Ar-23 Derivatives With High Endosomal Disrupting Ability Enhance Poly(L-lysine)-mediated Gene Transfection

Background (cid:0) pH-sensitive peptides are a relatively new strategy for conquering poor endosomal release of cationic polymer-mediated transfection. Modication of antimicrobial peptides (AMPs) by exchanging positively-charged residues with negatively-charged glutamic acid residues (Glu) greatly improved its lytic activity at the endosomal pH, which could improve cationic polymer-mediated transfection. Methods (cid:0) In the present study, we investigate 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 for enhancing cationic polymer-mediated transfection. Three analogs were synthesized by replacing the positively-charged residues in AR-23's sequence with Glu one-by-one. The pH-sensitive lysis ability of the peptides, the effect of peptides on the physicochemical characteristics, the intracellular tracking (cid:0) the transfection eciency, and the cytotoxicity of the polyplexes were determined. Results (cid:0) 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 AR-23's sequence greatly improved PLL-mediated gene transfection eciency while maintaining low cytotoxicity. Conclusion (cid:0) The results indicate that replacement of positively-charged residues with enough Glu residues may be considered a method for designing pH-sensitive peptides, which could be applied as potential enhancers for improving cationic polymer-mediated transfection. of their pH-sensitive cellular toxicity, we studied the membrane lytic activity of the derived peptides by hemolytic tests and the calcein acetoxymethyl ester (Calcein-AM) assays. The transfection eciency of nanoparticles containing PLL/DNA with or without the peptides was studied and compared with the transfection eciency of the commercial reagent Lipofectamine 2000 in different cell lines.


Introduction
Non-viral gene delivery vectors are one of the most potential alternatives to viral vectors because of their less immunogenicity, low toxicity, and easy productivity despite their lower capacity of gene transfection compared to that of viral vectors [1][2][3][4]. A major drawback limiting the transfection e ciency 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[7-9]. 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 e ciency in different cell lines [7,14,15]. Melittin, covalently attached to Polyethyleneimine(PEI), enables the e cient 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 endosomal escaping activity of melittin. For example, acidic modi cation 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 modi ed membrane disrupting antimicrobial peptides, LL-37, melittin, and bombolitin V by replacing all positivelycharged residues with glutamic acid. These analogs are pH-sensitive and cause endosomal disrupting activity with insigni cant cytotoxicity at pH 7.4 [18]. We also design endosomolytic peptides by replacing the positively-charged residues with Glu in melittin and RV-23, an AMP from Rana draytonii. The designed peptides showed pH-sensitive lytic activity, which promote endosomal release of PEI/DNA polyplexes.
The incorporation of pH-sensitive peptides into polyplexes enhanced the PEI-mediated transfection e ciency 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 e ciency of PEI/DNA polyplexes.
The aim of this study was to investigate the effect of the number of glutamic acids substituted for positively-charged residues on the endosomal escape activity of AR-23 , a melittin related peptide which shows 81% sequence identity but has less positively charged residues than melittin [19,20], and the ability of modi ed AR-23 for enhancing cationic polymer-mediated transfection. Since PEI itself has endosomal disrupting activity due to the "proton sponge effect"[21], poly(L-lysine) (PLL), which has high DNA condense ability but poor endosome release activity was used for study the ability of modi ed peptide in improving cationic polymer mediated transfection [22]. 1 to 3 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 studied the membrane lytic activity of the derived peptides by hemolytic tests and the calcein acetoxymethyl ester (Calcein-AM) assays. The transfection e ciency of nanoparticles containing PLL/DNA with or without the peptides was studied and compared with the transfection e ciency of the commercial reagent Lipofectamine 2000 in different cell lines.

Peptide synthesis
Peptides (Table 1) were synthesized using the standard Fmoc procedure puri ed by reverse-phase semipreparative high-performance liquid chromatography as described and were dissolved in 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 veri ed by analytical RP-HPLC and was further characterized by mass spectrometry in electrospray positive ion detection mode using an Agilent 1100 ESI/MS (Agilent Technologies, USA). Where, F p uorescence of the sample containing the peptide, F c uorescence of the sample without the peptide and F t uorescence of the sample after the addition of Triton X-100(0.1% nal 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 ( nal N/P ratio was 5, which determines the ratio of nitrogen (N) in the PLL to phosphate (P) in the nucleic acid) and incubated for 15 min at room temperature. Then, the solution was added to the same volume of the indicated amounts of peptides and incubated for 15 min 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 tra cking of polyplexes
The cellular uptake e ciency was evaluated in HeLa cells. Brie y, HeLa cells (1.5×10 4 cells/well) were seeded in a CVG 8-well chamber (NUNC Lab-TekTM). Polyplexes were prepared as above and using 50 nM FITC-ODN instead of 8 µg/ml plasmid DNA. 24 h later, the medium was replaced with 300 µl of polyplexes. After incubating at 37 °C for 1 h, the cells were washed three times with PBS and then incubated with serum-free DMEM containing 50 nMLyso-Tracker Red DND-99 for 1.5 h. The cells were then washed three times with PBS and xed with 4% paraformaldehyde. The cells were stained with the nuclear stain DAPI ( nal concentration 300 nM) for 5 min at room temperature and observed under a confocal laser scanning microscope using a 63 × objective (CLSM, ZEISS LSM880, Germany).
9. Physicochemical characterization of polyplexes 9.1 DNA gel retardation To test the effect of peptides on the DNA condensation ability of PLL, agarose gel electrophoresis was performed[27]. Ten microliters DNA, 10 µl PLL/DNA, or 10 µl PLL/DNA/peptide polyplexes were mixed with 10×loading buffer such that the amounts of DNA were identical (0.2 mg DNA/well). The samples were then loaded into the slots of a 0.3% agarose gel containing 0.5 mg/ml ethidium bromide.
Electrophoresis was carried out at 120 V for 30 min in the 1×TAE running buffer. DNA retardation was analyzed using a gel image system (Tanon 1600, China) to indicate the location of the DNA.
9.2 Characterization of particle sizes and zeta potential The size and zeta potential of the polyplexes were evaluated via laser light scattering using a Malvern NANO-ZS90 (Malvern Instruments, Malvern, UK).

Transfection assay
HeLa cells, HEK293 cells, U251 cells and COS7 cells were separately seeded in 96-well plates (Corning Inc., Lowell, MA, USA) at a density of 5×10 3 cells/well on the day before transfection. The mean±SD of the absorbance was calculated for each group. Lipofectamine 2000 were used as control groups.

Statistical analysis
Statistical signi cance of the differences among groups was determined using unpaired Student's t-tests.

Design of AMP-derived endosomolytic peptides
Replacement of the positively-charged residues of the naturally occurring membrane-permeabilizing antimicrobial peptide with Glu yielded the pH-sensitive endosomolytic peptides, which could enhance the PEI-mediated transfection e ciency [11]. To investigate the effect of the number of Glu substituted for positively-charged residues on the endosomal escape activity of AR-23 and the ability of modi ed AR-23 for enhancing cationic polymer-mediated transfection. The rst positively-charged residue of AR-23 K13 was replaced by Glu E to generated aAR1 and aAR2 was designed by replacing K13 and K21 of AR-23 with E. Finally, aAR3 was produced by substituting of K13, K21 and R23. in AR-23 with E.The close agreement between the measured and theoretical molecular weights of the peptides (Table 1), suggesting that the peptides had been successfully synthesized.

Circular dichroism (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 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 αhelical 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, while the α-helical content of aAR2 was slightly higher at pH 7.4 than that of at pH 5.0 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 levels30 (

pH-dependent hemolytic activity
Glu replacement was su cient 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, while aAR2 and aAR3 had an inverse tendency (Figure 2A and  2B). With the increasing number of glutamate residues, the hemolytic activities of the peptides were reduced at pH 7.4, for example, the hemolytic activities of the peptides were reduced at pH 7.4, for example, the hemolytic activity of AR-23, aAR1, aAR2 and aAR3 at concentrations of 10μM was 101.3%, 79.9%, 14.6 and 0.1% respectively. The hemolysis rates of AR-23 analogs at pH 5.0 were higher than that of AR-23, for example the hemolysis rates of AR-23, aAR1, aAR2, and aAR3 were (42.7%, 79.7%, 61.4% and 56.0%) respectively at concentrations of 20 μM. It was worth noting that the hemolysis rate of AR-23 and aAR1 at pH 7.4 was higher than that of them at pH 5.0 respectively while hemolysis rates of aAR2 and aAR3 was lower than that of them at the under pH 5.0 respectively. 40μM aAR3 induced 80.3% cell lysis at pH 5.0, while only 3.8% cell were lysis at pH 7.4.

hRBCs membrane integrity
Membrane permeabilization induced by the four peptides was measured by releasing entrapped calcein from hRBCs at pH 7.4 and pH 5.0. Like the results of hemolytic assays, the calcein release induced by peptides were reduced with the increasing number of the Glu at pH 7.4. aAR2 and aAR3 induced more calcein release at pH 5.0 ( Figure 2C and 2D). For example, 20 μM aAR3 induced 98.4% and 1.9% calcein release at pH 5.0 and pH 7.4, respectively. On the other hand, AR-23 and aAR1 induced higher calcein release at pH 7.4 than at pH 5.0.

Intracellular tra cking of the polyplexes
In this study, the FITC-ODN was used to evaluate the endosomal lytic activity of the peptides. Red uorescence indicates the integration of endolysosomes, whereas yellow uorescence suggests that FITC-ODN is in endolysosomes. As shown in Figure 3, FITC-ODN was restricted within intracellular vesicles and showed a punctate distribution of uorescence signal in the cytoplasm and the strong dotlike signal turned yellow in the overlapping pictures in PLL/DNA-only group. However, some PLL/DNA/aAR3-treated cells showed dispersive green uorescence signals in the cytoplasm, indicating late degradation of some endosomes/lysosomes. Moreover, some green uorescence signal was observed in the nuclei of PLL/DNA/aAR3-treated cells. The results indicated that aAR3 promoted FITC-ODN release from endosomes and were transferred into the cytoplasm and nuclei, suggesting that the acidic peptides exhibited strong endosomal lytic activity.
6. Physicochemical characteristics of the polyplexes 6.1. DNA gel retardation assays To investigate the effect of AR-23 and its analogs on the condensation capability of PEI with DNA, a DNA gel retardation experiment was performed for different concentrations of peptides mixed with PLL/DNA polyplexes. Free plasmid DNA was included as a control. Compared to control plasmid DNA, no migrated band was observed for PLL/DNA polyplexes mixed with peptides ( Figure s1). The results suggested that incorporation Glu-replaced AR-23 does not affect the binding ability of PLL to plasmid DNA.
6.2. Size and zeta potential of the polyplexes To investigate the effects of peptides on the physicochemical characteristics of PLL/DNA polyplexes, the size and zeta potential of the polyplexes were measured by dynamic light scattering. The size of the PLL/DNA polyplexes (N/P = 5) was 76 nm (Figure 4). Generally, minor changes in the size of the PLL/DNA polyplexes were observed when AR-23, aAR1or aAR2were incorporated (Figure 4). For example, the average size of PLL/DNA/AR-23, PLL/DNA/aAR1, and PLL/DNA/aAR2 polyplexes was mainly distributed between 65-85 nm. However, the average size of PLL/DNA/aA23 polyplexes increased with increasing peptide concentrations. For example, the average sizes of the PLL/DNA polyplexes were 171.7 nm and 116.1 nm when 40 μM and 20 μM aA23 were incorporated ( Figure 4D).
The zeta potential of the PLL/DNA polyplexes also slightly changed due to the incorporation of AR-23, aAR1or aAR2at different peptide concentrations. However, signi cant changes were observed when aAR3 were incorporated into the PLL/DNA polyplexes at different peptide concentrations (Figure 4).  (Figure 6). For example, the cell viability of U251 cells upon treatment with the PLL/DNA polyplexes was 63.3%, and incorporation of 40 μMaAR3 into the PLL/DNA polyplexes decreased the cell viability to 52.8%. However, the cell viability of U251 treated with PLL/DNA polyplexes incorporated with 40 μMaAR3 was still higher than that of U251 treated with Lipofectamine 2000.

Discussion
Viruses invade live cells via receptor-mediated endocytosis, and the internalized virus is tra cked to late endosomes [28]. 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[29]. The major envelope protein (E) 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 to maximum conformational change in the protein within seconds [30]. Several peptides derived from viral proteins also have been shown to exert endosomolytic activity. These peptides are enriched in glutamic acids (Glu), which may be related to their lytic activity at acidic pH. GALA and INF7 are glutamic acid-rich peptides that show signi cant toxicity at pH 5.0 but negligible toxicity at similar concentrations at pH 7.0[31, 32]. Glu is partially protonated at pH 5.0, which appears to be su cient for inducing the pH responsiveness of these peptides [17,18,33]. Replacement of the positively-charged residues of the naturally occurring membrane-permeabilizing antimicrobial peptide with Glu yielded the pH-sensitive endosomolytic peptides, which could enhance the PEI-mediated transfection e ciency [11].
The aim of this study was 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 modi ed AR-23 for enhancing cationic polymer mediated transfection. Glu replacement decreased the α-helical content of AR-23 in 50% TFE at pH 7.4 (Table 2); however, aAR3 showed higher α-helical content in 50% TFE at pH 5.0 than that at pH 7.4. The low Gibbs free energy of the peptide partition from water to a membrane interface (ΔG if ) indicates a strong membrane partition [34]. Increased the ΔG if of peptides was observed with the increased number of Glu replacement in AR-23 sequence at pH 7.4, indicating that Glu replacement decreased the binding ability of the peptides with eukaryotic membranes at neutral pH (Table 2). Quite opposite tendency was observed acidic pH. Indeed, AR-23 is an AMP with high lytic activity at neutral pH. 5 μM AR-23 induced 70% and 6.5% hRBCs lysis at pH 7.4 and pH 5.0, respectively, which means AR-23 was toxic peptide with low endosomal disrupting activity. However, after glutamate replacement, the hemolysis rate of various analogs at pH 7.4 was greatly reduced, especially aAR3.
Membrane permeabilization induced by the four peptides were like the results of hemolytic assays at pH 7.4, the calcein release and hRBCs lysis induced by peptides were reduced with the increasing number of the glutamate residues. However, the calcein release and hRBCs lysis induced by peptides were comparable at pH 5.0. The above data suggested aAR3 induced signi cantly more hRBCs membrane disruption at pH 5.0 than at pH 7.4, which allow pH-sensitive lysis of cell membranes and exert endosomolytic activity at acidic pH.
PLL/DNA polyplexes are primarily uptaken through endocytosis. Eventually, the polyplexes are enclosed in a low-pH environment of endosomes for degradation by the contained hydrolytic enzymes. FITC-ODN was used to evaluate the intracellular tra cking of the polyplexes and the endosomal lytic activity of acidic peptides. As shown in Figure 3, most FITC-ODN was restricted within intracellular vesicles and showed a punctate distribution in the PLL/DNA-only group and the PLL/DNA incorporated with Mel or RV groups. However, dispersive green uorescence signals were observed in the PLL/DNA/aAR3-treated cells, which indicates the degradation of some late endosomes/lysosomes ( Figure 3C and 3D However, aAR3 enhances PLL-mediated gene transfection much more than chloroquine. Furthermore, PLL/DNA/aAR3 polyplexes induced comparative luciferase activity and cytotoxicity to the Lipofectamine 2000 group. The above results indicated that the incorporation of the triple-Glu substituted peptide could promote appreciable disruption of the endosomal membrane and promote the entry of polyplexes into the cytoplasm, thus increasing transfection e ciency.

Conclusions
In summary, the 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 glutamic acids substituted for positively charged residues of AR-23 dramatically affects its ability for enhancing PLL-mediated transfection.
Triple-Glu substitution in the AR-23 sequence improved its lytic ability at acidic pH and decreased cytotoxicity at neutral conditions. The designed aAR3interactedwith PLL/DNA polyplexes and increased the particle size of the polyplexes. aAR3 showed higher endosomolytic activity and greatly improved PLLmediated gene transfection e ciency while maintaining low cytotoxicity. We suggest that enough glutamic acid residue replacement may be considered a method for designing pH-sensitive peptides, which could be applied as potential enhancers for improving the transfection e ciency of cationic polymers. However, more work remains to achieve synthetic virus-like particles that can transfect genes into speci c cells in an e cient, safe manner.
Declarations Figure 1 CD spectra of peptides acquired in 50% TFE at pH 5.0 and pH 7.4 at 25°C.All the peptides formed a wellde ned α-helical structure in the presence of 50% TFE. Figure 1 CD spectra of peptides acquired in 50% TFE at pH 5.0 and pH 7.4 at 25°C.All the peptides formed a wellde ned α-helical structure in the presence of 50% TFE.   The late endosomes and lysosomes are stained with Lyso-Tracker red, and cell nuclei are stained with DAPI.

Figure 3
Confocal microscopy images of HeLa cells incubated with A PLL/DNA, B PEI/DNA with 2.5 µM AR-23, C PEI/DNA with 40 µM aAR3. Images were acquired with a confocal laser scanning microscope (scale bar = 20 µm) at an additional 1.5 h after uptake and show the subcellular distribution of calcein uorescence.