Enhancing Membrane-Disruptive Activity via Hydrophobic

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Biological and Medical Applications of Materials and Interfaces

Enhancing Membrane-Disruptive Activity via Hydrophobic Phenylalanine and Lysine Tethered to Poly(aspartic acid) Bo Liu, Qifa Zhang, Fang Zhou, Lixia Ren, Yunhui Zhao, and Xiaoyan Yuan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b22721 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 1, 2019

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ACS Applied Materials & Interfaces

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Enhancing Membrane-Disruptive Activity via

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Hydrophobic Phenylalanine and Lysine Tethered to

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Poly(aspartic acid)

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Bo Liu, Qifa Zhang, Fang Zhou, Lixia Ren,* Yunhui Zhao and Xiaoyan Yuan*

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School of Materials Science and Engineering, and Tianjin Key Laboratory of Composite and

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Functional Materials, Tianjin University, Tianjin 300350, China

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KEYWORDS: membrane-disruptive activity; hydrophobic amino acids; poly(aspartic acid);

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pH-response; intracellular delivery

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ABSTRACT: Amphiphilic polymers with pH-responsive abilities have been widely used as

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carriers for intracellular delivery of bioactive substances, while their membrane-disruptive

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activity exerted on cells is a critical characteristic that determines the delivery efficiency. Herein,

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we present a novel method to prepare amphiphilic and pH-responsive polymers by chemically

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tethering L-phenylalanine methyl ester and followed by Nε-carbobenzyloxy-L-lysine benzyl ester

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to the side carboxylic acid groups of poly(aspartic acid). The obtained phenylalanine- and lysine-

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grafted polymer (PAsp-g-Phe)-g-Lys demonstrated enhanced membrane-disruptive activity at pH

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7.4 in comparison with PAsp-g-Phe. Moreover, the pH-responsive behavior of the grafted

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polymers caused by the significantly intensified hydrophobicity could be modulated by the

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tethered amount of hydrophobic amino acids with phenyl groups. The prepared amphiphilic

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(PAsp-g-Phe)-g-Lys could facilitate entry of calcein into NIH/3T3 and HeLa cells at the

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physiological pH values, possibly due to locally chemical destabilization of cell membranes by

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the interaction between the polymer and membrane bilayers. Therefore, we have provided a

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feasible approach to prepare pH-responsive polymers with enhanced membrane-disruptive

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activity, and the phenylalanine and lysine-grafted polymers could be a potential candidate for

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intracellular delivery of bioactive molecules in biomedical applications.

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INTRODUCTION

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Similar with traditional small substances, bioactive macromolecules such as plasmid DNA,

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mRNA, and antibodies can be introduced into cytoplasm by amphiphilic polymers through a

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diverse range of intracellular delivery methods.1-4 However, efficient cytoplasmic delivery of

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therapeutic agents, especially macromolecular proteins and nucleic acids, remains a key

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challenge due to the weak interaction of the polymers with cell membranes and possible

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degradation of cargo by enzymes.5,6 One of the most important approaches for intracellular

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delivery is disruption of the cell membrane to facilitate entry of cargo.2 Lots of membrane-

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disruptive methods have been explored to facilitate cytoplasmic delivery of bioactive molecules

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in past decades, including mechanical, electrical, optical and chemical strategies.2,7-10 Meanwhile,

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amphiphilic and pH-responsive polymers have been widely used as intracelluar delivery carriers,

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and the membrane-disruptive activity of the polymers is a critical characteristic that determines

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the delivery efficiency of cargo.1,2

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Among biodegradable carriers, stimuli-responsive polymers have attracted great attention in

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intracellular delivery of therapeutic agents.11-13 Some of the stimuli-responsive polymers, such as

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poly(ethylene imine), poly(L-lysine) (PLL), and poly(N,N-dimethylamino ethyl methacrylate)


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containing cationic groups, can be used for the mild negative cargo delivery.14,15 However, the

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cationic polymers generally suffer from cytotoxicity and relatively lower transduction

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efficiencies.16 In order to improve the transfection efficiency of PLL, “molecular string” p-

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toluylsulfonyl arginine (RT) was introduced onto the PLL backbone.17 Multiple interactions

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between the novel polycationic carrier PLL-RT and the cell membrane, including electrostatic

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interaction, hydrogen bond interaction and hydrophobic interaction, were beneficial to enhance

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the gene transfection.17

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Amphiphilic and pH-responsive polymers with biocompatibility and biodegradability carrying

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weakly ionizable groups such as anionic groups can respond to pH variation due to the

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protonation or deprotonation equilibrium in the aqueous solution,18-20 via mimicking the

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amphiphilic structure and the pH-dependent membrane-disruptive behavior of fusogenic viral

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peptides.21,22 Amphipathic cell-penetrating peptides can induce membrane penetration through

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the interactions of hydrophobic and hydrophilic groups with the membrane bilayer.23,24 A series

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of biodegradable poly(L-lysine iso-phthalamide) pseudopeptides with hydrophobic backbone and

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pendant carboxyl groups were ever reported by Slater et al., showing a pH-sensitivity and weak

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cell membrane-disruptive capacity.25,26 Modification of the pseudopeptides by grafting

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hydrophobic monomers, was conducted to improve the cell membrane-disruptive activity with

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almost trivial hemolysis at the physiological pH 7.4 value.22,26-31 Furthermore, the lysine-based

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hyperbranched polymers demonstrated pH-induced considerable membrane destabilization.32 It

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was considered that the amphiphilic and pH-responsive polymers with membrane-disruptive

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activity exhibited efficient cytoplasmic drug delivery.

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Amphipathic negative poly(amino acid)s and their derivatives were also candidates in the

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cytoplasmic delivery on account of their biodegradability and biocompatibility.33-37 In view of


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the improvement of the interaction between the polymer and the cells for intracellular delivery of

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biological molecules, poly(amino acid) derivatives were usually prepared by hydrophobic

3

modification with hydrophobic amino acids, aliphatic hydrocarbons, aromatic compounds and so

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on. Akagi et al. reported a series of poly(γ-glutamic acid) (γ-PGA) derivatives using various

5

types of hydrophobic amino acids and focused on the relationship between the hydrophobicity of

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side groups of γ-PGA and the cell membrane.38 Ma et al. synthesized temperature and pH-

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sensitive polyaspartamide derivatives containing pendant aromatic structures and ionizable

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tertiary amino groups for antitumor drug delivery.39

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Natural-occurring poly(aspartic acid) (PAsp) is plentiful, biodegradable, and conveniently

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available. As inspired by the publications above, in this study, amphiphilic and pH-responsive

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polymers were synthesized by chemically tethering two hydrophobic amino acids,

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phenylalanine methyl ester hydrochloride (Phe) and Nε-carboxyl-L-lysine benzyl ester

13

hydrochloride (Lys), to the side carboxylic acid groups of PAsp in sequence. The effect of Phe

14

and Lys on the pH-responsive properties of the obtained polymers and membrane destabilization

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were investigated. The cytotoxicity and ability of the prepared polymers to improve drug model

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entry into cells were also discussed by using NIH/3T3 and HeLa cells.

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EXPERIMENTAL SECTION

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L-

Materials. PAsp (average molecular weight 4500) was purchased from Shanghai Yiji

19

Biological

Reagent

Co.,

Ltd.,

China.

Calcein,

Hoechst

33342,

1-ethyl-3-(3-

20

dimethylaminopropyl) carbodiimide hydrochloride (EDCI), Phe and Lys were provided by

21

Tianjin Heowns Biochemical Technology Co., Ltd., China. Sodium hydroxide (NaOH), sodium

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hydrogen carbonate (NaHCO3), anhydrous ethanol (EtOH) and other solvents were supplied by


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Kermel Chemical Technology Co., Ltd., China. Texas Red hydrazide and LysoTracker Red

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DND-99 were purchased from Fisher Scientific (China). Fluorescein isothiocyanate-dextran

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(FITC-dextran, 4 kDa), Deuterium dimethyl sulfoxide (DMSO-d6) and deuterium oxide (D2O)

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were purchased from Sigma Aldrich (Energy Chemical, China). Dulbecco’s Modified Eagle

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Medium (DMEM) and fetal bovine serum, certified (FBS) were purchased from Gibco. Alamar

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Blue cell viability reagent was provided by Shanghai BestBio biotechnology Co., Ltd., China.

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All chemicals were used directly as received without further purification unless mentioned.

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Scheme 1.

Synthesis of PAsp-g-Phe (PF) and (PAsp-g-Phe)-g-Lys (PFK)

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Synthesis. As illustrated in Scheme 1, two modified polymers were prepared by the sequential

11

reactions of PAsp with Phe and Lys, adopted from the reference,38 and the products were

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designated as PAsp-g-Phe (PF) and (PAsp-g-Phe)-g-Lys (PFK), respectively. In detail, PAsp

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(1.0 g, 8.7 mmol [COOH]), Phe (2.25 g, 10.4 mmol [NH2]) and EDCI (1.2 molar equivalents of

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[COOH]) were dissolved in a 50 mM NaHCO3 (50 mL) to prepare PF. The reaction was allowed

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to proceed at 25 °C for 72 h. NaOH in anhydrous ethanol (5 wt%, 30 mL) was then added to

16

fulfill ester hydrolysis. Then, the product PF was further reacted with Lys to synthesize PFK in


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the same way. The resultants, PF and PFK, were purified via dialysis (molecular weight cut-off

2

1000) against deionized water for 3 days and lyophilized for 24 h before further uses. The

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detailed feed ratios and compositions of PF and PFK are shown in Table 1.

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Characterization. Chemical structures of PF and PFK were verified by analyses of 1H

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nuclear magnetic resonance (NMR, Bruker AV 400 MHz, Germany) and Fourier transform

6

infrared spectroscopy (FTIR, Perkin-Elmer Spectrum 100, USA). The 1H NMR measurement

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was performed via dissolving 5 mg of the specimen in DMSO-d6 or D2O. The FTIR spectra were

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obtained in the range from 4000 cm-1 to 400 cm-1 using the KBr pellet technique. The molecular

9

weight of PF and PFK, calculated by the integral of Phe and Lys signals via 1H NMR spectra

10

and the molecular weight of PAsp, are also shown in Table 1.

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Table 1. Compositions and Molecular Weights of the Synthesized PF and PFK

Abbreviation

12

a)

13 14

Sample

Feed ratio

Composition a)

[COOH]:[Phe]:[Lys]

[COOH]:[Phe]:[Lys]

(mol:mol:mol)

(mol:mol:mol)

Molecular weight b)

PF

PAsp-g-Phe

1:1.2:0

39:28: 0

8.6103

PFK

(PAsp-g-Phe)-g-Lys

1:1.2:1.2

39:28:16

1.3104

Stoichiometric molar ratio of Phe and Lys relative to the carboxylic acid group along the backbone of the original poly(aspartic acid).

b)

The molecular weights of PF and PFK were calculated via 1H NMR spectra by the integral

15

ratio of Phe and Lys signals and the original molecular weight of poly(aspartic acid).

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The optical transmittance of the polymer solution at 480 nm wavelength was acquired on a

17

UV-vis-NIR spectrophotometer (UV-3600 Plus, Shimadzu, Japan) using an absorption cell with


6

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a 4 cm path length.26,32 The zeta potentials and size distribution were measured by a dynamic

2

light scattering (DLS) method using a laser light scattering spectrometer (Zetasizer Nano Zs90,

3

Malvern, UK).30,40 The polymer solutions in the buffer at pH 3.07.4 were used for the DLS

4

measurement which was performed at an angle of 90º in a 10 mm diameter cell at 25 ºC. Eleven

5

scans were run for each measurement which was repeated in triplicate. The polymer solution at

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1.0 mgmL-1 was prepared in the buffer at a specific pH value and allowed to equilibrate for 48 h

7

before tests. The morphology of the polymer in solution was recorded using a transmission

8

electron microscope (TEM, JEM-2100f, Japan) at an accelerating voltage of 200 kV. The

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polymer solution in 10 µL was dropped on a copper grid coated with a carbon film and the

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sample was dried in air overnight before TEM observation.

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Hemolysis assay. Sterile defibrinated sheep red blood cells (RBCs) were provided by

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Guangzhou Future Biotechnology Co., Ltd., China, and were washed with single strength PBS

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solution (306 mOsm, pH 7.4, preparation of the buffer solution shown in the Supporting

14

Information) for three times. Then, RBCs were resuspended in polymer solutions at different

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pHs from 3.0 to 7.4. After being incubated at 37 ºC for 2 h, the optical density (OD) values of

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hemoglobin release were measured at 541 nm using the microplate reader (TECAN,

17

Switzerland). The sample of RBCs in buffers at the special pH value was taken as the negative

18

control, and the sample of RBCs lysed with deionized water was used as the positive control.

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The relative hemolytic rate of each sample was calculated by the following equation (1).22

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ODSample - ODNegative control

Hemolysis (%) = ODPositive control - ODNegative control × 100

(1)

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Membrane-disruptive activity. Hemolysis assay of RBCs was used as a model test to

22

examine the membrane-disruptive activity of the polymers. After washed with single strength

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PBS (306 mOsm, pH 7.4) for three times, RBCs (107 cells∙mL-1) were resuspended and cultured


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at 37 ºC for 30 min in the buffer solution (pH = 7.4) containing PF or PFK (0.1 mgmL-1), 10

2

µM FTIC-dextran and 1.5 µM Texas Red hydrazide. RBCs were cultured without polymers as

3

control. The images of cells were recorded using a confocal laser scanning microscope (CLSM,

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Leica SP8, Germany) at the excitation wavelengths of 488 nm for FTIC-dextran and 543 nm for

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Texas Red hydrazide, respectively.30,32

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RBCs before and after treatment with the polymer were also observed with a scanning electron

7

microscope (SEM, Hitachi su1510, Japan). The washed RBCs in 50 µL were added to 1 mL

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polymer solution (0.1 mgmL-1). After being incubated at 37 °C for 2 h, the RBCs suspensions

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were dropped onto the slide, followed by fixation with 2.5% glutaraldehyde in PBS overnight.

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The fixed RBCs were dehydrated with a series of graded ethanol solutions (30, 50, 60, 70, 80 90,

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95 and 100%, 15 min each), and then dried in air. The samples were gold-coated prior to SEM

12

observation.41

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Cytotoxicity. The cytotoxicity of the polymers was examined by using NIH/3T3 fibroblasts

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and cancerous HeLa cells via Alamar Blue assay.29,30,42 Cells were cultured in DMEM containing

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10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin in a humidified

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incubator with 5% CO2 at 37 °C for 24 h, and then collected by centrifugation at 1000 rpm for 3

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min and suspended in the medium. NIH/3T3 and HeLa cells were seeded into the 96-well plate

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(Corning, USA) containing medium (0.2 mL per well) at a density of 1×104 cells per well for 24

19

h, respectively. The outspent medium was replaced with 0.2 mL of the 0.22 μL filter sterilized

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serum-free DMEM containing PF or PFK at specific concentrations. After 24 h of incubation,

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the polymer-contained medium was replaced with the complete DMEM composed of 10% (v/v)

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Alamar Blue reagent. The plate was further incubated for 4 h according to the manufacturer’s

23

instruction, and the fluorescence of each well was measured using a microplate reader at the


8

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excitation wavelength of 525 nm and emission wavelength of 590 nm. The relative cell viability

2

was calculated from the fluorescence intensity reading values with the following equation (2).

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I590, Sample

Relative cell viability (%) = I590, Positive control × 100

(2)

4

where, the medium without polymer was used as the positive control, and each sample was

5

repeated in triplicate.

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Cytoplasmic delivery. Calcein as a model molecule was employed to evaluate the membrane-

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disruptive activity of PFK and cell entry in comparison with PF.43 Typically, 1 mL of NIH/3T3

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or HeLa cells suspension (1×105 cells∙mL-1) was seeded into a 20 mL glass-bottom culture dish

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and incubated for 24 h. The spent medium was then replaced by 1 mL of serum-free medium

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containing the polymer (0.1 mgmL-1) and calcein (2 mgmL-1). The cells were cultured with 2

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mgmL-1 calcein alone as control. After treatment with or without polymer for 1 h, the cells were

12

washed thrice with PBS and replenished with the complete DMEM for further incubation for 3 h.

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Cells were observed under CLSM after being stained endosomal/lysosome and nuclei by Lyso

14

Tracker (50 nM) and Hoechst 33342 (1 μg∙mL-1), respectively for 15 min.

15

Statistical analysis. All data points were repeated in triplicate. Reported results and graphical

16

data are mean values with standard deviation encompassing a 95% confidence interval.

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RESULTS AND DISCUSSION

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In this work, the amphiphilic and pH-responsive polymers were synthesized by modifying

19

PAsp with tethered Phe and Lys. While maintaining the backbone length of PAsp as a constant,

20

the structures of side amino acids residues could modulate the physicochemical properties and

21

biological activities of the obtained amphiphilic and pH-responsive products.


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Characterization of PF and PFK. As shown in Scheme 1, PF and PFK were obtained via

2

graft modification of PAsp by Phe and Lys, as well as subsequent ester hydrolysis in

3

NaOH/ethanol. The chemical structure, compositions and molecular weights of the synthesized

4

PF and PFK, are shown in Table 1 and Figure 1. As shown in Figure 1A, PAsp, PF and PFK

5

exhibited similar signals at  4.574.36 ppm and  2.842.45 in 1H-NMR spectra, assigned to -

6

CH- of backbone and -CH2- of pendant groups. The signals at  7.1 ppm in the spectra of PF

7

(Figure 1A) were attributed to the protons of phenyl of Phe residues. For PFK, the signals at 

8

7.47.0 were assigned to the phenyl group in Phe and Lys residues, and the signals at  1.90.6

9

ppm were attributed to -CH2- of Lys residues. Thus, the composition and molecular weight of

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PF and PFK could be calculated by the signal integrations of amino acid residues, as depicted in

11

Table 1. The stoichiometric molar ratio of the amino acid relative to the carboxylic acid group

12

along the backbone of PAsp was calculated from 1H-NMR spectra (PF, δ 4.64.3 ppm and δ 7.1

13

ppm; PFK, δ 2.10.9 ppm, δ 4.64.0 ppm, and δ 7.47.0 ppm). The molecular weights of PF

14

(8.6103) and PFK (1.3104) were estimated from the original molecular weight of PAsp

15

(average molecular weight 4500), while the calculated molar ratios of [COOH]:[Phe]:[Lys] were

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39:28:0 for PF and 39:28:16 for PFK (Table 1).

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FTIR spectra in Figure 1B revealed the typical phenyl groups for PF and PFK at 3058 cm-1

18

(C-H stretching), 1457 cm-1 (C=C stretching) and 702 cm-1 (C-H external bending). It could be

19

seen that the amide I and amide II bands at stretching vibration absorption were observed at 1650

20

cm-1 (C=O stretching) and 1532 cm-1 (C-N and N-H bending), respectively. The absorption peak

21

at 1394 cm-1 attributed to C-O-C band. Considering the results of 1H-NMR and FTIR in

22

combination, it could be confirmed that the grafted products PF and PFK were obtained

23

successfully.


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Figure 1. 1H NMR (A) and FTIR spectra (B) of PAsp, PF and PFK. The signals at  7.47.0

3

(A) and the absorption peaks at 3058 cm-1 (C-H stretching), 1457 cm-1 (C=C stretching) and 702

4

cm-1 (C-H external bending) (B) were assigned to the phenyl groups in Phe and Lys residues.

5

pH-responsive behavior. The pH-responsive behaviors of PF and PFK were investigated by

6

measuring the transmittance of the aqueous polymer solutions in 1.0 mgmL-1 concentration at

7

different pH values. It could be seen from Figure 2 that the PF solution showed a sharper

8

transition from pH 3.9 to 4.0, while a broader transition from pH 4.8 to 5.8 was observed for

9

PFK which held higher contents of hydrophobic groups in the tethered amino acids. It was

10

consistent with the observation that the pH-mediated aggregation of the modified PAsp was

11

concentration-dependent.26,31 As a control sample, the PAsp solution showed no transition in the

12

pH range of 3.07.4. It could also be found from the digital pictures (Figure 2B) that the PFK


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solution became transparent at pH 5.8, whereas the PF solution echoed the appearance from its

2

translucent state at pH 3.9 into transparency at pH 4.0.

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Figure 2. pH-dependent transmittance of the polymer solutions at 1.0 mgmL-1 (A) and digital

5

pictures showing the appearance of the PF and PFK solutions at specific pH values (B).

6

As for the emission spectra, the intensity ratio of the first (I373) to the third (I384) vibronic

7

peaks of pyrene (I373/I384) upon the different hydrophobic environments was also tested. As

8

shown in the Supporting Information Figure S1, the PFK solution had a more hydrophobic

9

association over pH range from 4.5 to 7.4. These results suggested that the transmittance

10

transition of polymer solutions was dependent on the hydrophobicity of polymers, resulting from

11

the conjugation of hydrophobic amino acids, Phe or Lys. It was assumed that higher amount of

12

hydrophobic amino acid pendant groups led to higher hydrophobicity of the polymers.26,30,44

13

However, when the hydrophobic amino acids were beyond a certain content, the modified PAsp

14

was almost insoluble (data not shown). At the lower pH region, the amphiphilic polymer was in

15

compact hydrophobic domains caused by the neutralization of carboxyl groups and hydrophobic


12

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groups,45 and the solution became turbid or precipitated. With the increase of pH value, irregular

2

micelles were formed. The micelles were composed of the hydrophobic compact core and the

3

extended negative charged chains around core. Further increase of pH value resulted in

4

transparent solutions.

5

The zeta potential and size distribution of PFK at 1.0 mgmL-1 were measured by DLS. As

6

shown in Figure 3A, the zeta potential of PAsp remained -12 0 mV in pH range from 3.0 to 7.4,

7

while those of PF and PFK was around -29 mV (pH 3.84.4) and -34 mV (pH 4.45.6),

8

respectively. The amphiphilic nature of PF and PFK provided an opportunity for self-

9

assembling into multimolecular aggregates, and the increase of the negative surface charges led

10

to more stable aggregates. The size distribution and aggregate morphology of PFK (1.0 mgmL-

11

1)

12

pH values from 4.8 to 5.5, the average particle sizes of PFK aggregates obtained by DLS were

13

304.71.19 nm (pH 4.8), 155.33.39 nm (pH 5.0), 180.51.55 nm (pH 5.2), and 349.02.93 nm

14

(pH 5.5), respectively (Figure 3B). When pH was 5.8, the size distribution curve of PFK became

15

flatter with a peak at 102.61.34 nm, and two main peaks at 243.65.26 and 53.72.21 nm were

16

detected at pH 6.0. The result of the particle size distribution of PFK at pH 6.0 could be caused

17

by the unstable state of the polymer due to its negative potential.

at different pH values were shown in Figure 3B and Figure 3C, respectively. With increased

18

To solve the delivery problem, polymeric or lipid-based nanoparticles of cargo delivery

19

systems have been developed.10,46 Because of the resolution difference, the pH-responsive

20

behaviors of the polymers at low concentrations was not detectable using the DLS method.

21

However, from the TEM images in Figure 3C, the nanoparticles average size of PFK obtained

22

from the 0.1 mg·mL-1 solution varied with pHs could be observed in about 88.1 nm (pH 5.0),

23

55.4 nm (pH 6.0) and 35.5 nm (pH 7.4), respectively. The size discrepancy obtained by DLS and


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TEM might be ascribed to the shrinkage of polymer chains in dry state, which was outstretched

2

in aqueous solution. It could be seen that the morphology of PFK evolved with the increased pH

3

value. When pH value was increased, the deprotonation of carboxyl groups enabled amphiphilic

4

PFK more hydrophilic, and then the electrostatic and intermolecular interaction would be

5

reduced. The peak of particle size distribution presented multimodal when PFK was more

6

hydrophilic and two size distribution peaks appeared at pH 6.0 (Figure 3B).

7 8

Figure 3. (A) Zeta potential of PAsp, PF and PFK in PBS; (B) hydrodynamic particle size

9

distributions of PFK at different pH values; (C) TEM images of PFK aggregates prepared from

10

a 0.1 mg·mL-1 solution at pH 5.0, 6.0 and 7.4, respectively.

11

Membrane-disruptive activity. Hemolysis assay is a rapid method with high-throughput

12

screen for testing the cytocompatibility and endosomolytic activity of intracellular drug delivery

13

systems. There are a few reports on the evaluation of the membrane-disruptive properties by the

14

hemolysis assay.22,26,30 In order to evaluate the interaction between the polymer and the cell


14

Page 15 of 32 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


1

membrane, the cell membrane-disruptive activity of PF and PFK was investigated by the

2

hemolysis absorbance at 541 nm of sheep RBCs before and after incubation with the polymers.

3

As shown in Figure 4A, the hemolysis of PFK at pH 7.4 was independent of time from 1 h to 7 h,

4

and remained almost unchanged with the time at different concentrations from 0.03 mgmL-1 to

5

0.1 mgmL-1. As Figure 4B exhibited, in the presence of PF (0.1 mgmL-1), almost no hemolysis

6

activity was detected within the pH range studied, while PFK showed a significant hemolysis

7

around 70%. In Figure 4C, as the polymer concentration increased, the hemolysis of PFK

8

boosted from about 10% to 80%, and those of PAsp and PF varied slightly with the polymer

9

concentration. In Figure 4D, after being incubated in PBS at 37 °C for 2 h, PAsp (0.1 mgmL-1)

10

and PF (0.1 mgmL-1) solutions had trivial hemolysis, while PFK (0.1 mgmL-1) exhibited

11

obvious hemolysis. After being washed with isotonic PBS, sheep RBCs were fixed and

12

dehydrated, and then observed under SEM. As shown in Figure 4E, the SEM images showed

13

spherical morphology of sheep RBCs after treatment in the PAsp solution, similar with those in

14

the control PBS solution, whereas sheep RBCs incubated with PF exhibited weak deformation.

15

The sheep RBCs morphologies being co-cultured with PFK showed irregular topography. The

16

phenomena were also observed by a digital optical microscope (VHX-2000c, Keyence, Japan) as

17

shown in the Supporting Information Figure S2. These results suggested that the interaction

18

between PFK and RBCs was stronger due to the hydrophobic groups. It was consistent with the

19

hemolysis results of PAsp, PF and PFK (Figure 4). The hemolysis was correlated with the

20

relative hydrophobicity and structures of tethered Phe and Lys residuals.35-37,39 As mentioned in

21

the section of pH-responsive behavior, PFK displayed significant hydrophobic association and

22

formed more hydrophobic and compact structures than PAsp and PF in aqueous solutions. Also,

23

it has been reported that the membrane-disruptive activity of poly(α-alkylacrylic acid)s can be


15


Page 16 of 32

1

modulated by changing the alkyl chain length of monomer units.44 The increased hydrophobicity

2

and compact structures could enhance polymer-membrane interactions and subsequent higher

3

levels of membrane disruption.17,22

4 5

Figure 4. (A) Hemolysis of sheep RBCs at pH 7.4 as the incubation time with different

6

concentrations of PFK; (B) hemolysis of sheep RBCs as a function of pH value with PFK and

7

PF; (C) concentration-dependent hemolysis with PAsp, PF and PFK at pH 7.4; (D) digital

8

pictures of RBC hemolytic phenomena with PAsp, PF, and PFK at 0.1 mgmL-1, pH 7.4, 37 °C

9

for 2 h in comparison with PBS; (E) SEM images of RBCs incubated with PBS and 0.1 mgmL-1

10

of PAsp, PF, and PFK, respectively at pH 7.4 for 2 h. Scale bar 2 µm.


16

Page 17 of 32 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


1

In this study, PFK was more hemolytic than PF. One of the possible reasons was that the

2

amount of aromatic groups in PFK was higher than that of PF. The π-π stacking interactions

3

between phenyl groups in L-phenylalanine and carbobenzyloxy-L-lysine of the polymers could

4

cause a dramatic increase in membrane-disruptive activity.22,47 Another possible reason may be

5

that the hydrophobic aromatic groups can act as hydrophobic anchors inserting into the lipid

6

bilayer of cells to facilitate membrane binding.26,45 Moreover, it could be seen from Figure 4B

7

that the hemolysis of PFK increased slightly with the pH decline. It is in good agreement with

8

the pH-responsive properties of PFK and PF due to neutralization of carboxyl groups and more

9

hydrophobicity with decrease of pH values. Disruption of the polymer acting on the cell

10

membrane could cause an increase in the quantity of hemoglobin released from RBCs. Therefore,

11

the membrane-disruptive activity of PFK could be enhanced under relative low pH values.

12

The mechanism of lipid membrane disruption was also investigated by CLSM. As we know,

13

RBCs have no endocytic ability without outside assistance. As shown in Figure 5, the sheep

14

RBCs showed no fluorescent molecule after being treated with PBS, PAsp or PF. It was

15

suggested that FITC-dextran (green color) and Texas Red hydrazide (red color) could not

16

penetrate the RBCs membrane. It might be that the interaction between PAsp and RBC

17

membrane was relatively weak. But for PFK, as shown in Figure 5D, the ghost RBCs

18

(highlighted with arrows), which was visible in the bright field, could be visualized through

19

staining with Texas Red hydrazide. By treating sheep RBCs with 10 µM FITC-dextran 4 kDa

20

(0.1 mgmL-1, pH 7.4), it was found that FITC-dextran 4 kDa could permeate through membrane,

21

and the difference between the fluorescence intensities inside and outside RBCs was reduced.

22

However, no ghost RBCs was visible by treating sheep RBCs with PBS (Figure 5A), PAsp

23

(Figure 5B) or PF (Figure 5C). The hydrophobic groups of PFK interacted with the RBC lipid


17


Page 18 of 32

1

bilayer, and the lipid membrane construction was disrupted, providing transient diffusion for

2

FITC-dextran and Texas Red hydrazide. Thus, it was assumed that modification by hydrophobic

3

amino acids could increase the polymer-membrane interactions, and subsequent higher

4

membrane-disruptive ability of PFK was obtained.

5 6

Figure 5. CLSM images of RBCs incubated with 10 µM FITC-dextran (4 kDa) and 1 µM

7

Texas Red hydrazide in the presence of PBS (A), or 0.1 mgmL-1 PAsp (B), PF (C) and PFK (D)

8

solutions at pH 7.4 for 2 h. Scale bar 10 µm.

9

In vitro cytotoxicity. The concentration dependent cytotoxic effects of PF and PFK toward

10

NIH/3T3 and HeLa cells were assessed by Alamar Blue assay, respectively. Figure 6 displayed

11

that the relative cell viability was over 80% after treatment with PF in the concentration range of

12

0~1.0 mgmL-1 at pH 7.4 for 24 h, whereas PFK showed trivial cytotoxicity with the relative cell


18

Page 19 of 32 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


1

viability over 80% at pH 7.4 within concentration less than 0.3 mg·mL-1 to NIH/3T3 and HeLa

2

cells. With further increase of the polymer concentration from 0.3 to 1.0 mgmL-1, PFK showed

3

significant cytotoxicity with the relative viability below 60% against both the cell lines.

4

Comparing the relative cell viability of two kinds of cells, it could be found that HeLa cells

5

showed higher level of tolerance to PFK. This suggested that PFK was biocompatible within the

6

concentration below 0.3 mgmL-1.

7 8

Figure 6. Concentration-dependent relative cell viability with PF and PFK at 37 °C for 24 h

9

determined by Alamar Blue assay. (A) NIH/3T3; (B) HeLa.

10

Generally, PLL and its derivatives exhibited significant cytotoxicity when interacting with

11

cells, but the cytotoxicity could be weakened by complexation with negative cargo. For example,

12

complexes of cationic PLL-RT and negative DNA did not show obvious cytotoxicity at various

13

mass ratios with the help of enhanced multiple interactions between the polymer and cell

14

membrane.17 In this study, both hemolysis and cytotoxicity results above suggested that the

15

membrane-disruptive activity of PFK was accomplished through direction penetration, in which

16

the right kind of pores on the plasma membrane could be formed with the help of PFK (0.1

17

mg·mL-1) to achieve the substantial delivery of cargo (discussed later in the next section) without


19


Page 20 of 32

1

cell perturbation associated with membrane damage.48 However, if the concentration of PFK

2

was higher over 0.1 mg·mL-1, the balance of both the membrane damage and cell membrane

3

recovery would be disturbed, thereby the cells would be unable to restore.2 Thus, the PFK

4

polymer at a certain concentration below 0.1 mg·mL-1 could be safe for eukaryotic cells such as

5

NIH/3T3 and HeLa cells, and relatively mild hemolysis for RBCs.

6

Cytoplasmic delivery. Membrane-impermeable calcein was used as an endocytosed tracer

7

molecule to evaluate whether PFK could facilitate entry of bioactive molecules into different

8

kinds of cells at physiological pH values. NIH/3T3 and HeLa cells were tested by incubating

9

with both the 0.1 mgmL-1 polymer solution and 2 mgmL-1 calcein, respectively. After cultured

10

at 37 °C for 1 h, the cells were further incubated for 3.5 h before imaging to allow maturation of

11

endosomes and transformation of the polymer to cytoplasm. The term membrane disruption

12

refers to the generation of any kind of pores that would increase the permeability of the plasma

13

membrane to cargo.2 As seen in Figure 7, when the NIH/3T3 cells were treated with calcein

14

alone (control sample), the green spotty dots were observed due to constitutive endocytosis of

15

external medium (Figure 7A). When the NIH/3T3 cells were incubated with calcein and PF, only

16

bright punctate spots were detected (Figure 7B), similar to the control sample. In contrast, Figure

17

7C showed some diffused green fluorescence, which was noted spreading over the cells after

18

being treated with PFK. Endosomes/lysosomes were stained by LysoTracker, while the cell

19

nuclei were stained by Hoechst. The morphologies and position of endosomes/lysosomes were

20

consistent with cell nuclei. The results suggested that the amphiphilic pH-mediated PFK

21

destabilized the bilayered membrane and facilitated entry of calcein into cells (Figure 7C). It

22

could be suggested that hydrophobic interactions between the resulting hydrophobic domains

23

and the phospholipid bilayer of cells led to strengthened polymer-membrane binding.22,30,32,49 In


20

Page 21 of 32 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


1

Figure 7D, it was assumed that the PFK-membrane interactions partly disrupted the upper leaflet

2

of the bilayer at the position of contact between the pH-responsive PFK and lipid bilayer, which

3

could then lead to enhanced permeability of calcein. As discussed in the references, direct

4

penetration across the membrane could induce endocytosis and/or generate membrane defects

5

including pores, inhomogeneities, tears, lesions, holes, and perforations of all sizes and

6

shapes.2,50,51 Disruption of the cell membrane could facilitate entry for efficient cytoplasmic

7

delivery. After cellular uptake to lysosome, the pH value inside the endosome changed from pH

8

7.0 to 5.0, and calcein might be released into cytoplasm under the membrane-disruptive activity

9

of pH-responsive PFK at the endosomal pH environments (Figure 7D).14,21,52

10 11

Figure 7. CLSM images of NIH/3T3 cells showing the intracellular distribution of calcein

12

fluorescence. (A) Cells were treated with 2.0 mgmL-1 calcein (Green) only; (B) cells were

13

treated with both 2.0 mgmL-1 calcein (Green) and 0.1 mgmL-1 PF; (C) cells were treated with

14

both 2.0 mgmL-1 calcein (Green) and 0.1 mgmL-1 PFK. The endosomes or lysosomes were

15

stained by LysoTracker (Red) and the cell nuclei were stained by Hoechst (Blue). Images were

16

collected after 1 h of uptake and 3 h of further incubation in fresh complete DMEM. (D)

17

Schematic representation of PFK facilitating transmembrane calcein delivery. Scale bar 10 µm.


21


Page 22 of 32

1

Analogously, when HeLa cells were treated with calcein only (Figure 8A) or calcein and PF

2

(Figure 8B), green spots around cells were observed, whereas diffuse green fluorescence

3

throughout the cell interior was observed when HeLa cells were incubated with calcein and PFK

4

(Figure 8C). The ability indicated the promising biomedical application of PFK to enhance entry

5

of calcein into the cytoplasm of NIH/3T3 and HeLa through membrane destabilization.

6 7

Figure 8. CLSM images of HeLa cells showing the intracellular distribution of calcein

8

fluorescence treated with 2.0 mgmL-1 calcein (Green) only (A), with both 2.0 mgmL-1 calcein

9

(Green) and 0.1 mgmL-1 PF (B), and with both 2.0 mgmL-1 calcein (Green) and 0.1 mgmL-1

10

PFK (C). Endosomes/lysosomes were stained by LysoTracker (Red) and the cell nuclei were

11

stained by Hoechst (Blue). Images were collected after 1 h of uptake and 3 h of further

12

incubation in fresh complete DMEM. Scale bar 10 µm.


22

Page 23 of 32 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


1

In this work, PAsp modified with hydrophobic amino acids demonstrated enhanced

2

membrane-disruptive activity and facilitated entry of the drug model calcein into cells. On basis

3

of in vitro results above, the applications of the Phe and Lys-grafted polymers could be further

4

tested in vivo for intracellular delivery of bioactive macromolecules to cope with tumor or

5

cardiovascular diseases. However, the structure of the modified polymer is not perfect, and it

6

could be further improved by introducing block polymer structures or cationic groups to enhance

7

its applicable functions.53 In addition, the polymer could be modified by polyethylene glycol or

8

RBC membrane coating for the long circulation as nanoscopic drug carriers.2,9,54

9

CONCLUSIONS

10

Amphiphilic polymers, PF and PFK, were synthesized by graft modification of PAps with the

11

hydrophobic amino acids, Phe and Lys. It was shown that the hydrophobicity of PFK was

12

significantly improved with the increasing amounts of phenyl groups in tethered Phe and Lys

13

residuals, and the pH-responsive range of PFK was shifted to 4.8~5.8 with respect to 3.9~4.0 of

14

PF. Meanwhile, PFK exhibited enhanced membrane-disruptive ability at pH 7.4, and showed

15

trivial cytotoxicity at pH 7.4 and 0.3 mgmL-1 concentration to HeLa or NIH/3T3 cells. PFK

16

could destabilize the lipid bilayer of the cell membrane and facilitated entry of calcein into the

17

cell cytoplasm. In comparison with PAsp and PF, the amphiphilic and pH-responsive PFK

18

demonstrated remarkable potential in enhanced membrane-disruptive ability for intracellular

19

delivery of bioactive molecules.

20

ASSOCIATED CONTENT

21

Supporting Information


23


Page 24 of 32

1

The Supporting Information is available free of charge including the preparation of buffer

2

solutions, the pH-dependent variations in I373/I384 of PF and PFK (Figure S1), and optical

3

microscope images of RBCs (Figure S2).

4

AUTHOR INFORMATION

5

Corresponding Author

6

*E-mail:

7

ORCID

8

Xiaoyan Yuan: 0000-0002-2895-3730; Lixia Ren: 0000-0001-7659-0025

9

Notes

[email protected]; [email protected]

10

The authors declare no competing financial interest.

11

ACKNOWLEDGEMENTS

12

This work is financially supported by the National Natural Science Foundation of China (No.

13

51773150).

14

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Enhancing Membrane-Disruptive Activity via Hydrophobic

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