A Thermoresponsive Cationic Comb-Type Copolymer Enhances

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A Thermoresponsive Cationic Comb-Type Copolymer Enhances Membrane Disruption Activity of an Amphiphilic Peptide Tsukuru Masuda, Naohiko Shimada, and Atsushi Maruyama* School of Life Science and Technology, Tokyo Institute of Technology, B-57 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan S Supporting Information *

ABSTRACT: Membrane active peptides (MAPs) have potential applications in drug delivery systems and as antimicrobials. We previously showed that a cationic comb-type copolymer, poly(allylamine)-graf t-dextran (PAA-g-Dex), forms a soluble interpolyelectrolyte complex with an anionic MAP, the E5 peptide, resulting in significant enhancement of the membrane disruption activity of E5. In this study, we designed a novel comb-type cationic copolymer composed of a PAA main chain and thermoresponsive poly(N-isopropylacrylamide) graft chains (PAA-g-PNIPAAm). We hypothesized that the thermoresponsive hydrophilic/hydrophobic transition of the grafted polymer would regulate the membrane disruption activity of E5 peptide. Both the binding affinity of the complex and the membrane disruption activity of E5/PAA-gPNIPAAm were found to be enhanced above the phase transition temperature of the grafted chain. Our analysis suggests that the hydrophilic/hydrophobic environment around the cationic polymer chain plays important roles in the enhancement of the activity of the anionic peptide.

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INTRODUCTION Amphiphilic molecules or materials are of great interest in various research fields, including physical chemistry, materials science, and life sciences, due to the structures they form and their surfactant behavior.1−5 Among them, the amphiphilic membrane active peptides (MAPs), which are composed of hydrophilic and hydrophobic amino acids, have membrane permeabilizing activity and play important roles in lipid membrane recognizing processes.6−8 MAPs have been investigated as tools for delivery of proteins or antibacterial agents.9−11 For biological applications, peptides intrinsically have limitations such as instability, both chemically and structurally, and low solubility. Thus, the molecular design and function control have been increasingly important with the progress in investigations for their applications. The E5 peptide is a MAP that mimics the N-terminal end of hemagglutinin, the surface protein of influenza virus.12−14 E5 peptide undergoes a transition from random coil to α-helix when the pH decreases from neutral to acidic conditions. The amphiphilic α-helix structure plays an important role in membrane disruption activity at acidic pH (Figure 1). Because of the hydrophobic structure, the peptide is poorly soluble in aqueous solution, resulting in low membrane disruption activity under biologically relevant conditions. In previous studies, we have reported that a cationic comb-type copolymer, poly(allylamine)-graft-dextran (PAA-g-Dex), forms a soluble interpolyelectrolyte complex with E5 peptide and induces the coilto-helix transition of the peptide. Moreover, PAA-g-Dex © XXXX American Chemical Society

Figure 1. Sequence of E5, helical wheel diagram, and illustration of coil-to-helix transition in response to pH.

enhances the membrane disruption activity of E5 peptide not only at acidic pH but also at neutral pH.15,16 Considering that E5 peptide contains hydrophobic amino acid residues, the hydrophobic environment of the polymer is likely an important factor in the binding affinity of the copolymer to the anionic peptide and the coil-to-helix transition. We also found that the formation of the amphiphilic helix of E5 peptide was induced in the presence of liposomes.15 Thus, we hypothesized that increasing the hydrophobicity of the cationic copolymer would increase the binding affinity of the copolymer to the anionic peptide. In order to control hydrophilic/hydrophobic properties of the cationic copolymer, Received: February 6, 2018 Revised: March 7, 2018 Published: March 12, 2018 A

DOI: 10.1021/acs.biomac.8b00197 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules we focused on poly(N-isopropylacrylamide) (PNIPAAm), which exhibits hydrophilic/hydrophobic phase transition behavior in aqueous media across the lower critical solution temperature (LCST)17 and is suitable for hybridization with biomolecules.18,19 It would be useful if the enhancement of the membrane disruption activity of E5 peptide could be regulated by external stimuli, and we reasoned that in complex with this thermally responsive cationic copolymer the membranedisrupting function of E5 could be regulated by temperature. In this study, we designed a novel thermoresponsive cationic comb-type copolymer composed of PAA main chain and thermoresponsive PNIPAAm graft chains (PAA-g-PNIPAAm) to enhance the membrane disruption activity of E5 peptide. The target copolymer was prepared by activators regenerated by electron-transfer atom-transfer radical polymerization (ARGET ATRP). This controlled radical polymerization proceeds in aqueous media without deoxygenation at low Cu concentration,20−22 which is a suitable for preparation of biomaterials. We investigated the interaction between E5 peptide and PAA-g-PNIPAAm and the membrane disruption behavior induced by E5 and PAA-g-PNIPAAm complex. Above the phase transition temperature of the grafted chain, PAA-gPNIPAAm enhanced E5 membrane-disrupting activity. The effect of the hydration/dehydration transition of PNIPAAm on this activity is discussed.

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Scheme 1. Preparation of the Thermoresponsive Cationic Comb-Type Copolymer: (1) Conjugation of ATRP Initiator to PAA; (2) Preparation of PNIPAAm Graft Chain by ARGET ATRP

containing CuBr2 (6.3 mg, 0.028 mmol) and Me6TREN (74.9 μL, 0.28 mmol) was added to the solution, and the solution was stirred for 15 min to form CuBr2/Me6TREN complex as a catalyst for ATRP. Aqueous solution (1 mL) containing ascorbic acid (49.3 mg, 0.28 mmol) was added to the solution, and the polymerization was conducted at 25 °C for 1 h. The reaction solution was dialyzed against first against 10 mM aqueous HCl and then against water using a cellulose membrane (MWCO: 3500). The polymer was obtained by freeze-drying. The composition of the obtained polymer was analyzed by 1H NMR measurement (Avance 400, Buruker) using D2O as a solvent. The number-averaged molecular weight (Mn) and the polydispersity index (the weight-averaged molecular weight (Mw) over Mn, i.e., Mw/Mn) of the obtained polymer were determined by a gel permeation chromatography−multiangle light scattering (GPCMALS) (JASCO). An aqueous solution containing CH3COOH (0.5 M) and NaNO3 (0.2 M) was used as an eluent, and the flow rate was 0.8 mL/min. Measurement of the Phase Transition Behavior of the Thermoresponsive Polymer. The optical transmittance of the PAAg-PNIPAAm was monitored at 500 nm using a UV−vis spectrophotometer (UV-1650 PC, Shimadzu) equipped with a Peltier temperature controller at scanning rate of 0.2 °C min−1. The hydrodynamic size was estimated by dynamic light scattering (DLS) using a ZetaSizer Nano ZS (Malvern; Worcestershire, UK). The polymer solution (5 mg mL−1) was prepared in 10 mM HEPES buffer (pH 7.4) containing 140 mM NaCl. Measurement of Circular Dichroism Spectra. The circular dichroism (CD) spectrum of E5 peptide (10 μM) was measured in 10 mM Tris-HCl (pH 7.4) containing 140 mM NaCl using a CD spectropolarimeter (J-820, Jasco; Tokyo, Japan) equipped with a Peltier temperature controller. Samples were placed in 5 mm quartz cells, and data were averaged over eight accumulations. Measurement of Fluorescence Spectra of E5 Peptide. The fluorescence spectra (λex = 280 nm) of E5 peptide (10 μM) in 10 mM HEPES (pH 7.4) containing 140 mM NaCl were measured using a spectrofluorometer (FP-6500, Jasco) in the presence and the absence of PAA-g-PNIPAAm. The temperature was controlled by using a Peltier temperature controller. Measurement of Leakage from Liposomes. The large unilamellar vesicles (LUVs) of DOPC containing CF as a fluorescence probe were prepared as follows. A dry lipid film of DOPC was hydrated with 3 mL of 10 mM HEPES (pH 7.4) containing 50 mM CF and 73 mM NaCl with vortex mixing for 2 min at 25 °C. The LUVs were extruded through a polycarbonate membrane (pore diameter: 0.1 μm). The free lipid and CF were removed by gel permeation chromatography on a Sephadex G-25 M column using 10 mM HEPES (pH 7.4) containing 140 mM NaCl as an eluent. The LUVs were suspended in 10 mM HEPES (pH 7.4) containing 140

EXPERIMENTAL SECTION

Materials. Poly(allylamine hydrochloride) (PAA; Mw: 5 × 103) was kindly provided by Nitto Boseki Co. (Tokyo, Japan) in aqueous solution; it was purified by precipitation from methanol. NIsopropylacrylamide (NIPAAm) was kindly provided by KJ Chemicals (Tokyo, Japan) and was purified by recrystallization in toluene/ hexane. 1-(3-(Dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) was purchased from Nacalai Tesque (Kyoto, Japan). E5 peptide was purchased from Genenet (Fukuoka, Japan). The concentration of E5 peptide in aqueous solution was determined by UV−vis spectroscopy (UV-1650PC, Shimadzu, Kyoto, Japan) using a molar extinction coefficient of 5500 M−1 cm−1 at 280 nm (expressed as tryptophan). Carboxyfluorescein (CF) was purchased from Tokyo Chemical Industry (Tokyo, Japan). 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) was purchased from NOF Corporation (Kawasaki, Japan). All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan). PAA-g-Dex was prepared and characterized as reported previously.16 The molecular weight of PAA main chain and the dextran content of PAA-g-Dex used in this study were 5K and 93 wt %, respectively. Preparation of ATRP Initiator-Conjugated PAA (PAA-I). The initiator-conjugated PAA was prepared as shown in Scheme 1(1). 2Bromoisobutyric acid (BIBA) (535 mg, 3.21 mmol), N-hydroxysuccinimide (NHS; 369 mg, 3.21 mmol), and EDC (768 mg, 4.01 mmol) were dissolved in DMF (5 mL), and the solution was stirred at 25 °C for 1 h to activate the carboxyl group of BIBA. PAA (1.0 g, 10.7 mmol of amino group) was dissolved in water (2 mL), the solution pH was adjusted to 8.0 by adding 1 M aqueous NaOH, and then 0.2 M borate buffer (pH 8.0) (6 mL) was added to the solution. DMF solution containing the activated BIBA was added to the aqueous solution containing PAA. After 4 h at 25 °C, the solution was dialyzed against water using a cellulose membrane (MWCO: 1000), and then the polymer was obtained by freeze-drying. The obtained polymer was dissolved in 1 M aqueous HCl and purified by precipitation from ethanol. The content of BIBA conjugated to PAA was determined by 1 H NMR measurement (Avance 400, Bruker, Rheinstetten, Germany) using D2O as a solvent. Preparation of PAA-g-PNIPAAm by ARGET ATRP. Preparation of the PNIPAAm graft chain was accomplished as shown in Scheme 1(2). PAA-I (322 mg, 0.28 mmol of initiator) and NIPAAm (2.8 mmol) were dissolved in water (38 mL). An aqueous solution (1 mL) B

DOI: 10.1021/acs.biomac.8b00197 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules mM NaCl (final concentration: 2.5 μM). The fluorescence intensities (λex = 490 nm and λem = 520 nm) of the suspensions in the presence of PAA-g-PNIPAAm and/or E5 peptide were measured using a spectrofluorometer (FP-6500, Jasco) as a function of time. The leakage was defined as follows: leakage (%) =

F(t ) − F(0) × 100 F(∞) − F(0)

where F(0), F(t), and F(∞) are the initial, the intermediate, and the final fluorescence intensities, respectively. The final fluorescence intensity was determined by measuring the fluorescence spectra of the suspension after rupturing of LUVs by adding Triton X-100. The dependence of E5 concentration on the kinetics of the leakage was analyzed as follows. The initial period of the leakage profile as a function of time was used to fit the following equation:

leakage (%) = 100 × [1 − exp(−kobst )] where kobs is the observed rate constant. The dependence of the kobs values on the E5 peptide concentration can be expressed as follows: ⎛ [E5] ⎞n kobs = A⎜ ⎟ ⎝ [lipid] ⎠ where n is the degree of the reaction, A is a constant, and [E5] and [lipid] correspond to the concentrations of E5 peptide and the lipid (DOPC), respectively. Thus, the following equation is obtained:

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⎛ [E5] ⎞ ln kobs = n ln⎜ ⎟ + ln A ⎝ [lipid] ⎠

Figure 2. (A) Temperature dependence of the optical transmittance of PNIPAAm and of PAA-g-PNIPAAm (5 mg mL−1) in 10 mM HEPES (pH 7.4) containing 140 mM NaCl. Inset: images of the (a) PNIPAAm and (b) PAA-g-PNIPAAm solutions above LCST. The aqueous solution were incubated at 37 °C. (B) Temperature dependence of the z-averaged size of PAA-g-PNIPAAm (5 mg mL−1) in 10 mM HEPES (pH 7.4) containing 140 mM NaCl.

RESULTS AND DISCUSSION Preparation and Characterization of the Thermoresponsive Cationic Comb-Type Copolymer. The target thermoresponsive cationic comb-type copolymer was prepared by conjugating ATRP initiator to PAA, followed by preparation of PNIPAAm grafted polymer by ARGET ATRP (Scheme 1). The ratio of AA units to Initiator to NIPAAm units was estimated to be 100:9:421 by 1H NMR (Figure S1). Thus, the number-averaged molecular weight (Mn) of PNIPAAm and the PNIPAAm contents were 1.0 × 104 and 94.3 wt %, respectively. In addition, Mn and Mw/Mn for the PAA-g-PNIPAAm estimated by GPC-MALS were 1.2 × 105 and 1.49, respectively (GPC profile shown as Figure S2). For the PAA-g-PNIPAAm comb-type copolymer, we first investigated the thermoresponsive behavior. Figure 2A shows temperature dependency of the optical transmittance for the PAA-g-PNIPAAm and PNIPAAm homopolymer solutions (5 mg mL−1) in 10 mM HEPES (pH 7.4) containing 140 mM NaCl. Above the LCST of PNIPAAm, a decrease in the optical transmittance of PAA-gPNIPAAm was observed as a function of temperature, which is attributed to the aggregation of dehydrated PNIPAAm chain. Above LCST, the transmittance of the PAA-g-PNIPAAm solution was larger than that of PNIPAAm solution, suggesting that PAA-g-PNIPAAm dispersed in the aqueous media. In addition, the thermoresponsive behavior of PAA-g-PNIPAAm was also investigated by DLS (Figure 2B; the details of the experiment are given as Figure S3). The sizes of the particles significantly increased with temperature to a maximum of 134 nm at 35 °C. We hypothesize that PAA-g-PNIPAAm forms a micelle-like structure as the PNIPAAm chain aggregates due to hydrophobic interactions above LCST with the cationic PAA acting as the hydrophilic segment. Interaction between E5 Peptide and the Thermoresponsive Cationic Comb-Type Copolymer. To investigate

the effect of PAA-g-PNIPAAm on the structure of E5 peptide at pH 7.4, the CD spectra of E5 peptide were monitored in the presence and absence of PAA-g-PNIPAAm at 25 °C, which is below the LCST. At pH 7.4, E5 peptide alone was unstructured without PAA-g-PNIPAAm (Figure 3A), which is in agreement with the previous report.15 We also confirmed that E5 peptide was unstructured in the presence of PNIPAAm homopolymer (Figure S4). In contrast, the CD spectrum of E5 peptide in the presence of PAA-g-PNIPAAm had minima at 208 and 222 nm (Figure 3A). As the concentration of PAA-g-PNIPAAm was increased, the molar residue ellipticity at 222 nm (θ222) decreased (Figure 3B). These results indicated that E5 formed a soluble inter-polyelectrolyte complex with PAA-g-PNIPAAm, resulting in the coil-to-helix transition of the E5 peptide at pH 7.4. For further investigation of the interaction between E5 peptide and the cationic copolymer, the fluorescence spectra derived from the tryptophan (W) residue in the E5 peptide were monitored below and above the LCST of PNIPAAm (25 and 35 °C, respectively). Note that the fluorescence spectra also confirmed that there was no interaction between E5 peptide and PNIPAAm homopolymer (Figure S5). As the concentration of PAA-g-PNIPAAm was increased, a blue-shift in the fluorescence from W was observed (Figure S6). Figure 4 shows the peak wavelength in the fluorescence from W as a function of the cationic copolymers. The peak wavelength shift as a function of the PAA-g-PNIPAAm concentration at 25 °C (Figure 4B) was similar to the θ222 change monitored by CD at C

DOI: 10.1021/acs.biomac.8b00197 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 3. (A) CD spectra of E5 peptide at the indicated concentrations of PAA-g-PNIPAAm in aqueous media at pH 7.4. The peptide concentration was 10 μM. The concentrations of the copolymer correspond to the concentrations of amino groups of AA unit. (B) Relationship between the concentration of PAA-g-PNIPAAm and θ222. Temperature: 25 °C.

Figure 4. Peak wavelength of the fluorescence spectrum of the W residue of E5 peptide as a function of the concentration of (A) PAA-gPNIPAAm and (B) PAA-g-Dex in 10 mM Tris (pH 7.4) containing 140 mM NaCl. The concentrations of the copolymer correspond to the concentrations of amino groups of AA unit.

25 °C (Figure 3B). These results indicate that the environment around W becomes more hydrophobic as the cationic copolymer concentration is increased; the W residue is on the hydrophobic side of the α-helix structure adopted by E5 peptide. The blue-shift in the spectrum of E5 was larger when PAA-gPNIPAAm was added above LCST than that below LCST; this was not the case when E5 was incubated in the presence of the nonthermoresponsive cationic copolymer PAA-g-Dex. This result suggests that the environment around E5 peptide is more hydrophobic in the complex of E5 and PAA-g-PNIPAAm at 35 °C than at 25 °C. By analyzing the wavelength shift as a function of the concentration of the cationic copolymer, the binding affinity between E5 and the cationic copolymers was estimated (Figure S7). Table 1 summarizes the equilibrium dissociation constant (Kd) values of E5 peptide and the cationic copolymers. For PAA-g-Dex, the Kd value at 35 °C was slightly larger than that at 25 °C. In contrast, for PAA-g-PNIPAAm, the Kd value at 35 °C was smaller than that at 25 °C, indicating that the binding affinity between E5 and PAA-g-PNIPAAm is higher at 35° than at 25 °C. When the PNIPAAm chains become dehydrated above LCST, the environment around the cationic main chain becomes hydrophobic, resulting in an increase in the interaction between the cationic copolymer and the hydrophobic residues of E5 peptide. In addition, electrostatic interactions are enhanced (or ion pairs become less dissociated) in hydrophobic or low polarity conditions.23−26 Previous studies of thermoresponsive polymer materials indicates that a weakly acidic group copolymerized in a thermoresponsive polymer has reduced acidity above the

Table 1. Dissociation Constants for Interaction between E5 Peptide and the Cationic Copolymers at 25 and 35 °C Kd/μM polymer

25 °C

35 °C

PAA-g-PNIPAAm PAA-g-Dex

23.0 27.5

5.9 32.3

LCST.25,26 In our system, the electrostatic interactions between E5 peptide and PAA-g-PNIPAAm are likely stronger at 35 °C than at 25 °C. It is also possible that there is an increase in the charge density of the cationic copolymer PAA-g-PNIPAAm above the LCST due to the dehydration and aggregation of the PNIPAAm chain, resulting in an increase in the binding affinity between E5 and PAA-g-PNIPAAm. Considering that complex formation between E5 and the cationic copolymer plays an important role in the membrane disruption,15,16 the difference in the binding affinities between E5 and PAA-g-PNIPAAm below and above LCST should influence the membrane disruption activity. Membrane Disruption Activity of E5 Peptide in the Presence of PAA-g-PNIPAAm. The membrane disruption activity of E5 peptide was monitored by using the selfquenching dye CF. CF-loaded liposomes have been used previously for detection of efflux from liposomes.27 Figure 5 shows the leakage behavior of CF from liposomes induced by E5 and the cationic copolymer (PAA-g-Dex or PAA-gPNIPAAm) at near-neutral pH. E5 alone did not induce the leakage from liposomes; however, E5 in the presence of the cationic copolymer did. Note that the cationic copolymer alone D

DOI: 10.1021/acs.biomac.8b00197 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 5. Membrane disruption activity of E5 with PAA-g-PNIPAAm in 10 mM HEPES (pH 7.4) containing 140 mM NaCl. E5 concentration: 0.3 μM. PAA-g-PNIPAAm concentration: 15 μM.

Figure 6. Effect of E5 peptide concentration on the liposome membrane disruption activity induced by PAA-g-PNIPAAm in 10 mM HEPES (pH 7.4) containing 140 mM NaCl. E5 concentration: 0.05, 0.1, 0.3, or 0.5 μM. PAA-g-PNIPAAm concentration: 15 μM.

had no membrane disruption activity (Figure S8 and the dotted lines in Figure 5). These results suggest that both PAA-g-Dex and PAA-g-PNIPAAm induce folding and activation of E5 peptide, resulting in efflux of CF from liposomes. The leakage induced by E5/PAA-g-Dex decreased at 35 °C (Figure 5A). In contrast, E5/PAA-g-PNIPAAm induced significantly more leakage at 35 °C than at 25 °C (Figure 5B). These temperature-dependent leakage behaviors can be understood based on the binding affinity between E5 peptide and cationic copolymer. For PAA-g-Dex, the leakage decreased at 35 °C relative to 25 °C due to the lower affinity between E5 and the polymer at the higher temperature. On the other hand, for PAA-g-PNIPAAm, the binding affinity increased above the LCST of PNIPAAm, resulting in an increase in the leakage at the higher temperature. To understand the membrane disruption induced by the complex of E5 with PAA-g-PNIPAAm, the effect of the E5 concentration on the membrane disruption was investigated both below and above LCST. Figure 6 shows the membrane disruption behaviors under various concentrations of E5 peptide at 25 and 35 °C. At both temperatures, the leakage increased with an increase in E5 peptide concentration. Figure 7 shows the dependency of the kobs values on E5 concentration: The rate constant kobs was proportional to the power of the concentration of E5 peptide. The power indexes (n values) at 25 and 35 °C were estimated to be 2.0 and 1.8, respectively.

Figure 7. Relationship between E5 concentration and kobs at 25 °C (black circles) and at 35 °C (white circles). R2 values at 25 and 35 °C were 0.9674 and 0.9795, respectively.

Thus, the kinetics of the membrane disruption strongly depend on E5 concentration, and there is positive cooperativity in the membrane disruption behavior induced by E5/PAA-g-PNIPAAm both below and above LCST. Thus, our data indicate that the assembly of multiple E5 peptides induces the membrane disruption. The kobs values at 35 °C were larger than those of 25 °C, suggesting that the affinity of the E5/PAAg-PNIPAAm complex for the membrane at 35 °C is higher than that at 25 °C. In addition, at lower peptide concentration ([E5] E

DOI: 10.1021/acs.biomac.8b00197 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules < 0.3 μM), the quantity of membrane disruption induced by E5/PAA-g-PNIPAAm at 35 °C was found to be larger than that at 25 °C, which is attributed to the higher kinetics of membrane disruption above LCST. Our data demonstrate that the membrane disruption activity of E5 peptide induced by thermoresponsive cationic copolymer can be regulated by temperature. This thermoregulation will be useful in applications of amphiphilic peptides such as drug delivery. In a previous study, we demonstrated that the membrane disruption activity of the E5/PAA-g-Dex complex allowed the transport of the fluorescence-labeled bovine serum albumin as a model macromolecule through the cellular membrane.16 By employing the thermoresponsive cationic copolymer, by coupling with the hyperthermia system, the membrane disruption activity of the E5/PAA-g-PNIPAAm complex would be locally enhanced, allowing the tumor-site specific drug transport. Furthermore, the regulation of the binding affinity in the soluble inter-polyelectrolyte complex coupled with the phase transition of PNIPAAm grafted chain would be applicable to other ionic biomolecules. We envision that rationally designed thermoresponsive cationic comb-type copolymers will be used to regulate the function of ionic biomolecules.

ORCID

Tsukuru Masuda: 0000-0001-6452-811X Atsushi Maruyama: 0000-0002-7495-2974 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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.), by 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.).

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CONCLUSIONS In this study, a novel cationic comb-type copolymer with thermoresponsive grafted chain (PAA-g-PNIPAAm) was designed to regulate the activity of the amphiphilic E5 peptide through coupling with the hydrophilic/hydrophobic transition of the grafted chain. CD spectra measurement revealed that E5 peptide formed an inter-polyelectrolyte complex with PAA-gPNIPAAm, resulting in coil-to-helix transition of E5 peptide at neutral pH. Furthermore, the interaction between E5 peptide and PAA-g-PNIPAAm at 35 °C (above LCST) was stronger than that at 25 °C (below LCST). These results suggest that the hydrophobicity of the environment around the cationic polymer chain influences complex formation. E5 peptide in the presence of PAA-g-PNIPAAm induced the leakage from liposomes, whereas E5 peptide alone did not. Importantly, E5/PAA-g-PNIPAAm induced more significant leakage at 35 °C than at 25 °C. The leakage behavior was strongly dependent on the concentration of the E5 peptide, and the analysis of the leakage kinetics revealed that the affinity of the E5/PAA-gPNIPAAm complex for the membrane at 35 °C is higher than that at 25 °C. We envision that thermoresponsive cationic comb-type copolymers will be useful materials for regulation of the functions of the amphiphilic peptides and will find applications in drug delivery systems.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.8b00197. 1 H NMR spectra and a GPC profile of the copolymers, DLS measurements, interaction between E5 peptide and the copolymers, and the leakage behavior from liposomes (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (A.M.). F

DOI: 10.1021/acs.biomac.8b00197 Biomacromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.biomac.8b00197 Biomacromolecules XXXX, XXX, XXX−XXX