RESUMO
RNA interference (RNAi) is a powerful therapeutic approach for messenger RNA (mRNA) level regulation in human cells. RNAi can be triggered by small interfering RNAs (siRNAs) which are delivered by non-viral carriers, e.g., dendriplexes. siRNA quantification inside carriers is essential in drug delivery system development. However, current siRNA measuring methods either are not very sensitive, only semi-quantitative or not specific towards intact target siRNA sequences. We present a novel reverse transcription real-time PCR (RT-qPCR)-based application for siRNA quantification in drug formulations. It enables specific and highly sensitive quantification of released, uncomplexed target siRNA and thus also indirect assessment of siRNA stability and concentration inside dendriplexes. We show that comparison with a dilution series allows for siRNA quantification, exclusively measuring intact target sequences. The limit of detection (LOD) was 4.2 pM (±0.2 pM) and the limit of quantification (LOQ) 77.8 pM (±13.4 pM) for uncomplexed siRNA. LOD and LOQ of dendriplex samples were 31.6 pM (±0 pM) and 44.4 pM (±9.0 pM), respectively. Unspecific non-target siRNA sequences did not decrease quantification accuracy when present in samples. As an example of use, we assessed siRNA complexation inside dendriplexes with varying nitrogen-to-phosphate ratios. Further, protection of siRNA inside dendriplexes from RNase A degradation was quantitatively compared to degradation of uncomplexed siRNA. This novel application for quantification of siRNA in drug delivery systems is an important tool for the development of new siRNA-based drugs and quality checks including drug stability measurements.
RESUMO
Prophylaxis and the treatment of surgical site infections (SSIs) with antibiotics frequently fail due to the antibiotic resistance of bacteria and the ability of bacteria to reside in biofilms (i.e., bacterial clusters in a protective matrix). Therefore, alternative antibacterial treatments are required to combat biofilm infections. The combination of diethyldithiocarbamate (DDC-) and copper ions (Cu2+) exhibited antibiofilm activity against the staphylococci species associated with SSIs; however, the formation of a water-insoluble Cu(DDC)2 complex limits its application to SSIs. Here, we describe the development and antibiofilm activity of an injectable gel containing a liposomal formulation of Cu(DDC)2 and Cu2+ (lipogel). Lyophilized liposomes were incorporated into a mixture of chitosan (CS) and beta-glycerophosphate (ßGP), and the thermosensitive gelling properties of CS-ßGP and the lipogel were determined. The liposomes remained stable after lyophilization over six months at 4-6 °C and -20 °C. The sol-gel transition of the gel and lipogel occurred between 33 and 39 °C, independently of sterilization or storage at -20 °C. CS-ßGP is biocompatible and the liposomes were released over time. The lipogel prevented biofilm formation over 2 days and killed 98.7% of the methicillin-resistant Staphylococcus aureus and 99.9% of the Staphylococcus epidermidis biofilms. Therefore, the lipogel is a promising new prophylaxis and treatment strategy for local application to SSIs.