A matrix reservoir for improved control of non-viral gene delivery. J Control Release
Network of Excellence in Functional Biomaterials, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland. Journal of Controlled Release
(Impact Factor: 7.71).
03/2009; 136(3):220-5. DOI: 10.1016/j.jconrel.2009.02.006
Non-viral gene delivery suffers from a number of limitations including short transgene expression times and low transfection efficiency. Collagen scaffolds have previously been investigated as in vitro DNA reservoirs, which allow sustained release of genetic information. Efficient viral gene-transfer from these scaffolds has previously been demonstrated. However, due to concerns about the safety of viral gene therapy, the use of non-viral vectors may be preferable. In this study a DNA-dendrimer complex embedded in a cross-linked collagen scaffold was investigated as a reservoir for non-viral delivery. Elution from the scaffolds and transfection of seeded rat mesenchymal stem cells were used to evaluate the scaffold's ability to act as a reservoir for the complexes. Elution from the scaffolds was minimal after 2 days with a total of 25% of the complexes released after 7 days. Extended transgene expression after DNA-dendrimer complex delivery from the scaffolds in comparison to direct delivery to cells was observed. The elongated transfection period and relatively high levels of reporter gene expression are significant advantages over other non-viral gene therapy techniques. This platform has the potential to be an effective method of scaffold-mediated gene delivery suitable for in vitro and in vivo applications.
Available from: Benjamin Chu
- "Other studies have also explored the use of nonviral delivery of siRNA, microRNAs, and shRNAs through polycation, liposomal, and biochemical chaperoning . Specifically, these studies used a variety of platforms to deliver the siRNA or shRNA and ranged from hydrogels – films , , nanoparticles –, sponges –, and even creams . Collectively, these studies targeted various genes that play a role in a variety of biological processes. "
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ABSTRACT: RNA interference (RNAi) is a promising approach for cancer treatment. Site specific and controlled delivery of RNAi could be beneficial to the patient, while at the same time reducing undesirable off-target side effects. We utilized electrospinning to generate a biodegradable scaffold capable of incorporating and delivering a bioactive plasmid encoding for short hairpin (sh) RNA against the cell cycle specific protein, Cdk2. Three electrospun scaffolds were constructed, one using polycaprolactone (PCL) alone (Control) and PCL with plasmid DNA encoding for either Cdk2 (Cdk2i) and EGFP (EGFPi, also served as a control) shRNA. Scaffold fiber diameters ranged from 1 to 20 µm (DNA containing) and 0.2-3 µm (Control). While the electrospun fibers remained intact for more than two weeks in physiological buffer, degradation was visible during the third week of incubation. Approximately 20-60 ng/ml (∼2.5% cumulative release) of intact and bioactive plasmid DNA was released over 21 days. Further, Cdk2 mRNA expression in cells plated on the Cdk2i scaffold was decreased by ∼51% and 30%, in comparison with that of cells plated on Control or EGFPi scaffold, respectively. This decrease in Cdk2 mRNA by the Cdk2i scaffold translated to a ∼40% decrease in the proliferation of the breast cancer cell line, MCF-7, as well as the presence of increased number of dead cells. Taken together, these results represent the first successful demonstration of the delivery of bioactive RNAi-based plasmid DNA from an electrospun polymer scaffold, specifically, in disrupting cell cycle regulation and suppressing proliferation of cancer cells.
Available from: Pim-On Rujitanaroj
- "Such prolonged and enhanced transfection efficiency by scaffolds is likely due to the sustained release and localized concentration of siRNA to cells. Similar findings were also observed previously  . The coencapsulation of siCOL1A1 and CADY within PCLEEP fibers resulted in similar COL1A1 knockdown as compared to siCOL1A1/TKO complexes. "
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ABSTRACT: The foreign body reaction often interferes with the long-term functionality and performance of implanted biomedical devices through fibrous capsule formation. While many implant modification techniques have been adopted in attempts to control fibrous encapsulation, the outcomes remained sub-optimal. Nanofiber scaffold-mediated RNA interference may serve as an alternative approach through the localized and sustained delivery of siRNA at implant sites. In this study, we investigated the efficacy of siRNA-poly(caprolactone-co-ethylethylene phosphate) nanofibers in controlling fibrous capsule formation through the down-regulation of collagen type I (COL1A1) in vitro and in vivo. By encapsulating complexes of COL1A1 siRNA with a transfection reagent (Transit TKO) or the cell penetrating peptides CADY or MPG within the nanofibers (550-650nm in diameter), a sustained release of siRNA was obtained for at least 28days (loading efficiency ∼60-67%). Scaffold-mediated transfection significantly enhanced cellular uptake of oligonucleotides and prolonged in vitro gene silencing duration by at least 2-3 times as compared to conventional bolus delivery of siRNA (14days vs. 5-7days by bolus delivery). In vivo subcutaneous implantation of siRNA scaffolds revealed a significant decrease in fibrous capsule thickness at weeks 2 and 4 as compared to plain nanofibers (p<0.05). Taken together, the results demonstrated the efficacy of scaffold-mediated siRNA gene-silencing in providing effective long-term control of fibrous capsule formation.
Available from: Carolyn Holladay
- "A 0.3w/v% type I atelocollagen solution was freeze-dried and crosslinked with 1- ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysuccinimide (EDC/ NHS) to make the collagen scaffolds, as described elsewhere . Interleukin-10 plasmid-dendrimer polyplexes (pIL-10) were prepared by incubating IL-10 plasmids with SuperFectÔ. 2 mg of plasmid complexed to 30 mg of dendrimer was added to each scaffold and the IL-10 polyplexes (pIL-10) were allowed to adsorb for 3 h. "
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ABSTRACT: Stem cell transplantation has been suggested as a treatment for myocardial infarction, but clinical studies have yet to demonstrate conclusive, positive effects. This may be related to poor survival of the transplanted stem cells due to the inflammatory response following myocardial infarction. To address this, a scaffold-based stem cell delivery system was functionalised with anti-inflammatory plasmids (interleukin-10) to improve stem cell retention and recovery of cardiac function. Myocardial infarction was induced and these functionalised scaffolds were applied over the infarcted myocardium. Four weeks later, stem cell retention, cardiac function, remodelling and inflammation were quantified. Interleukin-10 gene transfer improved stem cell retention by more than five-fold and the hearts treated with scaffold, stem cells and interleukin-10 had significant functional recovery compared to the scaffold control (scaffold: -10 ± 7%, scaffold, interleukin-10 and stem cells: +7 ± 6%). This improved function was associated with increased infarcted wall thickness and increased ratios of collagen type III/type I, decreased cell death, and a change in macrophage markers from mainly cytotoxic in the scaffold group to mainly regulatory in scaffold, stem cells and interleukin-10 group. Thus, treatment of myocardial infarction with stem cells and interleukin-10 gene transfer significantly improved stem cell retention and ultimately improved overall cardiac function.
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