Novel endosomolytic peptides for enhancing gene delivery in nanoparticles

Abstract

Trapping in the endosomes is currently believed to represent the main barrier for transfection. Peptides, which allow endosomal escape have been demonstrated to overcome this barrier, similarly to the entry of viruses. However, the design principles of such endosomolytic peptides remain unclear. We characterized three analogs derived from membrane disrupting antimicrobial peptides (AMP), viz. LL-37, melittin, and bombolitin V, with glutamic acid substituting for all basic residues. These analogs are pH-sensitive and cause negligible membrane permeabilization and insignificant cytotoxicity at pH7.4. However, at pH5.0, prevailing in endosomes, membrane binding and hemolysis of human erythrocytes become evident. We first condensed the emerald green fluorescent protein (emGFP) containing plasmid by protamine, yielding 115 nm diameter soluble nanoplexes. For coating of the nanoplex surface with a lipid bilayer we introduced a hydrophobic tether, stearyl-octa-arginine (SR8). The indicated peptides were dissolved in methanol and combined with lipid mixtures in chloroform, followed by drying at RT under a nitrogen flow. The dry residues were hydrated with nanoplexes in Hepes, pH7.4 yielding after a 30 min incubation at RT,rather monodisperse nanoparticles having an average diameter of 150-300 nm, measured by DLS and cryo-TEM. Studies with cell cultures showed the above peptides to yield expression levels comparable to those obtained using Lipofectamine 2000. However, unlike the polydisperse aggregates formed upon mixing Lipofectamine 2000 and plasmid, the procedure described yields soluble, and reasonably monodisperse nanoparticles, which can be expected to be suitable for gene delivery in vivo, using intravenous injection.

Antimicrobial Peptides in 2014

“…Additionally, glutamic acid substitution of basic residues in LL-37, melittin, and bombolitin V linked to lipid nanoparticles could be used for endosomal escape and efficient gene delivery using intravenous injections. These yield expression levels comparable to those obtained using Lipofectamine 2000 and the probable mode of action resembles viruses [ 155 ]. Antimicrobial peptides have also been shown to have superb drug releasing properties in PEG-PLGA microparticles [ 156 ].…”

Human Antimicrobial Peptides in Bodily Fluids: Current Knowledge and Therapeutic Perspectives in the Postantibiotic Era

“…[278][279][280] Alternatively, AMPs could be used as drug delivery systems in the form of conjugates in order to accurately target drugs and other agents to tumor sites or intended organs, as typified by monodisperse "endosomolytic" nanoparticles for in vivo gene delivery using intravenous injection, or by functionalized nanoparticles with higher membrane penetration targeted against gliomas. 281,282 Such functionalization may allow for deeper tissue penetration, increased antimicrobial potency, sustained release, and novel routes of administration to be achieved.…”

Design of pH‐sensitive peptides from natural antimicrobial peptides for enhancing polyethylenimine‐mediated gene transfection

“…Likewise, acidic modification of Mel by replacing neutral glutamines (Gln25 and Gln26) with glutamic acid residues greatly improved the gene transfer efficiency of C‐terminally linked PEI conjugates and truncated peptides consisting of the first 20 residues of Mel in which all of the positively‐charged residues at the C‐terminal were removed also induced higher lytic activity at endosomal pH and lower lytic activity at neutral conditions . Glu is partially protonated at pH 5.0, which appears to be sufficient for inducing pH responsiveness of these peptides …”

“…23 Glu is partially protonated at pH 5.0, which appears to be sufficient for inducing pH responsiveness of these peptides. 22,31,42,44,45 In the present study, we designed two pH-sensitive peptides by replacing the positively-charged amino acids in the sequences of Mel and RV with Glu. Glu replacement decreased the α-helical content of Mel and RV in 50% TFE at pH 7.4 ( Table 2).…”