Dual Peptide Nucleic Acid- and Peptide-Functionalized Shell Cross-Linked Nanoparticles Designed to Target mRNA toward the Diagnosis and Treatment of Acute Lung Injury

Abstract

In this work, multi-functional bio-synthetic hybrid nanostructures were prepared and studied for their potential utility in the recognition and inhibition of mRNA sequences for inducible nitric oxide synthase (iNOS), which are overexpressed at sites of inflammation, such as in cases of acute lung injury. Shell crosslinked knedel-like polymer nanoparticles (SCKs) that present peptide nucleic acids, for binding to complementary mRNAs, and cell penetrating peptides (CPPs), to gain cell entry, along with fluorescent labels and sites for radiolabeling, were prepared by a series of robust, efficient and versatile synthetic steps that proceeded from monomers to polymers to functional nanoparticles. Amphiphilic block graft copolymers having combinations of methoxy- and thioacetyl-terminated poly(ethylene glycol) (PEG) and DOTA-lysine units grafted from the backbone of poly(acrylic acid) (PAA) and extending with a backbone segment of poly(octadecyl acrylate-co-decyl acrylate) (P(ODA-co-DA)) were prepared by a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and chemical modification reactions, which were then used as the building blocks for the formation of well-defined SCKs decorated with reactive thiols accessible to the surface. Fluorescent labeling with Alexa Fluor 633 hydrazide was then accomplished by amidation with residual acrylic acid residues within the SCK shells. Finally, the PNAs and CPP units were covalently conjugated to the SCKs via Michael addition of thiols on the SCKs to maleimide units on the termini of PNAs and CPPs. Confirmation of the ability of the PNAs to bind selectively to the target iNOS mRNAs when tethered to the SCK nanoparticles was determined by in vitro competition experiments. When attached to the SCKs having a hydrodynamic diameter of 60 ± 16 nm, the Kd values of the PNAs were ca. an order of magnitude greater than the free PNAs, while the mismatched PNA showed no significant binding.

Single-step grafting of aminooxy-peptides to hyaluronan: A simple approach to multifunctional therapeutics for experimental autoimmune encephalomyelitis

“…[6-9] Today, peptides are routinely grafted to polymers or to colloids to target drug delivery, mediate endocytosis, promote tissue growth, modify immune response, and myriad other functions. [10-13] Grafted peptide number and density have now been recognized as key determinants of molecular response for many of these systems. [14-17]…”

Degradable Cationic Shell Cross-Linked Knedel-like Nanoparticles: Synthesis, Degradation, Nucleic Acid Binding, and in Vitro Evaluation

“…In the design of nucleic acid delivery vectors, a multitude of efforts has been made to overcome many of the extra- and intracellular obstacles by incorporation of hydrophilic poly(ethylene glycol) moieties to suppress the adsorption of serum proteins, 10–12 membrane destabilizing and pH-sensitive polymers for endosomal escape, 7,13–15 targeting ligands 16–18 or covalent crosslinking for stabilization against counter polyion exchange. 8,19–21 However, only a few studies has been focused on minimizing the overall toxicity of cationic carriers by integration of degradability and evaluating their long term fate.…”

Controlled Radical Polymerization: State-of-the-Art in 2014

“…In the former case, polymers grown from nanoparticles or flat surfaces, dramatically change particles stability, dispersibility and thermomechanical properties, in addition to tremendously enhancing lubricity, antifouling or antibacterial properties (53)(54)(55)(56)(57)(58)(59)(60)(61). Hybrids with proteins or nucleic acids increase stability of proteins in harsh environments, (including temperature, pH, and salts) assist in delivery of genes, and can be used in advanced drug delivery (45,(62)(63)(64)(65)(66)(67)(68)(69)(70)(71).…”