Cationic Copolymers Act As Chaperones of a Membrane-Active

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Cationic copolymers act as chaperones of a membraneactive peptide: Influence on membrane selectivity Wakako Sakamoto, Tsukuru Masuda, Takuro Ochiai, Naohiko Shimada, and Atsushi Maruyama ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01582 • Publication Date (Web): 20 Mar 2019 Downloaded from http://pubs.acs.org on March 20, 2019

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ACS Biomaterials Science & Engineering

Cationic copolymers act as chaperones of a membrane-active peptide: Influence on membrane selectivity

Wakako Sakamoto, Tsukuru Masuda, Takuro Ochiai, Naohiko Shimada, and Atsushi Maruyama*

School of Life Science and Technology, Tokyo Institute of Technology, B-57 4259 Nagatsutacho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan *E-mail: [email protected] KEYWORDS membrane-active peptides, cationic copolymer, chaperones, lipid membrane, membrane permeabilization

ABSTRACT Membrane-active peptides have potential as drug delivery tools for control of lipid bilayer structures in cells and liposomes. In a previous study, we reported that a cationic combtype copolymer,

poly(allylamine)-graft-dextran

(PAA-g-Dex), forms a soluble inter-

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polyelectrolyte complex with an anionic peptide, E5, and enhances its membrane-disrupting activity.

Furthermore, the E5/PAA-g-Dex complex augments the cellular membrane

permeability of other proteins. In this study, the affinities of the E5/PAA-g-Dex complex for lipid membranes with various compositions were determined. Secondary structure analysis of E5 and analyses of binding of E5 to liposomes revealed that lipid composition strongly influenced the interaction. No significant folding of E5 alone was observed at either pH 5.4 or pH 7.4 and folding into the functional conformation, which is both N-terminal and C-terminal helix, was observed only at pH 5.4 in the presence of liposomes having liquid-disordered phase (L ). PAA-g-Dex induced partial folding of E5, presumably at C-terminus, at both pH 5.4 and pH d

7.4. Folding of E5 into the functional structure was induced by the addition of liposomes having L phases at either pH 5.4 or pH 7.4. A leakage assay showed that PAA-g-Dex enhanced the d

membrane-permeabilizing activity of E5 by promoting the adsorption of E5 onto the surface of liposomes and/or E5 association with the lipid bilayer. These results indicated that E5 activated by PAA-g-Dex destabilizes the lipid membrane having L phase even when the lipid membrane d

has a heterogeneous phase separated structure. Hence, PAA-g-Dex serves as a chaperone for E5 without altering its membrane selectivity. The chaperoning activity of this comb-type copolymer may activate other ionic peptides with unstable structures and low solubility.

１. INTRODUCTION Biomembranes separate the cellular contents from the environment and are necessary for subcellular organelle organization. Membranes control the permeability of molecules that are exported and imported within the cell and between the cell and the environment. Membrane permeability must be considered during drug development.

Macromolecular drugs such as


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nucleic acids and peptides have attracted great attention owing to their high target specificity,1,2 however, they cannot pass through the cellular membranes. These drugs are endocytosed and then degraded through the endosome/lysosome pathway resulting in a low efficacy. Various drug delivery systems (DDS), including polyion complex micelles, polycation liposomes, oligoaminoethane polyplexes, and virus-like liposomes have been developed, to facilitate productive import of drugs into cells.3-7

A promising tool for DDS are membrane-active

peptides such as antimicrobial peptides and artificially designed peptides that cell typeselectively perturb membranes. These peptides have amphiphilic properties as they consist of both hydrophilic and hydrophobic amino acids and have membrane fusion and membrane disruption abilities. To date, various peptides including melittin, magainin-2, GALA, and KALA have been investigated.8-12 The E5 peptide (Fig. 1a) is an amphiphilic peptide that mimics the N-terminal end of hemagglutinin, a membrane fusogenic protein expressed on the surface of influenza virus.13,14 E5 converts from random coil to amphiphilic α-helix conformation upon acidification due to protonation of its glutamic acid residues, and thus acquires membrane-disrupting activity. The intrinsic hydrophobicity of E5 makes it difficult to use as a delivery tool due to its poor solubility and non-specific interactions with biological components that cause loss of its function. In previous studies, we reported that a cationic comb-type copolymer, poly(allylamine)graft-dextran (PAA-g-Dex) (Fig. 1b), forms a soluble inter-polyelectrolyte complex with E5. In the complex, the cationic main chain PAA of the copolymer shields the electronic repulsion of glutamic acids to stabilize the α-helix conformation, while the hydrophilic Dex side chain improves solubility of E5 in the helical conformation.15 Because it chaperones the coil-to-helix transition of E5, PAA-g-Dex augments the membrane-disruptive ability of E5 not only in acidic


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condition but also at neutral pH.15 Moreover, we have demonstrated that the E5/PAA-g-Dex complex augments the permeation of a model protein through a cellular membrane.16 Importantly, cellular membranes are heterogeneous and membrane composition varies depending on cell type, growth cycle, and differentiation stage.17-19 As membrane active properties of E5 depend on the composition of the membrane, in this study we assessed the effect of the lipid composition on the interaction between E5 and liposomes and the membrane-disruption ability of the E5 in the presence of the cationic copolymer.

Influence of the copolymer on the

membrane selectivity and activation mechanisms of E5 were discussed.

2. EXPERIMENTAL SECTION 2.1. Materials

1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) were obtained from NOF Corporation (Tokyo, Japan). Cholesterol, sodium

hydroxide

(NaOH),

hydrochloric

acid

(HCl),

and

4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid (HEPES) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Sodium chloride (NaCl), 28% ammonium aqueous solution,

chloroform, and tris(hydroxymethyl)aminomethane (Tris) were purchased from Nacalai Tesque (Kyoto, Japan). 2-(N-Morpholino)ethanesulfonic acid (MES) was purchased from DOJINDO Laboratories (Kumamoto, Japan).

Acid Red was purchased from FUJIFILM Wako Pure

Chemical Corporation (Osaka, Japan). E5 peptide was purchased from Genenet (Fukuoka, Japan). The concentration of E5 in 0.03% ammonium aqueous solution was determined by UV-


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vis spectroscopy UV-1650PC spectrophotometer from Shimadzu (Kyoto, Japan) using a molar extinction coefficient of 5500 M−1 cm−1 at 280 nm (derived from tryptophan). Poly(allylamine) hydrochloride (PAA·HCl) (Mw of PAA: 5,000 g/mol) in aqueous solution was kindly supplied by Nitto Boseki Co., Ltd. (Tokyo, Japan), and purified by reprecipitation from methanol. Dextran 8 (Mw: 10,000 g/mol) was obtained from Funakoshi Co., Ltd. (Tokyo, Japan). Because of difficulty in obtaining Dextran T-10 (Pharmacia Biotech, Uppsala, Sweden) which had been used in our previous study,15 we changed a source of dextran. PAA-g-Dex was synthesized and characterized according to a previous report.15

Briefly, Dextran 8 was

conjugated to amino groups of PAA by reductive amination. The copolymer was purified by ion exchange followed by dialysis against water and then lyophilized. The dextran content of PAAg-Dex, determined by 1H NMR, was 95 wt%. The average numbers of allylamine repeating units and dextran grafts of the copolymer were 88 and 11, respectively. The obtained PAA-g-Dex was referred to as 5k95D, where 5k and 95D represent the molecular weight of PAA and the dextran content, respectively.

2.2. Preparation of large and small unilamellar vesicles.

A chloroform solution containing phospholipids and/or cholesterol was dried in a recovery flask by rotary evaporation under vacuum, followed by vacuum drying in a desiccator overnight. The obtained lipid thin film was hydrated with 10 mM MES-NaOH (pH 5.4) or 10 mM HEPES-NaOH (pH 7.4) containing 100 mM NaCl and 40 mM Acid Red at above transition temperature for the leakage assay. The lipid suspension was extruded 11 times through a polycarbonate membrane with a mean pore diameter of 100 nm using an extruding device. In the case of DPPC/cholesterol (80/20) and DOPC/DPPC/cholesterol (40/40/20), the heatblock on


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the extruding device was maintained at 55 °C. The obtained large unilamellar vesicle (LUV) suspension was purified by gel permeation chromatography on a Sephadex G-25M column. The lipid concentration of the LUV suspension was determined using a phospholipid assay kit (FUJIFILM Wako Pure Chemical Corporation). For tryptophan fluorescence intensity measurements, the vesicle suspension was prepared in the same way using 10 mM MES-NaOH (pH 5.4) or 10 mM Tris-HCl (pH 7.4) containing 140 mM NaCl. Extrusion through a polycarbonate membrane with a mean pore diameter of 50 nm resulted in small unilamellar vesicles (SUVs).

2.3. Determination of dissociation constant using circular dichroism.

The circular dichroism (CD) spectra of E5 as a function of 5k95D concentration and in SUVs in the absence and presence of 5k95D were acquired using a J-820 CD spectrometer (Jasco, Tokyo, Japan). E5 solution (10 µM) was prepared in 10 mM MES-NaOH (pH 5.4) or 10 mM Tris-HCl (pH 7.4) containing 140 mM NaCl. Measurements were acquired using a quartz cuvette with a 2-mm path length. Each spectrum represents the average of 16 scans from 200 to 250 nm with a 0.1 nm resolution, obtained at 100 nm/min with a bandwidth of 1 nm at 25 °C. The equilibrium dissociation constants (Kd) of 5k95D and E5 were determined as described in a previous report.15 Helicity was calculated by using eq. 1: 20

Helicity (%) = −

𝜃/// + 2340 × 100 (eq. 1) 30300

where 𝜃/// is the mean molar residue ellipticity at 222 nm.


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2.4. Assessment of the interaction between the lipid bilayer and E5 based on tryptophan fluorescence.

The fluorescence spectra of E5 peptide was measured using a Jasco FP-6500 spectrofluorometer (Ex: 280 nm, Em: 250-450 nm) at 25 °C. Samples were prepared at final concentrations of 0-2 mM lipid and 10 µM E5 in 10 mM MES-NaOH (pH 5.4) or 10 mM TrisHCl (pH 7.4) containing 140 mM NaCl.

2.5. Leakage assay of incorporated fluorescent dye.

Acid Red-loaded LUVs were used to assay the membrane-disrupting activity of E5 in the presence or absence of 5k95D. For time course measurements, the LUVs were suspended in a plastic cuvette, and the fluorescence signal was monitored using a Jasco FP-6500 spectrofluorometer (Ex: 565 nm, Em: 585 nm). Fluorescence intensity from intact LUVs before addition of E5 or 5k95D, 𝐹(0), was defined as 0% leakage. An aqueous solution containing E5 or 5k95D at high concentration was added to the LUV suspension at 50 seconds, 200 seconds, or 1250 seconds (final concentration: 2.5 µM lipid, 0-2.5 µM E5, 5.6 µM 5k95D in 10 mM MESNaOH (pH 5.4) or 10 mM HEPES-NaOH (pH 7.4) containing 140 mM NaCl), and fluorescence was monitored at 25 °C. Vesicles were lysed with 0.1 w/v% TritonX-100 to obtain the maximum fluorescence intensity, 𝐹 (∞), corresponding to 100% leakage. Leakage as a function of time (t) was calculated by using eq. 2:

Leakage (%) =

𝐹(𝑡) − 𝐹(0) × 100 (eq. 2) 𝐹 (∞) − 𝐹(0)

To study the dependence on E5 concentration, the fluorescence intensities from Acid Red-loaded LUVs in the presence of various concentrations of E5 were measured using a microplate reader. E5 and/or 5k95D was added to the LUV suspension in wells of 96-well black


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plates, and the mixture was incubated at 25 °C for 20 minutes. The fluorescence intensity was measured by using TriStar2 (Berthold Technologies) (Ex: 570 nm, Em: 590 nm).

The

fluorescence intensities of untreated LUVs and LUVs treated with 0.1 w/v% TritonX-100 were used as 𝐹 (0) and 𝐹 (∞) in eq. 2. The effective concentration (EC50) and Hill coefficient (n) were determined by substituting leakage values (Y) and concentration of E5 (c) into eq. 4:21

𝑌 = 𝑌A +

log L

𝑌BCD − 𝑌A × 100 (eq. 3) 1 + (ECGA ⁄𝑐 )J

𝑌BCD − 𝑌 M = −𝑛 log 𝑐 + 𝑛 log ECGA (eq. 4) 𝑌 − 𝑌A

where Y0 and Ymax are 0 and 100, respectively.

3. RESULTS AND DISCUSSION 3.1 Effect of the cationic comb-type copolymer on the folding of E5 The effect of the prepared cationic copolymer, 5k95D on the structure of E5 peptide was investigated by CD. At neutral pH, E5 alone adopts a random coil structure due to electrostatic repulsion derived from anionic glutamic acid residues.

Upon acidification, glutamic acid

residues are protonated and E5 folds into a helical conformation and simultaneously aggregates.14 We have reported that a cationic comb-type copolymer, PAA-g-Dex (5k92D), promotes the helical folding of E5 both at acidic and at neutral pH without inducing aggregation.15 The cationic main chain PAA of the copolymer shields the glutamic acids to stabilize helical conformation while the hydrophilic Dex side chain improves solubility of helical E5. Herein, we used PAA-g-Dex with 95 wt% dextran grafts, higher than wt% of dextran in the


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previous study, and found no significant difference in interaction with E5. As summarized in Table 1 and Fig. 2, 5k95D, as well as 5k92D, increased helicity of E5 both at pH 5.4 and 7.4. This result indicated that an inter-polyelectrolyte complex, E5/5k95D, formed even when the dextran content in the cationic copolymer was 95 wt%. At pH 5.4, electrostatic interactions between E5 and 5k95D was expected to be weaker than that at pH 7.4 because of protonation of glutamic acid residues of E5. However, E5 interacted with 5k95D with Kd values the 10-7 M range at pH 5.4, which was comparable to the Kd at pH 7.4 (Kd = 0.5 µM and 0.9 µM at pH 5.4 and 7.4, respectively) (Figs. S1 and S2). Moreover, E5 formed slightly more stable complex with 5k95D than 5k92D (Kd with 5k92D = 1.5 µM).15

3.2 Cooperative folding of E5 in the presence of lipids and cationic copolymers We next investigated the structures of E5 in the presence of the lipid vesicles without the cationic copolymer. The CD spectra of E5 in the presence of SUVs composed of 100% DOPC at acidic pH and at neutral pH were measured. At 25 °C, DOPC liposomes are in a liquiddisordered (Ld) phase. At acidic pH, in the presence of DOPC liposomes, the helicity of E5 peptide was 35%, whereas the helicity of E5 alone was 3% (Fig. 2a). This result suggested that the coil-to-helix transition of E5 was promoted by the hydrophobic interaction between E5 and the liposomes. The hydrophobic interaction was monitored by measurement of the fluorescence of the tryptophan residue (Trp14), which is a component of the hydrophobic face upon helical folding (Fig. 1a). In response to hydrophilic-to-hydrophobic environmental changes induced by an increase in lipid concentration the emission spectrum of tryptophan undergoes a blue shift.


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The blue shift in the Trp14 emission was observed with an increase in DOPC liposome concentration and was saturated at 100 µM DOPC (Fig. S3, white symbol).

This result

suggested that Trp14 of E5 interacts with the hydrophobic region of lipid bilayer. At neutral pH in the presence of DOPC, α-helical signals are present in the CD spectrum of E5 that are not apparent in the absence of lipid (Fig. 2b). The blue shift in Trp14 fluorescence was also indicative of an interaction between E5 and the hydrophobic region of lipid bilayer (Fig. S3, black symbol). The change in helical CD signal and Trp14 blue shift induced by DOPC at neutral pH were weaker than those at acidic pH. These results suggest the partial folding of E5 at neutral pH in the presence of a DOPC membrane. Hsu et al. reported on the basis of an NMR study that E5 possesses two helical segments that are connected by a Gly12-Gly13 hinge (Fig. 1a).22 One helical segment is formed by Leu2-Glu11, and the other is by Trp14-Ile18. Only the Nterminal segment folds into a helical structure at neutral pH in the presence of SDS micelles. At lower pH in the presence of SDS micelles, C-terminal segment folding is observed.22 Hence, hydrophobic interactions drive N-terminal helix folding, whereas both hydrophobic interactions and acidification are required in the functional helical folding, which is both N- and C-terminal helix, and the resulting fusogenic activity. To evaluate the effect of the lipid membrane composition on E5 folding, the CD spectra of E5 were measured in the presence of liposomes having diverse compositions. In the presence of liposomes composed of DOPC/cholesterol (80/20) or DOPC/DPPC/cholesterol (40/40/20), E5 formed an α-helical structure at pH 7.4 (Figs. 2c, d). A blue shift of Trp14 fluorescence at pH 7.4, indicated that E5 interacted with these liposomes (Fig. S3, blue and red symbols). In contrast, in the presence of DPPC/cholesterol liposomes E5 remained in a random coil structure at pH 7.4 (Fig. 2e), and a blue shift was not observed (Fig. S3, green symbol). The DOPC/cholesterol


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(80/20) and the DPPC/cholesterol (80/20) liposome have a liquid-disordered (Ld) and liquidordered (Lo) phase, respectively.23 The membrane fluidity of the Lo phase is significantly lower than that of the Ld phase, which is correlated with the tight packing than Ld phase.23, 24 In the presence of DOPC/cholesterol (80/20), the helicity of E5 was slightly lower than that in the presence of DOPC (100). The membrane fluidity of DOPC/cholesterol membranes decreases with the increase in cholesterol contents.23 The decrease in helicity would be caused by the decrease in membrane fluidity in DOPC membrane by the addition of cholesterol.

We

hypothesize that the tight packing of DPPC/cholesterol (80/20) inhibited the folding of E5. In contrast, the DOPC/DPPC/cholesterol (40/40/20) has a phase-separated structure composed of DPPC-rich Lo and DOPC-rich Ld phases.23 The increase in helicity and blue shift observed for DOPC/DPPC/cholesterol resulted from the interaction of E5 with the Ld phase. We next investigated conformal change of E5 complexed with cationic copolymer upon addition to liposomes at pH 7.4. The helicity of E5 in the presence of 5k95D was higher than that in the absence of cationic copolymer when added to DOPC liposomes at neutral pH (Fig. 2b). Thus, the folding of E5 peptide was further enhanced by the interaction with DOPC liposomes even in the presence of the copolymer at neutral pH. The helicity of E5 with DOPC and the copolymer at neutral pH was as large as that of E5 with DOPC at acidic pH. These results indicate that the both N-terminal and C-terminal of E5 is estimated to be folded into the functional conformation in the presence of the copolymer at neutral pH, which is necessary to express the membrane disruption activity. Similarly, the helicity of E5 in the presence of DOPC/cholesterol and DOPC/DPPC/cholesterol liposomes was increased by complexation of E5 with 5k95D (Figs. 2c, d). However, E5 did not adopt a functional helical conformation in DPPC/cholesterol liposomes even in the presence of 5k95D (Fig. 2e).


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3.3 Membrane-disrupting activity of E5 is enhanced by cationic copolymer. In previous studies, the mechanisms of membrane disruption by amphiphilic peptides including GALA, melittin, and alamethicin have been characterized.25-27 Membrane disruption is proposed to occur in two steps; i) binding of the peptide onto the surface of lipid membranes and ii) association of peptides to form pores in the lipid bilayer. The steps are affected by the peptide to lipid ratio and the lipid composition. To evaluate the role of lipid composition on the membrane-disrupting activity of E5, LUVs were prepared that contained a self-quenching dye, Acid Red. The time course of leakage of Acid Red from LUV induced by E5 in the absence or presence of 5k95D was evaluated under various E5 concentrations at acidic and neutral conditions. At pH 5.4, E5 alone induced leakage of Acid Red from LUVs composed entirely of DOPC (Fig. 3a). This result was correlated with the effective helical folding of E5 on the surface of the DOPC lipid bilayer as previously reported.22, 28 The leakage rate increased with E5 concentration (Fig. 3a). The leakage rate depends on the rates of the adsorption of the peptide onto the surface of liposome and of pore formation in the lipid bilayer.29 At pH 7.4, E5 alone induced no leakage of Acid Red from LUVs of any of the compositions tested (Fig. 3b-e). Thus, at neutral pH, the folding of E5 on the surface of lipid bilayer is not sufficient to disrupt the bilayer. When 5k95D was added to the E5 and LUV mixture at acidic pH, more significant leakage was observed compared with E5 alone (Fig. 3a). 5k95D alone did not induce leakage (Fig. 3a). These results show that 5k95D enhances the membrane disruption activity of E5. Since 5k95D did not significantly change the structure of E5 at pH 5.4 (Fig. 2a), 5k95D


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presumably promotes the adsorption of E5 onto the liposome surface and/or its association to form pores in lipid bilayers. In neutral conditions, E5 in the presence of 5k95D caused significant leakage from LUVs composed of DOPC, DOPC/cholesterol, and DOPC/DPPC/cholesterol (Figs. 3b, c, d). No significant leakage was observed for LUV composed DPPC/cholesterol even at the high E5 concentration of 7 µM in the presence of 5k95D (Fig. 3e). This result was consistent with our observation that E5 did not interact with DPPC/cholesterol even in the presence of 5k95D.

3.4 Assessment of cooperativity of E5 in the presence of cationic copolymer. In the real-time monitoring of leakage, leakage of Acid Red increased as the E5 concentration increased. We hypothesize that several E5 molecules assemble in the membrane to form a pore. To evaluate the cooperativity of E5 in membrane disruption and the effects of 5k95D on this assembly, leakage was plotted as a function of E5 concentration under various conditions.

These plots were sigmoidal curves indicating an increase in leakage rate with

increasing concentration of E5 (Fig. 4).

Curves were fit to the Hill equation, and the

concentration of E5 necessary to induce 50% leakage (EC50) and the Hill coefficient were estimated (Table 2). At acidic pH, the Hill coefficient was approximately 3 in the both absence and presence of 5k95D. This suggests that E5 cooperatively interacted with liposomes and 5k95D did not affect the cooperativity. At pH 5.4, the EC50 of E5 in the presence of 5k95D was 0.14 µM, approximately 5 times lower than that of E5 alone (EC50 = 0.70 µM). EC50 is proportional relation with an apparent dissociation constant in the process of adsorption and association of E5 in liposomes. Thus, the decrease in EC50 indicates that 5k95D promotes adsorption and/or association but does not alter the degree of cooperativity in pore formation.


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At pH 7.4 in the presence of 5k95D, the EC50 value estimated for DOPC liposomes was 0.15 µM, which was similar with that in the presence of 5k95D at acidic pH, indicating that 5k95D eliminated the pH-dependence of E5 activity. Similar EC50 values were obtained for DOPC/cholesterol and DOPC/DPPC/cholesterol liposomes (EC50 = 0.18, and 0.14 µM, respectively). The Hill coefficient for DOPC/cholesterol was 5.8, higher than that for DOPC liposomes (Table 2). In a previous report, the number of amyloid β peptides per cluster was increased as cholesterol content in the membrane increased.30 The authors of this study proposed that the number of peptides involved in pore formation was determined by the hydrophobicity of bilayer surface and the thickness of bilayer.

In our system, the increase in hydrophobic

cholesterol content alters the hydrophobic environment of the membrane and presumably influences adsorption, association, and pore formation by E5.

The Hill coefficient for

DOPC/DPPC/cholesterol liposomes was similar to that for DOPC liposomes. Cholesterol is known to distribute more to saturated lipids than unsaturated lipids.31

In the

DOPC/DPPC/cholesterol liposomes, most of cholesterol is presumably associated with the Lo phase, which consists of DPPC.

As previously mentioned, E5 did not adsorb to the

DPPC/cholesterol (80/20) Lo lipid bilayer.

That E5 interacts with only Ld domain of

DOPC/DPPC/cholesterol explains why Hill coefficients are similar for DOPC and DOPC/DPPC/cholesterol liposomes.

4. CONCLUSIONS


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In this study, the interactions of the E5/PAA-g-Dex complex with lipid membranes with various compositions were evaluated. In the absence of the liposomes, the cationic copolymer promotes folding of E5 at both pH 5.4 and pH 7.4. Liposomes having Ld phases also promoted folding of E5 into a partially folded structure with an N-terminal helix, while a liposome with Lo phase did not. At near neutral pH of 7.4, the membrane-disrupting activity of E5 peptide was induced by the cooperative interactions with the cationic copolymer and the liposomes with Ld phases, even when the liposomes had the phase separated structure. Meanwhile, there was no significant membrane disruption activity for the liposomes with Lo phase in the presence of the cationic copolymer.

This cooperative behavior allowed preservation of lipid membrane

selectivity of E5 even in the presence of the copolymer. The membrane disruptive activity was considerably enhanced by the copolymer at both acidic pH and pH 7.4. The result implied that not only E5 folding but also adsorption to liposome surface and/or association of E5 on the liposome was promoted by the copolymer. As reported previously, the copolymer prevented aggregation of E5. The hydrophilic dextran side chains of the copolymer play an important role in preventing the aggregation. Interestingly, the interaction between the copolymer and E5 did not inhibit but rather promoted the interaction with liposomes or the association of E5 on the liposome. Lack of structural integrity and solubility are common issues in biomedical applications of peptides. In this study, we demonstrated that the PAA-g-Dex cationic copolymer solves these issues, leading to considerable argumentation of E5 peptide activity without loss of its membrane selectivity. In addition, E5/PAA-g-Dex exhibited the activity for the liposomes with phase separated heterogeneous structure as a model of cellular membrane.

Understanding of the

copolymer-mediated augmentation for lipid membrane with various compositions would enable


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us to design copolymers with better chaperone activity and DDS with the membrane selectivity of such cell type and organelle. We envision that the chaperone effect of the copolymer can be harnessed to construct carriers to deliver macromolecular drugs into cells.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. CD spectra of E5 as a function of 5k95D concentration, Fitting of experimental curves of molar ellipticity of E5 for determination of dissociation constant, Maximum fluorescence wavelength for assessment the interaction between the lipid bilayer and E5. (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Tsukuru Masuda: 0000-0001-6452-811X Naohiko Shimada: 0000-0002-1664-1721 Atsushi Maruyama: 0000-0002-7495-2974 Author Contributions The manuscript was written through contributions of all authors.

All authors have given

approval to the final version of the manuscript.


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Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported in part by in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant No. 15H01807 to A.M. and Grant No. 18K18384 to T.M.) and a grant from the Center of Innovation (COI) Program, Japan Science and Technology Agency (JST) and by the Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists (No. 17J04783 to T.M. and No. 18J13337 to W.S.). REFERENCES 1.

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Bakht O.; Pathak P.; London E. Effect of the structure of lipids favoring disordered

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Page 22 of 28

(a) E5

+ H+

H - GLFEAIAEFIEGGWEGLIEG - OH

- H+

Hydrophilic face

Fusogenic pH (C-terminus helix folding)

High pH (N-terminus helix folding) lipid bilayer

Hydrophobic face

hinge

N-terminus helix

(b)

C-terminus helix

Figure 1. (a) A sequence, a helical wheel representation and a cartoon representation at fusogenic pH of E5 used in this study.13,14,26 (b) Structural formula of PAA-g-Dex.

Figure 1. (a) Left: The sequence and helical wheel representation of E5. The sequence from left to right is from N- to C-terminus, and H- and -OH refer to unmodified terminal alpha amino group and C-terminus as free carboxylic acid, respectively. Right: A cartoon representation of 13, 14, 22 folding E5 at fusogenic pH. (b) Structural formula of PAA-g-Dex. Table 1 ofHelicity (%) of E5 in various condition. 5.4

7.4

lipid (-)

DOPC (100)

lipid (-)

DOPC (100)

DOPC/cholesterol (80/20)

DOPC/DPPC/cholesterol (40/40/20)

DPPC/cholesterol (80/20)

2.9

35.1

-1.6

19.9

13.0

4.0

-3.1

29.2

36.2

22.3

33.5

35.3

37.9

24.3


22

Page 23 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. CD spectra of E5 in the absence and presence of 5k95D in various liposome formulations. CD spectra at (a) pH 5.4 in 100% DOPC, (b) pH 7.4 in 100% DOPC, (c) pH 7.4 in DOPC/cholesterol (80/20), (d) pH 7.4 in DOPC/DPPC/cholesterol (40/40/20), and (e) pH 7.4 in DPPC/cholesterol (80/20). Final concentrations were 0 or 2000 µM total lipid, 10 µM E5, and 0 or 11.2 µM 5k95D in 10 mM MES-NaOH (pH 5.4) or Tris-HCl (pH 7.4) containing 140 mM NaCl. Spectra were recorded at 25 °C.


23


(a) pH 5.4, DOPC (100) 5k95D

E5

E5

100 90 80 70 60 50 40 30 20 10 0 -10

[E5] (μM)

100 90 80 70 60 50 40 30 20 10 0 -10

2.5 1.25 0.5 0.25 0.175 0.1 0.075 0.05 0

0

600

1200

1800

2400

0.5 0.25 0.175 0.1 0.075 0.05 0

5k95D

E5

110 100 90 80 70 60 50 40 30 20 10 0 -10

150 300 450 600 750 900

time (s)

(d) pH 7.4, DOPC/DPPC/cholesterol (40/40/20)

(c) pH 7.4, DOPC/cholesterol (80/20)

[E5] (μM) 0.25 0.175 0.1 0.075 0.05 0

150 300 450 600 750 900

Leakage (%)

Leakage (%)

0.5

0

[E5] (μM)

0

time (s)

E5

5k95D

Leakage (%)

Leakage (%)

(b) pH 7.4, DOPC (100)

5k95D

[E5] (μM)

100 90 80 70 60 50 40 30 20 10 0 -10

0.5 0.25 0.175 0.1 0.05 0.025 0.0175 0.01 0

0

time (s)

150 300 450 600 750 900

time (s)

(e) pH 7.4, DPPC/cholesterol (80/20) E5

5k95D

100 90 80 70 60 50 40 30 20 10 0 -10

[E5] (μM)

Leakage (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 28

7 2.5 0.5 0

0

150 300 450 600 750 900

time (s)

Figure 3. Kinetics of Acid Red leakage as a function of E5 concentration in the absence and presence of 5k95D at (a) pH 5.4 in 100% DOPC, (b) pH 7.4 in 100% DOPC, (c) pH 7.4 in DOPC/cholesterol (80/20), (d) pH 7.4 in DOPC/DPPC/cholesterol (40/40/20), and (e) pH 7.4 in Fig. 4 Kinetics of sulforhodamineB leakage with an increase of E5 concentration in the absence and DPPC/cholesterol (80/20). concentrations were 2.5sulforhodamine µM total Acid Red-encapsulated lipid, presence of 5k95D at acidic (a)Final and neutral (b-e) condition. (2.5 µM B encapsulated lipid,µM 0-2.5 E5, µM E5, 5.6 µM mM MES-NaOH (pH 5.4) or HEPES-NaOH 7.4), 140 mM 0-2.5 and 5.65k95D, µM 10 5k95D in 10 mM MES-NaOH (pH(pH5.4) or Tris-HCl (pH 7.4) NaCl. containing 140 mM NaCl. Time course intensity were recorded at 25 °C.


24

Page 25 of 28

(b)

(a)

5k95D (+) 5k95D (-)

120 110 100 90 80 70 60 50 40 30 20 10 0 -10

Leakage (%)

Leakage (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

0.5

1

1.5

2

2.5

DOPC (100) DOPC/cholesterol (80/20) DOPC/DPPC/cholesterol (40/40/20)

110 100 90 80 70 60 50 40 30 20 10 0 -10 0

0.1

[E5] (μM)

0.2

0.3

0.4

0.5

[E5] (μM)

Fig. 5 Leakage assay from LUV composed with (a) DOPC (100) at acidic condition, or LUV

Figure 4. Leakage of Acid from LUVs asand a function of E5 concentration in the absence and composed with (b) DOPC (100),Red DOPC/Chol (80/20) DOPC/DPPC/Chol (40/40/20) at neutral presence 5k95D at (a) pHchange 5.4 inin 100% DOPC, (b) pH 7.4 in 100% DOPC, (c) pH 7.4 in condition,of with E5 concentration the absence and presence of 5k95D. (2.5 µM sulforhodamine B encapsulated lipid, 5.6 µM 5k95D, 10 mM MES-NaOH (pH 5.4) DOPC/cholesterol (80/20), (d) pH0-2.5 7.4 µM in E5, DOPC/DPPC/cholesterol (40/40/20), andor(e) pH 7.4 in Tris-HCl (pH 7.4), 140 mM NaCl.) DPPC/cholesterol (80/20). Final concentrations were 2.5 µM total Acid Red-encapsulated lipid, 0-2.5 µM E5, and 5.6 µM 5k95D in 10 mM MES-NaOH (pH 5.4) or Tris-HCl (pH 7.4) containing 140 mM NaCl. Table 2 Hill coefficient and EC50 of E5 in DOPC (100), DOPC/Chol (80/20), DOPC/DPPC/Chol (40/40/20).

DOPC (100) DOPC/Chol (80/20) DOPC/DPPC/Chol (40/40/20)

pH

5k95D

n

EC50 / μM

R2

5.4 5.4

+

2.8 3.3

0.70 0.15

0.79 0.78

7.4

+

3.4

7.4 7.4

+ +

5.8 3.8

0.15 0.18 0.14

0.97 0.88 0.93


25


Page 26 of 28

Table 1. Helicity (%) of E5 in various conditions. pH

5.4

[CP]0 / μM

lipid (-)

0 11.2

2.9 29.2

DOPC (100) 35.1 36.2

lipid (-) -1.6 22.3

DOPC (100) 19.9 33.5

DOPC/cholesterol (80/20) 13.0 35.3

7.4 DOPC/DPPC/cholesterol (40/40/20) 4.0 37.9

DPPC/cholesterol (80/20) -3.1 24.3


26

Page 27 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. Hill coefficients and EC50 of E5 in liposomes of various compositions.

DOPC (100) DOPC/cholesterol (80/20) DOPC/DPPC/cholesterol (40/40/20)

pH 5.4 5.4 7.4 7.4 7.4

5k95D + + + +

n 2.8 3.3 3.4 5.8 3.8

EC50 / μM 0.70 0.15 0.15 0.18 0.14

R2 0.79 0.78 0.97 0.88 0.93


27


Page 28 of 28

For Table of Contents Use Only Title: Cationic copolymers act as chaperones of a membrane-active peptide: Influence on membrane selectivity Authors: Wakako Sakamoto, Tsukuru Masuda, Takuro Ochiai, Naohiko Shimada, and Atsushi Maruyama*


28

Cationic Copolymers Act As Chaperones of a Membrane-Active

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