Snail organs transfected with or without the fluorecent siRNA visualized by either light or fluorescence microscopy.

<p><i>Panels A and C</i>: Images of snail hepatopancreas and ovotestis tissues of juvenile snails that were either soaked in fluorescent siRNA (A) for 72 hrs or left untreated (C) viewed without fluorescence (10× magnification). <i>Panels B and D</i>: The same images of hepatopancreas and ovotestis regions shown in panels A and C, subjected to fluorescence microscopy as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001212#s2" target="_blank">Materials and Methods</a>. Note the very weak flourescence (red stain) in the hepatopancreas (B) indicating less uptake of the fluorescent siRNA into this tissue occurs without PEI (10× magnification).</p

Time course for optimum gene knockdown after soaking snails in either dsRNA/PEI or siRNA/PEI nanoparticles.

<p>(A) Relative expression of Cathepsin B gene in juvenile snails soaked for between 1 and 4 days in CathB dsRNA/PEI. (B) Relative expression of peroxiredoxin gene in snails soaked for between 1 and 4 days in Prx dsRNA/PEI. (C) Relative expression of Cathepsin B gene in snails soaked for 1 to 4 days in CathB1-siRNA/PEI. Note in all time course studies that the optimum knockdown of transcripts occurs at day 3 of incubating in either dsRNA/PEI or siRNA/PEI complexes.</p

Analysis of Cath B gene expression by real time qPCR.

<p>(A) Relative expression of Cathepsin B gene in CathB1 siRNA/jPEI, CathB2siRNA/jet PEI, ‘mock’ CathB siRNA, CathB1siRNA, and PEI treated snails <i>versus</i> untreated snails. Treatments were done at room temperature for 72 hrs. (B) Relative expression of Cathepsin B gene in CathB dsRNA/PEI, Myo dsRNA/PEI, CathB dsRNA, and PEI treated <i>versus</i> untreated juvenile snails. Treatments were done at room temperature for 72 hrs.</p

Analysis of Prx expression by real time qPCR and ELISA.

<p>(A) Relative expression of peroxiredoxin gene in Prx dsRNA/PEI, Myo dsRNA/PEI, Prx dsRNA alone and PEI treated snails <i>versus</i> untreated snails. Treatments were done at room temperature for 72 hrs. (B) Prx protein suppression analyzed by ELISA as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001212#s2" target="_blank">Materials and Methods</a> with recombinant <i>B. glabrata</i> Prx antibody. Each bar represents the geometric mean end point titre ± standard error (SE) of each experimental group as indicated on the y-axis.</p

Snail organs transfected with the jPEI/fluorescent siRNA nanoparticles visualized by light and fluorescence microscopy.

<p><i>Panels A and C</i>: Images of hepatopancreas and ovotestis regions of juvenile snails that were soaked either in jPEI/Alexa 555 siRNA for either 24 or 72 hrs viewed without fluorescence (10× magnification). <i>Panels B and D</i>: Images of the hepatopancreas and ovotestis tissues shown in panels A and C, subjected to fluorescence microscopy as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001212#s2" target="_blank">Materials and Methods</a>. Note the intense flourescence (red stain) in the hepatopancreas compared to the ovotestis, indicating preferential uptake of the jPEI/fluorecent siRNA nanoparticles into this tissue.</p

Surface-Modified HK:siRNA Nanoplexes with Enhanced Pharmacokinetics and Tumor Growth Inhibition

We characterized in this study the pharmacokinetics and antitumor efficacy of histidine-lysine (HK):siRNA nanoplexes modified with PEG and a cyclic RGD (cRGD) ligand targeting αvβ3 and αvβ5 integrins. With noninvasive imaging, systemically administered surface-modified HK:siRNA nanoplexes showed nearly 4-fold greater blood levels, 40% higher accumulation in tumor tissue, and 60% lower luciferase activity than unmodified HK:siRNA nanoplexes. We then determined whether the surface-modified HK:siRNA nanoplex carrier was more effective in reducing MDA-MB-435 tumor growth with an siRNA targeting Raf-1. Repeated systemic administration of the selected surface modified HK:siRNA nanoplexes targeting Raf-1 showed 35% greater inhibition of tumor growth than unmodified HK:siRNA nanoplexes and 60% greater inhibition of tumor growth than untreated mice. The improved blood pharmacokinetic results and tumor localization observed with the integrin-targeting surface modification of HK:siRNA nanoplexes correlated with greater tumor growth inhibition. This investigation reveals that through control of targeting ligand surface display in association with a steric PEG layer, modified HK: siRNA nanoplexes show promise to advance RNAi therapeutics in oncology and potentially other critical diseases