Screening Nylon-3 Polymers, a New Class of Cationic Amphiphiles, for siRNA Delivery

Abstract

Amphiphilic nucleic acid carriers have attracted strong interest. Three groups of nylon-3 copolymers (poly-β-peptides) possessing different cationic/hydrophobic content were evaluated as siRNA delivery agents in this study. Their ability to condense siRNA was determined in SYBR Gold assays. Their cytotoxicity was tested by MTT assays, their efficiency of delivering Alexa Fluor-488-labeled siRNA intracellularly in the presence and absence of uptake inhibitors was assessed by flow cytometry, and their transfection efficacies were studied by luciferase knockdown in a cell line stably expressing luciferase (H1299/Luc). Endosomal release was determined by confocal laser scanning microscopy and colocalization with lysotracker. All polymers efficiently condensed siRNA at nitrogen-to-phosphate (N/P) ratios of 5 or lower, as reflected in hydrodynamic diameters smaller than that at N/P 1. Although several formulations had negative zeta potentials at N/P 1, G2C and G2D polyplexes yielded >80% uptake in H1299/Luc cells, as determined by flow cytometry. Luciferase knockdown (20–65%) was observed after transfection with polyplexes made of the high molecular weight polymers that were the most hydrophobic. The ability of nylon-3 polymers to deliver siRNA intracellularly even at negative zeta potential implies that they mediate transport across cell membranes based on their amphiphilicity. The cellular uptake route was determined to strongly depend on the presence of cholesterol in the cell membrane. These polymers are, therefore, very promising for siRNA delivery at reduced surface charge and toxicity. Our study identified nylon-3 formulations at low N/P ratios for effective gene knockdown, indicating that nylon-3 polymers are a new, promising type of gene delivery agent.

The Impact of Nylon-3 Copolymer Composition on the Efficiency of siRNA Delivery to Glioblastoma Cells

“…Consequently, the siRNA encapsulation ability of polymers represents an important property in evaluating their suitability as siRNA carriers. Although the exact mechanism for complex formation between nylon-3 polymers and siRNA is still unknown, a combination of electrostatic and hydrophobic interactions due to the structural properties of polymers is implicated [24]. As previously described, polycationic structures cause dose-dependent toxicity; therefore, optimal polymer concentrations for efficient siRNA condensation and protection need to be evaluated [15].…”

“…Nylon-3 random copolymers were synthesized via the anionic ring-opening polymerization (ROP) of racemic β-lactams. Polymer DM 0.4 /CP 0.6 (DM as cationic monomer and CP as hydrophobic monomer) was synthesized as described previously [24,25]. The polymerization was conducted in the presence of ( ± )-7-(2-tritylthioacetyl)-7-azabicyclo[4,2,0]octan-8-one (I) as the co-initiator and lithium bis(trimethylsilylamide) (LiHMDS) as the base to afford the desired polymer with one N-terminal thiol-end group per polymer chain (Figure S1, Supplementary Materials) [23].…”

“…In addition, nylon-3 polymers, which have a similar backbone to biocompatible and biodegradable peptides, were tested for various medical/biological applications by Gellman and Liu et al Due to their high altering amenability, they were successfully used for mimicking antimicrobial host-defense peptides [20], lung surfactant [21], natural polysaccharides [22], and as cell adhesion promoters [23], which also laid promising foundations for application as a potential gene delivery agent. The first siRNA delivery performance study of these polymers was investigated in non-small cell lung cancer cells (H1299), indicating that hydrophobic structures that are made from a considerable amount of hydrophobic subunits might be superior siRNA delivery agents [24]. Nylon-3 polymers can be synthesized via anionic ring-opening polymerization (ROP) via the statistical copolymerization of various β-lactams using lithium bis(trimethylsilylamide) (LiHMDS) as the initiator to incorporate both cationic and lipophilic/hydrophobic subunits, providing the opportunity to obtain versatile and tailor-made polymer compositions by regulation of the monomer feed [25].…”

The impact of microfluidic mixing of triblock micelleplexes on in vitro $/$ in vivo gene silencing and intracellular trafficking

“…To study the route of micelleplex uptake, a modified in vitro cellular uptake was performed [4]. In short, 50 000 A549 cells were incubated with different types of uptake inhibitors such as chlorpromazine (10 μ g ml −1 ), nystatin (10 μ g ml −1 ), wortmannin (12 ng ml −1 ), and methyl- β -cyclodextrin (3 mg ml −1 ) for 1 h followed by incubation with PEI-g-PCL-b-PEG polyplexes containing AF488 siRNA for 4 h. Positive control cells were transfected with AF488 micelleplexes or LF lipoplexes for 4 h and untreated cells served as a blank control.…”

“…However, the transition of these potential therapies to clinical trials remains slow due to multiple barriers involved in the intracellular deposition of siRNA [3]. In order to overcome the hurdles associated with therapeutic siRNA, nanoparticles have long been the preferred method of condensation and protection with a large variety of formulations taking advantage of different delivery methods or carrier identity to achieve gene knockdown [4]. …”