faster than traditional methods (usually more than 24 h). This dual acoustic effect-based drug delivery strategy has the potential to save time and reduce the cost of drug screening, when combined with microfluidic technology for multi-concentration drug screening. This strategy offers enormous potential for use in multiple drug screening or efficient drug combination screening in individualized/personalized treatments, which can greatly improve efficiency and reduce costs.

Acoustic Droplet-Assisted Superhydrophilic-Superhydrophobic Microarray Platform for High-Throughput Screening of Patient-Derived Tumor Spheroids.

Cell-based high-throughput screening is a key step in the current disease-based research, drug development, and precision medicine. However, it is challenging to establish a rapid culture and screening platform for rare cells (patient-derived) due to the obvious differences between the traditional 2D cell model and the tumor microenvironment, as well as the lack of a low-consumption screening platform for low numbers of cells. Here, we developed an acoustic drop-assisted superhydrophilic-superhydrophobic microarray platform for the rapid culture and screening of a few cells. By employing hydrophilic and hydrophobic microarrays, we can automatically distribute the cell suspension into uniform droplets, and these cells can spontaneously form compact 3D cell spheroids within 36 h (similar to the microenvironment of tumors in vivo). By using the acoustic droplet ejection device, we can accurately inject a drug solution with a volume of ∼pL to ∼nL into the droplet, and the whole process can be completed within 20 ms (one print). By using three different cell lines (Caco-2, MCF-7, and HeLa) to optimize the platform, the culture and screening of five patients' colon cancer were subsequently realized. Using three conventional chemotherapeutics (5-fluorouracil, cetuximab, and panitumumab) of various concentrations, the best treatment was screened out and compared with the actual treatment effect of the patients, and the results were extremely similar. As a proof-of-concept application, we have proved that our platform can quickly cultivate patient samples and effectively screen the best treatment methods, highlighting its wide application in precision medicine, basic tumor research, and drug development.

Acoustic Droplet Vitrification Method for High-Efficiency Preservation of Rare Cells.

Cryopreservation is a key step for current translational medicine including reproductive medicine, regenerative medicine, and cell therapy. However, it is challenging to preserve rare cells for practical applications due to the difficulty in handling low numbers of cells as well as the lack of highly efficient and biocompatible preservation protocols. Here, we developed an acoustic droplet vitrification method for high-efficiency handling and preservation of rare cells. By employing an acoustic droplet ejection device, we can encapsulate rare cells into water-in-air droplets with a volume from ∼pL to ∼nL and deposit these cell-containing droplets into a droplet array onto a substrate. By incorporating a cooling system into the droplet array substrate, we can vitrify hundreds to thousands of rare cells at an ultrafast speed (about ∼2 s) based on the high surface to volume ratio of the droplets. By optimizing this method with three different cell lines (a human lung cancer cell line, A549 cells, a human liver cell line, L02 cells, and a mouse embryonic fibroblast cell line, 3T3-L1 cells), we developed an effective protocol with excellent cell viability (e.g., >85% for days, >70% for months), proliferation, and adhesion. As a proof-of-concept application, we demonstrated that our method can rapidly handle and efficiently preserve rare cells, highlighting its broad applications in species diversity, basic research, and clinical medicine.

Highly biocompatible and recyclable biomimetic nanoparticles for antibiotic-resistant bacteria infection.

Increasing number of resistant bacteria have emerged with the overuse of antibiotics, which indicates that the bacterial infection has become a global challenge. Furthermore, the pollution of antibiotics to the environment has become a serious threat to public health. It is known that toxins produced by bacteria are the main cause of bacterial infections. Photothermal therapy is an effective antibacterial approach. However, the photothermal reagents cannot eliminate bacterial toxins, and even some anti-bacterial materials are toxic. Here, we synthesized a biomimetic recycled nanoparticle, red blood cell (RBC) membrane-coated Fe3O4 nanoparticles (RBC@Fe3O4), as an antibacterial agent. The RBC@Fe3O4 nanoparticles act as nano-sponges to trap toxins and then kill them all with a photothermal effect. We can describe this process simply as a battle between two armies. Our strategy is to disarm the "enemy" so that we can easily kill the "enemy" who has no power, which results in enhancing the bactericidal efficacy. The toxin of methicillin-resistant Staphylococcus aureus (MRSA) was absorbed by RBC@Fe3O4in vitro. In addition, in vivo studies proved that the RBC@Fe3O4 nanoparticles confer obvious survival benefits against toxin-induced lethality by absorbing the toxin of MRSA. Furthermore, using a mouse model of MRSA wound infection, the RBC@Fe3O4 nanoparticles with laser irradiation were found to have a superior wound-healing effect. Simultaneously, the RBC@Fe3O4 nanoparticles could be recycled in a simple way without affecting the bactericidal efficacy. The highly biocompatible and recyclable RBC@Fe3O4 biomimetic nanoparticles based on photothermal therapy and bacterial toxin adsorption strategy are promising for treating bacterial infections.

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.

Efficient Detection and Single-Cell Extraction of Circulating Tumor Cells in Peripheral Blood.

Circulating tumor cells (CTCs) play an important role in cancer biology studies. To further elucidate the role of single CTCs in tumor metastasis and prognosis, effective methods must be developed to isolate and encapsulate single CTCs. In this work, a single CTC capture and encapsulation platform based on ZnO nanofibers and surface acoustic waves was constructed. We also demonstrated that this platform can capture and encapsulate single CTCs efficiently. We first validated that the dense ZnO nanofibers provide additional binding sites, resulting in an increased capture efficiency of up to 93.3%. We then demonstrated that the release efficiency was increased to 100% by dissolving the substrate with ultralow concentration (25 mM) phosphoric acid, and the activity of the released cells was not affected. Furthermore, the released cells were suspended in alginate solution and encapsulated single CTCs via droplet-based surface acoustic wave devices. The encapsulating rate of a single cell is up to 13% under a pulse width duration of 520 µs. Few technology based on nanofibers and acoustic tweezers for single-cell encapsulation via acoustic droplet vaporization has been reported. It has a wide range of potential applications to acquire single target cells, which may facilitate further early clinical diagnosis and treatment of cancer patients.

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.

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.

Rapid and efficient isolation and detection of circulating tumor cells based on ZnS:Mn2+ quantum dots and magnetic nanocomposites.

Rapid and non-destructive detection of circulating tumor cells (CTCs) with no disruption of their functions is of great significance for clinical tumor therapy. However, many existing methods for CTC detection commonly rely on conventional three-color immunofluorescence identification, which damages CTCs and easily causes loss of cells. Here, we employed a method to simultaneously capture and authenticate CTCs based on immunonanocomposites (ZnS:Mn2+ QDs and Fe3O4/SiO2) equipped with permanent fluorescent and magnetic properties. A multifunctional nanocomposite was synthesized by encapsulating ZnS:Mn2+ quantum dots (QDs) and Fe3O4 nanoparticles into SiO2 nanospheres and bio-conjugating tumor-specific anti-EpCAM antibodies onto the surface. The resulting nanocomposite had a high tumor cell binding ability, and the Fe3O4 nanoparticles had a rapid magnetic response that enabled capture of circulating tumor cells from patients' blood within minutes. In addition, the cell-immunonanocomposites complexes could be directly recognized by the yellow-orange light emitted by the ZnS:Mn2+ quantum dots, thus labeling cells without utilizing the complicated and destructive procedures involved in traditional CTCs identification. We successfully achieved a high capture efficiency of up to 90.8%, and the specific fluorescence labeling of CTCs was realized in 9 clinical breast cancer patients' samples. Furthermore, this simple, convenient and cell-friendly approach is significant for solving the problems of cell viability and enables non-destructive CTC detection, which marks an advance in cancer treatment and clinical 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.

Cancer Cell Membrane Camouflaged Nanoparticles to Realize Starvation Therapy Together with Checkpoint Blockades for Enhancing Cancer Therapy.

Although anti-PD-1 immunotherapy is widely used to treat melanoma, its efficacy still has to be improved. In this work, we present a therapeutic method that combines immunotherapy and starvation therapy to achieve better antitumor efficacy. We designed the CMSN-GOx method, in which mesoporous silica nanoparticles (MSN) are loaded with glucose oxidase (GOx) and then encapsulate the surfaces of cancer cell membranes to realize starvation therapy. By functionalizing the MSN's biomimetic surfaces, we can synthesize nanoparticles that can escape the host immune system and homologous target. These attributes enable the nanoparticles to have improved cancer targeting ability and enrichment in tumor tissues. Our synthetic CMSN-GOx complex can ablate tumors and induce dendritic cell maturity to stimulate an antitumor immune response. We performed an in vivo analysis of these nanoparticles and determined that our combined therapy CMSN-GOx plus PD-1 exhibits a better antitumor therapeutic effect than therapies using CMSN-GOx or PD-1 alone. Additionally, we used the positron emission tomography imaging to measuring the level of glucose metabolism in tumor tissues, for which we investigate the effect with the cancer therapy in vivo.

A digital acoustofluidic device for on-demand and oil-free droplet generation.

We report a digital acoustofluidic device for on-demand and oil-free droplet generation. By applying a programmed radio frequency signal to a circular interdigital transducer, the dynamic focused acoustic pressure profiles generated rise up and dispense sample liquids from a reservoir to dynamically eject the droplets into the air. Our device allows droplets to be dispensed on demand with precisely controlled generation time and sequence, and accurate droplet volume. Moreover, we also demonstrate the generation of a droplet with a volume of 24 pL within 10 ms, as well as the encapsulation of a single cell into droplets. This acoustofluidic droplet generation technique is simple, biocompatible, and enables the on-demand droplet generation and encapsulation of many different biological materials with precise control, which is promising for single cell sampling and analysis applications.

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.

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.

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.