PEG Bottle Brush Copolymers as Antimicrobial Mimics: Role of

Subscriber access provided by Iowa State University | Library

Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

PEG Bottle Brush Copolymers as Antimicrobial Mimics: Role of Entropic Templating in Membrane Lysis Amit Garle, and Bridgette Maria Budhlall Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00756 • Publication Date (Web): 15 Jan 2019 Downloaded from http://pubs.acs.org on January 20, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 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

Langmuir

PEG Bottle Brush Copolymers as Antimicrobial Mimics: Role of Entropic Templating in Membrane Lysis Amit L. Garle†, ‡ and Bridgette M. Budhlall†, †Department

of Plastics Engineering and Nanomanufacturing Center, 1 University Drive,

University of Massachusetts Lowell, Massachusetts, 01854. USA. ‡ Mayo

Clinic, Department of Internal Medicine, Endocrinology, 201 West Center Street, Rochester, Minnesota, 55902. USA.

* Corresponding author email: [email protected]

KEYWORDS: antimicrobial polymers, entropic templating, microbial membrane mimics

ACS Paragon Plus Environment

1

Langmuir 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 2 of 36

ABSTRACT

Novel polymers containing quaternary functional groups, with and without (control copolymer) PEG side chains were synthesized and characterized for their ability to lyse the phospholipid membranes of liposome vesicles. Calcein loaded unilamellar vesicles comprised of 1,2-dioleoylsn-glycero-3-phosphatidylcholine (DOPC) was used as a red-blood cell membrane mimic, and a 80:20 (mol/mol) mixture of 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanolamine (DOPE), and 1,2-dioleoyl-sn- glycero-3-[phospho-rac-(1-glycerol)] (DOPG), was used to mimic the outer cellmembrane of gram negative bacteria, E.coli. For DOPE:DOPG = 80:20 (mol/mol) liposome vesicles, the PEG “bottle-brush” copolymer caused leakage of the encapsulated Calcein dye whereas the control copolymer did not cause any leakage. Both the “bottle-brush” copolymer and the copolymer without PEG side chains had no effect on the zwitterionic DOPC liposome vesicles indicating that the “bottle-brush” architecture is needed for membrane lysis. The PEG “bottlebrush” copolymer did not affect the colloidal size of the DOPE:DOPG = 80:20 (mol/mol) liposome vesicles, but on the addition of Triton-X 100, the vesicles disappeared. This provided evidence that the dye leakage was caused by compromising the integrity of the vesicle membrane by the “bottle brush” polymer architecture. Such partial disruption was preceded by the entropic templating of lipid membranes by the PEG side chains of the “bottle-brush” copolymer. By careful comparison with non-PEGylated cationic polymers, Quart, the importance of PEG side chains in the membrane disrupting activity of the PEGylated cationic polymer, QPEG was demonstrated. This finding itself is interesting, and can contribute to the expansion of the design of membrane disrupting materials.


2

Page 3 of 36 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

Langmuir

INTRODUCTION

Infections pose serious concerns in a variety of settings, such as from medical implants, in hospitals, in water purification and in food packaging and storage. Hospital acquired infections are one of the leading causes of death in USA.1 In the United States alone, an estimated 100,000 deaths and $6.5 billion2 in cost is attributed to infections. The overuse of antibiotics has led to the development of drug resistant bacteria which are the primary cause of these infection related deaths.3 According to CDC,4 at least two million people are infected by drug resistant bacteria. Thus, novel approaches are urgently needed to treat these infections.

Nature has developed evolutionary weapons against bacterial infections in form of antimicrobial peptides (AMP). Both vertebrates and invertebrates, including humans, have genes which synthesize AMPs.5 The majority of AMPs are amphiphilic with a hydrophobic region (>30%) and a hydrophilic region comprising of cationic amino acids (+2 to +9) which on interaction with microbial cell membranes result in three dimensional rearrangement of the membrane structure.6 The cell membrane structure is also altered by changes in the membrane composition, the curvature of the membrane proteins and scaffolding.7 AMPs exploit this membrane pliability as a defense mechanism against various microbes. The loss of structural integrity of this protective cell membrane can lead to loss of ions, nutrients and entry of toxic agents that could destroy the intracellular organelles.

Selectivity of the AMPs to microbial membranes over erythrocytes is due to differences in membrane composition.8 Membranes of microbes have exposed negative charges which are binding sites for the AMPs.9 Clinical applications of AMPs have not been successful due to high


3

Langmuir 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 4 of 36

cost of manufacturing, short half-life and cytotoxicity.10 As a result, synthetic antimicrobial peptide mimics (SAMPM) have been investigated.

Most of the reported SAMPM are amphiphilic polymers with quaternary groups and lipidic or hydrophobic side chains.11 The architectures differ in arrangement of the components such as segregated, facially amphiphilic or same- centered.11 As one of these components, PEG is used to minimize hemolysis by the quaternary group and improve the biocompatibility SAMPM.12 It is well known that a polymer brush architecture on surfaces prevents the nonspecific binding of proteins.13 PEG polymers in particular have been shown to provide resistance to fouling.14,15 PEG prevents the adsorption of quaternary groups to membranes of RBC as well as preventing nonspecific protein binding by steric hindrance.16

Even though PEG is hydrophilic its interaction with lipids are known. For e.g. membrane modifications by PEG are also used for hybridoma production.17 In this procedure, highly concentrated PEG is used to fuse cells to create hybrid cells. The exact mechanism of the fusion is not known but it is hypothesized to be due to dehydration of cell membranes.18 Dehydration brings membranes closer and results in fusion. Triton-X 100, a nonionic surfactant containing PEG chains, is commonly used for cell lysis. For it to be effective, its concentration has to be above its critical micellar concentration (CMC). Below CMC, no detectable lysis occurs.19

This observation implies that a secondary structure causing aggregation of PEG chains is important for the membrane lysis. These examples suggest that hydrophilic PEG interacts with lipidic membranes but its role and mechanism is not clearly understood. Thus, to understand the role of PEG, the local concentrations of PEG near membranes was increased and their interaction with membrane mimics was investigated for the “bottle brush” architecture.


4

Page 5 of 36 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

Langmuir

Due to its hydrophilic nature, PEG is not compatible with lipids. But mixing between chemically incompatible polymers at the molecular level has been demonstrated in two dimensional systems.20 For a “bottle-brush” polymer architecture, mixing occurs due to high conformational stresses experienced by the dense packing of chains. The chains are highly stretched which on contact with particles or polymers chains capable of intercalating between the chains results in entropic gain which is much higher than the enthalpy. This results in mixing at the molecular level irrespective of the chemical nature of the intercalating moiety.21 This strategy was explored and reported here22 in order to induce membrane lysis or poration, which can be used to design novel antimicrobial therapeutics.

EXPERIMENTAL SECTION

Materials. Polyallylamine (Mw = 15 kDa) (Polysciences) was lyophilized and stored in the refrigerator. 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1,2-dioleoyl -sn-glycero-3phosphatidylethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) were purchased from Avanti Polar Lipids. Polyethylene glycol (PEG) (average Mn = 750), 4-

Methylmorpholine-2,6-dione

(MIDA),

Calcein,

N-(3-

Dimethylaminopropyl)-N’-

ethylcarbodiimide hydrochloride (EDC.HCl), N-Hydroxysuccinimide (NHS), Sodium phosphate dibasic (Na2HPO4), 2-(N-morpholino)ethanesulfonic acid (MES), Sephadex G-50 and Triton X100 were purchased from Sigma Aldrich. All chemicals were used, as received.

Synthesis of Polyethylene glycol 4-Methylmorpholine-2,6-dione ester (PEG-MIDA). Mono hydroxyl functional PEG (4.5 g, 6 mmol) was dried overnight at 60 ◦C under vacuum to


5

Langmuir 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 6 of 36

remove moisture and dissolved in 50 mL acetonitrile. MIDA (1 g, 7.8 mmol, 1.3 eq.) was added and the reaction temperature was raised to 70 ◦C and allowed to react for 24 hours under nitrogen atmosphere. After 24 hours, the reaction mixture was allowed to cool to room temperature and a white precipitate of unreacted MIDA was observed. The soluble fraction was evaporated under vacuum to obtain crude PEG-MIDA. The crude product was dissolved in deionized water (DI water) and dialyzed (MWCO = 100-500 Da) against water for 8 hours with water changes every 2 hours. The purified product was obtained by lyophilization. Yield = 90 %. The degree of functionalization was calculated to be ~100 %.1H NMR: δ 2.6 (-N(CH3)-), 3.4 (CH3O-), 3.4 (C(O)CH2N-), 3.6 (-N(CH3)CH2-), 3.65 - 3.75 (-OCH2CH2-), 4.35 (-CH2OC(O)-). PEGylation

of

Polyallylamine

with

PEG-MIDA

ester

(PAA-PEG-MIDA).

Polyallylamine (50 mg, 0.88 mmol) was dissolved in 50 mL of MES buffer (0.1 M, pH = 6.5). PEG-MIDA (850 mg. 0.96 mmol, 1.1 eq.) was added to it followed by EDC.HCl (500 mg, 2.63 mmol, 3 eq.) and NHS (200 mg, 1.75 mmol, 2 eq.) and stirred for 24 hours. After 24 hours, the reaction mixture was dialyzed (MWCO = 3500 Da.) against DI water and lyophilized to obtain the product as a waxy solid. Yield = 80 %. The degree of functionalization calculated was ~50 %. 1H NMR: δ 1.1, 1.2 (-CH2CH-), 1.8, 1.9 (-CH2CH-), 2.4 (-CH2NH2), 2.7 (-CH2NH-), 2.8 (-N(CH3)), 3.4 (CH3O-), 3.4 (-C(O)CH2N-), 3.6 (-N(CH3)CH2-), 3.6 - 3.7 (-OCH2CH2-)(PEG), 4.35 (CH2OC(O)-). Synthesis of Polyallylamine glycine amide (PAA-MIDA). Polyallylamine (200 mg, 3.5 mmol) was dissolved in 50 mL of MES buffer (0.1 M, pH = 6.5). Glycine (290 mg, 3.85 mmol, 1.1 eq.) was added to it followed by EDC.HCl (2 gm, 10.51 mmol, 3 eq.) and NHS (810 mg, 7.01 mmol, 2 eq.) and stirred for 24 hours. After 24 hours, the reaction mixture was dialyzed (MWCO = 3500 Da.) against DI water and lyophilized to obtain the product as an off-white powder. Yield


6

Page 7 of 36 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

Langmuir

= 80 %. The degree of functionalization calculated was ~65 %. 1H NMR: δ 1.2 (-CH2CH-), 1.7 (CH2CH-), 2.4 (-CH2NH2), 2.7 (-CH2NH-), 2.8 – 3.5 (-N(CH3)2-), 3.7 (-C(O)CH2N-). Quaternization of PAA-PEG-MIDA with ethyl iodide (QPEG). PAA-PEG-MIDA (350 mg) was dissolved in 20 mL ethanol under nitrogen atmosphere and heated to 60 ◦C. 1 mL of ethyl iodide was added to the reaction mixture and the reaction was continued for 24 hours in darkness. After the reaction, the reaction mixture was evaporated under vacuum to obtain the product as an amber colored rubbery solid. Yield = 70%. The degree of functionalization calculated was ~47 %. 1H

NMR: δ 1.1, 1.2 (-CH2CH-), 1.3 (-CH2CH3), 1.8, 1.9 (-CH2CH-), 2.4 (-CH2NH2), 2.8 (-

N(CH3)-), 3.1 (-CH2CH3, -CH2NH-), 3.4 (CH3O-), 3.4 (-C(O)CH2N-), 3.6 (-N(CH3)CH2-), 3.6 3.7 (-OCH2CH2-)(PEG), 4.35 (-CH2OC(O)-). Quaternization of PAA-MIDA with Ethyl Iodide (Quart). PAA-MIDA (100 mg) was dissolved in 20 mL ethanol under nitrogen atmosphere and heated to 60 ◦C. 1 mL of ethyl iodide was added to the reaction mixture and the reaction was continued for 24 hours in darkness. After the reaction, the reaction mixture was evaporated under vacuum to obtain the product as an amber colored solid. Yield = 70%. The degree of functionalization calculated was ~67 %. 1H NMR: δ 1.2 (-CH2CH-), 1.3 (-CH2CH3), 1.7 (-CH2CH-), 2.5 (-CH2NH2), 2.9 (-CH2NH-), 3.1 – 3.7 (-N(CH3)2-, -NCH2CH3), 4 (-C(O)CH2N-). Characterization Methods. NMR spectra were recorded on a Bruker & Spectrospin Avance DRX 500 MHz spectrometer. Chemical shifts are reported in δ (ppm) and are referenced to the residual proton peaks of the deuterated solvents. All NMR measurements were done using either deuterated chloroform (CDCl3) or deuterium oxide (D2O) as solvent. Fluorescence


7

Langmuir 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 8 of 36

measurements were performed on Agilent Cary Eclipse Fluorescence Spectrophotometer. Particle size and zeta potential analysis was performed using a Malvern Zetasizer (Nano ZS). For matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis, the PEG samples were dissolved in a (50:50 vol:vol) water:methanol solution. A 10 mg/mL 2, 5-dihydroxybenzoic acid (DHB) in methanol was used as a matrix. 1 mg/mL polymer in 1:1 methanol and water were mixed in 1:1 matrix: sample ratio. 0.5 μL aliquot was applied to a MALDI sample target for analysis. MALDI-TOF mass spectra were acquired using the reflectron mode on a Bruker UltrafleXtreme mass spectrometer (Bruker Daltonik, Germany). The instrument was equipped with a 355nm smartbeam laser. The mass spectrum acquisition rate was 1000 Hz. The instrument was previously calibrated with a standard peptide mix. Liposome Vesicle Preparation. Homogeneous large unilamellar vesicles (LUVs) were prepared by the extrusion method as reported in literature.23 The specific amount of phospholipids, whose composition mimics E. coli and red-blood cell (RBC) membranes are listed in Table 1. The lipids were first dissolved in 2 mL of CHCl3, slowly evaporated at reduced pressure and then they were placed under a vacuum at room temperature for more than 2 hours to prepare a thin lipid layer. The thin layer was then hydrated for 1 hour with 1 mL of buffer A (40 mM Calcein, 10 mM Na2HPO4, pH = 7). The resulting suspension was subjected to ten freeze-thaw cycles using dry ice to freeze and a warm water bath (37 ◦C) to thaw. An Avanti mini-extruder, with gas tight syringe (1 mL capacity) and a 200 nm pore diameter polycarbonate nucleopore membrane stacked between two pairs of membrane supports was used to extrude the liposomes. 1 mL of each batch of liposome suspension was extruded for an average of 12 times. The free or unencapsulated Calcein was


8

Page 9 of 36 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

Langmuir

removed by gel filtration (Sephadex G-50) using buffer B (10 mM Na2HPO4, 90 mM NaCl, pH = 7), and the resulting 1.0 mL of liposome vesicle solution was diluted with buffer B to 20 mL to give a Calcein loaded stock solution with final lipid concentration of ~0.5 mM.

Table 1. Composition of Lipids used to prepare Vesicles for Calcein Leakage Assay Mimics

Composition of Lipids (mol. %)

E. Coli

DOPE : DOPG = 80/20

RBC

DOPC = 100

Liposome Vesicle Leakage Assays. 20 µL of the Calcein-loaded liposomes were added to 1.98 mL of buffer B to dilute the lipid concentration to ~5.0 µM. Using a Agilent Cary-Eclipse Fluorescence spectrophotometer, the suspension was excited with wavelength λex = 495 nm and its fluorescence emission intensity, It (λem = 515 nm) was monitored as a function of time (t) continuously during the addition of 100 µL of polymer solution and 100 µL of 10% Triton X-100, respectively.

HYPOTHESIS Bottle-Brush Design Architecture Rationale and Proposed Membrane Interactions. To design a SAMPM using entropic templating for membrane poration or lysis, the architecture design should include features, which would enable attachment of the SAMPM to the membrane as well as induce conformational stress. Our hypothesis is that these features will be achieved by


9

Langmuir 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 10 of 36

a “bottle- brush” copolymer architecture, which has quaternary groups near the core or on backbone of the brush. The distance between the PEG side chains or grafting density should be such that the quaternary core is accessible to the membrane surface for binding. If too close, binding of the brush to the membrane surface is prevented but, if on the other-hand; PEG side chains are too far apart, there is less of an increase in entropy on binding to the membrane, resulting in less strain on the membrane interphase. For entropic templating, the two-dimensional arrangement of the chains forces them together. In three dimensions, the chains have a larger number of conformations, which reduces the entropic repulsion. This feature is useful as it allows for binding to the membrane. The quaternary groups distributed along the backbone allows for multi-site interaction of the “bottlebrush” polymer core with the membrane. As the polymer binds at multiple sites on the membrane, the degree-of- freedom of the PEG side chains decreases and the conformational stress increases. The stressed chains can exert stress on the membrane leading to disruption of the membrane or at least the formation of pores as illustrated in Scheme 1. Another requirement for entropic templating is small interactions between the interacting species. This requirement is fulfilled, as the PEG due to its ether linkage does not have strong interactions with the lipidic membrane. For side chains to be fully stretched, high solvation of the “bottle-brush” polymer is needed. As water is a very good solvent for PEG, it is highly solvated. In addition, at a higher degree of substitution, the distance between grafted chains is much smaller than the PEG chain length. This leads to repulsion between chains and thus stretching. Therefore, the proposed structure of the PEG copolymer with “bottle-brush” architecture fulfills this requirement.


10

Page 11 of 36 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

Langmuir

Attachment of Polymer due to electrostatic attraction

3-D chains are forced into 2D increasing the repulsion between chains of the brush

Membrane disruption either due to mixing with lipids or deformation of the membrane due to strain

Negative charge Quaternary amine PEG Chains PAA backbone

Scheme 1. Proposed mechanism for the disruption of the cell membrane by the cationic “bottle brush” polymer with PEG side chains.

The strategy for selecting the molecular weight of PEG to use in the current study, given that the goal is to perforate the vesicle’s membrane in order to cause leakage of its contents, is as follows. It is known that Triton X-100, which has 9 to 10 ethylene glycol units (PEG Mw ~500 g mol-1), causes the lysis of cell membranes, whereas PEG with a molecular weight of 1500 g mol-1 is generally used for fusion of cell membranes in hybridoma production.18 Therefore, an intermediate PEG molecular weight (~750 g mol-1), between these two extremes, was used in the current study. Longer PEG chains can be used but this would prevent the interaction of the membrane with the quaternary group at the base of PEG.24,25 Also, as the chain length increases, the stretching of PEG is less as more room is available for the PEG chain end. Thus, finding the optimum chain length would require a careful balance between the entropy of the chain and the binding efficiency.


11

Langmuir 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 12 of 36

Poly(allylamine) (PAA) was chosen to be the backbone of the “bottle-brush” polymer. The amine functionality allows for dense grafting of PEG chains. High grafting density and quaternization leads to a highly stretched backbone due to steric as well as charge repulsion. This creates structures with a high aspect ratio. It has been demonstrated that high aspect ratio antimicrobials have activity against Gram-negative bacteria and fungi which are not as negatively charged.26 Our hypothesis that a cationic polymer with “bottle-brush” architecture is needed for sequential binding by electrostatic attraction and membrane disruption by deformation of the phospholipid membranes was investigated with a quaternary PEG “bottle-brush” (QPEG) copolymer, as well as with a control (Quart) copolymer that has a quaternary group but no PEG brushes.


12

Page 13 of 36

RESULTS AND DISCUSSIONS Synthesis and Characterization of PEG Bottle Brush Copolymers. Mono hydroxyl PEG was reacted with MIDA to obtain a polymer with carboxylic and tertiary amine functional groups at the end of PEG chain. These dual functionalities help to couple the PEG to the poly(allylamine) as well as introduce a tertiary amine group at the base of the bottle brush chain.

n

A

Step I n

+

Step II

HN

H2 N

O N

O

N

B

QPEG

N

O

O

N

O

OH

O

Step I

O

H2 N

O

Ethanol, 24 hours Yield = 70 %

O

+

HN

O

O m

n

H2 N

Ethyl Iodide

MES Buffer, 24 hours Yield = 80 %

O O

EDC, HCL, NHS

n

m

H2 N

H2 N

m

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

Langmuir

Step II

n

EDC, HCL, NHS

Ethyl Iodide

NH

OH

MES Buffer, 24 hours Yield = 80 %

n

O

NH

Ethanol, 24 hours Yield = 70 %

N

O

Quart

N

Figure 1. (A) Synthesis of PEG bottle brush copolymer (QPEG) with PAA and PEG-MIDA using the grafting to approach followed by quaternization with ethyl iodide. (B) Synthesis of the control polymer, PAA functionalized with a quaternary glycine group and without PEG side chains (Quart).

The reaction was quantitative and as seen in the 1H NMR spectra shown in Figure 2A and the 13C-NMR spectra shown in Supporting Information Figure S2, a highly purified product (PEG-


13

Langmuir 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 14 of 36

MIDA) was obtained after dialysis. The attachment of PEG-MIDA to the polyallylamine polymer was accomplished by carbodiimide chemistry. A polymer with a grafting density of ~50% was synthesized and found to be soluble in water. The grafting density was calculated by integrating the area under the peak for secondary hydrogen of PAA at ~1.1 ppm and secondary hydrogens of the ethylene groups of PEG in PEG-MIDA ester at ~4.3 ppm (Supporting Information Figure S3). The ratio of the area under the peak for 100 % substitution should be 1 but based on NMR analysis, the ratio is 0.5. Hence, the grafting density of PEG-MIDA to PAA is ~50 %. Quaternization of PAA-PEG-MIDA was carried out in ethanol at 60 ◦C under a nitrogen atmosphere. The degree of quaternization was confirmed by comparing the protons of the PAA with the methyl group of ethyl iodide from the 1H-NMR shown in Figure 2B. Evidence that the quaternary PAA-PEG-MIDA (QPEG) copolymer with a PEG graft density of 50%, can adopt a “bottle-brush” architecture is gleaned from a consideration of the recent study,27 where it was shown tethered PEG chains adopted a mushroom conformation for less densely grafted PEGs with the PEG height equal to 2Rg. Now for PEG with a molecular weight of 750 g mol-1, this height is calculated to be approximately 15 nm.28 Theoretically, with a PEG chain height of 15nm and 50% grafting density then the average linear distance between the grafted chains needs to be less than 0.616 nm (4 carbon- carbon bonds, linear distance). These two factors, in addition to having a positive charge along the backbone, provides evidence that the PEG chains will be crowded and being highly solvated in water, will need to extend outward to minimize repulsion. This extension, it is postulated, will lead to the formation of a “bottle-brush” polymer architecture.


14

Page 15 of 36 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

Langmuir

Figure 2. 1H-NMR spectrum of A) PEG-MIDA polymer and B) quaternary PAA-PEG-MIDA copolymer.


15

Langmuir 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 16 of 36

The degree of quaternization was calculated from the ratio of the integral areas under these protons to be ~47 %. The quaternization reaction was found to be slow as reported in the literature.29 The corresponding 13C-NMR spectra of PAA-PEG-MIDA and QPEG are shown in the Supporting Information Figures S4 and S5, respectively. Functionalization of polyallylamine (PAA) with MIDA was not performed as PAA is not soluble in organic solvents and MIDA is not stable in water. Glycine was selected as the moiety to functionalize poly(allylamine) as it has reactive groups similar to those in the MIDA group. PAA-glycine was successful synthesized as seen in 1H-NMR spectra shown in the Supporting Information Figure S6. The degree of quaternization for PAA-MIDA was calculated to be ~67 % (as shown in the Supporting Information Figure S7). MALDI-TOF mass spectral analysis was performed on PEG and PEG MIDA polymers to confirm the presence of the MIDA end-groups. All samples were analyzed without adding a cationizing agent. As seen in Figure 3, the average repeat unit mass of the polymers can be determined from the individual MALDI-TOF mass spectra. Both PEG and PEG MIDA showed a backbone repeat unit with ~44 Da difference between the peaks. This value exactly matches the theoretical ethylene oxide repeat unit mass of ~44 Da.


16

Page 17 of 36 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

Langmuir

Figure 3. MALDI-TOF mass spectra of PEG and PEG-MIDA with full spectra (left) and expanded spectral region (right).

For a given number of repeat units (n), the theoretical molecular weight (Mn theory) of the polymer chain was calculated using equation (1) and the data shown in Table 2.

𝑀𝑛 𝑇ℎ𝑒𝑜 = 𝑛 × 𝐸𝐺1 + 𝐸𝐺2 + 𝐶

(1)

where, EG is Mn of the repeat unit, EG1 and EG2 are the molecular weights of end groups and C is the molecular weight of any “cationizing” cations, such as either a proton [M+H]+ or a


17

Langmuir 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 18 of 36

sodium [M+Na]+ cation. The presence of sodium ions in the polymers are typically due to absorption of sodium ions from the glass containers they were prepared and stored in.

Table 2. MALDI-TOF Mass Spectral Analysis for PEG and PEG-MIDA

Polymer

End-Group 1

End-Group 2

Mn (g/mol)

Mn (g/mol)

PEG

CH3 (15.024)

OH (17.003)

PEG-MIDA

CH3 (15.035)

MIDA (146.040)

Cation

Repeat unit #

Mn theory

Mn observe

(g/mol)

(g/mol)

Na (22.989)

18

847.708

847.7

Na (22.989)

18

955.020

954.7

18

977.010

976.8

H (1.008)

The theoretical and observed values of individual mass peaks for the PEG and PEG-MIDA polymers are listed in Table 2. The average difference between the theoretical and observed values is consistent once a proton and a sodium ion are taken into account. For a given repeat unit, the difference in molecular weights of PEG and PEG-MIDA corresponds to 129.1 Da, which is the molecular weight of the 4-methylmorpholine-2,6-dione (MIDA) functional group. These results provide additional evidence for the successful incorporation of the MIDA functional group in the PEG chains.

Interaction of PEG copolymers with the Liposome Vesicles used as Microbial Membrane Mimics. The outer membrane of cells protects the cytoplasm of the cells from the action of various antimicrobials. The key distinguishing feature that determines the action of


18

Page 19 of 36 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

Langmuir

antimicrobials is the composition of the cell membrane.30 Lipids are important constituents of cellular membranes. The concentration and composition of lipids in various cell membranes give rise to the selectivity of antimicrobial peptides. For antimicrobial peptides, the interaction with erythrocyte membranes is a weak hydrophobic interaction as the membrane lacks any anionic phospholipids.8 The outer erythrocyte membrane is composed of cholesterol, phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine lipids while the inner membrane is, in addition to the above lipids, include negatively charged lipids, phosphatidylserine. Bacterial and fungal cell membranes differ in both composition as well as structure. While gram-positive bacteria have a single lipid bilayer, gram-negative bacteria have two concentric lipid bilayer membranes9 with different lipid compositions in each membrane. The inner leaflet of the outer membrane has a similar composition of cytoplasmic membranes while the outer leaflet is mainly composed of lipopolysaccharides, which are highly negative in charge. In addition, grampositive bacteria have a thick layer of peptidoglycan with teichoic acid31 in the outermost portion. Even though the thickness of peptidoglycan layer is 20 to 80 nm, the peptidoglycan layer is more permeable to antibiotics than gram-negative bacteria. Due to the presence of teichoic acid in the membrane, the quaternary group of our “bottle-brush” polymer will be able to interact with the peptidoglycan layer and allow attachment of the brush polymer. One of the key features of our design is the polyvalent interaction of the quaternary groups of polymers (both QPEG and Quart) with the anionic groups on the surface of the lipid vesicles. Polyvalent interactions are characterized by simultaneous binding of multiple functional groups of polymer to functional groups on lipid vesicle.32 Polyvalent interactions of Quart allow it to bind to the vesicle surface and exert pressure on the membrane resulting in a decrease in size as observed in the Supporting Information Figure S8C. As Quart lacks the ability to intercalate with


19

Langmuir 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 20 of 36

the lipids in the vesicles, it does not affect the membrane integrity and thus there is no leakage of the vesicles. Polyvalent interactions of QPEG with DOPE:DOPG = 80:20 (mol:mol) results in crowding of the chains at the vesicle surface and causes conformational stress, which is relieved by intercalating of PEG with lipid membrane subsequently leading to leakage. Liposome Vesicle Leakage. The interaction of antimicrobials with the microbial membrane is critical for either the pore formation or complete disruption of the membrane. We investigated the interaction of our cationic PEG “bottle-brush” polymers with phospholipid vesicles using calcein dye leakage assays as reported in literature. Two model membrane systems were investigated: 100 % DOPC mimicking the outer leaflet membrane of RBC and DOPE:DOPG = 80:20 (mol:mol) mimicking the phospholipid membrane compositions of E.coli, a gram negative bacteria. As seen in Figure 4, both QPEG and Quart showed no membrane activity against 100 % DOPC. QPEG due to its hydrophilic nature as well as steric repulsion by PEG would not interact with DOPC. Similar observations have been reported previously.33 For DOPE:DOPG = 80:20 (mol:mol) liposome vesicles, QPEG caused leakage of Calcein dye as shown in Figure 4C. The PEG “bottle-brush” copolymer was able to interact with the lipid membrane through both ionic interactions and it is postulated that rearrangement of PEG chains with the resulting conformational stress causes leakage of the dye. This hypothesis was tested by systematically measuring the vesicles’ particle sizes before and after addition of QPEG and the results are shown in Figure 6. The contribution of steric repulsion to conformational stress in the copolymer is critical for membrane rupture as electrostatic interactions alone did not cause any leakage when the Quart was used with DOPE:DOPG = 80:20 (mol:mol) liposome vesicles (Figure 4D).


20

Page 21 of 36 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

Langmuir

Figure 4. Calcein dye leakage from various phospholipid vesicles upon the addition of copolymers, followed by the addition of 100 µL of a 10 wt% aqueous solution of Triton X-100 surfactant after 700 sec. (A) Leakage of dye from the DOPC vesicles (RBC mimic) by QPEG and Triton X-100, (B) leakage from the DOPC vesicles by Quart and Triton X-100, (C) leakage from PE:PG = 80:20 (mol:mol) vesicle (E. Coli mimic) by QPEG and Triton X-100 and (D) leakage from PE:PG = 80:20 (mol:mol) vesicles upon addition of the Quart polymer.


21

Langmuir 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 22 of 36

It has been observed that the interaction of amphiphilic molecules with lipid membranes cause formation of pores within the membrane bilayer.34 The unequal pressure experienced due to binding of the amphiphilic molecules causes the inner layer to rearrange to create transient pores. Thus, this proves our hypothesis that in order to disrupt the phospholipid membrane integrity, the chemical nature of the intercalating moiety is not important. Rather, any pliable molecule can force the membrane to rearrange as long as it is able to exert a pressure on the lipid membrane layers. In order to investigate the effect of QPEG concentration on the dye leakage, another experiment was conducted, where the amount of QPEG added to the liposome vesicles was doubled. This did not cause any substantial increase in the dye leakage rate or the amount of dye released as shown in Figure 5. This result implies that the dye leakage was caused by perforation of membrane layer rather than the complete disruption of membrane. Indeed, if QPEG was disrupting the membrane structure, then as its volume increased the number of disrupted vesicles and hence the amount of dye released should have increased. A probable explanation of these results is that as QPEG interacts with the membrane, it prevents further interaction of other QPEG polymers within the vicinity. Once the liposome vesicle surface was saturated with QPEG, further increase in its concentration does not result in an increase in dye leakage.


22

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

Langmuir

Normalized Fluorencence Intensity

Page 23 of 36

QPEG addition

Triton X addition

200

400

600

800

1000

200 L 100 L 1200 1400

Time (s)

Figure 5. Effect of QPEG copolymer concentration on Calcein dye leakage from PE:PG = 80:20 (mol:mol) liposome vesicles (E. Coli mimic) followed by addition of 100 µL of 10 wt% aqueous solution of Triton X-100 surfactant after 700 sec. Dynamic Light Scattering Analysis of PEG Copolymers Interacting with Liposome Vesicles. To further probe the dye leakage mechanism, the particle size of the individual components were measured by dynamic light scattering under the same conditions used for the Calcein dye leakage assay. The particle size of the PEG copolymers in the presence and absence of phospholipid vesicles and Triton X-100 surfactant were investigated in the buffer and the results shown in Figure 6. A bimodal particle size distribution was found for the QPEG copolymer with a z-average diameter of 277 nm and 50 nm. The particle size distribution of the Quart copolymer on the other hand, was unimodal with a z-average diameter of 23 nm (as shown in Supporting Information Figure S8C). The 80:20 molar ratio of DOPE:DOPG had z-average diameter of 307


23

Langmuir 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 36

nm. It was also demonstrated that the QPEG and Quart copolymers were cationic having zeta potential of 17.55 mV and 30 mV respectively. As seen in Figure 6C, the addition of QPEG copolymer to DOPE:DOPG = 80:20 (mol:mol) liposome vesicles (PGPE), results in a slight but significant decrease in the vesicle peak to 299 nm. This change is due to the averaging of the distributions of QPEG copolymer and the PGPE vesicles. When the PGPE vesicles and QPEG were mixed together, dye leakage occurred as shown in Figure 4C. The fact that a slight decrease in the vesicles’ particle size was observed provides evidence that rearrangement of the PEG chains during intercalation of the QPEG with the lipid molecules in the PGPE vesicles occurred. If no rearrangement of chains had occurred, the particle size of the PGPE vesicle would have increased. On addition of surfactant Triton X-100, complete disappearance of the peak at 299 nm along with new peaks at 11 nm and 166 nm (Figure 6D) were observed. As seen in Figure 6F, the 11 nm peak corresponds to Triton-X 100. The appearance of the new peak at 166 nm was investigated by the addition of Triton X-100 to the solution of QPEG copolymer. After the addition of Triton X-100, the particle size distribution looked similar to that of the mixture of PGPE and QPEG copolymer. What this means is that the addition of Triton X-100 surfactant to the PGPE liposome vesicles results in complete destruction of the vesicles (Supporting Information Figure S8F). Furthermore, these observations indicate that the QPEG copolymer disrupts the integrity of the vesicles’ membranes without affecting its structure, unlike Triton X-100. As shown in Figure 6E, a new peak at 149 nm was observed when QPEG and Triton X100 was mixed. This particle size is not much smaller than the structure at 166 nm, but much smaller than the partially disrupted membrane structure of PGPE and QPEG at 299 nm. It is


24

Page 25 of 36 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

Langmuir

speculated that the peak at 149 nm is due to remnants of broken PGPE vesicles entangled with QPEG and Triton X-100 molecules.


25

Langmuir 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 26 of 36

Figure 6. The particle size distributions of (A) QPEG copolymer only, (B) PG:PE phospholipid vesicle (E. Coli mimic) only, (C) PG:PE phospholipid vesicle in the presence of QPEG copolymer, (D) PG:PE phospholipid vesicle and QPEG copolymer after the addition of 100 µL of a 10 wt% aqueous solution of Triton X-100 surfactant, (E) QPEG copolymer in the presence of Triton X100 surfactant and (F) Triton X-100 surfactant only.


26

Page 27 of 36 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

Langmuir

Interestingly, repeating the same sequence of steps using the control copolymer (Quart) with no PEG brushes, results in a decrease in z- average particle size of PGPE liposome vesicles (Supporting Information Figure S8A). This reduction in size is attributed to interactions of the Quart copolymer with the liposome vesicles. It has been observed that the membrane radius or curvature changes when the head groups of the lipids changes.7 The interaction of cationic Quart with the anionic heads of DOPG lipids changes the diameter of the vesicles. This change occurs without causing any leakage of the dye. Even though, the Quart copolymer has a higher cationic charge than the QPEG, the Quart does not adversely affect the integrity of the vesicle membrane because no leakage was observed. To explain these results, we propose the following mechanism for vesicle membrane disruption by the PEG “bottle-brush” copolymer as illustrated in Scheme 2. The DLS experimental results suggests that the QPEG brush polymers binds to the vesicle surface and causes poration of their membranes via an entropic templating mechanism. This leads to leakage of the Calcein dye, a decrease in the size of the vesicles and finally collapse of the vesicles themselves. Increasing the concentration of QPEG does not lead to an increase in the dye release rates as bound QPEG prevents further attachment of additional QPEG molecules. This finding provides additional evidence that an ionic interaction of the copolymer with the liposome vesicles alone is not sufficient. That is, the “bottle-brush” copolymer architecture is also needed for lysis of the microbial membrane mimics.


27

Langmuir 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 28 of 36

Scheme 2. Proposed mechanism of vesicle membrane rupture: entropic templating of the QPEG “bottle-brush” copolymer onto the surface of the lipidic membrane leads to poration, Calcein dye leakage and a decrease in the vesicle particle sizes.

As reported by Sheiko et al.,20 mixing of a “bottle-brush” polymer with immiscible polymers was shown using Langmuir–Blodgett films (LB), which demonstrates two-dimensional (2D) mixing. The current study22 demonstrates this phenomenon in 3D. The driving force for mixing of the LB films was the compression pressure applied whereas in the current study a plausible explanation of the mixing is the multi-point interaction of the Quaternary groups of the “bottle-brush” polymer with the anionic groups on the surface of the vesicles. This interaction increases as more and more groups are attached to the vesicles. This leads to compression of PEG chains. To relieve the pressure, the chains intercalate with lipids in vesicles causing leakage. In summary, the novelty of this study is the finding that an amphiphilic structure is not a requirement for phospholipid membrane lysis. To the best of the authors’ knowledge, all antimicrobial polymers previously reported in the literature are amphiphilic with both a cationic and a lipidic group. This study provides the first example of a fully hydrophilic polymer (with no


28

Page 29 of 36 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

Langmuir

lipidic group) being capable of membrane lysis. Moreover, the unique architecture of the copolymer with PEG (a lipid-insoluble polymer) indicates it is capable of lipidic membrane lysis, whereas the non-PEGylated polymer did not cause any membrane disruption or vesicle leakage.


29

Langmuir 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 30 of 36

CONCLUSIONS A novel cationic PEG-based copolymer with “bottle-brush” architecture, QPEG as well as a control cationic polymer without PEG brushes, Quart was successfully synthesized and characterized. By careful comparison with non-PEGylated cationic polymers, Quart, the importance of PEG chains in the membrane disrupting activity of the PEGylated cationic polymer, QPEG was demonstrated. 1H-

and

13C-NMR,

MALDI-TOF mass spectral analysis, DLS and zeta potential

measurements were used to confirm their chemical and physical structures. The QPEG copolymer structure comprised of design features for binding to phospholipid cell membranes as well as for undergoing transformations that disrupt the cell wall of microbial mimics for selective antimicrobial activity. Using the QPEG copolymer with “bottle-brush” architecture, selective lysis of bacterial cell membrane mimics was successfully demonstrated with no effect on the membranes of RBC mimics. The interaction between the copolymers and the membranes of the microbial mimics was demonstrated using a Calcein dye release assay. DLS experiments provided additional evidence for the mechanism of dye leakage from liposome vesicles, whereby the cationic QPEG “bottle brush” copolymers partially disrupts the lipid membrane of anionic but not zwitterionic vesicles. In fact, the release of dye was caused by compromising the integrity of the membrane without affecting the structure of the vesicles. In conclusion, this study provides evidence that amphiphilic structures are not a requirement for phospholipid membrane lysis. Using the concept of entropic templating, hydrophilic architectures are shown herein for lysis of lipid membranes of E.coli mimics. This


30

Page 31 of 36 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

Langmuir

finding itself is interesting, and can contribute to an expansion in the design of membrane disrupting materials.

ASSOCIATED CONTENT Figures showing PEG MIDA synthesis scheme, 1H- and 13C-NMR of polymers and DLS analysis of composite systems are included in supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGEMENTS The authors are grateful to Prof. Jayant Kumar for use of the Fluorescence Spectrophotometer, Dr. Soujanya Muralidhara for assistance with the DLS measurements, NSF Award # DUE-1044363 for financial support and the Nanomanufacturing Center at the University of Massachusetts Lowell for facility support. Mass spectral data were obtained at the University of Massachusetts Amherst Mass Spectrometry Center. A.L.G. is also grateful to the department of Plastics Engineering for a Teaching Assistantship. The authors also thank the anonymous reviewers for their questions and suggestions to improve the quality of this paper.


31

Langmuir 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 32 of 36

REFERENCES (1)

Center for Disease Control and Prevention. National and State Healthcare Associated Infections Progress Report; CDC, 2016; pp 1–147.

(2)

Perencevich EN; Pittet D. Preventing Catheter-Related Bloodstream Infections: Thinking Outside the Checklist. JAMA 2009, 301 (12), 1285–1287. https://doi.org/10.1001/jama.2009.420.

(3)

Tavernise, S.; Grady, D. Infection Raises Specter of Superbugs Resistant to All Antibiotics. New York Times. New York, NY May 27, 2016, p A12.

(4)

Center for Disease Control and Prevention. Antibiotic Resistance Threats in the United States; CDC, 2013.

(5)

Shai, Y. Mode of Action of Membrane Active Antimicrobial Peptides. Pept. Sci. 2002, 66 (4), 236–248. https://doi.org/10.1002/bip.10260.

(6)

Hancock, R. E. W.; Sahl, H.-G. Antimicrobial and Host-Defense Peptides as New AntiInfective Therapeutic Strategies. Nat. Biotechnol. 2006, 24, 1551.

(7)

McMahon, H. T.; Gallop, J. L. Membrane Curvature and Mechanisms of Dynamic Cell Membrane Remodelling. Nature 2005, 438, 590.

(8)

Zasloff, M. Antimicrobial Peptides of Multicellular Organisms. Nature 2002, 415, 389.

(9)

Beveridge, T. J. Structures of Gram-Negative Cell Walls and Their Derived Membrane Vesicles. J. Bacteriol. 1999, 181 (16), 4725–4733.

(10) Nederberg, F.; Zhang, Y.; Tan, J. P. K.; Xu, K.; Wang, H.; Yang, C.; Gao, S.; Guo, X. D.; Fukushima, K.; Li, L.; Hedrick, J. L.; Yang, Y.-Y. Biodegradable Nanostructures with Selective Lysis of Microbial Membranes. Nat. Chem. 2011, 3, 409. (11) Engler, A. C.; Wiradharma, N.; Ong, Z. Y.; Coady, D. J.; Hedrick, J. L.; Yang, Y.-Y. Emerging Trends in Macromolecular Antimicrobials to Fight Multi-Drug-Resistant Infections. Nano Today 2012, 7 (3), 201–222. https://doi.org/10.1016/j.nantod.2012.04.003. (12) Morris, C. J.; Beck, K.; Fox, M. A.; Ulaeto, D.; Clark, G. C.; Gumbleton, M. Pegylation of Antimicrobial Peptides Maintains the Active Peptide Conformation, Model Membrane Interactions, and Antimicrobial Activity While Improving Lung Tissue Biocompatibility Following Airway Delivery. Antimicrob. Agents Chemother. 2012, 56 (6), 3298–3308. https://doi.org/10.1128/AAC.06335-11.


32

Page 33 of 36 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

Langmuir

(13) Chen, W.-L.; Cordero, R.; Tran, H.; Ober, C. K. 50th Anniversary Perspective: Polymer Brushes: Novel Surfaces for Future Materials. Macromolecules 2017, 50 (11), 4089–4113. https://doi.org/10.1021/acs.macromol.7b00450. (14) Trongsatitkul, T.; Budhlall, B. M. Temperature Dependence of Serum Protein Adsorption in PEGylated PNIPAm Microgels. Colloids Surf. B Biointerfaces 2013, 103, 244–252. https://doi.org/10.1016/j.colsurfb.2012.10.053. (15) Garle, A. L.; White, F.; Budhlall, B. M. Improving the Antifouling Properties of Polypropylene Surfaces by Melt Blending with Polyethylene Glycol Diblock Copolymers. J. Appl. Polym. Sci. 2018, 135 (15), 46122. https://doi.org/10.1002/app.46122. (16) Venkataraman, S.; Zhang, Y.; Liu, L.; Yang, Y.-Y. Design, Syntheses and Evaluation of Hemocompatible Pegylated-Antimicrobial Polymers with Well-Controlled Molecular Structures. Biomaterials 2010, 31 (7), 1751–1756. https://doi.org/10.1016/j.biomaterials.2009.11.030. (17) Lane, R. D. A Short-Duration Polyethylene Glycol Fusion Technique for Increasing Production of Monoclonal Antibody-Secreting Hybridomas. J. Immunol. Methods 1985, 81 (2), 223–228. https://doi.org/10.1016/0022-1759(85)90207-8. (18) Lentz, B. R. PEG as a Tool to Gain Insight into Membrane Fusion. Eur. Biophys. J. 2007, 36 (4), 315–326. https://doi.org/10.1007/s00249-006-0097-z. (19) Koley, D.; Bard, A. J. Triton X-100 Concentration Effects on Membrane Permeability of a Single HeLa Cell by Scanning Electrochemical Microscopy (SECM). Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (39), 16783–16787. https://doi.org/10.1073/pnas.1011614107. (20) Sheiko, S. S.; Zhou, J.; Arnold, J.; Neugebauer, D.; Matyjaszewski, K.; Tsitsilianis, C.; Tsukruk, V. V.; Carrillo, J.-M. Y.; Dobrynin, A. V.; Rubinstein, M. Perfect Mixing of Immiscible Macromolecules at Fluid Interfaces. Nat. Mater. 2013, 12, 735. (21) Szleifer, I. Macromolecular Mixing: Entropic Templating. Nat Mater 2013, 12 (8), 693– 694. (22) Garle, A. L.; Budhlall, B. M. PEG Bottle Brush Copolymers for Lysis of Microbial Membrane Mimics by Entropic Templating. Annu. Meet. Soc. Biomater. 2017, 38, 340. (23) AL-Badri, Z. M.; Som, A.; Lyon, S.; Nelson, C. F.; Nüsslein, K.; Tew, G. N. Investigating the Effect of Increasing Charge Density on the Hemolytic Activity of Synthetic Antimicrobial Polymers. Biomacromolecules 2008, 9 (10), 2805–2810. https://doi.org/10.1021/bm800569x.


33

Langmuir 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 34 of 36

(24) Pozzi, D.; Colapicchioni, V.; Caracciolo, G.; Piovesana, S.; Capriotti, A. L.; Palchetti, S.; De Grossi, S.; Riccioli, A.; Amenitsch, H.; Lagana, A. Effect of Polyethyleneglycol (PEG) Chain Length on the Bio-Nano-Interactions between PEGylated Lipid Nanoparticles and Biological Fluids: From Nanostructure to Uptake in Cancer Cells. Nanoscale 2014, 6 (5), 2782–2792. https://doi.org/10.1039/C3NR05559K. (25) Cruz, L. J.; Tacken, P. J.; Fokkink, R.; Figdor, C. G. The Influence of PEG Chain Length and Targeting Moiety on Antibody-Mediated Delivery of Nanoparticle Vaccines to Human Dendritic Cells. Biomaterials 2011, 32 (28), 6791–6803. https://doi.org/10.1016/j.biomaterials.2011.04.082. (26) Fukushima, K.; Tan, J. P. K.; Korevaar, P. A.; Yang, Y. Y.; Pitera, J.; Nelson, A.; Maune, H.; Coady, D. J.; Frommer, J. E.; Engler, A. C.; Huang, Y.; Xu, K.; Ji, Z.; Qiao, Y.; Fan, W.; Li, L.; Wiradharma, N.; Meijer, E. W.; Hedrick, J. L. Broad-Spectrum Antimicrobial Supramolecular Assemblies with Distinctive Size and Shape. ACS Nano 2012, 6 (10), 9191– 9199. https://doi.org/10.1021/nn3035217. (27) Tockary, T. A.; Osada, K.; Chen, Q.; Machitani, K.; Dirisala, A.; Uchida, S.; Nomoto, T.; Toh, K.; Matsumoto, Y.; Itaka, K.; Nitta, K.; Nagayama, K.; Kataoka, K. Tethered PEG Crowdedness Determining Shape and Blood Circulation Profile of Polyplex Micelle Gene Carriers. Macromolecules 2013, 46 (16), 6585–6592. https://doi.org/10.1021/ma401093z. (28) Lee, H.; Venable, R. M.; MacKerell, A. D.; Pastor, R. W. Molecular Dynamics Studies of Polyethylene Oxide and Polyethylene Glycol: Hydrodynamic Radius and Shape Anisotropy. Biophys. J. 2008, 95 (4), 1590–1599. https://doi.org/10.1529/biophysj.108.133025. (29) Ramadan, A. M.; Mosleh, S.; Gawish, S. M. Grafting and Quaternization of 2(Dimethylamino) Ethyl Methacrylate onto Polyamide-6 Fabric Pretreated with Acetone. J. Appl. Polym. Sci. 2001, 81 (10), 2318–2323. https://doi.org/10.1002/app.1672. (30) Gabriel, G. J.; Som, A.; Madkour, A. E.; Eren, T.; Tew, G. N. Infectious Disease: Connecting Innate Immunity to Biocidal Polymers. Mater. Sci. Eng. R Rep. 2007, 57 (1), 28–64. https://doi.org/10.1016/j.mser.2007.03.002. (31) Xia, G.; Kohler, T.; Peschel, A. The Wall Teichoic Acid and Lipoteichoic Acid Polymers of Staphylococcus Aureus. Pathophysiol. Staphylococci Post-Genomic Era 2010, 300 (2), 148–154. https://doi.org/10.1016/j.ijmm.2009.10.001. (32) Colak, S.; Nelson, C. F.; Nüsslein, K.; Tew, G. N. Hydrophilic Modifications of an Amphiphilic Polynorbornene and the Effects on Its Hemolytic and Antibacterial Activity. Biomacromolecules 2009, 10 (2), 353–359. https://doi.org/10.1021/bm801129y.


34

Page 35 of 36 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

Langmuir

(33) DeGrado, W. F.; Musso, G. F.; Lieber, M.; Kaiser, E. T.; Kézdy, F. J. Kinetics and Mechanism of Hemolysis Induced by Melittin and by a Synthetic Melittin Analogue. Biophys. J. 1982, 37 (1), 329–338. https://doi.org/10.1016/S0006-3495(82)84681-X. (34) Arnt, L.; Tew, G. N. Cationic Facially Amphiphilic Poly(Phenylene Ethynylene)s Studied at the Air−Water Interface. Langmuir 2003, 19 (6), 2404–2408. https://doi.org/10.1021/la0268597.


35

Langmuir 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 36 of 36

TABLE OF CONTENTS


36

PEG Bottle Brush Copolymers as Antimicrobial Mimics: Role of

Recommend Documents