Liposome Surface-Initiated ARGET ATRP: Surface Softness

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

Liposome Surface-Initiated ARGET ATRP: Surface Softness Generated by “Grafting from” Polymerization Tsukuru Masuda, Naohiko Shimada, and Atsushi Maruyama Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00450 • Publication Date (Web): 30 Mar 2019 Downloaded from http://pubs.acs.org on March 31, 2019

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Liposome Surface-Initiated ARGET ATRP: Surface Softness Generated by ÒGrafting fromÓ Polymerization

Tsukuru Masuda, Naohiko Shimada, Atsushi Maruyama* School of Life Science and Technology, Tokyo Institute of Technology, B-57 4259 Nagatsutacho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan *E-mail: [email protected]

ABSTRACT

Liposomes are self-assembled vesicles of amphiphilic lipid molecules, which have been investigated as models of cells, or tools for drug delivery systems. In these systems, the surface property of the liposomes plays an important role.

In this study, we demonstrated a novel

polymer modification of liposome surfaces using a controlled radical polymerization, Òactivators regenerated by electron transfer for atom transfer radical polymerization (ARGET ATRP)Ó, in

ACS Paragon Plus Environment

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aqueous media without a deoxygenation step.

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Dynamic light scattering and 1H NMR

measurement indicated the successful modification of the polymer on the liposome surface. The molecular weight of the grafted polymer chain was systematically controlled by changing the monomer concentrations in the Ògrafting fromÓ polymerization. Moreover, the modification resulted in a notable increase in surface softness as indicated by electrophoretic behavior, which was comparable to the surface of cells.

The preparation method and the characterization

presented in this study would be a helpful guideline in designing polymer/liposome hybrid having target surface properties.

INTRODUCTION Phospholipid bilayers such as liposomes or bicelles are self-assembled structures of amphiphilic lipid molecules. Because of their similarity to the biological membranes and their biocompatibility, phospholipid bilayer systems are used as models of cells, as platforms for analysis of membrane proteins, and as tools for drug delivery.[1-7] The types of polymer chains on the surface of a phospholipid bilayer tune the surface properties. For example, uptake of molecules into cells can be regulated using temperature-responsive or pH-responsive liposomes.[6,7] In addition, modification of cellular membranes by polymer chains is of interest in biomaterials development and tissue engineering.[8,9]

In these systems, the systematic

control of the surface structure is important to obtain the target property. Atom transfer radical polymerization (ATRP) is a controlled radical polymerization technique. Surface-initiated ATRP is widely used in design of polymer-modified surfaces on


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solid substrates.[10,11]

Recently, activators regenerated by electron transfer (ARGET) for

ATRP have been developed to overcome the need for a high concentration of catalyst and oxygen sensitivity of the originally report method.[12,13]

Moreover, ARGET ATRP can

proceed in aqueous media under ambient conditions and thus is useful for introduction of polymer chains into materials in water by the Ògrafting fromÓ method.[14,15] For instance, Averick et al. reported preparation of polymer-protein hybrids using the Ògrafting fromÓ method under biologically relevant conditions.[14]

Matsukawa et al. developed a surface-grafted

hydrogel in which the surface structure dominates the thermo-responsive behavior.[15] Modifications of polymer chains on the surfaces of liposomes are typically installed using a Ògrafting toÓ approach.[9] We hypothesized that ARGET ATRP could be used to modify a liposome surface by the Ògrafting fromÓ method. In this study, we investigated ARGET ATRP initiated on the surface of liposomes as a model of Ògrafting fromÓ phospholipid bilayers (Fig. 1). This approach is powerful because polymerization can proceed in aqueous media without the need for a deoxygenation step, such as gas-bubbling and freeze-and-thaw steps, that causes damages to liposomes and their formulations. The effects of polymer chain modifications on the surface properties of the liposome were also evaluated.


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bromoisobutyric acid (BIBA-NHS) was purchased from Aldrich (St. Louis, MO). 1,2-Dioleoylsn-glycero-3-phosphoethanolamine (DOPE) was purchased from Tokyo Chemical Industry (Tokyo, Japan). 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) was purchased from NOF Corporation (Kawasaki, Japan).

All other chemical reagents were obtained from

FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) unless otherwise noted.

Synthesis and characterization of ATRP initiator-conjugated DOPE (DOPE-I) DOPE (74.4 mg, 0.1 mmol) and BIBA-NHS (52.8 mg, 0.2 mmol) were dissolved in chloroform (1 mL), and the reaction solution was incubated at 25 ¡C for 24 h (Scheme S1). The resulting lipid was purified by Bligh-Dyer method[16] as follows. Aqueous solution (1 mL) containing NaHCO3 (0.1 M) was added to the chloroform solution, followed by adding methanol (2 mL). Subsequently, chloroform (1 mL) and water (1 mL) were added, and the separated lower phase (chloroform solution) was obtained. The solvent was evaporated under nitrogen gas flow, followed by vacuum drying, to obtain the target lipid. The obtained lipid was characterized by 1H NMR measurement (Avance 400, Buruker) using chloroform-d as a solvent at 25 ¡C.

Modification of PDMAAm on the surface of liposome by ARGET ATRP The large unilamellar vesicles (LUVs) composed of DOPC and DOPE-I were prepared as follows. A dry lipid film of DOPC/DOPE-I = 90/10 (mol/mol) was hydrated with aqueous solution containing glucose (200 mM) by vortex mixing for 2 min. The liposomes were extruded through a polycarbonate membrane (pore size: 0.1 µm). The PDMAAm grafted LUVs with various chain lengths were prepared by changing feed monomer concentrations in ARGET ATRP (Table S1 in Supporting Information). The typical


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procedure of liposome surface-initiated ARGET ATRP was as follows. Aqueous solution (500 µL) containing DMAAm (200 mM), CuBr2 (0.4 mM), Me6TREN (4 mM) and glucose (50 mM) was mixed with LUV dispersion (500 µL, total lipid concentration: 2mM). The obtained ATRP reaction solution (1 mL) contains DMAAm (100 mM), CuBr2 (0.2 mM), Me6TREN (2 mM), glucose (122.5 mM), and LUVs, where the ATRP initiator concentration on the outer surface of liposomes is 0.05 mM. To initiate ATRP, aqueous solution (10 µL) containing ascorbic acid (200 mM) was added, and the reaction dispersion was incubated at 25 ¡C for 50 min. During the polymerization, an aliquot (20 µL) of the reaction dispersion was sampled for dynamic light scattering measurement. After the polymerization, the liposome dispersion was dialyzed against 200 mM glucose aqueous solution using a cellulose membrane (MWCO: 14k) for 2 days to remove unreacted monomers. To analyze the obtained polymer, the mixture of the polymer and lipids were extracted by Bligh-Dyer method,[16] and analyzed by 1H NMR (Avance 400) using chloroform-d as a solvent at 25 ¡C.

Particle size and electrophopertic mobility of the particles The hydrodynamic diameters of unmodified or PDMAAm-modified LUVs were estimated by dynamic light scattering (DLS) using ZetaSizer Nano ZS (Malvern, Worcestershire, UK). The sample dispersion (lipid concentration: 20 µM) was prepared in 200 mM glucose aqueous solution. The electrophopretic mobility of non-modified or PDMAAm-modified LUVs were measured by using ZetaSizer Nano ZS (Malvern).

The sample dispersion (lipid

concentration: 40 µM) was prepared in aqueous solution containing NaCl and glucose to be isotonic (200 mOsm).


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Membrane fluidity of liposomes The membrane fluidity of liposomes was estimated by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a fluorescent dye.

An aliquot (10 µL) of ethanol

solution containing 100 µM DPH was added to the liposome dispersion (0.15 mM lipid and 200 mM glucose), followed by incubation at 30 ¡C for 30 min. The fluorescence of DPH (!ex = 360 nm, !em = 430 nm) was monitored by using a spectrofluorometer (FP-6500, Jasco) at 25 ¡C. The fluorescence polarization (P) was calculated by the following equation: P = (IVV - GIVH)/(IVV + GIVH) where IVV and IVH represent the intensities of the parallel polarized fluorescence and vertically polarized fluorescence, respectively. The instrumental correction factor, G, was 0.864.

Transmission electron microscopy (TEM) observation TEM observations for the unmodified liposome and the polymer-grafted liposome were performed by using a JEM-1400 plus (JEOL) with carbon-coated copper grids. The samples were negatively strained with gadolinium acetate.

RESULTS AND DISCUSSION An ATRP initiator with an activated ester group was covalently conjugated through an amide bond to the head group of DOPE to yield DOPE-I (Fig. 1b). The obtained DOPE-I was purified by the Bligh-Dyer method.[16] Analysis of the 1H NMR spectrum confirmed that


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DOPE-I was successfully prepared because the peak derived from the ATRP initiator (-COC(CH3)2-Br) was detected at ! = 1.9 (Figures S1 and S2 in Supporting Information). The procedure for liposome surface-initiated ARGET ATRP involves (1) preparation of mixed liposomes composed of DOPC/DOPE-I (90:10, mol/mol) and (2) ARGET ATRP from the ATRP initiator displayed on the surface of the DOPC/DOPE-I liposome (Fig. 1a). Note that ARGET ARTP can proceed without the need for a deoxygenation step, such as gas-bubbling and freezeand-thaw steps, which causes damages to liposomes and their formulations (an example shown as Fig. S3 in Supporting Information). In this procedure, only the polymer chains on the outer surface of the liposome are modified because the ionic Cu(II)/Me6TREN complex, the catalyst for ATRP, cannot penetrate through the lipid bilayer. Liposomes were prepared as LUVs using the extrusion technique under isotonic conditions (200 mOsm), where the osmotic pressure was calculated on the basis of the concentrations of solutes. The hydrodynamic diameter (z-average size) was determined to be 135 nm by DLS measurements. In this study, DMAAm, a conventional hydrophilic vinyl monomer was polymerized in aqueous solution containing 100 mM DMAAm, 0.2 mM CuBr2, 2 mM Me6TREN, 2 mM ascorbic acid, and 122.5 mM glucose when the feed monomer concentration was 100 mM. In the polymerization reaction, the concentration of lipid was adjusted to 1 mM, and the concentration of DOPE-I on the outer surface was 0.05 mM. Monomer consumption during the polymerization results in a lower osmotic pressure in the solution outside the liposomes relative to the inside; therefore, ARGET ATRP was initiated under hypertonic conditions (outer solution: 222.5 mOsm). If the osmotic pressure inside the liposome becomes higher than that outside, solvent penetrates into the liposome, which causes the rupture of the liposome.


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Fig. 2 shows the hydrodynamic sizes and the normalized scattering light intensity monitored by DLS as a function of time during ARGET ATRP with various monomer concentrations. For the liposome composed of DOPC/DOPE-I, both the hydrodynamic size and the light scattering intensity increased during ARGET ATRP. Moreover, the increments in the hydrodynamic size and the light scattering intensity became larger as the feed monomer concentration increased. For the liposome composed of only DOPC, no significant changes in the hydrodynamic size or the light scattering intensity were observed even when the monomer, catalyst, and reducing agent were present in the outer solution. This result indicated that the polymerization proceeded from the surface of the liposome containing DOPE-I, and the polymer chain was modified on the outer surface of the liposome. Fig. 3 shows the size distributions of the as-prepared DOPC/DOPE-I liposomes and the DOPC/DOPE-I liposomes after ARGET ATRP and dialysis under isotonic condition.

After ARGET ATRP and dialysis, the size

distribution remained unimodal. In addition, for the unmodified and polymer-grafted liposomes, spherical structures were observed by TEM (Figure S4 in Supporting Information). The sizes were approximately similar to those estimated by DLS measurements.


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Table 1. Characterizations of the unmodified liposome and the PDMAAm-grafted liposomes prepared by ARGET ATRP. Feed monomer / mM

Mn b)

Unmodified liposome

-

P2.3k-liposome

Sample a)

Polymer Hydrodynamic content / wt% diameter / nm

Fluorescence polarization, P d)

b)

c)

-

-

135

0.058

25

2.3 ! 103

12.5

137

0.063

P4.8k-liposome

50

4.8 ! 103

32.0

143

0.068

P11k-liposome

100

1.1 ! 104

41.5

164

0.075

a) The PDMAAm-grafted liposomes are referred as Px-liposomes where x represents the number-averaged molecular weight of the grafted PDMAAm. b) Determined by 1H NMR measurement. c) Determined by DLS measurement. d) Determined by fluorescence measurement of DPH.

We hypothesize that the grafted PDMAAm chain would alter the surface properties of the liposome. To test this, the electrophoretic mobility (µ) of the unmodified and PDMAAmmodified liposomes was measured as a function of ionic strength (Fig. 5). The µ values for each liposome sample were negative because these liposomes contain anionic DOPE-I. The absolute values of µ for PDMAAm-modified liposomes were smaller than those of the unmodified liposomes. Of important, the absolute values of µ decreased as the Mn of the grafted PDMAAm became larger. This may be due to a decrease in charge density and/or the outer electrolyte solution penetration due to the surface modification of PDMAAm chain. Morimoto et al. reported that surface modification of a self-assembled nanogel containing cationic poly(L-lysine) by enzymatic polymerization with a neutral saccharide chain caused masking of the cationic charge of the nanogel.[19] In our system, the neutral PDMAAm chain likely masks the anionic


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charge of phosphate group. We also investigated the effect of fetal bovine serum (FBS) addition on the property of the unmodified and polymer-grafted liposomes. For the unmodified liposome, significant decrease in the absolute value of µ as well as aggregation occurred in the presence of 5% FBS, while those for the polymer-grafted liposomes did not (Table S2 and Table S5 in Supporting Information).

This result indicates the polymer modification on the surface of

liposomes inhibits the interaction between the liposomes and serum components. Theoretical models were used to further analyze the electrophoretic behaviors of the PDMAAm-modified and unmodified liposomes.

For the unmodified liposome, the

electrophoretic mobility is related to the zeta potential (") by SmoluchowskiÕs formula: !! ! !

!! !! !

(1)

!

where #r is the relative permittivity, #0 is the permittivity of a vacuum, and $ is the viscosity of the dispersion medium. The " value for the unmodified liposome at ionic strength of 10 mM was -46.6 mV. For the PDMAAm-modified liposome, " cannot be defined because the solvent and electrolytes can permeate in the grafted polymer layer. According to OhshimaÕs theory [20], the electrophoretic mobility of ÒsoftÓ particles is related to the charge density (charge amount per volume, ZN) and the softness parameter (%-1) as follows: !! ! !

!"# !!!

!! ! !

! ! !!!!!!!! !

!!!!!!!

(2)

where & is DebyeÕs parameter and e is the elementary electric charge. The parameter % is defined as % = ('/$)1/2, where ' and $ are the friction coefficient of the grafted polymer and the viscosity of the solvent, respectively.

These parameters would be useful as indexes to

characterize liposomes modified with soft materials. Among the polymer-grafted liposomes, the electrophoretic mobility for the P11k-liposome as a function of ionic strength can be calculated


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For comparison, Table 2 summarizes the ZN and %-1 values for various systems that can be modelled as soft particles.[20-23] The ZN value of the PDMAAm-modified liposome was smaller than those of human blood cells and HL-60 RG cells. The surfaces of these cells are covered with saccharide chains including negatively charged sialic acid.

In contrast, the

PDMAAm chains covering the surface of liposomes are non-ionic, resulting in the lower charge density for the liposomes compared to the cells. The %-1 value of the P11k-liposome was approximately equivalent to that of the V. alginolyticus bacterial cell. Thus, the analysis on the basis of soft particles theory[20] also indicates successful modification of polymer chain onto the liposome surface by Ògrafting fromÓ approach. Because the structure of the polymer can be systematically controlled by using surface-initiated ATRP,[10] the polymer-modified liposomes prepared by the Ògrafting fromÓ approach could serve as cellular models of the property of cells, and we envision that surface modification of liposomes by a Ògrafting fromÓ approach will be useful in designing polymer/liposome hybrids with specific surface properties.

Table 2. The charge density (ZN) and softness parameter (!-1) values for the various ÒsoftÓ particles Sample

Temp. / ¡C

ZN / mM

!-1 / nm

Reference

P11k-liposome

25

-0.95

5.5

This study

PNIPAAm gel-latex beads

25

-1.5

1.2

20

35

-30

0.9

20

Human red blood cell

37

-15

2.5

21

HL-60 RG

37

-14

2.1

22

Bacteria, V. alginolyticus

25

-3.8

6.4

23


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CONCLUSIONS In summary, we demonstrated modification of polymer chains on a liposome surface by an ARGET ATRP-based Ògrafting fromÓ method in aqueous media without deoxygenation. DLS and 1H NMR measurements revealed that the polymerization of DMAAm proceeded from the ATRP initiator displayed on the surface of the liposomes. The hydrodynamic size of the liposome possessing the ATRP initiator increased during ARGET ATRP, whereas that of the liposome composed of DOPC only did not. The molecular weight of the grafted polymer chain was controlled by the feed monomer concentration in ARGET ATRP. Moreover, we found that the electrophoretic mobility decreased upon modification of the polymer chain; thus was attributed to the decrease in the charge density and the electrolyte solution penetration. This Ògrafting fromÓ approach and the characterization presented in this study will guide design of polymer/liposome hybrids having desired surface properties.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Synthesis of ATRP initiator-conjugated DOPE (DOPE-I), the reaction conditions of ARGET ATRP, 1H NMR spectra of DOPE and DOPE-I, an example the effect of freeze-and-thaw step on liposomes and the inclusion, TEM images, and effect of FBS addition on the properties of liposomes. (PDF) AUTHOR INFORMATION


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Corresponding Author *E-mail: [email protected] ORCID Tsukuru Masuda: 0000-0001-6452-811X Naohiko Shimada: 0000-0002-1664-1721 Atsushi Maruyama: 0000-0002-7495-2974 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes There are no conflicts of interest to declare.

ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant No. 15H01807 to A.M. and Grant No. 18K18384 to T.M.), by Center of Innovation (COI) program, Japan Science and Technology Agency (JST), and by the Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists (No. 17J04783 to T.M.). We are grateful to Ph.D. student, Ms. Wakako Sakamoto for helpful advices.

We thank the Biomaterials Analysis

Division, Tokyo Institute of Technology for technical assistance in transmission electron microscopy.


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(15) Matsukawa, K.; Masuda, T.; Akimoto, A. M.; Yoshida, R. A surface-grafted theroresponsive hydrogel in which the surface structure dominates the bulk properties. Chem. Commun. 2016, 52, 11064 - 11067. (16) Bligh, E. G.; Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959, 37, 911 - 917. (17) Jovanovic, A. A.; Balanc, B. D.; Ota, A.; Grabnar, P. A.; Djordjevic, V. B.; Savikin, K. P.; Bugarski, B. M.; Nedovic, V. A.; Ulrih, N. P. Comparative Effects of Cholesterol and !Sitosterol on the Liposome Membrane Characteristics. Eur. J. lipid Sci. Technol. 2018, 120, 1800039. (18) Suga, K.; Umakoshi, H. Detection of Nanosized Ordered Domains in DOPC/DPPC and DOPC/Ch Binary Lipid Mixture Systems of Large Unilamellar Vesicles Using a Tempo Quenching Method. Langmuir 2013, 29, 4830 Ð 4838. (19) Morimoto, N.; Yamazaki, M.; Tamada, J.; Akiyoshi, K. Polysaccharide-Hair Cationic Polypeptide Nanogels: Self-Assembly and Enzymatic Polymerization of Amylose PrimerModified Cholesteryl Poly(L-lysine). Langmuir 2013, 29, 7509 - 7514. (20) Ohshima, H. Electrophopretic mobility of soft particles. Electrophoresis 1995, 16, 1360 1363. (21) Kawahata, S.; Ohshima, H.; Muramatsu, N.; Kondo, T. Charge Distribution in the Surface Region of Hyman Erythrocytes as Estimated from Electrophoretic Mobility Data. J. Colloid and Interface Sci. 1990, 138, 182 - 186.


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Liposome Surface-Initiated ARGET ATRP: Surface Softness

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