Antimicrobial peptide AR‐23 derivatives with high endosomal disrupting ability enhance poly(l‐lysine)‐mediated gene transfer

Abstract Background pH‐sensitive peptides are a relatively new strategy for conquering the poor endosomal release of cationic polymer‐mediated transfection. Modification of antimicrobial peptides by exchanging positively‐charged residues with negatively‐charged glutamic acid residues (Glu) greatly improves its lytic activity at the endosomal pH, which could improve cationic polymer‐mediated transfection. Methods In the present study, we investigated the effect of the number of Glu substituted for positively‐charged residues on the endosomal escape activity of AR‐23 and the ability of mutated AR‐23 with respect to enhancing cationic polymer‐mediated transfection. Three analogs were synthesized by replacing the positively‐charged residues in the AR‐23 sequence with Glu one‐by‐one. Results The pH‐sensitive lysis ability of the peptides, the effect of peptides on the physicochemical characteristics, the intracellular trafficking, the transfection efficiency and the cytotoxicity of the polyplexes were determined. Increased lytic activity of peptides was observed with the increased number of Glu replacement in the AR‐23 sequence at acidic pH. The number of Glu substituted for positively‐charged residues of AR‐23 dramatically affects its lysis ability at neutral pH. Triple‐Glu substitution in the AR‐23 sequence greatly improved poly(l‐lysine)‐mediated gene transfection efficiency at the same time as maintaining low cytotoxicity. Conclusions The results indicate that replacement of positively‐charged residues with sufficient Glu residues may be considered as a method for designing pH‐sensitive peptides, which could be applied as potential enhancers for improving cationic polymer‐mediated transfection.


| INTRODUCTION
Non-viral gene delivery vectors are potential alternatives to viral vectors because of their reduced immunogenicity, low toxicity and easy producibility, despite their lower capacity of gene transfection compared to that of viral vectors. 1-4 A major drawback limiting the transfection efficiency of non-viral gene delivery vectors has historically been the poor release of DNA from endosomal compartments. 5,6 Given the challenges associated with traditional gene delivery methodology, there is clearly a need for gene delivery technology to enable endosomal escape with minimal cytotoxicity. Strategies to achieve endosomolysis have traditionally been based on osmotic agents, fusogenic lipids and fusogenic peptides. [7][8][9] The addition of pH-sensitive peptides has been shown to potentially enhance gene expression of non-viral gene delivery vectors. 10,11 Melittin, a 26-residue peptide from the venom of Apis mellifera honeybees, is a well-studied endosomal disrupting peptide. 12,13 Melittin or its analogues have been incorporated into polyplex formulations to increase transfection efficiency in different cell lines. 7,14,15 Melittin, covalently attached to polyethyleneimine (PEI), enables the efficient release of PEI/DNA polyplexes from endosomes, increasing its nuclear localization and subsequently enhancing the transfection activity of PEI/DNA polyplexes in a broad range of cell lines. 16 However, inherent lytic activity at neutral pH also provokes high cytotoxicity as a result of cell membrane damage. Increasing negatively-charged residues could also improve the endosomal escaping activity of melittin. For example, acidic modification of melittin by exchanging neutral glutamines (Gln25 and Gln26) with negatively-charged Glu greatly improved its lytic activity at the endosomal pH of 5.0 with lower lytic activity at neutral condition. 17 Moreover, Ahmad et al. 18 modified membrane disrupting antimicrobial peptides, LL-37, melittin and bombolitin V, by replacing all positively-charged residues with glutamic acid. These analogs are pH-sensitive and cause endosomal disrupting activity with insignificant cytotoxicity at pH 7.4. 18 We also designed endosomolytic peptides by replacing the positively-charged residues with Glu in melittin and RV-23, an antimicrobial peptide (AMP) from Rana draytonii. The designed peptides showed pH-sensitive lytic activity, which promotes endosomal release of PEI/DNA polyplexes. The incorporation of pH-sensitive peptides into polyplexes enhanced the PEI-mediated transfection efficiency, corresponding to up to 42-fold higher luciferase activity compared to that of PEI alone. 11 The results indicate that replacement of positively-charged residues with glutamic acid residues in the AMP sequence yields pH-sensitive peptides, which enhance the transfection efficiency of PEI/DNA polyplexes.
AR-23, a frog skin-derived AMP from Rana tagoi, is a melittin related peptide which shows 81% sequence identity. 19,20 Furthermore, AR-23 has similar positively-charged residues distribution pattern at the positions 1, 8 and 17 position to melittin and RV-23. The present study aimed to investigate the effect of the number of glutamic acids substituted for positively-charged residues on the endosomal escape activity of AR-23 and the ability of modified AR-23 for enhancing cationic polymer-mediated transfection.
Because PEI itself has endosomal disrupting activity as a result of the 'proton sponge effect', 21 poly(L-lysine) (PLL), a cationic polymer with high DNA condense ability but poor endosome release activity, was used to investigate the ability of the modified peptide to enhance cationic polymer-mediated transfection. 22 One to three positively-charged residues (lysine or arginine) of AR-23 were replaced by Glu, respectively. To explore the molecular basis of their pH-sensitive cellular toxicity, we investigated the membrane lytic activity of the derived peptides by hemolytic tests and calcein acetoxymethyl ester (Calcein-AM) assays.

| Peptide synthesis
Peptides (Table 1) were synthesized using the standard Fmoc procedure purified by reverse-phase (RP) semi-preparative highperformance liquid chromatography (HPLC) and were dissolved in dimethylsulfoxide (DMSO) to yield a 1000 μM stock solution for further use. The purity of the synthetic peptide was greater than 95%.
The purity of the peptides was verified by analytical RP-HPLC and was further characterized by mass spectrometry in electrospray positive ion detection mode using an Agilent 1100 ESI/MS system (Agilent Technologies, Santa Clara, CA, USA). (v/v) FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin unless specified otherwise. The cells were trypsinized using trypsin-ethylenediaminetetraacetic acid and maintained in a humidified incubator with 5% CO 2 at 37 C.

| Hemolytic activity of the peptides
The study protocol and acquisition of blood samples were respectively. The percentage of hemolysis was calculated according to: 2.6 | Assay for hRBCs membrane integrity was calculated as follows 26 : where F p is the fluorescence of the sample containing the peptide, F c is the fluorescence of the sample without the peptide and F t is the fluorescence of the sample after the addition of Triton X-100 (0.1% final concentration).

| Preparation of polyplexes
All the PLL/DNA and PLL/peptide/DNA polyplexes were freshly prepared before use. Polyplexes were prepared by adding PLL solution to equal volumes of plasmid solution (final N/P ratio of 5, which determines the ratio of nitrogen (N) in the PLL to phosphate (P) in the nucleic acid) and incubated for 15 minutes at room temperature.
Then, the solution was added to the same volume of the indicated amounts of peptides and incubated for 15 minutes at room temperature before characterization and gene transfection experiments.
Polyplexes for physicochemical characterization and transfection experiments were prepared in double-distilled water and serum-free DMEM, respectively.

| Intracellular trafficking of polyplexes
The cellular uptake efficiency was evaluated in HeLa cells.   The mean ± SD of the absorbance was calculated for each group.
Lipofectamine 2000 were used as a control group.

| Statistical analysis
Statistical significance of the differences among groups was determined using unpaired Student's t tests. Glu. The close agreement between the measured and theoretical molecular weights of the peptides suggesting that the peptides had been successfully synthesized (Table 1).

| CD spectroscopic analysis of the peptides
The secondary structure of peptides in different pH environments (in 50% TFE pH 5.0 and pH 7.4) were determined by the CD spectra.
As shown in Figure 1, all of the peptides exhibited a typical α-helix spectrum with double minima at 208 nm and 222 nm in the two pH solutions with 50% TFE. Substitution of positively-charged residues with Glu decreased the α-helical content, and aAR2 exhibited the lowest α-helical content of the four peptides at pH 7.4 ( Table 2). Similar α-helical content of AR-23 at different pH was observed and so was aAR1, whereas the α-helical content of aAR2 was slightly higher at pH 5.0 than that of at pH 7.4 and aAR3 possessed the highest α-helical content of the four peptides at pH 5.0, which was 54.3%.
The Gibbs free energy of the peptide partition from water to a membrane interface (ΔG if ) was calculated by MPEx according to the helicities of the peptides at different pH levels 28 (

| pH-dependent hemolytic activity
Glu replacement was sufficient to induce the pH sensitivity of AMPs.
The impact of the number of Glu substituted for positively-charged residues on the membrane lytic activity of AR-23 was investigated by hemolytic assays at pH 5.0 and 7.4 using hRBCs. The hemolytic activity of AR-23 and aAR1 at pH 7.4 was higher than that of them at pH 5.0, whereas aAR2 and aAR3 had an inverse tendency (Figure 2A and

| Intracellular trafficking of the polyplexes
In the present study, FITC-ODN was used to evaluate the endosomal lytic activity of the peptides. As shown in Figure 3

| DISCUSSION
Viruses invade live cells via receptor-mediated endocytosis, and the internalized virus is trafficked to late endosomes. 29 The acidic endosomal environment induces membrane fusion between the virus and endosomes via a conformational change of surface proteins, and the viral genome is released into the cytoplasm of the target cells, thus enabling escape from the endosomes. 30 The major envelope protein of the West Nile virus is another example of a fusogenic agent that exerts its endosomal disruptive activities at an upper threshold of pH 7.0 and has a maximum activity at pH 6.4 and below, which leads

| CONCLUSIONS
In summary, the increased lytic activity of peptides was observed with an increased number of Glu replacements in the AR-23 sequence at acidic pH. The number of glutamic acids substituted for positively-charged residues of AR-23 dramatically affects the ability to enhance PLL-mediated transfection. Triple-Glu substitution in the AR-23 sequence improved lytic ability at acidic pH and decreased cytotoxicity at neutral conditions. The designed aAR3 interacted with PLL/DNA polyplexes and increased the particle size of the polyplexes. aAR3 showed higher endosomolytic activity and greatly improved PLLmediated gene transfection efficiency at the same time as maintaining low cytotoxicity. We suggest that sufficient glutamic acid residue replacement may be considered as a method for designing pHsensitive peptides, which could be applied as potential enhancers for improving the transfection efficiency of cationic polymers. However, more work is required to achieve synthetic virus-like particles that can transfect genes into specific cells in an efficient and safe manner.