In vivo assessment of caries excavation with a fluorescence camera compared to direct bacteriological sampling and quantitative analysis using flow cytometry.

The aim of this study was to assess the performance of the fluorescence camera device VistaCam iX (VC) for the control of caries excavation compared to visual assessment and bacteriological evaluation. Twenty-three patients with 32 dentinal carious lesions were included in the study. The lesions were classified using the International Caries Detection and Assessment System. Fluorescence images were taken at the surface of each lesion using the VC before excavation (stage 1), during (stage 2) and after excavation (stage 3). At each stage, dentine samples were collected. All cavities were closed after excavation using permanent fillings. Bacteria were counted within each dentine sample using flow cytometry. Significant moderate correlation between the fluorescence readings and the bacteria count was found at stage 3 (Spearman correlation coefficient rs = 0.40, p = 0.027). Area under the ROC curve (AUC) was calculated for fluorescence readings and bacteria count using the clinical status at stage 3 as a reference. AUC was 0.954 for the VC readings and 0.797 for bacteria count. Using the median bacteria count at stage 3 as a reference, the AUC for the fluorescence readings was 0.753. The results indicate that the fluorescence camera appears to be a useful supplementary tool in assessing the endpoint of caries excavation with the advantage of enabling data to be digitally visualized and stored. When an experienced dental clinician determined the excavation endpoint, the performance with respect to remaining bacteria count was good.

Polyethylenimine PEI F25-LMW allows the long-term storage of frozen complexes as fully active reagents in siRNA-mediated gene targeting and DNA delivery.

BACKGROUND: Polyethylenimines (PEIs) are synthetic, charged polymers which function as transfection reagents based on their ability to compact DNA into complexes. Recently, PEI-mediated delivery of nucleic acids has been extended towards small interfering RNAs (siRNAs) which are instrumental in the induction of RNA interference (RNAi). Since RNAi represents a powerful method for specific gene silencing, the PEI-based delivery of siRNAs is a promising tool for novel putative therapeutic strategies. AIM: For therapeutic use, major requirements are the development of formulations which (i) are sufficiently stable in the presence of serum, and which can be (ii) easily and reproducibly manufactured and (iii) stored for a prolonged time with full retention of their integrity and bioactivity. In this paper, we explore the potential of PEI F25-LMW, a low-molecular weight PEI with superior transfection efficacy and low toxicity, towards these goals. RESULTS: We have systematically analyzed and determined optimal DNA and siRNA complexation conditions with regard to various parameters including buffer concentration, ionic strength, pH and incubation time. As opposed to 22kDa linear PEI (L-PEI), the low-molecular weight (4-10kDa) PEI F25-LMW performs DNA transfection and siRNA gene targeting with identical efficacies in the presence of serum, thus emphasizing its usefulness in vivo. Furthermore, in contrast to other polyethylenimines, PEI F25-LMW-based DNA or siRNA complexes allow freeze/thawing and frozen storage for several months. Their activity is fully retained without requiring specific buffer conditions or the addition of any lyoprotectant. Physicochemical analysis and atomic force microscopy reveal a distinct size pattern with the presence of two complex subgroups and show that frozen PEI F25-LMW complexes remain stable with little increase in complex size, no changes regarding their zeta potential and cytotoxicity, and full retention of nucleic acid protection. CONCLUSIONS: Frozen PEI F25-LMW-based complexes represent efficient and stable ready-to-use formulations of DNA- or siRNA-based gene therapy products.

RNA interference-mediated gene silencing of pleiotrophin through polyethylenimine-complexed small interfering RNAs in vivo exerts antitumoral effects in glioblastoma xenografts.

RNA interference (RNAi) is a powerful strategy to inhibit gene expression through specific mRNA degradation mediated by small interfering RNAs (siRNAs). In vivo, however, the application of siRNAs is severely limited by their instability and poor delivery into target cells and target tissues. Glioblastomas are the most frequent and malignant brain tumors with, so far, limited treatment options. To develop novel and more efficacious therapies, advanced targeting strategies against glioblastoma multiforme (GBM)-relevant target genes must be established in vivo. Here we use RNAi-based targeting of the secreted growth factor pleiotrophin (PTN), employing a polyethylenimine (PEI)/siRNA complex strategy. We show that the complexation of chemically unmodified siRNAs with PEI leads to the formation of complexes that condense and completely cover siRNAs as determined by atomic force microscopy (AFM). On the efficient cellular delivery of these PEI/siRNA complexes, the PTN downregulation in U87 glioblastoma cells in vitro results in decreased proliferation and soft agar colony formation. More importantly, in vivo treatment of nude mice through systemic application (subcutaneous or intraperitoneal) of PEI-complexed PTN siRNAs leads to the delivery of intact siRNAs into subcutaneous tumor xenografts and a significant inhibition of tumor growth without a measurable induction of siRNA-mediated immunostimulation. Likewise, in a clinically more relevant orthotopic mouse glioblastoma model with U87 cells growing intracranially, the injection of PEI-complexed PTN siRNAs into the CNS exerts antitumoral effects. In conclusion, we present the PEI complexation of siRNAs as a universally applicable platform for RNAi in vitro and in vivo and establish, also in a complex and relevant orthotopic tumor model, the potential of PEI/siRNA-mediated PTN gene targeting as a novel therapeutic option in GBM.

A low molecular weight fraction of polyethylenimine (PEI) displays increased transfection efficiency of DNA and siRNA in fresh or lyophilized complexes.

RNA interference (RNAi) represents a powerful method for specific gene silencing. It is mediated through small double-stranded RNA molecules (small interfering RNAs, siRNAs) which sequence-specifically trigger the cleavage and subsequent degradation of their target mRNA. One critical factor that determines the success of RNAi is the ability to deliver intact siRNAs into target cells. Polyethylenimines (PEIs) are synthetic polymers with a high cationic charge density which function as transfection reagents based on their ability to compact DNA or RNA into complexes. This paper describes the application of lyophilized PEI/siRNA complexes based on a novel polyethylenimine. By fractionation of a commercially available 25-kDa PEI using gel permeation chromatography, a low molecular weight polyethylenimine (PEI F25-LMW) with superior transfection efficacy and low toxicity in various cell lines is obtained. Complexes formed in 5% glucose, but not in 150 mM NaCl, can be lyophilized and reconstituted without loss of transfection efficacy. Furthermore, PEI F25-LMW is able to complex and fully protect siRNAs against nucleolytic degradation, and delivers siRNAs into cells where they display bioactivity. Upon lyophilization and reconstitution of PEI F25-LMW-based siRNA complexes, siRNAs are still able to efficiently induce RNAi. To further demonstrate their applicability, lyophilized PEI/siRNA complexes are employed for targeting of the growth factor VEGF. Treatment of PC-3 prostate carcinoma cells with fresh or with lyophilized complexes results in decreased cell proliferation in different assays due to the siRNA-mediated downregulation of VEGF. In conclusion, siRNAs can be applied in lyophilized formulations, and lyophilized PEI F25-LMW-based siRNA complexes represent a powerful, inexpensive, non-toxic and simple ready-to-use platform for the specific and efficient targeting of genes in vitro.