Optimization of micelle-encapsulated extremely small sized iron oxide nanoparticles as a T1 contrast imaging agent: biodistribution and safety profile.

BACKGROUND: Iron oxide nanoparticles (IONPs) have been cleared by the Food and Drug Administration (FDA) for various clinical applications, such as tumor-targeted imaging, hyperthermia therapy, drug delivery, and live-cell tracking. However, the application of IONPs as T1 contrast agents has been restricted due to their high r2 values and r2/r1 ratios, which limit their effectiveness in T1 contrast enhancement. Notably, IONPs with diameters smaller than 5 nm, referred to as extremely small-sized IONPs (ESIONs), have demonstrated potential in overcoming these limitations. To advance the clinical application of ESIONs as T1 contrast agents, we have refined a scale-up process for micelle encapsulation aimed at improving the hydrophilization of ESIONs, and have carried out comprehensive in vivo biodistribution and preclinical toxicity assessments. RESULTS: The optimization of the scale-up micelle-encapsulation process, specifically employing Tween60 at a concentration of 10% v/v, resulted in ESIONs that were uniformly hydrophilized, with an average size of 9.35 nm and a high purification yield. Stability tests showed that these ESIONs maintained consistent size over extended storage periods and dispersed effectively in blood and serum-mimicking environments. Relaxivity measurements indicated an r1 value of 3.43 mM- 1s- 1 and a favorable r2/r1 ratio of 5.36, suggesting their potential as T1 contrast agents. Biodistribution studies revealed that the ESIONs had extended circulation times in the bloodstream and were primarily cleared via the hepatobiliary route, with negligible renal excretion. We monitored blood clearance and organ distribution using positron emission tomography and magnetic resonance imaging (MRI). Additionally, MRI signal variations in a dose-dependent manner highlighted different behaviors at varying ESIONs concentrations, implying that optimal dosages might be specific to the intended imaging application. Preclinical safety evaluations indicated that ESIONs were tolerable in rats at doses up to 25 mg/kg. CONCLUSIONS: This study effectively optimized a scale-up process for the micelle encapsulation of ESIONs, leading to the production of hydrophilic ESIONs at gram-scale levels. These optimized ESIONs showcased properties conducive to T1 contrast imaging, such as elevated r1 relaxivity and a reduced r2/r1 ratio. Biodistribution study underscored their prolonged bloodstream presence and efficient clearance through the liver and bile, without significant renal involvement. The preclinical toxicity tests affirmed the safety of the ESIONs, supporting their potential use as T1 contrast agent with versatile clinical application.

Circulation Time-Optimized Albumin Nanoplatform for Quantitative Visualization of Lung Metastasis via Targeting of Macrophages.

The development of molecular imaging probes to identify key cellular changes within lung metastases may lead to noninvasive detection of metastatic lesions in the lung. In this study, we constructed a macrophage-targeted clickable albumin nanoplatform (CAN) decorated with mannose as the targeting ligand using a click reaction to maintain the intrinsic properties of albumin in vivo. We also modified the number of mannose molecules on the CAN and found that mannosylated serum albumin (MSA) harboring six molecules of mannose displayed favorable pharmacokinetics that allowed high-contrast imaging of the lung, rendering it suitable for in vivo visualization of lung metastases. Due to the optimized control of functionalization and surface modification, MSA enhanced blood circulation time and active/passive targeting abilities and was specifically incorporated by mannose receptor (CD206)-expressing macrophages in the metastatic lung. Moreover, extensive in vivo imaging studies using single-photon emission computed tomography (SPECT)/CT and positron emission tomography (PET) revealed that blood circulation of time-optimized MSA can be used to discern metastatic lesions, with a strong correlation between its signal and metastatic burden in the lung.

iPSCs in NK Cell Manufacturing and NKEV Development.

Natural killer (NK) cell immunotherapies for cancer can complement existing T cell therapies while benefiting from advancements already made in the immunotherapy field. For NK cell manufacturing, induced pluripotent stem cells (iPSCs) offer advantages including eliminating donor variation and providing an ideal platform for genome engineering. At the same time, extracellular vesicles (EVs) have become a major research interest, and purified NK cell extracellular vesicles (NKEVs) have been shown to reproduce the key functions of their parent NK cells. NKEVs have the potential to be developed into a standalone therapeutic with reduced complexity and immunogenicity compared to cell therapies. This review explores the role iPSC technology can play in both NK cell manufacturing and NKEV development.

Pulmonary delivery of a recombinant RAGE antagonist peptide derived from high-mobility group box-1 in a bleomycin-induced pulmonary fibrosis animal model.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterised by irreversible fibrosis and destruction of the alveolar structure. Receptor for advanced glycation end products (RAGE) has been identified as one of the key molecules involved in IPF pathogenesis. A RAGE-antagonist peptide (RAP) was developed based on the RAGE-binding domain of high mobility group box-1 (HMGB-1). Anti-IPF effects of RAP were evaluated in a bleomycin-induced mouse model of IPF. Bleomycin was administered intratracheally, and then RAP was administrated twice by intratracheal instillation, 1 and 3 d after bleomycin challenge. Seven days after the bleomycin challenge, the mice were sacrificed and the lungs were harvested. The results showed that pulmonary hydroxyproline was reduced in mice administered RAP compared with the control group. Tumour growth factor-ß (TGF-ß), α-smooth muscle actin (α-SMA) and collagen were also reduced by RAP administration in a dose-dependent manner. Longer-term effects of RAP were investigated in mice challenged with bleomycin. RAP was administered intratracheally every 7 d for 28 d, after which lung samples were harvested and analysed. The results showed that hydroxyproline, TGF-ß, α-SMA and collagen were reduced by repeated RAP administration. Taken together, the results suggest that RAP is useful for treatment of IPF.

High-quantum yield alloy-typed core/shell CdSeZnS/ZnS quantum dots for bio-applications.

BACKGROUND: Quantum dots (QDs) have been used as fluorophores in various imaging fields owing to their strong fluorescent intensity, high quantum yield (QY), and narrow emission bandwidth. However, the application of QDs to bio-imaging is limited because the QY of QDs decreases substantially during the surface modification step for bio-application. RESULTS: In this study, we fabricated alloy-typed core/shell CdSeZnS/ZnS quantum dots (alloy QDs) that showed higher quantum yield and stability during the surface modification for hydrophilization compared with conventional CdSe/CdS/ZnS multilayer quantum dots (MQDs). The structure of the alloy QDs was confirmed using time-of-flight medium-energy ion scattering spectroscopy. The alloy QDs exhibited strong fluorescence and a high QY of 98.0%. After hydrophilic surface modification, the alloy QDs exhibited a QY of 84.7%, which is 1.5 times higher than that of MQDs. The QY was 77.8% after the alloy QDs were conjugated with folic acid (FA). Alloy QDs and MQDs, after conjugation with FA, were successfully used for targeting human KB cells. The alloy QDs exhibited a stronger fluorescence signal than MQD; these signals were retained in the popliteal lymph node area for 24 h. CONCLUSION: The alloy QDs maintained a higher QY in hydrophilization for biological applications than MQDs. And also, alloy QDs showed the potential as nanoprobes for highly sensitive bioimaging analysis.

Extracellular Vesicles Induce an Aggressive Phenotype in Luminal Breast Cancer Cells Via PKM2 Phosphorylation.

BACKGROUND: Aerobic glycolysis is a hallmark of glucose metabolism in cancer. Previous studies have suggested that cancer cell-derived extracellular vesicles (EVs) can modulate glucose metabolism in adjacent cells and promote disease progression. We hypothesized that EVs originating from cancer cells can modulate glucose metabolism in recipient cancer cells to induce cell proliferation and an aggressive cancer phenotype. METHODS: Two breast cancer cell lines with different levels of glycolytic activity, MDA-MB-231 cells of the claudin-low subtype and MCF7 cells of the luminal type, were selected and cocultured as the originating and recipient cells, respectively, using an indirect coculture system, such as a Transwell system or a microfluidic system. The [18F]fluorodeoxyglucose (FDG) uptake by the recipient MCF7 cells was assessed before and after coculture with MDA-MB-231 cells. Proteomic and transcriptomic analyses were performed to investigate the changes in gene expression patterns in the recipient MCF7 cells and MDA-MB-231 cell-derived EVs. RESULTS: FDG uptake by the recipient MCF7 cells significantly increased after coculture with MDA-MB-231 cells. In addition, phosphorylation of PKM2 at tyrosine-105 and serine-37, which is necessary for tumorigenesis and aerobic glycolysis, was highly activated in cocultured MCF7 cells. Proteomic profiling revealed the proliferation and dedifferentiation of MCF7 cells following coculture with MDA-MB-231 cells. Transcriptomic analysis demonstrated an increase in glycolysis in cocultured MCF7 cells, and the component analysis of glycolysis-related genes revealed that the second most abundant component after the cytoplasm was extracellular exosomes. In addition, proteomic analysis of EVs showed that the key proteins capable of phosphorylating PKM2 were present as cargo inside MDA-MB-231 cell-derived EVs. CONCLUSIONS: The phenomena observed in this study suggest that cancer cells can induce a phenotype transition of other subtypes to an aggressive phenotype to consequently activate glucose metabolism via EVs. Therefore, this study could serve as a cornerstone for further research on interactions between cancer cells.

Synthesis and Application of Silica-Coated Quantum Dots in Biomedicine.

Quantum dots (QDs) are semiconductor nanoparticles with outstanding optoelectronic properties. More specifically, QDs are highly bright and exhibit wide absorption spectra, narrow light bands, and excellent photovoltaic stability, which make them useful in bioscience and medicine, particularly for sensing, optical imaging, cell separation, and diagnosis. In general, QDs are stabilized using a hydrophobic ligand during synthesis, and thus their hydrophobic surfaces must undergo hydrophilic modification if the QDs are to be used in bioapplications. Silica-coating is one of the most effective methods for overcoming the disadvantages of QDs, owing to silica's physicochemical stability, nontoxicity, and excellent bioavailability. This review highlights recent progress in the design, preparation, and application of silica-coated QDs and presents an overview of the major challenges and prospects of their application.

Neural stem cell delivery using brain-derived tissue-specific bioink for recovering from traumatic brain injury.

Traumatic brain injury is one of the leading causes of accidental death and disability. The loss of parts in a severely injured brain induces edema, neuronal apoptosis, and neuroinflammation. Recently, stem cell transplantation demonstrated regenerative efficacy in an injured brain. However, the efficacy of current stem cell therapy needs improvement to resolve issues such as low survival of implanted stem cells and low efficacy of differentiation into respective cells. We developed brain-derived decellularized extracellular matrix (BdECM) bioink that is printable and has native brain-like stiffness. This study aimed to fabricate injured cavity-fit scaffold with BdECM bioink and assessed the utility of BdECM bioink for stem cell delivery to a traumatically injured brain. Our BdECM bioink had shear thinning property for three-dimensional (3D)-cell-printing and physical properties and fiber structures comparable to those of the native brain, which is important for tissue integration after implantation. The human neural stem cells (NSCs) (F3 cells) laden with BdECM bioink were found to be fully differentiated to neurons; the levels of markers for mature differentiated neurons were higher than those observed with collagen bioinkin vitro. Moreover, the BdECM bioink demonstrated potential in defect-fit carrier fabrication with 3D cell-printing, based on the rheological properties and shape fidelity of the material. As F3 cell-laden BdECM bioink was transplanted into the motor cortex of a rat brain, high efficacy of differentiation into mature neurons was observed in the transplanted NSCs; notably increased level of MAP2, a marker of neuronal differentiation, was observed. Furthermore, the transplanted-cell bioink suppressed reactive astrogliosis and microglial activation that may impede regeneration of the injured brain. The brain-specific material reported here is favorable for NSC differentiation and suppression of neuroinflammation and is expected to successfully support regeneration of a traumatically injured brain.

Extracellular vesicle-associated miR-135b and -135a regulate stemness in Group 4 medulloblastoma cells by targeting angiomotin-like 2.

BACKGROUND: Extracellular vesicles (EVs) secreted by tumours, including exosomes, are important factors that regulate cell-cell interactions in oncogenesis. Although EV studies are ongoing, the biological understanding of EV-miRNAs derived from brain tumour spheroid-forming cells (BTSCs) of medulloblastoma is poor. PURPOSES: We explored the specific cellular miRNAs and EV-miRNAs in medulloblastoma BTSCs to determine their potential biological function. METHODS: Bulk tumor cells (BTCs) and BTSCs were cultured under different conditions from medulloblastoma tissues (N = 10). RESULTS: Twenty-four miRNAs were simultaneously increased in both cells and EVs derived from BTSCs in comparison to BTCs. After inhibition of miR-135b or miR135a which were the most significantly increased in BTSCs, cell viability, self-renewal and stem cell marker expression decreased remarkably. Through integrated analysis of mRNAs and miRNAs data, we found that angiomotin-like 2 (AMOTL2), which was significantly decreased, was targeted by both miR-135b and miR-135a. STAT6 and GPX8 were targeted only by miR-135a. Importantly, low expression of AMOTL2 was significantly associated with overall poor survival in paediatric Group 3 and Group 4 medulloblastoma patients. CONCLUSION: Our results indicated that inhibition of miR-135b or miR-135a leads to suppress stemness of BTSC through modulation of AMOTL2.

Size-Dependent EPR Effect of Polymeric Nanoparticles on Tumor Targeting.

Passive targeting of large nanoparticles by the enhanced permeability and retention (EPR) effect is a crucial concept for solid tumor targeting in cancer nanomedicine. There is, however, a trade-off between the long-term blood circulation of nanoparticles and their nonspecific background tissue uptake. To define this size-dependent EPR effect, near-infrared fluorophore-conjugated polyethylene glycols (PEG-ZW800s; 1-60 kDa) are designed and their biodistribution, pharmacokinetics, and renal clearance are evaluated in tumor-bearing mice. The targeting efficiency of size-variant PEG-ZW800s is investigated in terms of tumor-to-background ratio (TBR). Interestingly, smaller sized PEGs (≤20 kDa, 12 nm) exhibit significant tumor targeting with minimum to no nonspecific uptakes, while larger sized PEGs (>20 kDa, 13 nm) accumulate highly in major organs, including the lungs, liver, and pancreas. Among those tested, 20 kDa PEG-ZW800 exhibits the highest TBR, while excreting unbound molecules to the urinary bladder. This result lays a foundation for engineering tumor-targeted nanoparticles and therapeutics based on the size-dependent EPR effect.

Systemic delivery of microRNA-21 antisense oligonucleotides to the brain using T7-peptide decorated exosomes.

Antisense miRNA oligonucleotides against miR-21 (AMO-21) have a therapeutic potential for treatment of glioblastoma. However, glioblastoma-targeted delivery through systemic injection requires development of an efficient targeting carrier. For this purpose, a glioblastoma-targeting carrier was developed using the T7 peptide and exosomes. The transferrin receptor is overexpressed on the surface of glioblastoma cells, and T7 is a transferrin receptor-binding peptide. A T7 peptide-decorated exosome (T7-exo) was produced by incorporation of T7 into the exosome membrane as a fusion protein of T7 and Lamp2b. As a control, rabies virus glycoprotein (RVG) peptide targeting brain neuron cells was incorporated into the exosome membrane. AMO-21 was loaded into the exosomes by electroporation. In vitro studies of AMO-21 delivery showed that T7-exo had a higher delivery efficiency to C6 glioblastoma cells than unmodified exosome (Unmod-exo) and RVG-decorated exosome (RVG-exo). For in vivo delivery studies, T7-exo with AMO-21 was delivered into intracranial glioblastoma rat models by intravenous injection through the tail vein. The results showed that T7-exo delivered AMO-21 into the brain more efficiently than Unmod-exo and RVG-exo. In addition, delivery of AMO-21 using T7-exo reduced the miR-21 level in the glioblastoma efficiently. Reduction of miR-21 by AMO-21 induced the expression of PDCD4 and PTEN in tumors, resulting in reduction of tumor sizes. Taken together, these findings indicate that T7-exo is an efficient carrier of AMO-21 into the glioblastoma and may be useful in development of glioblastoma therapy.

Delivery of High Mobility Group Box-1 siRNA Using Brain-Targeting Exosomes for Ischemic Stroke Therapy.

Ischemic strokes are caused by decreased blood flow into the brain, due to narrowed cerebral arteries. In the ischemic brain, high-mobility group box 1 (HMGB1) is released into extracellular spaces and induces inflammatory reactions. In this study, HMGB1 small interfering RNA (siRNA) was delivered into ischemic brains by intravenous administration using rabies virus glycoprotein (RVG) peptide-decorated exosomes. A fusion protein of RVG and Lamp2b was expressed in 293T cells. Since Lamp2b is an exosome membrane-integral protein, RVG-Lamp2b is integrated into the exosomes, producing RVG-decorated exosomes (RVG-Exo). HMGB1-siRNA was loaded into RVG-Exo and unmodified exosomes (Unmod-Exo) by electroporation. The exosomes were homogenous with a size of less than 50 nm and a negative surface charge. In vitro delivery assays showed that RVG-Exo showed higher efficiency to Neuro2A cells than Unmod-Exo. Also, HMGB1 levels were reduced more effectively by RVG-Exo/HMGB1-siRNA. In vivo delivery efficiency and therapeutic effects of RVG-Exo/HMGB1-siRNA were evaluated in a middle cerebral artery occlusion (MCAO) model. RVG-Exo/HMGB1-siRNA, Unmod-Exo/HMGB1-siRNA, and PEI25k/HMGB1-siRNA were administrated into the MCAO model intravenously through the tail vein. The results showed that HMGB1, tumor necrosis factor-α (TNF-α), and apoptosis levels in the brain were reduced in the RVG-Exo/HMGB1-siRNA group more efficiently than the other groups. In addition, the infarct size was decreased in the RVG-Exo/HMGB1 group more effectively than the other groups. These results suggest that RVG-Exo with HMGB1-siRNA may have potential as a therapeutic system for the treatment of ischemic strokes.

Graphene oxide-quenching-based fluorescence in situ hybridization (G-FISH) to detect RNA in tissue: Simple and fast tissue RNA diagnostics.

FISH-based RNA detection in paraffin-embedded tissue can be challenging, with complicated procedures producing uncertain results and poor image quality. Here, we developed a robust RNA detection method based on graphene oxide (GO) quenching and recovery of fluorescence in situ hybridization (G-FISH) in formalin-fixed paraffin-embedded (FFPE) tissues. Using a fluorophore-labeled peptide nucleic acid (PNA) attached to GO, the endogenous long noncoding RNA BC1, the constitutive protein ß-actin mRNA, and miR-124a and miR-21 could be detected in the cytoplasm of a normal mouse brain, primary cultured hippocampal neurons, an Alzheimer's disease model mouse brain, and glioblastoma multiforme tumor tissues, respectively. Coding and non-coding RNAs, either long or short, could be detected in deparaffinized FFPE or frozen tissues, as well as in clear lipid-exchanged anatomically rigid imaging/immunostaining-compatible tissue hydrogel (CLARITY)-transparent brain tissues. The fluorescence recovered by G-FISH correlated highly with the amount of miR-21, as measured by quantitative real time RT-PCR. We propose G-FISH as a simple, fast, inexpensive, and sensitive method for RNA detection, with a very low background, which could be applied to a variety of research or diagnostic purposes.

Intra-Cardiac Release of Extracellular Vesicles Shapes Inflammation Following Myocardial Infarction.

RATIONALE: A rapid and massive influx of inflammatory cells occurs into ischemic area after myocardial infarction (MI), resulting in local release of cytokines and growth factors. Yet, the mechanisms regulating their production are not fully explored. The release of extracellular vesicles (EVs) in the interstitial space curbs important biological functions, including inflammation, and influences the development of cardiovascular diseases. To date, there is no evidence for in situ release of cardiac EVs after MI. OBJECTIVE: The present study tested the hypothesis that local EV generation in the infarcted heart coordinates cardiac inflammation after MI. METHODS AND RESULTS: Coronary artery ligation in mice transiently increases EV levels in the left ventricle when compared with sham animals. EVs from infarcted hearts were characterized as large vesicles (252±18 nm) expressing cardiomyocyte and endothelial markers and small EVs (118±4 nm) harboring exosomal markers, such as CD (cluster of differentiation) 63 and CD9. Cardiac large EVs generated after MI, but not small EVs or sham EVs, increased the release of IL (interleukin)-6, CCL (chemokine ligand) 2, and CCL7 from fluorescence-activated cell-sorted Ly6C+ cardiac monocytes. EVs of similar diameter were also isolated from fragments of interventricular septum obtained from patients undergoing aortic valve replacement, thus supporting the clinical relevance of our findings in mice. CONCLUSIONS: The present study demonstrates that acute MI transiently increases the generation of cardiac EVs characterized as both exosomes and microvesicles, originating mainly from cardiomyocytes and endothelial cells. EVs accumulating in the ischemic myocardium are rapidly taken up by infiltrating monocytes and regulate local inflammatory responses.

Panobinostat, a histone deacetylase inhibitor, suppresses leptomeningeal seeding in a medulloblastoma animal model.

Leptomeningeal seeding is a strong negative prognostic factor for medulloblastoma (MB). The mechanism of leptomeningeal seeding is unclear but may involve epigenetic regulation. In this study, we evaluated the feasibility of a histone deacetylase (HDAC) inhibitor, panobinostat, in the suppression of MB leptomeningeal seeding. Panobinostat decreased the cell viability and proliferation, inducing cell cycle arrest and apoptosis in MB cell lines. The migration and adhesion capabilities were significantly decreased. Panobinostat effectively down-regulated protein expression of CCND1 and ID3 which has been associated with leptomeningeal seeding of MB. After panobinostat treatment, neurophil-like cellular processes developed and expression of synaptophysin and NeuroD1 was increased, indicating neuronal differentiation. In MB leptomeningeal seeding in vivo model, the panobinostat-treated group showed significantly decreased spinal leptomeningeal seeding and a survival benefit. The findings demonstrate that panobinostat suppresses MB leptomeningeal seeding through the down-regulation of ID3 and the induction of neuronal differentiation. An HDAC inhibitor might be a potent treatment option for the treatment of MB patients with leptomeningeal seeding.

Fibrin-Targeted and H2O2-Responsive Nanoparticles as a Theranostics for Thrombosed Vessels.

A thrombus (blood clot) is formed in injured vessels to maintain the integrity of vasculature. However, obstruction of blood vessels by thrombosis slows blood flow, leading to death of tissues fed by the artery and is the main culprit of various life-threatening cardiovascular diseases. Herein, we report a rationally designed nanomedicine that could specifically image obstructed vessels and inhibit thrombus formation. On the basis of the physicochemical and biological characteristics of thrombi such as an abundance of fibrin and an elevated level of hydrogen peroxide (H2O2), we developed a fibrin-targeted imaging and antithrombotic nanomedicine, termed FTIAN, as a theranostic system for obstructive thrombosis. FTIAN inhibited the generation of H2O2 and suppressed the expression of tumor necrosis factor-alpha (TNF-α) and soluble CD40 ligand (sCD40L) in activated platelets, demonstrating its intrinsic antioxidant, anti-inflammatory, and antiplatelet activity. In a mouse model of ferric chloride (FeCl3)-induced carotid thrombosis, FTIAN specifically targeted the obstructive thrombus and significantly enhanced the fluorescence/photoacoustic signal. When loaded with the antiplatelet drug tirofiban, FTIAN remarkably suppressed thrombus formation. Given its thrombus-specific imaging along with excellent therapeutic activities, FTIAN offers tremendous translational potential as a nanotheranostic agent for obstructive thrombosis.

Simultaneous Detection of EGFR and VEGF in Colorectal Cancer using Fluorescence-Raman Endoscopy.

Fluorescence endomicroscopy provides quick access to molecular targets, while Raman spectroscopy allows the detection of multiple molecular targets. Using a simultaneous fluorescence-Raman endoscopic system (FRES), we herein demonstrate its potential in cancer diagnosis in an orthotopically induced colorectal cancer (CRC) xenograft model. In the model, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) were targeted with antibody-conjugated fluorescence and surface-enhanced Raman scattering (F-SERS) dots. FRES demonstrated fast signal detection and multiplex targeting ability using fluorescence and Raman signals to detect the F-SERS dots. In addition, FRES showed a multiplex targeting ability even on a subcentimeter-sized CRC after spraying with a dose of 50 µg F-SERS dots. In conclusion, molecular characteristics of tumor cells (EGFR in cancer cell membranes) and tumor microenvironments (VEGF in the extracellular matrix) could be simultaneously investigated when performing a colonoscopy.

Radio-graphene in Theranostic Perspectives.

Owing to its unique physicochemical properties such as high surface area, notable biocompatibility, robust mechanical strength, high thermal conductivity, and ease of functionalization, 2D-layered graphene has received tremendous attention as a futuristic nanomaterial and its-associated research has been rapidly evolving in a variety of fields. With the remarkable advances of graphene especially in the biomedical realm, in vivo evaluation techniques to examine in vivo behavior of graphene are largely demanded under the hope of clinical translation. Many different types of drugs such as the antisense oligomer and chemotherapeutics require optimal delivery conveyor and graphene is now recognized as a suitable candidate due to its simple and high drug loading property. Termed as 'radio-graphene', radioisotope-labeled graphene approach was recently harnessed in the realm of biomedicine including cancer diagnosis and therapy, contributing to the acquisition of in vivo information for targeted drug delivery. In this review, we highlight current examples for bioapplication of radiolabeled graphene with brief perspectives on future strategies in its extensive bio- or clinical applications.

Repositioning disulfiram as a radiosensitizer against atypical teratoid/rhabdoid tumor.

BACKGROUND: Atypical teratoid/rhabdoid tumor (AT/RT) is one of the most common malignant brain tumors in infants. Although cancer stem cells of AT/RT express aldehyde dehydrogenase (ALDH), effective chemotherapies against AT/RT have not been established. Here, we examined radiosensitizing effects of disulfiram (DSF), an irreversible inhibitor of ALDH against AT/RT for a novel therapeutic method. METHODS: Patient-derived primary cultured AT/RT cells (SNU.AT/RT-5 and SNU.AT/RT-6) and established AT/RT cell lines (BT-12 and BT-16) were used to assess therapeutic effects of combining DSF with radiation treatment (RT). Survival fraction by clonogenic assay, protein expression, immunofluorescence, and autophagy analysis were evaluated in vitro. Antitumor effects of combining DSF with RT were verified by bioluminescence imaging, tumor volume, and survival analysis in vivo. RESULTS: The results demonstrated that DSF at low concentration enhanced the radiosensitivity of AT/RT cells with reduction of survival fraction to 1.21‒1.58. DSF increased DNA double-strand break (γ-H2AX, p-DNA-PKcs, and p-ATM), apoptosis (cleaved caspase-3), autophagy (LC3B), and cell cycle arrest (p21) in irradiated AT/RT cells, while it decreased anti-apoptosis (nuclear factor-kappaB, Survivin, and B-cell lymphoma 2 [Bcl2]). In vivo, DSF and RT combined treatment significantly reduced tumor volumes and prolonged the survival of AT/RT mouse models compared with single treatments. The combined treatment also increased γ-H2AX, cleaved caspase-3, and LC3B expression and decreased ALDH1, Survivin, and Bcl2 expression in vivo. CONCLUSIONS: DSF and RT combination therapy has additive therapeutic effects on AT/RT by potentiating programmed cell death, including apoptosis and autophagy of AT/RT cells. We suggest that DSF can be applied as a radiosensitizer in AT/RT treatment.

In vivo visualization of endogenous miR-21 using hyaluronic acid-coated graphene oxide for targeted cancer therapy.

Oncogene-targeted nucleic acid therapy has been spotlighted as a new paradigm for cancer therapeutics. However, in vivo delivery issues and uncertainty of therapeutic antisense drug reactions remain critical hurdles for a successful targeted cancer therapy. In this study, we developed a fluorescence-switchable theranostic nanoplatform using hyaluronic acid (HA)-conjugated graphene oxide (GO), which is capable of both sensing oncogenic miR-21 and inhibiting its tumorigenicity simultaneously. Cy3-labeled antisense miR-21 peptide nucleic acid (PNA) probes loaded onto HA-GO (HGP21) specifically targeted CD44-positive MBA-MB231 cells and showed fluorescence recovery by interacting with endogenous miR-21 in the cytoplasm of the MBA-MB231 cells. Knockdown of endogenous miR-21 by HGP21 led to decreased proliferation and reduced migration of cancer cells, as well as the induction of apoptosis, with enhanced PTEN levels. Interestingly, in vivo fluorescence signals markedly recovered 3 h after the intravenous delivery of HGP21 and displayed signals more than 5-fold higher than those observed in the HGPscr-treated group of tumor-bearing mice. These findings demonstrate the possibility of using the HGP nanoplatform as a cancer theranostic tool in miRNA-targeted therapy.