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.

Thermally Managed, Injectable Optoelectronic Probe with Heat Dissipation Guide for Photodynamic Therapy.

The development of fabrication technologies and appearance of new materials has resulted in dramatic increase in the performance of electronic devices, while the overall size has decreased. Recent electronic devices made of micro/nano-size components show high efficiency and outstanding performance with compact size, but these devices have revealed several fatal problems. In particular, the isolated heat that is generated by numerous components concentrated in a limited small area at high density, such as bio-integrated devices, is an issue that needs to be urgently addressed, because it is closely related to the performance and lifetime of electronic devices. To solve these problems, the microscale light emitting diode (µLED)-based neural probe is introduced on an injectable heat dissipation guide. The heat dissipation guide is made of boron nitride (BN) nanomaterials with high thermal conductivity. The heat management noticeably improves the optical output performance of the µLEDs, in which BN effectively dissipates heat, and allows enhanced lighting from the LEDs to be transmitted through brain tissue without thermal damage. Moreover, it shows remarkable improvement in the therapeutic effect of photodynamic therapy of mouse cancer cells.

In vivo toxicity evaluation of tumor targeted glycol chitosan nanoparticles in healthy mice: repeated high-dose of glycol chitosan nanoparticles potentially induce cardiotoxicity.

BACKGROUND: Glycol chitosan nanoparticles (CNPs) have emerged as an effective drug delivery system for cancer diagnosis and treatment. Although they have great biocompatibility owing to biodegradable chemical structure and low immunogenicity, sufficient information on in vivo toxicity to understand the potential risks depending on the repeated high-dose have not been adequately studied. Herein, we report the results of in vivo toxicity evaluation for CNPs focused on the number and dose of administration in healthy mice to provide a toxicological guideline for a better clinical application of CNPs. RESULTS: The CNPs were prepared by conjugating hydrophilic glycol chitosan with hydrophobic 5ß-cholanic acid and the amphiphilic glycol chitosan-5ß-cholanic acid formed self-assembled nanoparticles with its concentration-dependent homogeneous size distributions (265.36-288.3 nm) in aqueous condition. In cell cultured system, they showed significantly high cellular uptake in breast cancer cells (4T1) and cardiomyocytes (H9C2) than in fibroblasts (L929) and macrophages (Raw264.7) in a dose- and time-dependent manners, resulting in severe necrotic cell death in H9C2 at a clinically relevant highly concentrated condition. In particular, when the high-dose (90 mg/kg) of CNPs were intravenously injected into the healthy mice, considerable amount was non-specifically accumulated in major organs (liver, lung, spleen, kidney and heart) after 6 h of injection and sustainably retained for 72 h. Finally, repeated high-dose of CNPs (90 mg/kg, three times) induced severe cardiotoxicity accompanying inflammatory responses, tissue damages, fibrotic changes and organ dysfunction. CONCLUSIONS: This study demonstrates that repeated high-dose CNPs induce severe cardiotoxicity in vivo. Through the series of toxicological assessments in the healthy mice, this study provides a toxicological guideline that may expedite the application of CNPs in the clinical settings.

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.

Short-Term Cessation of Dabigatran Causes a Paradoxical Prothrombotic State.

OBJECTIVE: It is unclear if stopping treatment with dabigatran, a new oral anticoagulant (NOAC), induces a paradoxical rebound prothrombotic state. We investigated if short-term (1-3 days) dabigatran cessation is associated with a higher thrombus volume than expected from a simple reversal of the anticoagulant effect. METHODS: Ten-week-old C57Bl/6 mice (n = 338) received one of the following oral treatments: phosphate-buffered saline (PBS), dabigatran for 7 days with or without 1 to 4 day cessation, and aspirin in either a single dose or daily for 7 days. Some of the animals that ceased dabigatran for 1 to 3 days received single-dose aspirin. Thereafter, we induced FeCl3 -mediated carotid thrombosis in 130 mice, after which we performed micro computed tomography thrombus imaging. The other 208 mice underwent coagulation assays or platelet function tests. As an explorative pilot study, we reviewed the medical records of 18 consecutive patients with NOAC cessation-related cerebral infarction in a large acute stroke cohort. RESULTS: We observed a ~ 40% higher volume of carotid thrombus after dabigatran cessation at 1 to 3 days than after vehicle treatment and showed that this effect could be prevented by single-dose aspirin pretreatment. Dabigatran cessation unduly increased platelet aggregability for 2 days after drug cessation, an effect mediated through thrombin or arachidonic acid, which effect was significantly attenuated by single-dose aspirin pretreatment. In patients, short-term (≤ 3 days) cessation of NOAC therapy, compared with longer-term (≥ 5 days) cessation, tended to be associated with relatively high stroke severity. INTERPRETATION: We provide the first preclinical evidence that a rebound prothrombotic state follows short-term cessation of dabigatran therapy. ANN NEUROL 2021;89:444-458.

Preclinical development of carrier-free prodrug nanoparticles for enhanced antitumor therapeutic potential with less toxicity.

BACKGROUND: Nanomedicine has emerged as a promising strategy for cancer treatment. The most representative nanomedicine used in clinic is PEGylated liposomal doxorubicin DOXIL®, which is first FDA-approved nanomedicine. However, several shortcomings, such as low drug loading capacity, low tumor targeting, difficulty in mass production and potential toxicity of carrier materials, have hindered the successful clinical translation of nanomedicines. In this study, we report a preclinical development process of the carrier-free prodrug nanoparticles designed as an alternative formulation to overcome limitations of conventional nanomedicines in the terms of technical- and industrial-aspects. RESULTS: The carrier-free prodrug nanoparticles (F68-FDOX) are prepared by self-assembly of cathepsin B-specific cleavable peptide (FRRG) and doxorubicin (DOX) conjugates without any additional carrier materials, and further stabilized with Pluronic F68, resulting in high drug loading (> 50%). The precise and concise structure allow mass production with easily controllable quality control (QC), and its lyophilized powder form has a great long-term storage stability at different temperatures (- 4, 37 and 60 °C). With high cathepsin B-specificity, F68-FDOX induce a potent cytotoxicity preferentially in cancer cells, whereas their cytotoxicity is greatly minimized in normal cells with innately low cathepsin B expression. In tumor models, F68-FDOX efficiently accumulates within tumor tissues owing to enhanced permeability and retention (EPR) effect and subsequently release toxic DOX molecules by cathepsin B-specific cleavage mechanism, showing a broad therapeutic spectrum with significant antitumor activity in three types of colon, breast and pancreatic cancers. Finally, the safety of F68-FDOX treatment is investigated after single-/multi-dosage into mice, showing greatly minimized DOX-related toxicity, compared to free DOX in normal mice. CONCLUSIONS: Collectively, these results provide potential preclinical development process of an alternative approach, new formulation of carrier-free prodrug nanoparticles, for clinical translation of nanomedicines.

Light-Activated Monomethyl Auristatin E Prodrug Nanoparticles for Combinational Photo-Chemotherapy of Pancreatic Cancer.

Pancreatic cancer is a highly fatal disease that is becoming an increasingly leading cause of cancer-related deaths. In clinic, the most effective approach to treat pancreatic cancers is the combination treatment of several chemotherapeutic drugs, including fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX), but this approach is not adequate to manage patients due to their severe toxic side effects. Herein, we proposed light-activated monomethyl auristatin E (MMAE) prodrug nanoparticles for combinational photo-chemotherapy and optimized its applications for pancreatic cancer treatment. The photosensitizer (Ce6) and chemotherapeutic drug (MMAE) were conjugated through caspase-3-specific cleavable peptide (KGDEVD). The resulting CDM efficiently promoted the reactive oxygen species (ROS) under visible light irradiation and thereby induced caspase-3 overexpression in pacreatic cancers, which subsequently released the MMAE from the system. Importantly, MMAE released from CDM further amplified the activation of CDM into MMAE by inducing extensive apoptotic cell death in tumor microenvironment for treatment of tumor cells in deep in the tumor tissues as far visible light cannot reach. In addition, CDM formed prodrug nanoparticles via intermolecular π-π stacking and hydrophobic interactions, allowing durable and reliable treatment by preventing fast leakage from the pancreatic cancers via the lymphatic vessels. The CDM directly (intratumoral) injected into pancreatic cancers in orthotopic models through an invasive approach significantly delayed the tumor progression by combinational photo-chemotherapy with less toxic side effects. This study offers a promising and alternative approach for safe and more effective pancreatic cancer treatment via prodrug nanoparticles that combine photodynamic therapy and chemotherapy.

Intracellular Uptake Mechanism of Bioorthogonally Conjugated Nanoparticles on Metabolically Engineered Mesenchymal Stem Cells.

Nanoparticles have been used for effectively delivering imaging agents and therapeutic drugs into stem cells. However, nanoparticles are not sufficiently internalized into stem cells; thus, new delivery method of nanoparticles into stem cells is urgently needed. Herein, we develop bicyclo[6.1.0]nonyne (BCN)-conjugated gold nanoparticles (BCN-AuNPs), which can be bioorthogonally conjugated to azide (-N3) groups on the surface of metabolically engineered stem cells via bioorthogonal click chemistry. For incorporating azide groups on the cell surface, first, human adipose-derived mesenchymal stem cells (hMSCs) were metabolically engineered with N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz). Second, clickable BCN-AuNPs were bioorthogonally conjugated to azide groups on Ac4ManNAz-treated hMSCs. Importantly, a large amount of BCN-AuNPs was specifically conjugated to metabolically engineered hMSCs and then internalized rapidly into stem cells through membrane turnover mechanism, compared to the conventional nanoparticle-derived endocytosis mechanism. Furthermore, BCN-AuNPs entrapped in endosomal/lysosomal compartment could escape efficiently to the cytoplasm of metabolically engineered stem cells. Finally, BCN-AuNPs in stem cells were very safe, and they did not affect stem cell functions, such as self-renewal and differentiation capacity. These bioorthogonally conjugated nanoparticles on metabolically engineered stem cells can enhance the cellular uptake of nanoparticles via bioorthogonal conjugation mechanism.

Extracellular Vesicles as Potential Therapeutics for Inflammatory Diseases.

Extracellular vesicles (EV) deliver cargoes such as nucleic acids, proteins, and lipids between cells and serve as an intercellular communicator. As it is revealed that most of the functions associated to EVs are closely related to the immune response, the important role of EVs in inflammatory diseases is emerging. EVs can be functionalized through EV surface engineering and endow targeting moiety that allows for the target specificity for therapeutic applications in inflammatory diseases. Moreover, engineered EVs are considered as promising nanoparticles to develop personalized therapeutic carriers. In this review, we highlight the role of EVs in various inflammatory diseases, the application of EV as anti-inflammatory therapeutics, and the current state of the art in EV engineering techniques.

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.

Recent Trends in In Situ Enzyme-Activatable Prodrugs for Targeted Cancer Therapy.

Enzyme-activatable anticancer prodrugs are modified medications that are composed of an anticancer drug, cleavable linker, and functional moiety. The purpose of such a prodrug structure is to generate multipurpose functions that traditional drugs cannot perform and to reduce the toxicity of conventional anticancer drugs by the mask of the cleavable linker. Once the cleavable linker is degraded via a specific chemical reaction in the cancer microenvironment, the cytotoxicity of the degraded prodrugs is selectively recovered. Among many factors that cleave the linker, we focus on the overexpressed enzymes in cancer. Because of the selective enzymatic degradation of the cleavable linker and the high local concentration of specific enzymes in cancer, the enzyme-activatable prodrugs could show low toxicity in normal tissues, while showing comparable anticancer effect in tumors. In addition, some prodrugs provide additional features, such as cancer imaging, drug release monitoring, tumor targeting, and enhanced stability, which conventional anticancer drugs cannot possess. In this review, we summarize currently developed enzyme-activatable prodrugs according to their activating enzymes, and categorize them by their additional functions, e.g. targeting, imaging, and delivery. This summary of enzyme-activatable prodrugs may help in the design of anticancer prodrugs, and in the establishment of a personalized cancer treatment strategy.

In Situ One-Step Fluorescence Labeling Strategy of Exosomes via Bioorthogonal Click Chemistry for Real-Time Exosome Tracking In Vitro and In Vivo.

Exosomes are cellular components with promising uses in cancer diagnostics and therapeutics, and their imaging and tracking are essential to study their biological properties. Herein, we report on an in situ one-step fluorescence labeling strategy for exosomes via bioorthogonal click chemistry. First, exosome donor cancer cells were treated with tetraacetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) to generate unnatural azide groups (-N3) on their surface via metabolic glycoengineering. Then, the azide groups were labeled with near-infrared fluorescent dye-conjugated dibenzylcyclooctyne (DBCO-Cy5) via bioorthogonal click chemistry. After 2 days of incubation, the DBCO-Cy5-labeled exosomes (Cy5-Exo) were successfully secreted from the donor cancer cells and were isolated via classical ultracentrifugation, providing a high-yield of fluorescent dye-labeled exosomes. This in situ one-step bioorthogonal click chemistry offers improved labeling efficiency, biocompatibility, and imaging sensitivy compared to standard exosomes (ST-Exo), purified with classical ultracentrifugation or carbocyanine lipophilic dye (DiD)-labeled exosomes (DiD-Exo) in vitro. In particular, the Cy5-Exo were successfully taken up by A549 cells in a time-dependent manner, and they could escape from lysosome confinement, showing their possible use as a delivery carrier of therapeutic drugs or imaging agents. Finally, intraveneously injected Cy5-Exo were noninvasively tracked and imaged via near-infrared fluorescence (NIRF) imaging in tumor-bearing mice. This new fluorescence labeling strategy for natural exosomes may be useful to provide better understanding of their theranostic effects in many biomedical applications.

A Comparative Study on Albumin-Binding Molecules for Targeted Tumor Delivery through Covalent and Noncovalent Approach.

Various types of albumin-binding molecules have been conjugated to anticancer drugs, and these modified prodrugs could be effective in cancer treatments compared to free anticancer drugs. However, the tumor targeting of albumin-binding prodrugs has not been clearly investigated. Herein, we examined the in vitro and in vivo tumor-targeting efficiency of three different albumin-binding molecules including albumin-binding peptide (DICLPRWGCLW: PEP), fatty acid (palmitic acid: PA), and maleimide (MI), respectively. In order to characterize the different targeting efficiency of albumin-binding molecules, PEP, PA, or MI was chemically labeled with near-infrared fluorescence (NIRF) dye, Cy5.5, in resulting PEP-Cy5.5, PA-Cy5.5, and MI-Cy5.5. These NIRF dye-labeled albumin-binding molecules were physically or chemically bound to albumin via gentle incubation in aqueous conditions in vitro. Notably, PA-Cy5.5 with reversible and multivalent binding affinities formed stable albumin complexes, compared to PEP-Cy5.5 and MI-Cy5.5, confirmed via surface plasmon resonance measurement, gel electrophoresis assay, and albumin-bound column-binding test. In tumor-bearing mice model, the different albumin-binding affinities of PA-Cy5.5, PEP-Cy5.5, and MI-Cy5.5 greatly contributed to their tumor-targeting ability. Even though the binding affinity of PEP-Cy5.5 and MI-Cy5.5 to albumin is higher than that of PA-Cy5.5 in vitro, intravenous PA-Cy5.5 showed a higher tumor-targeting efficiency in tumor-bearing mice compared to that of PEP-Cy5.5 and MI-Cy5.5. The reversible and multivalent affinities of albumin-binding molecules to native serum albumin greatly increased the pharmacokinetics and tumor-targeting efficiency in vivo.

Quantitative Imaging of Cerebral Thromboemboli In Vivo: The Effects of Tissue-Type Plasminogen Activator.

BACKGROUND AND PURPOSE: Quantitative imaging for the noninvasive assessment of thrombolysis is needed to advance basic and clinical thrombosis-related research and tailor tissue-type plasminogen activator (tPA) treatment for stroke patients. We quantified the evolution of cerebral thromboemboli using fibrin-targeted glycol chitosan-coated gold nanoparticles and microcomputed tomography, with/without tPA therapy. METHODS: We injected thrombi into the distal internal carotid artery in mice (n=50). Fifty-five minutes later, we injected fibrin-targeted glycol chitosan-coated gold nanoparticles, and 5 minutes after that, we treated animals with tPA or not (25 mg/kg). We acquired serial microcomputed tomography images for 24 hours posttreatment. RESULTS: Thrombus burden at baseline was 784×103±59×103 µm2 for the tPA group (n=42) and 655×103±103×103 µm2 for the saline group (n=8; P=0.37). Thrombus shrinkage began at 0.5 to 1 hour after tPA therapy, with a maximum initial rate of change at 4603±957 µm2/min. The rate of change lowered to ≈61% level of the initial in hours 1 to 2, followed by ≈29% and ≈1% in hours 2 to 3 and 3 to 24, respectively. Thus, 85% of total thrombolysis over 24 hours (≈500 µm2, equivalent to 64% of the baseline thrombus burden) occurred within the first 3 hours of treatment. Thrombus burden at 24 hours could be predicted at around 1.5 to 2 hours. Saline treatment was not associated with significant changes in the thrombus burden. Infarct size was smaller in the tPA group versus saline group (18.1±2.3 versus 45.8±3.3 mm2; P<0.01). Infarct size correlated to final thrombus burden (r=0.71; P<0.01). Time to thrombolysis, completeness of thrombolysis, and tPA therapy were independent predictors of infarct size. CONCLUSIONS: Thromboembolic burden and the efficacy of tPA therapy can be assessed serially, noninvasively, and quantitatively using high-resolution microcomputed tomography and a fibrin-binding nanoparticle imaging agent.

Systemic PEGylated TRAIL treatment ameliorates liver cirrhosis in rats by eliminating activated hepatic stellate cells.

UNLABELLED: Liver fibrosis is a common outcome of chronic liver disease that leads to liver cirrhosis and hepatocellular carcinoma. No US Food and Drug Administration-approved targeted antifibrotic therapy exists. Activated hepatic stellate cells (aHSCs) are the major cell types responsible for liver fibrosis; therefore, eradication of aHSCs, while preserving quiescent HSCs and other normal cells, is a logical strategy to stop and/or reverse liver fibrogenesis/fibrosis. However, there are no effective approaches to specifically deplete aHSCs during fibrosis without systemic toxicity. aHSCs are associated with elevated expression of death receptors and become sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death. Treatment with recombinant TRAIL could be a potential strategy to ameliorate liver fibrosis; however, the therapeutic application of recombinant TRAIL is halted due to its very short half-life. To overcome this problem, we previously generated PEGylated TRAIL (TRAILPEG ) that has a much longer half-life in rodents than native-type TRAIL. In this study, we demonstrate that intravenous TRAILPEG has a markedly extended half-life over native-type TRAIL in nonhuman primates and has no toxicity in primary human hepatocytes. Intravenous injection of TRAILPEG directly induces apoptosis of aHSCs in vivo and ameliorates carbon tetrachloride-induced fibrosis/cirrhosis in rats by simultaneously down-regulating multiple key fibrotic markers that are associated with aHSCs. CONCLUSION: TRAIL-based therapies could serve as new therapeutics for liver fibrosis/cirrhosis and possibly other fibrotic diseases. (Hepatology 2016;64:209-223).

Inorganic Nanoparticles for Image-Guided Therapy.

Recently, nanotechnology has provided significant advances in biomedical applications including diagnosis and therapy. In particular, nanoparticles have emerged as valuable outcomes of nanotechnology due to their unique physicochemical properties based on size, shape, and surface properties. Among them, a large amount of research has reported imaging and therapeutic applications using inorganic nanoparticles with special properties. Inorganic nanoparticles developed for imaging and therapy contain metal (Au), metal oxide (Fe3O4, WO3, WO2.9), semiconductor nanocrystal (quantum dots (QDs)), and lanthanide-doped upconversion nanoparticles (UCNPs). Based on their intrinsic properties, they can generate heat, reactive oxygen species (ROS), or energy transfer, so that they can be used for both imaging and therapy. In this review, we introduce biocompatible inorganic nanoparticles for image-guided thermal and photodynamic therapy, and discuss their promising results from in vitro and in vivo studies for biomedical applications.

Artificial Chemical Reporter Targeting Strategy Using Bioorthogonal Click Reaction for Improving Active-Targeting Efficiency of Tumor.

Biological ligands such as aptamer, antibody, glucose, and peptide have been widely used to bind specific surface molecules or receptors in tumor cells or subcellular structures to improve tumor-targeting efficiency of nanoparticles. However, this active-targeting strategy has limitations for tumor targeting due to inter- and intraheterogeneity of tumors. In this study, we demonstrated an alternative active-targeting strategy using metabolic engineering and bioorthogonal click reaction to improve tumor-targeting efficiency of nanoparticles. We observed that azide-containing chemical reporters were successfully generated onto surface glycans of various tumor cells such as lung cancer (A549), brain cancer (U87), and breast cancer (BT-474, MDA-MB231, MCF-7) via metabolic engineering in vitro. In addition, we compared tumor targeting of artificial azide reporter with bicyclononyne (BCN)-conjugated glycol chitosan nanoparticles (BCN-CNPs) and integrin αvß3 with cyclic RGD-conjugated CNPs (cRGD-CNPs) in vitro and in vivo. Fluorescence intensity of azide-reporter-targeted BCN-CNPs in tumor tissues was 1.6-fold higher and with a more uniform distribution compared to that of cRGD-CNPs. Moreover, even in the isolated heterogeneous U87 cells, BCN-CNPs could bind artificial azide reporters on tumor cells more uniformly (∼92.9%) compared to cRGD-CNPs. Therefore, the artificial azide-reporter-targeting strategy can be utilized for targeting heterogeneous tumor cells via bioorthogonal click reaction and may provide an alternative method of tumor targeting for further investigation in cancer therapy.

Cytokine Response to Diet and Exercise Affects Atheromatous Matrix Metalloproteinase-2/9 Activity in Mice.

BACKGROUND: The aim of this study is to identify the principal circulating factors that modulate atheromatous matrix metalloproteinase (MMP) activity in response to diet and exercise.Methods and Results:Apolipoprotein-E knock-out (ApoE-/-) mice (n=56) with pre-existing plaque, fed either a Western diet (WD) or normal diet (ND), underwent either 10 weeks of treadmill exercise or had no treatment. Atheromatous MMP activity was visualized using molecular imaging with a MMP-2/9 activatable near-infrared fluorescent (NIRF) probe. Exercise did not significantly reduce body weight, visceral fat, and plaque size in either WD-fed animals or ND-fed animals. However, atheromatous MMP-activity was different; ND animals that did or did not exercise had similarly low MMP activities, WD animals that did not exercise had high MMP activity, and WD animals that did exercise had reduced levels of MMP activity, close to the levels of ND animals. Factor analysis and path analysis showed that soluble vascular cell adhesion molecule (sVCAM)-1 was directly positively correlated to atheromatous MMP activity. Adiponectin was indirectly negatively related to atheromatous MMP activity by way of sVCAM-1. Resistin was indirectly positively related to atheromatous MMP activity by way of sVCAM-1. Visceral fat amount was indirectly positively associated with atheromatous MMP activity, by way of adiponectin reduction and resistin elevation. MMP-2/9 imaging of additional mice (n=18) supported the diet/exercise-related anti-atherosclerotic roles for sVCAM-1. CONCLUSIONS: Diet and exercise affect atheromatous MMP activity by modulating the systemic inflammatory milieu, with sVCAM-1, resistin, and adiponectin closely interacting with each other and with visceral fat.

Long-Circulating Au-TiO2 Nanocomposite as a Sonosensitizer for ROS-Mediated Eradication of Cancer.

Although sonodynamic therapy (SDT) has emerged as a potential alternative to conventional photodynamic therapy, the low quantum yield of the sonosensitizer such as TiO2 nanoparticles (NPs) is still a major concern. Here, we have developed hydrophilized Au-TiO2 nanocomposites (HAu-TiO2 NCs) as sonosensitizers for improved SDT. The physicochemical properties of HAu-TiO2 NCs were thoroughly studied and compared with their counterparts without gold deposition. Upon exposure of HAu-TiO2 NCs to ultrasound, a large quantity of reactive oxygen species (ROS) were generated, leading to complete suppression of tumor growth after their systemic administration in vivo. Overall, it was evident that the composites of gold with TiO2 NPs significantly augmented the levels of ROS generation, implying their potential as SDT agents for cancer therapy.

Non-invasive stem cell tracking in hindlimb ischemia animal model using bio-orthogonal copper-free click chemistry.

Labeling of stem cells aims to distinguish transplanted cells from host cells, understand in vivo fate of transplanted cells, particularly important in stem cell therapy. Adipose-derived mesenchymal stem cells (ASCs) are considered as an emerging therapeutic option for tissue regeneration, but much remains to be understood regarding the in vivo evidence. In this study, a simple and efficient cell labeling method for labeling and tracking of stem cells was developed based on bio-orthogonal copper-free click chemistry, and it was applied in a mouse hindlimb ischemia model. The human ASCs were treated with tetra-acetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) to generate glycoprotein with unnatural azide groups on the cell surface, and the generated azide groups were fluorescently labeled by specific binding of dibenzylcyclooctyne-conjugated Cy5 (DBCO-Cy5). The safe and long-term labeling of the hASCs by this method was first investigated in vitro. Then the DBCO-Cy5-hASCs were transplanted into the hindlimb ischemia mice model, and we could monitor and track in vivo fate of the cells using optical imaging system. We could clearly observe the migration potent of the hASCs toward the ischemic lesion. This approach to design and tailor new method for labeling of stem cells may be useful to provide better understanding on the therapeutic effects of transplanted stem cells into the target diseases.