1. Nanoparticle-mediated Gene Silencing Confers Radioprotection to Salivary Glands In Vivo 
Szilvia Arany, Danielle SW Benoit, Stephen Dewhurst, Catherine E Ovitt 
Molecular Therapy 
Volume 21, Issue 6, Pages (June 2013)
DOI: /mt
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
In vivo administration of siRNA–nanoparticle complexes into mouse submandibular gland. (a) Retroductal injection was performed through an ultra-thin, pre-made polyurethane tube, inserted into the orifice of the Wharton duct draining the left SMG (×30 original magnification). (b) Colocalization of the early endosomal antigen 1, EEA1 (green) with Cy3-tagged siRNA–nanoparticle complexes (red) in duct cells at 3 hours after injection (arrowheads). (c,f) Phase contrast and red fluorescent image of Lipofectamine-mediated transfection of Cy3-siRNA (red) in SMG at 8 hours post-injection. (d,g) Phase contrast and fluorescent images of nanoparticle-complexed Cy3-siRNA injected into SMG, at 8 hours post-injection. (e,h) Cy3-siRNA–nanoparticle complexes are detected in the acinar compartment (ac), as well as in duct cells (du), at 6 hours post-injection. For b, bar = 20 μm; for c,f, bar = 500 μm; for d,g, bar = 100 μm; for e,h, bar = 50 μm. DAPI, 4′,6-diamidino-2-phenylindole; siRNA, small-interfering RNA; SMG, submandibular gland.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Retrograde injection of siRNA–nanoparticle complexes mediates in vivo knockdown of Nkcc1 gene expression in mouse SMG. Real-time quantitative PCR measurements of Nkcc1 mRNA expression were conducted at different time points after (a) single or (b) triplicate retrograde injections of siRNA–nanoparticle complexes. Quantification represents data in each sample group relative to mRNA levels determined from Nkcc1+/− littermates, which were set at 100% (all error bars represent SEM; n = 3–5; *P < 0.05) (scr siRNA, scrambled siRNA; NP, nanoparticles). siRNA, small-interfering RNA; SMG, submandibular gland.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
In vivo Nkcc1 protein expression is significantly decreased following nanoparticle-mediated delivery of siRNA. (a,b) Sections from control and Nkcc1−/− SMGs stained with antibody to Nkcc1. (c,d) SMG sections isolated at 2 days post-injection from mice following a single injection of (c) scrambled or (d) Nkcc1 siRNA, respectively. (e) Measure of Nkcc1 protein knockdown over time as a result of a single injection of siRNA complexed with nanoparticles, expressed as the ratio of Nkcc1-positive cells to total cell number. (f) Measure of Nkcc1 protein knockdown over time following triplicate nanoparticle siRNA injections, expressed as a ratio of Nkcc1-positive cells to total cell number. Calculations for e and f were determined from 10 randomly selected areas (330 × 430 μm) on sections from three animals per sample group. (Means ± SEM; *P < 0.05) (scr siRNA, scrambled siRNA; NP, nanoparticles; 3×, triplicate). DAPI, 4′,6-diamidino-2-phenylindole; siRNA, small-interfering RNA; SMG, submandibular gland; wt, wild-type.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Saliva secretion rates decrease following retrograde injection of saline or siRNA–nanoparticle complexes. Secretagogue-stimulated saliva was collected from cannulated SMGs for a 20-minute period. The saliva output is expressed as a ratio of secretion volume per wet gland weight, and values are normalized to those from untreated control Nkcc1+/− littermates. (a) Retroductal injections of saline only were used to establish “background” reduction in saliva secretion presumably resulting from the reverse hydrodynamic pressure generated during retrograde injection. Saliva secretion was stimulated at time points indicated and collected. (b) Changes in saliva secretion rates were measured at the time points indicated after single (striped bars) or triplicate (hatched bars) siRNA–nanoparticle injections, and normalized to the secretion levels measured from non-injected animals (data were corrected for the nonspecific reduction of ± 7.95% in saliva output occurring after saline retrograde injection). The secretion rate determined in SMG from Nkcc1−/− mice is shown for comparison (black bar). (Means ± SEM, n = 3–5, *P < 0.05, **P < 0.001). (scr siRNA, scrambled siRNA; 3× scr siRNA, triple injection of scrambled siRNA; Nkcc1 siRNA, single injection of Nkcc1 siRNA; 3× Nkcc1 siRNA, triple injection of Nkcc1 siRNA). siRNA, small-interfering RNA; SMG, submandibular gland.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Analysis of mouse SMG for off-target effects following siRNA–nanoparticle retroductal injections. (a–c) Apoptosis was measured by staining for caspase-3–positive cells at 1 week post-injection. Sections were prepared from (a) SMG of untreated control, (b) SMG injected with scrambled (scr) siRNA–nanoparticle complexes, and (c) SMG injected with Nkcc1 siRNA–nanoparticle complexes. Fluorescent microscopic images of TUNEL apoptotic assay on (d) SMG of untreated control (green; arrow), (e) SMG injected with scrambled siRNA–nanoparticle complexes (green; arrow), and (f) SMG injected with Nkcc1 siRNA–nanoparticle complexes. (g) Quantification of TUNEL-positive cells (green in d–f) counted from 10 random microscopic fields per gland. The percentage of apoptotic to total cells (labeled by DAPI; blue) is plotted (means ± SEM). (h) Immunohistochemistry with antibody to Mist1, an acinar cell-specific transcription factor (red), and (i) antibody to sABP, a cytoplasmic marker for acinar cells (red), demonstrates normal acinar cell gene expression in SMGs treated with Nkcc1 siRNA–nanoparticles at 1 week post-injection. Nuclei are stained with DAPI (blue). For a–c, bar = 200 μm; for d–f,h,l, bar = 100 μm. (scr siRNA, scrambled siRNA). DAPI, 4′,6-diamidino-2-phenylindole; siRNA, small-interfering RNA; SMG, submandibular gland.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
siRNA–nanoparticle injection elicits only limited local and systemic immune responses. Expression of cytokine or interferon pathway immunoresponse genes, (a) IFIT1, (b) Mx1, (c) Oas1, and (d) STAT1 analyzed at 3 and 6 hours post-injection by quantitative RT-PCR of SMG tissue (Data pooled from three separate quantitative PCR experiments on all samples, and compared with untreated controls; means ± SEM, *P < 0.05). (e) Serum levels of IL-6 were measured using ELISA assay on serum collected at 6 and 24 hours after injection with saline, scrambled siRNA–nanoparticles, or Nkcc1 siRNA–nanoparticles. Initial measurements at “0” time point were obtained from serum collected before siRNA injection. (f) Serum levels of IFN-γ were measured using ELISA assay on serum collected at 6 and 24 hours after injection with saline, scrambled siRNA–nanoparticles, or Nkcc1 siRNA–nanoparticles. Initial measurements at “0” time point were obtained from serum collected before siRNA injection. (Means ± SEM; n = 3) (scr siRNA, scrambled siRNA; NP, nanoparticles). ELISA, enzyme-linked immunosorbent assay; IFN, interferon; IL, interleukin; RT-PCR, reverse transcription-PCR; siRNA, small-interfering RNA; SMG, submandibular gland.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
In vivo application of siRNA blocks radiation-induced increase of the proapoptotic protein Pkcδ. Pkcδ knockdown was achieved through a single retrograde injection of siRNAs targeting the Pkcδ gene. All siRNA injections were done in complex with nanoparticles. (a) RNA samples were prepared from five sample groups to compare Pkcδ gene expression changes between control SMG and SMGs receiving scrambled (scr) or Pkcδ siRNAs, with or without radiation exposure (10.0 Gy), as well as those receiving radiation alone. Results are based on quantitative RT-PCR, and the level of Pkcδ mRNA measured in untreated control samples was set at 100% (means ± SEM, n = 3–5, *P < 0.05). (b) Pkcδ protein expression levels in each sample were analyzed on western blots at 48 hours after treatment. The membrane was stripped and reprobed with β-actin antibody as control. (c) Pkcδ protein expression was quantified based on protein band intensities measured on western blots (shown in b) using Image J. The level of Pkcδ protein measured in untreated control samples was set at 100% (means ± SEM, n = 3–5, *P < 0.05). (d) The number of apoptotic cells per total cells detected on sections (n = 10) of irradiated SMGs was established using the TUNEL assay, and compared with the numbers detected after pre-administration of scrambled (scr) or siRNAs targeting Pkcδ. (e) The ratio of apoptotic cells to total cells was also determined at 48 hours after irradiation by staining for active caspase-3 in nonirradiated SMG (untreated control), and in irradiated SMG, either non-injected (10.0 Gy), or pretreated with scrambled siRNA (scr) or Pkcδ siRNA (means ± SEM, *P < 0.05). RT-PCR, reverse transcription-PCR; siRNA, small-interfering RNA; SMG, submandibular gland.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

9. Figure 8
Targeted in vivo delivery of Pkcδ siRNA confers radioprotection to the SMG. (a) Timeline of experimental procedure. Scrambled or Pkcδ siRNA–nanoparticle complexes were administered by retrograde injection into the SMG 3 days before irradiation (10.0 Gy). Phenotypic changes were assessed at various time points using morphological and immunohistochemical methods. H/E staining of (b) untreated, normal gland, (c) 10.0 Gy irradiated gland, and (d) Pkcδ siRNA-treated, irradiated gland at 3 months postirradiation (black arrow: cytoplasm vacuolization; black arrowhead: acinar cell lysis; white arrowhead: pycnotic nucleus). (e–g) Alcian-blue/PAS-stained sections at 3 months postirradiation (acinar cells (ac), blue; ducts (du), pink; nuclei, dark blue) from (e) untreated gland, (f) irradiated gland, and (g) Pkcδ siRNA-treated, irradiated SMG. (h–j) Immunohistochemical detection of AQP5 at 3 months postirradiation. AQP5 expression (red) in (h) untreated, (i) irradiated only, and (j) Pkcδ siRNA-treated, irradiated SMG at 3 months postirradiation. (k) Relative AQP5 protein expression at 3 months postirradiation was calculated by dividing AQP5-positive (red fluorescent) areas by total DAPI-positive (blue) areas in 10 randomly selected SMG regions from three animals per sample group. (l) Changes in the SMG saliva flow rate, 3 months after ionizing radiation. Pilocarpine-stimulated saliva was collected for 20 minutes and normalized to SMG weight (n = 3–5). (All error bars represent SEM; *P < 0.05; for a–c,h–j, bar = 100 μm; for e–g, bar = 200 μm; scr siRNA, single injection of scrambled siRNA–nanoparticles; Pkcδ siRNA, single injection of Pkcδ siRNA–nanoparticles; 10.0 Gy, ionizing radiation dose). AQP5, aquaporin 5; DAPI, 4′,6-diamidino-2-phenylindole; H/E, hematoxylin/eosin; PAS, periodic acid-Schiff staining; siRNA, small-interfering RNA; SMG, submandibular gland.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions