Article pubs.acs.org/Macromolecules
Effects of the Size, Shape, and Structural Transition of Thermosensitive Polypeptides on the Stability of Lipid Bilayers and Liposomes Hwankyu Lee,*,† Hyun Ryoung Kim,*,‡ Ronald G. Larson,§ and Jae Chan Park‡ †
Department of Chemical Engineering, Dankook University, Yongin, 448-701, South Korea Bio Research Center, Samsung Advanced Institute of Technology (SAIT), Samsung Electronics Co., Ltd., Yongin, 446-712, South Korea § Departments of Chemical Engineering, Biomedical Engineering, Mechanical Engineering, and Macromolecular Science and Engineering Program, University of Michigan, Ann Arbor, Michigan 48109, United States ‡
ABSTRACT: We performed all-atom and coarse-grained (CG) molecular dynamics (MD) simulations of lipid bilayers grafted with elastin-like polypeptides (ELPs; [VPGVG]n). All-atom simulations of a single ELP in water show that ELPs become more collapsed and folded as the temperature increases from 293 up to 353 K, in agreement with experiments. All-atom simulations of lipid bilayers composed of dipalmitoylglycerophosphocholine (DPPC), cholesterol, and fatty acids grafted with ELPs show that ELPs insert into the bilayer and significantly disorder lipids, to an extent that depends on the ELP length over the temperature range 293−323 K. In the bilayer, ELPs are mainly, but not entirely, random coil in character at temperatures between 293 and 315 K and, in contrast to the behavior in water, become increasing random coil and extended in length over the range 315−323 K, over which the bilayer is in the disordered liquid phase. The insertion of ELPs into the lipid-tail region is mediated by the interaction of hydrophobic Pro and Val residues with lipid tails, which become stronger at increased temperature, but the insertion is incomplete because of the interaction between hydrophilic backbones of Gly residues and the lipid headgroups. Longer time CG simulations of the transition from ordered gel to disordered liquid bilayer at 315 K in a liposome are able to capture cholesterol flip-flops between bilayer leaflets, leading to an increase in the number of cholesterols in the inner layer, which helps the bilayer accommodate the reduced membrane curvature resulting from the expansion of the bilayer area driven by the phase transition. Our findings indicate that lipid bilayers can be disrupted more effectively by the stronger hydrophobic interaction of the random coils of ELPs at 315−323 K than by the compact ELPs at 293−310 K, which helps explain the experimental observation that ELP-conjugated liposomes are stable at 310 K, but become unstable and release drugs at 315 K.
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INTRODUCTION Liposomes, which are artificially prepared vesicles composed of lipid bilayers containing biocompatible phospholipids, have the potential to encapsulate drugs and transport them across cell membranes.1−8 In addition, liposomes can be easily prepared and modified to increase the targeting efficiency. For example, ligands and soluble polymers can be attached to the liposome surface,9−11which can target the liposome and its contents at specific cancer cells. For these biomedical applications, the encapsulated drug should be released only at the desired site, and the release rate needs to be controlled. To achieve these goals, temperature changes (hyperthermia), light (photodynamic therapy), and ultrasound have been applied to directly trigger the drug release and control the release rate.12−14 For example, the phase behavior of the liposome membrane, and therefore its permeability to the drug, can be easily modulated by temperature, leading to extensive studies of temperaturesensitive liposomes.15 © 2012 American Chemical Society
Experimentally, the Needham group studied and optimized the temperature-sensitive phase behavior of liposomes composed of various lipids.15 Since the gel-to-liquid transition temperature for dipalmitoylglycerophosphocholine (DPPC) lipids is 41.5 °C,16 DPPC liposomes are in the stable gel phase at the physiological temperature (37 °C) but become disordered and unstable above 41.5 °C. To develop liposomes that can release drugs above 37 °C, DPPC and other phospholipids were mixed with poly(ethylene glycol) (PEG)conjugated lipids (i.e., PEGylated lipids) at controlled ratios; these showed a rapid release of 50% of the encapsulated doxorubicin (DOX; a drug for cancer) at 42 °C.15 To increase the efficiency of drug delivery, Park et al. recently conjugated elastin-like polypeptides (ELPs), which consist of repeated Received: June 27, 2012 Revised: August 11, 2012 Published: August 23, 2012 7304
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units [VPGVG], to fatty acids that are then incorporated into liposomes; these showed a rapid drug release of nearly 100% of DOX at 42−45 °C.17 Although the ELP-conjugated liposomes show a more efficient drug release, the mechanism of this action and the influence of the ELPs on the liposome membrane structure are still not well understood. Intriguingly, ELPs are known to reduce the extent of their random-coil structure at increased temperature,18 producing a so-called “inverse-temperature transition” (ITT). This is opposite to the folding behavior of other typical proteins such as lysozyme, SNase, or RNase A. This paradoxical temperature-induced conformational change may modulate the interaction between ELPs and membrane. To explore this, the conformation properties of ELPs and their interactions with lipid bilayers at different temperatures need to be studied at nearly the atomic scale, as can be done using molecular dynamics (MD) simulations. In this work, we therefore perform all-atom MD simulations of flat, periodic, lipid bilayers grafted with ELPs and coarsegrained (CG) MD simulations of a whole liposome grafted with ELPs. We first simulate atomistically a single ELP in water and analyze its size, structure, and diffusivity at different temperatures. Second, ELP-grafted lipid bilayers are simulated atomistically, which reveals the effects of ELP on the lipid disorder and bilayer properties at different temperatures. The size, shape, and secondary structures of the ELP in lipid bilayers are analyzed, and the behavior is rationalized by considering the specific interactions between individual amino acids of ELP and lipids. Finally, we perform CG simulations of an ELP-grafted liposome, which reveals flip-flops of cholesterol in the fluid state above the transition temperature, which helps accommodate the phase change to the fluid state and its effect on liposome curvature.
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Figure 1. Structures of DPPC, cholesterol, C18-ELP3, and DSPE-PEG for all-atom (left) and CG (right) models. Hydrocarbons are colored in light blue. For all-atom models, red, brown, dark-blue, and white colors respectively represent O, P, N, and H atoms. For CG models, green, brown, dark-blue, purple, red, pink, and yellow beads respectively represent glycerol, phosphate, choline, steroid, PEG, ELP backbone, and side chains. The images were created with Visual Molecular Dynamics.61 chains were represented by one or multiple beads. Simulations with the MARTINI amino acid FF have successfully captured protein− protein and protein−membrane interactions, although the secondary structure of the protein needs to be assigned and held fixed,47,48 so that changes in secondary structure cannot be modeled. In this work, backbone beads of CG ELP chains were modeled using the potentials for the random coil because all-atom simulations showed that ELPs interacting with lipid bilayers in the fluid state (above the transition temperature) predominantly have the random-coil structure. Computational facilities were supported by the Supercomputing Center/ Korea Institute of Science and Technology Information with supercomputing resources including technical support [KSC-2012C2-53]. All-Atom Simulations of Lipid Bilayers Grafted to ELP. Coordinates of ELP3 ([VPGVG]3) and ELP6 ([VPGVG]6) were generated in an initially α-helical structure using Swiss-Pdb Viewer.49 ELP3 (or ELP6) were simulated in TIP4P water at 293−323 K, and the equilibrated final configurations were used as the starting states for simulations of C18-ELP3 interacting with bilayers. The simulated bilayer consists of 856 DPPC (428 DPPC/leaflet), 288 cholesterol, 8 C18-ELP3, and ∼57 000 TIP4P water molecules in a periodic box of size 15.6 × 15.6 × 13 nm3 (Figure 2, top), leading to a DPPC:cholesterol:C18-ELP3 ratio of 107:36:1, close to the experimental ratio (100:36:1). Here, the tail group of C18-ELP3 was initially inserted into the bilayer, with ELP3 positioned with a distance of 2.5 nm in the z direction between the centers of ELP3 and of the bilayer. To investigate the effect of ELP, bilayer systems without ELP were also generated. A real space cutoff of 11 Å was applied for electrostatic forces with the inclusion of particle mesh Ewald for longrange electrostatics.50,51 The LJ forces were smoothly switched to zero between 8 and 11 Å. A pressure of 1 bar, and temperatures of 293, 310, 315, or 323 K were maintained by applying a Berendsen thermostat52 in the NPxyPzT ensemble (Table 1). Simulations were performed for 140 ns with a time step of 2 fs, and the last 30 ns was used for analyses. CG Simulations of Liposomes Grafted to ELP. 8800 DPPC lipids, 3200 cholesterols, 320 DSPE-PEGs, and 88 C18-ELP3 were randomly distributed in a periodic box of 40 nm/side, leading to a DPPC:cholesterol:DSPE-PEG:C18-ELP ratio of 100:36.4:3.6:1, close to the experimental condition. Here, DSPE-PEGs were included because they were used with the same ratio in the experiment to
METHODS
All simulations and analyses were performed using the GROMACS4.5.3 simulation package.19−21 Structures for all-atom and CG models are shown in Figure 1. For all-atom simulations, the “Berger lipid” force field (FF),22 which has GROMOS atom types with OPLS partial charges, was used for DPPC lipids. Since the Berger lipid FF is not suitable for proteins, mixtures of proteins and lipids have been typically simulated with the combination of the Berger lipid FF and other FFs by adjusting the scaling factors of Lennard-Jones (LJ) and Coulomb interactions, which have successfully captured the experimentally observed structure and dynamics of proteins and lipids.23−28 Here, different FFs were tested for ELP, showing that only the OPLS all-atom FF29,30 successfully predicts the experimentally observed secondary structures of ELP at different temperatures, as will be presented in the Results and Discussion section. Therefore, cholesterols and ELPs were modeled with the OPLS FF, and their interactions with DPPC lipids were modeled by combining the OPLS and Berger FFs. ELP3 and ELP6 respectively consist of 3 and 6 repeats of [VPGVG], and each ELP chain was grafted to an 18-hydrocarbon alkyl chain, named C18-ELP. For CG simulations, models for DPPC, cholesterol, PEG, and C18-ELP were taken from the MARTINI lipid and amino acid FF,31−33 which lumps a few (three or four) heavy atoms into each CG bead. This CG FF has been successfully applied to our previous simulations of lipids interacting with dendrimers and single-walled carbon nanotubes.34−43 In particular, simulations with a previously parametrized CG PEG FF showed self-assembled liposomes, bicelles, and micelles at the expected ratios of lipids and PEGylated lipids, in agreement with experiment.44−46 The same PEGylated lipid (45 monomers; PEG Mw = 2000) was used in this work, and its interactions with DPPC, cholesterol, and C18-ELP were modeled using the MARTINI FF. For ELP, the backbone atoms of each amino acid residue were lumped into a single bead, and side 7305
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different temperatures are explained in the Results and Discussion section.
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RESULTS AND DISCUSSION Size, Structure, and Diffusivity of ELPs in Water. Experimentally, Nicolini et al. measured secondary structure of ELP using circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopies, showing that in water the extent of the random-coil (unfolded) structure decreases with increasing temperature up to 353 K.18,53 Thus, this polypeptide exhibits an inverse-temperature folding transition, which is the opposite of typical peptides. To test the ability of different FFs to predict this structural change of ELP, a single ELP3 was simulated in water at 293, 315, and 323 K for 300 ns using the OPLS all-atom FF and different versions of GROMOS96 FFs (43a2, 45a3, 53a5, 53a6). The secondary structure of ELP3 was calculated using the DSSP program,54 which showed that only the OPLS FF predicts the inverse-temperature folding transition. Therefore, the OPLS FF was chosen for other allatom simulations in this work. Table 2 shows that fractions of Table 2. End-to-End distances (⟨h2⟩1/2), Radii of Gyration (Rg), Diffusion Coefficients (D), and Fractions of RandomCoil Structure of ELP3 in Water
Figure 2. Snapshots at the beginning (0 ns, top) and end (140 ns, rows 2−5) of all-atom simulations of C18-ELPs with lipid bilayers. For the initial configuration, the top view (top, left) and the side view (top, right) are shown, while only side views are shown for final configurations. Note that side views show only one cross section of the system and cannot capture all C18-ELPs. Blue and red chains represent ELPs and their hydrocarbon tails, respectively. Light blue represents the tails of DPPC, cholesterol, and DSPE-PEG. P atoms of DPPC and O atoms of cholesterol are highlighted in brown and red dots, respectively. For clarity, water beads are omitted.
fraction of the random-coil structure
increase solubility and circulating lifetime. The mixture was then solvated with ∼360 000 CG water beads (representing ∼1 440 000 real waters) and 320 Na+ counterions to achieve neutrality. A constant temperature and a pressure of 1 bar were maintained by applying a Berendsen thermostat in the NPT ensemble.52 A cutoff of 12 Å was used for the LJ potential and electrostatic interactions. The LJ potential was smoothly shifted to zero between 9 and 12 Å, and the Coulomb potential was smoothly shifted to zero between 0 and 12 Å. Because of inclusion of PEG dihedral potentials, a time step of 8 fs was used instead of the typical time step of 20−40 fs for the MARTINI FF. The last 100 ns was used for analyses. The simulation procedures at
temp (K)
⟨h2⟩1/2 (nm)
Rg (nm)
D (10−5 cm2 s−1)
this work
exp18
293 315 323
1.64 ± 0.16 1.41 ± 0.08 1.47 ± 0.12
0.74 ± 0.03 0.68 ± 0.01 0.66 ± 0.03
0.35 ± 0.01 0.47 ± 0.02 1.08 ± 0.01
0.53 0.50 0.45
0.55 0.50 0.47
the random-coil structure in ELP3 agree quantitatively with those obtained from experiments.18 End-to-end distances (⟨h2⟩1/2) and radii of gyration (Rg) were also calculated, and these showed that the ELP3 is larger at 293 K than at higher temperatures, 315 and 323 K. This indicates that the ELP3 shrinks at higher temperature because of an increase in the folded (turn and sheet) structure, which supports previous simulations47 and experiments.18 To obtain the diffusion coefficients (D), the slopes of the mean-square displacements
Table 1. List of All All-Atom and CG Simulationsa no. of molecules
all-atom (planar bilayer)
CG (liposome) a
name
DPPC
cholesterol
ELP0-20 ELP0-37 ELP0-42 ELP0-50 ELP3-20 ELP3-37 ELP3-42 ELP3-50 ELP6-20 ELP6-37 ELP6-42 ELP6-50
856 856 856 856 856 856 856 856 856 856 856 856 8800
288 288 288 288 288 288 288 288 288 288 288 288 3200
DSPEPEG
320
C18ELP
8 8 8 8 8 8 8 8 88
no. of ELP monomers (VPGVG)
temp (K)
simulation time (ns)
3 3 3 3 6 6 6 6 3
293 310 315 323 293 310 315 323 293 310 315 323 300 → 273 → 315
140 140 140 140 140 140 140 140 140 140 140 140 1020
Planar lipid bilayers and the self-assembled liposome were simulated using all-atom and CG models, respectively. 7306
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(MSD) of the center of mass (COM) of ELP3 versus time were calculated (DPBC) and corrected for finite size effects using the formula55 D = DPBC + KBTξ/6πηL, where KB is Boltzmann’s constant, L is the cubic box length, ξ = 2.837 297 (which has been found to be the coefficient for the correction factor for particles on a cubic lattice interacting electrostatically via Poisson’s equation with Ewald summation), and viscosity η = 0.75, 0.71, and 0.69 cP respectively at 293, 315, and 323 K for CG water.56 Table 2 shows the higher diffusion coefficients at 315 and 323 K, apparently because of the decreased size as well as the reduced solvent viscosity at the higher temperatures. All-Atom Simulations: Insertion and Disruption of ELPs in Lipid Bilayers. To investigate the effect of ELP on the lipid bilayer, bilayers consisting of DPPC, cholesterol, and C18ELP3 (or C18-ELP6) were simulated at 293, 310, 315, and 323 K for 140 ns. Simulated systems are named in Table 1, where the first and second numbers describe the number of ELP monomers and temperature in °C, respectively. For example, “ELP3-20” designates a bilayer with C18-ELP3 at 293 K, and “ELP0-20” indicates that the simulated system does not include C18-ELP. Figure 2 shows snapshots from the beginning (top) and end of all-atom simulations with C18-ELPs (rows 2−5). ELP3 molecules insert into the lipid bilayer at all simulated temperatures, but ELP6 molecules do not at 293 K, presumably because the large ELP6 cannot penetrate into the orderedphase bilayer. ELP6 only partially inserts at 310 K and starts to more deeply insert at 315 K, indicating the effect of the ELP size on their insertion into the bilayer. The DPPC tail conformational order can be quantified by the order parameter SCC = 3/2⟨cos2 θz⟩ − 1/2, where θz is the angle that the vector connecting carbons Cn−1 to Cn+1 makes with the z-axis. The bracket indicates averaging over time and over all molecules in the simulation. Order parameters can vary between 1 (perfect orientation in the interface normal direction) and −1/2 (perfect orientation perpendicular to the normal).57 Here, the order parameters of the DPPC tails were calculated within 2 nm of the COM of each ELP. Figure 3 shows that the bilayers with ELP0, ELP3, and ELP6 become more disordered at higher temperatures. At each temperature, lipid tails are significantly disordered around ELPs. At 293 K, only ELP3, and not ELP6, disorders the lipids, consistent with the insertion of ELP3, but not ELP6, as observed in Figure 2. This lipid disordering accompanying ELP insertion modulates bilayer dimensions. Figure 4 shows the xy-plane areas (which equal the bilayer surface areas) at 323 K as functions of time. The areas reach steady-state values at around 100 ns, and decrease in the order of ELP6-50 > ELP3-50 > ELP0-50. Autocorrelation functions for the areas, C(t), yield relaxation times below ∼5 ns, defined as the time at which C(t) = 1/e, indicating that the values are equilibrated. This indicates that larger ELPs expand the bilayer more. Figure 5 compares average equilibrated areas of the bilayers for all simulated systems. At 293 K, ELP3 shows an expanded area relative to ELP0, while the area for ELP6 is almost unchanged from ELP0. This is consistent with the fact that the relatively small ELP3 can insert even into the orderedphase bilayer, but the larger ELP6 cannot, as is shown in Figures 2 and 3. Bilayers with ELP3 and ELP6 have almost the same areas at 310−315 K, but at 323 K the ELP6-containing bilayer has larger area than does the ELP3-containing bilayer, indicating the dependence on the ELP size. The ELP insertion and bilayer properties were further quantified by calculating the bilayer thicknesses and mass
Figure 3. Order parameters of DPPC tails. For the system with ELPs, lipids within 2 nm of the COM of each ELP were calculated.
Figure 4. Areas of lipid bilayers at 323 K (top) and their autocorrelation functions (C(t); bottom), as functions of time.
densities. To calculate the bilayer thickness, the xy-plane parallel to the bilayer surface was equally divided into 900 voxels by using a grid (30 × 30 grid). For each voxel, the z component (normal to the bilayer) of the COM values of the P atoms of DPPC was averaged for each leaflet, and the difference 7307
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while ELP6 does not at 293 K but increasingly inserts over the temperature range 310−323 K, again indicating that ELP6 can penetrate only into the disordered lipid bilayer, consistent with Figures 2, 3, and 5. These results indicate that ELPs disorder lipids and insert into the bilayer, but the extent of insertion and disorder significantly depends on the ELP length and temperature. Size, Shape, and Secondary Structure of ELPs Interacting with Lipid Bilayers. Figure 7 shows average
Figure 5. Areas of lipid bilayers as a function of temperature.
in average z values between leaflets was taken to be the bilayer thickness. Figure 6a shows that bilayers are thinner at higher
Figure 7. Average end-to-end distances (⟨h2⟩1/2) and radii of gyration (Rg) of ELP3 in lipid bilayers at simulated temperatures.
end-to-end distance ⟨h2⟩1/2, which is the root-mean-squared distance between the C atom of carboxy (C-) terminus and the N atom of amino (N-) terminus, and Rg of ELP3 in lipid bilayers. The values do not change at 293 and 310 K but start to increase at 315 K, indicating an expanded conformation of ELP3 at a higher temperature. Note that this swelling at higher temperature is opposite to the simulated behavior in water, where ELPs become more ordered and smaller at higher temperature. This indicates that ELPs behave differently in water than they do in lipid bilayers. To understand their shapes, we computed the aspect ratios, Iz/Ix and Iz/Iy, where Iz, Iy, and Ix are the principal moments of inertia (Iz > Iy > Ix) averaged over the last 30 ns of the simulations, and obtained from these the relative shape anisotropy, κ2 (κ2 = 1 − 3I2/I12, where I1 and I2 are the first and second invariants of the radius of gyration tensor (I1 = Ix + Iy + Iz, I2 = IxIy + IyIz + IxIz)).58 A linear array of skeletal atoms is characterized by κ2 = 1, while a molecule with tetrahedral or spherical symmetry is characterized by κ2 = 0. In Table 3, ELP3-20 and ELP3-37 show aspect ratios of 1.18−2.61 and shape anisotropies of 0.04−0.06, indicating that ELP3's are modestly ellipsoidal in shape. However, ELP3-42 and ELP3-50 show much higher aspect ratios and shape anisotropies, indicating that these swollen ELP3's have more linear conformations, implying an unfolded structure. Figure 8 shows that ELP3-20 and ELP3-37 retain β-bridges, β-bends, and turns for whole simulation time, while the random-coil structure becomes much more prominent in ELP3-42 and ELP3-50, indicating that the swollen ELP3 chains at 315 and 323 K mostly have the random-coil (unfolded) structure. Interactions between ELPs and Lipid Bilayers. ELPs consist of three amino acids, namely proline (Pro), valine (Val), and glycine (Gly). The side chains of Pro and Val are hydrophobic, while Gly does not have a side chain. These amino acids interact differently with lipid headgroups and tails, which may determine the final conformation of ELPs inserted
Figure 6. (a) Bilayer thickness (top, 140 ns) and (b) mass density profiles (bottom, averaged for the last 30 ns). Bilayer thickness is represented by different colors, which were created with GridMATMD.62 The ELP names and temperatures for density profiles are arranged in the same order of the bilayer-thickness profiles.
temperatures for ELP0, ELP3, and ELP6 because lipids are more disordered at higher temperature. In particular, ELP3 shows some blue spots (representing a thinner bilayer) at all temperatures, while ELP6 shows these spots at 310−323 K. This thickness change is also shown in the density profiles in Figure 6b. ELP3 inserts into the bilayer at all temperatures, 7308
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Table 3. Average Values of the Principal Moments of Inertia, Aspect Ratios, and Relative Anisotropies of ELPs for ELP3-20, -37, -42, and -50 principal moments of inertia (amu nm2) Iz ELP3-20 ELP3-37 ELP3-42 ELP3-50
534 664 937 791
± ± ± ±
Iy 2 5 15 24
452 539 848 704
± ± ± ±
aspect ratio Ix
2 5 9 9
265 254 212 217
± ± ± ±
2 2 1 6
Figure 8. Secondary structure profiles of ELP3 as a function of time (ns).
Iz/Iy
Iz/Ix
relative shape anisotropy
1.18 1.23 1.10 1.12
2.02 2.61 4.42 3.65
0.04 0.06 0.12 0.10
Figure 9. Radial distribution functions between DPPC (choline, phosphate, glycerol, and carbon tail) and individual amino acids of ELP3-50.
into the bilayer. Note that in Figures 2 and 6 (mass densities) ELPs insert into the bilayer, but not completely into the tail region. Instead, ELPs are positioned broadly in both lipid headgroup and tail regions. To understand the interactions between ELPs and lipids, we calculated radial distribution functions (RDFs) of individual amino acids of ELPs with respect to the different regions of DPPC lipids, such as cholines, phosphates, glycerols, and carbon tails. Figure 9 shows that Gly has the highest peak of all the polar headgroup groups (choline, phosphate, and glycerol), indicating that the interaction is strongest between Gly and lipid headgroups. For the lipid tail region, Gly shows the lowest peak, indicating the weak interaction of Gly with lipid tails. These results indicate that hydrophobic amino acids, Val and Pro, tend to have hydrophobic interactions with lipid tails, while Gly residues strongly interact with the polar lipid headgroups because Gly does not have a side chain, and so the polar backbone is easily exposed and so interacts with polar headgroups. Since ELP chains interact with both lipid headgroups and tails, they do not completely insert into the tail region, as observed in Figures 2 and 6. Figure 10 shows that the interactions of DPPC tails with both Pro and Val residues of ELP3 become stronger at increased temperature. This
Figure 10. RDFs of DPPC tails with respect to Pro and Val residues of ELP3.
indicates that the ELP3 chains with the higher extent of random coil character interact more strongly with DPPC tails, which can disrupt the bilayer more effectively. 7309
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These results, combined with the observations in Figures 7 and 8 and Table 3, may help explain the mechanism of the ELP-triggered drug release from the liposome at 315 K, which was observed in the experiment by Park et al.17 Our simulations show that ELP3 chains disorder lipids and insert into the bilayer at 293−323 K, but the size, shape, and secondary structure of ELP3 only significantly change between 310 and 315 K, where the drug release occurs in the experiment. In particular, ELP3's in lipid bilayers show a compact conformation with small size at 310 K and a linearly swollen conformation with mostly random-coil conformation at 315 K. This implies that the liposome membrane may be disrupted more effectively by the random coils of ELP3 at 315 K, rather than by the compact ELP3 at 310 K, and this supports the experimental observations that the liposome with ELP3 was stable at 310 K but became unstable and released drug molecules at 315 K. CG Simulations of the Self-Assembled Liposome: FlipFlops of Cholesterols. As discussed above, all-atom simulations reveal the effects of ELPs on the bilayer properties and the mechanism of the ELP insertion and disruption of the bilayer. However, because of computational limitations on system size and time scale, pore formation in the disordered bilayer was not captured by all-atom simulations within the simulated time. More importantly, the bilayer of a liposome has curvature and thus which is not the case in a planar lipid bilayer. To simulate more realistically the bilayer of the liposome, we performed CG MD simulations of the self-assembled liposome. Figure 11 shows snapshots of CG simulations. The fluid−gel transition temperature for MARTINI DPPC flat bilayers is 295 K, which is lower than the experimental value by ∼20 K.59 Also, experiments have shown that the transition temperature is lower for smaller liposomes (
