No abstract

To develop stable and inhalable dry powder formulations with long shelf life, polyplexes consisting of siRNA and a polyethylenimine (PEI)‐based block copolymer in presence of mannitol or trehalose are spray dried. The effect of inlet (T‐In) and outlet (T‐Out) temperature on the recovery of siRNA and adsorption effects within the tubing material are investigated. Choosing a low abrasion silicon tubing prevented siRNA loss due to adsorption. Mannitol and trehalose formulations preserved siRNA integrity regardless of excipient concentration and temperature at T‐Out below the siRNA melting temperature. Trehalose formulations allowed full siRNA recovery whereas mannitol formulations resulted in spray drying induced losses of ≈20% siRNA and of 50–60% polymer. Mannitol formulations showed optimal aerodynamic characteristics as confirmed by next generation impaction analysis based upon siRNA content. All spray dried formulations resulted in green fluorescent protein (GFP) silencing comparable or better than freshly prepared polyplexes. To test if the observed results could be transferred, formulations of siRNA and transferrin‐PEI conjugates are spray dried, characterized, and used to transfect primary human T cells ex vivo. Results confirmed successful silencing of the transcription factor GATA3 in primary CD4+ T cells with spray dried formulations as a potential treatment for severe asthma.

Polyethylenimine (PEI) is a commonly used cationic polymer for small‐interfering RNA (siRNA) delivery due to its high transfection efficiency at low commercial cost. However, high molecular weight PEI is cytotoxic and thus, its practical application is limited. In this study, different formulations of low molecular weight PEI (LMW‐PEI) based copolymers polyethylenimine‐g‐polycaprolactone (PEI–PCL) (800 Da–40 kDa) and PEI–PCL–PEI (5–5–5 kDa) blended with or without polyethylene glycol‐b‐polycaprolactone (PEG–PCL) (5 kDa‐4 kDa) are investigated to prepare nanoparticles via nanoprecipitation using a solvent displacement method with sizes ≈100 nm. PEG–PCL can stabilize the nanoparticles, improve their biocompatibility, and extend their circulation time in vivo. The nanoparticles composed of PEI–PCL–PEI and PEG–PCL show higher siRNA encapsulation efficiency than PEI–PCL/PEG–PCL based nanoparticles at low N/P ratios, higher cellular uptake, and a gene silencing efficiency of ≈40% as a result of the higher molecular weight PEI blocks. These results suggest that the PEI–PCL–PEI/PEG–PCL nanoparticle system could be a promising vehicle for siRNA delivery at minimal synthetic effort.

Platinum-based chemotherapy remains a mainstay treatment for the management of advanced non-small cell lung cancer. A key cellular factor that contributes to sensitivity to platinums is the 5′-3′ structure-specific endonuclease excision repair cross-complementation group 1 (ERCC1)/ xeroderma pigmentosum group F (XPF). ERCC1/XPF is critical for the repair of platinum-induced DNA damage and has been the subject of intense research efforts to identify small molecule inhibitors of its nuclease activity for the purpose of enhancing patient response to platinum-based chemotherapy. As an alternative to small molecule inhibitors, small interfering RNA (siRNA) has often been described to be more efficient in interrupting protein–protein interactions. The goal of this study was therefore to determine whether biocompatible nanoparticles consisting of an amphiphilic triblock copolymer (polyethylenimine-polycaprolactone-polyethylene glycol (PEI-PCL-PEG)) and carrying siRNA targeted to ERCC1 and XPF made by microfluidic assembly are capable of efficient gene silencing and able to sensitize lung cancer cells to cisplatin. First, we show that our PEI-PCL-PEG micelleplexes carrying ERCC1 and XPF siRNA efficiently knocked down ERCC1/XPF protein expression to the same extent as the standard siRNA transfection reagent, Lipofectamine. Second, we show that our siRNA-carrying nanoparticles enhanced platinum sensitivity in a p53 wildtype model of non-small cell lung cancer in vitro. Our results suggest that nanoparticle-mediated targeting of ERCC1/XPF is feasible and could represent a novel therapeutic strategy for targeting ERCC1/XPF in vivo.