Melittin-grafted HPMA-oligolysine based copolymers for gene delivery

Abstract

Non-viral gene delivery systems capable of transfecting cells in the brain are critical in realizing the potential impact of nucleic acid therapeutics for diseases of the central nervous system. In this study, the membrane-lytic peptide melittin was incorporated into block copolymers synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The first block, designed for melittin conjugation, was composed of N-(2-hydroxypropyl)methacrylamide (HPMA) and pyridyl disulfide methacrylamide (PDSMA) and the second block, designed for DNA binding, was composed of oligo-L-lysine (K10) and HPMA. Melittin modified with cysteine at the C-terminus was conjugated to the polymers through the pyridyl disulfide pendant groups via disulfide exchange. The resulting pHgMelbHK10 copolymers are more membrane-lytic than melittin-free control polymers, and efficiently condensed plasmid DNA into salt-stable particles (~ 100–200 nm). The melittin-modified polymers transfected both HeLa and neuron-like PC-12 cells more efficiently than melittin-free polymers although toxicity associated with the melittin peptide was observed. Optimized formulations containing the luciferase reporter gene were delivered to mouse brain by intraventricular brain injections. Melittin-containing polyplexes produced about 35-fold higher luciferase activity in the brain compared to polyplexes without melittin. Thus, the melittin-containing block copolymers described in this work are promising materials for gene delivery to the brain.

Synergistic effect of pendant hydroxypropyl and pyrrolidine moieties randomly distributed along polymethacrylamide backbones on in vitro DNA-transfection

“…This partial complexation can be attributed to the environmental pH, where most of the amine groups are deprotonated and surrounded by neutral HPMA units, and thus unable to interact with the DNA. On the other hand, the copolymers EPA 30 and EPA 10 were not able to bind DNA in any of the studied conditions (data not shown).…”

“…Their galactosylated form has been evaluated in hepatocyte targeting gene therapy. Pun et al copolymerized HPMA with oligolysine methacrylamides to obtain oligocations randomly grafted on a neutral backbone, which exhibited a good cytotoxicity/transfection-efficiency balance, close to control PEI [9][10][11].…”

Dual Responsive, Stabilized Nanoparticles for Efficient In Vivo Plasmid Delivery

“…We have a long-standing interest in developing materials for gene delivery to the central nervous system (CNS) for potential treatment of neurodegenerative diseases. [11,45,46] Therefore, these three polyplexes containing the luciferase reporter gene were injected into the right lateral ventricle of mice. All the formulations were well tolerated and no significant gross morbidities or mortalities were observed due to polyplex injection.…”

High-content analysis for drug delivery and nanoparticle applications

“…Importantly, the IC50 values for the cell parameters were lower than those obtained in the MTT and rat erythrocyte hemolytic assays, suggesting that these cellular changes ultimately brought about cell death. Although these data suggested that melittin and its analogs cannot be further developed Page 6 of 26 A c c e p t e d M a n u s c r i p t as oral PEs per se, prodrug modifications have recently enabled melittin to be used as part of a successful gene delivery construct when grafted onto N-(2-hydroxypropyl)methacrylamide (HPMA) and pyridyl disulfide methacrylamide (PDSMA) block copolymers, following intracerebral injection to mouse brain [44]. Elsewhere, formulation of melittin in the lipid membrane of a perfluorocarbon NP reduced systemic toxicity and permitted localization in mouse tumour cells [45].…”