Exosomes in diagnostic and therapeutic applications of ovarian cancer.

Ovarian cancer accounts for more deaths than any other female reproductive tract cancer. The major reasons for the high mortality rates include delayed diagnoses and drug resistance. Hence, improved diagnostic and therapeutic options for ovarian cancer are a pressing need. Extracellular vesicles (EVs), that include exosomes provide hope in both diagnostic and therapeutic aspects. They are natural lipid nanovesicles secreted by all cell types and carry molecules that reflect the status of the parent cell. This facilitates their potential use as biomarkers for an early diagnosis. Additionally, EVs can be loaded with exogenous cargo, and have features such as high stability and favorable pharmacokinetic properties. This makes them ideal for tumor-targeted delivery of biological moieties. The International Society of Extracellular Vesicles (ISEV) based on the Minimal Information for Studies on Extracellular Vesicles (MISEV) recommends the usage of the term "small extracellular vesicles (sEVs)" that includes exosomes for particles that are 30-200 nm in size. However, majority of the studies reported in the literature and relevant to this review have used the term "exosomes". Therefore, this review will use the term "exosomes" interchangeably with sEVs for consistency with the literature and avoid confusion to the readers. This review, initially summarizes the different isolation and detection techniques developed to study ovarian cancer-derived exosomes and the potential use of these exosomes as biomarkers for the early diagnosis of this devastating disease. It addresses the role of exosome contents in the pathogenesis of ovarian cancer, discusses strategies to limit exosome-mediated ovarian cancer progression, and provides options to use exosomes for tumor-targeted therapy in ovarian cancer. Finally, it states future research directions and recommends essential research needed to successfully transition exosomes from the laboratory to the gynecologic-oncology clinic.

Harnessing small extracellular vesicles for pro-oxidant delivery: novel approach for drug-sensitive and resistant cancer therapy.

Multidrug resistance (MDR) is an inevitable clinical problem in chemotherapy due to the activation of abundant P-glycoprotein (P-gp) that can efflux drugs. Limitations of current cancer therapy highlight the need for the development of a comprehensive cancer treatment strategy, including drug-resistant cancers. Small extracellular vesicles (sEVs) possess significant potential in surmounting drug resistance as they can effectively evade the efflux mechanism and transport small molecules directly to MDR cancer cells. One mechanism mediating MDR in cancer cells is sustaining increased levels of reactive oxygen species (ROS) and maintenance of the redox balance with antioxidants, including glutathione (GSH). Herein, we developed GSH-depleting benzoyloxy dibenzyl carbonate (B2C)-encapsulated sEVs (BsEVs), which overcome the efflux system to exert highly potent anticancer activity against human MDR ovarian cancer cells (OVCAR-8/MDR) by depleting GSH to induce oxidative stress and, in turn, apoptotic cell death in both OVCAR-8/MDR and OVCAR-8 cancer cells. BsEVs restore drug responsiveness by inhibiting ATP production through the oxidation of nicotinamide adenine dinucleotide with hydrogen (NADH) and inducing mitochondrial dysfunction, leading to the dysfunction of efflux pumps responsible for drug resistance. In vivo studies showed that BsEV treatment significantly inhibited the growth of OVCAR-8/MDR and OVCAR-8 tumors. Additionally, OVCAR-8/MDR tumors showed a trend towards a greater sensitivity to BsEVs compared to OVCAR tumors. In summary, this study demonstrates that BsEVs hold tremendous potential for cancer treatment, especially against MDR cancer cells.

Gut Dysbiosis: A New Avenue for Stroke Prevention and Therapeutics.

A stroke is a serious life-threatening condition and a leading cause of death and disability that happens when the blood vessels to part of the brain are blocked or burst. While major advances in the understanding of the ischemic cascade in stroke was made over several decades, limited therapeutic options and high mortality and disability have caused researchers to extend the focus toward peripheral changes beyond brain. The largest proportion of microbes in human body reside in the gut and the interaction between host and microbiota in health and disease is well known. Our study aimed to explore the gut microbiota in patients with stroke with comparison to control group. Fecal samples were obtained from 51 subjects: 25 stroke patients (18 hemorrhagic, 7 ischemic) and 26 healthy control subjects. The variable region V3-V4 of the 16S rRNA gene was sequenced using the Illumina MiSeq platform. PICRUSt2 was used for prediction of metagenomics functions. Our results show taxonomic dysbiosis in stroke patients in parallel with functional dysbiosis. Here, we show that stroke patients have (1) increased Parabacteroides and Escherichia_Shigella, but decreased Prevotella and Fecalibacterium; (2) higher transposase and peptide/nickel transport system substrate-binding protein, but lower RNA polymerase sigma-70 factor and methyl-accepting chemotaxis protein, which are suggestive of malnutrition. Nutrients are essential regulators of both host and microbial physiology and function as key coordinators of host-microbe interactions. Manipulation of nutrition is expected to alleviate gut dysbiosis and prognosis and improve disability and mortality in the management of stroke.

Differential responses to double-stranded RNA injection and feeding in Mormon cricket (Orthoptera: Tettigoniidae).

The Mormon cricket, Anabrus simplex, is a flightless katydid, one of the major devastating rangeland pests in several states of the western United States. During the past few years, their sudden and periodic outbreaks into massive migratory bands caused significant economic losses to the rangeland forage and agricultural crops, particularly grain crops. Current population management methods rely heavily on broad-spectrum chemical insecticides, which could be toxic to nontargets, and even the targeted species might develop resistance in the long run. Therefore, we assessed the potential of RNA interference (RNAi)-based alternative management strategies that could supplement the current methods. In insects, RNAi efficiency varies with the method of double-stranded RNA (dsRNA) delivery. We tested 2 different methods of dsRNA delivery: injection and oral feeding of dsRNA. The results showed that Mormon crickets are sensitive to injection of dsRNA in a dose-dependent manner, but refractory to the oral feeding of dsRNA. Further, we confirmed the high nuclease activity in the insect midgut. In order to protect the dsRNA from the dsRNase activity and facilitate its uptake in the midgut, we encapsulated dsRNA inside poly lactic-co-glycolic acid (PLGA) nanoparticles and studied its release kinetics and RNAi efficiency by oral feeding. The release kinetics clearly suggested that the PLGA nanoparticle permeates from the insect digestive system to the hemolymph; however, it failed to induce an efficient RNAi response of the targeted genes. In conclusion, our findings suggest the different responses to dsRNA delivery methods in Mormon crickets, and further investigations involving dsRNA stability and its uptake mechanism are required to use RNAi as an alternative Mormon cricket population management strategy.

Magnetically Enhanced Intracellular Uptake of Superparamagnetic Iron Oxide Nanoparticles for Antitumor Therapy.

Superparamagnetic iron oxide nanoparticles (SPIONs) have been widely employed in biomedical fields, including targeted delivery of antitumor therapy. Conventional magnetic tumor targeting has used simple static magnetic fields (SMFs), which cause SPIONs to linearly aggregate into a long chain-like shape. Such agglomeration greatly hinders the intracellular targeting of SPIONs into tumors, thus reducing the therapeutic efficacy. In this study, we investigated the enhancement of the intracellular uptake of SPIONs through the application of rotating magnetic fields (RMFs). Based on the physical principles of SPION chain disassembly, we investigated physical parameters to predict the chain length favorable for intracellular uptake. Our prediction was validated by clear visualization of the intracellular distributions of SPIONs in tumor cells at both cellular and three-dimensional microtissue levels. To identify the potential therapeutic effects of enhanced intracellular uptake, magnetic hyperthermia as antitumor therapy was investigated under varying conditions of magnetic hyperthermia and RMFs. The results showed that enhanced intracellular uptake reduced magnetic hyperthermia time and strength as well as particle concentration. The proposed method will be useful in the development of techniques to determine the optimized physical conditions for the enhanced intracellular uptake of SPIONs in antitumor therapy.

Understanding of Ovarian Cancer Cell-Derived Exosome Tropism for Future Therapeutic Applications.

Exosomes, a subtype of extracellular vesicles, ranging from 50 to 200 nm in diameter, and mediate cell-to-cell communication in normal biological and pathological processes. Exosomes derived from tumors have multiple functions in cancer progression, resistance, and metastasis through cancer exosome-derived tropism. However, there is no quantitative information on cancer exosome-derived tropism. Such data would be highly beneficial to guide cancer therapy by inhibiting exosome release and/or uptake. Using two fluorescent protein (mKate2) transfected ovarian cancer cell lines (OVCA4 and OVCA8), cancer exosome tropism was quantified by measuring the released exosome from ovarian cancer cells and determining the uptake of exosomes into parental ovarian cancer cells, 3D spheroids, and tumors in tumor-bearing mice. The OVCA4 cells release 50 to 200 exosomes per cell, and the OVCA8 cells do 300 to 560 per cell. The uptake of exosomes by parental ovarian cancer cells is many-fold higher than by non-parental cells. In tumor-bearing mice, most exosomes are homing to the parent cancer rather than other tissues. We successfully quantified exosome release and uptake by the parent cancer cells, further proving the tropism of cancer cell-derived exosomes. The results implied that cancer exosome tropism could provide useful information for future cancer therapeutic applications.

Tumor-targeted exosomes for delivery of anticancer drugs.

Exosomes are small phospholipid bilayer vesicles that are naturally produced by all living cells, both prokaryotes and eukaryotes. The exosomes due to their unique size, reduced immunogenicity, and their ability to mimic synthetic liposomes in carrying various anticancer drugs have been tested as drug delivery vehicles for cancer treatment. An added advantage of developing exosomes as a drug carrier is the ease of manipulating their intraluminal content and their surface modification to achieve tumor-targeted drug delivery. In the past ten-years, there has been an exponential increase in the number of exosome-related studies in cancer. Preclinical studies demonstrate exosomes-mediated delivery of chemotherapeutics, biologicals and natural products produce potent anticancer activity both, in vitro and in vivo. In contrast, the number of exosome-based clinical trials are few due to challenges in the manufacturing and scalability related to large-scale production of exosomes and their storage and stability. Herein, we discuss recent advances in exosome-based drug delivery for cancer treatment in preclinical and clinical studies and conclude with challenges to be overcome for translating a larger number of exosome-based therapies into the clinic.

Grabody B, an IGF1 receptor-based shuttle, mediates efficient delivery of biologics across the blood-brain barrier.

Effective delivery of therapeutics to the brain is challenging. Molecular shuttles use receptors expressed on brain endothelial cells to deliver therapeutics. Antibodies targeting transferrin receptor (TfR) have been widely developed as molecular shuttles. However, the TfR-based approach raises concerns about safety and developmental burden. Here, we report insulin-like growth factor 1 receptor (IGF1R) as an ideal target for the molecular shuttle. We also describe Grabody B, an antibody against IGF1R, as a molecular shuttle. Grabody B has broad cross-species reactivity and does not interfere with IGF1R-mediated signaling. We demonstrate that administration of Grabody B-fused anti-alpha-synuclein (α-Syn) antibody induces better improvement in neuropathology and behavior in a Parkinson's disease animal model than the therapeutic antibody alone due to its superior serum pharmacokinetics and enhanced brain exposure. The results indicate that IGF1R is an ideal shuttle target and Grabody B is a safe and efficient molecular shuttle.

Immunogenicity and Durability of Antibody Responses to Homologous and Heterologous Vaccinations with BNT162b2 and ChAdOx1 Vaccines for COVID-19.

During the COVID-19 pandemic, vaccines were developed based on various platform technologies and were approved for emergency use. However, the comparative analysis of immunogenicity and durability of vaccine-induced antibody responses depending on vaccine platforms or vaccination regimens has not been thoroughly examined for mRNA- or viral vector-based vaccines. In this study, we assessed spike-binding IgG levels and neutralizing capacity in 66 vaccinated individuals prime-boost immunized either by homologous (BNT162b2-BNT162b2 or ChAdOx1-ChAdOx1) or heterologous (ChAdOx1-BNT162b2) vaccination for six months after the first vaccination. Despite the discrepancy in intervals for the prime-boost vaccination regimen of different COVID-19 vaccines, we found stronger induction and relatively rapid waning of antibody responses by homologous vaccination of the mRNA vaccine, while weaker boost effect and stable maintenance of humoral immune responses were observed in the viral vector vaccine group over 6 months. Heterologous vaccination with ChAdOx1 and BNT162b2 resulted in an effective boost effect with the highest remaining antibody responses at six months post-primary vaccination.

Inhibition of KIF20A suppresses the replication of influenza A virus by inhibiting viral entry.

The influenza A virus (IAV) has caused several pandemics, and therefore there are many ongoing efforts to identify novel antiviral therapeutic strategies including vaccines and antiviral drugs. However, influenza viruses continuously undergo antigenic drift and shift, resulting in the emergence of mutated viruses. In turn, this decreases the efficiency of existing vaccines and antiviral drugs to control IAV infection. Therefore, this study sought to identify alternative therapeutic strategies targeting host cell factors rather than viruses to avoid infection by mutated viruses. Particularly, we investigated the role of KIF20A that is one of kinesin superfamily proteins in the replication of IAV. The KIF20A increased viral protein levels in IAV-infected cells by regulating the initial entry stage during viral infection. Furthermore, the KIF20A inhibitor significantly suppressed viral replication, which protected mice from morbidity and mortality. Therefore, our findings demonstrated that KIF20A is highly involved in the viral replication process and viral propagation both in vitro and in vivo, and could thus be used as a target for the development of novel antiviral drugs.

Multi-target cell therapy using a magnetoelectric microscale biorobot for targeted delivery and selective differentiation of SH-SY5Y cells via magnetically driven cell stamping.

Cell therapy refers to a treatment that involves the delivery of cells or cellular material by means of injection, grafting, or implantation in order to replace damaged tissue and restore its function, or to aid the body in fighting disease. However, limitations include poor targeting delivery and low therapeutic efficacy due to low cell survival. Hence, novel approaches are required to increase cell delivery efficiency and enhance therapeutic efficacy via selective cell differentiation at target areas. Here, we present a stamping magnetoelectric microscale biorobot (SMMB) consisting of neuron-like cell spheroids loaded with magnetoelectric nanoparticles. The SMMB enables not only effective targeted delivery of cells to multiple target areas (via minimally invasive stamping employing magnetic actuation) but also facilitates selective neuronal differentiation via magnetoelectric (ME) stimulation. This ensures rapid colonization and enhances efficacy. SMMBs were fabricated using SH-SY5Y cells. Magnetoelectric nanoparticles for ME stimulation responded to an alternating magnetic field that ensured targeted cell differentiation. Multi-target cell therapy facilitated the targeted delivery and selective differentiation of SH-SY5Y cells to multiple regions using a single SMMB with rotating and alternating magnetic fields for delivery and ME stimulation. This promising tool may overcome the limitations of existing cell therapy for neurodegenerative diseases.

Pseudo-Isolated α-Helix Platform for the Recognition of Deep and Narrow Targets.

Although interest in stabilized α-helical peptides as next-generation therapeutics for modulating biomolecular interfaces is increasing, peptides have limited functionality and stability due to their small size. In comparison, α-helical ligands based on proteins can make steric clash with targets due to their large size. Here, we report the design of a monomeric pseudo-isolated α-helix (mPIH) system in which proteins behave as if they are peptides. The designed proteins contain α-helix ligands that do not require any covalent chemical modification, do not have frayed ends, and importantly can make sterically favorable interactions similar to isolated peptides. An optimal mPIH showed a more than 100-fold increase in target selectivity, which might be related to the advantages in conformational selection due to the absence of frayed ends. The α-helical ligand in the mPIH displayed high thermal stability well above human body temperature and showed reversible and rapid folding/unfolding transitions. Thus, mPIH can become a promising protein-based platform for developing stabilized α-helix pharmaceuticals.

Intraocular RGD-Engineered Exosomes and Active Targeting of Choroidal Neovascularization (CNV).

PURPOSE: To assess the transretinal penetration of intravitreally injected retinal multicell-derived exosomes and to develop exosome-based active targeting of choroidal neovascularization (CNV) by bioengineering with ASL, which is composed of a membrane Anchor (BODIPY), Spacer (PEG), and targeting Ligands (cyclic RGD peptide). METHODS: Retinal multicell-derived exosomes were recovered from a whole mouse retina using differential ultracentrifugation. Their size, number, and morphology were characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Exosome markers were confirmed using an exosome detection antibody array. Intravitreal injection of fluorescent (PKH-26)-labeled or engineered ASL exosomes (1 × 106 exosomes/µL) were given to the wild-type mouse or laser-induced CNV mouse model. Retinal uptake of exosomes was assessed by in vivo retinal imaging microscopy and histological staining with DAPI, GSA, and anti-integrin αv for retinal sections or choroid/RPE flat mounts. Active targeting of CNV was assessed by comparing retinal uptake between areas with and without CNV and by colocalization analysis of ASL exosomes with integrin αv within CNV. Staining with anti-F4/80, anti-ICAM-1, and anti-GFAP antibodies on retinal sections were performed to identify intracellular uptake of exosomes and immediate reactive retinal gliosis after exosome treatment. RESULTS: An average of 2.1 × 109 particles/mL with a peak size of 140 nm exosomes were recovered. Rapid retinal penetration of intravitreally injected exosomes was confirmed by retinal imaging microscopy at 3 and 24 h post-injection. Intravitreally delivered PKH-26-labeled exosomes reached inner and outer retinal layers including IPL, INL, OPL, and ONL at 1 and 7 days post-injection. Intravitreally injected ASL exosomes were predominantly delivered to the area of CNV including ONL, RPE, and choroid in laser-induced CNV mouse models with 89.5% of colocalization with integrin αv. Part of exosomes was also taken intracellularly to vascular endothelial cells and macrophages. After intravitreal injection, neither naive exosomes nor ASL exosomes induced immediate reactive gliosis. CONCLUSIONS: Intravitreally delivered retinal multicell-derived exosomes have good retinal penetration, and ASL modification of exosomes actively targets CNV with no immediate reactive gliosis. ASL exosomes have a great potential to serve as an intraocular drug delivery vehicle, allowing an active targeting strategy.

Conformation-specific Antibodies Targeting Aggregated Forms of α-synuclein Block the Propagation of Synucleinopathy.

Abnormal aggregation of α-synuclein is a key element in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. α-synuclein aggregation spreads through various brain regions during the course of disease progression, a propagation that is thought to be mediated by the secretion and subsequent uptake of extracellular α-synuclein aggregates between neuronal cells. Thus, aggregated forms of this protein have emerged as promising targets for disease-modifying therapy for PD and related diseases. Here, we generated and characterized conformation-specific antibodies that preferentially recognize aggregated forms of α-synuclein. These antibodies promoted phagocytosis of extracellular α-synuclein aggregates by microglial cells and interfered with cell-to-cell propagation of α-synuclein. In an α-synuclein transgenic model, passive immunization with aggregate-specific antibodies significantly ameliorated pathological phenotypes, reducing α-synuclein aggregation, gliosis, inflammation, and neuronal loss. These results suggest that conformation-specific antibodies targeting α-synuclein aggregates are promising therapeutic agents for PD and related synucleinopathies.

Unique behaviour of the α-helix in bending deformation.

The maximum degree of bending that can be tolerated by the rigid rod-like α-helix remains unknown; however, it should be very difficult or even impossible to make α-helices with varying degrees of curvature in folded proteins. As an experimentally tractable model, here we utilize cyclic proteins and peptides to determine the maximum possible bending in the α-helix. We artificially enforced bending in the α-helices by using variously sized macrocycles and compared the structural characteristics of the macrocycles with those of their linear counterparts. This differential analysis reveals that the radius of curvature (RC) for the maximally bent α-helix is approximately 10 times smaller than those of typical α-helices found in natural proteins. Together with the novel finding of the limit of α-helix deformation, excessively bent α-helices can be further utilized in designing de novo peptides and proteins with unique structures and peculiar functions.

P=O Functionalized Black Phosphorus/1T-WS2 Nanocomposite High Efficiency Hybrid Photocatalyst for Air/Water Pollutant Degradation.

Research on layered two-dimensional (2D) materials is at the forefront of material science. Because 2D materialshave variousplate shapes, there is a great deal of research on the layer-by-layer-type junction structure. In this study, we designed a composite catalyst with a dimension lower than two dimensions and with catalysts that canbe combined so that the band structures can be designed to suit various applications and cover for each other's disadvantages. Among transition metal dichalcogenides, 1T-WS2 can be a promising catalytic material because of its unique electrical properties. Black phosphorus with properly controlled surface oxidation can act as a redox functional group. We synthesized black phosphorus that was properly surface oxidized by oxygen plasma treatment and made a catalyst for water quality improvement through composite with 1T-WS2. This photocatalytic activity was highly efficient such that the reaction rate constant k was 10.31 × 10-2 min-1. In addition, a high-concentration methylene blue solution (20 ppm) was rapidly decomposed after more than 10 cycles and showed photo stability. Designing and fabricating bandgap energy-matching nanocomposite photocatalysts could provide a fundamental direction in solving the future's clean energy problem.

A Benzenesulfonamide-Based Mitochondrial Uncoupler Induces Endoplasmic Reticulum Stress and Immunogenic Cell Death in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies and requires new therapeutic strategies to improve clinical outcomes. EOC metastasizes in the abdominal cavity through dissemination in the peritoneal fluid and ascites, efficiently adapt to the nutrient-deprived microenvironment, and resist current chemotherapeutic agents. Accumulating evidence suggests that mitochondrial oxidative phosphorylation is critical for the adaptation of EOC cells to this otherwise hostile microenvironment. Although chemical mitochondrial uncouplers can impair mitochondrial functions and thereby target multiple, essential pathways for cancer cell proliferation, traditional mitochondria uncouplers often cause toxicity that precludes their clinical application. In this study, we demonstrated that a mitochondrial uncoupler, specifically 2,5-dichloro-N-(4-nitronaphthalen-1-yl)benzenesulfonamide, hereinafter named Y3, was an antineoplastic agent in ovarian cancer models. Y3 treatment activated AMP-activated protein kinase and resulted in the activation of endoplasmic reticulum stress sensors as well as growth inhibition and apoptosis in ovarian cancer cells in vitro Y3 was well tolerated in vivo and effectively suppressed tumor progression in three mouse models of EOC, and Y3 also induced immunogenic cell death of cancer cells that involved the release of damage-associated molecular patterns and the activation of antitumor adaptive immune responses. These findings suggest that mitochondrial uncouplers hold promise in developing new anticancer therapies that delay tumor progression and protect patients with ovarian cancer against relapse.

Metabolic and immunomodulatory control of type 1 diabetes via orally delivered bile-acid-polymer nanocarriers of insulin or rapamycin.

Oral formulations of insulin are typically designed to improve its intestinal absorption and increase its blood bioavailability. Here we show that polymerized ursodeoxycholic acid, selected from a panel of bile-acid polymers and formulated into nanoparticles for the oral delivery of insulin, restored blood-glucose levels in mice and pigs with established type 1 diabetes. The nanoparticles functioned as a protective insulin carrier and as a high-avidity bile-acid-receptor agonist, increased the intestinal absorption of insulin, polarized intestinal macrophages towards the M2 phenotype, and preferentially accumulated in the pancreas of the mice, binding to the islet-cell bile-acid membrane receptor TGR5 with high avidity and activating the secretion of glucagon-like peptide and of endogenous insulin. In the mice, the nanoparticles also reversed inflammation, restored metabolic functions and extended animal survival. When encapsulating rapamycin, they delayed the onset of diabetes in mice with chemically induced pancreatic inflammation. The metabolic and immunomodulatory functions of ingestible bile-acid-polymer nanocarriers may offer translational opportunities for the prevention and treatment of type 1 diabetes.

Magnetically Actuated Drug Delivery Helical Microrobot with Magnetic Nanoparticle Retrieval Ability.

Therapeutic drug delivery microrobots capable of accurate targeting using an electromagnetic actuation (EMA) system are being developed. However, these drug delivery microrobots include a large number of magnetic nanoparticles (MNPs) for accurate EMA targeting, which causes side effects, such as problems with membrane integrity and normal cell apoptosis. Here, a biocompatible and hydrolyzable PEGDA-based drug delivery helical microrobot capable of MNP retrieval is proposed in which doxorubicin (DOX), an anticancer drug, is encapsulated and MNPs are conjugated by a disulfide bond. After being accurately delivered to the lesion of cancer cells through magnetic field manipulation, the fabricated microrobot provides rapid MNP separation and retrieval from the microrobot because of the use of dithiothreitol (DTT), a reducing agent, as an environment similar to the surrounding cancer cells and near-infrared (NIR) as an external stimulus. The characteristics of the fabricated microrobot are analyzed, and fundamental tests for active electromagnetic field manipulation, separation/retrieval of MNPs from the microrobot, and its hydrolysis are discussed. The therapeutic performance of the fabricated microrobot is verified through an in vitro test using tumor cells. Consequently, by use of an integrated system of microscope, eight-coil EMA, and NIR it is shown that the proposed microrobot can be moved to the target site by electromagnetic manipulation. The MNPs conjugated to the microrobot can be separated and retrieved, and the therapeutic effect on tumor cells by the encapsulated drug can be seen.