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We evaluated modulation of the immunosuppressive tumor microenvironment in both local and liver metastatic colorectal cancer (LMCC), focusing on tumor-associated macrophages, which are the predominant immunosuppressive cells in LMCC. We developed an orally administered metronomic chemotherapy regimen, oral CAPOX. This regimen combines capecitabine and a nano-micelle encapsulated, lysine-linked deoxycholate and oxaliplatin complex (OPt/LDC-NM). The treatment effectively modulated immune cells within the tumor microenvironment by activating the cGAS-STING pathway and inducing immunogenic cell death. This therapy modulated immune cells more effectively than did capecitabine monotherapy, the current standard maintenance chemotherapy for colorectal cancer. The macrophage-modifying effect of oral CAPOX was mediated via the cGAS-STING pathway. This is a newly identified mode of immune cell activation induced by metronomic chemotherapy. Moreover, oral CAPOX synergized with anti-PD-1 antibody (αPD-1) to enhance the T-cell-mediated antitumor immune response. In the CT26. CL25 subcutaneous model, combination therapy achieved a 91 % complete response rate with a confirmed memory effect against the tumor. This combination also altered the immunosuppressive tumor microenvironment in LMCC, which αPD-1 monotherapy could not achieve. Oral CAPOX and αPD-1 combination therapy outperformed the maximum tolerated dose for treating LMCC, suggesting metronomic therapy as a promising strategy., Competing Interests: Declaration of competing interest There are no known competing financial interests or personal relationships that could have potentially influenced the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The exclusion of social play within an adolescent group interferes with learning and the acquisition of essential social behavior during development and can cause modulations in the social brain areas. However, despite the importance of social play in adolescence, an in-depth explanation of its physiological mechanisms is limited because of the lack of experimental animal models that embody social play exclusion in human society. To determine the mechanism of social play in adolescence, we identified differences in emotional behavior and brain activity in animal models of social play exclusion that mimicked human society. Emotional changes in the social play exclusion and non-exclusion groups were examined by tracking social play-related social interaction behavior, social play-related space preference, social play-related locomotor behavior, and anxiety-like behavior using a behavioral data analysis program. Differences in brain activity among groups were identified using immunohistochemical staining. During the social play exclusion model, the rats preferred the partition zone to the other areas in the test chamber. The exclusion group preferred the partition and the center zone over the non-exclusion group. When comparing before and after the social play exclusion, the exclusion group showed a decrease in mobility and an increase in anxiety-like behavior compared to the non-exclusion group. We found that c-Fos expression in the dentate gyrus (DG) of the exclusion group was lower than that in the non-exclusion group, whereas c-Fos expression in the lateral habenula (LHb) of the exclusion group was higher than that in the non-exclusion group. Taken together, in adolescence, exclusion from social play with peers can increase anxiety-like behavior in the exclusion group and change the neuronal activity of the DG and LHb, suggesting that exclusion from social play is linked to modifications in the DG and LHb, which are regions associated with mood regulation., Competing Interests: Declaration of Competing Interest The authors report no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Senescence is a phenomenon defined by alterations in cellular organelles and is the primary cause of aging and aging-related diseases. Recent studies have shown that oncogene-induced senescence is driven by activation of serine/threonine protein kinases (AKT1, AKT2 and AKT3). In this study, we evaluated twelve AKT inhibitors and revealed GDC0068 as a potential agent to ameliorate senescence. Senescence-ameliorating effect was evident from the finding that GDC0068 yielded lysosomal functional recovery as observed by reduction in lysosomal mass and induction in autophagic flux. Furthermore, GDC0068-mediated restoration of lysosomal function activated the removal of dysfunctional mitochondria, resulting in restoration of mitochondrial function. Together, our findings revealed a unique mechanism by which senescence is recovered by functional restoration of lysosomes and mitochondria through modulation of AKT activity., Competing Interests: Competing interest The authors declare no competing financial interests., (Copyright © 2023. Published by Elsevier Inc.)

While conventional approaches for PTEN-loss cancers mainly focus on turning off growth promoting process through modulation of PI3K/AKT pathways, no effective therapeutic treatments that target PTEN-loss cancer cells have yielded results. Moreover, conventional targeted therapies, which are potent against only a subset of cancer cells with limited specificity, bring on temporary response. Here, we report the development of albumin-binding caspase-3 cleavable peptide-drug conjugate (PDC), which utilizes the enhanced albumin metabolism pathway in PTEN-loss cancer cells to enhance the intracellular delivery of chemotherapeutic payload that could exert a bystander killing effect. Albumin metabolism-mediated apoptosis triggered expression of caspase-3 allows the continuous activation of the PDC, accumulation of payloads, sustained upregulation of tumoral caspase-3, and intensified in-situ apoptosis. Importantly, PDC strategy exerts potent therapeutic efficacy against PTEN-loss metastatic triple-negative breast cancer, the highly aggressive and heterogenous nature of which remains a challenge conventional targeted therapies need to overcome. This study thus presents a conceptually novel approach to treat PTEN-loss cancer and creates new translational perspectives of exploiting PTEN-loss for providing an avenue to advance current targeted therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In conventional chemotherapy, maximum tolerated dose approach is considered as a first-line medication for cancer treatment in clinics. In contrast to the conventional chemotherapy which has heavy tumor burdens arising from high dose treatment, metronomic chemotherapy (MCT) engages relatively low dose without drug-free breaks, and is recognized as a promising strategy for a long-term management of the disease. Although doxorubicin (DOX), an anthracycline anti-cancer drug, showed a potential of maintenance effect in vitro, further study on in vivo-relevant concentration to achieve tumor suppression with no toxicity is required to apply the MCT in clinicals. Therefore, the objective of this study was to identify an optimal MCT regimen of DOX by determining concentration-response relationships of tumor suppression (pharmacodynamic; PD) and cardiac toxicity (toxicodynamic; TD). Utilizing an oral DOX formulation complexed with deoxycholic acid (DOX/DOCA complex) which has enhanced bioavailability, physiologically-based pharmacokinetic (PBPK) model was linked to TD and PD models to generate drug profiles from the combined PK, TD, and PD parameters. The integrated model was validated for various scenarios of administration route, formulation, dose, and frequency. The established mathematical model facilitated calculations of adequate in vivo-relevant dosages and intervals, suggesting the optimum oral metronomic regimen of DOX. It is expected to serve as a useful guideline for the design and evaluation of oral DOX formulations in future preclinical/clinical studies., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Growth factors (GF) regulate normal development to cancer progression. GFs interact with extracellular matrix (ECM) biomolecules, such as heparin sulfate (HS) glycosaminoglycan (GAG), to enhance their stability and angiogenic signaling. Biomaterials that modulate GF activity by mimicking interactions observed in the native ECM could be designed as an effective treatment strategy. However, these materials failed to attenuate angiogenic signaling site-specifically without sparing normal tissues. In this work, we investigated the effect of a GAG-based biomaterial, which binds to the tumor endothelial cells (TEC), on the interaction among vascular endothelial growth factor (VEGF), its receptors-VEGFR2 and HS-and angiogenesis. Heparin-bile acid based conjugates, as ECM-mimicking component, were synthesized to selectively target the TEC marker doppel and doppel/VEGFR2 axis. The most effective compound LHbisD4 (low molecular weight heparin conjugated with 4 molecules of dimeric dexocholic acid) reduced tumor volume concentrated over doppel-expressing EC, and decreased tumor-interstitial VEGF without affecting its plasma concentration. Doppel-destined LHbisD4 captured VEGF, formed an intermediate complex with doppel, VEGFR2, and VEGF but did not induce active VEGFR2 dimerization, and competitively inhibited HS for VEGF binding. We thus show that GAG-based materials can be designed to imitate and leverage to control tumor microenvironment via bio-inspired interactions., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In this study, we investigated the immune-modulating effects of a novel metronomic chemotherapy (MCT) featuring combined oral oxaliplatin (OXA) and pemetrexed (PMX) for colon cancer. OXA and PMX were ionically complexed with lysine derivative of deoxycholic acid (DCK), and incorporated into nanoemulsions or colloidal dispersions, yielding OXA/DCK-NE and PMX/DCK-OP, respectively, to improve their oral bioavailabilities. MCT was not associated with significant lymphotoxicity whereas the maximum tolerated dose (MTD) afforded systemic immunosuppression. MCT was associated with more immunogenic cell death and tumor cell MHC-class I expression than was MTD. MCT improved the tumor antigen presentation of dendritic cells and increased the number of functional T cells in the tumor. MCT also helped to enhance antigen-specific memory responses both locally and systemically. By combining MCT with anti-programmed cell death protein-1 (αPD-1) therapy, the tumor volume was suppressed by 97.85 ± 84.88% compared to the control, resulting in a 95% complete response rate. Upon re-challenge, all tumor-free mice rejected secondary tumors, indicating the induction of a tumor specific memory response. Thus, MCT using an OXA and PMX combination, together with αPD-1, successfully treated colon cancer by activating both innate and adaptive immune cells and elicited tumor-specific long-term immune memory while avoiding toxicity caused by MTD treatment., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Crizotinib is used in the clinic for treating patients with ALK- or ROS1-positive non-small-cell lung carcinoma. The objective of the present study was to determine if crizotinib enantiomers could induce changes to the properties of cancer and cancer stem cell (CSC)-like cells at a high concentration (∼ 3 μM). While (R)-crizotinib induced changes in morphologies or sizes of cells, (S)-crizotinib did not. Pretreatment with (R)-crizotinib suppressed the proliferation of cancer or CSC-like cells in vitro and tumor growth in vivo. In vivo administration of (R)-crizotinib inhibited the growth of tumors formed from CSC-like cells by 72%. %. Along with the morphological changes induced by (R)-crizotinib, the expression levels of CD44 (NCI-H23 and HCT-15), ALDH1 (NCI-H460), nanog (PC-3), and Oct-4A (CSC-like cells), which appear to be specific marker proteins, were greatly changed, suggesting that changes in cellular properties accompanied the morphological changes in the cells. The expression levels of Snail, Slug, and E-cadherin were also greatly altered by (R)-crizotinib. Among several signal transduction molecules examined, AMPK phosphorylation appeared to be selectively inhibited by (R)-crizotinib. BML-275 (an AMPK inhibitor) and AMPKα2 siRNA efficiently induced morphological changes to all types of cells examined, suggesting that (R)-crizotinib might cause losses of characteristics of cancer or CSCs via inhibition of AMPK. These results indicate that (R)-crizotinib might be an effective anticancer agent that can cause alteration in cancer cell properties., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Here we report a novel combination of a caspase-cleavable peptide-doxorubicin conjugate (MPD-1) with CD47-antagonizing nanocage therapeutics for the treatment of microsatellite-stable (MSS) colorectal cancer (CRC). MPD-1 (i) upregulated markers of immunogenic cell death (ICD) in tumor, and increased co-stimulatory markers on dendritic cells (DCs), (ii) enhanced CD8 + T cell infiltration and antigen presenting cell (APC) activation, and (iii) showed negligible off-target immune-related toxicity compared to free dox. Then, the CD47 antagonist FS nanocage, a SIRPα-expressing ferritin nanocage, was co-administered with MPD-1 that resulted in 95.2% (p < 0.001) tumor growth inhibition in an established CRC model. T cell-mediated elimination of tumors was also confirmed by the tumor-specific activation of T cells detected by IFNγ and tumor-free mice were observed (95%) that bared a memory response when re-challenged. The strategically developed MPD-1 is an ideal adjuvant to immunotherapy and the combination with FS nanocage triggers potent immunity against MSS CRC. In summary, we present an approach to initiate and stimulate immune-mediated eradication of cancer cells using synergistic immunogenic agents targeting the MSS CRC., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The therapeutic efficacy of photodynamic therapy (PDT) in cancer treatment is attributed to the conversion of tumor oxygen into reactive singlet oxygen ( 1 O 2 ) using photosensitizers. However, poor tissue penetration and rapid oxygen depletion have limited the effectiveness of PDT. Therefore, we have developed visible light-induced apoptosis activatable nanoparticles of the photosensitizer (Ce6)-caspase 3 cleavable peptide (Asp-Glu-Val-Asp, DEVD)-anticancer drug monomethyl auristatin E (MMAE) conjugate, resulting in Ce6-DEVD-MMAE nanoparticles. The average size of self-assembled Ce6-DEVD-MMAE nanoparticles was 90.8 ± 18.9 nm. Compared with conventional PDT based on high-energy irradiation, the new therapy uses lower-energy irradiation to induce apoptosis of cancer cells, and activation of caspase 3 to successfully cleave the anticancer drug MMAE from the Ce6-DEVD-MMAE nanoparticles, resulting in strong cytotoxic effects in cancer cells. Notably, the one-time activation of MMAE in the Ce6-DEVD-MMAE nanoparticles further amplified the cytotoxic effect resulting in additional cell death in the absence of visible light irradiation. Furthermore, Ce6-DEVD-MMAE nanoparticles passively accumulated in the targeted tumor tissues via enhanced permeation and retention (EPR) effect in mice with squamous cell carcinoma (SCC7). The high levels of toxicity were retained after exposure to lower-energy irradiation. However, Ce6-DEVD-MMAE nanoparticles did not show any toxicity in the absence of exposure to visible light irradiation, in contrast to the toxicity of free MMAE (1-10 nM). Thus, the light-induced therapeutic strategy based on apoptotic activation of Ce6-DEVD-MMAE nanoparticles can be used to treat solid tumors inaccessible to conventional PDT., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Ferumoxytol is the only clinically available ultrasmall superparamagnetic iron oxide. However, the labeling efficacy of islet magnetic resonance imaging (MRI) using ferumoxytol is not suitable for use in clinical pancreatic islet transplantation (PIT). We evaluated the feasibility of pancreatic islet MRI using ferumoxytol through multi-layer surface modification. A four-layer nanoshield with poly (ethylene) glycol (PEG, 2 layers), ferumoxytol, and heparin was formed on the pancreatic islets. We compared pancreatic islet function, viability, and labeling efficacy of control, ferumoxytol alone-labeled, heparin-PEGylated, and ferumoxytol-heparin-PEGylated islets. With optimization of the ferumoxytol concentration during the ferumoxytol-heparin-PEGylation process, the labeling contrast in ex vivo MRI of ferumoxytol-heparin-PEGylated pancreatic islets was stronger than that of pancreatic islets labeled with ferumoxytol alone, without decreasing ex vivo islet viability or function. In a syngeneic mouse renal subcapsular PIT model, heparin-PEGylation and ferumoxytol-heparin-PEGylation delayed the revascularization of pancreatic islet grafts but did not impair glucose tolerance or revascularization of pancreatic islet grafts four weeks post-transplantation. Pancreatic islet visibility after labeling was also confirmed in a syngeneic mouse intraportal PIT model and in preliminary analysis of a non-human primate intraportal PIT model. In conclusion, multi-layer islet surface modification is a promising option for pancreatic islet MRI in intraportal PIT., (Copyright © 2019. Published by Elsevier Ltd.)

Despite the emergence of advanced therapeutics such as targeted therapy and immunotherapy in the modern oncology, cytotoxic chemotherapy still remains as the first-line treatment option in a wide range of cancers attributing to its potency. Many endeavors have been made to overcome the toxicity issues of cytotoxic chemotherapy by improving the specific delivery to the tumor, with active tumor targeting being one of the most popular approaches. However, such an approach has been challenged by the intratumor heterogeneity and the lack of valid molecular target in many types of cancer. Here, we introduce a novel albumin-binding prodrug MPD02 that could specifically deliver highly potent cytotoxin monomethyl auristatin E (MMAE) to the tumor as an important component of chemoradiotherapy for the treatment of triple-negative breast cancer (TNBC). MPD02 was synthesized by conjugating MMAE to the C-terminus of the KGDEVD peptide via self-eliminating linker and introducing a maleimide group to the Lys side chain of the peptide. MPD02 was able to bind albumin after administration via maleimide group for an extended circulation time and metabolized into MMAE in tumor-specific manner by reacting with the caspase-3 upregulated in tumor by radiotherapy, exerting a highly potent anticancer effect with good safety profile in two different TNBC xenograft models., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Metronomic chemotherapy, which is defined as a low-dose and frequent administration of cytotoxic drugs without drug-free breaks, has been recently emerged as an alternative to traditional MTD therapy and has shown therapeutic benefit in breast cancer patients in numbers of clinical studies. Unlike MTD, metronomic chemotherapy acts by multiple mechanisms including antiangiogenic effect and immunomodulation, but the direct cytotoxic effect only playing a minor role due to the lowered dose. In this light, within the limits of p53-deficient breast cancer, we demonstrate the enhanced anticancer effect of metronomic chemotherapy using doxorubicin when combined with Chk1 inhibitor MK-8776 by specifically augmenting the direct cytotoxic effect on cancer cells. Since the oral drug is greatly favored in metronomic chemotherapy due to the frequent and potential long-term administration, we prepared an oral doxorubicin by producing an ionic complex with deoxycholic acid, which showed sufficient bioavailability and anticancer effect when administered orally. MK-8776 selectively enhanced the cytotoxic effect of low-concentration doxorubicin in p53-deficient breast cancer cells by abrogating the Chk1-dependent cell cycle arrest in vitro. Consistently, combining MK-8776 significantly improved the anticancer effect of the daily administered oral doxorubicin in p53-deficient breast cancer xenografts especially in a lower dose of doxorubicin without evident systemic toxicities. Combination therapy of MK-8776 and metronomic oral doxorubicin would be thus promising in the treatment of p53-deficient breast cancer benefited from the augmented direct cytotoxic effect and low risk of toxicities., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Intraportal pancreatic islet transplantation incurs huge cell losses during its early stages due to instant blood-mediated inflammatory reactions (IBMIRs), which may also drive regulation of the adaptive immune system. Therefore, a method that evades IBMIR will improve clinical islet transplantation. We used a layer-by-layer approach to shield non-human primate (NHP) islets with polyethylene glycol (nano-shielded islets, NSIs) and polyethylene glycol plus heparin (heparin nano-shielded islets; HNSIs). Islets ranging from 10,000 to 20,000 IEQ/kg body weight were transplanted into 19 cynomolgus monkeys (n = 4, control; n = 5, NSI; and n = 10, HNSI). The mean C-peptide positive graft survival times were 68.5, 64 and 108 days for the control, NSI and HNSI groups, respectively (P = 0.012). HNSI also reduced the factors responsible for IBMIR in vitro. Based on these data, HNSIs in conjunction with clinically established immunosuppressive drug regimens will result in superior outcomes compared to those achieved with the current protocol for clinical islet transplantation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This study aims to develop a novel surface modification technology to prolong the survival time of pancreatic islets in a xenogenic transplantation model, using 3,4-dihydroxyphenethylamine (DOPA) conjugated poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) nanoparticles (DOPA-NPs) carrying immunosuppressant FK506 (FK506/DOPA-NPs). The functionalized DOPA-NPs formed a versatile coating layer for antigen camouflage without interfering the viability and functionality of islets. The coating layer effectively preserved the morphology and viability of islets in a co-culture condition with xenogenic lymphocytes for 7 days. Interestingly, the mean survival time of islets coated with FK506/DOPA-NPs was significantly higher as compared with that of islets coated with DOPA-NPs (without FK506) and control. This study demonstrated that the combination of surface camouflage and localized low dose of immunosuppressant could be an effective approach in prolonging the survival of transplanted islets. This newly developed platform might be useful for immobilizing various types of small molecules on therapeutic cells and biomaterial surface to improve the therapeutic efficacy in cell therapy and regenerative medicine., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The development of highly funtional and orally available nanoparticles is the ultimate goal in nanoparticle delivery. Various functional nanoparticles have been studied to that end but there has yet to be an oral nanoparticle that can be successfully applied. Here, we describe for the first time a novel bile acid conjugated nanoparticle that can be selectively absorbed by bile acid transporters in the small intestine. The bile acid conjugate nanoparticles that were first treated with enterocytes were successfully attached to the cell surface and then internalized inside the cells. We show that bile acid based interaction between a nanoparticle and its transporter induces its endocytosis and cellular uptake. This feature of cellular activity, described here for the first time, could be well utilized in the uptake of nanoparticles or macromolecules inside epithelial cells for oral delivery. In animal studies, bile acid conjugated self-assembling nanocomplexes successfully interacted with bile acid transporters in the ileum and were subsequently taken up into the epithelial cells. Considering the importance of orally deliverable nanoparticles, this nanotechnology using bile acid conjugation and transporter mediated endocytosis could be a crucial method for the successful application of various nanoparticles., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Clinical studies have found that the incidence of cancer metastasis through the lymphatic vessels are 3-5 times higher than that through the blood vessels. These findings suggest the potency of anti-lymphangiogenic therapy in reducing the incidence of cancer metastasis. Previously, we reported LHbisD4, which is the conjugate of low molecular weight heparin (LMWH) and four bis-deoxycholates as a potent anti-angiogenic drug with less toxicity and orally active property. Here, we show that LHbisD4 could also suppress the formation of new lymphatic vessels and attenuate the incidence of metastasis by blocking VEGF-C signaling pathway. LHbisD4 significantly enhanced binding affinity with VEGF-C when compared with LMWH, which enables LHbisD4 to suppress the proliferation, migration and formation of tubular structures of human dermal lymphatic endothelial cells(HDLECs) in in vitro condition even in the presence of excessive amounts of VEGF-C. Similarly, we found that the density of lymphatic vessels in the primary tumor tissue in breast cancer bearing mice was significantly diminished when LHbisD4 was administered compared with the control group. Also, the incidence of axillary lymph nodes and distant organ metastasis was significantly reduced in the LHbisD4 administered group, which demonstrates that LHbisD4 could successfully lower the incidence of metastasis through blocking VEGF-C induced lymphangiogenesis. Based on these results, we propose LHbisD4 as a potent anti-cancer drug that can reduce the incidence of metastasis by suppressing lymphangiogenesis through blocking VEGF-C signaling pathway., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Caspase-activated prodrug chemotherapy is introduced and demonstrated using the composite nanoparticles (NPs), which deliver doxorubicin (DOX) and DEVD-S-DOX together to the tumor tissue. DEVD-S-DOX, DOX linked to a peptide moiety (DEVD), is a prodrug that is cleaved into free DOX by caspase-3 upon apoptosis. DEVD-S-DOX has no therapeutic efficacy, but it changes into free DOX with the expression of caspase-3. With the accumulation of the composite NPs in the tumor tissue by the enhanced permeation and retention (EPR) effect, a small exposure of DOX in the tumor cells initiated apoptosis in a localized area of the tumor tissue, which induced caspase-3 activation. Cleavage of DEVD-S-DOX into free DOX by caspase-3 continued with repetitive activation of caspase-3 and cleavage of DEVD-S-DOX at the tumor site. The composite NPs were characterized with transmittance electron microscopy (TEM) and particle size analyzer. We then evaluated the nanoparticle drug release, therapeutic efficacy, and in vivo biodistribution for tumor targeting using a non-invasive live animal imaging technology and the quantification of DOX with high performance liquid chromatography. DOX-induced apoptosis-targeted chemotherapy (DIATC) was verified by in vitro/in vivo DEVD-S-DOX response to free DOX and cellular uptake behavior of the composite NPs with flow cytometry analysis. Significant antitumor efficacy with minimal cardiotoxicity was also observed, which supported DIATC for improved chemotherapy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Existence of the genomically and epigenomically diverse subclones in a tumor severely limits the therapeutic efficacy of targeted agents. To overcome such a limitation, we prepared a novel targeted prodrug, EMC-DEVD-S-DOX, which comprises two important features: radiation-induced apoptosis targeting and albumin-binding properties. In particular, the prodrug binds circulating albumin after intravenous administration and then activated by caspase-3, which is upregulated from apoptotic cells that responded to radiotherapy. The prodrug was designed to bind circulating albumin to extend half-life and facilitate tumor accumulation in order to increase the possibility of contacting caspase-3, which is only transiently upregulated during apoptosis. Our results showed that EMC-DEVD-S-DOX had a prolonged half-life with enhanced tumor accumulation, which clearly benefited the therapeutic effect of the prodrug. Also, agreeing with the in vitro studies that showed ignorable cytotoxic effect in the absence of caspase-3, the prodrug was effective only when combined with radiotherapy without any noticeable systemic toxicity in vivo. Due to the highly selective action of EMC-DEVD-S-DOX independent to the complex genomic profiles of tumor, the prodrug would overcome the limitation of current targeted therapy and potentiate radiotherapy in the clinical oncology., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study suggests a novel method of stabilizing fragile porcine islets to prevent the dissociation after isolation and reducing immune cell invasion in a combination therapy of 'surface camouflaging' and immunosuppressive drugs (FK506, Rapamycin, MR-1, anti-CD19 mAb, and Clodrosome(®)) to effectively alleviate overall immune reactions against xenotransplanted porcine islets. The surface camouflage of pancreatic islets using biocompatible materials improved stabilization of pancreatic islet and prevented the infiltration of immune cells. Firstly, the surface of porcine islets was camouflaged by SH-6-arm-PEG-lipid and gelatin-catechol (artificial extracellular matrix) in order to stabilize the fragile isolated islets. Secondly, three different PEG layers (6-arm-PEG-SH, 6-arm-PEG-catechol, and linear PEG-SH) were chemically conjugated onto the surface of the stabilized porcine islets. Both artificial extracellular matrix (artificial ECM) and PEGylation effectively covered the surface of porcine islets without increasing the size of the whole islet. In addition, the viability and functionality of the islets were not affected by this multi-layer surface modification. The multi-layer modification significantly reduced the attachment of human serum albumin, fibronectin, and immunoglobulin G in comparison to the control collagen surface. The combination effect of multi-layer PEGylation and cocktailed immunosuppressive drugs on the survival time of the transplanted islets was assessed in a xenogeneic porcine-to-mouse model. The median survival time (MST) of 'artificial ECM + PEGylation' group was 4-fold increased compared to that of control group. In addition, the MST of 'artificial ECM + PEGylation + drug' group was 2.16-fold increased, compared to the 'control + drug' group. In conclusion, we proposed a novel porcine islet transplantation protocol using surface multi-layer modification and cocktailed immunosuppressive drugs, for stabilization and immunoprotection against xenogeneic immune reactions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Targeting multiple stages in metastatic breast cancer is one of the effective ways to inhibit metastatic progression. To target human metastatic breast cancer as well as improving patient compliance, we developed an orally active low molecular weight heparin (LMWH)-taurocholate conjugated with tetrameric deoxycholic acid, namely LHTD4, which followed by physical complexation with a synthetic bile acid enhancer, DCK. In breast cancer, both transforming growth factor-β1 (TGF-β1) and CXCL12 exhibit enhanced metastatic activity during the initiation and progression stages of breast cancer, thus we direct the focus on investigating the antimetastatic effect of LHTD4/DCK complex by targeting TGF-β1 and CXCL12. Computer simulation study and SPR analysis were performed for the binding confirmation of LHTD4 with TGF-β1 and CXCL12. We carried out in vitro phosphorylation assays of the consecutive receptors of TGF-β1 and CXCL12 (TGF-β1R1 and CXCR4, respectively). Effects of LHTD4 on in vitro cell migration (induced by TGF-β1) and chemotaxis (mediated by CXCL12) were investigated. The in vivo anti-metastatic effect of LHTD4 was evaluated in an accelerated metastasis model and an orthotopic MDA-MB-231 breast cancer model. The obtained KD values of TGF-β1 and CXCL12 with LHTD4 were 0.85 and 0.019 μM respectively. The simulation study showed that binding affinities of LHTD4 fragment with either TGF-β1 or CXCL12 through additional electrostatic interaction was more stable than that of LMWH fragment. In vitro phosphorylation assays of TGF-β1R1 and CXCR4 showed that the effective inhibition of receptor phosphorylation was observed with the treatment of LHTD4. The expressions of epithelial to mesenchymal transition (EMT) marker proteins such as vimentin and Snail were prevented by LTHD4 treatment in in vitro studies with TGF-β1 treated MDA-MB-231 cells. Moreover, we observed that LHTD4 negatively regulated the functions of TGF-β1 and CXCL12 on migration and invasion of breast cancer cell. In several advanced orthotopic and experimental breast cancer metastasis murine models, the treatment with LHTD4 (5 mg/kg daily, p.o.) significantly inhibited metastasis compared to the control. Overall, LHTD4 exhibited anti-metastatic effects by inhibiting TGF-β1 and CXCL12, and the clinically relevant dose of orally active LHTD4 was found to be effective in preclinical studies without any apparent toxicity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Despite the therapeutic benefits of the angiogenesis inhibitors shown in the clinics, they have encountered an unexpected limitation by the occurrence of acquired resistance. Although the mechanism of the resistance is not clear so far, the upregulation of alternative angiogenic pathways and stabilization of endothelium by mural cells were reported to be responsible. Therefore, blocking multiple angiogenic pathways that are crucial in tumor angiogenesis has been highlighted to overcome such limitations. To develop an angiogenesis inhibitor that could block multiple angiogenic factors, heparin is an excellent lead compound since wide array of angiogenic factors are heparin-binding proteins. In previous study, we reported a heparin-derived angiogenesis inhibitor, LHT7, as a potent angiogenesis inhibitor and showed that it blocked VEGF signaling pathway. Here we show that LHT7 could block the fibroblast growth factor 2 (FGF2) and platelet-derived growth factor B (PDGF-B) in addition to VEGF. Simultaneous blockade of these angiogenic factors resulted in inhibition of multiple stages of the angiogenic process, including initial angiogenic response to maturation of the endothelium by pericyte coverage in vitro. In addition, the treatment of LHT7 in vivo did not show any sign of vascular normalization and directly led to decreased blood perfusion throughout the tumor. Our findings show that LHT7 could effectively inhibit tumor angiogenesis by blocking multiple stages of the angiogenesis, and could potentially be used to overcome the resistance., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Angiogenesis, the formation of new blood vessels, plays a pivotal role in tumor progression and for this reason angiogenesis inhibitors are an important class of therapeutics for cancer treatment. Heparin-based angiogenesis inhibitors have been newly developed as one of such classes of therapeutics and possess a great promise in the clinical context. Taurocholate conjugated low molecular weight heparin derivative (LHT7) has been proven to be a potent, multi-targeting angiogenesis inhibitor against broad-spectrum angiogenic tumors. However, major limitations of LHT7 are its poor oral bioavailability, short half-life, and frequent parenteral dosing schedule. Addressing these issues, we have developed an oral formulation of LHT7 by chemically conjugating LHT7 with a tetrameric deoxycholic acid named LHTD4, and then physically complexing it with deoxycholylethylamine (DCK). The resulting LHTD4/DCK complex showed significantly enhanced oral bioavailability (34.3 ± 2.89%) and prolonged the mean residence time (7.5 ± 0.5 h). The LHTD4/DCK complex was mostly absorbed in the intestine by transcellular pathway via its interaction with apical sodium bile acid transporter. In vitro, the VEGF-induced sprouting of endothelial spheroids was significantly blocked by LHTD4. LHTD4/DCK complex significantly regressed the total vessel fractions of tumor (77.2 ± 3.9%), as analyzed by X-ray microCT angiography, thereby inhibiting tumor growth in vivo. Using the oral route of administration, we showed that LHTD4/DCK complex could be effective and chronically administered as angiogenesis inhibitor., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Two different protein nanoparticles that are totally different in shape and surface structure, i.e. Escherichia coli DNA-binding protein (eDPS) (spherical, 10 nm) and Thermoplasma acidophilum proteasome (tPTS) (cylindrical, 12 × 15 nm) were engineered for in vivo optical tumor detection: arginine-glycine-aspartic acid (RGD) peptide (CDCRGDCFC) was genetically inserted to the surface of each protein nanoparticle, and also near-infrared fluorescence dye was chemically linked to the surface lysine residues. The specific affinity of RGD for integrin (αvβ3) facilitated the uptake of RGD-presenting protein nanoparticles by integrin-expressing tumor cells, and also the protein nanoparticles neither adversely affected cell viability nor induced cell damage. After intravenously injected to tumor-bearing mice, all the protein nanoparticles successfully reached tumor with negligible renal clearance, and then the surface RGD peptides caused more prolonged retention of protein nanoparticles in tumor and accordingly higher fluorescence intensity of tumor image. In particular, the fluorescence of tumor image was more intensive with tPTS than eDPS, which is due presumably to longer in vivo half-life and circulation of tPTS that originates from thermophilic and acidophilic bacterium. Although eDPS and tPTS were used as proof-of-concept in this study, it seems that other protein nanoparticles with different size, shape, and surface structure can be applied to effective in vivo tumor detection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Here, we report a double-stranded, dual-anchored, fluorescent aptamer on reduced graphene oxide (rGO) for the sensitive, selective, and speedy detection of a target protein in biological samples. This nano detector is composed of a target protein-specific fluorescent aptamer with BHQ1 as one anchoring moiety that forms double-stranded sequences with a complementary oligonucleotide sequence with BHQ1 as the other anchoring moiety, anchored to rGO nanosheets. The double-stranded and dual-anchored aptamer on rGO nanosheets (DAGO) exhibited 7.3-fold higher fluorescence intensities compared to a single-stranded, single-anchored fluorescent aptamer on rGO. As a model target protein, interferon-γ was used. DAGO detected the target protein, with linearity over a five-orders-of-magnitude concentration range (0.1 ng/ml-10 μg/ml) in buffer and human serum. DAGO was highly specific for the target protein, exhibiting little changes in fluorescence intensity in response to the non-target proteins, interleukin-2 and tumor necrosis factor-α. Moreover, DAGO allowed rapid quantification of the target protein in human immunodeficiency virus-positive patient serum samples. DAGO-based detection was complete in less than 10 min. Our results indicate that the DAGO provides new opportunities for the rapid and specific detection of target proteins in biological samples and could be widely applied to quantitate various target proteins by replacing the aptamer sequences., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The safe and effective systemic delivery of siRNA is a prerequisite for the successful development of siRNA-based cancer therapeutics. For the enhanced delivery of siRNA, cationic lipids and polymers have been widely used as siRNA carriers to form electrolyte complexes with anionic siRNA. However, the considerable toxicity of strong cationic-charged molecules hampers their clinical use. In this study, we utilized human serum albumin (HSA), which is the most abundant of the plasma proteins, as a siRNA carrier for systemic tumor-targeted siRNA delivery. Both HSA and siRNA molecules were thiol-introduced to improve the binding affinity for each other. The resulting thiolated HSA (tHSA) and polymerized siRNA (psi) formed stable nanosized complexes (psi-tHSAs) by chemical crosslinking and self-crosslinking. After internalization, the psi-tHSAs showed target gene silencing activity in vitro comparable to conventional Lipofectamine™-siRNA complexes, without remarkable cytotoxicity. After intravenous injection in tumor-bearing mice, psi-tHSAs accumulated specifically at the tumor sites, leading to efficient gene silencing in the tumors in a sequential manner. The therapeutic VEGF siRNA was loaded into psi-tHSAs, which significantly inhibited tumor-related angiogenesis in PC-3 tumor xenografts and resulted in retarding the growth of PC-3 tumors. The results showed that self-crosslinked psi-tHSA nanocarriers might provide a promising approach for the systemic siRNA therapy of various human cancers., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

To improve the survival of transplanted human adipose-derived stem cells (ADSCs), a liposome preparation containing the apoptosome inhibitor, NS3694, was formulated and co-delivered with ADSCs in fibrin gel scaffolds. Liposomes provided enhanced effect on ADSC proliferation in vitro as compared to free drug. Exposure of ADSCs to liposomal NS3694 for 7 days did not affect the surface marker expression profile. NS3694 encapsulated in negatively charged liposomes composed of phosphatidylcholine, phosphatidylglycerol, and cholesterol was evaluated in vivo following subcutaneous transplantation in mice. Survival of ADSCs co-delivered with liposomal NS3694 was significantly higher than that of untreated ADSCs or ADSCs treated with free NS3694 or empty liposomes. An immunohistochemical analysis revealed a higher number of human nucleus-positive cells after treatment with liposomal NS3694 than following treatment with free NS3694. Similarly, liposomal NS3694 significantly enhanced survival of transplanted ADSCs in rabbits compared to other treatments. Taken together, our results indicate the potential of liposomal NS3694 co-delivered with ADSCs using fibrin gel systems as an in vivo-survival enhancer., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A method for the sustained delivery of exenatide was proposed using nanoparticles (NPs) with a core/shell structure. The interactions between lipid bilayers and Pluronics were utilized to form various NPs using a layer-by-layer approach. Transmittance electron microscopy and dynamic light scattering were used to examine the morphology of the NPs. The in vitro release pattern was observed as a function of changes in the structure of the NPs, and the structural integrity of exenatide released was examined by SDS-PAGE analysis. Pharmacokinetics and antidiabetic effects were also observed with the structural change of NPs using in vivo animal models. In vitro-in vivo correlation was discussed in relation to manipulation of the NP structures., (© 2013 Elsevier Ltd. All rights reserved.)

Various angiogenesis inhibitors and apoptosis-targeting agents have been therapeutically applied in preclinical cancer models, some of which have been tested in clinical trials. In a previous study, we demonstrated that LHT7, a low molecular weight heparin (LMWH)-taurocholate conjugate, has strong antiangiogenic and tumor-suppressive activity and diminished anticoagulant properties. In this study, we developed LHT7-ApoPep-1, an apoptosis-homing peptide-conjugated variant of LHT7. LHT7-ApoPep-1 exhibited antiangiogenic activity in endothelial cell tube-formation assays and apoptotic cell-targeting ability in tumor cell binding assays; it also showed little toxicity toward healthy cells. Administration of LHT7-ApoPep-1 in mouse xenograft models of breast carcinoma delayed tumor growth compared to LHT7-only, and histological evaluations revealed decreased vessel formation and increased apoptotic area in tumor tissues. Moreover, an examination of LHT7-ApoPep-1-Cy7.5 localization within the body using in vivo live imaging showed accumulation at the tumor site of tumor-bearing mice, with a more prolonged circulation time and enhanced intensity compared to LHT7-Cy7.5. Inspection of the tumor microenvironment revealed that Cy5.5-labeled LHT7-ApoPep-1 was located on and near CD31-positive vessels in tumor tissue. We conclude that LHT7-ApoPep-1 has antiangiogenic and apoptosis-targeting properties and exerts antitumor effects by suppressing tumor vessel growth and homing to apoptotic cells within the tumor., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

This study proposed a double-layer shielding method of using 8-arm-PEG-catechol (PEG(8)) and N-hydroxysuccinimidyl-linked unfractionated heparin (UFH-NHS) for the prevention of instant blood-mediated inflammatory reaction (IBMIR) and immune reactions against transplanted pancreatic islets. The surface of islet was evenly covered by PEG(8) and UFH-NHS. Both viability and functionality of islets were evaluated in vitro, and the anti-coagulation effect of conjugated heparin on the islet surface was also evaluated. The inhibition effects of PEG(8)/UFH double-layer shielding system on immune reactions and IBMIR induced by transplanted islets were evaluated in an allograft model. When pancreatic islets of Sprague-Dawley (SD) rats were transplanted in the liver of F344 rats, the mean survival time (MST) of PEG(8)/UFH double-layer shielded islets (6.8 ± 1.6 days) was statistically increased, compared to that of unmodified islets (3.6 ± 1.1 days). Furthermore, when 0.5 mg/kg of FK506 was daily administered, the MST of double-layer shielded islet (15.0 ± 2.1 days) was increased by two-fold, compared to that of unmodified islets treated with the same dose of FK506 (8.0 ± 2.4 days). Therefore, a newly developed strategy of combining the PEG(8)/UFH double-layer shielding system with FK506 would certainly be effective for preventing immune activation and IBMIR against allotransplanted islets., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Heparin, a potent anticoagulant used for the prevention of venous thromboembolism, has been recognized as a tumor angiogenesis inhibitor. Its limitation in clinical application for cancer therapy, however, arises from its strong anticoagulant activity, which causes associated adverse effects. In this study, we show the structural correlation of LHT7, a previously developed heparin-based angiogenesis inhibitor, with its influence on VEGF blockade and its decreased anticoagulant activity. LHT7 was characterized as having average seven molecules of sodium taurocholates conjugated to one molecule of low-molecular-weight heparin (LMWH). This study showed that the conjugation of sodium taurocholates selectively blocked interaction with antithrombin III (ATIII) while enhancing the binding with VEGF. This resulted in LHT7 to have negligible anticoagulant activity but potent anti-angiogenic activity. Following up on this finding, we showed that the bidirectional effect of sodium taurocholate conjugation was due to its unique structure, that is, the sterane core hindering the ATIII-binding pentasaccharide unit of LMWH with its bulky and rigid structural characteristics while the terminal sulfate group interacts with VEGF to produce stronger binding. In addition, we showed that LHT7 was localized in the tumor, especially on the endothelial cells. One explanation for this might be that LHT7 was delivered to the tumor via platelets., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The chemical conjugate of low molecular weight heparin with taurocholate (LHT7) was previously designed to offer anticancer activity while minimizing the anticoagulant activity. In the present study, we found that the systemic administration of LHT7 in nanolipoplex could substantially enhance tumor vasculature targeting and anticancer effects. Moreover, we found that co-delivery of LHT7 with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, in nanolipoplex could provide synergistic antitumor effect. LHT7/SAHA nanolipoplex was formulated by encapsulating SAHA inside cationic liposomes, followed by complexation of negatively charged LHT7 onto the cationic surfaces of SAHA-loaded liposomes (SAHA-L). LHT7/SAHA nanolipoplex was positively charged with a mean diameter of 117.6 nm, and stable in serum. The nanolipoplex form of LHT7 could alter its pharmacokinetics and biodistribution. Compared to the free form of LHT7, LHT7 in the nanolipoplex showed 1.9-fold higher mean residence time, and higher tumor vasculature accumulation after its intravenous administration. LHT7/SAHA nanolipoplex showed highest antitumor efficacy in SCC-bearing mice, compared to LHT7, SAHA-L and sequential co-administration of LHT7 and SAHA-L. Consistent with the enhanced antitumor effect, the reduction of abnormal vessels in the tumor site was also the highest in the LHT7/SAHA nanolipoplex-treated group. These results suggested the potential of LHT7/SAHA nanolipoplex for enhanced tumor vasculature targeting, and the importance of nanolipoplex-mediated co-delivery with a histone deacetylase inhibitor for maximal anticancer effect., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

This study proposes a combination method of using 3,4-dihydorxy-l-phenylalanine (DOPA) conjugated heparin (heparin-DOPA) and a low dose of anti-CD154 monoclonal antibody (MR-1) treatment to improve the survival time of intrahepatic islet xenograft. To inhibit instant blood mediated inflammatory reactions, heparin-DOPA was directly grafted to the pancreatic islet surface. The surface coverage of heparin-DOPA, the viability and functionality of heparin-DOPA grafted islets were evaluated. In addition, the combined effect of grafted heparin-DOPA and a low dose of MR-1 (a T-cell targeting immunosuppressive drug) on the survival of islet was evaluated in a xenograft model. Both unmodified islets and heparin-DOPA grafted islets were completely rejected within 2 weeks after intraportal transplantation. However, when 0.1 mg/mouse of MR-1 was administered (at day 0, 2, 4, 6 of transplantation) to 11 mice that had heparin-DOPA grafted islets transplanted to, seven out of the recipients maintained normoglycemia over 60 days. Therefore, we propose that a developed combinatory immunoprotection protocol of surface modification of pancreatic islets using heparin-DOPA with a low dose of MR-1 can be effective in prolonging the survival rate of transplanted islets in a xenograft model., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This study proposes a new combination method of using 6-arm-PEG-catechol to enhance the PEG effect on one hand and another combination of using low doses of Tacrolimus (FK506) and anti-CD154 mAb (MR1) with PEGylation for effective immunoprotection on the other in a xenogenic islet transplantation model. The surface coverage of PEG, viability and functionality of islets were evaluated in vitro, and the effect of surface camouflage on immunoprotection for transplanted islets was evaluated. In addition, the synergistic effects of surface camouflaged islets with low doses of immunosuppressant drugs, such as FK506 and MR1, were evaluated in the xenotransplantation model. The median survival time (MST) of 6-arm-PEG-catechol grafted islets (12.0 ± 1.1 days) was not significantly increased, compared to that of unmodified islets (10.5 ± 1.3 days). However, when 0.2 mg/kg of FK506 was daily administered, the MST of 6-arm-PEG-catechol grafted islet (21.0 ± 1.9 days) was increased twice, compared to that of unmodified islets treated with 0.2 mg/kg of FK506 (10.0 ± 0.9 days). Interestingly, when the recipients of 6-arm-PEG-catechol grafted islets were treated with 0.2 mg/kg of FK506 and 0.1 mg/mouse of MR1, normoglycemia was maintained up to 50 days of transplantation without any fluctuation of glucose level. Therefore, a newly developed protocol using 6-arm-PEG-catechol with FK506 and MR1 would certainly be an effective combination therapy for the treatment of type 1 diabetes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Tissue plasminogen activator (tPA), a widely used thrombolytic agent, has an application limit due to short half-life. To prolong the half-life of tPA, liposomes composed of egg phosphatidylcholine (EPC), cholesterol (CHOL) and sodium cholesterol-3-sulfate (CS) were prepared by lipid film method. In addition, distearolyphosphatidyl ethanolamine-N-poly(ethylene glycol) 2000 (DSPE-PEG 2000) was included to give steric barrier to liposomes. Physicochemical characteristics such as particle size, zeta potential, entrapment efficiency and long-term storage stability at 4 degrees C were investigated. The fibrinolytic activity of tPA-loaded in liposomes was confirmed by fibrin clot lysis assay. In vivo pharmacokinetic properties of tPA and the effect of PEG on the blood circulation of tPA-loaded in liposomes in circulation were also evaluated. Both conventional liposomes (EPCL) and PEGylated liposomes (EPC-PEGL) were proper as an injectable formulation with small particle size. Loading process of tPA into liposomes did not alter fibrinolytic activity of intact tPA. Encapsulation of tPA into EPCL and EPC-PEGL prolonged half-life of tPA by 16 and 21 folds compared with free tPA, respectively. Therefore, the use of liposomes could prolong the circulation lifetimes and longevity effect of liposomes on tPA was increased by PEG.

The goal of this study was to develop anti-cancer drug conjugates with increased anti-tumor effect and reduced toxicity. In this regard, we utilized the physiological characteristics of tumors such as angiogenesis, the expression of matrix metalloproteinases (MMPs) and the enhanced permeability and retention (EPR) effect, and designed MMPs-specific PEGylated peptide-DOX conjugate micelles containing doxorubicin. These conjugates were prepared by using two peptides, GPLGV and GPLGVRG (P5D and P7D, respectively), and doxorubicin was loaded into micelles formed by each conjugate. P5D and P7D were specifically cleaved by active MMP-2 and all conjugates showed significantly better cell viability than doxorubicin at equivalent concentrations. In vivo, animals treated with PEGylated peptide-DOX conjugate micelles showed approximately 50% of the tumor growth of the control, and doxorubicin-loaded conjugates micelles inhibited tumor growth up to about 72% compared with the control, which matched the effect of doxorubicin. Doxorubicin-loaded PEGylated peptide-DOX conjugate micelles exhibited longer half-lives and maintained higher concentrations of doxorubicin in plasma than PEGylated peptide-DOX conjugate micelles alone. Doxorubicin-loaded PEGylated peptide-DOX conjugate micelles might offer a cancer therapy with an activity that is similar to that of the parent drug but with reduced toxicity.

The inhibitory efficacies of new bile acid acylated-heparin derivative (heparin-DOCA) were evaluated on experimental lung metastasis. We evaluated the effect of heparin-DOCA on intercellular interactions including those between B16F10 and thrombin-activated platelets and TNF-alpha-activated HUVECs, and between B16F10 and immobilized mouse P-selectin. In addition, the inhibitory effects of heparin-DOCA on adhesion and invasion of B16F10 to Matrigel were studied. In an animal mouse study, the blood clot formation and the retention of red fluorescence protein (RFP)-B16F10 in lungs were assessed after heparin-DOCA and RFP-B16F10 intravenous administration. Furthermore, we investigated the anti-metastatic effect of heparin-DOCA against lung metastasis induced by B16F10 and SCC7. Heparin-DOCA inhibited intercellular interactions between B16F10 and activated platelets or activated HUVECs by blocking P- and E-selectin-mediated interactions. Moreover, it reduced adhesion and invasion of B16F10 to ECM, thereby affecting the reduction of early retention of B16F10 in the lung. Heparin-DOCA attenuated lung colony formation on the surfaces and in interior of the lung, and attenuated metastasis by B16F10 and SCC7. These results suggest that heparin-DOCA may have potentials as therapeutic agent that prevents tumor metastasis and progression.

Pancreatic islet transplantation is one of the most promising strategies for patients suffering from type 1 diabetes mellitus, but several therapeutic immunosuppressive medications must be administered simultaneously to protect transplanted islets in the long-term, and these expose patients to the risk of serious complications. Thus, we developed chemically modified islets with a protective poly(ethylene glycol) (PEG) layer, which reduces immunogenicity by preventing cellular immune reactions. We report here that PEG-based chemical immunomodulation can provide a semi-permanent effective therapy that protects transplanted islets at least for 1 year when accompanied by cyclosporine. Moreover, this combinatorial approach appears to avoid the toxicities associated with immunosuppressive medications because of the reduced amounts of medication required. Also, the conjugated PEG molecules were found to be continuously present at the transplanted islets. However, unmodified islets (control) were completely eliminated within 2 weeks even when CsA was administered. These results strongly suggest that this new combinatorial therapy provides a semi-permanent, effective clinical means of attenuating transplanted islet immunogenicity for a long time, whilst avoiding the toxicities associated with therapeutic levels of immunosuppressants owing to the minimized immunosuppressant.

Although drug-eluting stents (DESs) have become the most effective means of treating coronary artery disease, safety concerns regarding their thrombogenicities remain to be surmounted. Here, we report on a novel type of DES capable of preventing restenosis and thrombosis. The DES was prepared by coating a bare metal stent with echinomycin (an anti-proliferative drug) in polyurethane by a spray drying method. Hydrophobic heparinized polymer was then topcoated onto stent over echinomycin/PU layer by dipping to improve hemocompatibility. The two-layered stent was characterized regarding surface and cross-sectional morphology, drug release pattern, platelet adhesion in vitro, and restenosis in vivo. It was found that the heparin topcoat acts as a diffusion barrier that allows the controlled release of drug in a sustained manner. Also, the heparin coated layer effectively reduced platelet adhesion, indicating excellent hemocompatibility. From the animal test using pigs, it was evident that the developed DESs can minimize neointimal proliferation and thrombus formation. The devised hydrophobic heparinized polymer-coated DES effectively reduced both restenosis and thrombosis, suggesting that they have potential as tools for the treatment of coronary artery diseases.

The development of more selective delivery systems for cancer diagnosis and chemotherapy is one of the most important goals of current anticancer research. The purpose of this study is to evaluate various self-assembled nanoparticles as candidates to shuttle radionuclide and/or drugs into tumors and to investigate the mechanisms underlying the tumor targeting with self-assembled nanoparticles. By combining different hydrophobic moieties and hydrophilic polymer backbones, various self-assembled nanoparticles were prepared, and their in vivo distributions in tumor-bearing mice were studied by radionuclide imaging. One type of nanoparticles (fluorescein isothiocyanate-conjugated glycol chitosan (FGC) nanoparticles) exhibited highly selective tumoral localization. Scintigraphic images obtained 1 day after the intravenous injection of FGC nanoparticles clearly delineated the tumor against adjacent tissues. The mechanisms underlying the tumor targeting with self-assembled nanoparticles were investigated in terms of the physicochemical properties of nanoparticles and tumor microenvironments. FGC nanoparticles were preferentially localized in perivascular regions, implying their extravasation to tumors through the hyperpermeable tumor vasculature. The magnitude and pattern of tumoral distribution of self-assembled nanoparticles were influenced by several key factors--(i) in vivo colloidal stability: nanoparticles should maintain their intact nanostructures in vivo for a long period of time, (ii) particle size, (iii) intracellular uptake of nanoparticle: fast cellular uptake greatly facilitates the tumor targeting, (iv) tumor angiogenesis: pathological angiogenesis permits access of nanoparticles to tumors. We believe that this work can provide insight for the engineering of nanoparticles and be extended to cancer therapy and diagnosis, so as to deliver multiple therapeutic agents and imaging probes at high local concentrations.

Five novel 3-carboranyl thymidine analogues (3CTAs) were designed and synthesized for boron neutron capture therapy (BNCT) of cancer. Phosphorylation of all five 3CTAs was catalyzed by recombinant human thymidine kinase (hTK1) using adenosine triphosphate (ATP) as the phosphate donor. The obtained phosphorylation rates ranged from 4% to 64.5% relative to that of thymidine. The compound with the most favorable hTK1 binding properties had a k(cat)/K(M) value of 57.4% relative to that of thymidine and an IC(50) of inhibition of thymidine phosphorylation by hTK1 of 92 microM. Among the five synthesized 3CTAs, this agent had also the overall most favorable physicochemical properties. Therefore, it may have the potential to replace N5-2OH, the current lead 3CTA, in preclinical studies. An in silico model for the binding of this compound to hTK1 was developed.

We report a novel method of surface grafting a polymeric phospholipid system containing an acryloyl end group (1stearoyl-2-[12-(acryloyloxy)-dodecanoyl]-sn-glycero-3-phosphocholine) onto medical grade silicone catheters. The surface of silicone catheters was functionalized in a sequence of steps: plasma polymerization of allyl alcohol on the catheter surface, grafting acryloyl moieties and in situ polymerization of the pre-assembled acryloyl terminated phospholipids on the acryloyl functionalized catheter surface. The surface morphological changes analyzed by scanning electron microscopy (SEM) and atomic force microscopy (AFM), a sharp decrease in water contact angle, and appearance of N1s peak in XPS analysis indicated a successful monolayer grafting of the phospholipid. In platelet adhesion tests performed using platelets isolated from rabbit plasma, the phospholipid grafted surface showed fewer adhered platelets, without emerging pseudopodes or aggregation. However, ungrafted catheter surface showed large number of platelets in extensively spread and aggregated states. Thus, this modified phospholipid system and its simple grafting technique was very effective with regard to suppressing in vitro platelet adhesion on the silicon catheter surface.

We validated the application of the surface plasmon resonance (SPR) technique to reliably determine adhesion of drugs to the intestinal wall using heparin-DOCA conjugates, developed to enhance the oral absorption of poorly absorbed heparin. In this study, heparin conjugates, or deoxycholyl-heparin (H-DOCA) and bisdeoxycholyl-heparin (H-bis-DOCA), were synthesized by covalently coupling the synthesized succinimido deoxycholate (DOCA-NHS) or succinimido bis-deoxycholyl-L-lysine (DOCA-bis-NHS) to amine groups of heparin, and their physicochemical and biological properties were evaluated. To mimic the duodenal and ileal surfaces, duodenal and ileal brush border membrane (BBM) vesicles isolated from Sprauge-Dawley (SD) rats were immobilized onto a biosensor chip composed of dextran derivatives with modified lipophilic residues. The adhesion of heparin conjugates on the BBM surface was evaluated by measuring the SPR response signal. The adhesion of heparin conjugates was significantly dependent on the conjugated DOCA molecules: that is, they showed higher adhesion signal on the ileal BBM surface than that on the duodenal BBM surface. In particular, the solubilized heparin conjugates in DMSO solution presented significantly increased adhesion affinity on the ileal BBM surface. The adhesion of heparin conjugates on the intestinal surfaces was successfully assayed using the surface plasmon resonance technique with the sensor chip on which BBM vesicles were immobilized.

This paper describes a strategy for designing a chemically anchored phospholipid monolayer that could be used as coating materials for biomedical implants. To make a chemically anchored phospholipid monolayer on the polymer substrate, we prepared the mono-acrylated phospholipid (1-palmitoyl-2-[12-(acryloyloxy)-dodecanoyl]-sn-glycero-3-phosphocholine; acryloyl-PC) and the acrylated polymer (poly(octadecylacrylate-co-4-acryloyloxy butylacrylate)), which was synthesized by the acrylation of poly(octadecyl acrylate-co-hydroxybutyl acrylate, poly(OA-co-HA)) with acryloyl chloride. The chemically anchored phospholipid monolayer was prepared by using in situ photopolymerization of a pre-assembled phospholipid monolayer, produced by lipid vesicle fusion, onto the acrylated polymer coated silicon wafer. Optimal condition of vesicle fusion and irradiation time was determined from the degree of hydrophilicity rendered by the polymerized phospholipid surface. The physicochemical properties of polymerized phospholipid monolayer on the substrate were evaluated using water contact angle, field-emission scanning electron micrograph (FE-SEM), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). These results confirmed that the polymerized phospholipid monolayer was chemically anchored on the acrylated polymer substrate. The chemically anchored phospholipid monolayer was stable in aqueous condition for 2 weeks, but the physically adsorbed phospholipid monolayer got removed within 1 day. Moreover, the polymerized phospholipid monolayer also suppressed albumin absorption and platelet adhesion, in vitro. This polymerized phospholipid monolayer provides a new biomimetic system for coating medical devises.

Eleven 3-carboranyl thymidine analogues (3CTAs) containing highly hydrophilic and flexible ethyleneoxide moieties were synthesized as potential agents for boron neutron capture therapy (BNCT) and their biochemical and physicochemical properties were evaluated. Based on specific structural features, this library of 3CTAs was divided into three subgroups. The first group contained 3CTAs with 1-4 ethyleneoxide units between the thymidine (Thd) scaffold and a carborane cluster. The second group of 3CTAs contained a pentylene spacer between Thd and the carborane and 2-4 ethyleneoxide units additionally attached to the carborane cluster. The third group contained three 3CTAs all with pentylene spacers and four ethylene units but with different carborane cages. The ethyleneoxide modified 3CTAs were good substrates of thymidine kinase 1 (TK1) and poor substrates of human mitochondrial thymidine kinase 2 (TK2) as determined in phosphoryl transfer assays. In the first group of 3CTAs, all the compounds were efficiently phosphorylated regardless of varying spacer lengths (37-42% of the activity of Thd). The second group of 3CTAs was less effectively phosphorylated (17-26% of the activity of Thd) probably due to a less favorable sterical orientation of Thd within the active site of TK1 and/or an increased lipophilicity compared with the first group. In the third group of structural isomers, no significant differences in phosphorylation rates were observed (17-25%). A structure-function hypothesis explaining these results is presented.

Graft rejection is the major limiting factor in islet transplantation and is closely related with the recruitment and activation of T cells and macrophages against the graft. To reduce the immunogenicity of islets, we have grafted biocompatible polyethylene glycol (PEG) onto the collagen capsule of islets without changing the morphology and function of islets. In this study, we evaluated whether the grafted PEG molecules on the collagen capsule of islet could prevent the activation of immune cells, and investigated factors that are mainly related to the immune reaction in vitro. During the co-culture with lymphocytes, the morphology and viability of PEG-grafted islets were not damaged, and the amounts of IL-2 and TNF-alpha secreted from lymphocytes co-cultured with PEG-grafted islets were significantly lower than that of free islets. However, when both kinds of islets were cultured with macrophages, there were no significant differences in morphology, viability and the secreted amounts of cytokines and nitric oxide. In conclusion, the grafted PEG could inhibit activation of lymphocytes, which are essential in initiating the graft rejection process. However, the grafted PEG molecules could not completely prevent the infiltration of cytotoxic molecules into the islets.

We have previously demonstrated phosphorylcholine monolayer chemically grafted onto a methacryloyl-terminated solid substrate by in situ polymerization. The in situ polymerization was carried out at the interface between a pre-assembled acrylated phospholipid monolayer produced by vesicle fusion and a methacryloyl-terminated substrate using a water-soluble initiator, 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPD). Herein, we examined the biostability and biocompatibility of a surface-grafted phospholipid monolayer (poly-PC) on a methacryloyl-terminated substrate using a "wash off' test, in vitro protein adsorption and in vivo cage implantation for time intervals of 4, 7, 14 and 21 days, respectively. In order to compare the biostability and biocompatibility of phospholipid surfaces on solid substrates, we used two types of phospholipid surfaces: a physically adsorbed phospholipid monolayer (PC) and a poly-PC. Atomic force microscopy and water contact angle measurements indicated that the poly-PC surface was more stable in PBS, Triton X-100 and to EO gas sterilization than the PC surface. The adsorption of proteins such as albumin, fibrinogen, IgG and human plasma proteins on the poly-PC surfaces were significantly reduced, in vitro. Moreover, the poly-PC surface greatly reduced macrophage adhesion and the formation of foreign body giant cells, in vivo.

Sterilization procedure is one of the most important obstacles in the clinical applications of biodegradable microspheres. The microspheres prepared with poly(alpha-hydroxy acid) were severely aggregated during ethylene oxide (EO) gas sterilization, and could not be used in clinical applications. In this study, the effects of EO gas sterilization on the poly(L-lactide) (PLLA) microspheres were analyzed by nuclear magnetic resonance spectroscopy (1H-NMR), differential scanning calorimetry (DSC), gel permeation chromatography (GPC), scanning electron microscope (SEM) and size fractionation. The aggregation between the microspheres might be stimulated by high mobility of amorphous regions of PLLA on the microsphere surfaces since both water vapor and gas mixture can reduce glass transition temperature (Tg) of PLLA below the sterilization temperature. During EO gas sterilization, there were no changes in the molecular structure and the molecular weight of PLLA in microspheres, but there were changes in the crystallinity of PLLA in microspheres. In this study, poly(L-lactide)-poly(ethylene glycol) diblock copolymers (PLE) were blended with PLLA homopolymers in various ratios to design the microsphere suitable for EO gas sterilization. Aggregation of PLLA microspheres was markedly prevented when more than 4wt% of PLE was blended in the microspheres. This inhibition effect on aggregation may be due to the increased initial crystallinity of the microspheres, which help to maintain the microsphere morphology during EO gas sterilization.

Escherichia coli O157:H7 produces two forms of verotoxin (VT), VT1 and VT2, which cause hemorrhagic colitis with development, in some cases, of hemolytic uremic syndrome. These toxins consist of an enzymatically active A subunit and pentamers of B subunit responsible for their binding to host cells. We used the secretion-expression system of Bacillus brevis to produce recombinant VT1B and VT2B. The secreted B subunits were purified and sequenced to verify their structure. Receptor-binding showed that rVT1B but not rVT2B bound to Gb3-receptor. When mice were nasally immunized with rVT1B or rVT2B together with a nontoxic mutant of cholera toxin (mCT) or native cholera toxin (nCT) as adjuvants, serum IgG and mucosal IgA antibody responses to VT1B were induced. The VT1B-specific antibodies prevented VT1B binding to its Gb3 receptor. In contrast, poor serum and no mucosal VT2B-specific antibodies but brisk CTB-specific antibody responses were induced by nasal immunization with rVT2B in the presence of mCT or nCT. These results show that nasal immunization with rVTB and mCT as a nontoxic mucosal adjuvant is an effective regimen for the induction of VT1B but not VT2B antibody responses which inhibit VT1B binding to Gb3 receptor.