Abstract
Glioblastoma multiforme is a devastating disease that has attracted enormous attention due to poor prognosis and high recurrence. Small interfering RNA (siRNA) in principle offers a promising therapeutic approach by the downregulation of disease-related genes via RNA interference. For efficient siRNA delivery to target sites, cationic polymers are often used in preclinical studies for the protection of siRNA and complex formation based on electrostatic interactions. In an effort to develop biocompatible and efficient nanocarriers with a translational outlook for optimal gene silencing at reduced toxicity, we synthesized two sets of nylon-3 copolymers with variable cationic content (DM or NM monomer) and hydrophobic subunits (CP monomer) and evaluated their suitability for in vitro siRNA delivery into glioblastoma cells. DM0.4/CP0.6 and NM0.4/CP0.6 polymers with similar subunit ratios were synthesized to compare the effect of different cationic subunits. Additionally, we utilized NM0.2/CP0.8 polymers to evaluate the impact of the different hydrophobic content in the polymer chain. The siRNA condensation ability and polymer–siRNA complex stability was evaluated by unmodified and modified SYBR gold assays, respectively. Further physicochemical characteristics, e.g., particle size and surface charge, were evaluated by dynamic light scattering and laser Doppler anemometry, whereas a relatively new method for polyplex size distribution analysis—tunable resistive pulse sensing—was additionally developed and compared to DLS measurements. Transfection efficiencies, the route of cell internalization, and protein knockdown abilities in glioblastoma cells were investigated by flow cytometry. Furthermore, cellular tolerability was evaluated by MTT and LDH assays. All the polymers efficiently condensed siRNA at N/P ratios of three, whereas polymers with NM cationic subunits demonstrated smaller particle size and lower polyplex stability. Furthermore, NM0.2/CP0.8 polyplexes with the highest hydrophobic content displayed significantly higher cellular internalization in comparison to more cationic formulations and successful knockdown capabilities. Detailed investigations of the cellular uptake route demonstrated that these polyplexes mainly follow clathrin-mediated endocytotic uptake mechanisms, implying high interaction capacity with cellular membranes. Taken together with conducive toxicity profiles, highly hydrophobic nylon-3 polymers provide an appropriate siRNA delivery agent for the potential treatment of glioblastoma.
Highlights
Glioblastoma multiforme (GBM) is the most common devastating type of primary malignant tumor of the central nervous system
DM0.4/CP0.6 and NM0.4/CP0.6 polymers with similar subunit ratios were synthesized to compare the effect of different cationic subunits
Successful Small interfering RNA (siRNA) delivery and the silencing of glioma-related genes, e.g., B-cell lymphoma/leukemia-2 (Bcl-2), was enabled by dendrimer—or polyethylenimine (PEI)—entrapped gold nanoparticles by the groups of Qiu and Shi, respectively [11,12]. In these treatments and in most other cases of siRNA delivery, limitations in the application of naked siRNA caused by rapid degradation, immune response, and low passive cell uptake [13] are bypassed by using suitable delivery systems to encapsulate the nucleic acids by electrostatic interactions in order to shield them from the environment and assist cellular internalization
Summary
Glioblastoma multiforme (GBM) is the most common devastating type of primary malignant tumor of the central nervous system. Successful siRNA delivery and the silencing of glioma-related genes, e.g., B-cell lymphoma/leukemia-2 (Bcl-2), was enabled by dendrimer—or polyethylenimine (PEI)—entrapped gold nanoparticles by the groups of Qiu and Shi, respectively [11,12] In these treatments and in most other cases of siRNA delivery, limitations in the application of naked siRNA caused by rapid degradation, immune response, and low passive cell uptake [13] are bypassed by using suitable delivery systems to encapsulate the nucleic acids by electrostatic interactions in order to shield them from the environment and assist cellular internalization. Cellular internalization, the route of uptake, and gene knockdown efficiency were assessed by flow cytometry, and cell tolerability was examined by MTT and LDH assays Based on these findings, the most hydrophobic nylon-3 polymers. Nanomaterials 2019, 9, 986 provide optimal properties regarding particle characteristics and the ability to fuse with cell membranes, leading to excellent transfection efficiencies and successful gene knockdown in glioblastoma cells with minimal cytotoxic effects
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