The triblock copolymer polyethylenimine-polycaprolactone-polyethylene glycol (PEI-PCL-PEG) has been shown to spontaneously assemble into nano-sized particulate carriers capable of complexing with nucleic acids for gene delivery. The objective of this study was to investigate micelleplex characteristics, their in vitro and in vivo fate following microfluidic preparation of siRNA nanoparticles compared to the routinely used batch reactor mixing technique. Herein, PEI-PCL-PEG nanoparticles were prepared with batch reactor or microfluidic mixing techniques and characterized by various biochemical assays and in cell culture. Microfluidic nanoparticles showed a reduction of overall particle size as well as a more uniform size distribution when compared to batch reactor pipette mixing. Confocal microscopy, flow cytometry and qRT-PCR displayed the subcellular delivery of the microfluidic formulation and confirmed the ability to achieve mRNA knockdown. Intratracheal instillation of microfluidic formulation resulted in a significantly more efficient ( p < 0.05) knockdown of GAPDH compared to treatment with the batch reactor formulation. The use of microfluidic mixing techniques yields an overall smaller and more uniform PEG-PCL-PEI nanoparticle that is able to more efficiently deliver siRNA in vivo . This preparation method may prove to be useful when a scaled up production of well-defined polyplexes is required.

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.

The use of cationic polymers to interact with negatively charged siRNA via charge complexation to form polyelectrolyte complexes has been widely studied ever since the 1998 report on RNA interference. These polyelectrolyte complex formulations aim to overcome the many pitfalls associated with the use of RNA interference as a potential cancer therapy. The triblock copolymer polyethylenimine-polycaprolactone-polyethylene glycol (PEI-PCL-PEG) contains the cation PEI and has been shown to be an efficient carrier capable of complexing with nucleic acids for gene delivery. This copolymer system also allows for targeting moieties to be linked to the micelleplex, thereby exploiting overexpressed receptors (such as the folate receptor) located within tumors. Additionally, we demonstrated recently that microfluidic mixing of PEI-PCL-PEG nanoparticles allows for the rapid, scaled-up production of micelleplexes while maintaining small and uniform particle distributions. The preparation of small and reproducible particles is imperative for clinical translation of nanomedicine and for tumor targeting via systemic administration. Furthermore, to enable tracing of its deposition in vivo after its administration, micelleplexes can be radiolabeled. To assess tumor targeting over time, the noninvasive imaging technique single-photon emission computed tomography (SPECT) offers the ability to examine the same subject at multiple time points and generate biodistribution profiles. Since the biodistribution and tumor targeting of the therapeutic load of micelleplexes is of foremost interest, we recently described an approach to modify siRNA with a DTPA (diethylenetriaminepentaacetic acid) chelator. Herein, we explain the details of encapsulating indium-labeled siRNA via microfluidic mixing in PEI-PCL-PEG nanoparticles with a folic acid targeting ligand for assessment of their in vivo tumor targeting in an orthotopic ovarian cancer model.