Melting Behavior of Zipper-Structured Lipopeptides in Lipid Bilayer

Subscriber access provided by Fudan University

Article

Melting behavior of zipper-structured lipopeptides in lipid bilayer Sijia Wang, Xia Han, Danyang Liu, Mengya Li, Shouhong Xu, and Honglai Liu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04080 • Publication Date (Web): 18 Jan 2017 Downloaded from http://pubs.acs.org on January 19, 2017

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 free 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 accessible to all readers and 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.

Langmuir 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 25

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

Melting behavior of zipper-structured lipopeptides in lipid bilayer SijiaWang, Xia Han, Danyang Liu, Mengya Li, Shouhong Xu*, Honglai Liu* Key Laboratory for Advanced Materials and School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, 200237

Abstract: A zipper-structured lipopeptide is expected to play a role of “intelligent valve” in the lipid bilayer. In this paper, a series of zipper-structured lipopeptides have been designed for preparing thermo-controllable hybrid liposomes. Their conformational transition as a function of temperature in lipid bilayer has been investigated for understanding the influences of molecular structure and bilayer property on bio-function. The melting temperatures Tm of the lipopeptides have been found to depend on their molecular structures. When the lipopeptides have been doped in bilayer, an increase of size of alkyl chain increases the stability of the α-helix resulting in a decrease in fluidity of lipid bilayer. However, an increase of amino groups at N-terminal is found to decrease the stability of the spatial structure. The thermo-controllability of the “valve” in lipid bilayer is confirmed by drug release experiments under different temperatures. Meanwhile, effects of bilayer properties on the thermo-sensitivity of lipopeptides have also been investigated. Results show the Tm of lipopeptide doped in bilayer decreases with the increase of membrane fluidity. Furthermore, the reversibility of the thermo-controlled “valve” is also proved by release drug under intermittent temperatures. It could be concluded that the molecular structure of the lipopeptide, as well as the property of bilayer, give great influence on the bio-function of the hybrid liposomes. Keywords:

Peptide

amphiphile,

zipper

structure,

lipid

bilayer,

thermo-controllability, drug delivery.

Introduction Peptide amphiphile (PA), which is an amphiphile composed of a peptide and a hydrophobic group, has attracted much attention due to their outstanding potential in biomaterial applications.1-6 Two parts of PAs could be designed and combined freely 1

ACS Paragon Plus Environment

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

to obtain excellent bio-macromolecules with various functions. Castelletto.V et al. synthesized several PAs containing RGD and found that the RGD contained PAs selectively interacted with endothelial cells.7 Melittin is an antibacterial peptide which has been widely applied on clinic.8 Their antibacterial properties have been found to be improved obviously after modification by functional groups.9,10 Cuixia Chen et al. designed and synthesized a series of PAs, e.g. G(IIKK)nI-NH2, which have been identified their more excellent antibacterial activity in vitro and in vivo.11,12 In cancer therapy field, quick and accurate transport of DNA or bio-macromolecules into pathological cell is still a great challenge. Cationic PAs also have been proved to improve cellular internalization for gene delivery.13,14 Most of functional PAs existing in biological bodies are in α-helix and β-sheet structures.15,16 However, the amino acid sequence and the balance between hydrophilicity and hydrophobicity both lead to different secondary structures.17-21 So modifying these PAs to obtain better function has attracted more attention. N-terminal acetylation and C-terminal amidation are usually used in terminal modification. For α-helical PAs, Azad M. A. had proved that incorporating glutamic acid (Gly) at the N-terminus could stabilize PAs’ helicity.22 However, Blondelle S. E. found that the absence of Gly of melittin increased the helicity of peptide.23 So, N-terminal amino acids had different effects on the secondary structure of peptides. Similarly, C-terminal amidation had the same result with N-terminal acetylation. Park C.B. group had reported that deletion of four amino acids from the C-terminal end of buforin II resulted in a reduction of helical content.24 But Chen et al. found that the addition of Gly at the C-terminal of (IIKK)3-NH2 decreased the helicity of peptide.12 Meanwhile, attachment of alkyl chains was found to stabilize the secondary structure of PAs.25-28 As reported by Forns P. et al.,29 a series of PAs with varying alkyl chain lengths from C6 to C16 monoalkyl were synthesized. The helix-coil melting temperature was found to be increased with the increase of alkyl chain length. Leucine zipper peptides have typical α-helix structure. They always contain heptad repeats (abcdefg)n in which positions a and d are occupied by hydrophobic residues.30 The obvious feature of leucine peptides differentiating from others is their thermo-sensitivity. When the temperature is above their melting temperatures, leucine peptides will complete the transition of configuration from an α-helical dimmer to a single random coil.31,32 The thermo-sensitivity makes leucine zipper peptide be used widely in designing various thermo-controllable smart materials.33-35 We have originally designed a series of thermo-sensitive lipopeptides (LPe), 2


Page 2 of 25

Page 3 of 25

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

which belong to a kind of PA. They are composed of different sizes of alkyl chain and leucine zipper peptide. They can parallelly arrange with phospholipids in the lipid bilayer through self-organized behavior and then act as “intelligent valves” in bilayer (Lipo-LPe) (Scheme1a). Then, drug release amount of Lipo-LPe would expected to be accelerated and anti-tumor effect could be improved. However, how to achieve the best thermo-sensitivity of Lipo-LPe has been considered from two respects: 1) to lengthen the lipopeptide, especially the hydrophobic block, for increasing the open-close power of “valve”; 2) to adjust the lipid bilayer properties, maybe induce a more serious disturbance in the bilayer. In this paper, we have studied the effect of lipopeptides’ molecular structure on the physic-chemical properties and bio-functions of lipid bilayer. Meanwhile, their melting behaviors in bilayer have been investigated to understand the effect of membrane property. The lipopeptides doped in lipid bilayer have been proved to be a promising thermo-sensitive and thermo-controllable “intelligent valve” in the lipid membrane. This study provides a theoretical basis for the design of bio-functional PA and then environmental stimuli-controllable drug delivery system.

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

Scheme 1. (a) Thermo-sensitivity of lipopeptides as “intelligent valves” in the lipid bilayer. (b) Molecular structure of LPe1, LPe2, LPe3 (Upper) and LPe4 (Below). d: The distance between two alkyl chains. (c) Sequence of LPe1, LPe2, LPe3 and LPe4. 4


Page 4 of 25

Page 5 of 25

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

Experimental section Materials and regents. Self-designed lipopeptides (purity＞95%) with leucine zipper structure were purchased from commercial synthetics (Synpeptide, China). The lipopeptides were composed of alkyl chains with various alkyl numbers n from 5 to 9 and structured peptides (denoted as LPe1, LPe2 LPe3 and LPe4, separately) (Scheme 1b,c). 1,2-distearoyl–sn-glycero-3-phosphocholine (DSPC, purity＞98%), and 1,2– dipalmitoyl–sn–glycero-3-phosphoethanolamine

(DPPE,

purity ＞ 98%)

were

purchased from Lipoid (Germany). 3β-(N-(N',N'-Dimethylaminoethane)-carbamoyl) cholesterol (DC-Chol) was purchased from Avanti (America). Tris (hydroxymethyl) aminomethane, doxorubicin hydrochloride (DOX) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were obtained from J & K Chemical Co.Ltd (China). Sephadex G-50 was purchased from Shang Hai Haoran Biological Technology Co.Ltd. Other chemical solvents and reagents, such as chloroform, methanol and ethanol were all of analytical grades (China Reagent and Instrument).

Characterization of pure liposome and Lipo-LPe. Pure liposome (Lipo) and hybrid liposome (hybrid of lipopeptide and phospholipid, (Lipo-LPe)) were prepared by thin lipid film hydration method. Six kinds of liposomes with different lipid compositions were prepared: Lipo1 (DSPC:DPPE:DC-Chol =4:0.2:2), Lipo2 (DSPC:DPPE:DC-Chol=4:0.2:1.33), Lipo3 (DSPC:DPPE:DC-Chol =4:0.2:1), Lipo4 (DOPC:DSPC:DPPE:DC-Chol=0.4:4:0.2:1), DSPC:DPPE:DC-Chol=0.8:4:0.2:1)

and

Lipo5 Lipo6

(DOPC:

(DOPC:DSPC:DPPE:DC:Chol

=2:4:0.2:1). Firstly, DSPC, DPPE, DOPC and DC-Chol (a certain amount) were dissolved in a chloroform/methanol mixture (7:3) in a 250mL round-bottom flask. For the preparation of Lipo-LPe, lipopeptides were dissolved together with phospholipid to form the lipid film. Then the organic solvents were evaporated by a rotary evaporator (R206D, SENCO, Shanghai) at 45.0℃, and then lipid film was hydrated with 5mL of Tris buffer solution (10mM, pH=7.4) at 48 ℃ using a sonicator (KQ-100TDB, AK17, Kunshan) to get the liposome suspension (6mM). Finally, liposomes were extruded at 48℃ through 200nm polycarbonate membranes 11 times using a mini-extruder (LiposoFast-Basic, Avestin, Canada). The sizes and zeta-potentials of liposomes were evaluated by using dynamic light scanning (DLS) (Zetasizer Nano-ZS, Malvern, UK), and the polydispersity index (PDI) which indicates the size distribution of the liposome were determined. The secondary structures of lipopeptides were measured by circular dichroism (CD) thermal scan. These measurements were performed on a Chirascan spectrometer 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 25

(Chirascan, Applied Photophysics Ltd, UK) supplied with a thermoelectric temperature control system. Temperature-dependent conformational changes of lipopeptides (50µM) in lipid bilayer were measured in Tris buffer (10mM, pH=7.4). CD spectra of the samples were recorded from 260 to 190 nm using a 1mm quartz cuvette at 20.0℃, then the temperature increased from 20.0 to 70.0 ℃ at 1.0℃/min heating rate and 2.0℃/step. Finally, the sample was cooled to 20.0℃ and the CD spectrum was recorded after equilibration for 10min. The melting temperature (Tm) of lipopeptide was determined with Global Analysis T-Ramp software. The membrane fluidity of liposomes were studied by anisotropy measurements using a fluorophotometer (LS-55, PerkinElmer, USA) equipped with an automated polarizer and thermostatic cell holder connected to a water bath. 200µL of DPH solution (2×10-6 mol/L, Tris buffer) was added into liposome suspensions. The mass ratio of lipid to DPH was 800/1. The mixtures were shaken at room temperature for 12h before measurement. The anisotropy measurements were carried out in the range of 20.0℃ to 70.0℃ (5.0℃/step) with an excitation slit and emission slit both of 10 nm (excitation and emission wavelengths were 360 and 430 nm). Fluorescence anisotropy was measured and calculated based on the following equation:36,37

r=

ivv − Givh ivv + 2Givh

(1)

where r is the fluorescence anisotropy. G is a factor calculated to correct the instrument polarization which is defined by ihv/ ihh. Here, the fluorescence emission intensity is denoted by i, where the subscripts hv and hh indicate the orientation of the excitation polarizer and the emission polarizer, respectively.

DOX loading experiment. DOX loaded liposomes were prepared by ammonium sulfate gradient method. Briefly, liposomes were hydrated with 5mL ammonium sulfate buffer solution (200mM) instead of Tris buffer at 48.0℃. Then the liposome suspension was dialysed against 200 times volume of physiological saline (0.9%wt) at 4.0℃, and the dialyzate was replaced once an hour ten times. Then, DOX solution (2.5mg/mL) was added into liposome (DOX:lipids=1:5 mol/mol) and hatched at 50.0℃in a water bath for 45min. Finally, the unloaded DOX was separated by gel filtration through a sepharose column. The samples separated and not separated by gel filtration were both dealt with 10% TritonX-100 and measured by an ultraviolet spectrophotometer (UV) (UV-2450, Shimadzu, Japan). The UV absorption intensity of 485nm A1 and A0 were obtained. The encapsulation efficiency was calculated as A1/A0, and it was as high as 80% in this experiment. 6


Page 7 of 25

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

DOX release from liposome in vitro. DOX release experiments were carried out at 37.0℃ and 45.0℃. 1mL of DOX loaded sample was put in a dialysis bag (3500) and placed in 25mL of Tris buffer (10mM, pH=7.4). 3mL buffer was withdrawn and measured by UV once an hour, and the buffer was always put back. The UV absorption intensity of 485nm at various intervals At was then obtained. Then the percentage of DOX release was calculated as At/A1. In this study, every experiment has been repeated for 3~5 times, the statistical error shown in the figures and tables was the standard deviation of repeated measurements.

Results and discussion Physico-chemical properties of Lipo3-LPe. Four kinds of hybrid liposomes Lipo3-LPe were prepared by incorporating the four lipopeptides into Lipo3 (DSPC:DPPE:DC-Chol=4:0.2:1) with a fixed lipopeptide/phospholipid molar ratio (1:100). The phase transition temperature Ttr of liposome mainly composed of DSPC was about 54.0℃ by DSC measurement38 which was higher than the temperatures (37.0 ℃ and 45.0 ℃ ) used in the following experiments, then the influence of temperature on fluidity of membrane itself would be small. DLS method was used to investigate particle sizes and zeta-potentials of liposomes. The pure liposome and four Lipo3-LPe were incubated at 20.0℃ and 70.0℃ separately for 15min and their DLS results were shown in Table 1. The Lipo3 and Lipo3-LPe were all around 170nm in diameter and were all positively charged (around +45mV) at 20.0℃, indicating that the incorporation of the lipopeptides into lipid bilayer didn’t change their sizes and charged amounts. When the temperature increased to 70.0℃, their zeta potentials were almost the same as those at 20.0℃, and their sizes slightly increased due to the phase transition of lipids.39 The size distributions of Lipo3 and Lipo3-LPe under 20.0 and 70.0℃ were showed (Figure S1). The peaks overlapped under both conditions, indicating their vesicle structures had not been destroyed in the temperature range.

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 25

Table 1. The Tm values, diameters, polydispersity index and zeta-potentials of liposomes.

d/nm

PDI

Zeta/mV

Composition

20℃

70℃

20℃

70℃

Lipo3

172.0±2.6

180.2±1.1

0.167

0.216

—

Lipo3-LPe1

167.4±1.3

179.6±3.3

0.192

0.230

45.0℃

Lipo3-LPe2

172.0±3.6

195.3±1.6

0.209

0.243

Lipo3-LPe3

170.8±5.0

201.9±0.3

0.224

0.254

44.1℃

Lipo3-LPe4

175.2±5.0

200.5±1.4

0.217

0.254

43.7℃

+45.0

70℃

Tm/℃

20℃

+41.8

45.0℃

Thermo-sensitivity of Lipo3-LPe. CD spectrum was used to investigate the melting temperature (Tm) of lipopeptides doped in lipid bilayer, at which temperature their α-helix structure would change to a disorder state. The Tm values of these four lipopeptides doped in lipid bilayer were also listed in Table1 with DLS results. As shown in Figure 1, at room temperature, the four lipopeptides all adopted predominantly α-helix structure with well-defined characteristic negative bands at 208, 222 nm and a positive band at 192 nm. When the temperature increased to 70.0℃, the negative bands and the positive band of LPe1 and LPe2 weakened to represent a transition to disordered conformation (Figure1a,b). The Tm value was obtained to be 45.0℃ for both LPe1 and LPe2 in Lipo3. The same Tm value showed the increase of alkyl number from 5 to 7 had no effect on lipopeptide’s secondary structure and the phase transition. However, for LPe3, it showed very little structural change even the temperature increased to 70.0℃. The Tm 44.1℃ just represented the fastest change from an α-helix to a little weak α-helix, so the Tm value obtained by the same method was then thought to be meaningless. When incorporated in bilayer, LPe3 with longer alkyl chain could reach the second lipid layer since its length was estimated to be larger than the thickness of lipid monolayer. So originally, a greater disturbance in the bilayer had been expected to be caused by the LPe3. However, the structural transition of LPe3 in lipid bilayer was blocked due to the longer alkyl chain. It was thought the increase of alkyl chain length had improve the stability of lipopeptides, which agreed with literature27. Meanwhile, the hydrophobic force between the carbon chains was somewhat large and kept the zipper structure from splitting. Then, we designed the fourth lipopeptide LPe4, which had the same alkyl chain as LPe2 and a longer peptide with two extra amino acids Ser-Ala (Ser was hydrophilic 8


Page 9 of 25

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

and Ala was hydrophobic) at the N-terminal of peptide. The length of hydrophobic block was almost the same with LPe3. And meanwhile, the distance between two alkyl chains in a zipper structure became large (as shown in Scheme 1b) for weakening the hydrophobic force between the two tails. As shown in Figure 1d, LPe4 had an obvious conformational transition from α-helix partly to β-sheet with a Tm value of 43.7℃. β-sheet structure was more thermodynamically stable and extended than α-helix,16,40 which was different from α-helix to provide a nano-channel in the lipid bilayer. Meanwhile, the smaller value of Tm meant the decrease of α-helical stability. It showed that the modification of Ser-Ala at N-terminal weakened the stability of α-helix and then enhanced its thermo-sensitivity. In short, LPe1,2 and 4 in bilayer had better thermo-sensitivity than LPe3. To verify the reversibility of lipopeptides’ conformational transition, the CD spectrum was recorded 15 min after the temperature fell back to 20.0℃. As shown in Figure 1 (Right), the four lipopeptides doped in lipid bilayer all showed better reversible thermo-sensitivity. This result also proved that these lipopeptides could be used as great thermo-controllable biomaterials.

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

Figure 1. Temperature-dependent conformational changes (Left) and thermo-reversibility (Right) of (a) Lipo3-LPe1, (b) Lipo3-LPe2, (c) Lipo3-LPe3 and (d) Lipo3-LPe4. 10


Page 10 of 25

Page 11 of 25

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

Membrane fluidity of Lipo3-LPe. DPH is a hydrophobic membrane-bound fluorescence probe and it will stay at the hydrophobic region of the lipid bilayer after mixing with liposomes. The anisotropy of DPH reflects the perturbations in the hydrophobic region of lipid bilayer. The influence of lipopeptides’ insertion on membrane fluidity of liposome was studied by fluorescence anisotropy method with DPH as probe. The temperature-dependent anisotropy curves of DPH in pure liposome and hybrid liposomes were shown in Figure 2. All of liposomes had almost the same trend to exhibit a decrease in anisotropy values r with temperature increasing. A Ttr value could be obtained from the temperature at where the anisotropy decreased most rapidly. From Figure 2, the value of Ttr was found to be near 50℃for pure liposome and a little increase for hybrid liposomes, reflecting that the insertion of lipopeptides improved the lipid stability. Meanwhile, the anisotropy r values of various liposomes were almost the same at hypothermia, but showed a little higher for hybrid liposomes than pure liposome at hyperthermia. The results suggested the very low amount of lipopeptide (1%, mol/mol) in liposomes might not change the membrane fluidity at hypothermia. However, when temperature increased above Ttr, the effect of lipopeptide’s insertion became more and more apparent. This might relate to the phase state of membrane under the different temperatures. The lipid bilayer was a gel-like phase below Ttr, which usually had very low fluidity and not easy to be interrupted. But at hyperthermia, the membrane stability could be improved by inserting lipopeptide into a liquid crystal phase even the lipopeptide had transformed from α-helix zipper structure to random coil state. Contrastively, the r of Lipo3-LPe4 increased most, indicating LPe4 with longer hydrophobic block seemed to decrease membrane fluidity better. The Ttr values (about 50.0-54.0℃) obtained from anisotropy measurement were related to the movement of phospholipid tails in the bilayer. While

Tm was the melting temperature of lipopeptide, which was about 43.7-45.0℃for lipopeptides in lipid bilayer used here. Thus, in the following DOX release experiments performed under 37.0℃ or 45.0℃,the membrane fluidity of the various liposomes was almost the same under either of the two temperatures. Their differences in release amount should mainly induced by the conformational transition of lipopeptides from zipper structure to random coils.

11


Langmuir

Lipo3 Lipo3-LPe1 Lipo3-LPe2 Lipo3-LPe3 Lipo3-LPe4

0.40 0.35 Anisotropy

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 25

0.30 0.25 0.20 0.15 20

30

40 50 T/℃ ℃

60

70

Figure 2. Fluorescence anisotropy of DPH in various liposomes measured as a function of temperature from 20.0℃ to 70.0℃.

DOX release in vitro. DOX was encapsulated into liposomes by using ammonium sulfate gradient method. DOX release experiments in vitro were carried out at 37.0℃ and 45.0℃ over 24h. The effects of lipopeptides on DOX release had been investigated. As shown in Figure 3, the start point on the x-axis corresponds to the beginning of heating triggering. DOX released as a function of time under a constant temperature. For pure liposome (Lipo3), DOX released relatively quickly at first 6h and then released slowly and gradually. It reached the release percentages of 24.5% at 37.0 ℃ and 42.9% at 45.0 ℃ over 24h. The 18.4% difference of DOX release percentage between the two temperatures was thought to be caused by the better permeability of bilayer under a higher temperature. For the hybrid liposomes, their DOX release amounts were almost the same with pure liposome when released at 37.0℃, which was consistent with the result of membrane fluidity. As shown in Figure 2, their values of anisotropy were almost equal which indicated their membrane fluidity was the same. However, under 45.0℃, the differences in release amount of the liposomes became obvious. The maximum release percentage of DOX was 78.2% for Lipo3-LPe4 while the minimum was 42.9% for Lipo3. The drug release depended on permeability of membrane and appearance of nano-channel. From Figure 2, the anisotropy values of the hybrid liposomes at 45℃ were almost the same, suggesting the membrane fluidity was the same under this temperature. Then 12


Page 13 of 25

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

the difference in release amount was resulted from the appearance of nano-channels on the membrane and not the difference in membrane fluidity. The results told that LPe4 incorporated in liposome showed the best effect on stimulating drug release. Table S1 listed the final release percentages and their differences of the release percentage between 37.0 ℃ and 45.0 ℃

which were used to evaluate the

thermo-sensitivity of drug release. The difference value of Lipo3-LPe4 was about 54.6% showing the best thermo-sensitivity among these liposomes. And, Lipo3-LPe3 had the worst thermo-sensitive effect which was easy to understand from its CD result. The thermo-sensitivity of lipopeptide in lipid bilayer was thought to be depended on two facts. One was that the length of the hydrophobic alkyl chain which could go deep into the bilayer to give membrane larger disturbance when the lipopeptide denatured. However in this experimental condition, the denaturation of lipopeptide at 45.0℃ did not influence the membrane fluidity according to Figure 2. The other was the ability or the strength of the conformational transition. According to mentioned above, the increase in DOX release was mainly induced by the conformational transition of the lipopeptide, which might produce channel-like structure on lipid bilayer. A powerful conformational transition would provide stable and/or large channels for more DOX release. For LPe4, comparing with LPe3, two methyl groups were replaced by two amino acids and enlarged the distance between the two alkyl chains. The zipper structure of LPe4 could be embedded deeply into the bilayer like LPe3 but easier to separate during conformational transition. This result was consisted with the result of membrane fluidity showed in Figure 2 and the thermo-sensitivity showed in Figure 1. The kinetics of DOX release at 45℃ were quite different from these at 37℃. We took Lipo3-LPe1 as an example and analyzed its DOX release at 37℃ and 45℃ (Figure S2). The DOX release at 37℃ belonged to Higuchi model41 for diffusion and belonged to Ambiexponent model42 at 45℃ for the nano-channel in lipid bilayer.

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

Figure 3. The percentage of DOX release from pure liposome (Lipo3) and four hybrid liposomes at (a) 37.0℃ ℃ and (b) 45.0℃ ℃ over 24h.

Effect of membrane property on thermo-sensitivity. To investigate the effect of membrane property on Lipo-LPe’s thermo-sensitivity, six kinds of liposomes with different lipid compositions were prepared. The temperature-dependent anisotropy curves of DPH in these liposomes were shown in Figure 4a,b. DC-Chol was reported that it usually acted as a “buffer” of the membrane order. When incorporated into liposome, it could increase the thermo-stability of liposome and resulted in a decrease in the enthalpy of phase transition (gel to liquid crystal).38 Then, the membrane fluidity of the liposomes usually be increased below Ttr and decreased above Ttr due to the addition of DC-Chol. The increase of DC-Chol increased the mechanical strength and decreased the permeability of the membrane. Still, the characteristic for fluid phospholipid bilayers were maintained.43 Comparing the results of Lipo1-3 shown in Figure 4a, a decrease in enthalpy of phase transition could be speculated when DC-Chol increased, indicating the membrane thermo-stability increased. Lipo1-3 used here were mainly composed of DSPC, which had a high Ttr value44 and showed the similar varying tendency to the reference. The Lipo1, who had more DC-Chol, showed a more flexible membrane state but higher thermo-stability than Lipo3 in the temperature range of 20.0-50.0℃. As for Lipo4-6, it was certainly that the fluidity of liposomes increased and Ttr decreased a lot with the increase of DOPC (Ttr value of pure DOPC is about-20.0℃45). It was known that the saturated carbon bond made the model membrane more rigid, 14


Page 14 of 25

Page 15 of 25

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

while the presence of unsaturated carbon bond increases its fluidity.46,47 The double bonds in the hydrophobic chain affects it geometrical structure48 which changed the distance between lipids and prevented them from packing tightly. So the addition of DOPC made the membrane less stable and the membrane fluidity increased. In Figure 4b, the comparison between pure liposomes and hybrid liposomes had been shown. In this section, LPe1 was chosen as an example to investigate the effect of membrane property on thermo-sensitivity of Lipo-LPe. It was found the r values of Lipo6 and Lipo6-LPe1 were almost the same in the whole temperature range, suggesting the addition of lipopeptide gave no influence on membrane fluidity irrespective of the temperature. However, Lipo1 and Lipo1-LPe1 had the most significant difference which were almost the same under low temperature and grew apart from 30.0℃ on. The addition of LPe1 resulted in a decrease in membrane fluidity. Taken together, it was obvious that a lipid membrane, such as the membrane of Lipo6, having high fluidity and low Ttr might be flexible enough and then was difficult to be disturbed by lipopeptides, even the lipopeptides had a conformational transition. The thermo-sensitivities of LPe1 doped in different lipid bilayers were evaluated by CD. The results of Lipo1, Lipo3 and Lipo6 were chosen as examples to shown in Figure 4c for discussing the effect of lipopeptide. As shown in Figure 4c, the temperature-dependent curves of the three hybrid liposomes almost showed the same tendency, which indicated that LPe1 in the different bilayers yielded similar conformational transition. Their values of Tm were also obtained and found to decrease a little with the decrease of membrane stability. This result reflected that a quiet stable and solidified bilayer would impede the conformation transition and then increase the Tm value of the lipopeptide. The results were consistent with those mentioned in Figure 4b.

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 25

Figure 4. Fluorescence anisotropy of DPH in (a) pure Lipo1~Lipo6 and (b) the comparison between pure liposomes and hybrid liposomes measured as a function of temperature. (c) Temperature-dependent

conformational

changes

of

Lipo1-LPe1,

Lipo3-LPe1

and

Lipo6-LPe1, and their Tm values.

To intuitively study the effect of membrane stability on thermo-sensitivity, experiments of drug release from the hybrid liposomes in vitro over 24h were carried out (Figure 5). At 37.0℃, the total amounts of DOX release increased gradually with the decrease of lipid thermo-stability. Lipo1-LPe1 showed to be somewhat higher in the membrane fluidity than Lipo3-LPe1 but lower DOX release amount. The less DOX leakage suggested that the drug release depended more on the membrane permeability and thermo-stability in this experimental condition.43 When temperature increased to 45.0℃, the DOX release amount increased a lot for all hybrid liposomes except for Lipo1-LPe1. This was because the release temperature (45.0℃) was lower than Tm of LPe1 in Lipo1 (45.6℃). Then, we added 16


Page 17 of 25

the DOX release experiment of Lipo1-LPe1 at 46.0℃. As shown in Figure S3, the percentage of DOX release at 46.0℃ improved to 57.9% and the difference of DOX release reached to 49.9%. In addition, as shown in Figure 4b, the membrane stability of Lipo1-LPe1 around 45-46℃ increased a lot than that of Lipo1, suggesting the membrane permeability might become lower when lipopeptide inserted in bilayer. Then, the increase in DOX release at 46.0℃ proved DOX release from Lipo-LPe depended more on nano-channel rather than permeability. From Figure 5, the differences in DOX release of these hybrid liposomes between the two temperatures were in the range of 0-54.6%. For liposomes with very high membrane fluidity, such as Lipo6-LPe1, they usually had serious drug leakage under 37.0℃. And meanwhile, the conformational transition of lipopeptides could not produce nano-channels on the lipid surface due to the too fluidic bilayer, or the nano-channels were filled by surrounding lipids and disappeared immediately. This resulted in a relative low release amount under 45.0℃. Among the six hybrid liposomes, Lipo3-LPe1 achieved the highest DOX release amount and the best effect of thermo-sensitivity. In conclusion, a suitable lipid bilayer would benefit the realization of thermo-sensitivity of Lipo-LPe1 for drug delivery.

100 DOX Release %

37.0℃ ℃ 45.0℃ ℃

80 60 40 20 0 Li po 1LP e1 Li po 2LP e1 Li po 3LP e1 Li po 4LP e1 Li po 5LP e1 Li po 6LP e1

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

Decrease of thermo-stability Figure 5. The total percentage of DOX release from hybrid liposomes with different fluidities at 37.0℃ ℃ and 45.0℃ ℃ over 24h.

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

Intermittent release behavior of Lipo-LPe. Since the conformational transition of the lipopeptides were reversible, DOX release experiments undergoing intermittent heating were performed. The release medium was kept under 45.0℃-37.0℃-45.0℃ alternately for 4h at each temperature. The intermittent release experiments lasted for 24h. As shown in Figure 6a, the DOX in all Lipo3-LPe released rapidly at 45.0℃ but hardly release when the temperature decreased to 37.0℃. The stepped release behavior suggested that the DOX release of these hybrid liposomes could be controlled by temperature reversibly. However, the step of release amount was found to become smaller with the time. As it proved in Figure S4, the α-helix content of lipopeptides decreased after several temperature cycles and then the reversibility of the thermo-sensitivity of the lipopeptieds in the bilayer faded away. From the release results, it was found that, except for Lipo3-LPe4, their accumulated amounts of intermittent release in 12h under 45.0℃ were almost equal to those amounts of continuous release for same period (Figure 3). The release amount of Lipo3-LPe4 obtained through intermittent mode was about 28% less than that of continuous release for the same period. This might be related to the molecular structure of LPe4, whose distance between two alkyl chains was large. The zipper structured lipopeptides were easy to separate and difficult to go back to the double α-helix, resulting in the gradual disappearance of gradient release after 2 temperature cycles. Then, the release interval time of the intermittent experiments for Lipo3-LPe4 had been shortened to be 2 hours under each temperature. The release results were shown in Figure 6b. The DOX released intermittently from hybrid liposome controlled by the temperature and the final release amount was calculated to be near 100%. Lipo3-LPe4 was found to achieve a complete release of its cargo, although the step of release amount became lower after 3 temperature cycles. Compared with the result shown in Figure 6a, the release amount doubled. It could be thought that 4 hours of hyperthermia induced the motion of lipids, then the zipper structured lipopeptides were separated by lipids and difficult to combine again. However, a shorten time of hyperthermia decreased the risk. These results suggested that the liposomes acquired the thermo-sensitivity through inserting zipper structured lipopeptides. Their effects of thermo-controllable release were related to the molecular structure of lipopeptides, membrane properties and even the release mode.

18


Page 18 of 25

Page 19 of 25

Lipo3-LPe2

Lipo3-LPe1

Lipo3-LPe3

Lipo3-LPe4

60

a

DOX Release %

40

20

0

0 4 8 12 16 20 24 0 4 8 12 16 20 24 0 4 8 12 16 20 24 0 4 8 12 16 20 24

t/h

t/h

t/h

t/h

Lipo3-LPe4

100

b

DOX Release %

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 60 40 20 0

1 2 3 4 5 6 7 8 Temperature cycle times

Figure 6. (a) Drug release from Lipo3-LPe with temperature varying intermittently between 37.0℃ ℃ and 45.0℃ ℃ for 4h each. Grey area: 37.0℃ ℃, Red area: 45.0℃ ℃. (b) Drug release plotted against temperature cycle times from Lipo3-LPe4 with temperature varying intermittently between 37.0℃ ℃ and 45.0℃ ℃ for 2h each.

Conclusions Various zipper structured lipopeptides were designed and inserted into lipid bilayer as “intelligent valves”. The effects of lipopeptides’ molecular structure and membrane properties on drug release were investigated. The Tm values of the four lipopeptides inserted into lipid bilayer were influenced by molecular structures. The increase of alkyl chain’s length increased the stability of the α-helix resulting in a decrease in fluidity of lipid bilayer. However, an increase of amino groups at N-terminal was found to decrease the stability of the spatial structure. The highest 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 25

difference in DOX release amount of hybrid liposomes between 37.0℃ and 45.0℃ reached about 54.6% for Lipo3-LPe4. Meanwhile, the membrane property also affected the thermo-sensitivity of lipopeptides. A stable and solidified bilayer would impede the conformation transition and then increase the Tm value of the lipopeptide. Finally,

an

intermittent

release

mode

also

show

the

good

reversible

thermo-controllability of the hybrid liposomes. Through the intermittent mode, Lipo3-LPe4 realized a complete release when selected a proper interval time. The designed lipopeptides have been proved to be a promising thermo-controllable “intelligent valve” in lipid bilayer, which have been expected to apply in design and manufacture of smart drug carrier. This study have provided a theoretical basis for designing and preparing various novel smart drug carriers.

Association content Supporting information Figures of size distribution of liposomes (Figure S1), kinetics of DOX release from Lipo3-LPe1 (Figure S2), DOX release from Lipo1-LPe1 (Figure S3), CD spectra of Lipo3-LPe1 (Figure S4) and table of total release percentage of DOX from liposomes (Table S1). Author information Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. Tel. & Fax: 86-21-64252921. Notes The authors declare no competing financial interest. Acknowledgement Financial support for this work is provided by the National Natural Science Foundation of China (No. 21276074, No. 21376073), the 111 Project (No. B08021) of China and the Fundamental Research Funds for the Centre Universities of China.

References (1) Berns, E. J.; Alvarez, Z.; Goldberger, J. E.; Boekhoven, J.; Kessler, J. A.; Kuhn, H. G.; Stupp, S. I. A tenascin-C mimetic peptide amphiphile nanofiber gel promotes 20


Page 21 of 25

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

neurite outgrowth and cell migration of neurosphere-derived cells. Acta Biomater 2016, 37, 50-58. (2) Mumcuoglu, D.; Sardan Ekiz, M.; Gunay, G.; Tekinay, T.; Tekinay, A. B.; Guler, M. O. Cellular Internalization of Therapeutic Oligonucleotides by Peptide Amphiphile Nanofibers and Nanospheres. ACS Appl Mater Interfaces 2016, 8, 11280-11287. (3) Liang, X.; Shi, B.; Wang, K.; Fan, M.; Jiao, D.; Ao, J.; Song, N.; Wang, C.; Gu, J.; Li, Z. Development of self-assembling peptide nanovesicle with bilayers for enhanced EGFR-targeted drug and gene delivery. Biomaterials 2016, 82, 194-207. (4) Choi, H.; Jeena, M. T.; Palanikumar, L.; Jeong, Y.; Park, S.; Lee, E.; Ryu, J. H. The HA-incorporated nanostructure of a peptide-drug amphiphile for targeted anticancer drug delivery. Chemical communications 2016, 52, 5637-5640. (5) Zhao, X.; Pan, F.; Xu, H.; Yaseen, M.; Shan, H.; Hauser, C. A.; Zhang, S.; Lu, J. R. Molecular self-assembly and applications of designer peptide amphiphiles. Chemical Society reviews 2010, 39, 3480-3498. (6) Versluis, F.; Marsden, H. R.; Kros, A. Power struggles in peptide-amphiphile nanostructures. Chemical Society reviews 2010, 39, 3434-3444. (7) Castelletto, V.; Gouveia, R. M.; Connon, C. J.; Hamley, I. W. New RGD-peptide amphiphile mixtures containing a negatively charged diluent. Faraday Discussions 2013, 166, 381. (8) Maher, S.; Feighery, L.; Brayden, D. J.; McClean, S. Melittin as a permeability enhancer II: in vitro investigations in human mucus secreting intestinal monolayers and rat colonic mucosae. Pharmaceutical research 2007, 24, 1346-1356. (9) Liu, M.; Wang, H.; Liu, L.; Wang, B.; Sun, G. Melittin-MIL-2 fusion protein as a candidate for cancer immunotherapy. Journal of translational medicine 2016, 14, 155. (10) Lyu, Y.; Yang, Y.; Lyu, X.; Dong, N.; Shan, A. Antimicrobial activity, improved cell selectivity and mode of action of short PMAP-36-derived peptides against bacteria and Candida. Scientific reports 2016, 6, 27258. (11) Chen, C.; Hu, J.; Zeng, P.; Chen, Y.; Xu, H.; Lu, J. R. High cell selectivity and low-level antibacterial resistance of designed amphiphilic peptide G(IIKK)(3)I-NH(2). ACS Appl Mater Interfaces 2014, 6, 16529-16536. (12) Chen, C.; Chen, Y.; Yang, C.; Zeng, P.; Xu, H.; Pan, F.; Lu, J. R. High Selective Performance of Designed Antibacterial and Anticancer Peptide Amphiphiles. ACS Appl Mater Interfaces 2015, 7, 17346-17355. (13) Wiradharma, N.; Tong, Y. W.; Yang, Y. Y. Self-assembled oligopeptide nanostructures for co-delivery of drug and gene with synergistic therapeutic effect. Biomaterials 2009, 30, 3100-3109. (14) Guo, X. D.; Tandiono, F.; Wiradharma, N.; Khor, D.; Tan, C. G.; Khan, M.; Qian, Y.; Yang, Y. Y. Cationic micelles self-assembled from cholesterol-conjugated oligopeptides as an efficient gene delivery vector. Biomaterials 2008, 29, 4838-4846. (15) Fjell, C. D.; Hiss, J. A.; Hancock, R. E.; Schneider, G. Designing antimicrobial peptides: form follows function. Nature reviews. Drug discovery 2012, 11, 37-51. (16) Ulijn, R. V.; Smith, A. M. Designing peptide based nanomaterials. Chemical Society reviews 2008, 37, 664-675. (17) Chen, C.; Hu, J.; Zeng, P.; Pan, F.; Yaseen, M.; Xu, H.; Lu, J. R. Molecular 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

mechanisms of anticancer action and cell selectivity of short alpha-helical peptides. Biomaterials 2014, 35, 1552-1561. (18) Miravet, J. F.; Escuder, B.; Segarra-Maset, M. D.; Tena-Solsona, M.; Hamley, I. W.; Dehsorkhi, A.; Castelletto, V. Self-assembly of a peptide amphiphile: transition from nanotape fibrils to micelles. Soft Matter 2013, 9, 3558. (19) Fu, I. W.; Markegard, C. B.; Chu, B. K.; Nguyen, H. D. Role of hydrophobicity on self-assembly by peptide amphiphiles via molecular dynamics simulations. Langmuir 2014, 30, 7745-7754. (20) Fleming, S.; Ulijn, R. V. Design of nanostructures based on aromatic peptide amphiphiles. Chemical Society reviews 2014, 43, 8150-8177. (21) Cui, H.; Webber, M. J.; Stupp, S. I. Self-assembly of peptide amphiphiles: from molecules to nanostructures to biomaterials. Biopolymers 2010, 94, 1-18. (22) Azad, M. A.; Huttunen-Hennelly, H. E.; Ross Friedman, C. Bioactivity and the first transmission electron microscopy immunogold studies of short de novo-designed antimicrobial peptides. Antimicrobial agents and chemotherapy 2011, 55, 2137-2145. (23) Blondelle, S. E.; Houghten, R. A. Hemolytic and antimicrobial activities of the twenty-four individual omission analogues of melittin. Biochemistry 1991, 30, 4671-4678. (24) Park, C. B.; Yi, K. S.; Matsuzaki, K.; Kim, M. S.; Kim, S. C. Structure-activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: the proline hinge is responsible for the cell-penetrating ability of buforin II. Proceedings of the National Academy of Sciences of the United States of America 2000, 97, 8245-8250. (25) Meijer, J. T.; Roeters, M.; Viola, V.; Löwik, D. W. P. M.; Vriend, G.; van Hest, J. C. M. Stabilization of Peptide Fibrils by Hydrophobic Interaction. Langmuir 2007, 23, 2058-2063. (26) Löwik, D. W.; Garcia-Hartjes, J.; Meijer, J. T.; van Hest, J. C. Tuning secondary structure and self-assembly of amphiphilic peptides. Langmuir : the ACS journal of surfaces and colloids 2005, 21, 524-526. (27) Hamley, I. W. Self-assembly of amphiphilic peptides. Soft Matter 2011, 7, 4122-4138. (28) Chu-Kung, A. F.; Bozzelli, K. N.; Lockwood, N. A.; Haseman, J. R.; Mayo, K. H.; Tirrell, M. V. Promotion of Peptide Antimicrobial Activity by Fatty Acid Conjugation. Bioconjugate Chemistry 2004, 15, 530-535. (29) Forns, P.; Lauer-Fields, J. L.; Gao, S.; Fields, G. B. Induction of protein-like molecular architecture by monoalkyl hydrocarbon chains. Biopolymers 2000, 54, 531-546. (30) Shen, W.; Lammertink, R. G. H.; Sakata, J. K.; Kornfield, J. A.; Tirrell, D. A. Assembly of an artificial protein hydrogel through leucine zipper aggregation and disulfide bond formation. Macromolecules 2005, 38, 3909-3916. (31) Mason, J. M.; Hagemann, U. B.; Arndt, K. M. Role of Hydrophobic and Electrostatic Interactions in Coiled Coil Stability and Specificity. Biochemistry 2009, 48, 10380-10388. (32) Lai, J. R.; Fisk, J. D.; Weisblum, B.; Gellman, S. H. Hydrophobic Core Repacking in a Coiled-Coil Dimer via Phage Display: Insights into Plasticity and 22


Page 22 of 25

Page 23 of 25

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

Specificity at a Protein−Protein Interface. J. Am. Chem. Soc. 2004, 126, 10514-10515. (33)Al-Ahmady, Z. S.; Al-Jamal, W. T.; Bossche, J. V.; Bui, T. T.; Drake, A. F.; Mason, A. J.; Kostarelos, K. Lipid–Peptide Vesicle Nanoscale Hybrids for Triggered Drug Release by Mild Hyperthermia in Vitro and in Vivo. ACS Nano 2012, 6, 9335-9346. (34) Wang, S.; Shen, Y.; Zhang, J.; Xu, S.; Liu, H. A designed lipopeptide with a leucine zipper as an imbedded on/off switch for lipid bilayers. Phys. Chem. Chem. Phys. 2016, 18, 10129-10137. (35) Xu, X.; Xiao, X.; Xu, S.; Liu, H. Computational insights into the destabilization of alpha-helical conformations formed by leucine zipper peptides in response to temperature. Phys. Chem. Chem. Phys. 2016, 18, 25465-25473. (36) Soto-Arriaza, M. A.; Olivares-Ortega, C.; Lissi, E. A. Effect of the addition of alkanols of different topology to dipalmitoyl-phosphatidylcholine vesicles in the presence of gramicidin. J Colloid Interface Sci 2012, 385, 48-57. (37) Hurjui, I.; Neamtu, A.; Dorohoi, D. O. The interaction of fluorescent DPH probes with unsaturated phospholipid membranes: A molecular dynamics study. Journal of Molecular Structure 2013, 1044, 134-139. (38) McMullen, T. P. W.; McElhaney, R. N. Differential Scanning Calorimetric Studies of the Interaction of Cholesterol with Distearoyl and Dielaidoyl Molecular Species of Phosphatidylcholine, Phosphatidylethanolamine, and Phosphatidylserine. Biochemistry 1997, 36, 4979-4986. (39) Knecht, V.; Mark, A. E.; Marrink, S. J. Phase Behavior of a Phospholipid/Fatty Acid/Water Mixture Studied in Atomic Detail. J. Am. Chem. Soc. 2006, 128, 2030-2034. (40) Shimada, T.; Lee, S.; Bates, F. S.; Hotta, A.; Tirrell, M. Wormlike Micelle Formation in Peptide-Lipid Conjugates Driven by Secondary Structure Transformation of the Headgroups. J. Phys. Chem. B 2009, 113, 13711-13714. (41) Aznar, E.; Sancenon, F.; Marcos, M. D.; Martinez-Manez, R.; Stroeve, P.; Cano, J.; Amoros, P. Delivery modulation in silica mesoporous supports via alkyl chain pore outlet decoration. Langmuir 2012, 28, 2986-2996. (42) Feng, R.; Zhu, W.; Song, Z.; Zhao, L.; Zhai, G. Novel star-type methoxy-poly(ethylene glycol) (PEG)–poly(ε-caprolactone) (PCL) copolymeric nanoparticles for controlled release of curcumin. Journal of Nanoparticle Research 2013, 15, 1748-1759. (43) Ohvo-Rekilä, H.; Ramstedt, B.; Leppimäki, P.; Peter Slotte, J. Cholesterol interactions with phospholipids in membranes. Progress in Lipid Research 2002, 41, 66-97. (44) Khan, D. R.; Rezler, E. M.; Lauer-Fields, J.; Fields, G. B. Effects of drug hydrophobicity on liposomal stability. Chemical biology & drug design 2008, 71, 3-7. (45) Bui, T. T.; Suga, K.; Umakoshi, H. Roles of Sterol Derivatives in Regulating the Properties of Phospholipid Bilayer Systems. Langmuir 2016, 32, 6176-6184. (46) van Blitterswijk, W. J.; van der Meer, B. W.; Hilkmann, H. Quantitative Contributions of Cholesterol and the Individual Classes of Phospholipids and Their Degree of Fatty Acyl (Un) Saturation to Membrane Fluidity Measured by Fluorescence Polarization. Biochemistry 1987, 26, 1746-1756. 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

(47) Zehmer, J. K.; Hazel, J. R. Thermally induced changes in lipid composition of raft and non-raft regions of hepatocyte plasma membranes of rainbow trout. The Journal of Experimental Biology 2005, 208, 4283-4290. (48) Hac-Wydro, K.; Wydro, P. The influence of fatty acids on model cholesterol/ phospholipid membranes. Chemistry and physics of lipids 2007, 150, 66-81.

24


Page 24 of 25

Page 25 of 25

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

Abstract Graphic

25


Melting Behavior of Zipper-Structured Lipopeptides in Lipid Bilayer

Recommend Documents