Glycopolycation–DNA Polyplex Formulation N/P Ratio Affects Stability, Hemocompatibility, and in Vivo Biodistribution

Abstract

Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose-block-2-methylaminoethyl methacrylate) [P(MAG-b-MAEMt)-1, -2, -3]. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG-b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG-b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG-b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG-b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG-b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG-b-MAEMt) N/P = 5 and P(MAG-b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG-b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG-b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.

Polymeric Delivery of Therapeutic Nucleic Acids

“…115 Along with avoiding hemolysis or altering blood coagulation, nonviral gene delivery vehicles must not activate the complement system to be considered hemocompatible. 116 Complement activation has been observed for liposomes, naked phosphorothioate oligonucleotides, and polyplexes, as well. 112,116,117 While naked polycations such as PLL, poly(amidoamine) (PAMAM) dendrimers, and PEI can all strongly activate the complement system, this activation is greatly diminished by charge neutralization with nucleic acid cargo.…”

“…116 Complement activation has been observed for liposomes, naked phosphorothioate oligonucleotides, and polyplexes, as well. 112,116,117 While naked polycations such as PLL, poly(amidoamine) (PAMAM) dendrimers, and PEI can all strongly activate the complement system, this activation is greatly diminished by charge neutralization with nucleic acid cargo. 117−119 In addition, it was found that complement activation was strongly dependent on polymer chain length, with cationic oligomers showing a weak activation.…”

Assembly and Characterization of Polyelectrolyte Complex Micelles

“…Recent work by our group and others in the field has begun to address this problem by developing structure-property, and in some cases structure-property-function relationships for PCMs formed from nucleic acids and various cationic-neutral polymers 7,[25][26][27] . Two consistent themes that have emerged from these studies are the importance of developing well-controlled, repeatable protocols for PCM assembly and the benefit of using multiple techniques to characterize the resulting nanoparticles.…”

Guanidinopropyl end‐modified poly(ethylene glycol) to form highly compact plasmid DNA mono‐ion complexes by thermal treatment

“…In 2019, naked plasmid DNA (pDNA) encoding human hepatocyte growth factor (HGF) gene was first approved for clinical gene therapy in Japan 1–3 . Compared with other candidates, pDNA can synthesize various copies of emerging nucleic acid agents, such as short interfering RNA, messenger RNA, and non‐coding RNA, as well as proteins 4–9 . However, pDNA needs to overcome many barriers for clinical application of gene therapy.…”

“…[1][2][3] Compared with other candidates, pDNA can synthesize various copies of emerging nucleic acid agents, such as short interfering RNA, messenger RNA, and non-coding RNA, as well as proteins. [4][5][6][7][8][9] However, pDNA needs to overcome many barriers for clinical application of gene therapy. In Asayama's recent review, 10 various molecular design of polymer-based carriers as supramolecular systems for pDNA delivery in vitro and in vivo has been reported, [11][12][13][14] including ours.…”