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Abstract 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 5β-cholanic acid and the amphiphilic glycol chitosan-5β-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.

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 5β-cholanic acid and the amphiphilic glycol chitosan-5β-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. [ABSTRACT FROM AUTHOR]

Ji Young Yhee,1 Hong Yeol Yoon,2 Hyunjoon Kim,3 Sangmin Jeon,2 Polla Hergert,1 Jintaek Im,1 Jayanth Panyam,3 Kwangmeyung Kim,2,4 Richard Seonghun Nho1 1Department of Medicine, University of Minnesota, Minneapolis, MN, USA; 2Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea; 3Department of Pharmaceutics, University of Minnesota, Minneapolis, MN, USA; 4Korea University-Korea Institute of Science and Technology (KU-KIST) Graduate School of Converging Science and Technology, Korea University, Seoul, Republic of Korea Abstract: Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate chronic lung diseases including idiopathic pulmonary fibrosis (IPF). IPF is a fatal lung disease characterized by persistent fibrotic fibroblasts in response to type I collagen-rich extracellular matrix. As a pathological microenvironment is important in understanding the biological behavior of nanoparticles, in vitro cellular uptake of glycol chitosan nanoparticles (CNPs) in human lung fibroblasts was comparatively studied in the presence or absence of type I collagen matrix. Primary human lung fibroblasts from non-IPF and IPF patients (n=6/group) showed significantly increased cellular uptake of CNPs (>33.6–78.1 times) when they were cultured on collagen matrix. To elucidate the underlying mechanism of enhanced cellular delivery of CNPs in lung fibroblasts on collagen, cells were pretreated with chlorpromazine, genistein, and amiloride to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Amiloride pretreatment remarkably reduced the cellular uptake of CNPs, suggesting that lung fibroblasts mainly utilize the macropinocytosis-dependent mechanism when interacted with collagen. In addition, the internalization of CNPs was predominantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor in IPF fibroblasts, indicating that enhanced PI3K activity associated with late-stage macropinocytosis can be particularly important for the enhanced cellular delivery of CNPs in IPF fibroblasts. Our study strongly supports the concept that a pathological microenvironment which surrounds lung fibroblasts has a significant impact on the intracellular delivery of nanoparticles. Based on the property of enhanced intracellular delivery of CNPs when fibroblasts are made to interact with a collagen-rich matrix, we suggest that CNPs may have great potential as a drug-carrier system for targeting fibrotic lung fibroblasts. Keywords: cellular uptake, glycol chitosan nanoparticles, idiopathic pulmonary fibrosis, macropinocytosis, type I collagen matrix

Enhanced drug-loading and therapeutic efficacies are highly essential properties for nanoparticles as tumor-targeting drug carriers. Herein, we developed the glycol chitosan nanoparticles with hydrotropic oligomers (HO-CNPs) as a new tumor targeting drug delivery system. For enhancing drug-loading efficiency of paclitaxel in drug carriers, hydrotropic 2-(4-(vinylbenzyloxy)-N,N-diethylnicotinamide) (VBODENA-COOH) oligomers, that were used for enhancing the aqueous solubility of paclitaxel, were directly conjugated to glycol chitosan polymers. The amphiphilic conjugates readily formed nanoparticle structure (average size=302±22nm) in aqueous condition. Water-insoluble paclitaxel (PTX) was readily encapsulated into HO-CNPs with a high drug-loading amount up to 24.2wt.% (2.4 fold higher than other polymeric nanoparticles) by a simple dialysis method. The PTX encapsulated HO-CNPs (PTX-HO-CNPs; average size=343±12nm) were very stable in aqueous media up to 50days. Also, PTX-HO-CNPs presented rapid cellular uptake and lower cytotoxicity in cell culture system, compared to Cremophor EL/ethanol formulation of PTX. In tumor-bearing mice, the extravasation and accumulation of PTX-HO-CNPs in tumor tissue were precisely observed by intravital fluorescence imaging techniques. Furthermore, PTX-HO-CNPs showed the higher therapeutic efficacy, compared to Abraxane®, a commercialized PTX-formulation. These overall results demonstrate its potential as a new nano-sized PTX carrier for cancer treatment. [ABSTRACT FROM AUTHOR]

Abstract: We designed a cancer-cell specific photosensitizer nano-carrier by synthesizing pheophorbide a (PheoA) conjugated glycol chitosan (GC) with reducible disulfide bonds (PheoA-ss-GC). The amphiphilic PheoA-ss-GC conjugates self-assembled in aqueous condition to form core–shell structured nanoparticles (PheoA-ss-CNPs) with good colloidal stability and switchable photoactivity. The photoactivity of PheoA-ss-CNPs in an aqueous environment was greatly suppressed by the self-quenching effect, which enabled the PheoA-ss-CNPs to remain photo-inactive and in a quenched state. However, after the cancer cell-specific uptake, the nanoparticular structure instantaneously dissociated by reductive cleavage of the disulfide linkers, followed by an efficient dequenching process. Compared to non-reducible PheoA-conjugated GC-NPs with stable amide linkages (PheoA–CNPs), PheoA-ss-CNPs rapidly restored their photoactivity in response to intracellular reductive conditions, thus presenting higher cytotoxicity with light treatment. In addition, the PheoA-ss-CNPs presented prolonged blood circulation in vivo compared to free PheoA, demonstrating enhanced tumor specific targeting behavior through the enhanced permeation and retention (EPR) effect. The enhanced tumor accumulation of PheoA-ss-CNPs enabled tumor therapeutic efficacy that was more efficient than free PheoA in tumor-bearing mice. Based on the enhanced intracellular release for cytosolic high dose and switchable photoactivity mechanism for reduced side effects, these results suggest that PheoA-ss-CNPs have good potential for photodynamic therapy (PDT) in cancer treatment. [Copyright &y& Elsevier]

Abstract: To evaluate the tumor targeting efficiency of self-assembled polymeric nanoparticles, four glycol chitosan nanoparticles (CNPs) with different degrees of hydrophobic substitution were prepared by coupling 7.5, 12, 23, and 35wt.% of 5β-cholanic acid to hydrophilic glycol chitosan polymer (GC). The sizes and zeta-potentials of different CNPs in aqueous condition were not significantly different, but their stability and deformability were greatly dependent upon the degree of substitution (DS) of 5β-cholanic acid. With an increase in hydrophobicity, CNPs became more stable and rigid, as characterized by SDS-PAGE and filtration tests. To compare with CNPs, linear GC and polystyrene nanoparticles (PSNPs) were employed as controls. In vivo tumor accumulation of Cy5.5-labeled linear GC, polystyrene nanoparticles (PSNPs) and CNPs were monitored in flank tumors and liver tumor-bearing mice models using near-infrared fluorescence (NIRF) imaging systems. CNPs displayed higher tumor accumulation than GC and PSNPs via the enhanced permeability and retention (EPR) effect. Interestingly, CNPs containing 23wt.% of 5β-cholanic acid (CNP-23%) showed the highest tumor-targeting efficiency compared to other CNPs. As exemplified in this study, the stability of CNP-23% is better than CNP-7.5% and CNP-12% containing 7.5wt.% and 12wt.% of 5β-cholanic acid, respectively, and the deformability of CNP-23% is better than that of CNP-35% containing 35wt.% of 5β-cholanic acid. We proposed that the superior tumor-targeting efficiency of CNP-23% is mainly due to their balanced stability and deformability in vivo. This study demonstrates that the degree of hydrophobic substitution of self-assembled nanoparticles could determine their stability and deformability. Importantly, they were founded to be the key factors which affect their tumor-targeting efficiency in vivo, and so that these factors should be highly considered during developing nanoparticles for tumor-targeted imaging or drug delivery. [Copyright &y& Elsevier]

Abstract: Antiangiogenic peptide drugs have received much attention in the fields of tumor therapy and tumor imaging because they show promise in the targeting of integrins such as αvβ3 on angiogenic endothelial cells. However, systemic antiangiogenic peptide drugs have short half-lives in vivo, resulting in fast serum clearance via the kidney, and thus the therapeutic effects of such drugs remain modest. In this study, we prepared self-assembled glycol chitosan nanoparticles and explored whether this construct might function as a prolonged and sustained drug delivery system for RGD peptide, used as an antiangiogenic model drug in cancer therapy. Glycol chitosan hydrophobically modified with 5β-cholanic acid (HGC) formed nanoparticles with a diameter of 230nm, and RGD peptide was easily encapsulated into HGC nanoparticles (yielding RGD-HGC nanoparticles) with a high loading efficiency (>85%). In vitro work demonstrated that RGD-HGC showed prolonged and sustained release of RGD, lasting for 1 week. RGD-HGC also inhibited HUVEC adhesion to a βig-h3 protein-coated surface, indicating an antiangiogenic effect of the RGD peptide in the HGC nanoparticles. In an in vivo study, the antiangiogenic peptide drug formulation of RGD-HGC markedly inhibited bFGF-induced angiogenesis and decreased hemoglobin content in Matrigel plugs. Intratumoral administration of RGD-HGC significantly decreased tumor growth and microvessel density compared to native RGD peptide injected either intravenously or intratumorally, because the RGD-HGC formulation strongly enhanced the antiangiogenic and antitumoral efficacy of RGD peptide by affording prolonged and sustained RGD peptide delivery locally and regionally in solid tumors. [Copyright &y& Elsevier]

The objective of the present study is synthesis of glycol chitosan coated selenium nanoparticles (GC-Se NPs) and evaluation of oxidative stress and ginsenoside accumulation in P. ginseng C. A. Meyer. We synthesized (Se NPs and GC-Se NPs) and characterized using various spectroscopic analyses. The highest concentration (20 mg L−1) of GC-Se NPs induced moderate ROS (O 2 − and H 2 O 2) accumulation and upregulation of PgSOD and PgCAT showing good biocompatibility and less toxicity at the highest concentration. Furthermore, ginsenoside biosynthetic pathway genes (PgHMGR , PgSS , PgSE , PgDDS) also showed significant upregulation upon 20 mg L−1 GC-Se NPs treatment. At 20 mg L−1 GC-Se NPs treatment, ginsenoside accumulated upto 217.47 mg/mL and 169.86 mg/mL mainly due to the increased proportion of Rb1 and Re ginsenosides. Altogether, our results suggested that ecofriendly conjugation of GC with Se NPs could be used as a bio fortifier to enhance the ginsenoside profile and to increase the quality of ginseng roots. • GC-Se NPs can induce ROS accumulation. • GC-Se NPs can induce ginsenoside content. • GC-Se NPs (20 mg L−1) is optimum concentration and can be used as a biofortifier to improve the quality of P. ginseng. [ABSTRACT FROM AUTHOR]

Development of biopolymer-based imaging agents which can access rapidly and provide detailed information about the diseases has received much attention as an alternative to conventional imaging agents. However, development of biopolymer-based nanomaterials for tumor imaging still remains challenging due to their low sensitivity and image resolution. To surmount of these limitations, multimodal imaging agents have been developed, and they were widely utilized for theranostic applications. Herein, iodine containing echogenic glycol chitosan nanoparticles are developed for x-ray computed tomography (CT) and ultrasound (US) imaging of tumor diagnosis. X-ray CT/US dual-modal imaging probe was prepared by following below two steps. First, iodine-contained diatrizoic acid (DTA) was chemically conjugated to the glycol chitosan (GC) for the CT imaging. DTA conjugated GC (GC-DTA NPs) formed stable nanoparticles with an average diameter of 315 nm. Second, perfluoropentane (PFP), a US imaging agent, was physically encapsulated into GC-DTA NPs by O/W emulsion method yielding GC-DTA-PFP nanoparticles (GC-DTA-PFP NPs). The GC-DTA-PFP NPs formed nanoparticles in physiological condition, and they presented the strong x-ray CT, and US signals in phantom test in vitro. Importantly, GC-DTA-PFP NPs were effectively accumulated on the tumor site by enhanced permeation and retention (EPR) effects. Moreover, GC-DTA-PFP NPs showed x-ray CT, and US signals in tumor tissues after intratumoral and intravenous injection, respectively. Therefore, GC-DTA-PFP NPs indicated that x-ray CT/US dual-modal imaging using iodinated echogenic nanoparticles could be provided more comprehensive and accurate diagnostic information to diagnosis of tumor., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.