Journal: Scientific Reports

Article Title: Defective CFTR leads to aberrant β-catenin activation and kidney fibrosis

doi: 10.1038/s41598-017-05435-5

Figure Lengend Snippet: Overexpression of CFTR alleviates fibrotic phenotype in UUO model. ( a ) Immunohistochemical staining showing the dramatically increased expression of β-catenin in delta F508 mice compared to wild type mice after UUO. The black box marks the area enlarged, and the black arrows indicate the nuclear localization of β-catenin, scale bar = 50 µm; ( b–d ) Real time-PCR analysis showing higher expression levels of Axin-2, c-jun and Mmp7 in the delta F508 mice than that in the wild type mice after UUO. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3; ( e–h ) CFTR overexpression abrogates kidney fibrosis in mice. ( e–g ) Real time-PCR analysis showing the decreased expression of α-SMA, collagen I and FN in peGFP-CFTR overexpression kidneys. *p < 0.05, quantification analysis represents data from 9 control and 10 CFTR overexpressed UUO kidneys. ( h ) Representative western blot showing the decreased expression of matrix protein and α-SMA in CFTR overexpressed UUO kidneys (peGFP-CFTR) comparing to control UUO kidneys (peGFP-C3) and mock UUO kidney (injected same volume of microbubbles without any DNA). (Full-length blot is shown in Supplementary Figure .)

Article Snippet: Before injection, 200 μg of peGFP-C3 or peGFP-CFTR plasmid were mixed with sulphur hexafluoride microbubbles (Bracco Imaging B.V., Geneva, Switzerland) at a ratio of 1:1 (v/v).

Techniques: Over Expression, Immunohistochemical staining, Staining, Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Injection

Journal: Theranostics

Article Title: Janus USPION modular platform (JUMP) for theranostic ultrasound-mediated targeted intratumoral microvascular imaging and DNA/miRNA delivery

doi: 10.7150/thno.78454

Figure Lengend Snippet: In vitro analysis of Janus nanoparticle (jNP) targeting and carrier functions. (A) Schematic diagram of stepwise self-assembly and directional orientation of jNPs on 1 µm diameter microbubbles (MB) with zeta potential average -3.2 ± 0.4, forming jNP-DNA-MBs with tunable amount of jNPs added per MB (e.g., 1 x 10 4 or 5 x 10 4 jNPs/MB). DEspR-jNP, targeting jNP via anti-DEspR antibody face; IgG-jNP, control non-targeting with isotype IgG targeting face. (B) Representative flow cytometry analysis of double-fluorescent jNP-DNA-MBs distinguished from single-fluorescent and non-fluorescent MBs, using 5 x 10 2 jNPs/MB. Y-axis: red-fluorescence intensity; X-axis, green-fluorescence; Control-1: non-fluorescent microbubbles (MBs) in quadrant 4 (Q4), red-fluorophore labeled jNPs (jNP), green-fluorophore labeled single strand 50-nt oligoDNA (DNA); 4-quadrants with differential fluorescence attained by MBs: ± bound DNA, ± bound jNPs: Q1-Q4. (C) Representative flow cytometry analysis of jNP-DNA-MB assembly: Y-axis, fluorescence intensity of jNPs with fluorescent antibody layer; X-axis, forward scatter representing size. Left-panel: non-fluorescent microbubbles (MB); Middle-panel: fluorescent jNP-DNA-MBs with 10 3 jNPs/MB; Right panel: fluorescent jNP-DNA-MBs with 5 x 10 4 jNPs/MB. Fluorescence intensity > 10 3 above red horizontal line. (D) Contingency group analysis graph of jNP concentration-dependent self-assembly of jNP-DNA-MBs: fluorescent self-assembled jNP-DNA-MBs (solid red bars), non-fluorescent, non-assembled or free DNA-MBs (solid black bars); contingency chi square analysis, P < 0.0001. (E) Flow cytometry analysis of DEspR-targeting jNP-DNA-MBs binding to pancreatic tumor (Panc1) cells using different cell-to-[jNP-DNA-MB] ratios, X-axis: size indicator forward scatter area. Control-1, non-fluorescent MB only, Y-axis: side scatter granularity. Control-2, red-fluorescently labeled jNP-DNA-MBs only; Control-3: panc1 tumor cells only; cell-complex formation with 1:1 ratio of Panc1 cells to DEspR-targeting jNP-DNA-MBs; and with 1:5 cell-complex ratio using 1 x 10 4 DEspR-targeting jNP-DNA-MBs (panels with Y-axis: fluorescence intensity of jNPs from labeled antibody layer). Free jNPs gated (dashed red triangle with corresponding % in dashed rectangle); free cells below the red horizontal line; jNP-DNA-MB bound cells: fluorescent = above red line. (F) Contingency group analysis graph of flow cytometry results comparing % bound vs % free cells exposed to jNP-DNA-MBs at 0, 1:1, and 1:5 ratio of cells-to-jNP-DNA-MBs: % bound cells (solid red bars jNP-MB [+] ), and % free cells (solid black bars). Chi square analysis, n = 5000 cells, P < 0.0001. (G) Representative fluorescence microscopy images of a Panc1 tumor cell with multiple bound jNP-DNA-MBs (~ 1 µm diameter MBs). Red: fluorescently-labeled jNP-DNA-MBs, blue: Hoechst nuclear stain, bar = 5 µm. (H) Contingency group analysis graph of % bound cells [+] with bound DEspR-targeting jNP-DNA-MBs (solid red bar DEspR-jNP [+] ), or with non-specific bound non-targeting isotype (IgG) jNP-DNA-MBs (open red bar: IgG-jNP [+] ); compared with free cells (solid black bar: DEspR-jNP [-] cells); open black bar: IgG-jNP [-] cells); contingency chi-square analysis: P < 0.0001; n = 80 cells exposed to DEspR jNP-DNA-MBs); n= 30 cells exposed to IgG jNP-DNA-MBs. (I) Comparison of cell-targeting showing maximum number (max #) of fluorescently labeled jNP-DNA-MBs bound to Panc1 tumor cells comparing DEspR-targeting jNP-DNA-MBs (solid red bar) vs isotype IgG non-targeting jNP-DNA-MBs. Mann Whitney test: P = 0.0006; DEspR-targeting (solid red bar) n = 14 cells; non-targeting IgG-isotype (open red bar) n = 6 cells (cells with no jNP-DNA-MBs excluded here).

Article Snippet: The DNA payload in DNA-MBs was measured as follows: After suspension of microbubbles (MBs, MicroMarker microbubbles, Visual Sonics Inc.), 10 8 MBs were mixed with 30 μg dsDNA in 1 mL saline solution with gentle agitation until analysis timepoints.

Techniques: In Vitro, Zeta Potential Analyzer, Control, Flow Cytometry, Fluorescence, Labeling, Concentration Assay, Binding Assay, Microscopy, Staining, Comparison, MANN-WHITNEY

Journal: Theranostics

Article Title: Janus USPION modular platform (JUMP) for theranostic ultrasound-mediated targeted intratumoral microvascular imaging and DNA/miRNA delivery

doi: 10.7150/thno.78454

Figure Lengend Snippet: In vivo analysis of jNP-MB theranostic functionality: contrast-enhanced ultrasound molecular-imaging and delivery of reporter-RFP minigene. (A) Diagram of molecular imaging sequence, with key events marked #1-#5 in series. Arrows connect to corresponding representative contrast-enhanced ultrasound images: overlay of B-mode (grey-scale image) and contrast-enhanced images (pseudo-colored green image) of spontaneous rat mammary tumors showing baseline (#1), during bolus infusion (#2) comparing control DEspR-targeted MB-DNA (Targeted MB-DNA, yellow line) and DEspR-targeted jNP-DNA-MBs (red line) during adherence (#4) and after disruption (#5) of MBs. Free MBs, dotted blue line are typically cleared by 5 min. (B) Corresponding time intensity curve generated during infusion (#2), average (green line), individual signals (blue dots). (C) Diagram depicting regions of interest (ROI) for time-intensity analysis of molecular imaging done on mammary tumors: ROI of intratumoral microvessels (mv, red vessels, dashed red oval in A#4, A#5) and of tumor feeder vessels at base of tumor (boxed yellow here and in A). (D) Representative time-intensity curves of background-subtracted contrast intensity signals (CIS) in designated tumor-ROIs at pre-destruct (pre) and post-destruct (post) comparing DEspR-targeting jNP-DNA-MBs (Targeted jNP-DNA-MBs) and control DEspR-targeting (biotin-avidin) MBs (Targeted MB-DNA) in extra-tumoral feeder vessels (fv) and intratumoral microvessels (mv). Timepoint of high-power ultrasound MB-destruct sequence (dashed line) demarcating pre- and post-destruct CIS. Green line, average of background-subtracted contrast intensity signals (CIS, blue dots) representing contrast-enhanced signals from adherent targeted-MBs in pre-destruct phase, and confirmation of adherent MBs after MB-destruction in post-destruct phase, determined via VisualSonics Contrast software. (E) Quantitative analysis of average CIS in the 10 s pre-destruct sequence, comparing DEspR-targeting jNP-DNA-MBs (jNP, open red circles) vs control (C) non-jNP DEspR-targeting MB-DNA microbubbles (C, open black circles) in two ROIs: extratumoral feeder vessels (fv) and intratumoral microvessels (mv). At t-20 (and t-30 min), CIS-levels represent mostly if not only DEspR-bound adherent MBs, as shown at the end of post-destruct level. One-way ANOVA P < 0.0001; ****, Tukey's multiple pairwise comparison P < 0.0001, 8 groups, n = 10 average CIS-levels/group representing 3-4 per second averages during pre-destruct phase, from 3 independent experiments using spontaneous mammary tumor rat model. Average CIS values taken from both tumor-ROIs: extra-tumoral feeder vessels (fv) and intra-tumoral microvessels (vs) at two imaging sessions (t20- and t30 min). (F) Diagram of key events in sonoporation of targeting jNP-DNA-MBs in intratumoral microvessels: pre-sonoporation #1-#4: #1, sonoporator; #2, endothelial cells in microvessel; 3, adherent jNP-DNA-MBs after clearance of unbound MBs; 4: tumor cells in cancer-microvascular niche; ➔, after sonoporation #5-#8: #5, non-injured endothelial cells; #6, disassembled jNPs and MBs and disrupted insonated MBs; #7, jNP-DNA released from MBs and direct entry into cytosol through transient “sonopores” that seal subsequently in conditions with no acoustic injury; #8, heterogeneous tumor cells in perivascular cancer niche transfected with jNP-DNA functional RFP-minigene (red inverted triangles). (G) Graph of IVIS-generated peak reporter-function fluorescence in vivo comparing DEspR-targeting jNP-DNA-MBs (open red circles, n = 5 tumors, min 4.7 x 10 7 to 1.4 x 10 9 photons/s/area) and control DEspR-targeting MB-DNA (solid black squares, n = 3 tumors); *, p = 0.036 two-tailed Mann Whitney test. Peak fluorescence units from published reports of in vivo delivery using CMBs are noted as relative reference points (REFs) with arrows: E, F, G, H - , respectively. Reference F is ICAM-1 targeted; E, G, H utilize default liver-uptake. (H) Comparison of number of MBs used per gram body weight (#MBs: 4 x 10 5 /g BW for jNP-MBs and control MB-DNA) used for in vivo delivery of reporter function genes comparing jNP-MBs used in 200-250 g rat models, with published CMBs (REFs C, E, G, H are , , , , respectively, used in 20-25 g mouse models.

Article Snippet: The DNA payload in DNA-MBs was measured as follows: After suspension of microbubbles (MBs, MicroMarker microbubbles, Visual Sonics Inc.), 10 8 MBs were mixed with 30 μg dsDNA in 1 mL saline solution with gentle agitation until analysis timepoints.

Techniques: In Vivo, Imaging, Sequencing, Control, Disruption, Generated, Avidin-Biotin Assay, Software, Comparison, Transfection, Functional Assay, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Experimental and Therapeutic Medicine

Article Title: Experimental study of TNF-α receptor gene transfection by ultrasound-targeted microbubble destruction to treat collagen-induced arthritis in rats in vivo

doi: 10.3892/etm.2019.7158

Figure Lengend Snippet: Enhanced green fluorescent protein analysis of groups 1 to 5 at different time points. The fluorescence intensity at 2 weeks was higher than that at 4 and 8 weeks in all groups. The intensity in the PL+MB+US (muscle) and PL+MB+US (joint) was significantly greater than that in the other groups at the corresponding time points. No significant difference was identified between the PL+MB+US (muscle) and PL+MB+US (joint) groups and no significant difference was identified among the remaining groups. *P<0.05 vs. the PL+US, PL+MB or PL groups. PL, plasmid; MB, microbubble; US, ultrasound.

Article Snippet: The SonoVue microbubble suspensions (Bracco Imaging S.p.A, Milano, Italy) were prepared at a density of 2–5×10 8 /ml and a concentration of 5 mg/ml using 0.9% saline solution.

Techniques: Fluorescence, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: Experimental study of TNF-α receptor gene transfection by ultrasound-targeted microbubble destruction to treat collagen-induced arthritis in rats in vivo

doi: 10.3892/etm.2019.7158

Figure Lengend Snippet: Fluorescence microscopy examination of EGFP expression and DAPI staining of the tibialis anterior and synovium of the rat ankle at 2, 4 and 8 weeks (magnification, ×200). Group 1, plasmid + microbubble + ultrasound (muscle group); Group 2, plasmid + microbubble + ultrasound (joint group); Group 3, plasmid + ultrasound; Group 4, plasmid + microbubble; Group 5, plasmid only. EGFP, enhanced green fluorescent protein.

Article Snippet: The SonoVue microbubble suspensions (Bracco Imaging S.p.A, Milano, Italy) were prepared at a density of 2–5×10 8 /ml and a concentration of 5 mg/ml using 0.9% saline solution.

Techniques: Fluorescence, Microscopy, Expressing, Staining, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: Experimental study of TNF-α receptor gene transfection by ultrasound-targeted microbubble destruction to treat collagen-induced arthritis in rats in vivo

doi: 10.3892/etm.2019.7158

Figure Lengend Snippet: Arthritis scores at different time points for each group. For all treatment groups, the scores decreased with increasing treatment time. A significant reduction occurred at 2 weeks; at which point the scores stabilized. The score reduction in the PL+MB+US (muscle) and PL+MB+US (joint) groups was greater than in other groups (P<0.05); however, the muscle injection group showed no significant difference when compared with the joint injection group (P>0.05). *P<0.05 vs. the PL+US, PL+MB, PL or control groups. PL, plasmid; MB, microbubble; US, ultrasound.

Article Snippet: The SonoVue microbubble suspensions (Bracco Imaging S.p.A, Milano, Italy) were prepared at a density of 2–5×10 8 /ml and a concentration of 5 mg/ml using 0.9% saline solution.

Techniques: Injection, Control, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: Experimental study of TNF-α receptor gene transfection by ultrasound-targeted microbubble destruction to treat collagen-induced arthritis in rats in vivo

doi: 10.3892/etm.2019.7158

Figure Lengend Snippet: Hematoxylin and eosin staining of the synovium in groups 1 to 6. In groups 1–5, cellular infiltration at 2 weeks was greater than at 4 and 8 weeks. Groups 1 and 2 exhibited milder inflammation compared to the remaining groups at all corresponding time points, and no marked difference between groups 1 and 2 were observed. The control group demonstrated the most severe inflammatory cell infiltration (magnification, ×200). Group 1, plasmid + microbubble + ultrasound (muscle group); Group 2, plasmid + microbubble + ultrasound (joint group); Group 3, plasmid + ultrasound; Group 4, plasmid + microbubble; Group 5, plasmid only; Group 6, untreated control.

Article Snippet: The SonoVue microbubble suspensions (Bracco Imaging S.p.A, Milano, Italy) were prepared at a density of 2–5×10 8 /ml and a concentration of 5 mg/ml using 0.9% saline solution.

Techniques: Staining, Control, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: Experimental study of TNF-α receptor gene transfection by ultrasound-targeted microbubble destruction to treat collagen-induced arthritis in rats in vivo

doi: 10.3892/etm.2019.7158

Figure Lengend Snippet: Quantitative analysis of TNF-α expression by immunohistochemical staining. PL+MB+US (muscle) and PL+MB+US (joint) groups exhibited significantly lower staining intensities at all corresponding time points when compared with the other groups (P<0.05). No significant difference between the PL+MB+US (muscle) and PL+MB+US (joint) groups was observed. No significant difference among the PL+US, PL+MB and PL groups was also observed (P>0.05). *P<0.05 vs. the PL+US, PL+MB, PL or control groups; # P<0.05 vs. the control group. TNF-α, tumor necrosis factor-α; PL, plasmid; MB, microbubble; US, ultrasound.

Article Snippet: The SonoVue microbubble suspensions (Bracco Imaging S.p.A, Milano, Italy) were prepared at a density of 2–5×10 8 /ml and a concentration of 5 mg/ml using 0.9% saline solution.

Techniques: Expressing, Immunohistochemical staining, Staining, Control, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: Experimental study of TNF-α receptor gene transfection by ultrasound-targeted microbubble destruction to treat collagen-induced arthritis in rats in vivo

doi: 10.3892/etm.2019.7158

Figure Lengend Snippet: (A) Western blot analysis of TNF-α protein expression in the synovial tissues of the (B) PL+MB+US (muscle) group and the (C) PL+MB+US (joint) group at 2, 4 and 8 weeks. TNF-α expression in both groups decreased at 4 and 8 weeks post-treatment compared with the controls, and TNF-α levels gradually decreased over time and reached their lowest point at 8 weeks following treatment. *P<0.05 vs. Control. TNF-α, tumor necrosis factor-α; PL, plasmid; MB, microbubble; US, ultrasound.

Article Snippet: The SonoVue microbubble suspensions (Bracco Imaging S.p.A, Milano, Italy) were prepared at a density of 2–5×10 8 /ml and a concentration of 5 mg/ml using 0.9% saline solution.

Techniques: Western Blot, Expressing, Control, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: Experimental study of TNF-α receptor gene transfection by ultrasound-targeted microbubble destruction to treat collagen-induced arthritis in rats in vivo

doi: 10.3892/etm.2019.7158

Figure Lengend Snippet: Serum levels of TNF-α at different time points. A reduction in serum TNF-α levels in the PL+MB+US (muscle) and PL+MB+US (joint) groups was greater than that in the other groups at all corresponding time points. *P<0.05 vs. the PL+US, PL+MB, PL or control groups; # P<0.05 vs. the control group. TNF-α, tumor necrosis factor-α; PL, plasmid; MB, microbubble; US, ultrasound.

Article Snippet: The SonoVue microbubble suspensions (Bracco Imaging S.p.A, Milano, Italy) were prepared at a density of 2–5×10 8 /ml and a concentration of 5 mg/ml using 0.9% saline solution.

Techniques: Control, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Nanoparticles as Theranostic Vehicles in Experimental and Clinical Applications—Focus on Prostate and Breast Cancer

doi: 10.3390/ijms18051102

Figure Lengend Snippet: Nanoparticle-based drugs for PCa and BC, approved or under clinical evaluation. Listed are also examples of drugs for solid cancers in general, since they also might be applicable to PCa and BC in the future.

Article Snippet: , US enhancement imaging , Phospholipid microbubbles , - , SonoVue® , Pilot , Bracco Imaging Inc..

Techniques: Imaging, Plasmid Preparation