Effects of Membrane Defects and Polymer Hydrophobicity on

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Effects of Membrane Defects and Polymer Hydrophobicity on Networking Kinetics of Vesicles Yan Xia, Hyun-Sook Jang, Zhiqiang Shen, Geoffrey D. Bothun, Ying Li, and Mu-Ping Nieh Langmuir, Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 20, 2017

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Effects of Membrane Defects and Polymer Hydrophobicity on Networking Kinetics of Vesicles Yan Xia,1 Hyun-Sook Jang,2,6 Zhiqiang Shen,3 Geoffrey D. Bothun,4 Ying Li,3 Mu-Ping Nieh1, 5 ,6* 1

Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT

06269, USA 2

Center for Soft and Living Matter (CSLM), Institute for Basic Science (IBS), Ulju-gun, Ulsan

689-798, Republic of Korea 3

Department of Mechanical Engineering, University of Connecticut, Storrs, CT 06269, USA

4

Department of Chemical Engineering, University of Rhode Island, Kingston, RI, 02881, USA

5

Department of Biomedical Engineering, University of Connecticut, Storrs, CT 06269, USA

6

Polymer Program, Institute of Materials Science, University of Connecticut, Storrs, CT 06269,

USA

ACS Paragon Plus Environment

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ABSTRACT: The kinetics of clustering unilamellar vesicles induced by inverse Pluronics, poly(propylene oxide)m–poly(ethylene oxide)n–poly(propylene oxide)m, POm-EOn-POm, were investigated via experiments and molecular dynamic simulations. Two important factors for controlling the networking kinetics are the membrane defects presumably located at the interfacial region between two lipid domains induced by acyl chain mismatch, and the polymer hydrophobicity. As expected, the clustering rate increases significantly with increasing bilayer defects on the membrane where the insertion of PPO is likely to take place because of the reduced energy barrier for the insertion of PO. The hydrophobic interaction between the PO blocks and membranes with the defects region dictates the “anchoring” kinetics which is controlled by the association-dissociation of PO with the lipid membrane. As a result, the dependence of clustering rate on polymer concentration is strongly influenced by the hydrophobicity of the PO blocks. Nevertheless, longer PO blocks show stronger association with the membrane, resulting in faster consumption of the “active” sites made of these defect regions (causing mostly “invalid” insertions) with increasing polymer concentration, hence inhibiting the formation of large networking clusters, while shorter PO blocks undergo more frequent association with/dissociation from the defects, allowing continuous formation of larger clusters with increasing polymer concentration. This study provides important insights into how biomembrane’s organization and dynamics influence its interaction with foreign amphiphilic molecules.


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INTRODUCTION Biomembranes define the boundary of the enclosed biological compartments, serving as a selective barrier that regulates the transport of foreign molecules.1 The membrane-associate biomolecules (e.g., proteins, cholesterol, oligosaccharides, etc) not only change the physicochemical properties of the membranes, but also provide important biological functions. For instance, it has been reported that the association of proteins containing intrinsically disordered domains can induce membrane bending because of their large hydrodynamic radii that generates steric pressure,2, 3 which provides mechanistic explanation for the exclusion of transmembrane cargo from the clathrin-coated pits. Moreover, certain lipid “rafts” that contain large portions of cholesterol, sphingolipids, and other protein and lipid constituents formed by lateral segregation of liquid ordered (Lo) and disordered phases (Ld) have important implications in signal transduction and membrane trafficking.1 For simplicity, Lo and Ld in this paper refer to the lipid phases at temperatures below and above the main transition temperature (Tm). .4, 5, 6, 7, 8 Though lipid phase segregation is often observed in the presence of cholesterol,9, 10, 11 it can also be induced by different physical properties of the acyl chains such as chain length, Tm and molecular architecture.12,

13

While the binding of certain molecules onto the membrane can oppress such lipid separation and achieve lipid mixing through generating crowding pressure.3, 14

Fig. 1. Schematic of a cluster formed by vesicles “strung” by POm-EOn-POm inverse pluronics. The red and green segments represent EO and PO blocks, respectively.


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The advancing development of synthetic chemistry enables the investigations of selective interactions between model membrane and biomolecules of interest.15,

16, 17, 18

One such

important interaction is the hydrophobic interaction that is present in the biomembrane, which drives amphiphilic molecules into bilayer structures.19 It plays an important role in controlling the dynamics of biomolecules and host membranes. The understanding of how polymer hydrophobicity, lipid architecture (fluidity or crystallinity) and mismatch of bilayer thickness collectively affect their interactions is thus of significance. In this report, triblock copolymers are used to “string” lipid bilayers to form clusters of unilamellar vesicles (ULVs). Investigation on the clustering mechanism provides fundamental insights into how kinetically- and energeticallycontrolled processes compete with each other, leading to different clustering rates and final cluster sizes. The polymer linkers are inverse Pluronics composed of a middle hydrophilic poly(ethylene oxide), PEO block, and two identical hydrophobic poly(propylene oxide), PPO ends with a chemical structure of POm-EOn-POm. This non-ionic surfactant has been widely used to form mesoporous polymers,20 colloidosomes21 and microemulsion of water.22 When the two PPO blocks of one inverse Pluronics insert into different bilayers due to hydrophobic interactions, the stringing of ULVs eventually forms a cluster (Fig. 1). Such networks have been observed by using other linkers, such as hydrophobically modified chitosan,23, 24 cholesterol-modified PEO25, 26

and NeutrAvdinTM.27 Here, the spontaneous formation and associated time scale of the system

allows us to investigate detailed interactions between the linkers and the host membranes, which will advance our understanding on the dynamics of membrane-associated molecules with the lipid bilayers. Recently, the interaction of Pluronic polymers (EOn-POm-EOn) with lipid bilayers has received considerable attention.28,

29, 30

Experimental and theoretical work have been conducted to


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establish a correlation between the PEO or PPO block lengths with its permeability into the lipid membranes.29,

31, 32, 33, 34, 35

The hydrophobicity of the Pluronics was found to dictate their

functions distinctly from a membrane sealant to a membrane disruptor.30 It has been reported that the PPO block with a comparable length as that of the lipid acyl chain is more likely to be inserted into the bilayer.33, 34 To the best of our knowledge, no prior study on the interaction between POm-EOn-POm and lipids has been reported. The inverse Pluronics belong to another category of amphiphilic molecules and are expected to behave differently from the regular Pluronics.


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EXPERIMENTAL SECTION Materials 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

(DPPC),

1,2-dimyristoyl-sn-glycero-3-

phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. Pluronic® 17R4 (PO14-EO24-PO14) and 31R1 (PO25-EO7-PO25) were purchased from SigmaAldrich (St. Louis, MO). PO26-EO20-PO26 was customized at Advanced Polymer Materials Inc. (Montreal, QC, Canada) and used without further purification. Preparation of Unilamellar vesicles (ULV) and Polymer Solutions Lipid mixtures of DPPC/DHPC at the DH/DP molar ratios of 0, 0.1 and 0.2 and DMPC/DHPC at DH/DM molar ratios of 0.1 and 0.2 were prepared individually. Dry lipid powders were first homogenized in chloroform. The chloroform was then removed under a stream of nitrogen at around ambient temperature followed by further sample drying under vacuum overnight. The lipid films were then hydrated to a lipid concentration of 0.5 wt.% in DI water. ULVs were produced by multi-pass extrusions (31 passes through the filter) through 100 nm-sized polycarbonate membranes (Avanti Polar Lipids, Alabaster, AL) at a temperature above Tm of the long-chain lipid (either DPPC or DMPC) after several freeze-dry cycles and vigorous vortex. 31R1 and PO26-EO20-PO26 were dissolved in filtered DI water using a 0.22 m Nylon filter at a concentration of 1 wt.%, and 17R4 was prepared in the same fashion at a concentration of 5 wt.%.. All the polymer solutions were subjected to magnetic stirring for overnight before use. Time-resolved UV-vis Absorption


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The UV-vis absorptions were conducted using a commercial PerkinElmer Lambda 1050 UV/Vis/NIR spectrophotometer. The apparent UV absorbance is proportional to the turbidity of solution, which increases as the big particles form. The UV spectra were also collected on polymer and vesicle solutions individually with an interval of 1 nm and scan speed of 324.44 nm/min. The aggregation kinetics of vesicles was monitored using time-resolved UV absorbance at 500 nm where no characteristic absorbance is observed in the UV spectrum of neither vesicle nor polymer solutions. Multi-angle Dynamic Light Scattering The aggregation kinetics of vesicles were also studied using time-resolved dynamic light scattering (DLS) by monitoring the change of hydrodynamic radius (RH) on an ALV compact goniometer systems with 4-detectors (CG3-3MD) (Hessen, Germany). The source is a 22 mW He-Ne laser (emitting vertically polarized light with a wavelength of 632.8 nm). Four avalanche photo diode (APD) detectors are equally spaced (34° apart) on an arc of a tray driven by a goniometer. For DLS, the field autocorrelation functions were collected by an ALV-7004 digital multiple tau correlator, yielding data in the range as short as 25 ns. We measured the RH at four different scattering angles, namely 32, 64, 96 and 128°. The RH is yielded using Einstein-Stokes 𝑘 𝑇

𝐵 relation: 𝑅𝐻 = 6𝜋𝜂𝐷 , where 𝑘𝐵 , T, 𝜂 and D are Boltzmann constant, the absolute temperature, the

viscosity of solvent and the diffusion coefficient of aggregates, respectively. Cryo-Transmission Electron Microscopy (Cryo-TEM) Cryo-TEM samples were prepared at 25 °C using a Vitrobot (FEI Company), which is a PCcontrolled robotic assembly for sample vitrification. Quantifoil grids were used with 2 μm carbon holes on 200 square mesh copper grids (Electron Microscopy Sciences, Hatfield, PA).


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The sample was first equilibrated within the Vitrobot at 25 oC and 100% humidity for 30 min. After immersing the grid into the sample, it was then removed, blotted to reduce film thickness, and vitrified in liquid ethane. The sample was then transferred to liquid nitrogen for storage. Imaging was performed at -170 oC in a cooled stage (model 626 DH, Gatan Inc., Pleasanton, CA) at 200 kV using a JEOL JEM-2100 TEM (Peabody, MA). Confocal Microscopy The aggregation of vesicles was monitored using laser scanning confocal microscopy (Leica TCS SP8 STED; Leica Microsystems). Standard confocal images were obtained using a 100×/1.4 NA oil-immersion objective lens for optical color correction and lipophilic but hydrophobic dye of DiI (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate, Ex/ Em = 549 /565 nm) with 0.01mol %. The white laser light (WLL) was used to excite the dye with the wavelength of 549 nm and embedded acousto-optical beam splitter (AOBS) selected emission detection ranges from 595 nm to 660 nm. Finally, intensity was collected via 3x HyD hybrid detector with low noise with high signal. Acquired raw data was further processed with Leica LAS X if needed. Molecular Dynamic Simulation Due to the limited approachable time and length scales of all-atom molecular dynamics (MD) simulations, MARTINI coarse-grained MD simulations have been performed to study the interaction between POm-EOn-POm polymers and DPPC (DPPC/DHPC) membranes. The lipid models for DPPC and DHPC are established based on the molecular structures that DPPC and DHPC have an identical head group attached to the acyl chains with different lengths which are described by two identical strings, each having four beads for DPPC and two beads each for DHPC.36, 37 In the mapping for inverse Pluronics, both the PPO and PEO monomers are


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represented by single particles in coarse-grained simulations. The potential parameters between PPO and PEO monomers and their interactions with lipids are described in literature.31,

38

Standard MARTINI water model was adopted in our simulations.39 All simulations have been performed under NPT ensemble. Both temperature and pressure are maintained to target values. The pressure was set at 1 bar and coupled control within the plane of the bilayer and independently controlled along the out-of-plane direction of the bilayer, guaranteeing the zero bilayer tension. Time step of 10 fs is taken in all simulations. Bilayers in simulations consist of 1352 lipid molecules. 64799 water molecules were added into the simulation box to ensure the suitable lipid molecule concentration. All the beads in our simulation shared the same constant mass of 72 amu for efficiency.40, 41 All the coarse-grained MD simulations have been performed by using Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) package.42 Considering the difference in phase transition temperature between model DPPC and real DPPC,36 we monitored the tail order parameters29 of DPPC in equilibrated bilayers in four series of temperature of 270K, 280K, 290K, and 300K. It is found that the tail orderness of DPPC increases dramatically between 290K and 280 K, which indicates that the liquid-to-gel phase transition occurs between 280K and 290K for the bilayer consisting of pure DPPC lipids. To ensure the gel state of the DPPC lipids in simulation by closely mimicking experiments, we maintained the system temperature at 280K in the following simulations. To avoid the crystallization of the non-polarize water molecules under that temperature, 10% of the water beads in the simulation box were changed to antifreeze particles.39 To test the interaction between polymers and bilayers, we firstly fully equilibrated the polymers and bilayers independently. In the subsequent simulations, to avoid the influence of


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thermal fluctuation and facilitate the insertion, the hydrophobic parts of polymers were put just above the lipid tails in bilayers. As shown in the main text, we found that the polymers had only been absorbed to the DPPC bilayers without any defects after 1 μs simulation. In comparison, the same polymers can insert into the DPPC/DHPC bilayers in a short time (10 ns). To determine the free energy difference between inserted and un-inserted polymers, we tried to pull the inserted polymers outside bilayers and calculated the potential of mean force (PMF) as a function of the distance between the center of mass of the polymers and bilayers using 1

umbrella sampling method.43 A harmonic potential 𝑈 = 2 𝑘(𝜉 − 𝜉0 )2 was applied on the centerof-mass of polymers, with the force constant 𝑘 = 2.16𝑘𝑐𝑎𝑙 ⁄ (𝑚𝑜𝑙/Å2 ). 𝜉 denotes the distance between the center-of-mass of polymers and lipid bilayer. A series of windows were performed at different values of 𝜉0 . The width of window is set to be 0.1 nm to ensure the consecutiveness of the PMF profile. Each window in the simulation lasted 50 ns for fully equilibrium. Weighted histogram analysis method (WHAM)44 was adopted to calculate the PMF profile during the pulling process. As shown in the main text, the free energy difference between the inserted and un-inserted polymers increases with the hydrophobicity of polymers.


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RESULTS AND DISCUSSION The effect of lipid composition of the bilayers on the clustering rate of ULV

DH/DP = 0 DH/DP = 0.1 DH/DP = 0.2

6

Normalized absorbance

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5

network was first investigated using

4

time-resolved UV-vis absorbance, as the

3

turbidity of the solution increases with

2

the growth of the clusters. The tested 1 0

100

200

Time (min)

ULVs were composed of DPPC (di-C16) and DHPC (di-C6) at different DHPC-to-

Fig. 2. Normalized UV absorbance as a function of time for

DPPC molar ratios (DH/DP). All ULVs

®

ULV/Pluronic 17R4 mixture with P/L of 2.5 at different DH/DP values

were obtained through extrusion, yielding

an average hydrodynamic radius, RH of ~50 nm. ULVs of the three series (DH/DP = 0, 0.1 and 0.2) were mixed with the Pluronic® 17R4 (PO14-EO24-PO14) individually to form a final polymer-to-lipid weight ratio, P/L of 2.5. A constant lipid concentration of 0.2 wt.% was used throughout the experiment. The time-resolved UV absorbance was collected at 25°C and normalized by the absorbance of ULV in the absence of 17R4. The UV absorbance of the ULV sample with DH/DP = 0 remains constant, suggesting no apparent clustering. Addition of the shorter-chain DHPC facilitates the cluster formation, which shows the highest increment of UV absorbance at the highest DHPC concentration, i.e., DH/DP = 0.2. Most recently, the spontaneous transfer rate constant of DMPC (di-C14) in the case of DMPC/DHPC discoidal bicelles was two orders of magnitude enhanced compared to that of DMPC vesicles, presumably due to the formation of interfacial defects between liquid-ordered (Lo) DMPC-rich and liquiddisordered (Ld) DHPC-rich domains owing to their acyl chain mismatch.45, 46, 47 The enhanced


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clustering rate here is presumably attributed to the “defects” of such interfaces between DPPC and DHPC, which provide ‘active sites’ for the insertion of POs with a reduced energy cost. The formation of large ULV clusters was confirmed using confocal microscopy (Fig. S1, Supporting Information). The micrograph of the ULV clusters was also obtained using cyrogenic transmission electron microscopy (Fig. S2, Supporting Information), where almost no multilamellar vesicles (MLVs) were observed, indicating the increased UV absorbance is mainly contributed by the large ULV clusters, instead of MLVs. Coarse-grained (a)

(b)

molecular dynamic (MD) DPPC

simulations also revealed phase segregation between DHPC

DPPC- and DHPC- rich PO

domains, y

EO

with

the tail

z

z y x

x

Fig. 3. Molecular Simulation result (a): the bilayer only contains pure DPPC (grey), where no insertion of polymer was found after 1 μs simulation, (b) bilayer is composed of both DPPC and DHPC (blue), where the insertion of polymer was observed at 10 ns. Models of DPPC, DHPC and polymer used in the molecular simulations are given at the left. Upper and lower panels represent the side and top views, respectively. For clarity, the lipids tails are made invisible in the top panels.

order

parameter

being

reduced significantly at the interfaces

(Fig.

S3,

Supporting

Information),

signifying the gel-to-fluid transition. More importantly, the polymer insertion into the bilayer was not observed in pure DPPC bilayer even after 1 μs simulation, while the insertion of the PPO took place after 10 ns at the interfacial region for DH/DP = 0.2 bilayer (Fig. 3). Since DPPC (Tm = 41 oC) is in Lo phase at 25°C, it is also possible that the PPO insertion for DH/DP = 0 bilayer is inhibited by the more ordered acyl chains of DPPC rather than the


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hydrophobic mismatch. To differentiate the two origins, ULVs composed of Ld-phase DMPC (Tm = 24 °C) were examined with 17R4 under the same condition. The solution remained clear and the time-resolved RH did not increase over a span of 24 hours (Fig. 1 and Fig. S4 in Supporting Information), suggesting that the inhibition of PPO insertion is independent of the lipid phases. More revealing results were obtained as the same P/L ratio was applied to the

(a) 1.0

Without polymer With polymer

w/o polymer w/ polymer

0.2

0.6 0.4

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0.2 0.0

0.0 10-1 100

Population

0.8

0.6 0.4

Without polymer With polymer

(b) 1.0

0.8

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101

102

103

104

105

Hydrodynamic radius (nm)

106

107

10-1

100 101 102 103 Hydrodynamic radius (nm)

104

Fig. 4. RH distribution of the DMPC/DHPC vesicles in the presence (red) and absence (black) of PO 14EO24–PO14 at (a) 20°C (4 hours after the mixing); (b) 28°C (9 hours after the mixing).

DMPC/DHPC ULVs with DH/DM = 0.2 at 20 (< Tm, DMPC) and 28 (> Tm, DMPC) °C, respectively. An increase in RH and turbidity was observed in the mixtures at 20 °C (Fig. 4(a)), while no significant change at 28 °C (Fig. 4(b)). It is anticipated that lipid segregation between DMPCand DHPC- rich domains occurs when T is below Tm,DMPC. In contrast, the bilayer becomes more homogeneous at T > Tm,DMPC. The experimental results of DPPC and DMPC samples confirm that such lateral segregation is required in order for the PPO to insert into the bilayer. Therefore, both acyl chain mismatch and lateral segregation are critical for clustering ULV. It should be mentioned that PPO block is likely to insert into the bilayer instead of weakly associating onto the membrane surface during the clustering process as suggested by the simulation work, which


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showed the quick insertion of PPO block into the acyl chain region once it associates onto the membrane that might take longer time.31 Polymers with different PPO and PEO lengths can interact differently with the bilayers,31,

32

and are therefore expected to alter the clustering

kinetics. Here, we examined the ULV clustering in the presence of 17R4, PO25–EO7–PO25 (Pluronic® 31R1) and PO26–EO20–PO26, respectively. Fig.5 shows the time-resolved UV absorbance of different polymer/ULV systems at various P/L ratios. The most striking outcome is the distinct clustering kinetics with respect to the increased P/L ratios. In the case of the most hydrophilic polymer, 17R4, the UV absorbance increased with increased P/L in general, suggesting that higher P/L ratios increase the clustering rate (Fig 5(a)). Eventually, ULV clusters precipitate for all P/L ratios other than 0 (Fig 5(d) and Fig S6). However, for more hydrophobic polymer linkers (with longer PPO lengths), i.e., 31R1 and PO26–EO20–PO26, lower P/L ratios (i.e., 1/100 and 1/50) slowly drive the ULVs into large-size clusters (Figs 5 (b) and 5(c)) and produces discrete precipitates separating from the solution (Figs 5(e) and 5(f)). This is consistent with the gradual increase of the observed turbidity of the polymer/ULV mixtures (Figs. S8 and S10 in Supporting Information). Note that in Fig. 5(c), part of the data from P/L of 1/2, the green curve, was removed because that part of data was collected when there were precipitations in the cuvette. After we gently shook the samples, the UV absorbance recovered nicely. The ULV clustering in samples with higher P/L ratios (e.g., 1/2 ~ 1/10), on the contrary, was initially rapid but quickly plateaued (Figs 5 (b) and 5(c)) with no distinct precipitations. The time-resolved RH profiles from DLS measurements were also consistent with the UV data, confirming the formation of larger clusters as well as the dependence of P/L ratios (Figs. S5, S7 and S9, Supporting Information). Two questions need to be addressed in order to advance our understanding on the interactions between bilayers and inverse Pluronics. First, why are the


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8 6

(d)

3

1 to 2 1 to 20 1 to 50 1 to 100

2

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Blank

1:100

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1:50

1:10

1:2

(e)

20

Time (min)

4

(f) 2 0

100

200

300

Time (min)

400

500

Fig. 5. Normalized UV absorption as a function of time for the mixtures of ULV and different polymers (a) 17R4; (b) 31R1; (c) PO26–EO20–PO26 (the missing data of the 1 to 100 sample in green is because of the precipitation of clusters. After ~ 7 hs, the sample was gently shaken and re-measured. Photos of (d) 17R4, (e) 31R1 and (f) PO26–EO20–PO26 were taken when the clustering approaching equilibrium (after ~ 10 hours), showing formation of large clusters (precipitates) at high P/L ratios for 17R4, but low P/L for 31R1 and PO26–EO20–PO26.

characteristic curves of ULV clustering different between low and high P/L ratios in the case of hydrophobic polymer linkers? Second, why is the dependence of clustering ULVs on P/L ratios different for hydrophilic and hydrophobic polymer linkers? Here, we propose a stringing/clustering mechanism that rationalizes all the aforementioned observations. The growth of ULV clusters requires the two PPO blocks on one inverse Pluronics


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inserting into bilayers of different ULVs (or ULV clusters). Earlier we have shown that over the time scale studied PPO only interacts with the defects (‘active sites’) on the bilayer. We anticipate that availability of the ‘active sites’ and concentration of the free linkers are important for the stringing kinetic. At a fixed DH/DP ratio, there are only limited numbers of ‘active sites’. In the case of the hydrophilic polymer (i.e., 17R4), the clustering rate increases with increasing P/L, indicating that such ‘active sites’ are constantly available on the ULVs, otherwise further networking of ULVs would cease. For the higher-hydrophobicity linkers (i.e., 31R1 and PO26–EO20–PO26), the rapid initial increment of UV absorbance observed in the first several data points at large P/L [insets in Figs 5(b) and 5(c)], indicates the fast insertion of PPO (also confirmed by the MD simulation shown in Fig. S11 in the Supporting Information) and subsequent clustering of the ULVs. Moreover, the initial increase of absorbance of the ULV mixture with 31R1 or PO26–EO20–PO26 is more drastic compared to that with 17R4. This eventually leads to the quick consumption of either available linkers or active sites as evident in the nearly no growth of UV absorbance at later stage (Fig. 5(b) and (c)). Obviously, high P/L ratios enhance the initial rate of overall PPO insertion; however not all the PPO insertions are valid for clustering. The enhanced initial rate also increases the probability of “invalid” insertions which account for the events of only one PPO end anchoring or both PPO ends anchoring on the same cluster or ULV (“looping”), prohibiting the growth of clusters at the later stage. This explains why the final sizes of the major clusters at higher P/L (e.g., 1/2) samples tend to be smaller. To validate the presumption, we titrated the nearly equilibrated polymer/ULV system with additional hydrophobic polymer linkers. There is no further increase of UV absorbance (Fig. S12, Supporting Information), implying that no ‘active sites’ at the exterior of clusters are available for further stringing.


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The aforementioned mechanism, however, does not explain the discrepancy in the critical P/L ratios that use up the available ‘active sites’ in the cases of hydrophilic 17R4 and the other two more hydrophobic inverse Pluronics. This could be explained by the weaker hydrophobic interaction of the shorter PPO chains of 17R4 with the bilayer, leading to a low-energy association/dissociation process, which constantly frees up the active sites available for other PPO to anchor, thus allowing further ULV stringing. In the cases of 31R1 and PO26–EO20–PO26, which have longer PPO, the dissociation of anchored PPO is less favorable; hence the active sites have lower probability to be freed and re-occupied by other PPO. The potential of mean forces to pull out the PPO blocks from the bilayer (as shown in Fig 6) estimated by umbrella samplings in MD simulation indicates higher energy is needed for PO25 (68 kcal/mole) and PO26 (76 kcal/mole) than PO17 (48 kcal/mol), consistent with this proposed mechanism. Another MD simulation on the interaction between DPPC and two regular Pluronics (EO25-PO7-EO25 and EO26-PO40-EO26) have also been conducted. It shows that the shorter-PPO Pluronic (EO25-PO7EO25) does not insert into the DPPC bilayer 100

(Fig. S13 in Supporting Information) in

PO14-EO24-PO14 PO25-EO7-PO25

80

PMF [kcal/mol]

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

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contrast to the prediction for the interaction

PO26-EO20-PO26 60

between the longer-PPO Pluronic (EO26-

40

PO40-EO26) and DPPC bilayer (Fig. S14 in

20

Supporting Information). The fact that this

0 20

40

60

80

100

120

140

160

Distance [Å]

outcome is consistent with the short-time Pluronic/lipid

Fig. 6. Potential of mean force (PMF) of the pulling of different polymers as a function of the distance between polymer’s center of mass to the bilayer.

interaction

reported

in

literature further validates the approach of our MD simulation.28-30


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A possible scenario should not be overlooked that the higher-hydrophobicity polymers could form aggregates (or micelles) at lower concentrations (i.e., lower “critical micellation or aggregation concentrations”) and the dissociation of polymer linkers from such aggregates could be slow, thus becoming the bottleneck for the PPO insertion into the bilayer, and consequently the ULV clustering. We therefore mixed the ULV with the polymer at different initial polymer stock concentrations, yet achieving the same final polymer and lipid concentrations. The fact that the time-resolved UV absorbance showed no clear dependence on the stock polymer concentration negates this possibility (Fig. S15, Supporting Information). However, it does not preclude that the polymer aggregates may slow down their insertion into the bilayer at the initial clustering stage in a much shorter time scale as reported in simulation work.31


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CONCLUSION In summary, we have clearly demonstrated that the bilayer defects (“active” sites) of the lipid segregated domains induced by the acyl chain mismatch allow the fast insertion of hydrophobic segments of the inverse Pluronics, leading to the clustering of ULVs. The consumption of such active sites and the association/dissociation rate of polymers (related to segmental hydrophobicity) with the sites dictate the clustering kinetics. For more hydrophilic polymer linker (e.g., 17R4), the clustering rate increases with the polymer concentrations as expected. However, an opposite trend was observed for polymers with longer PPO (e.g., 31R1 and PO 26– EO20–PO26) which is rationalized by rapid consumption of the active sites and increasing probability of the stringing events invalid for ULV clustering, thus inhibiting the growth of large clusters. This study provides fundamental insights into the interactions of biomolecules with lipid lateral domains on biomembranes and implies the existence of various lipids with different chain lengths and fluidity is required for enhancing biologically-relevant interactions. ACKNOWLEDGEMENT M-PN, YX, and H-SJ acknowledge the support from NSF (CBET 1433903 and CBET 1510468). YL and ZQS are grateful for the support from the Department of Mechanical Engineering at University of Connecticut. ZQS would like to thank the partial financial support from GE Fellowship for innovation. This research benefited in part from the computational resources and staff contributions provided for the Booth Engineering Center for Advanced Technology (BECAT) at the University of Connecticut. SUPPORTING INFORMATION AVAILABLE


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The supporting information is composed of eight sessions (S1 – S8), which contain 15 figures (Figs. S1 – S15), as described in the following. S1. Confocal and transmission electron microscopies, S2. Coarse grained MD simulation of different-composition bilayers, S3. Interactions between DMPC vesicles with 17R4 at 25 °C, S4. Aggregation kinetics of vesicles with different polymers measured by DLS, S5. Insertion of different polymers into bilayer by coarse grained MD simulations, S6. Polymer titration, S7. Interaction between Pluronics (EOnPOm-EOn) and bilayer (DPPC) at temperature T = 37 oC by MD simulations, and S8. Stock polymer concentration dependence on the aggregation dependence. This information is available free of charge via the Internet at http://pubs.acs.org/.


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Table of Contents

DH/DP = 0 DH/DP = 0.1 DH/DP = 0.2

6


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

5 4 3 2 1 0

100

200

Time (min)


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Effects of Membrane Defects and Polymer Hydrophobicity on

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