Cancer cell-specific and pro-apoptotic SMAC peptide-doxorubicin conjugated prodrug encapsulated aposomes for synergistic cancer immunotherapy.

BACKGROUND: Immunogenic cell death (ICD) is a crucial approach to turn immunosuppressive tumor microenvironment (ITM) into immune-responsive milieu and improve the response rate of immune checkpoint blockade (ICB) therapy. However, cancer cells show resistance to ICD-inducing chemotherapeutic drugs, and non-specific toxicity of those drugs against immune cells reduce the immunotherapy efficiency. METHODS: Herein, we propose cancer cell-specific and pro-apoptotic liposomes (Aposomes) encapsulating second mitochondria-derived activator of caspases mimetic peptide (SMAC-P)-doxorubicin (DOX) conjugated prodrug to potentiate combinational ICB therapy with ICD. The SMAC-P (AVPIAQ) with cathepsin B-cleavable peptide (FRRG) was directly conjugated to DOX, and the resulting SMAC-P-FRRG-DOX prodrug was encapsulated into PEGylated liposomes. RESULTS: The SMAC-P-FRRG-DOX encapsulated PEGylated liposomes (Aposomes) form a stable nanostructure with an average diameter of 109.1 ± 5.14 nm and promote the apoptotic cell death mainly in cathepsin B-overexpressed cancer cells. Therefore, Aposomes induce a potent ICD in targeted cancer cells in synergy of SMAC-P with DOX in cultured cells. In colon tumor models, Aposomes efficiently accumulate in targeted tumor tissues via enhanced permeability and retention (EPR) effect and release the encapsulated prodrug of SMAC-P-FRRG-DOX, which is subsequently cleaved to SMAC-P and DOX in cancer cells. Importantly, the synergistic activity of inhibitors of apoptosis proteins (IAPs)-inhibitory SMAC-P sensitizing the effects of DOX induces a potent ICD in the cancer cells to promote dendritic cell (DC) maturation and stimulate T cell proliferation and activation, turning ITM into immune-responsive milieu. CONCLUSIONS: Eventually, the combination of Aposomes with anti-PD-L1 antibody results in a high rate of complete tumor regression (CR: 80%) and also prevent the tumor recurrence by immunological memory established during treatments.

Fluorescence-Based Mono- and Multimodal Imaging for In Vivo Tracking of Mesenchymal Stem Cells.

The advancement of stem cell therapy has offered transformative therapeutic outcomes for a wide array of diseases over the past decades. Consequently, stem cell tracking has become significant in revealing the mechanisms of action and ensuring safe and effective treatments. Fluorescence stands out as a promising choice for stem cell tracking due to its myriad advantages, including high resolution, real-time monitoring, and multi-fluorescence detection. Furthermore, combining fluorescence with other tracking modalities-such as bioluminescence imaging (BLI), positron emission tomography (PET), photoacoustic (PA), computed tomography (CT), and magnetic resonance (MR)-can address the limitations of single fluorescence detection. This review initially introduces stem cell tracking using fluorescence imaging, detailing various labeling strategies such as green fluorescence protein (GFP) tagging, fluorescence dye labeling, and nanoparticle uptake. Subsequently, we present several combinations of strategies for efficient and precise detection.

Self-Assembled Nanostructures of Homo-Oligopeptide as a Potent Ice Growth Inhibitor.

This study reports the formation of self-assembled nanostructures with homo-oligopeptides consisting of amino acids (i.e., alanine, threonine, valine, and tyrosine), the resulting morphologies (i.e., spherical shape, layered structure, and wire structure) in aqueous solution, and their potential as ice growth inhibitors. Among the homo-oligopeptides investigated, an alanine homo-oligopeptide (n = 5) with a spherical nanostructure showed the highest ice recrystallization inhibition (IRI) activity without showing a burst ice growth property and with low ice nucleation activity. The presence of nanoscale self-assembled structures in the solution showed superior IRI activity compared to an amino acid monomer because of the higher binding affinity of structures on the growing ice crystal plane. Simulation results revealed that the presence of nanostructures induced a significant inhibition of ice growth and increased lifetime of hydrogen bonding compared with unassembled homo-oligopeptide. These results envision extraordinary performance for self-assembled nanostructures as a desirable and potent ice growth inhibitor.

Photothermal Effect of Gold Nanoparticles as a Nanomedicine for Diagnosis and Therapeutics.

Gold nanoparticles (AuNPs) have received great attention for various medical applications due to their unique physicochemical properties. AuNPs with tunable optical properties in the visible and near-infrared regions have been utilized in a variety of applications such as in vitro diagnostics, in vivo imaging, and therapeutics. Among the applications, this review will pay more attention to recent developments in diagnostic and therapeutic applications based on the photothermal (PT) effect of AuNPs. In particular, the PT effect of AuNPs has played an important role in medical applications utilizing light, such as photoacoustic imaging, photon polymerase chain reaction (PCR), and hyperthermia therapy. First, we discuss the fundamentals of the optical properties in detail to understand the background of the PT effect of AuNPs. For diagnostic applications, the ability of AuNPs to efficiently convert absorbed light energy into heat to generate enhanced acoustic waves can lead to significant enhancements in photoacoustic signal intensity. Integration of the PT effect of AuNPs with PCR may open new opportunities for technological innovation called photonic PCR, where light is used to enable fast and accurate temperature cycling for DNA amplification. Additionally, beyond the existing thermotherapy of AuNPs, the PT effect of AuNPs can be further applied to cancer immunotherapy. Controlled PT damage to cancer cells triggers an immune response, which is useful for obtaining better outcomes in combination with immune checkpoint inhibitors or vaccines. Therefore, this review examines applications to nanomedicine based on the PT effect among the unique optical properties of AuNPs, understands the basic principles, the advantages and disadvantages of each technology, and understands the importance of a multidisciplinary approach. Based on this, it is expected that it will help understand the current status and development direction of new nanoparticle-based disease diagnosis methods and treatment methods, and we hope that it will inspire the development of new innovative technologies.

Nano-Biotechnology for Bacteria Identification and Potent Anti-bacterial Properties: A Review of Current State of the Art.

Sepsis is a critical disease caused by the abrupt increase of bacteria in human blood, which subsequently causes a cytokine storm. Early identification of bacteria is critical to treating a patient with proper antibiotics to avoid sepsis. However, conventional culture-based identification takes a long time. Polymerase chain reaction (PCR) is not so successful because of the complexity and similarity in the genome sequence of some bacterial species, making it difficult to design primers and thus less suitable for rapid bacterial identification. To address these issues, several new technologies have been developed. Recent advances in nanotechnology have shown great potential for fast and accurate bacterial identification. The most promising strategy in nanotechnology involves the use of nanoparticles, which has led to the advancement of highly specific and sensitive biosensors capable of detecting and identifying bacteria even at low concentrations in very little time. The primary drawback of conventional antibiotics is the potential for antimicrobial resistance, which can lead to the development of superbacteria, making them difficult to treat. The incorporation of diverse nanomaterials and designs of nanomaterials has been utilized to kill bacteria efficiently. Nanomaterials with distinct physicochemical properties, such as optical and magnetic properties, including plasmonic and magnetic nanoparticles, have been extensively studied for their potential to efficiently kill bacteria. In this review, we are emphasizing the recent advances in nano-biotechnologies for bacterial identification and anti-bacterial properties. The basic principles of new technologies, as well as their future challenges, have been discussed.

Recent Studies and Progress in the Intratumoral Administration of Nano-Sized Drug Delivery Systems.

Over the last 30 years, diverse types of nano-sized drug delivery systems (nanoDDSs) have been intensively explored for cancer therapy, exploiting their passive tumor targetability with an enhanced permeability and retention effect. However, their systemic administration has aroused some unavoidable complications, including insufficient tumor-targeting efficiency, side effects due to their undesirable biodistribution, and carrier-associated toxicity. In this review, the recent studies and advancements in intratumoral nanoDDS administration are generally summarized. After identifying the factors to be considered to enhance the therapeutic efficacy of intratumoral nanoDDS administration, the experimental results on the application of intratumoral nanoDDS administration to various types of cancer therapies are discussed. Subsequently, the reports on clinical studies of intratumoral nanoDDS administration are addressed in short. Intratumoral nanoDDS administration is proven with its versatility to enhance the tumor-specific accumulation and retention of therapeutic agents for various therapeutic modalities. Specifically, it can improve the efficacy of therapeutic agents with poor bioavailability by increasing their intratumoral concentration, while minimizing the side effect of highly toxic agents by restricting their delivery to normal tissues. Intratumoral administration of nanoDDS is considered to expand its application area due to its potent ability to improve therapeutic effects and relieve the systemic toxicities of nanoDDSs.

Perspectives for Improving the Tumor Targeting of Nanomedicine via the EPR Effect in Clinical Tumors.

Over the past few decades, the enhanced permeability and retention (EPR) effect of nanomedicine has been a crucial phenomenon in targeted cancer therapy. Specifically, understanding the EPR effect has been a significant aspect of delivering anticancer agents efficiently to targeted tumors. Although the therapeutic effect has been demonstrated in experimental models using mouse xenografts, the clinical translation of the EPR effect of nanomedicine faces several challenges due to dense extracellular matrix (ECM), high interstitial fluid pressure (IFP) levels, and other factors that arise from tumor heterogeneity and complexity. Therefore, understanding the mechanism of the EPR effect of nanomedicine in clinics is essential to overcome the hurdles of the clinical translation of nanomedicine. This paper introduces the basic mechanism of the EPR effect of nanomedicine, the recently discussed challenges of the EPR effect of nanomedicine, and various strategies of recent nanomedicine to overcome the limitations expected from the patients' tumor microenvironments.

Mesenchymal Stem Cell-Mediated Deep Tumor Delivery of Gold Nanorod for Photothermal Therapy.

Gold nanoparticles (AuNPs) with various sizes and morphologies have been extensively investigated for effective photothermal therapy (PTT) against multiple cancer types. However, a highly dynamic and complex tumor microenvironment (TME) considerably reduces the efficacy of PTT by limiting deep tumor penetration of AuNPs. Herein, we propose a mesenchymal stem cell (MSC)-mediated deep tumor delivery of gold nanorod (AuNR) for a potent PTT. First, MSCs are treated with tetraacylated N-azidomannosamine (Ac4ManNAz) to introduce modifiable azide (N3) groups on the cell surface via metabolic glycoengineering. Then, AuNRs modified with bio-orthogonal click molecules of bicyclo[6.1.0]nonyne (AuNR@BCN) are chemically conjugated to the N3 groups on the MSC surface by copper-free click chemistry reaction, resulting in AuNR@MSCs. In cultured MSCs, the appropriate condition to incorporate the AuNR into the MSCs is optimized; in addition, the photothermal efficiency of AuNR-MSCs under light irradiation are assessed, showing efficient heat generation in vitro. In colon tumor-bearing mice, intravenously injected AuNR@MSCs efficiently accumulate within the tumor tissues by allowing deep tissue penetration owing to the tumor homing effect by natural tumor tropism of AuNR@MSCs. Upon localized light irradiation, the AuNR@MSCs significantly inhibit colon tumor growth by the enhanced photothermal effect compared to conventional AuNRs. Collectively, this study shows a promising approach of MSCs-mediated deep tumor delivery of AuNR for effective PTT.

How Did Conventional Nanoparticle-Mediated Photothermal Therapy Become "Hot" in Combination with Cancer Immunotherapy?

One of the promising cancer treatment methods is photothermal therapy (PTT), which has achieved good therapeutic efficiency through nanoparticle-based photoabsorbers. Because of the various functions of nanoparticles, such as targeting properties, high light-to-heat conversion, and photostability, nanoparticle-mediated PTT successfully induces photothermal damage in tumor tissues with minimal side effects on surrounding healthy tissues. The therapeutic efficacy of PTT originates from cell membrane disruption, protein denaturation, and DNA damage by light-induced heat, but these biological impacts only influence localized tumor areas. This conventional nanoparticle-mediated PTT still attracts attention as a novel cancer immunotherapy, because PTT causes immune responses against cancer. PTT-induced immunogenic cell death activates immune cells for systemic anti-cancer effect. Additionally, the excellent compatibility of PTT with other treatment methods (e.g., chemotherapy and immune checkpoint blockade therapy) reinforces the therapeutic efficacy of PTT as combined immunotherapy. In this review, we investigate various PTT agents of nanoparticles and compare their applications to reveal how nanoparticle-mediated PTT undergoes a transition from thermotherapy to immunotherapy.

Electrochemically driven optical and SERS immunosensor for the detection of a therapeutic cardiac drug.

Cardiovascular diseases pose a serious health risk and have a high mortality rate of 31% worldwide. Digoxin is the most commonly prescribed pharmaceutical preparation to cardiovascular patients particularly in developing countries. The effectiveness of the drug critically depends on its presence in the therapeutic range (0.8-2.0 ng mL-1) in the patient's serum. We fabricated immunoassay chips based on QD photoluminescence (QDs-ELISA) and AuNP Surface Enhanced Raman Scattering (SERS-ELISA) phenomena to detect digoxin in the therapeutic range. Digoxin levels were monitored using digoxin antibodies conjugated to QDs and AuNPs employing the sandwich immunoassay format in both the chips. The limit of detection (LOD) achieved through QDs-ELISA and SERS-ELISA was 0.5 ng mL-1 and 0.4 ng mL-1, respectively. It is demonstrated that the sensitivity of QDs-ELISA was dependent on the charge transfer mechanism from the QDs to the antibody through ionic media, which was further explored using electrochemical impedance spectroscopy. We demonstrate that QDs-ELISA was relatively easy to fabricate compared to SERS-ELISA. The current study envisages replacement of conventional methodologies with small immunoassay chips using QDs and/or SERS-based tags with fast turnaround detection time as compared to conventional ELISA.

Sustained and Long-Term Release of Doxorubicin from PLGA Nanoparticles for Eliciting Anti-Tumor Immune Responses.

Immunogenic cell death (ICD) is a powerful trigger eliciting strong immune responses against tumors. However, traditional chemoimmunotherapy (CIT) does not last long enough to induce sufficient ICD, and also does not guarantee the safety of chemotherapeutics. To overcome the disadvantages of the conventional approach, we used doxorubicin (DOX) as an ICD inducer, and poly(lactic-co-glycolic acid) (PLGA)-based nanomedicine platform for controlled release of DOX. The diameter of 138.7 nm of DOX-loaded PLGA nanoparticles (DP-NPs) were stable for 14 days in phosphate-buffered saline (PBS, pH 7.4) at 37 °C. Furthermore, DOX was continuously released for 14 days, successfully inducing ICD and reducing cell viability in vitro. Directly injected DP-NPs enabled the remaining of DOX in the tumor site for 14 days. In addition, repeated local treatment of DP-NPs actually lasted long enough to maintain the enhanced antitumor immunity, leading to increased tumor growth inhibition with minimal toxicities. Notably, DP-NPs treated tumor tissues showed significantly increased maturated dendritic cells (DCs) and cytotoxic T lymphocytes (CTLs) population, showing enhanced antitumor immune responses. Finally, the therapeutic efficacy of DP-NPs was maximized in combination with an anti-programmed death-ligand 1 (PD-L1) antibody (Ab). Therefore, we expect therapeutic efficacies of cancer CIT can be maximized by the combination of DP-NPs with immune checkpoint blockade (ICB) by achieving proper therapeutic window and continuously inducing ICD, with minimal toxicities.

Attomolar Sensitive Magnetic Microparticles and a Surface-Enhanced Raman Scattering-Based Assay for Detecting SARS-CoV-2 Nucleic Acid Targets.

Highly sensitive, reliable assays with strong multiplexing capability for detecting nucleic acid targets are significantly important for diagnosing various diseases, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The nanomaterial-based assay platforms suffer from several critical issues such as non-specific binding and highly false-positive results. In this paper, to overcome such limitations, we reported sensitive and remarkably reproducible magnetic microparticles (MMPs) and a surface-enhanced Raman scattering (SERS)-based assay using stable silver nanoparticle clusters for detecting viral nucleic acids. The MMP-SERS-based assay exhibited a sensitivity of 1.0 fM, which is superior to the MMP-fluorescence-based assay. In addition, in the presence of anisotropic Ag nanostructures (nanostars and triangular nanoplates), the assay exhibited greatly enhanced sensitivity (10 aM) and excellent signal reproducibility. This assay platform intrinsically eliminated the non-specific binding that occurs in the target detection step, and the controlled formation of stable silver nanoparticle clusters in solution enabled the remarkable reproducibility of the results. These findings indicate that this assay can be employed for future practical bioanalytical applications.

Copper-Free Click Chemistry: Applications in Drug Delivery, Cell Tracking, and Tissue Engineering.

Traditionally, organic chemical reactions require organic solvents, toxic catalysts, heat, or high pressure. However, copper-free click chemistry has been shown to have favorable reaction rates and orthogonality in water, buffer solutions, and physiological conditions without toxic catalysts. Strain-promoted azide-alkyne cycloaddition and inverse electron-demand Diels-Alder reactions are representative of copper-free click chemistry. Artificial chemical reactions via click chemistry can also be used outside of the laboratory in a controllable manner on live cell surfaces, in the cytosol, and in living bodies. Consequently, copper-free click chemistry has many features that are of interest in biomedical research, and various new materials and strategies for its use have been proposed. Herein, recent remarkable trials that have used copper-free click chemistry are described, focusing on their applications in molecular imaging and therapy. The research is categorized as nanoparticles for drug delivery, imaging agents for cell tracking, and hydrogels for tissue engineering, which are rapidly advancing fields based on click chemistry. The content is based primarily on the experience with click chemistry-based biomaterials over the last 10 years.

Precise control over the silica shell thickness and finding the optimal thickness for the peak heat diffusion property of AuNR@SiO2.

Silica-coated gold nanorods (AuNRs) exhibit significantly enhanced photothermal effects and photoacoustic (PA) signal intensities, which is beneficial for various nanophotonic applications in materials science. However, the silica shell thickness for optimum enhancement is not fully understood and is even controversial depending on the physical state of the silica shell. This is because of the lack of systematic investigations of the nanoscale silica shell thickness and the photothermal effect. This study provides a robust synthetic method to control the silica shell thickness at the nanoscale and the physical state-dependent heat diffusion property. The selected base and solvent system enabled the production of silica-coated AuNRs (AuNR@SiO2) with silica shell thicknesses of 5, 10, 15, 20, 25, 30, 35, and 40 nm. AuNRs with a 20 nm silica shell showed the highest photothermal effect with a 1.45-times higher photothermal efficiency than that of AuNRs without a silica shell. The low density of the silica shell on the AuNRs showed a low photothermal effect and photostability. It was found that the disruption of cetyltrimethyl ammonium bromide (CTAB) layers on the AuNRs was responsible for the low photostability of the AuNRs. The simulation study for the heat diffusion property showed facilitated heat diffusion in the presence of a 20 nm silica shell. In a cell-based study, AuNRs with a 20 nm silica shell showed the most sensitive photothermal effect for cell death. The results of this robust study can provide conclusive conditions for the optimal silica shell thickness to obtain the highest photothermal effect, which will be useful for the future design of nanomaterials in various fields of application.

Thiol-Responsive Gold Nanodot Swarm with Glycol Chitosan for Photothermal Cancer Therapy.

Photothermal therapy (PTT) is one of the most promising cancer treatment methods because hyperthermal effects and immunogenic cell death via PTT are destructive to cancer. However, PTT requires photoabsorbers that absorb near-infrared (NIR) light with deeper penetration depth in the body and effectively convert light into heat. Gold nanoparticles have various unique properties which are suitable for photoabsorbers, e.g., controllable optical properties and easy surface modification. We developed gold nanodot swarms (AuNSw) by creating small gold nanoparticles (sGNPs) in the presence of hydrophobically-modified glycol chitosan. The sGNPs assembled with each other through their interaction with amine groups of glycol chitosan. AuNSw absorbed 808-nm laser and increased temperature to 55 °C. In contrast, AuNSw lost its particle structure upon exposure to thiolated molecules and did not convert NIR light into heat. In vitro studies demonstrated the photothermal effect and immunogenic cell death after PTT with AuNSW. After intratumoral injection of AuNSw with laser irradiation, tumor growth of xenograft mouse models was depressed. We found hyperthermal damage and immunogenic cell death in tumor tissues through histological and biochemical analyses. Thiol-responsive AuNSw showed feasibility for PTT, with advanced functionality in the tumor microenvironment.

Theragnostic Glycol Chitosan-Conjugated Gold Nanoparticles for Photoacoustic Imaging of Regional Lymph Nodes and Delivering Tumor Antigen to Lymph Nodes.

Lymph node mapping is important in cancer immunotherapy because the morphology of lymph nodes is one of the crucial evaluation criteria of immune responses. We developed new theragnostic glycol-chitosan-coated gold nanoparticles (GC-AuNPs), which highlighted lymph nodes in ultrasound-guided photoacoustic (US/PA) imaging. Moreover, the ovalbumin epitope was conjugated GC-AuNPs (OVA-GC-AuNPs) for delivering tumor antigen to lymph node resident macrophage. In vitro studies proved the vigorous endocytosis activity of J774A.1 macrophage and consequent strong photoacoustic signals from them. The macrophages also presented a tumor antigen when OVA-GC-AuNPs were used for cellular uptake. After the lingual injection of GC-AuNPs into healthy mice, cervical lymph nodes were visible in a US/PA imaging system with high contrast. Three-dimensional analysis of lymph nodes revealed that the accumulation of GC-AuNPs in the lymph node increased as the post-injection time passed. Histological analysis showed GC-AuNPs or OVA-GC-AuNPs located in subcapsular and medullar sinuses where macrophages are abundant. Our new theragnostic GC-AuNPs present a superior performance in US/PA imaging of lymph nodes without targeting moieties or complex surface modification. Simultaneously, GC-AuNPs were able to deliver tumor antigens to cause macrophages to present the OVA epitope at targeted lymph nodes, which would be valuable for cancer immunotherapy.

Solution-Based One-Step Preparation of Three-Dimensional Self-Assembled Octadecyl Silica Nanosquare Plate and Microlamella Structures for Superhydrophobic and Icephobic Surfaces.

Icephobic surfaces have gained immense attention owing to their significant roles in decreasing the energy consumption of refrigerators and in improving safety issues by preventing the formation of ice on them. Superhydrophobic surfaces incorporating micro- or nanoscale roughness and hydrophobic functional groups have been shown to prevent ice accumulation. Herein, we report a simple, low-cost, and solution-based one-step process for the production of superhydrophobic surfaces with three-dimensional (3D) self-assembled structures. The controlled hydrolysis and polycondensation of n-octadecyltrichlorosilane (OTS-Cl) in an acetone solution produced a highly uniform superhydrophobic surface on various substrates such as glass, metals, and polymers without the limitation of the surface curvature structure. The as-prepared 3D self-assembled surface exhibited a very high contact angle of 161.7° and a low contact hysteresis of 1.47°. The solvent type, H2O content in acetone, and carbon chain length of the silane compound were critical in the formation of self-assembled nanostructures. The thickness of the superhydrophobic 3D self-assembled structure could be varied by controlling the surface properties of the glass substrate. In addition, a novel octadecyl silica nanosquare plate structure was formed as an intermediate for the microlamella structure. The water drop impact experiments on the 3D self-assembled superhydrophobic glass substrates at low temperatures (T < -25 °C) showed that the as-prepared superhydrophobic glass possessed a high impalement threshold for water contact, resulting in excellent and stable icephobic properties. The preparation method proposed in this study is scalable and can be used on a flat glass surface or in a glass vial inside a glass tube. Moreover, it can be applied to various substrates such as metals and polyurethane surfaces with curvature. Therefore, the solution-based self-assembly method proposed in this study is a promising approach to produce superhydrophobic and icephobic surfaces on a wide range of substrates regardless of their structure and properties.

Predicting in vivo therapeutic efficacy of bioorthogonally labeled endothelial progenitor cells in hind limb ischemia models via non-invasive fluorescence molecular tomography.

Human embryonic stem cells-derived endothelial progenitor cells (hEPCs) were utilized as cell therapeutics for the treatment of ischemic diseases. However, in vivo tracking of hEPCs for predicting their therapeutic efficacy is very difficult. Herein, we developed bioorthogonal labeling strategy of hEPCs that could non-invasively track them after transplantation in hind limb ischemia models. First, hEPCs were treated with tetraacylated N-azidomannosamine (Ac4ManNAz) for generating unnatural azide groups on the hEPCs surface. Second, near-infrared fluorescence (NIRF) dye, Cy5, conjugated dibenzocylooctyne (DBCO-Cy5) was chemically conjugated to the azide groups on the hEPC surface via copper-free click chemistry, resulting Cy5-hEPCs. The bioorthogonally labeled Cy5-hEPCs showed strong NIRF signal without cytotoxicity and functional perturbation in tubular formation, oxygen consumption and paracrine effect of hEPCs in vitro. In hind limb ischemia models, the distribution and migration of transplanted Cy5-hEPCs were successfully monitored via fluorescence molecular tomography (FMT) for 28 days. Notably, blood reperfusion and therapeutic neovascularization effects were significantly correlated with the initial transplantation forms of Cy5-hEPCs such as 'condensed round shape' and 'spread shape' in the ischemic lesion. The condensed transplanted Cy5-hEPCs substantially increased the therapeutic efficacy of hind limb ischemia, compared to that of spread Cy5-hEPCs. Therefore, our new stem cell labeling strategy can be used to predict therapeutic efficacy in hind limb ischemia and it can be applied a potential application in developing cell therapeutics for regenerative medicine.