Additional file 1 of In vivo toxicity evaluation of tumor targeted glycol chitosan nanoparticles in healthy mice: repeated high-dose of glycol chitosan nanoparticles potentially induce cardiotoxicity

Additional file 1: Figure S1. Synthetic route to prepare the glycol chitosan and 5β-cholanic acid conjugates. Figure S2. Detail information of average size of different concentrations of CNPs in the mouse serum. Figure S3. Detail information of average size of different concentrations of CNPs in the mouse serum (n=5). Figure S4. Flow cytometric results showing H9C2 cells stained with Annexin V/PI after treatment with CNPs for 24 h. Figure S5. Fluorescence image of major organs from mice treated with 90 mg/kg of CNPs for 7 days. Fluorescence intensities were normalized with the results of Figure 3B and 3C. Figure S6. Excretion profile of Cy5.5-CNPs after 90 mg/kg treatment. The urines were collected from the mice at the indicated time points, followed by analysis of Cy5.5 fluorescence intensity using HPLC. Figure S7. Detail information of hematological parameters on day 7 after single- or multi-dose of 10, 22.5 or 90 mg/kg CNPs. Figure S8. Detail information of complete cell count results on day 7 after single- or multi-dose of 10, 22.5 or 90 mg/kg CNPs (n=5). Figure S9. Uncropped images of western blot results in Figure 5E

Rational Design of Inflammation-Responsive Inflatable Nanogels for Ultrasound Molecular Imaging

Microbubbles are clinically used as an imaging agent for contrast-enhanced ultrasound image. Beyond the preformed microbubbles, nanoscale gas-generating chemical systems that are capable of stimulus-responsive inflation to microbubbles have recently been employed as a new echogenic strategy for ultrasound molecular imaging. Here, we report a peroxamide-based ultrasound contrast agent as a H2O2-responsive gas (CO2)-generating system for diagnostic ultrasound imaging of inflammatory diseases. A hydrolytic degradation-resistant peroxamide nanogel was constructed by nanoscopic cross-linking of polymeric aliphatic amines (branched polyethyleneimine) with oxalyl chloride, which intrinsically offers highly concentrated peroxamides as a reactive cross-linking point for H2O2-responsive CO2-generation by the peroxalate chemiluminescence reaction that is intrinsically catalyzed by the polyamine-derived intraparticle basic environment. It was experimentally revealed that the interior of the peroxamide-concentrated nanogel colloid serves as an optimal nanoscale catalytic reactor for the H2O2-responsive gas generation as well as a gas reservoir capable of nano-to-micro inflation. We demonstrate that the peroxamide-concentrated nanogels are indeed capable of enhancing the ultrasound contrast in response to H2O2, which allows us to perform diagnostic ultrasound imaging of H2O2-overproducing inflammatory diseases in mouse models. Along with the biocompatibility of the peroxamide nanogels revealed by the animal toxicity study, our design strategy for the inflatable nanoparticles would contribute to the advancement of activatable contrast agents for ultrasound molecular imaging

Tumor-Targeting Transferrin Nanoparticles for Systemic Polymerized siRNA Delivery in Tumor-Bearing Mice

Transferrin (TF) is widely used as a tumor-targeting ligand for the delivery of anticancer drugs because the TF receptor is overexpressed on the surface of various fast-growing cancer cells. In this article, we report on TF nanoparticles as an siRNA delivery carrier for in vivo tumor-specific gene silencing. To produce siRNA carrying TF nanoparticles (NPs), both TF and siRNA were chemically modified with sulfhydryl groups that can build up self-cross-linked siRNA-TF NPs. Self-polymerized 5′-end thiol-modified siRNA (poly siRNA, psi) and thiolated transferrin (tTF) were spontaneously cross-linked to form stable NPs (psi-tTF NPs) under optimized conditions, and they could be reversibly degraded to release functional monomeric siRNA molecules under reductive conditions. Receptor-mediated endocytosis of TF induced rapid tumor-cell-specific uptake of the psi-tTF NPs, and the internalized NPs resulted in a downregulation of the target protein in red-fluorescent-protein-expressing melanoma cancer cells (RFP/B16F10) with negligible cytotoxicity. After systemic administration, the psi-tTF NPs showed marked accumulation at the tumor, leading to successful target-gene silencing in vivo. This psi-tTF NP system provided a safe and effective strategy for in vivo systemic siRNA delivery for cancer therapy

Rational Design of Inflammation-Responsive Inflatable Nanogels for Ultrasound Molecular Imaging

Microbubbles are clinically used as an imaging agent for contrast-enhanced ultrasound image. Beyond the preformed microbubbles, nanoscale gas-generating chemical systems that are capable of stimulus-responsive inflation to microbubbles have recently been employed as a new echogenic strategy for ultrasound molecular imaging. Here, we report a peroxamide-based ultrasound contrast agent as a H2O2-responsive gas (CO2)-generating system for diagnostic ultrasound imaging of inflammatory diseases. A hydrolytic degradation-resistant peroxamide nanogel was constructed by nanoscopic cross-linking of polymeric aliphatic amines (branched polyethyleneimine) with oxalyl chloride, which intrinsically offers highly concentrated peroxamides as a reactive cross-linking point for H2O2-responsive CO2-generation by the peroxalate chemiluminescence reaction that is intrinsically catalyzed by the polyamine-derived intraparticle basic environment. It was experimentally revealed that the interior of the peroxamide-concentrated nanogel colloid serves as an optimal nanoscale catalytic reactor for the H2O2-responsive gas generation as well as a gas reservoir capable of nano-to-micro inflation. We demonstrate that the peroxamide-concentrated nanogels are indeed capable of enhancing the ultrasound contrast in response to H2O2, which allows us to perform diagnostic ultrasound imaging of H2O2-overproducing inflammatory diseases in mouse models. Along with the biocompatibility of the peroxamide nanogels revealed by the animal toxicity study, our design strategy for the inflatable nanoparticles would contribute to the advancement of activatable contrast agents for ultrasound molecular imaging

Rational Design of Inflammation-Responsive Inflatable Nanogels for Ultrasound Molecular Imaging

Microbubbles are clinically used as an imaging agent for contrast-enhanced ultrasound image. Beyond the preformed microbubbles, nanoscale gas-generating chemical systems that are capable of stimulus-responsive inflation to microbubbles have recently been employed as a new echogenic strategy for ultrasound molecular imaging. Here, we report a peroxamide-based ultrasound contrast agent as a H2O2-responsive gas (CO2)-generating system for diagnostic ultrasound imaging of inflammatory diseases. A hydrolytic degradation-resistant peroxamide nanogel was constructed by nanoscopic cross-linking of polymeric aliphatic amines (branched polyethyleneimine) with oxalyl chloride, which intrinsically offers highly concentrated peroxamides as a reactive cross-linking point for H2O2-responsive CO2-generation by the peroxalate chemiluminescence reaction that is intrinsically catalyzed by the polyamine-derived intraparticle basic environment. It was experimentally revealed that the interior of the peroxamide-concentrated nanogel colloid serves as an optimal nanoscale catalytic reactor for the H2O2-responsive gas generation as well as a gas reservoir capable of nano-to-micro inflation. We demonstrate that the peroxamide-concentrated nanogels are indeed capable of enhancing the ultrasound contrast in response to H2O2, which allows us to perform diagnostic ultrasound imaging of H2O2-overproducing inflammatory diseases in mouse models. Along with the biocompatibility of the peroxamide nanogels revealed by the animal toxicity study, our design strategy for the inflatable nanoparticles would contribute to the advancement of activatable contrast agents for ultrasound molecular imaging

Rational Design of Inflammation-Responsive Inflatable Nanogels for Ultrasound Molecular Imaging

Microbubbles are clinically used as an imaging agent for contrast-enhanced ultrasound image. Beyond the preformed microbubbles, nanoscale gas-generating chemical systems that are capable of stimulus-responsive inflation to microbubbles have recently been employed as a new echogenic strategy for ultrasound molecular imaging. Here, we report a peroxamide-based ultrasound contrast agent as a H2O2-responsive gas (CO2)-generating system for diagnostic ultrasound imaging of inflammatory diseases. A hydrolytic degradation-resistant peroxamide nanogel was constructed by nanoscopic cross-linking of polymeric aliphatic amines (branched polyethyleneimine) with oxalyl chloride, which intrinsically offers highly concentrated peroxamides as a reactive cross-linking point for H2O2-responsive CO2-generation by the peroxalate chemiluminescence reaction that is intrinsically catalyzed by the polyamine-derived intraparticle basic environment. It was experimentally revealed that the interior of the peroxamide-concentrated nanogel colloid serves as an optimal nanoscale catalytic reactor for the H2O2-responsive gas generation as well as a gas reservoir capable of nano-to-micro inflation. We demonstrate that the peroxamide-concentrated nanogels are indeed capable of enhancing the ultrasound contrast in response to H2O2, which allows us to perform diagnostic ultrasound imaging of H2O2-overproducing inflammatory diseases in mouse models. Along with the biocompatibility of the peroxamide nanogels revealed by the animal toxicity study, our design strategy for the inflatable nanoparticles would contribute to the advancement of activatable contrast agents for ultrasound molecular imaging

Rational Design of Inflammation-Responsive Inflatable Nanogels for Ultrasound Molecular Imaging

Microbubbles are clinically used as an imaging agent for contrast-enhanced ultrasound image. Beyond the preformed microbubbles, nanoscale gas-generating chemical systems that are capable of stimulus-responsive inflation to microbubbles have recently been employed as a new echogenic strategy for ultrasound molecular imaging. Here, we report a peroxamide-based ultrasound contrast agent as a H2O2-responsive gas (CO2)-generating system for diagnostic ultrasound imaging of inflammatory diseases. A hydrolytic degradation-resistant peroxamide nanogel was constructed by nanoscopic cross-linking of polymeric aliphatic amines (branched polyethyleneimine) with oxalyl chloride, which intrinsically offers highly concentrated peroxamides as a reactive cross-linking point for H2O2-responsive CO2-generation by the peroxalate chemiluminescence reaction that is intrinsically catalyzed by the polyamine-derived intraparticle basic environment. It was experimentally revealed that the interior of the peroxamide-concentrated nanogel colloid serves as an optimal nanoscale catalytic reactor for the H2O2-responsive gas generation as well as a gas reservoir capable of nano-to-micro inflation. We demonstrate that the peroxamide-concentrated nanogels are indeed capable of enhancing the ultrasound contrast in response to H2O2, which allows us to perform diagnostic ultrasound imaging of H2O2-overproducing inflammatory diseases in mouse models. Along with the biocompatibility of the peroxamide nanogels revealed by the animal toxicity study, our design strategy for the inflatable nanoparticles would contribute to the advancement of activatable contrast agents for ultrasound molecular imaging

Rational Design of Inflammation-Responsive Inflatable Nanogels for Ultrasound Molecular Imaging

Microbubbles are clinically used as an imaging agent for contrast-enhanced ultrasound image. Beyond the preformed microbubbles, nanoscale gas-generating chemical systems that are capable of stimulus-responsive inflation to microbubbles have recently been employed as a new echogenic strategy for ultrasound molecular imaging. Here, we report a peroxamide-based ultrasound contrast agent as a H2O2-responsive gas (CO2)-generating system for diagnostic ultrasound imaging of inflammatory diseases. A hydrolytic degradation-resistant peroxamide nanogel was constructed by nanoscopic cross-linking of polymeric aliphatic amines (branched polyethyleneimine) with oxalyl chloride, which intrinsically offers highly concentrated peroxamides as a reactive cross-linking point for H2O2-responsive CO2-generation by the peroxalate chemiluminescence reaction that is intrinsically catalyzed by the polyamine-derived intraparticle basic environment. It was experimentally revealed that the interior of the peroxamide-concentrated nanogel colloid serves as an optimal nanoscale catalytic reactor for the H2O2-responsive gas generation as well as a gas reservoir capable of nano-to-micro inflation. We demonstrate that the peroxamide-concentrated nanogels are indeed capable of enhancing the ultrasound contrast in response to H2O2, which allows us to perform diagnostic ultrasound imaging of H2O2-overproducing inflammatory diseases in mouse models. Along with the biocompatibility of the peroxamide nanogels revealed by the animal toxicity study, our design strategy for the inflatable nanoparticles would contribute to the advancement of activatable contrast agents for ultrasound molecular imaging

Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes <i>In Vivo</i>

Establishment of an appropriate cell labeling and tracking method is essential for the development of cell-based therapeutic strategies. Here, we are introducing a new method for cell labeling and tracking by combining metabolic gylcoengineering and bioorthogonal copper-free Click chemistry. First, chondrocytes were treated with tetraacetylated N-azidoacetyl-d-mannosamine (Ac₄ManNAz) to generate unnatural azide groups (-N₃) on the surface of the cells. Subsequently, the unnatural azide groups on the cell surface were specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented strong NIRF signals with relatively low cytotoxicity and the amounts of azide groups and DBCO-650 could be easily controlled by feeding different amounts of Ac₄ManNAz and DBCO-650 to the cell culture system. For the <i>in vivo</i> cell tracking, DBCO-650-labeled chondrocytes (1 × 10⁶ cells) seeded on the 3D scaffold were subcutaneously implanted into mice and the transplanted DBCO-650-labeled chondrocytes could be effectively tracked in the prolonged time period of 4 weeks using NIRF imaging technology. Furthermore, this new cell labeling and tracking technology had minimal effect on cartilage formation <i>in vivo</i>

Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes <i>In Vivo</i>

Establishment of an appropriate cell labeling and tracking method is essential for the development of cell-based therapeutic strategies. Here, we are introducing a new method for cell labeling and tracking by combining metabolic gylcoengineering and bioorthogonal copper-free Click chemistry. First, chondrocytes were treated with tetraacetylated N-azidoacetyl-d-mannosamine (Ac₄ManNAz) to generate unnatural azide groups (-N₃) on the surface of the cells. Subsequently, the unnatural azide groups on the cell surface were specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented strong NIRF signals with relatively low cytotoxicity and the amounts of azide groups and DBCO-650 could be easily controlled by feeding different amounts of Ac₄ManNAz and DBCO-650 to the cell culture system. For the <i>in vivo</i> cell tracking, DBCO-650-labeled chondrocytes (1 × 10⁶ cells) seeded on the 3D scaffold were subcutaneously implanted into mice and the transplanted DBCO-650-labeled chondrocytes could be effectively tracked in the prolonged time period of 4 weeks using NIRF imaging technology. Furthermore, this new cell labeling and tracking technology had minimal effect on cartilage formation <i>in vivo</i>