Figure 6.

In vivo analysis of jNP-mediated targeted delivery of miRNA and contrast-enhanced MR-imaging. (A) Plot of melting temperatures (Tm) derived from real-time qRT-PCR analyses of miRNA-126 corroborating miRNA-126-specific amplification to detect miRNA-126 in rat xenograft tumors 48-h after sonoporation with DEspR-targeted jNP[miRNA-126]MBs: using 10⁸ MBs, 10¹² jNPs, 27 µg of ds[miRNA-126]-mimic compared to negative control tumors (non-sonoporated, non-infused). T_m plots from different samples are identical and consistent with expected Tm for miRNA-126 ~ 75.4°C. (B) Real-time qRT-PCR cycle threshold (Ct) plots of miRNA-126 comparing sonoporated breast (red: MB-231-CSC) and pancreatic (blue: Panc1-CSC) xenograft tumors and negative control (grey: non-sonoporated, non-infused) tumors; low Ct indicate high miRNA-126 levels. (C) Bar graph of Ct values, means ± sd; **, P < 0.001 one-way ANOVA followed by Holms Sidak multiple pairwise comparison of control tumor (tmr) vs tumor tissues individually, *, P < 0.05. Control tumor (non-treated, solid black bar), control normal kidney and liver (n = 4/group) (open bars); sonoporated for miRNA-126 delivery: MB 231 TNBC-mammary xenograft tumor (n = 4) (solid red bar), Panc1 pancreatic cancer xenograft subcutaneous tumor (n = 6) (solid blue bar). (D) Representative Western blot analysis of miRNA-126's target KRAS protein shows decreased KRAS level 48 h after delivery of miRNA-126 by sonoporation; b-actin protein levels serve as internal control. (E) jNP-MB miR-126 in vivo testing in a xenograft tumor model of pancreatic peritoneal metastasis. Diagram of experimental timeline of tumor establishment and miR-126 delivery. Representative necropsy pictures at study endpoint comparing control mock-treated and jNP-MB miR-126 treated rat with xenograft Panc1-CSC derived peritoneal metastasis. Yellow arrows point to tumors. (F) Magnetic resonance studies of gradient-echo signal intensity versus echo time (TE), from 10-100 milliseconds (ms), for IgG-jNPs (solid red circles) and precursor PEG-uspion phantoms (solid black circles), both at 5 x 10¹⁰/mL in 1% agar, and control blank 1% agar phantom (solid blue circles). Data points show mean ± s.d. over 60 pixels in each phantom. Each curve was normalized so that peak signal at TE = 4 ms is equal to 1. jNPs exhibited shorter T2* values (mean ± sd: 35.2 ± 1.3 ms) compared with precursor PEG-USPIONs (57.6 ± 2.9 ms) and control blanks (82.2 ± 4.6 ms), *** P < 0.001, two-way ANOVA (subtype x time) repeated measures. (G) Representative ex vivo magnetic resonance (MR)-images (MRI) of pancreatic peritoneal tumors obtained using identical MRI settings and digital image settings 24-h after infusion of jNPs compared with control no jNPs infused. Regions containing high concentrations of jNPs showed hypointense (dark) signals at TE=6.5 ms, and this effect was amplified at TE=13 ms (compared area in dashed yellow and red circles, and area with red arrow). The control samples (no jNPs) did not show similar signal dropouts indicating presence of USPIONs in jNPs. In all images, t = tumor. (H) ELISA levels of key cytokines/chemokines (IL-1a, -6, -4, -10, -12, 13: interleukins; TNFa: tumor necrosis factor alpha, GM-CSF: granulocyte macrophage colony stimulating factor, IFN-g: interferon gamma, RANTES: Regulated on Activation, Normal T Cell Expressed and Secreted (or CCL5) produced by cells which are exposed to jNPs in the circulation such as ECs, endothelial cells, MNC, monocytes, T- and B-cell leukocytes, PMNs, neutrophils; Mast, complement-activating mast cells. Statistics performed: two-way (jNP-dose x cytokine levels across different cytokines) ANOVA (ns, not significant); n = 2 rats/group x 3 groups: 10¹² jNP infusion (red hashed bars), 10¹³ jNPs infusion (solid red bars), and no jNP-infusion (solid black bars) negative control rats. RANTES and IL-13 show elevation but not significantly different between groups likely due to rat-specific wide-variations.