Trehalose-Based Block Copolycations Promote Polyplex Stabilization for Lyophilization and in Vivo pDNA Delivery

Abstract

The development and thorough characterization of nonviral delivery agents for nucleic acid and genome editing therapies are of high interest to the field of nanomedicine. Indeed, this vehicle class offers the ability to tune chemical architecture/biological activity and readily package nucleic acids of various sizes and morphologies for a variety of applications. Herein, we present the synthesis and characterization of a class of trehalose-based block copolycations designed to stabilize polyplex formulations for lyophilization and in vivo administration. A 6-methacrylamido-6-deoxy trehalose (MAT) monomer was synthesized from trehalose and polymerized via reversible addition–fragmentation chain transfer (RAFT) polymerization to yield pMAT43. The pMAT43 macro-chain transfer agent was then chain-extended with aminoethylmethacrylamide (AEMA) to yield three different pMAT-b-AEMA cationic-block copolymers, pMAT-b-AEMA-1 (21 AEMA repeats), -2 (44 AEMA repeats), and -3 (57 AEMA repeats). These polymers along with a series of controls were used to form polyplexes with plasmids encoding firefly luciferase behind a strong ubiquitous promoter. The trehalose-coated polyplexes were characterized in detail and found to be resistant to colloidal aggregation in culture media containing salt and serum. The trehalose-polyplexes also retained colloidal stability and promoted high gene expression following lyophilization and reconstitution. Cytotoxicity, cellular uptake, and transfection ability were assessed in vitro using both human glioblastoma (U87) and human liver carcinoma (HepG2) cell lines wherein pMAT-b-AEMA-2 was found to have the optimal combination of high gene expression and low toxicity. pMAT-b-AEMA-2 polyplexes were evaluated in mice via slow tail vein infusion. The vehicle displayed minimal toxicity and discouraged nonspecific internalization in the liver, kidney, spleen, and lungs as determined by quantitative polymerase chain reaction (qPCR) and fluorescence imaging experiments. Hydrodynamic infusion of the polyplexes, however, led to very specific localization of the polyplexes to the mouse liver and promoted excellent gene expression in vivo.

Trehalose Glycopolymer Enhances Both Solution Stability and Pharmacokinetics of a Therapeutic Protein

“…22, 28 Others have shown that polymers with trehalose side chains can inhibit amyloid formation and form stable nanoparticles for nucleic acid delivery. 29–31 We hypothesized that trehalose glycopolymers would also stabilize proteins in vivo similar to PEG. In the study described here, for the first time we demonstrate that the trehalose glycopolymer can maintain the plasma protein concentration within the therapeutic window over an extended period of time and also stabilize a protein therapeutic at elevated temperature and under mechanical stress.…”

Reversible Deactivation Radical Polymerization: State-of-the-Art in 2017

“…Consequently, it was proposed that the clam shell conformation, bending of the anomeric position of trehalose disaccharide leading to the two glucose ring coming into close proximity, taken up the trehalose may be more important than the regiochemistry of the vinyl benzyl ether. Interestingly, a trehalose block copolycation consisting of 6-methacrylamido-6-deoxy trehalose-co-N(2-aminoethyl) methacrylamide (pMAT-b-AEMA) was an effective polyplex with short interfering RNA (siRNA) (244) for gene knockdown in cultured glioblastoma (U87), and pT2/CaL plasmid DNA (pDNA) (245) for gene delivery in U87 and human liver carcinoma (HepG2) cell lines. In both cases high stability and minimum toxicity were reported for the polyplexes.…”

Trehalose-Rich, Degradable Hydrogels Designed for Trehalose Release under Physiologically Relevant Conditions

“…Since this, covalent incorporation of trehalose in macromolecules has received continuous interest with more than fifty known publications available (Scopus database). Polymers containing trehalose were obtained using various polymerization and post-polymerization techniques and were studied as e.g., excipients for protein stabilization under deactivating conditions [14][15][16][17], nonviral nucleic acid carriers [18][19][20], thermogelling hydrogel matrices for 3D cancer cell culture [21], and magnetic nanoparticles for selective interactions with mycobacteria [22]. In our recent studies, we presented the utilization of trehalose derivatives in hydrogels synthesis as hydrolytically-labile crosslinkers [23][24][25][26][27].…”