Supplemental Material - The dialectics of leadership identity construction: Case studies from Indigenous women leaders

Supplemental Material for The dialectics of leadership identity construction: Case studies from Indigenous women leaders by Mariana de Santibañes, Sonia Ospina, Seulki Lee, Angela Santamaria, Michelle M Evans, Dunen Muelas, and Nazareth Guerrero in Leadership</p

Rational Design of Matrix Metalloproteinase-13 Activatable Probes for Enhanced Specificity

Because of the important roles that matrix metalloproteinases (MMPs) play in tumor invasion and metastasis, various activatable optical probes have been developed to visualize MMP activities <i>in vitro</i> and <i>in vivo</i>. Our recently developed MMP-13 activatable probe, l-MMP-P12, has been successfully applied to image the expression and inhibition of MMPs in a xenografted tumor model [Zhu, L., et al. (2011) <i>Theranostics 1</i>, 18–27]. In this study, to further optimize the <i>in vivo</i> behavior of the proteinase activatable probe, we tracked and profiled the metabolites by a high-resolution liquid chromatography–mass spectrometry (LC–MS) system. Two major metabolites that contributed to the fluorescence recovery were identified. One was specifically cleaved between glycine (G⁴) and valine (V⁵) by MMP, while the other one was generated by nonspecific cleavage between glycine (G⁷) and lysine (K⁸). To visualize the MMP activity more accurately and specifically, a new probe, d-MMP-P12, was designed by replacing the l-lysine with d-lysine in the MMP substrate sequence. The metabolic profile of the new probe, d-MMP-P12, was further characterized by <i>in vitro</i> enzymatic assay, and no nonspecific metabolite was found by LC–MS. Our <i>in vivo</i> optical imaging also demonstrated that d-MMP-P12 had a tumor-to-background ratio (TBR, 5.55 ± 0.75) significantly higher than that of l-MMP-P12 (3.73 ± 0.31) 2 h postinjection. The improved MMP activatable probe may have the potential for drug screening, tumor diagnosis, and therapy response monitoring. Moreover, our research strategy can be further extended to study other protease activatable probes

Precise Targeting of Liver Tumor Using Glycol Chitosan Nanoparticles: Mechanisms, Key Factors, and Their Implications

Herein, we elucidated the mechanisms and key factors for the tumor-targeting ability of nanoparticles that presented high targeting efficiency for liver tumor. We used several different nanoparticles with sizes of 200–300 nm, including liposome nanoparticles (LNPs), polystyrene nanoparticles (PNPs) and glycol chitosan-5β-cholanic acid nanoparticles (CNPs). Their sizes are suitable for the enhanced permeation and retention (EPR) effect in literature. Different <i>in vitro</i> characteristics, such as the particle structure, stability, and bioinertness, were carefully analyzed with and without serum proteins. Also, pH-dependent tumor cell uptakes of nanoparticles were studied using fluorescence microscopy. Importantly, CNPs had sufficient stability and bioinertness to maintain their nanoparticle structure in the bloodstream, and they also presented prolonged circulation time in the body (blood circulation half-life <i>T</i>_1/2 = about 12.2 h), compared to the control nanoparticles. Finally, employing liver tumor bearing mice, we also observed that CNPs had excellent liver tumor targeting ability <i>in vivo</i>, while LNPs and PNPs demonstrated lower tumor-targeting efficiency due to the nonspecific accumulation in normal liver tissue. Liver tumor models were produced by laparotomy and direct injection of HT29 tumor cells into the left lobe of the liver of athymic nude mice. This study provides valuable information concerning the key factors for the tumor-targeting ability of nanoparticles such as stability, bioinertness, and rapid cellular uptake at targeted tumor tissues

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>

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>

Suppression of HCC invasion by 3-BP, BSO, and a combination treatment.

<p>(A) Invasion capability of AR Huh-BAT cells significantly suppressed by BSO (200 μM), 3-BP (20 μM) and a combination by invasion assay using Boyden chambers (quantified at right panels). (B) Invasion capability of AR HepG2 cells significantly suppressed by BSO (200 μM), 3-BP (20 μM) and a combination by invasion assay using Boyden chambers (quantified at right panels). The concentrations of 3-BP (20 μM) and BSO (200 μM) which did not kill cancer cells were used in this assay. 3-BP and BSO was treated for 72 hours. In a combination treatment in this assay, 3-BP (20 μM) was treated for 72 hours with 24-hour pre-treatment of BSO (200 μM). *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001. Abbreviation: AR, anoikis-resistant; BSO, buthionine sulfoximine; 3-BP, 3-bromopyruvate.</p

Nuclear Mapping of Nanodrug Delivery Systems in Dynamic Cellular Environments

Nanoformulations have shown great promise for delivering chemotherapeutics and hold tremendous clinical relevance. However nuclear mapping of the chemodrugs is important to predict the success of the nanoformulation. In this study fluorescence microscopy and a subcellular tracking algorithm were used to map the diffusion of chemotherapeutic drugs in cancer cells. Positively charged nanoparticles efficiently carried the chemodrug across the cell membrane. The algorithm helped map free drug and drug-loaded nanoparticles, revealing a varying nuclear diffusion pattern of the chemotherapeutics in drug-sensitive and -resistant cells in a live dynamic cellular environment. While the drug-sensitive cells showed an exponential uptake of the drug with time, resistant cells showed random and asymmetric drug distribution. Moreover nanoparticles carrying the drug remained in the perinuclear region, while the drug accumulated in the cell nuclei. The tracking approach has enabled us to predict the therapeutic success of different nanoscale formulations of doxorubicin

<i>In vivo</i> anti-tumor effects of 3-BP, sorafenib, and a combination treatment in xenograft nude mice bearing AR Huh-BAT cells.

<p>(A) Tumor growth rates in the combination treatment group were significantly lower than those in the control, sorafenib, or 3-BP treatment group (upper panel). Gross images of tumors before treatment, tumors from the control group, and tumors from the combination treatment group are shown (lower panel). (B) <i>In vivo</i> demonstration of the apoptosis-inducing efficacy in the control, 3-BP, sorafenib, and combination treatment group was shown: H & E and TUNEL staining of tumor tissues in the control, sorafenib, 3-BP, and combination-treated mice (×40 magnification). (C) TUNEL-positive cell percentages (apoptotic index) were determined in six different high power (×400 magnification) fields. (D) There was a significant difference in body weight between the control group and the combination treatment group (<i>P</i><0.05). (E) After matrix detachment, anoikis-resistant cancer cells decrease intracellular ROS levels through inducing enzymes involved in the glycolysis and antioxidant systems for their survival. The Warburg effect can be modulated by increased intracellular ROS levels. Increased ROS levels induce HK II expression and make cancer cells sensitive to 3-BP treatment, and thereby promote cell death via ROS-mediated apoptosis (the black box indicates monocarboxylate transporter-1, and the white box indicates monocarboxylate transporter-4). *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001. Abbreviation: AR, anoikis-resistant; BSO, buthionine sulfoximine; 3-BP, 3-bromopyruvate.</p

Changes of lactic acid levels following 3-BP, BSO, or a combination treatment.

<p>(A) Lactic acid production in Huh-BAT, HepG2, and the corresponding AR cells was significantly increased after BSO treatment compared to the control. (B) Lactic acid production in Huh-BAT, HepG2, and the corresponding AR cells was significantly suppressed after 3-BP treatment compared to the control. (C) Lactic acid production was significantly suppressed after a combination treatment of 3-BP and BSO in AR Huh-BAT and AR HepG2 cells; there was a significant difference among baseline, 2, and 12 hours exposure to the combination treatment. Each assay and western blotting was performed at condition of 3-BP treatment 40 μM, 12 hour; BSO 200 μM, 24 hours; a combination treatment at 3-BP 40 μM, 12 hours after 24 hours pre-exposure of BSO 200 μM. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001. Abbreviation: AR, anoikis-resistant; BSO, buthionine sulfoximine; CTL, control; HK II, hexokinase II; rGCS, gamma-glutamylcysteine synthetase; ROS, reactive oxygen species; 3-BP, 3-bromopyruvate.</p