Evaluation of EGCG loading capacity in DMPC membranes

Subscriber access provided by KEAN UNIV

Biological and Environmental Phenomena at the Interface

Evaluation of EGCG loading capacity in DMPC membranes Filipa Pires, Vananélia P. N. Geraldo, Bárbara Rodrigues, António de Granada-Flor, Rodrigo F. M. de Almeida, Osvaldo Novais Oliveira, Bruno L. Victor, Miguel Machuqueiro, and Maria Raposo Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00372 • Publication Date (Web): 21 Apr 2019 Downloaded from http://pubs.acs.org on April 21, 2019

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

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

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Evaluation of EGCG loading capacity in DMPC membranes †

Filipa Pires,

Granada-Flor,

¶

Vananélia P.N. Geraldo,

‡

Rodrigo F.M. de Almeida,

Victor,

¶

Bárbara Rodrigues,

¶,§

António de

Osvaldo N. Oliveira Jr.,

∗,¶,k

Miguel Machuqueiro,

†

and Maria Raposo

‡

Bruno L.

∗,†

†CEFITEC,

Departamento de Física, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica ‡Instituto de Física de São Carlos, Universidade de São Paulo, Brazil ¶Centro de Química e Bioquímica, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal §Centro de Química Estrutural, Faculty of Sciences, University of Lisboa, Campo Grande, C8 bdg, 1749-016 Lisboa, Portugal kBioISI - Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisboa, Campo Grande, C8 bdg, 1749-016 Lisboa, Portugal E-mail: [email protected]; [email protected] Phone: +351 217500112; +351 212948576 . Fax:

+351 21 294 85 49

Abstract Catechins are molecules with potential use in dierent pathologies such as diabetes and cancer, but their pharmaceutical applications are often hindered by their instability in the bloodstream. This issue can be circumvented by using liposomes as their nanocarriers for

in vivo

delivery. In this work, we studied the molecular details 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

Page 2 of 36

of (-)-epigallocatechin-3-gallate (EGCG) interacting with 1,2-dimyristoyl-sn -glycero-3phosphocholine (DMPC) monolayer/bilayer systems to understand the catechin loading ability and liposome stability, using experimental and computational techniques. The molecular dynamics simulations show the EGCG molecules deep inside the lipid bilayer, positioned below the lipid ester groups, generating a concentration-dependent lipid condensation. This eect was also inferred from the surface pressure isotherms of DMPC monolayers. In the PM-IRRAS assays, the predominant eect at higher concentrations of EGCG (e.g. 20 mol%) was an increase in lipid tail disorder. The steady-state uorescence data conrmed this disordered state, indicating that the catechin-induced liposome aggregation out-weights the condensation eects. Therefore, by adding more than 10 mol% EGCG to the liposomes, a destabilization of the vesicles occurs with the ensuing release of entrapped catechins. The loading capacity for DMPC seems to be limited by its disordered lipid arrangements, typical of a uid phase. To further increase the liposomes clinical usefulness, lipid bilayers with more stable and organized assemblies should be employed to avoid aggregation at large concentrations of catechin.

Introduction Epigallocatechin-3-gallate (EGCG; Figure 1) is the most abundant catechin in the

Camellia

sinensis green tea, whose potential for cancer therapy has been studied owing to its ability to modulate membrane organization and coordinate intracellular signalling pathways in the various steps of tumorigenesis.

1,2

EGCG is known to partition favourably to lipid bilayers,

and can even induce membrane disruption at high concentrations.

3

35

Also, EGCG can lead

to aggregation of phosphatidylcholine (PC) liposomes, rapidly potentiating the cargo release from the liposome in a concentrationdependent manner.

6

Using phase-contrast uorescence

microscopy, it has been shown that EGCG can induce calcein leakage from egg PC giant unilamellar vesicles, where their prolate shape changed to two spheres connected by a narrow neck.

3,7

EGCG modulates biological pathways

2

811

by altering membrane properties and


Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

acting as pan-assay interference compounds (PAINS).

12,13

Adsorption kinetics studies of dif-

ferent catechins on DMPC phospholipid surface showed that the catechins bearing gallate moiety bind more strongly to lipid membranes than non-gallolylated catechins, due to the additional phenolic OH groups.

14

Additionally, the stereochemical structure also seems to

aect the kinetic behaviour of catechins.

15

The

cis congurations have more favourable con-

formations for hydrogen bonding since the trihydroxyl groups are better oriented and closer to the bilayer surface.

Figure 1: Schematic chemical structure of Epigallocatechin-3-gallate (EGCG).

From the clinical point of view, the major limitation in the oral administration of EGCG is its poor bioavailability in the bloodstream. The susceptibility of its phenolic groups to deprotonation, oxidation and metabolic reactions within the living system echin very unstable. Fortunately, drug carriers including liposomes, and microparticles,

22,23

1719

16

makes this cat-

nanoparticles

20,21

can be used to overcome this problem, and therefore loading EGCG

into nanocarriers is a promising natural chemopreventive and chemotherapeutic agent for brain,

24,25

breast,

26,27

prostate,

28,29

lung,

30,31

and skin

32,33

cancers. Many reports have fo-

cused on the design of nanocarrier formulations and the use of new production techniques to improve encapsulation eciency and enhance the cellular uptake of liposomes.

3


3437

These

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 36

developments should be guided by molecularlevel information regarding the interaction of EGCG in lipidic model systems.

For instance, details on the catechin preferred location,

membrane stability and other monolayer/bilayer structural properties are useful to evaluate and improve encapsulation eciency. This type of information can be obtained by Molecular Dynamics (MD) simulations, which have been used to investigate interactions between membranes and small drugs, larger compounds,

40,41

peptides,

4244

and catechins.

4,15,4549

This

38,39

in silico technique can pro-

vide a detailed picture of the molecular interactions and allow the prediction of several properties that are usually very dicult to tackle using experimental techniques.

50

MD studies

on EGCG insertion into lipid bilayers showed that these molecules adopted a more ordered conguration in membranes composed of phosphatidyl ethalonamine: phosphatidyl choline (PE:PC) lipids (1:1 ratio),

15,45,46

which is signicantly dierent from pure PC. The molecu-

lar interactions of four green tea catechin compounds, including EGCG, with a POPC pure bilayer have also been studied, where EGCG was found to establish hydrogen bonds with the lipids ester region, inducing only a small local condensing eect on the bilayer, probably due to the presence of only one catechin molecule per leaet.

4

Recently, EGCG was observed to

insert into two dierent membrane models, resembling either a plasma membrane or a late endosome membrane, without a clear tendency to aggregate.

49

Furthermore, the ability of

EGCG to insert into neutral and anionic membrane models was modulated by the presence of salt, especially divalent Ca

++

and Mg

++ 47,48 . Despite growing evidence of the health benets

of catechins, EGCG has not been thoroughly studied using Langmuir monolayers models. These experimental membrane models mimic half a liposome/cell membrane, providing valuable information on drug-lipid interactions, particularly its impact on the physicochemical properties of the system under study (degree of molecular packing, molecular organization, and surface potential).

5153

In this work, we carried out a detailed study of the molecular interactions between EGCG and DMPC model membranes. The carrier loading capacity and the eects of the catechin on

4


Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

the membrane were studied using a multidisciplinary approach combining Langmuir monolayers, infrared and uorescence spectroscopies, and MD simulations. The results obtained provide an accurate evaluation of liposomes as nanocarriers and catechins encapsulation agents, which is relevant for future use in food packaging, cosmetics, and cancer therapeutics.

Experimental Materials EGCG (M.W. 458.4 g/mol) and dimyristoylphosphatidylcholine (DMPC) phospholipid (M.W. 677.93 g/mol) were purchased from SigmaAldrich and Avanti Polar Lipids, respectively. 4(2-[6-(dioctylamino)-2-naphthalenyl]ethyl]-1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS) was purchased from Biotium.

Langmuir Monolayers Studies The Langmuir monolayers were prepared using the co-spreading methodology.

Firstly,

DMPC and EGCG were dissolved in chloroform at concentrations of 0.5 mg/ml and 0.3 mg/ml, respectively, leading to the same molar concentration in each solution. Langmuir monolayers were obtained after spreading the pure DMPC or EGCG/DMPC mixtures, (1.6, 5, 10, and 20 mol%), on an aqueous subphase (Milli-Q water at 297

±

1 K). Surface pressure mea-

surements started after the solvent was allowed to evaporate for 10 min. The experiments were carried out in a Langmuir minitrough from KSV Instruments (model 2000, KSV Nima, Helsinki, Finland), in a class 10,000 clean room and were repeated at least three times to ensure reproducibility of the isotherms.

During lm compression, two symmetric barriers

moved at a constant speed of 10 mm.min

−1

.

In the isotherms analyses, the EGCG molecules were considered to also occupy an area on the Langmuir monolayer, in addition to the DMPC molecules. Therefore, the area per lipid

5


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 36

(i.e. per DMPC molecule) (Al ) was calculated by dividing the trough area by the number of DMPC molecules after subtracting the estimated area occupied by the EGCG molecules. This procedure was necessary because the area occupied by each EGCG molecule varied with its concentration, since not all EGCG molecules are expected to be at the interface. We also used the results from MD simulations to estimate the EGCG area at the interface. Finally, the area of DMPC was calculated from the total area of the Langmuir minitrough occupied by the DMPC phospholipid (area of the Langmuir minitrough minus the total area occupied by EGCG molecules) and the number of DMPC molecules spread in each condition. The compressional modulus (Cs

−1

π -Al compression isotherms.

) and the minimum area per lipid (Al ) were estimated from the

Cs

−1

was calculated using:

Cs−1 = −Al

∂π ∂Al

, where

π is the

T

monolayer surface pressure. The minimum area per lipid was derived by extrapolating the tangent to the inection point on the condensed region of the isotherm, indicating the area occupied by one lipid molecule at the air/water interface. The surface potential and surface pressure-area isotherms were recorded simultaneously in a Langmuir minitrough equipped with a surface potential sensor (KSV Kelvin probe, Biolin Scientic Oy, Helsinki, Finland), during the compression of the spread mixed EGCG/DMPC monolayers with selected ratios. The surface potential sensor measures the potential dierence between the vibrating plate (placed roughly 12 mm above the monolayer) and the reference electrode immersed into the water subphase, thus reecting the changes in surface potential.

54

PM-IRRAS Studies Polarization-modulated infrared reection absorption spectra (PM-IRRAS) measurements were performed using a KSV PMI 550 instrument (KSV Instruments Ltd, Helsinki, Finland) at an incidence angle of subphase at 297

80◦ .

The EGCG/DMPC mixtures were spread on an aqueous

± 1 K, and then compressed up to 30 mN m−1 .

The spectra were measured

for s- and p-polarizations at a high frequency. All the spectra were obtained by subtracting the spectrum of a pure water subphase at the same temperature as that of samples. Spectra

6


Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

of lipid samples were cut to a frequency range between 3000 and 900 cm

−1

and were baseline

corrected with a straight line. Spectra deconvolution using Gaussian lineshape was used to reveal the components of the phosphate and carbonyl bands as well as the band position and bandwidth of these components. The phosphate region of DMPC consists of two bands from the phosphate hydrogen-bonding to water (1237 cm

−1

) and "free" non-bound phosphates

−1 (1261 cm ). The phosphate hydration degree of DMPC was assessed from the ratio between the area under the band at 1237 cm

−1

and the total area under the curve. The carbonyl

band was deconvoluted into two Gaussian bands at 1737 cm

−1

and 1746 cm

−1

, which can

be attributed to the hydrogen-bonded ester carbonyl groups and free ester carbonyl groups, respectively. Within the same rationale, the hydration degree of the carbonyl groups was calculated from the ratio between the area of the band at 1737 cm under the curve.

−1

and the total area

The orientational order parameter of the CH2 lipid hydrocarbon chains

was obtained from the band area ratios A(2920 cm

−1

)/A(2855 cm

−1

).

55

Fluorescence Studies Steady-state uorescence anisotropy measurements were performed for EGCG in DMPC unilamellar vesicles (LUVs) suspensions. Briey, the required amount of lipid to obtain a nal lipid concentration in the LUVs suspension of 0.25 mM

56

was dissolved in chloroform and

the solvent was evaporated using a gentle stream of nitrogen, followed by vacuum desiccation for 4h to remove the last traces of solvent. The dried phospholipid lms were dispersed in buer (10 mM HEPES, 0.1 mM EDTA, 100 mM NaCl, pH 7.4) at 308 K (above the gel to liquid-crystalline transition temperature of DMPC, which is ca. 297 K

57

) and vortex-mixed.

The resulting multilamellar vesicle suspensions were subjected to ve freeze/thaw cycles and extruded 21 times through polycarbonate membranes with a pore diameter of 100 nm to form the LUVs and left to equilibrate overnight. Then, EGCG (at 5, 10, 20 or 30 mol%) was added to the sample at 303 K and incubated for 2h before the measurements. Fluorescence spectroscopy measurements were performed in a Horiba Jobin Yvon Spex

7


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 36

Fluorolog 3-22 spectrouoremeter (Kyoto, Japan) with excitation at 275 nm (slit width 5 nm) and emission at 353 nm (slit width 5 nm), i.e.

at the maximum of the excitation

and emission spectra under our conditions. The uorescence anisotropy,

hri,

was calculated

using equation 1:

hri =

IV V − GIV H IV V + 2GIV H

(1)

where the uorescence emission I had subscripts V and H for the vertical and horizontal orientations of the polarizers. The G-factor is a correction factor for detector sensitivity and is calculated as

G=

IHV . A total of seven readings for each intensity component were taken IHH

per sample using an integration time of 0.1 s. For each intensity reading, the background obtained for a DMPC suspension (without EGCG) was subtracted. All measurements were performed for 3 independent samples at 303 K, which was conrmed directly inside the cuvette using a temperature probe in a thermostated cuvette holder. To detect the changes in the bilayer dipole potential, the lipid suspensions were labelled with di-8-ANEPPS (1:500 probe/lipid mole ratio

41

) overnight. The ratio of the uorescence

intensity at 630 nm of di-8-ANEPPS excited at 420 nm to that excited at 520 nm was used to evaluate the membrane dipole potential.

58,59

Computational Details Molecular Dynamics MD simulations were performed with GROMACS 2016.4 eld.

61,62

60

and the GROMOS 54A7 force

Initial parameters for the EGCG molecule were obtained with the Automated

Topology Builder (ATB) and Repository

63,64

and manually curated. The pairs section were

modied to exclude 1-4 interactions in the aromatic rings. The charge set was obtained from a RESP

65

tting protocol, using the electrostatic potential calculated with Gaussian 09,

the B3LYP functional

6769

and 6-31G*

70

66

basis set. We have not explicitly accounted for the

8


Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

anionic form of EGCG (pKa

∼7.7) 71

since the neutral form is the most abundant. Addition-

ally, when interacting with lipid bilayers, anionic groups have their neutral forms stabilized.

43

Two types of MD simulations were performed: the insertion and the concentration sets. In the insertion simulations, two EGCG molecules were introduced to a pre-equilibrated 128 lipid DMPC membrane patch, either outside (in the water phase), or inside the membrane (Figure 2A). These two molecules were not allowed to interact (they are always at distances larger than 1.0 nm, which is the PME cuto ) and were analyzed as independent replicates. For the concentration simulations set, we created systems with 0, 2, 6, 12 and 24 EGCG molecules, corresponding to 0, 1.6, 4.7, 9.4, and 18.8 mol% of EGCG. Note that these molar fractions will slightly dier from the molar fractions in the experiments. Furthermore, the choice for DMPC was based on its small size, which leads to faster simulations, while keeping the correct lipid uid phase. The simulations were performed using the v-rescale heat bath

72

at 298 K with separate

couplings for water and solute (DMPC and EGCG), with a relaxation time of 0.1 ps. Parrinello-Rahman

73

semi-isotropic pressure couple was used to keep the pressure at 1 bar

with isothermal compressibility of

4.5 × 10−5

bar

−1

, and a relaxation time of 2.0 ps.

bonds were constrained with the parallel version of the LINCS algorithm, algorithm

75

A

was used for water (SPC

76

74

and the SETTLE

). A time step of 2 fs were used in the integration

of the equations of motion, with the neighbor lists being updated every 10 steps. Particle-Mesh Ewald (PME)

77

All

The

electrostatics was applied using 0.12 nm for the maximum

grid spacing of the Fast Fourier Transform and a cuto distance of 1.0 nm for Lennard-Jones and Coulomb interactions. The interpolation order for PME was 4 (cubic). Energy minimization of all systems was performed using two steps with the steepest descent algorithm, with LINCS constraints turned on in the second step. The initialization was achieved in a 250 ps MD simulation with harmonic restraints in both DMPC and EGCG, followed with 500 ps with restraints only on DMPC phosphorous atoms, always with a

2 restraint force of 1000 kJ/(mol nm ). We ran 5 replicate simulations (3 in the insertion set)

9


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 36

of 200 ns and all equilibrium properties were obtained from the last 100 ns. In the insertion set, we doubled our replicates by following the two EGCG molecules independently.

Analyses and error calculations The computational analyses were performed using in-house scripts and GROMACS analysis utilities. All simulations and experiments were done with replicates. The error bars represent the standard error of the mean and were determined with a leave-one-out resampling method.

Results and discussion EGCG insertion into DMPC bilayers We used MD simulations to study the interaction of EGCG with a lipid bilayer at the molecular level. We setup dierent systems where EGCG (catechin) was introduced either in the water phase or at the bilayer hydrophobic region (Figure 2A). Regardless of the starting positions, all EGCG molecules accumulate in the membrane at

∼10

Å below the

average phosphorous atoms positions (Figure 2B-C). There is only a small energy barrier around the phosphate region, which is easily overcome by the molecules within 100 ns. The preferred position is in good agreement with the minimum region of the reported potential of mean force,

13

even though completely dierent force elds were used. In another separate

set of MD simulations, Sirk

et al did not observe the complete insertion of EGCG into POPC

membranes, probably due to the lack of sampling on their simulations where the catechins were placed at the water region.

4

The large number of hydrogen bond donor groups in EGCG molecule makes it ideal to interact with the lipids ester region, where there are plenty of hydrogen bond acceptors. This feature and the signicant hydrophobicity of EGCG explain its membrane location and the most commonly observed conformations where its OH groups preferentially interact with the lipid esters (Figure 2D).

10


Page 11 of 36

15

Water Membrane

10 5

Probability Density

Membrane Insertion (Å)

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

C

0 −5

1.6 %EGCG 4.7 %EGCG 9.4 %EGCG 18.8 %EGCG

0.2

0.1

−10

D

−15

0 0

50

100

150

200

−15

−10

−5

0

5

Membrane Insertion (Å)

Time (ns)

Figure 2: Initial (A) and nal (B) conformations of the EGCG membrane insertion process. The catechin molecules were added near either the water/membrane interface (blue sticks) or the membrane center (pink sticks).

Even though the catechin molecules started from

the water phase, we observe full insertion in all simulations (B). The membrane is shown as thin grey sticks with the phosphorous atoms in spheres to highlight the polar interface. Membrane insertion of all EGCG molecules over time (C) and the corresponding histograms for the dierent molar ratios of the EGCG/DMPC systems (D). The catechin molecules tend to accumulate within the membrane (in each monolayer), regardless of its original position. Negative insertion values correspond to positions below the average phosphorous atoms positions, which were used as reference.

11


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 36

Eects of increasing EGCG concentration on DMPC lipid organization In the MD simulations, we also built EGCG/DMPC mixtures at dierent molar ratios: 0, 1.6, 4.7, 9.4, and 18.8, corresponding to 0, 2, 6, 12, and 24 lipids, respectively.

∼

EGCG molecules in 128

∼DMPC

The EGCG molecules always remained mainly at the membrane pre-

ferred position (∼10 Å below the average position of the phosphorous atoms). Only for 9.4 and 18.8 molar ratios did we observe some catechin exchange between solvent and membrane (Figure 2D). The

Al values calculated specically for DMPC indicate signicant condensation

with increasing concentrations of EGCG (Figure 3A), even though the system is expanded due to the presence of the catechin (Figure S1 of Supporting Information).

The catechin

specic area per molecule was calculated from the MD simulations to be 82.6, 88.2, 96.8, and

2 89.6 Å , for the 1.6, 5, 10, and 20 mol% EGCG mixtures, respectively. The surface pressure 2 of DMPC Langmuir monolayers displays a gaseous (10080 Å ) and a liquid-expanded (LE) 2 phase (molecular areas lower than 80 Å ), without a well-dened LE/LC transition (Figure 3B). The surface pressure increased monotonically up to compressional modulus of

∼96

Al

mN/m, reaching values of

mN/m (Figure 3C). On further compression, the monolayer

collapses to a three-dimensional state at The

∼49

πc ∼49

mN/m.

values from Figure 3B are in good agreement with those obtained from MD simu-

lations (Figure 3A). The addition of EGCG to the phospholipid monolayers caused a shift in the isotherms towards lower

Al ,

a trend similar to the condensing eect due to the presence

of cholesterol in PC membranes at the air-water interface.

8184

As an example, in DMPC

monolayers at 30 mN/m, cholesterol concentrations of 4%, 8% and 21% promote molecular

2 2 2 85 area contractions to 53 Å , 50 Å and 48 Å , respectively. Therefore, it seems that EGCG has an even larger condensing eect on DMPC than cholesterol.

Interestingly, although

EGCG causes condensation of DMPC monolayers, this monotonic dependence on concentration was not observed in the compressional modulus. The surface pressure isotherms in Figure 3C indicate that small amounts of EGCG (1.6 and 5 mol%) turn the DMPC mem-

12


Page 13 of 36

64

experimental simulation

Al (Å2)

60 56 52 48

A 0

5

10

15

20

mol% EGCG/DMPC

Surface Pressure (mN/m)

50

DMPC 1.6%EGCG 5%EGCG 10%EGCG 20%EGCG

40 30 20 10

B 0 20

Compressional Modulus (mN/m)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

40

60 Al (Å2)

80

100

100 80 60 40 20

C 0 0


10 20 30 40 Surface Pressure (mN/m)

50

Figure 3: Area per lipid (Al ) values of DMPC calculated in the presence of dierent amounts

A).

of EGCG (

The simulation

Al

values were calculated using GridMAT-MD,

78

while the

experimental details are given in the Experimental section. The gray shaded region corresponds to the values measured experimentally for pure DMPC in the uid phase.

79,80

Surface

pressure isotherms for monolayers of neat DMPC (black), 1.6% (blue), 5% (green), 10% (orange) and 20% w/w EGCG in DMPC (red) are shown (

B).

The respective compressional

C).

modulus as a function of surface pressure at the air/water interface are also shown ( 13


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 36

brane more rigid as reected by the increased compressional modulus. This agrees with the MD simulations, in particular with the observed increase in the lipid tail order parameter (Figure 4A). These results are also consistent with the literature where a slight, approximately linear, decrease of uidity at the acyl chain level was observed in DMPC LUVs in the uid phase with increasing EGCG.

86

In addition, the rigidifying eects of EGCG are

believed to be responsible for the anti-migratory and anti-metastatic eects on lung cancer cells.

8791

Rigidication slows down disintegration of drug-loaded liposomes, thus improving

stability although compromising drug entrapment eciency.

92

However, for higher concen-

trations of EGCG, the compressional modulus decreases, i.e. the DMPC monolayer becomes more compressible (membrane uidization). The latter change was not captured in the MD simulation, probably because the short time scales used are inadequate to probe lipid lateral diusion (several hundreds of nanoseconds are needed

93

to observe EGCG aggregation).

Additionally, the small membrane patch used (128 lipids) may not capture EGCG-induced phase separation and/or membrane curvature phenomena. When a large number of EGCG molecules are inserted into the DMPC monolayer, an anisotropic molecular packing is likely to occur upon the formation of EGCG aggregates. The preferred location of the EGCG molecules in the lipid bilayer, as inferred from the MD simulations (Figure 2D), indicates that catechin can have dierent concentrationdependent eects on the lipid head and tail groups. The membrane thickness and the order parameter of lipid aliphatic tails can provide decoupled information on which region is most aected by the presence of the catechin. In pure bilayers, these properties are usually highly coupled to the

Al values.

In mixtures, however, specic parts of the lipids can be altered while

little or no impact is observed in the overall

Al

values. The DMPC membrane thickness can

be measured by following the distance between average position of the phosphorous atoms in each monolayer. In our simulations, there are no signicant dierences in the membrane thickness values, even in the presence of higher amounts of EGCG (Figure S2 of Supporting Information). A membrane condensation eect, as suggested by the

14


Al

values (Figure 3A),

Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

is often associated with an increase in the membrane thickness values, which is not observed. Therefore, these simulations suggest that EGCG could be accommodated in the DMPC bilayer, without perturbing too much the lipid head groups. The changes in the DMPC membrane ordering, caused by increasing molar ratios of EGCG, can be analyzed by calculating the deuterium order parameter on the lipid tails and by PM-IRRAS spectroscopy of DMPC+EGCG monolayers. The deuterium order parameter values (−SCD ) increase signicantly with the molar fraction of EGCG (Figure S3 of Supporting Information). The

−SCD

plateau, usually observed at the 6th methylene group of

aliphatic chains, highlights the EGCG induced gain of order in this membrane region (Figure 4A). These results indicate that the condensing eect observed in the MD simulations can be attributed to a better packing of the lipid hydrocarbon chains in the presence of EGCG. In the experiments using Langmuir monolayers, the arrangement of the acyl chains was assessed by analyzing the shifts and shape changes of the peaks assigned to the ing modes. As shown in Figure 4B, the positions of the bands at 2855 assigned to the symmetric and anti-symmetric stretching of

CH2

cm−1

CH2

stretch-

and 2921

cm−1

in the DMPC monolayer,

respectively, were not signicantly aected by the incorporation of EGCG. However, the experimental hydrocarbon ordering parameter (Figure 4C), calculated using the ratio between the

CH2

anti-symmetric and symmetric stretching bands, revealed that EGCG may have a

disordering eect on the trans-gauche conformational equilibrium of DMPC. Indeed, even though at lower concentrations we see no signicant eect on the

CH2

order parameter (the

values fall within their error bars), at a larger EGCG concentration (20 mol%) a clear loss of lipid tail ordering is observed in the monolayer. This disordering eect is not consistent with the

−SCD

data from the MD simulations, probably due to EGCG aggregation eects,

which were not accounted for in the

in silico studies even at higher concentrations.

The EGCG molecules intrinsic uorescence is environment sensitive,

56

which allows one

to monitor its interaction with the membrane. The uorescence anisotropy of the catechin

15


Langmuir

A

0.003 Absorbance

0.26

−SCD

0.24

DMPC 1.6%EGCG 20%EGCG

νas CH2

νs CH2

0.002

0.001

0.22

0.2

B

0

5 10 15 mol% EGCG/DMPC

0 2950

20

2890

2860

2830

−1

Wavenumber (cm ) 1

D

0.16

C

2

2920

CH2 Order

0.8 0.12 1.5

0.6 0.08 0.4 0.04

1

0.2

0 0

5 10 15 EGCG Molar Fraction

0 0

20

Total intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 36

10 20 EGCG molar fraction

30

Figure 4: Lipid tails order in the presence of dierent molar ratios of EGCG. Deuterium order parameter (−SCD ) values for the 6th methylene group in DMPC ( correspond to a plateau in the

−SCD

A). The 6th positions

proles (Figure S3 of Supporting Information). The

gray shaded region corresponds to the experimental value (0.212± 0.005) for pure DMPC in uid phase.

79

PM-IRRAS measurements for dierent EGCG molar fractions in the region

B), from which the asymmetric/symmetric bands ratios C). EGCG uorescence anisotropy hri in the presence of DMPC vesicles (D).

of DMPC methylene (CH2 ) groups ( were obtained (

Total uorescence intensity corresponds to the sum of measurements, corrected for inner lter eects sample. bars =

94

IV V +2GIV H

retrieved from anisotropy

and normalized for the max value in each

All values correspond to the average of, at least, 3 independent samples.

±

standard deviation.

16


Error

Page 17 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

in the presence of DMPC vesicles remains unaltered at dierent mol% values (Figure 4D). However, the catechin uorescence intensity increases signicantly up to 10 mol%, which indicates that most catechin molecules are incorporated in the liposomes, since EGCG quantum yield is larger in the lipid bilayer than in the water phase.

56

This increase is reverted at

higher EGCG concentrations (20 and 30 mol%), probably due to instabilities and the increase of scattering from the suspension associated with catechin and liposome aggregation (observable with the naked eye after sample preparation) and/or a change of the catechin microenvironment. These results at high EGCG concentrations are consistent with the disordering eect observed in the phospholipid monolayers, pointing to the role of aggregation in limiting the catechin loading capacity of DMPC. In fact, from the MD simulations, which exclude the aggregation eects, one could expect a higher loading capacity according to the increased

−SCD

values (Figure 4A) and the low catechin exchange from the membrane to

the water phase, which is only present at 18.8 mol% (Figure 2D).

Eects of increasing EGCG concentration on DMPC dipolar properties Membrane dynamical properties are usually strongly coupled to the electric eld generated by the anisotropic organization of the charged, polar head groups.

This will include the

glycerol ester moiety, the rst water solvation shells, and even the solution electrolytes, acting as counterions. The transmembrane potential, the surface potential and the dipole potential are the three types of membrane electrical potential, which are known to play important roles in the structure and permeability of biological membranes.

59,9597

A surface

potential (∆V) is generated due to the charge dierence that exists between the aqueous interface (ions in bulk water) and the membrane monolayer surface (charged head groups of lipids).

95,98

We assessed the eect of EGCG on DMPC membrane potential using the lipid monolayer/bilayer as a model. Figure 5 shows the surface potential isotherms. In pure DMPC,

17


1 mN/m 10 mN/m 20 mN/m 25 mN/m 30 mN/m

1.8

µ/µ0

400

Page 18 of 36

2.2


600

200

1.4

1

A

0

20

40

60 80 100 Alipid (Å2)

120

B

0.6

140

0

5 10 15 EGCG molar fraction

2.5

phosphate carbonyl fits

0.6

Ratio (420/520)

0.5 Hydration

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

Surface Potential (mV)

Langmuir

0.4 0.3

20

exp. data fit

2

1.5

1

D

C

0.2

0.5 0

5 10 15 EGCG Molar Fraction

Figure 5: Surface potential at dierent in DMPC monolayers (

A).

20

Al

0

10 20 EGCG molar fraction

30

values for the dierent EGCG molar fractions

The ratio between the apparent dipole moment of EGCG and

DMPC (µ) and neat DMPC (µ0 ) when varying the EGCG molar fractions for dierent surface pressures (

B).

DMPC carbonyl and phosphate hydration degrees (

C)

obtained from

their carbonyl and asymmetric phosphate bands (see Figure S4A-B from Supporting Information), respectively. Eect of EGCG concentration on the uorescence ratio reecting the membrane dipole potential of DMPC vesicles labeled with di-8-ANEPPS (

D). The exponen-

tial functions, which are also shown (black), were tted to the data points. Error bars = standard deviation of 4 independent samples.

18


±

Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

contributions to the surface potential arise from the intrinsic dipoles of choline, phosphate and ester (carbonyl) groups, and the interfacial water molecules in the lipid headgroup region.

98,99

Surface potential becomes non zero at large

Al

2 values (∼ 104 Å ) due to the initial

assembling of the lipid molecules in the liquid expanded state, which leads to an increase in the normal component of the dipole moment (black curve in Figure 5A). At

Al of ∼ 58 Å2 the

surface potential increases further, probably related to a LE/LC transition (not completely clear from the isotherm), where dipoles are even more aligned to the membrane normal. At too small

∼

Al

values, the monolayer collapses with the highest surface potential observed of

530 mV. The

Al

vs.

∆V

curves for the EGCG/DMPC mixtures have similar proles to

pure DMPC. However, some dierences can be noted, namely, the negative high

∆V

values at

Al , or the signicantly higher ∆V values observed for 5 and 10% EGCG/DMPC systems

(∼ 610 mV), at monolayer pre-collapsing

Al

values (Figure 5A). It seems that EGCG con-

tributes with up to 80 mV to the total membrane surface potential. The potential functions do not exhibit a monotonic behavior due to the distinct eect of low and large amounts of EGCG in the packing of DMPC phospholipids molecules, as should be expected from the PM-IRRAS and steady-state uorescence measurements. For the 20% EGCG/DMPC mixture, the membrane surface potential decreases drastically at small

Al , being only ∼ 410 mV

near the monolayer collapse. This is probably due to the signicant increase in the physical distance between DMPC head groups, promoted by the presence of EGCG, which surpasses any condensing eect that may have occurred at lower mol% values. This extra space between the choline/phosphate groups is generated by the presence of the catechin molecules (Figure S1 of Supporting Information), which are usually inserted at higher depth (Figure 2D), and allows for signicant bending of the head group, leading to a decrease in its total membrane surface potential (Figure S5 of Supporting Information). This eect is also captured in the apparent dipole ratios between EGCG/DMPC and neat DMPC at dierent EGCG molar fractions, for dierent surface pressures (Figure 5B). Indeed, the predominant eect at small amounts of EGCG seems to be the lipid monolayer condensation, which is

19


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 36

indicated by the compressional modulus (Figure 3C) and, to some extent, by the PM-IRRAS measurements (Figure 4C). This seems to be related to an increase in the apparent dipole moment. However, at larger EGCG mol% fractions, the dipole moment decreases probably due to the DMPC head group bending, as suggested above.

+ (N (CH3 )3 ) asymmetric stretching band from 978 to 963

cm−1

Shifts in the choline group with 1.6 mol% EGCG in the

PM-IRRAS measurements (Figure S4C of Supporting Information) also suggest a condensation eect, since a shift of this band to a lower wavenumber indicates a decrease in the choline group bending. (969

100

The asymmetric stretching band observed for the 20 mol% fraction

cm−1 ) suggests a larger choline bending when compared with the 1.6 mol% system, but

still not fully consistent with the pure DMPC measurements. Furthermore, the head group bending can also explain why the membrane thickness prole does not change signicantly in the MD simulations, even though the

Al

of DMPC decreases with increased EGCG con-

centration. The limitations of our MD simulations to describe the membrane stability are the main reason why these data were not used further to help interpret the surface potential and PM-IRRAS results. Considering the relationship between lipid hydration and membrane dipole potential, the region between 1100-1800

cm−1

in the PM-IRRAS spectra was analyzed in more detail.

This analysis provides information about the spatial arrangement of the water molecules in the vicinity of the ester (carbonyl) and phosphate groups of lipids, and about its hydrogen bond network (Figures S4A-B of Supporting Information).

The absorption band

assigned to the carbonyl group (C=O) was deconvoluted with two components at 1737 and 1746

cm−1

cm−1

corresponding to the water-bound ester carbonyl groups and free carbonyl

groups, respectively.

101

At lower concentrations (1.65 mol% EGCG) there is a shift to

higher wavenumbers, reecting a reduction in hydrogen bonding to carbonyl groups. The hydration of DMPC carbonyl groups decreases in Figure 5C, probably due to replacement of water molecules by EGCG, which can act as hydrogen bond donors to the oxygen atoms in the DMPC carbonyl groups. Similar results were obtained for the phosphate region in Fig-

20


Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

ure 5C, where the absorption band assigned to the phosphate group (P=O) antisymmetric stretching was deconvoluted with two components at 1232

cm−1

and 1261

ing to water hydrogen bonding and free phosphate groups, respectively.

cm−1 102

correspond-

The hydration

level of DMPC phosphate groups also decreased upon addition of EGCG, following a similar exponential decay trend. The dipole potential changes in the presence of EGCG measured in DMPC monolayers were compared with similar data in DMPC bilayers using uorescence spectroscopy.

The

DMPC vesicles were labeled with di-8-ANEPPS, a probe that is very sensitive to the dipole potential changes in the membrane.

58

The addition of EGCG leads to a decrease of the dipole

potential of DMPC vesicles (Figure 5D), following an exponential decrease that is more analogous to the monolayer dehydration proles (Figure 5C) than to their dipole moment values (Figure 5B). In fact, this decrease in dipole moment is paralleled with the reduced polarity/hydration at the hydrophilic-hydrophobic interface in DMPC vesicles assessed through the generalized polarization of Laurdan.

86

This suggests that the displacement of the wa-

ter molecules by catechin in lipid bilayers is a key factor in the dipole moment decrease. Moreover, the addition of EGCG induces DMPC headgroup bending due to increased P P distances or lipid disorder, which could be related with liposomes aggregation at higher EGCG concentrations.

Conclusion The eects of embedding EGCG into DMPC membranes were studied using Langmuir monolayer techniques, lipid vesicles and MD simulations of lipid bilayers.

This interdis-

ciplinary study showed that EGCG accumulates at the lipids ester region, displacing the water molecules and establishing hydrogen bond interactions with all available hydrogen bond acceptors (phosphate and ester groups) of DMPC lipids.

The presence of EGCG induces

changes in several physical properties of DMPC monolayers/bilayers.

21


The computational

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 36

data suggests that the main eect is a condensation, reminiscent of what has been described for cholesterol.

8184

The PM-IRRAS data on lipid monolayers showed that such an eect ap-

pears only at low catechin concentrations, since at higher values the predominant eect was of increased disorder, which could be conrmed by the lowering of the dipole moments. The steady-state uorescence data at higher EGCG concentrations (> 10 mol%) corroborated that a catechin-induced liposome aggregation process overtakes any condensation eect. Furthermore, the membrane dipole potential of DMPC vesicles, followed using a di-8-ANEPPS label, shows that EGCG induces lipid bending in the bilayer and conrms that catechin loading capacity in DMPC liposomes is

∼10

mol%.

This loading capacity indicates that

liposomes are promising as nanocarriers and catechins encapsulation agents. Nonetheless, it may be desirable to develop formulations that stably retain larger molar fractions of EGCG. From our results we infer that more stable and organized liposomes (e.g. DPPC),

103

may

circumvent the aggregation process at larger concentrations of catechin. Finally, this work contributes to better understanding the extent of molecular interactions between EGCG and PC lipids and reveal how these interactions inuence the loading eciency and stability of liposomes.

EGCG encapsulation in PC-based liposomes can promote its antioxidant and

antimicrobial activities by triggering its sustained and controlled release over time. Looking ahead, these liposomal systems can be included in: (1) food packaging as an active coating that extends the food shelf life by preventing oxidation; (2) cosmetic formulations with increased EGCG absorption into human skin; and (3) cancer therapies as vehicles that provide higher EGCG accumulation within tumour cells.

Acknowledgement The authors acknowledge the nancial support from FEDER, through Programa Operacional Factores de Competitividade COMPETE and Fundação para a Ciência e a Tecnologia through research project grants PEst-OE/FIS/UI0068/2011, UID/FIS/00068/2013,

22


Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

UID/FIS/00068/2019, UID/MULTI/00612/2019, UID/MULTI/04046/2019, PTDC/FIS-NAN/0909/2014, PTDC/QEQ-COM/5904/2014, and PTDC/BBB-BQB/6071/2014. The Conselho Nacional de Desenvolvimento Cientíco e Tecnológico and Fundação de Amparo à Pesquisa do Estado de São Paulo are also acknowledged. MM acknowledges fellowship SFRH/BPD/110491/2015 and contract CEECIND/02300/2017 grants, and FP acknowledges the fellowship grant PD/BD/106036/2015 from RABBIT Doctoral Programme (Portugal).

Supporting Information Available Al ,

membrane thickness, deuterium order parameter, and PM IRRAS measurements fo-

cused on the ester, phosphate, and choline grops. Representative conformations of DMPC headgroup bending in the presence of EGCG.

References (1) Crous-Masó, J.; Palomeras, S.; Relat, J.; Camó, C.; Martínez-Garza, Ú.; Planas, M.; Feliu, L.; Puig, T. (-)-Epigallocatechin 3-Gallate Synthetic Analogues Inhibit Fatty Acid Synthase and Show Anticancer Activity in Triple Negative Breast Cancer.

Molecules 2018, 23, 1160. (2) Luo, K.-W.; Lung, W.-Y.; Chun-Xie, X.-L. L.; Huang, W.-R. EGCG inhibited bladder cancer T24 and 5637 cell proliferation and migration via PI3K/AKT pathway.

Oncotarget 2018, 9, 12261. (3) Tamba, Y.; Ohba, S.; Kubota, M.; Yoshioka, H.; Yoshioka, H.; Yamazaki, M. Single GUV method reveals interaction of tea catechin (-)-epigallocatechin gallate with lipid membranes.

Biophys. J. 2007, 92, 31783194.

(4) Sirk, T. W.; Brown, E. F.; Friedman, M.; Sum, A. K. Molecular binding of catechins

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

to biomembranes: relationship to biological activity.

Page 24 of 36

J. AAgric. Food Chem. 2009,

57, 67206728. (5) Sun, Y.; Hung, W.-C.; Chen, F.-Y.; Lee, C.-C.; Huang, H. W. Interaction of tea catechin ()-epigallocatechin gallate with lipid bilayers.

Biophys. J. 2009, 96, 1026

1035.

(6) Ikigai, H.; Nakae, T.; Hara, Y.; Shimamura, T. Bactericidal catechins damage the lipid bilayer.

Biochem. Biophys. Acta, Biomembr. 1993, 1147, 132136.

(7) Yamazaki, M. The single GUV method to reveal elementary processes of leakage of internal contents from liposomes induced by antimicrobial substances.

Adv. Planar

Lipid Bilayers Liposomes 2008, 7, 121142. (8) Shimizu, M.; Weinstein, I. B. Modulation of signal transduction by tea catechins and related phytochemicals.

Mutat. Res. 2005, 591, 147160.

(9) Khan, N.; Afaq, F.; Saleem, M.; Ahmad, N.; Mukhtar, H. Targeting multiple signaling pathways by green tea polyphenol (-)-epigallocatechin-3-gallate.

Cancer Res. 2006,

66, 25002505. (10) Nagle, D. G.; Ferreira, D.; Zhou, Y.-D. Epigallocatechin-3-gallate (EGCG): chemical and biomedical perspectives.

Phytochemistry 2006, 67, 18491855.

(11) Prasad, S.; Phromnoi, K.; Yadav, V. R.; Chaturvedi, M. M.; Aggarwal, B. B. Targeting inammatory pathways by avonoids for prevention and treatment of cancer.

Planta

Med. 2010, 76, 10441063. (12) Baell, J.; Walters, M. A. Chemical con artists foil drug discovery.

Nature 2014, 513,

481.

(13) Ingólfsson, H. I. et al. Phytochemicals perturb membranes and promiscuously alter protein function.

ACS Chem. Biol. 2014, 9, 17881798. 24


Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(14) Kamihira, M.; Nakazawa, H.; Kira, A.; Mizutani, Y.; Nakamura, M.; Nakayama, T. Interaction of tea catechins with lipid bilayers investigated by a quartz-crystal microbalance analysis.

Biosci. Biotechnol. Biochem. 2008, 72, 13721375.

(15) Sirk, T. W.; Brown, E. F.; Sum, A. K.; Friedman, M. Molecular dynamics study on the biophysical interactions of seven green tea catechins with lipid bilayers of cell membranes.

J. AAgric. Food Chem. 2008, 56, 77507758.

(16) Long, L. H.; Clement, M. V.; Halliwell, B. Artifacts in cell culture: rapid generation of hydrogen peroxide on addition of (-)-epigallocatechin,(-)-epigallocatechin gallate,(+)catechin, and quercetin to commonly used cell culture media.

Biochem. Biophys. Res.

Commun. 2000, 273, 5053. (17) Riaz, M. K.; Riaz, M. A.; Zhang, X.; Lin, C.; Wong, K. H.; Chen, X.; Zhang, G.; Lu, A.; Yang, Z. Surface Functionalization and Targeting Strategies of Liposomes in Solid Tumor Therapy: A Review.

Int. J. Mol. Sci. 2018, 19, 195.

(18) Olusanya, T. O.; Haj Ahmad, R. R.; Ibegbu, D. M.; Smith, J. R.; Elkordy, A. A. Liposomal drug delivery systems and anticancer drugs.

Molecules 2018, 23, 907.

(19) Torchilin, V. P. Immobilization of specic proteins on liposome surface: systems for drug targeting.

Liposome Technol. 2018, 3, 7594.

(20) Hu, B.; Ting, Y.; Yang, X.; Tang, W.; Zeng, X.; Huang, Q. Nanochemoprevention by encapsulation of (-)-epigallocatechin-3-gallate with bioactive peptides/chitosan nanoparticles for enhancement of its bioavailability.

ChemComm 2012, 48, 24212423.

(21) Liang, J.; Yan, H.; Puligundla, P.; Gao, X.; Zhou, Y.; Wan, X. Applications of chitosan nanoparticles to enhance absorption and bioavailability of tea polyphenols: A review.

Food Hydrocoll. 2017, 69, 286292.

25


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 36

(22) Isteni£, K.; Cerc Koro²ec, R.; Poklar Ulrih, N. Encapsulation of (-)-epigallocatechin gallate into liposomes and into alginate or chitosan microparticles reinforced with liposomes.

J. Sci. Food Agric. 2016, 96, 46234632.

(23) Gómez-Mascaraque, L. G.; Sanchez, G.; López-Rubio, A. Impact of molecular weight on the formation of electrosprayed chitosan microcapsules as delivery vehicles for bioactive compounds.

Carbohydr. Polym. 2016, 150, 121130.

(24) Hong, W.; Zhao, Y.; Guo, Y.; Huang, C.; Qiu, P.; Zhu, J.; Chu, C.; Shi, H.; Liu, M. PEGylated Self-Assembled Nano-Bacitracin A: Probing the Antibacterial Mechanism and Real-Time Tracing of Target Delivery in Vivo.

ACS Appl. Mater. Interfaces 2018,

10, 1068810705. (25) Zhang, Y.; Wang, S.-X.; Ma, J.-W.; Li, H.-Y.; Ye, J.-C.; Xie, S.-M.; Du, B.; Zhong, X.Y. EGCG inhibits properties of glioma stem-like cells and synergizes with temozolomide through downregulation of P-glycoprotein inhibition.

J. Neuro-Oncol. 2015, 121,

4152.

(26) Hajipour, H.; Hamishehkar, H.; Nazari Soltan Ahmad, S.; Barghi, S.; Marou, N. F.; Taheri, R. A. Improved anticancer eects of epigallocatechin gallate using RGDcontaining nanostructured lipid carriers.

Artif Cells Nanomed. Biotechnol. 2018, 110.

(27) Ramadass, S. K.; Anantharaman, N. V.; Subramanian, S.; Sivasubramanian, S.; Madhan, B. Paclitaxel/epigallocatechin gallate coloaded liposome: a synergistic delivery to control the invasiveness of MDA-MB-231 breast cancer cells.

Colloid. Surface. B

2015, 125, 6572. (28) Sanna, V.; Singh, C. K.; Jashari, R.; Adhami, V. M.; Chamcheu, J. C.; Rady, I.; Sechi, M.; Mukhtar, H.; Siddiqui, I. A. Targeted nanoparticles encapsulating (-)epigallocatechin-3-gallate for prostate cancer prevention and therapy.

7, 41573. 26


Sci. Rep. 2017,

Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(29) Tsai,

L.-C.;

Hsieh,

H.-Y.;

Lu,

K.-Y.;

Wang,

S.-Y.;

Mi,

F.-L.

EGCG/gelatin-

doxorubicin gold nanoparticles enhance therapeutic ecacy of doxorubicin for prostate cancer treatment.

Nanomedicine 2016, 11, 930.

(30) Dhatwalia, S. K.; Kumar, M.; Dhawan, D. K. Role of EGCG in Containing the Progression of Lung TumorigenesisA Multistage Targeting Approach.

Nutr. Cancer

2018,

70, 334349. (31) Ma, Y.; Yang, J.; Li, S.; Yu, W. Synergic Inhibition of Lung Carcinoma 95-D Cell Proliferation and Invasion by Combination with (-)-Epigallocatechin-3-Gallate and Ascorbic Acid.

Wuhan Univ. J. Nat. Sci. 2018, 23, 270276.

(32) Ramadon, D.; Goldie, A. W.; Anwar, E. Novel Transdermal Ethosomal Gel Containing Green Tea (Camellia sinensis L. Kuntze) Leaves Extract: Formulation and In vitro Penetration Study.

J. Young Pharm. 2017, 9, 336.

(33) Zhang, J.; Lei, Z.; Huang, Z.; Zhang, X.; Zhou, Y.; Luo, Z.; Zeng, W.; Su, J.; Peng, C.; Chen, X. Epigallocatechin-3-gallate (EGCG) suppresses melanoma cell growth and metastasis by targeting TRAF6 activity.

Oncotarget 2016, 7, 79557.

(34) Akhavan, S.; Assadpour, E.; Katouzian, I.; Jafari, S. M. Lipid nano scale cargos for the protection and delivery of food bioactive ingredients and nutraceuticals.

Trends

Food Sci. Technol. 2018, (35) Zoghi, A.; Khosravi-Darani, K.; Omri, A. Process Variables and Design of Experiments in Liposome and Nanoliposome Research.

Mini-Rev. Med. Chem. 2018, 18, 324344.

(36) Esfanjani, A. F.; Assadpour, E.; Jafari, S. M. Improving the bioavailability of phenolic compounds by loading them within lipid-based nanocarriers.

Technol. 2018,

27


Trends Food Sci.

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 36

(37) Gonçalves, R. F.; Martins, J. T.; Duarte, C. M.; Vicente, A. A.; Pinheiro, A. C. Advances in nutraceutical delivery systems: From formulation design for bioavailability enhancement to ecacy and safety evaluation.

Trends Food Sci. Technol. 2018,

(38) Kope¢, W.; Telenius, J.; Khandelia, H. Molecular dynamics simulations of the interactions of medicinal plant extracts and drugs with lipid bilayer membranes.

FEBS J.

2013, 280, 27852805. (39) Vila-Viçosa, D.; Victor, B. L.; Ramos, J.; Machado, D.; Viveiros, M.; Switala, J.; Loewen, P. C.; Leitao, R.; Martins, F.; Machuqueiro, M. Insights on the mechanism of action of INH-C10 as an antitubercular prodrug.

Mol. Pharm. 2017, 14, 45974605.

(40) Wong-Ekkabut, J.; Baoukina, S.; Triampo, W.; Tang, I.-M.; Tieleman, D. P.; Monticelli, L. Computer simulation study of fullerene translocation through lipid membranes.

Nat. Nanotechnol. 2008, 3, 363.

(41) Filipe, H. A.; Sousa, C.; Marques, J. T.; Vila-Vicosa, D.; de Granada-Flor, A.; Viana, A. S.; Santos, M. S. C.; Machuqueiro, M.; de Almeida, R. F. Dierential targeting of membrane lipid domains by caeic acid and its ester derivatives.

Free

Radic. Biol. Med. 2018, 115, 232245. (42) Cardenas, A. E.; of

a

Peptide:

It

Shrestha, R.; is

Better

to

Webb, L. J.; be

Positive.

Elber, R. Membrane Permeation

J. Phys. Chem. B

2015,

DOI:

10.1021/acs.jpcb.5b02122.

(43) Teixeira, V. H.; Vila-Viçosa, D.; Reis, P. B. P. S.; Machuqueiro, M. pKa Values of Titrable Amino Acids at the Water/Membrane Interface.

J. Chem. Theory Comput.

2016, 12, 930934. (44) Vila-Viçosa, D.; Silva, T. F.; Slaybaugh, G.; Reshetnyak, Y. K.; Andreev, O. A.; Machuqueiro, M. Membrane-Induced pKa shifts in

wt-pHLIP

J. Chem. Theory Comput. 2018, 14, 32893297. 28


and its L16H Variant.

Page 29 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(45) Zhu, W.; Xiong, L.; Peng, J.; Deng, X.; Gao, J.; Li, C.-m. Molecular Insight into Anities of Gallated and Nongallated Proanthocyanidins Dimers to Lipid Bilayers.

Sci. Rep. 2016, 6, 37680. (46) Zhu, W.; Khalifa, I.; Peng, J.; Li, C. Position and orientation of gallated proanthocyanidins in lipid bilayer membranes: inuence of polymerization degree and linkage type.

J. Biomol. Struct. Dyn. 2017, 114.

(47) Laudadio, E.; Mobbili, G.; Minnelli, C.; Massaccesi, L.; Galeazzi, R. Salts inuence cathechins and avonoids encapsulation in liposomes: A molecular dynamics investigation.

Mol. Inform. 2017, 36, 1700059.

(48) Laudadio, E.; Minnelli, C.; Amici, A.; Massaccesi, L.; Mobbili, G.; Galeazzi, R. Liposomal formulations for an ecient encapsulation of epigallocatechin-3-gallate: An in-silico/experimental approach.

Molecules 2018, 23, 441.

(49) Villalaín, J. Epigallocatechin-3-gallate location and interaction with late endosomal and plasma membrane model membranes by molecular dynamics.

J. Biomol. Struct.

Dyn. 2018, 113. (50) Hospital, A.; Goñi, J. R.; Orozco, M.; Gelpí, J. L. Molecular dynamics simulations: advances and applications.

Adv. Appl. Bioinforma. Chem. 2015, 8, 37.

(51) Elderd, M.; Sikorski, A. F. Langmuir-Monolayer Methodologies for Characterizing Protein-Lipid Interactions.

Chem. Phys. Lipids 2018,

(52) Nobre, T. M.; Pavinatto, F. J.; Caseli, L.; Barros-Timmons, A.; Dynarowicz-¡tka, P.; Oliveira Jr, O. N. Interactions of bioactive molecules & nanomaterials with Langmuir monolayers as cell membrane models.

Thin Solid Films 2015, 593, 158188.

(53) Giner-Casares, J. J.; Brezesinski, G.; Möhwald, H. Langmuir monolayers as unique physical models.

Curr. Opin. Colloid Interface Sci. 2014, 19, 176182. 29


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 36

(54) Oliveira, O. N.; Taylor, D. M.; Lewis, T. J.; Salvagno, S.; Stirling, C. J. Estimation of group dipole moments from surface potential measurements on Langmuir monolayers.

J. Chem. Soc. Faraday Trans. 1989, 85, 10091018. (55) Levin, I.; Thompson, T.; Barenholz, Y.; Huang, C.-h. Two types of hydrocarbon chain interdigitation in sphingomyelin bilayers.

Biochemistry 1985, 24, 62826286.

(56) Caturla, N.; Vera-Samper, E.; Villalaín, J.; Mateo, C. R.; Micol, V. The relationship between the antioxidant and the antibacterial properties of galloylated catechins and the structure of phospholipid model membranes.

Free Radic. Biol. Med. 2003, 34,

648662.

(57) Mabrey, S.; Sturtevant, J. M. Investigation of phase transitions of lipids and lipid mixtures by sensitivity dierential scanning calorimetry.

Proc. Natl. Acad. Sci. USA

1976, 73, 38623866. (58) Clarke, R. J. The dipole potential of phospholipid membranes and methods for its detection.

Adv. Colloid Interface Sci. 2001, 89, 263281.

(59) Khmelinskaia, A.; Ibarguren, M.; de Almeida, R. F.; López, D. J.; Paixão, V. A.; Ahyayauch, H.; Goñi, F. M.; Escribá, P. V. Changes in membrane organization upon spontaneous insertion of 2-hydroxylated unsaturated fatty acids in the lipid bilayer.

Langmuir 2014, 30, 21172128. (60) Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.; Hess, B.; Lindahl, E. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers.

SoftwareX

2015, 1-2, 19 25.

(61) Schmid, N.; Eichenberger, A. P.; Choutko, A.; Riniker, S.; Winger, M.; Mark, A. E.; Van Gunsteren, W. F. Denition and testing of the GROMOS force-eld versions 54A7 and 54B7.

Eur. Biophys. J. 2011, 40, 843856. 30


Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(62) Huang, W.; Lin, Z.; van Gunsteren, W. F. Validation of the GROMOS 54A7 Force Field with Respect to

β -Peptide

Folding.

J. Chem. Theory Comput. 2011, 7, 1237

1243.

(63) Malde, A. K.; Zuo, L.; Breeze, M.; Stroet, M.; Poger, D.; Nair, P. C.; Oostenbrink, C.; Mark, A. E. An automated force eld topology builder (ATB) and repository: version 1.0.

J. Chem. Theory Comput. 2011, 7, 40264037.

(64) Koziara, K. B.; Stroet, M.; Malde, A. K.; Mark, A. E. Testing and validation of the Automated Topology Builder (ATB) version 2.0: enthalpies.

prediction of hydration free

J. Comput. Aided Mol. Des. 2014, 28, 221233.

(65) Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. A Well Behaved Electrostatic Based Method Using Charge Restraints For Deriving Atomic Charges: Model.

The RESP

J. Phys. Chem. 1993, 97, 1026910280.

(66) Frisch, M. et al. Gaussian 09, Revision A.02. Gaussian, Inc., Wallingford, CT, 2016.

(67) Vosko, S. H.; Wilk, L.; Nusair, M. Accurate spin-dependent electron liquid correlation energies for local spin density calculations: a critical analysis.

Can. J. Phys. 1980,

58, 12001211. (68) Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density.

Phys. Rev. B 1988, 37, 785.

(69) Becke, A. D. Density-functional thermochemistry. III. The role of exact exchange.

J.

Chem. Phys. 1993, 98, 56485652. (70) Hariharan, P. C.; Pople, J. A. The inuence of polarization functions on molecular orbital hydrogenation energies.

Theor. Chim. Acta 1973, 28, 213222.

(71) Muzolf, M.; Szymusiak, H.; Gliszczy«ska-wigªo, A.; Rietjens, I. M.; Tyrakowska, B.

31


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 36

pH-dependent radical scavenging capacity of green tea catechins.

J. Agric. Food Chem.

2008, 56, 816823. (72) Bussi, G.; Donadio, D.; Parrinello, M. Canonical sampling through velocity rescaling.

J. Chem. Phys. 2007, 126, 014101. (73) Parrinello, M.; Rahman, A. Polymorphic transitions in single crystals: A new molecular dynamics method.

J. Appl. Phys. 1981, 52, 71827190.

(74) Hess, B. P-LINCS: A Parallel Linear Constraint Solver for Molecular Simulation.

J.

Chem. Theory Comput. 2008, 4, 116122. (75) Miyamoto, S.; Kollman, P. A. SETTLE: An analytical version of the SHAKE and RATTLE algorithm for rigid water models.

J. Comput. Chem. 1992, 13, 952962.

(76) Hermans, J.; Berendsen, H. J. C.; van Gunsteren, W. F.; Postma, J. P. M. A Consistent Empirical Potential for Water-Protein Interactions.

Biopolymers 1984, 23, 15131518.

(77) Darden, T.; York, D.; Pedersen, L. Particle mesh Ewald: Ewald sums in large systems.

An Nlog(N) method for

J. Chem. Phys. 1993, 98, 1008910092.

(78) Allen, W. J.; Lemkul, J. A.; Bevan, D. R. GridMAT-MD: A grid-based membrane analysis tool for use with molecular dynamics.

J. Comput. Chem. 2009, 30, 1952

1958.

(79) Petrache, H. I.; Dodd, S. W.; Brown, M. F. Area per lipid and acyl length distributions in uid phosphatidylcholines determined by

2

H-NMR spectroscopy.

Biophys. J. 2000,

79, 31723192. (80) Nagle, J. F.; Tristram-Nagle, S. Structure of lipid bilayers.

Rev. Biomembr. 2000, 1469, 159195.

32


Biochem. Biophys. Acta,

Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(81) Wydro, P.; Hac-Wydro, K. Thermodynamic description of the interactions between lipids in ternary Langmuir monolayers: the study of cholesterol distribution in membranes.

J. Phys. Chem. B 2007, 111, 24952502.

(82) Su, Y.; Li, Q.; Chen, L.; Yu, Z. Condensation eect of cholesterol, stigmasterol, and sitosterol on dipalmitoylphosphatidylcholine in molecular monolayers.

Colloids Surf.

A 2007, 293, 123129. (83) Gong, K.; Feng, S.-S.; Go, M. L.; Soew, P. H. Eects of pH on the stability and compressibility of DPPC/cholesterol monolayers at the airwater interface.

Colloids

Surf. A 2002, 207, 113125. (84) Sabatini, K.; Mattila, J.-P.; Kinnunen, P. K. Interfacial behavior of cholesterol, ergosterol, and lanosterol in mixtures with DPPC and DMPC.

Biophys. J. 2008, 95,

23402355.

(85) Miyoshi, T.; Kato, S. Detailed analysis of the surface area and elasticity in the saturated 1, 2-diacylphosphatidylcholine/cholesterol binary monolayer system.

Langmuir

2015, 31, 90869096. (86) Colina, J. R.; Suwalsky, M.; Manrique-Moreno, M.; Petit, K.; Aguilar, L. F.; JemiolaRzeminska, M.; Strzalka, K. Protective eect of epigallocatechin gallate on human erythrocytes.

Colloid. Surface. B 2019, 173, 742750.

(87) Takahashi, A.; Watanabe, T.; Mondal, A.; Suzuki, K.; Kurusu-Kanno, M.; Li, Z.; Yamazaki, T.; Fujiki, H.; Suganuma, M. Mechanism-based inhibition of cancer metastasis with (-)-epigallocatechin gallate.

Biochem. Biophys. Res. Commun. 2014, 443,

16.

(88) Luo, Q.; Kuang, D.; Zhang, B.; Song, G. Cell stiness determined by atomic force microscopy and its correlation with cell motility.

Biochem. Biophys. Acta 2016, 1860,

19531960.

33


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(89) Suganuma,

M.;

Takahashi,

A.;

Watanabe,

Page 34 of 36

T.;

Iida,

K.;

Matsuzaki,

T.;

Yoshikawa, H. Y.; Fujiki, H. Biophysical approach to mechanisms of cancer prevention and treatment with green tea catechins.

Molecules 2016, 21, 1566.

(90) Tsuchiya, H.; Nagayama, M.; Tanaka, T.; Furusawa, M.; Kashimata, M.; Takeuchi, H. Membrane-rigidifying eects of anti-cancer dietary factors.

Biofactors 2002, 16, 45

56.

(91) Tsuchiya, H. Eects of green tea catechins on membrane uidity.

Pharmacol. 1999,

59, 3444. (92) Holzschuh, S.; Kaeÿ, K.; Bossa, G. V.; Decker, C.; Fahr, A.; May, S. Investigations of the inuence of liposome composition on vesicle stability and drug transfer in human plasma: a transfer study.

J. Liposome Res. 2018, 28, 2234.

(93) Vaz, W. L. C.; Clegg, R. M.; Hallmann, D. Translational diusion of lipids in liquid crystalline phase phosphatidylcholine multibilayers. A comparison of experiment with theory.

Biochemistry-US 1985, 24, 781786.

(94) Kubista, M.; Sjöback, R.; Eriksson, S.; Albinsson, B. Experimental correction for the inner-lter eect in uorescence spectra.

Analyst 1994, 119, 417419.

(95) Wang, L. Measurements and implications of the membrane dipole potential.

Annu.

Rev. Biochem. 2012, 81, 615635. (96) Lakhdar-Ghazal, F.; Tichadou, J.-L.; Tocanne, J.-F. Eect of pH and monovalent cations on the ionization state of phosphatidylglycerol in monolayers.

Eur. J. Biochem.

1983, 134, 531537. (97) Oliveira Jr, O.; Riul Jr, A.; Ferreira, G. L. Surface potentials of mixed Langmuir lms: a model consistent with a domain-structured monolayer. 239242.

34


Thin Solid Films 1994, 242,

Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(98) Brockman, H. Dipole potential of lipid membranes.

Chem. Phys. Lipids

1994,

73,

5779.

(99) Peterson, U.; Mannock, D. A.; Lewis, R. N.; Pohl, P.; McElhaney, R. N.; Pohl, E. E. Origin of membrane dipole potential: chains.

Contribution of the phospholipid fatty acid

Chem. Phys. Lipids 2002, 117, 1927.

(100) Terakosolphan, W.;

Trick, J. L.;

Royall, P. G.;

Rogers, S. E.;

Lamberti, O.;

Lorenz, C. D.; Forbes, B.; Harvey, R. D. Glycerol solvates DPPC headgroups and localises in the interfacial regions of model pulmonary interfaces altering bilayer structure.

Langmuir 2018,

(101) Dicko, A.; Bourque, H.; Pézolet, M. Study by infrared spectroscopy of the conformation of dipalmitoylphosphatidylglycerol monolayers at the airwater interface and transferred on solid substrates.

Chem. Phys. Lipids 1998, 96, 125139.

(102) Levinger, N. E.; Costard, R.; Nibbering, E. T.; Elsaesser, T. Ultrafast energy migration pathways in self-assembled phospholipids interacting with conned water.

J. Phys.

Chem. A 2011, 115, 1195211959. (103) Adler-Moore, J.; Prott, R. T. AmBisome: Lipsomal formulation, structure, mechanism of action and pre-clinical experience.

35

J. Antimicrob. Chemother. 2002, 49, 2130.


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

Graphical TOC Entry

36


Page 36 of 36

Evaluation of EGCG loading capacity in DMPC membranes

Recommend Documents