Multifunctional Magnetic Nanocarriers for Image-Tagged SiRNA Delivery to Intact Pancreatic Islets

Abstract

Background With the ultimate hope of finding a cure for diabetes, researches are looking into altering the genetic profile of the beta cell as a way to manage metabolic dysregulation. One of the most powerful new approaches for the directed regulation of gene expression utilizes the phenomenon of RNA interference. Methods Here, we establish the feasibility of a novel technology centered around multifunctional magnetic nanocarriers, which concurrently deliver siRNA to intact pancreatic islets and can be detected by magnetic resonance (MR) and optical imaging. Results In the proof-of-principle studies described here, we demonstrate that, following in vitro incubation, magnetic nanoparticles carrying siRNA designed to target the model gene for enhanced green fluorescent protein, are efficiently taken up by murine pancreatic islets, derived from egfp transgenic animals. This uptake can be visualized by magnetic resonance imaging (MRI) and near-infrared fluorescence optical imaging and results in down-regulation of the target gene. Conclusions These results illustrate the value of our approach in overcoming the challenges associated with genetic modification of intact pancreatic islets in a clinically acceptable manner. Furthermore, an added advantage of our technology derives from the combined capability of our magnetic nanoparticles for siRNA delivery and magnetic labeling of pancreatic islets.

Comparison of Cationic and Amphipathic Cell Penetrating Peptides for siRNA Delivery and Efficacy

“…Green fluorescent protein (GFP)-targeting siRNA (siGFP, sense: 5’-CAAGCUGACCCUGAAGUUdTdT-3’; antisense: 5’-GAACUUCAGGGUCAGCUUdTdT-3’) 22 and fluorophore-labeled negative control siRNA (siDY547, sense: 5’-DY547-UUCUCCGAACGUGUCACGUdTdT-3’; antisense: 5’-ACGUGACACGUUCGGAGAAdTdT-3’) 23 were obtained from Dharmacon (Lafayette, CO). Unlabeled negative control siRNA (siNC) and phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-targeting siRNA (siPTEN) were purchased from Ambion (Foster City, CA) and Cell Signaling Technology (Danvers, MA), respectively.…”

MRI as a tool to monitor islet transplantation

“…In addition, we showed that this uptake can be visual ized by MRI (Figure 5a) and results in down regulation of the target gene (Figure 5b). 60 These studies lay the groundwork for future applications, in which genes that are implicated in islet graft loss (for example, those associated with allorejection, islet cell death and auto immune attack) can be similarly targeted to improve graft outcome. Furthermore, this approach enables non invasive tracking of the ironoxide conjugated siRNA and facilitates investigations of its relationship with graft fate.…”

“…Constant transverse relaxivity (r2) at field strengths >0.5 T ■ 32 and high nanomolar sensitivity of detection 63 Proven clinical safety record ■ Can be used (through modifications of the nanoparticles' size and surface ■ coating) to detect and characterize specific cell subpopulations and/or biochemical processes 60,[64][65][66][67] Disadvantages:…”

“…59 siRNA-conjugated nanoparticles Some of our most recent work has employed a multi functional rationale to explore the possibility for con currently imparting imaging and therapeutic capabilities to transplanted pancreatic islets. 60 we took advantage of the fact that pancreatic islets avidly take up dextran coated superparamagnetic iron oxide nanoparticles to design a probe that consisted of a dextrancoated iron oxide core conjugated to a small interfering RNA (siRNA). we speculated that the capability of siRNAs to selectively silence the expression of essentially any gene of choice with single nucleotide specificity might be useful in the context of islet transplantation.…”

Transgene and islet cell delivery systems using nano-sized carriers for the treatment of diabetes mellitus

“…The injection site of the pancreas was then sandwiched with a pair of tweezer-type electrodes, and electroporation was carried out using a square-pulse generator [24]. Magnetic nanoparticles comprising super-paramagnetic iron oxide and bearing short interfering RNA (siRNA) have been reported to transfect successfully siRNA into cultured pancreatic islet cells [59]. The internalization of magnetic nanoparticles into target cells is possible via manipulation of energy-dependent endocytosis [60,61].…”