Polymeric Nanocarriers for siRNA Delivery to Murine Macrophages

Abstract

This work investigates the interactions of a polycationic nanocarrier with siRNA and with cells in order to better understand the capabilities and limitations of the carrier. The polycationic nanocarriers are cross-linked copolymer nanoparticles synthesized in a single-step reaction using ARGET ATRP (activators regenerated by electron transfer atom transfer radical polymerization). The polycationic nanocarriers efficiently bind siRNA for polymer/siRNA mass ratios less than 1. A method to prepare fluorescently labeled polycationic nanocarriers is presented. The fluorescently labeled polycationic nanocarriers are used to investigate cellular internalization with RAW264.7 murine macrophage cells. Flow cytometry demonstrates that the uptake increased with nanoparticle concentration and incubation time. Confocal microscopy confirmed internalization of fluorescently labeled nanoparticles. The investigation of siRNA-induced knockdown demonstrates that higher concentrations of nanoparticles and siRNA are associated with increased knockdown. For the conditions tested in the knockdown experiments, the ARGET ATRP polycationic nanocarriers outperformed a commercially available Lipofectamine control.

Stimulus-responsive hydrogels: Theory, modern advances, and applications

“…pH-responsive hydrogel systems have been widely used for the controlled drug delivery of a variety of therapeutics ranging from proteins [63], to small molecule drugs [64], chemotherapeutics [65], and genetic material such as RNA and DNA [66]. These therapeutic molecules can be dissolved or encapsulated within the hydrogel network [54], such as in protein loaded hydrogels [67], or electrostatically bound to the charged hydrogel network, as in the case of genetic material [68].…”

“…Forbes et al were able to exhibit tighter control of final cationic nanogel parameters by switching from a UV-initiated polymerization to ARGET-ATRP initiated polymerization [109,110]. Furthermore, these cationic nanogels were able to be successfully complexed with siRNA, and promote cellular uptake in RAW264.7 macrophages and HEK293T cells [66,110]. It is argued that the pH-triggered swelling of the nanogel in response to the decrease in pH within the endosome results in the so-called proton-sponge effect, which ends in the bursting of the vesicle due to excessive water imbibition due to an osmotic pressure imbalance [111].…”

“…Once the vesicle bursts, the siRNA is no longer complexed with the now deionized cationic nanoparticle and is distribution into the cellular cytosol where it can exert therapeutic effect. The particles developed by Forbes et al do indeed accomplish siRNA-mediated gene silencing, which indicates that the siRNA and associated nanogel undergo some extent of endosomal escape [66,110]. These results have powerful implications for the treatment of a variety of disorders that are caused by gene overexpression including several autoimmune disorders and types of cancer.…”

Polymer nanocarriers for MicroRNA delivery

“…With respect to size, nanoscale materials are ideally suited for RNA delivery because they can protect RNA from degradation, extend circulation half‐life, facilitate cellular entry, and increase therapeutic index . Indeed, various nanoparticle (NP) delivery systems have shown considerable promise for the delivery of plasmid DNAs, antisense oligonucleotides, small interfering RNAs (siRNAs), and miRNAs to diseased cells in vitro and in vivo . When considering size as a design parameter for miRNA mimic delivery vehicles, it is important to note that NPs with diameters less than ∼5 nm rapidly undergo renal clearance upon intravenous administration and those with diameters greater than ~200 nm exhibit splenic filtration due to the 200–500 nm size range of interendothelial cell slits .…”

Enzymatic Biodegradation of Hydrogels for Protein Delivery Targeted to the Small Intestine

“…As the next generation of biomaterials, these "intelligent" networks are able to respond to or mimic biological environments and processes such as vascularization 5,6 , tumor physiology 7, 8 , endosomal compartments [9][10][11] , or the extracellular matrix 12,13 . This capability could be instrumental in achieving various biomedical advances, including tissue regeneration and controlled delivery of biological therapeutics 14,15 .…”