PERMEABILITY OF GEL PHASE PHOSPHOLIPID

BILAYERS

by

Yamini Sravanthi Ammoor

14th May,2015

A thesis submitted to the

Faculty of the Graduate School of
the University at Buffalo, State University of New York
in partial fulfillment of the requirements of the

degree of

MASTER OF SCIENCE

Department of Chemical & Biological Engineering

 UMI Number: 1594687

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Table of Contents
Abstract ......................................................................................................................................................... v
Chapter 1: Introduction ................................................................................................................................. 1
1.1 Phospholipids Bilayers ........................................................................................................................ 1
1.2 Cholesterol .......................................................................................................................................... 3
1.3 Phase Behavior.................................................................................................................................... 4
1.4 Reason to study the gel phase ............................................................................................................. 5
1.5 Lipid Surface Density ......................................................................................................................... 6
1.6 Permeability ........................................................................................................................................ 7
1.7 Overview of the Thesis ....................................................................................................................... 8
Chapter 2: Bilayer Structural Parameters...................................................................................................... 9
2.1 Thickness of the phospholipid bilayer .............................................................................................. 10
2.2 Volume of the phospholipid bilayers ................................................................................................ 13
2.3 Area per phospholipid and lipid surface density ............................................................................... 14
2.4 Chain tilt............................................................................................................................................ 17
2.5 Effect of cholesterol .......................................................................................................................... 18
2.5.1 Condensation effect.................................................................................................................... 22
2.6 Summary ........................................................................................................................................... 24
Chapter 3: Phase Behavior of the Bilayer ................................................................................................... 25
3.1 Effect of cholesterol .......................................................................................................................... 27
3.2 Ripple phase ...................................................................................................................................... 29
3.3 Comparison of published results ....................................................................................................... 30
3.4 Summary ........................................................................................................................................... 33
Chapter 4: Analysis of Permeability Data for Various Solutes................................................................... 34
4.1 Ordering Effects of Cholesterol ........................................................................................................ 35
4.2 Barrier- domain Solubility Diffusion Model..................................................................................... 36
4.3 Permeability correlation for pure phospholipid bilayers in the gel phase ......................................... 38

ii
 4.3.1 Calculation of partition coefficient Kdec ..................................................................................... 39
4.3.2 Plotting of the data ..................................................................................................................... 40
4.3.3 Correlations for Permeability coefficient ................................................................................. 44
4.3.4 Comparison of Fluid Phase Permeability correlations in gel-phase .......................................... 45
4.4 Summary ........................................................................................................................................... 46
Chapter 5: Intercalation of Cholesterol ....................................................................................................... 47
5.1 Estimation of Permeability relation in 2-phase coexistence region .................................................. 47
5.2 Prediction for fluid phase permeability coefficient at Gel phase temperatures ................................ 53
5.3 Prediction of Liquid-ordered phase permeability coefficient ........................................................... 56
5.4 Summary ........................................................................................................................................... 56
6. Conclusions ............................................................................................................................................. 57
References:.................................................................................................................................................. 70
Nomenclature .............................................................................................................................................. 83
Appendix ..................................................................................................................................................... 84
Estimation of the permeability coefficient .............................................................................................. 84
Choice of DPPC ...................................................................................................................................... 84

iii
 ACKNOWLEDGEMENT

I would like to use this opportunity to its maximum potential, to extend my gratitude to the

individuals who helped me in overcoming the challenges of academic life and in my personal life

shaped my life and its complex decisions.

First and foremost, I would like to thank Dr. Johannes M Nitsche for being my mentor, advisor

and giving me this opportunity to learn from his vast knowledge and experience. He always

allowed me to fly in colors like a free bird. His vast knowledge, experience, kindness and patience

with my short comings, is very much appreciated. His guidance has helped my thesis rise out of

the limbo and reach the pinnacle of great workmanship that it has reached today. I would like to

thank Dr. Sriram Neelamegham and Dr. Stelios Andreadis for their service as my committee

members.

I would also like to thank Dr. J. Errington, Dr. Carl R Lund, Dr. David Kofke and Dr. M. Tsianou.

Our interactions have been limited to academic and courses related but have contributed to my

growth as an individual and as a chemical engineer. I would also like to thank members of Dr.

Kofke’s research group to share their lab and their assistance directly or indirectly, helped me

during my work on my thesis.

I am happy to have friends who have supported me during this endeavor. I would like to thank my

friends of 118 Englewood Ave for their support during difficult times. My family has always been

a great source of motivation and support. I would like to thank my parents, grandparents, my

brother and sister-in-law for their continual love. I would also like to thank all my friends and

family all around the globe whose love, friendship and support have been a very important factor

in making what Yamini Sravanthi is today.

iv
Abstract

The membranes of viable cells are boundaries that control the transit of material into and out of

the cell. Pivotal components of these membranes are bilayers of phospholipids with intercalated

cholesterol, embedded membrane proteins and other constituents. The quantitative study of the

permeability of phospholipid bilayers is key to understanding the permeation of various molecules

through cell walls. Permeability depends on structural properties of these bilayers.

This thesis presents a detailed study of the permeability coefficients of phospholipid bilayers in

their gel phase with the aim of informing modelling effects in such areas as drug and chemical

absorption through the stratum corneum (SC) layer of skin, and permeation of cryoprotectant

molecules into erythrocytes in the freezing of blood. In both the cases, the operative temperature

is below the gel-fluid transition temperature. The study addresses: (a) permeability coefficients of

various solutes through the phospholipid bilayers; (b) the key structural properties that mostly

strongly influence the permeability coefficients (i.e. lipid biophysics); and (c) the phase behavior

of the pure phospholipids and phospholipids with cholesterol. The main motive for studying phase

behavior is the need to know the conditions under which the bilayer is in the gel phase. The

character of this investigation is a thorough compilation and assessment of data reported in the

lipid biophysics and permeability literatures. It covers phosphatidylcholines broadly representative

of viable cell membranes.

■ Permeability coefficient data for various phospholipids at different temperatures and cholesterol

mole fractions are collected from the literature. The data show great variations among various

solutes and phospholipids and are difficult to interpret as is. ■ Aspects of lipid biophysics are

v
described in detail, particularly the area per phospholipids A, and the normalized surface density

σ. These parameters quantify the packing density of the fatty acid chains in the bilayer (i.e. the

hydrophobic part), which determines the free volume available in the trans-bilayer diffusion

process. The dependence of A and σ on temperature and cholesterol content are also discussed. ■

Changes in phase with changing temperature and cholesterol content are quantified in terms of

phase diagrams for phospholipids. ■ Theoretical relationships are developed that make variations

in permeability explicable when expressed in terms of a chain ordering factor following ideas of

Xiang & Anderson. (J. Membrane Biol. 148, 157-167 (1995))

Key words: Permeability, gel-phase, phospholipid bilayers, cholesterol, phase behavior, lipid

biophysics

vi
Chapter 1: Introduction

Pivotal components of the membranes of viable cells are bilayers of phospholipids with

intercalated cholesterol, embedded membrane proteins and other constituents. Cell membranes

have layers additional to the bilayer, such as the glycocalyx, comprising glycoproteins and

polysaccharides and conferring mechanical stability [1, 2]. Nevertheless, the bilayer forms the

primary barrier that controls the diffusion of various molecules into and out of the cell. Thus,

understanding of the permeability of phospholipid-cholesterol bilayers, the key constituent and

“background material” for other membrane species, is central to the quantification of cell

membrane permeability [3]. The latter property is of great interest, for example in assessing drug

and chemical bioavailability in drug development and chemical risk assessment [4-8].

1.1 Phospholipids Bilayers

Bilayers comprise two layers of lipid molecules, which have a hydrophilic head group and two

hydrophobic tails attached to the glycerol backbone by ester linkages as shown in Fig. 1.1.

Phospholipid molecules have a phosphate group as a part of the head group attached to the glycerol

backbone and thence the phospholipids. Of all the phospholipids, phosphatidylcholines (PC), with

a choline moiety attached to the phosphate group, represent the most abundant species in natural

lipid bilayers. Thus, PC molecules, upon which this thesis concentrates, are composed of a choline

molecule and a phosphate group that together comprise the head group, glycerol as the backbone,

and two saturated or unsaturated fatty acids as tails attached to the backbone.

1
 The fatty chains usually contain an even number of carbon atoms typically between 14 and

18 [9, 10]. For example, the major fatty acids in human epidermal cells are palmitic acid (28% by

weight), steric acid (16% by weight) and oleic acid (37% by weight) [11]. In this thesis, we

primarily discuss the following PCs: DLPC [Dilauroylphosphatidylcholine, di(12:0)PC], DMPC

[Dimyristoylphosphatidycholine, di(14:0)PC], DPPC [Dipalmitoylphosphatidylcholine,

di(16:0)PC], DSPC [Distearoylphosphatiducholine, di(18:0)PC], DOPC

[Dioleoylphosphatidylcholine, di(18:1)PC], and POPC [Palmitoyloleoylphosphatidylcholine,

16:0-18:1PC]. The notation used indicates the number of carbon atoms in the fatty acid chains

before the colon, and the number of double bonds after the colon. Thus, DOPC has two

monosaturated chains, with the double bond between the 9th and 10th carbon atoms, and POPC is

a mixed chain species with one 16-carbon saturated chain as in DPPC and one 16-carbon

monounsaturated chain as in DOPC. The presence of double bonds in the fatty acid chains hinders

the free rotation about the C-C bonds and causes a kink in the chain. This kink prevents the

molecules from packing closely together. Double bonds in unsaturated fatty acid chains cause

many changes in structural parameters of the bilayer as compared to saturated ones.

Fig 1.1: Phospholipid Bilayer Structure

(a) Cartoon representing the 3-D structure of a phospholipid bilayer

(Reproduced from http://www.biologycorner.com/APbiology/cellular/notes_cell_membrane.html)

2
(b) Individual phospholipid molecule (PC shown is DMPC [Dimyristoylphosphatidycholine, di(14:0)PC])

1.2 Cholesterol
Bilayers of cell membranes generally have various sterols intercalated between the fatty acid

chains. Of all the sterols, cholesterol is the major sterol present in the plasma membrane of

eukaryotic cells, and is present at concentration as high as 40 mol % [12]. As noted in a published

report [3] : “The cholesterol content of cell plasma membranes is typically assessed at 30–40 mole

% [13, 14] and sometimes as high as 50% [15, 16] . We adopt 40 mole % as a reasonable nominal

value.”

Cholesterol molecules consist of a fused four-ring structure with a polar hydroxyl group

and isooctyl side chain; the structure is included in Fig. 1.2 [12]. Cholesterol is a key membrane

constituent that provides the cell with a means to modify essential membrane properties. A detailed

understanding of the effect of cholesterol on the structure and organization of lipid bilayers is

essential for a thorough comprehension of membrane permeabilities.

3
Fig 1.2: Cholesterol in the bilayer.

(a) Cartoon showing molecules intercalated between fatty acid chains in a bilayer.

(b) Structure of a cholesterol molecule

H3C

CH3
CH3
CH3
H3C

HO

1.3 Phase Behavior

Bilayers of pure PCs can exist in several states analogous to the phases of bulk materials [3, 17].

The transition of primary interest to subsequent developments is the “melting transition” from a

low-temperature “solid” gel phase to a higher-temperature “fluid” liquid crystal phase. The gel-

fluid transition temperatures of the PCs named above are listed in Table 1. The phase behavior is

largely established by van der Waals attraction and short range repulsive forces. The effects of

these forces are in turn determined by the length of the alkyl chain present, partly because bilayers

with longer chains have more area for the chains to interact. This explains the fact that the longer

alkyl chain lipid bilayers are in the gel phase (i.e. solid) at room temperature. Unsaturated alkyl

4
bonds also affect the phase behavior due to distortion of the structure i.e., disruption of packing of

the hydrophobic part of the lipid molecule. Unsaturation causes a significant decrease in the gel-

fluid transition temperature.

1.4 Reason to study the gel phase

This report specifically addresses bilayers in the gel phase, with the aim of informing the

modelling of: (i) drug and chemical absorption through the stratum corneum (SC) layer of skin;

and (ii) permeation of cryoprotectant molecules into erythrocytes in the freezing of blood. In both

cases the lipid constituents of the biological membrane are in the gel phase because the operative

temperatures are below the gel-fluid transition.

1.4.1 Stratum Corneum

The stratum corneum (SC) is the primary barrier layer (~15 µm thick) of the skin lying at its

surface. It comprises 10-20 layers of flattened partially dessicated and keratined cells separated by

~0.1 µm thick layers of lipid [2]. The lipid comprises ceramides, cholesterol and free fatty acids

[18, 19]. Although the contributions of various molecular pathways through the SC are subject to

debate, SC lipids are generally regarded as furnishing the barrier properties of the SC. Norlen

proposed novel models for the barrier and formation of the SC as the “single gel-phase model”

and “the membrane-folding model” in 2001 [20]. These models suggest that the skin lipid barrier

exists as a single and coherent lamellar gel phase. This membrane structure is stabilized by a

particular lipid composition and lipid chain length distribution of the stratum corneum intercellular

space, and has no phase boundaries. The main factors stabilizing gel phases [18, 19, 21-23][18,

19, 21-23][18, 19, 21-23][18, 19, 21-23][18, 19, 21-23] are: (i) extensive compositional

heterogeneity; (ii) almost complete dominance of saturated, very long hydrocarbon chains; and

5
(iii) a large amount of cholesterol. The fact of gel-phase behavior of SC lipid motivates the study

of lipid permeability in the gel phase. The phospholipids fluidize the SC lipid bilayers and facilitate

the transdermal drug diffusion [24]. Permeability correlations for gel-phase phospholipid bilayers

will inform the prediction of SC permeability by providing a key parameter needed for microscopic

diffusion model.

1.4.2 Low Temperature phospholipids

As for other viable cells, the plasma membrane bounding red blood cells (RBC’s, erythrocytes)

comprise phospholipids and cholesterol, which provide a permeability barrier between the external

environment and the red cell cytoplasm [25]. The plasma membranes of mammalian red blood

cells (erythrocytes) have been useful as a model for studies of membrane structure. Phospholipids

comprise about 50-60% of the mass of the RBC cell membrane, and phosphatidylcholines (PC)

about 28-30% of the total phospholipids presents [26-29]. The permeation of cryoprotectant

molecules into erythrocytes is studied down to ultra-low temperatures (below about -100ºC) [30-

32]. At such low temperatures the phospholipids form a glassy matrix and definitely exist in the

gel phase.

1.4.3 Conclusion

In the case of the SC, gel phase behavior results from a long lipid chain length at physiological

temperatures (~32 - 37 0C). In the case of cryobiology applications, gel phase behavior study

results from low temperatures. In either case, the operational state is the gel phase.

1.5 Lipid Surface Density

Xiang and Anderson have shown that the area per phospholipid (often quantified in terms of a

normalized surface density discussed below) is the structural parameter most directly affecting

6
bilayer permeability via the so-called chain-ordering effect [33-36]. The presence of cholesterol

promotes the ordering of fatty acid chains and reduces the area per phospholipid. It is therefore

crucial to develop a convenient quantitative representation of the manner in which this parameter

depends upon physical variables like temperature and cholesterol content of the bilayer.

1.6 Permeability

The permeability of a given solute diffusing through the bilayer is quantified by the permeability

coefficient of the solute. As a basis for quantifying the permeability coefficient a suitable

solvent is required to mimic the chemical environment presented by the bilayer. We consider two

different solvents in this report, namely octanol and 1,9-decadiene. The basic theatrical framework

describing passive membrane permeability is the bulk solubility-diffusion model [37], which

regards the bilayer as a homogeneous slab of organic material. The permeability coefficient,

for a given solute combines

(i) The lipid/water partition coefficient ( )

(ii) the diffusion constant ( ) and

(iii) the lipid thickness ( )

The solubility diffusion model is not literally valid because the lipid molecules exist in an ordered

state instead of as molecules as bulk liquid. Due to the chain ordering effect observed in the bilayer,

the partition coefficients and the diffusion coefficients differ from the bulk solvent values. Also, it

is sufficient to focus attention on the region of the bilayer that is the most tightly packed and

ordered and therefore, the greater mass transfer resistance. In this report, we aim in establishing

7
numerical formulae to predict the permeability coefficient of gel phase phospholipid bilayers at

various temperature and cholesterol concentrations.

1.7 Overview of the Thesis

The contents of the thesis are as follows:

Chapter 2 discusses the key structures required to quantify the permeability of the phospholipid

bilayers. In particular, develops convenient formulas giving structural parameters like normalized

surface density. In Chapter 3, the phase behavior of the lipid molecules is discussed in detail. It

covers the phase transitions of the lipid molecules due to the presence of cholesterol. Chapter 4

presents a regression analysis of the data collected describing the dependence of Plip|w on the

normalized surface density of the phospholipid bilayer and the molecular weight and lipophilicity

of the solute. All structural data, phase behavior and the permeability data for various solutes and

PC molecules are gathered from various literature sources. Lastly, Chapter 5 presents a further

analysis for quantifying the permeability coefficient for a solute through a bilayer comprising a

binary mixture of phospholipid bilayer and cholesterol.

8
Chapter 2: Bilayer Structural Parameters

Phospholipid molecules arrange themselves as a bilayer representing the overall minimum energy

configuration for the mixture of themselves and the surrounding water molecules. The spatial

extent, thickness and density of this lamellar structure, comprising a hydrocarbon core sandwiched

between two head group layers in contact with the surrounding water, depends on temperature. At

temperatures well above , the fatty acid chains are in a state of considerable motion that partly

overcomes the strong van der Waals attractions between them. Thus, they spread out laterally and

occupy relatively large area per phospholipid, , and the head groups are relatively far apart. As

the temperature is reduced, the hydrocarbon layer contracts laterally as the fatty acid chains

become more tightly packed, and the head groups get closer to each other. When the temperature

is further decreased, contact between the head groups tends to enforce a minimum area per

phospholipid determined by their lateral packing. To accommodate further thermal contraction,

the fatty acid chains begin to tilt so that they can pack closer together. The chain tilt effect is [38]

discussed in the later part of the present chapter. This progression of changes in the lipid molecules

with temperature is described in Figure 2.1. Because part of the hydrocarbon layer usually

represents the primary barrier layer, we focus on the density of this layer as the primary

determinant of the permeability coefficient.

9
Fig 2.1: Cartoon depicting the behavior of lipid molecules at various temperatures with respect to the melting
temperature.

2.1 Thickness of the phospholipid bilayer

Primary structural parameters of the phospholipid bilayer are various measures of its thickness,

such as head-to-head thickness ( ),hydrocarbon thickness ( ),overall Luzzati thicknes( )

[17, 39]. Such thicknesses are determined experimentally using small and wide angle scattering

X-ray (SAXS & WAXS) and neutron scattering. The structural parameters of phospholipid

bilayers comprising various saturated and unsaturated fatty acids, at several temperatures have

been analyzed using these scattering techniques by Nagle, Kucerka and various co-workers [17,

40-44].

The overall approach is to match the measured diffraction patterns with those predicted

from detailed models of the bilayer that account for differing electron densities and other attributes

of the hydrocarbon and head group regions. Thus, for instance, the two head groups might be

represented as symmetrically positioned Gaussian peaks of electron density, with the center point

and width as adjustable parameters (see Figures 2.2 and 2.3). Matching of the measured diffraction

pattern establishes best-fit values of these structural parameters, which translate directly into head

group to head group thickness . The hydrocarbon and head group regions are usually broken

10
down into sub-components for greater details and accuracy. For example, one model distinguishes

methylene and terminal methyl components for the hydrocarbon region and glycerol backbone,

phosphate and choline components for the head group region [42].

X-ray and neutron scattering techniques provide complimentary data about the structure of

the bilayer as they are differentially sensitive to different parts of the bilayer. The electron dense

phosphate groups and the lower density hydrocarbon region are contrasted easily in X-ray

diffraction whereas, neutron scattering accurately measure the total thickness of the bilayer [45].

The X-ray interaction with the water molecules is very weak and hence the bulk samples can be

easily analyzed with easy preparatory methods. A limitation of X-ray scattering is its limited

interaction with small molecules. In contrast, neutron scattering is not sensitive to the size of the

molecules and provides isotopic contrast by preparing samples with mixtures of H2O and 2H2O

(water and deuterated water)[46].

SAXS is a scattering technique which measures the inhomogeneities in the sample by

measuring the scattering intensities at low angles close to 00 and WAXS refers to the analysis of

Bragg’s peaks scattered to wide angles, larger than 50. SAXS is measured by continuously rotating

the sample uniformly to an exposure of X-ray and obtaining the intensities of the reflections with

the same normalization which is perpendicular to the plane of the bilayer. These electron density

profiles can be analyzed to extract the values of and [39, 40]. Neutron scattering

accurately determines the bilayer thickness. Being electrically neutral, neutrons penetrate more

deeply into the bilayers. The repeat distance calculated using them yields the thickness of the

bilayer.

11
 Molecular dynamic and Monte Carlo simulations are heavily involved in the data analysis

that elucidates the structure of bilayers. These simulations, are based on input parameters like

head-to-head thickness or area per phospholipid, and constrained to match measured properties

such as specific volume of the bilayer [47, 48] Fitting the calculated diffraction patterns to the

measured ones produce the best-fit values of the structural parameters.

Fig 2.2 A snapshot of the electron density profile of various components in a PC bilayer from a X-Ray Scattering

Experiment [17, 45]. The figure shows the electron density profile of the dense head groups as two symmetric peaks

in blue and the electron density profile for the hydrocarbon region of the phospholipid bilayers as the plateau region

in black is the electron density profile for the hydrocarbon region of the phospholipid bilayers. The distance between

the peaks is used in calculating the value of .

12
Fig 2.3 Snapshot of the total electron density of various components in a pure PC bilayer from X-Ray scattering[17].

This snapshot details about the different components of the bilayer where the peak is the phosphate group and the

trough is the terminal methyl groups present in fatty acid acyl chains [17, 39-41, 44, 49, 50].

2.2 Volume of the phospholipid bilayers

The volume per lipid is the most accurately determinable physical property of phospholipid

bilayers[47, 48]. As the product of the bilayer area per phospholipid and the thickness of the

bilayer, it effectively connects these two parameters. Values of the volume per lipid are obtained

by neutral buoyancy centrifugation [45]. Neutral buoyancy centrifugation is based on the principle

that the lipid dispersed in mixtures of H2O and 2H2O (varying the density) can neither float nor

sink in the solvent having the same density or specific volume. Thus, from the concentration of

water and deuterated water mixtures the specific volumes of the lipid are calculated [51]. Various

researchers like Nagle and Wilkinson, Kucerka, Greenwood have developed useful empirical

formulas predicting the volumes of the lipids as a function of temperature and also cholesterol

content [17, 48, 52, 53]. As the cholesterol is intercalated in to the bilayers, the concept of partial

13
molar volumes enters the picture which is discussed in the later part of the chapter [48]. These

parameters have provided insight into how temperature and hydrocarbon chain length affect

phospholipid bilayer properties. An important application is that the study of molecular volume

with temperatures provides the phase behavior of the lipid bilayers and the head group interactions

[52] .

2.3 Area per phospholipid and lipid surface density

For bilayers, the area per molecule is the quantity of central interest and provides an overall

measure of lateral organization and determines how much free space is available to a given solute

for diffusion. The lipid area assists in understanding the intermolecular interactions taking place

within the phospholipid bilayers and in between the intercalated proteins, sterols and the lipids

[17, 39, 49, 53]. The area per phospholipid is calculated from bilayer thickness and volume data

according to the equation [40, 41].

2.1

where is the volume of the lipid and is the thickness of the lipid bilayer. Thus, area

and thickness are not independent parameters, but must satisfy the constraint imposed by this

equation in the models used. This equation can also be cast in a form that refers only to the

hydrocarbon (hydrophobic) layer of the bilayer as

2.2

The area per phospholipid is often quantified in terms of a so-called normalized surface

density given by the symbol, σ [34, 54]. Various studies have shown that the minimum area of

14
lipid molecule per crystal, is always constant [3, 55] with value of 40.8 Å2. The normalized

(relative) surface density, σ , is defined in terms of A0 as

2.3

where is the area per phospholipid.

Table 2 lists the selected values of σ of pure lipids at different temperatures reported in

various publications by Nagle, Xiang & Anderson and other various authors [34, 56, 57]. Some of

the values in this table are calculated using equation 2.3 and the remaining values are obtained

from the normalized density data provided by Xiang & Anderson [34]. The studies have shown

that there is steric interaction between the head groups in the gel phase and A mostly remains

constant. The areal thermal expansion is found to be in the range of 2 × 10-4 to 6 × 10-4 (ºC)-1 which

is almost 10 times less than in the fluid phase[17]. Using data collected from papers by Nagle et

al. [17, 38, 47, 49] and Xiang and Anderson[33, 34, 36] , we tried correlating the area of pure

phospholipid with the temperature to find out the apparent coefficient of thermal area expansion

coefficient. Figure 2.4 shows a semi-log plot of the area per phospholipid as a function of

temperature.

15
 3.91
y = 0.007x + 3.6221
3.9
R² = 0.9841
3.89

3.88

3.87
ln A

3.86

3.85

3.84

3.83

3.82
0 10 20 30 40 50
T

Fig 2.4 Plot of lnA as a function of T (in 0C) [34, 49, 50]

The straight line fit with reasonably good correlation coefficient of R2=0.98 indicates that

the area of the phospholipid varies exponentially with the temperature. However, the apparent

coefficient of thermal area expansion is suspect, considerably exceeding the range stated above.

The reason may have to do with the fact that the data shown cover the ripple-to-gel phase transition

— a transition between gel sub-phases below the main transition temperature [56] temperature

plot with ripple and gel written on it. Hence, we rather consider a reference datum for the area per

phospholipid bilayer, Å2 at 25oC and use the value α (the expansion coefficient) 2 × 10-
4 o
( C)-1 to correct this value for temperature changes. Based on the above, we calculate the area per

phospholipid (and from it the normalized surface density) for DPPC bilayers at any temperature

according to the equation

2.4

where = A at temperature = 250C = 47.3Å2 [38].

16
2.4 Chain tilt

At lower temperatures, the head groups of the contracting bilayer start to touch and cannot get

closer together. The fatty acid chains tilt due to their tendency to stick together and collectively

contract further with decreasing temperature, subject to the constraint of having an almost constant

number of chains per area dictated by the touching of headgroups. As the temperature increases,

the chain packing increases and allows reduction in the chain tilting, keeping the head group

spacing almost constant [50]. This physical phenomenon is described in Fig 2.5. The chain area,

Ac, increases at a faster pace with the temperature than A; hence, θc decreases with T. For longer

chain lengths, θc is large due to attractive van der Waals interactions and the value of Ac decreases

[17]. The chain tilt angle along with the direction of tilt for the fatty acid chains of the PC bilayers

under various temperatures and hydration regimes have been measured by the X-ray diffraction

and other physical techniques [38, 58, 59]. The carbonyl-to-carbonyl peak in the electron density

map provides the chain tilt in PC bilayers [58].

Fig 2.5: Cartoon showing the tilting of the alkyl lipid chains of the bilayer intercalated with cholesterol.

This phenomena seen in the gel phase also assists in determining the area per phospholipid

molecule using the relation [38]

17
 2.5

where is the chain area of the phospholipid measured perpendicular to the chain and is the

chain tilt angle measured.

2.5 Effect of cholesterol

Elucidating the effect of cholesterol on phospholipid bilayers is required towards rationalizing

their structural and functional role in the biological cell membranes. Cholesterol affects bilayer

structure in two ways. The rigid structure of cholesterol increases the chain order of lipid

molecules, thus increasing the bilayer thickness. Also, it is expected that the cholesterol would

decrease the thickness of lipid chain bilayers whose hydrophobic thickness is greater than the

hydrophobic length of cholesterol molecule. But it is observed otherwise. In mixtures of saturated

and unsaturated lipids, cholesterol preferentially partitions into the saturated part and forms a

liquid ordered phase. Cholesterol’s rigid sterol ring is accommodated by the mobile lipid chains

(high entropy) thereby reducing lipid chain entropy [60] . With the concomitant increase in

cholesterol content in the bilayers, we observe progressive increase in the thickness of the bilayers.

This is due to the concept called hydrophobic matching which explains the structural flexibility of

lipid hydrocarbon chains adjusting its thickness to minimize the thermodynamic interactions

between the water and hydrophobic surface of the lipid chain molecule [60]. The addition of

cholesterol up to a particular concentration (~ 5-10%) causes reduction in the lipid transition

temperature, reduction in the heat of transition, and an increase in the bound water.

The effect of cholesterol on the structure of PC bilayers in both the gel phase and the liquid

crystalline phase has been studied using the X-ray diffraction techniques. To study the electron

distribution across the bilayers it is necessary to find the phase of each reflection. To find the phase

18
information, one needs to determine the Fourier transform of the structure factor of PC/Cholesterol

bilayers by swelling experiments. The difference in the wide angle scattering patterns between gel

and liquid crystalline phase are marked by the decrease in the total thickness of the bilayer, and

increase in the terminal methyl trough. The addition of the cholesterol has several effects on the

electron density profile. The width of the bilayer is increased, as cholesterol reduces the

conformations of the phospholipid chains and increases the trans conformations. Cholesterol also

increases the density in the hydrocarbon region. Further, the width of the bilayer (i.e. head group-

to-head group separation) of the phospholipids at full hydration decreases due to the chain tilt

introduced by the addition of more water. And the addition of water to PC bilayers in both gel and

liquid crystalline phase has the effect of changing the density profile of head group from two

distinct bumps to a single broad peak.

The electron density of cholesterol shows that the steroid rings of cholesterol have greater

electron density than the alkyl chains and extend the head group region density profile towards the

center of the bilayer. Wide angle patterns indicate that the lipid alkyl chains are perpendicular to

the plane of the bilayer and also that the long axis of the cholesterol aligns itself parallel to the

fatty acid alkyl chains. Thus, the electron density profiles localize cholesterol in the PC bilayers

[53, 61, 62].

The effect of cholesterol on bilayer width depends on the nature of the phospholipid.

Cholesterol forms a tight complex with DMPC, which greatly reduces bilayer compressibility and

permeability compared to liquid-crystalline-state bilayers, but maintains a liquid-like (fluid) state,

in that no surface shear rigidity is observed, even at temperatures well below the acyl chain

crystallization temperature of the phospholipid The outer regions of the acyl chains therefore

appear to be condensed, thereby tightening up the surface, but the central bilayer region seems to

19
remain fluid [63]. The electron density of the methylene chain region is increased by the

incorporation of cholesterol, consistent with the localization of cholesterol in this region of the

bilayer. In DPPC, cholesterol extends to a few Angstrom units to the center of the bilayer and

some kinking in the DPPC fatty acid chains to accommodate the cholesterol as shown in Fig 2.6.

In the longer chained DSPC, the fatty acid chains extend beyond the cholesterol rings to form a

region of gel phase lipid in the center of the bilayer as shown in Fig 2.7. The cholesterol removes

the chain tilt in the pure hydrated DPPC, hence the DPPC/Cholesterol bilayers are much wider.

On the other hand, the width of DSPC/Chol bilayers is reduced because the longer acid chains in

DSPC become more kinked to accommodate the cholesterol molecules [61].

Fig 2.6 Snapshot of electron density distribution of pure DPPC and DPPC/Chol mixture [61].

20
Fig 2.7 Snapshot of electron density distribution of pure DSPC and DSPC/Chol mixture [61].

The presence of cholesterol does not affect the head group region in PC/Chol mixtures as

the width of the head group regions is same as the electron density profile of the pure PC bilayers.

The head group is oriented parallel to the plane of the bilayer in gel and liquid crystalline phase,

and this orientation is not changed due to the addition of cholesterol.

Due to the addition of cholesterol, the chain ordering dominates over the hydrophobic

matching between the length of the cholesterol molecule and the hydrocarbon thickness of the

bilayer. The effect of cholesterol on lipid bilayer properties also depends on the number of

unsaturated chain in the lipids. When both the chains are saturated, the bending modulus has

increased drastically due to the addition of cholesterol, while when both the chains are unsaturated,

the bending modulus was found to be same upto a certain amount of cholesterol concentration

(upto Xc = 0.4) [64]. WAXS from oriented lipid multilayers is the most widely used technique to

21
study the effect of adding cholesterol to PC bilayers [62]. As the concentration of the cholesterol

increases, there is an abrupt increase in width of WAXS peak and it spreads out over a wide range

of temperature. The WAXS studies have also shown that the cholesterol partitions more favorably

into the vesicles of saturated lipids than the vesicles of unsaturated lipids. The LAXS results are

used to obtain the thickness, bending modulus and structure of the phospholipid bilayers whereas

the WAXS results are used to obtain the order parameter of the mixtures of cholesterol with the

PC molecules [40, 43, 53, 62].

We have discussed the effect of cholesterol on the structure of the phospholipid bilayer so

far. The effect on permeability of the PC bilayers due to the presence of cholesterol is analyzed

with the relative normalized surface density. Cholesterol increases the ordering of fatty acids and

simultaneously decrease the average area occupied by each phospholipid molecule [3]. The

dependence of A upon cholesterol mole fraction Xchol can be quantified in terms of density ratio

[3] as

2.6

2.5.1 Condensation effect

When intercalated into bilayers, cholesterol molecules produce significant changes in structure and

properties [65]. It was observed first in monolayer experiments at the water interface that the area

per phospholipid decreases in presence of cholesterol, and similarly in the bilayers too, in the fluid

phase. Levine and Wilkins [66] have investigated this effect and found that cholesterol molecules

tend to make the hydrocarbon chains to orient perpendicular to the plane of the bilayer due to

increase in the orientational order parameter and also increase the head to head distance. This

22
thickening has been studied by number of researchers [67-70] who have concluded that this is

more than the molecular interactions between the cholesterol and the phospholipid. The phase

diagrams of PC-cholsterol mixtures (explained in Chapter3) also point towards the concept of the

complex formation (not a chemical reaction) between the cholesterol and the PC molecule fatty

acid chains. It is also explained by the fact that the lipid chains and cholesterol are present in the

same area as the pure phospholipid bilayers. Thus, cholesterol forces the bilayers to condense and

tightens the packing of the lipid bilayers [71]. This phenomenon is known as the condensation

effect of the cholesterol. This lateral condensation effect of induced by cholesterol can explain the

fact of reduced permeability of small solutes and water across the bilayer. The reduced fluid-phase

permeability of small solutes and water across the bilayer is due to the decreasing space between

fatty acid chains induced by cholesterol In short, the condensation effect is manifested as a

decrease in the surface area occupied by the mixed bilayers of PC molecules and cholesterol. The

condensation effect can be quantified in terms of the free area or free volume of lipid bilayers.

Studies have shown that the area and volume occupied by the cholesterol molecule varies as

function of distance from the bilayer center [17]. A strong decrease in the free volume and voids

is observed in the lipid bilayers in the vicinity of the cholesterol rings and they tend to orient

parallel to the membrane. The change in membrane thickness is also a consequence of the

condensation effect [72]. It has also been observed that the reduction [isn’t it increase?] in

thickness is [isn’t it less] pronounced in when the chain length is longer than 18 carbons due to the

fact that cholesterol is shorter than the fatty acid chains. This effect is also observed in phase

behavior of mixture of different saturated and unsaturated lipid bilayers along with cholesterol.

In the gel phase cholesterol tends to fluidize bilayers, which can increase permeability.

23
2.6 Summary

In this chapter, we have discussed about the major structural factors which affect the permeability

of solutes across the lipid bilayers, including area per phospholipid (or, equivalently, normalized

surface density), and the effects of cholesterol intercalated between fatty acid chains. The major

consequence of cholesterol to the intercalation of cholesterol are areduction in the passive

permeability of small molecules in the fluid phase, and an increase in permeability in the gel phase.

In the next chapter, we detail about the phase behavior of phospholipids and the changes observed

due to the presence of cholesterol.

24
Chapter 3: Phase Behavior of the Bilayer
Since phospholipid bilayers serving as a barrier to exchange of materials with the external

environment are often in the gel phase, it is vital to understand the permeability of various solutes

through the phospholipid bilayers when present in this phase. This goal drives a supporting survey

of the phase behavior of the lipid bilayer in order to: (i) identify the conditions under which it

exists in the gel phase; and (ii) inform the analysis of permeability with information on other

ordered phases the bilayer might be in at low temperature. The chain melting temperature is of

fundamental importance since it represents a turning point in the amount of orientational disorder

in the lipid bilayers [73]. The permeability of solutes also depends not only on the structural

properties of lipid bilayers but also the physical properties and is discussed below.

In the absence of cholesterol, phospholipid bilayers can exist in either of two physical

states, a rigid solid phase called the gel-phase at lower temperatures, or a fluctuating

liquid-crystalline phase known as the fluid phase higher temperatures [74]. The fluid

phase is also called the liquid disordered phase, to distinguish it clearly from the so-called liquid

ordered phase discussed below. In the state the phospholipid hydrocarbon chains are fully

extended, the cross-section area of the lipid molecules is a minimum, and the thickness of the

phospholipid bilayer is a maximum, thus making the intermolecular and intramolecular motion

restricted. In contrast, in the state, the cross sectional area of the lipid molecules increases

substantially due to intermolecular and intramolecular motion [75]. The main gel fluid transition

temperatures, for a number of PCs are listed in Table 1. The phase behavior is largely

established by the intermolecular van der Waals attraction and short-range repulsive forces. These

25
forces are in turn determined by the length of the alkyl chain present. The lipid bilayers with longer

chains have more area to interact [76], thus reducing the mobility of the lipids. This explains the

fact that the longer alkyl chain lipid bilayers are in gel phase (i.e. solid) at room temperature. The

unsaturated alkyl bonds also affect the phase behavior due to the distortion in the structure of the

hydrophobic part of the lipid molecule. Unsaturation produces drastic decreases in the transition

temperature from gel phase to fluid phase [50, 76, 77]. Because most natural lipid bilayers are

mixtures of various PCs along with cholesterol, we observe properties which are in-between the

properties of individual components, such as the formation of cholesterol-lipid complex formation.

Thus, we observe phase coexistence and partitioning of lipid molecule into more than one phase

in the presence of cholesterol [78, 79].

Fig 3.1: Generic phase behavior diagram for phospholipid intercalated with cholesterol

26
3.1 Effect of cholesterol

Numerous researchers have worked on the construction of phase diagram of cholesterol and

phospholipids binary mixtures with the excess of water (i.e., at full hydration) at various

compositions and temperatures. The phase boundary diagram proposed by Vist & Davis using

DSC and 2H-NMR spectroscopy [74] (refer to Figs 3.1 (qualitative) and Fig. 3.2 (quantitative)

below) is the most cited phase diagram for the binary mixtures. Three different phases appear when

the mole fraction of cholesterol is varied from 0 to 0.5 mole fraction, depending on the temperature.

As the mole fraction of cholesterol is varied from 0 to 0.05 at temperatures above Tm, the

phospholipid transitions from the fluid phase to a liquid ordered (Lo) phase. At temperatures below

Tm, and at cholesterol concentrations between 0.05 and 0.23 mole fraction, the gel phase coexists

with the liquid ordered (Lo) phase.

A huge literature exists on the effect upon phase behavior of the addition of cholesterol for various

phospholipids [78, 80-83]. Cholesterol has a dual nature, i.e., in fluid state it promotes the packing,

ordering and the rigidity of the bilayer membranes, whereas in the gel phase it tends to fluidize the

bilayer [72]. Some of the salient effects which have been observed [74] are as follows: (i) extended

and gradual elimination of the “phase transition” from the liquid-crystalline phase to the gel phase;

(ii) eventual elimination of the pre-transition; and (iii) a significant decrease in the permeability of

the lipid bilayer. Thus, he addition of increasing amounts of cholesterol generally converts the

and phases to an intermediate phase of properties intermediate between the gel and fluid phase,

the liquid ordered (Lo) phase [74].

Cholesterol plays a significant role in the behavior of the lipid bilayer. The major effects

are to: (i) increase ordering of the fluid phase; and (ii) increase fluidity of the gel phase. (i) The

27
thickening of fluid-phase bilayers due to the presence of cholesterol leads to the reduction in the

area per molecule of the phospholipid. This reduction in area per lipid molecule is known as the

condensation effect [41, 50, 80, 84]. This effect also makes the acyl chain of the phospholipids

more ordered. With the increase in the cholesterol concentration, the free phospholipid component

forms small ordered crystalline domains while the loosely and tightly associated phospholipid

form a disordered phase [63]. It is observed that each cholesterol molecule interacts with one

phospholipid molecule strongly forming a rigid complex while the remaining uncomplexed

phospholipid molecules are either free or loosely associated. (ii) In the low temperature region,

the free phospholipid bilayers freezes to form an ordered gel phase. The gal phase transitions to

the liquid ordered phase with the addition of sufficient cholesterol [63].

Addition of cholesterol to the bilayer reduces the melting temperature, i.e. the transition

temperature from gel phase to fluid phase. The fluidity of the hydrocarbon chains is controlled by

the cholesterol presence by the disruption in the crystalline lattice of the gel phase and by inhibiting

the flexibility of the fatty acid acyl chains in the dispersed liquid crystalline phase [85]. Cholesterol

also reduces the heat of transition and the increases the bound water molecules in the phospholipid

bilayers [85].

The rigid planar structure of cholesterol stabilizes the bilayer membranes by ordering the

lipid acyl chains. Also, when the cholesterol partitions in the solid domains of the lipid bilayer, the

structure of the lipid bilayer is disordered. Due to this dual effect, a new phase is observed in

created in the bilayers. Thus, due to the presence of cholesterol, the following phases are observed:

(i) solid ordered phase, (ii) liquid ordered phase and (iii) liquid disordered phase [86].

28
 As a complement to the wealth of experimental data available, the detailed molecular

structure, phase behavior and intermolecular interactions are also elucidated using the Monte Carlo

(MC) and Molecular Dynamics (MD) simulations. Two bilayer systems are built in CHARMM

(Chemistry at Harvard Macromolecular Mechanics) at different concentrations, taken from the

published experimental results. Each bilayer is simulated in the constant particle number, pressure

and temperature (NPT) ensemble and equilibrated. The produced results are used to study inter

and intramolecular interactions between the phospholipid bilayers and cholesterol. The change in

membrane area with the change in ambient temperature at constant pressure is used to map out the

lipid phase transitions. Thus, simulations on a nanoscale provide a detailed picture of the phase

behavior in the binary and ternary mixtures of lipids in the bilayer membranes [45, 60, 75, 87, 88].

3.2 Ripple phase

Some lipids undergo a low-enthalpy phase transition below , which is known as the pre-

transition [74, 89-93]. This phase is called the ripple phase because it is associated with the

formation of ripples on the membrane surface. Though this phase has been observed

experimentally and in various computational methods, the basis for the origin and molecular

details of this phase is not fully understood till date [42]. The stability of ripple phase stretches

from the pre-transition state to the main transition state.

At low temperatures, below the pre-transition, the hydrocarbon chains are fully extended

and tilted with respect to the plane of the lipid bilayer. The hydrocarbon chain packing displays a

temperature dependence and the angle of tilt of the hydrocarbon chains decreases with increasing

temperature. The pre-transition is associated with a structural transformation from a one- to two-

dimensional monoclinic lattice consisting of lipid lamellae distorted by a periodic undulation or

29
ripple where the hydrocarbon chains remain tilted in the temperature range between the pre-

transition and chain melting transition [94].

The ripple phase occurs when the phospholipids are surrounded by water molecules. The

structures associated with this transition are dependent on the nature of the phospholipid bilayers

and are studied with the help of X-Ray diffraction. This anomalous swelling in the ripple phase is

due to the conformational changes in the hydrophobic fatty acid acyl chains. The period of ripple

decreases with the increase in the intercalation of water molecules. Various results from the DSC

(Differential Scanning Calorimeter) have shown that the interaction between the intercalated water

molecules and the choline group of the phospholipid is responsible for the structural transformation

and the formation of ripple phase [94]. It has been published that the ripple phase doesn’t occur in

the bilayers consisting of phospholipids with smaller head groups [63].

3.3 Comparison of published results

Various researchers have studied the phase behavior of the PC bilayers with cholesterol. In this

report, we establish the differences published by various researchers. As stated earlier, Vist and

Davis studied the phase behavior using NMR and DSC, and published a precise phase diagram for

temperatures around the melting temperature as shown in Fig 3.2. The data included the cholesterol

concentration from 0 to 27 mole percent for DPPC lipids.

The phase behavior diagram shows the transition from the gel phase to the liquid ordered phase

with the coexistence of the gel & liquid ordered phase from cholesterol concentration from 6% to

22%. Above the melting temperature and as the cholesterol concentration is increased we observe

the phase coexistence of the fluid phase and liquid ordered phase. The coexistence disappears

above a critical point [48, 74].

30
Fig3.2 Phase behavior plot for the binary mixture of DPPC bilayers and cholesterol[74].

Xiang and Anderson also studied the permeability of various solutes in the binary mixtures

of DPPC bilayers and cholesterol using NMR method. Phase transitions were induced due to the

changes in the temperature and cholesterol concentrations [95]. The addition of cholesterol

induced very little change in the permeability coefficient in the gel phase, but reduced the

permeability coefficient significantly in the liquid phase. Corners in the semi-logarithmic plots of

and cholesterol concentrations occurred at the phase boundaries between the gel liquid ordered

phases, and between the fluid and liquid disordered phases. The phase diagrams were generated

from these break points in the semi-logarithmic plots at various temperatures. Thy resemble the

generic phase behavior, but differed in quantitative details from, Vist and Davis’s results as shown

in Fig 3.3 [95]. The coexistence of the gel and liquid-disordered phase near the melting temperature

was not elucidated from the permeability plots. Also, the region of phase coexistence of the liquid-

31
disordered and liquid–ordered at higher temperatures was generated as a broad region is not similar

to the narrow presented by Vist & Davis[74].

Fig3.3 Phase behavior plot for the binary mixture of DPPC bilayers and cholesterol[95].

Chiang et al. published the phase behavior of binary mixtures of DPPC and cholesterol to

study the dynamic structure of the binary mixture using 2D-Electron electron double resonance;

the phase diagram consisted of the gel phase, liquid-ordered, liquid-disordered phases and the

phase coexistence regions as shown in Fig 3.4 [96]. The phase boundaries and the phase

coexistence regions were not determined exactly in either of the studies presented and are

dependent on the physical methods employed. An interesting feature in the phase behavior

presented by Chiang et al. is the broadening of the coexistence of the liquid ordered and liquid-

disordered phase [96] as compared to the Vist and Davis [74]. The dissimilarity observed, the start

32
of two-phase coexistence is at high cholesterol concentration (i.e 17%) as compared to the other

results published.

Fig3.4 Phase behavior plot for the binary mixture of DPPC bilayers and cholesterol[96].

3.4 Summary

In this chapter we have seen that, in absence of cholesterol, the bilayer membranes exists in either

the solid ‘gel’ phase or the liquid crystalline ‘fluid’ phase. Cholesterol stabilizes the bilayer

membrane by ordering the lipid acyl chains, thus forming a new phase, the liquid ordered phase.

We have compared the results published by three groups and concluded that the binary phase

boundary behavior published by Vist and Davis is probably the most quantitatively reliable.

33
Chapter 4: Analysis of Permeability Data for
Various Solutes
Passive permeation of various chemical solutes to their respective site of action through

phospholipid bilayers is driven by the gradient of chemical potential. Hence, understanding and

analyzing the physicochemical factors that determine the permeability across the lipid bilayers is

required from the perspective of research as well as for the development of transdermal drug

delivery [3, 33, 97]. The solubility-diffusion model is the most commonly employed model to

describe the passive transport of solutes across the bilayers. It relates the solute’s permeability

coefficient to its ability to diffuse across and partition into the membrane, treating the lipid bilayer

membrane as a bulk lipid solvent [33, 34]. Xiang, Anderson, and coworkers state that according

to solubility-diffusion theory and Overton’s rule, the permeability coefficient is directly

proportional to the product of the partition coefficient and the diffusion coefficient of the

permeant in a bulk liquid hydrocarbon divided by the thickness of the hydrocarbon region

4.1

The dependence of permeability on diffusion coefficient and partition coefficient motivates the

study of these quantities in detail. For the evaluation of partition coefficients, a mimic for

phospholipid-cholesterol bilayers has been suggested in different publications [3, 33, 34, 36].

Various experimental works have focused on finding the solvent that best mimics the chemical

environment presented by the bilayer. Based on the studies presented by Xiang, Anderson, and

coworkers, 1,9-decadiene is identified as the solvent that best mimics the chemical selectivity of

34
the barrier domain in egg lecithin bilayers and other phospholipids [33, 34, 36]. However, the

model of solubility-diffusion based on the mimic for phospholipid-cholesterol mixture bilayers

fails to account for the effect of ordering of the fatty acid chains [33, 34]. Following Xiang and

Anderson, the present chapter aims to address this limitation by introducing a scaling factor that

modifies Overton’s rule, called the chain ordering factor F, as well as a correlating variable χ

representing a ratio of solute size to free space between fatty acid chains, to quantify chain ordering

effects on permeability.

4.1 Ordering Effects of Cholesterol

The cholesterol when intercalated increases the ordering of fluid-phase PC fatty acid acyl chains.

It is well recognized as ‘the ordering effect of cholesterol,’ and is generic in the lipid bilayers in

the fluid phase. These ordering effects have been quantified by various authors using experimental

techniques like NMR, X-ray scattering, spectroscopy and others [38, 51, 64, 98]. Even the results

from MD and Monte Carlo simulations have shown an increase in the ordering of the acyl chains

in PC/Chol mixtures relative to the pure PC bilayers [45, 60, 61, 63, 88]. This effect is manifested

as a straightening of the tails of the neighboring DPPC molecules (i.e. more ordering) when

cholesterol is introduced, increasing the thickness of the lipid bilayer and reducing the membrane

area per phospholipid. The change in the chain tilt observed in gel-phase PC/Chol mixtures

compared to pure PC bilayers is also a consequence of the ordering effects of the cholesterol [72].

The ordering effect due to the presence of cholesterol reduces the permeability of the

solutes in fluid-phase bilayers in a way that depends on the free surface area in the membrane and

the solute volume or cross-section area [75, 99]. The free volume in the bilayer is a critical variable

determining molecular diffusion and chemical potential. It plays an important role in estimating

35
the permeability of the solutes. [100-102]. The free volume theory [34] is modified in terms of free

surface area model as the trans-bilayer permeability depends on the two-dimensional packing of

the lipids (i.e. surface area of the lipid bilayer). Similar to the free- volume, the average free area

per lipid molecule is defined as af ;

4.2

The ordering effect observed in bilayers causes an increase in the phospholipid surface density,

which reduces the free area or volume available to the solute, which lies in the plane of the bilayer

and is aligned perpendicularly to it [34].

4.2 Barrier- domain Solubility Diffusion Model

The prediction of passive permeability across lipid bilayers based only on the structure/size of the

solute and the chemical composition of the lipid bilayer membrane has been the subject of research

in biophysics and transdermal drug delivery for many decades. Xiang and Anderson have

sharpened the solubility diffusion model by assuming that the permeability is rate limited by

barrier domain within the bilayer. The barrier domain exhibits a chemical selectivity similar to the

hydrocarbon alkyl chain region in liquid crystalline phase and varies with the bilayer phase

behavior [33, 34, 36, 97]. Accounting for the chain ordering and the permeant size on the

permeability, Xiang and Anderson developed barrier domain solubility diffusion model and is

defined by [97]

4.3

where f is a scaling factor due to the chain ordering .

36
 In a membrane made of a bulk solvent, the value of f would be 1.0 whereas, in all bilayers

composed of a randomly oriented chains, f is <1.0. The barrier domain model proposed that

permeability can be predicted from solubility-diffusion theory considering the chain ordering

effect which is a function of free-surface area, regardless of whether changes in free surface area

arise from variations in the temperature, composition or phase of the bilayer [34, 97].

The partition and diffusion coefficients follow Arrhenius dependence on temperature, and

the diffusion coefficient is also dependent on the solute size. Including these dependencies into the

relation we obtain a new precise relation

4.4

4.5

Substituting these equations into equation 4.3, permeability coefficient, Pm is given as

4.6

Also according to Wilke-Chang correlation [103]

4.7

where is the molar volume of solute as liquid at its normal boiling point, and is a constant

which depends on the solvent (decadiene) properties at T = 25 °C. For ease of calculation, the

molecular volume is best represented as a power law of solute molecular weight (MW). We use

the scaling presented by Ibrahim et al [104]:

4.8

The partition and diffusion coefficients account for the temperature changes and they do not

consider the ordering effects. The ordering effect in the bilayer membrane is quantified in terms

of a correlating parameter  that combines the molecular weight MW of the solute and the free

37
area available for the solute in the phospholipid bilayer membrane to diffuse through it, the latter

expressed in terms of the normalized surface density[105]

4.9

The best value of the exponent n, which indicates the best measure of solute size, is as yet unknown

and discussed below. Substituting all the dependent variables (from relations 4.4 - 4.9), we deduce

the form of the correlation for the permeability coefficient across the lipid bilayer:

4.10

where

An estimate for the overall activation energy for the product of for acetic

acid is 11 kcal/mol [34]. It is assumed as a generic value for other solutes as well to obtain a broad

correlation for the activation energy. The universal gas constant is 1.987 cal/(mol K) which

yields ~ 5.5 x 103 K.

4.3 Permeability correlation for pure phospholipid bilayers

in the gel phase

Based on this theory, we culled literatures and gathered the data on permeability of a series

of carboxylic acid solutes like acetic acid, butyric acid, formic acid, isovaleric acid, trimethyl acetic

acid and valeric acid, with the aim of predicting their permeability coefficients across the

phospholipid bilayers. The data from various sources has been tabulated in Table 3 and Table 4

[33, 34, 36, 95]. Using the data collected [33, 34, 36, 95], we effectively obtain the dependence

upon molecular size and phospholipid surface density of and in terms of the

38
correlating variable χ. Using χ, we aim to establish a relationship between the permeability of

solutes and lipid chain packing (free surface area) encompassing the effect of solute size,

phospholipid chain length, temperature, cholesterol concentration [34]. We gathered data 115

observations for for six different carboxylic acid solutes in DPPC bilayers in gel phase, out of

which 48 observations are for acetic acid and 45 are for trimethyl acetic acid. The data points are

read from the published permeability data as detailed in the Appendix.

4.3.1 Calculation of partition coefficient Kdec

The chemical selectivity of the barrier-domain has been shown to be best mimicked by the

reference solvent 1,9- decadiene. We base our theory on this solvent, but also carry out the analysis

with octanol for comparison. Octanol/waterpartition coefficients as ( ) are obtained

from EPS Suite [106, 107] (Estimation Program Interface), U.S. Environmental Protection Agency

accessed through ChemSpider, and from ACD/Labs’ LogP Calculator [108]. The ACD/Labs’

LogP value is used in absence of an experimental value. Values of the 1,9-decadiene partition

coefficients are taken from a published paper of Nitsche and Kasting [3] whenever listed. and In

the absence of data, it is predicted from a linear free energy relationship (LFER) as a function of

and Abraham solvation parameters [109, 110], (hydrogen bond activity),

(polarity/polarizability), (excess molar refraction) and (McGowan characteristic volume) [3].

For easy notation is henceforth referred to as . The Abraham solvation

parameters are obtained using the Absolv prog from ACD/Labs’ ADME Suite [106, 108]. The

LFER is as follows:

4.13

39
4.3.2 Plotting of the data

Due to chain ordering in the barrier domain, the primary dependence of and

is due to the molecular size and phospholipid surface density. The correlation for

permeability coefficient is thus expressed as

4.14

The function F(χ) quantifies the chain ordering in terms of the ratio of molecular size of the solute

(i.e. molecular weight) and the void region in the barrier domain region (expressed in terms of

normalized surface density). For spherical solutes, the molecular size quantified in terms of

projected would scale roughly as MW2/3, and for linear molecules it would be independent of chain

length (i.e., would scale as MW0). Quantified in terms of volume, the molecular size would scale

with 0.94 power of MW (roughly MW1). Hence, we consider the scale in the range of 0-2/3 on the

basis of free surface area theory, and around 1 according to the free volume theory. We performed

an analysis varying the power factor from 0 to 1 and fitting the dependence of log10 F as a function

of χ for several values of n. The chain ordering factor F (as opposed to Pm) exhibits rationallt

understandable trends when considered in terms of  and σ, covering variations in phospholipid

bilayer type, T and Xchol.

The permeability data for the pure DPPC bilayers at various conditions for different solutes

are plotted for different values of χ, varying the value of n (power of the MW) tabulated in Table

5. Of all the plots, we have observed that for n=0.10 is the optimum value for the representation

of the data. The semi-log plot of and χ, chain ordering factor at n=0.10 is shown in Fig

4.1. In the graphs, the blue diamond and square points represents the normalized permeability data

sets for pure DPPC bilayers where the reference solvent is 1,9 decadiene and octanol respectively.

40
The green diamond and square points represents the normalized permeability data sets for pure

DMPC bilayers where the reference solvent is 1,9 decadiene and octanol respectively. The purple

diamond and square points represents the normalized permeability data sets for pure DSPC

bilayers where the reference solvent is 1,9 decadiene and octanol respectively. The fluid phase

permeability data sets at gel phase proposed by Nitsche and Kasting is shown by the thick lines.

The orange thick line represents the chain ordering factor when n = 0.87 and the red thick line

represents the chain ordering factor when n =0.33.

a) n=0.1

n=0.1

4
y = -6.47x + 59.599
-1 R² = 0.8285
ln (Pm/(Kdeca*fsolv))

-6

-11

-16

-21

-26
8.5 9.0 9.5 10.0 10.5 11.0 11.5
χ

41
b) n=0.33

n=0.33
4

-1 y = -0.709x + 16.119
R² = 0.7622
ln (Pm/(Kdeca*fsolv))

-6

-11

-16

-21

-26
20 25 30 35 40 45 50
χ

c) n=0.67

n=0.67
4
y = -0.0793x + 6.7857
R² = 0.7407
-1
ln (Pm/(Kdeca*fsolv))

-6

-11

-16

-21

-26
50 100 150 200 250 300 350 400
χ

42
 d) n=0.87

n=0.87
4

y = -0.0256x + 4.738
-1 R² = 0.734
ln (Pm/(Kdeca*fsolv))

-6

-11

-16

-21

-26
100 300 500 700 900 1100 1300
χ

e) n=0.94

n=0.94
4

y = -0.0174x + 4.2301
-1 R² = 0.7319
ln (Pm/(Kdeca*fsolv))

-6

-11

-16

-21

-26
0 500 1000 1500 2000
χ

43
 f) n=1

n=1
4

y = -0.0126x + 3.8522
-1
R² = 0.7303
ln (Pm/(Kdeca*fsolv))

-6

-11

-16

-21

-26
0 1000 χ 2000 3000

Fig 4.1 Semi-log plot of permeability coefficients for various carboxylic acids across pure phospholipid bilayers at

various values of n, the power of molecular weight of solute in chain ordering factor, χ

4.3.3 Correlations for Permeability coefficient

Based on the numerical analysis performed, we have observed that for n=0.10 is the best value for

the exponent value of the molecular weight of the solute in the chain ordering factor, χ. Thus, the

chain ordering factor relation,

The chain ordering factor for various value n, from 0.1 to 0.94 are listed in table 6.Thus, we can

predict the passive permeability coefficient for any solute at any given condition with the

phospholipid bilayer parameters.

44
4.3.4 Comparison of Fluid Phase Permeability correlations in gel-phase

Permeability data for liquid crystalline phospholipid bilayers in their fluid phase was analyzed

using a mathematical model accounting for the free surface area, chain ordering effects and the

solute size by researchers Nitsche & Kasting [3]. A correlating variable χ is used to relate the

different membrane systems and the molecular weight of the solute. The best fit for χ at n=0.87

made the chain ordering factor more closer to the ratio of molecular free volume than the surface

areas [3]. These results have helped in estimating the passive permeability of bilayer membranes

as a function of temperature and cholesterol content. In this report, we validate the results

published in fluid-phase also applicable to gel-phase bilayer membranes. The predicted values of

passive permeability in fluid phase are also included and the behavior in gel phase is similar to the

fluid-phase as shown in Fig 4.2. The sudden decrease in with temperature in gel phase is due

to the molecular packing of the acyl chains from hexagonal to orthorhombic or monoclinic [3, 36,

95].

n=0.87
4

-1
y = -0.0256x + 4.738
ln (Pm/(Kdeca*fsolv))

-6 R² = 0.734

-11

-16

-21

-26
100 300 500 700 900 1100 1300
χ

Fig 4.2 Semi-log plot of permeability coefficients for various carboxylic acids across pure phospholipid bilayers.

45
4.4 Summary

In this chapter, we have established correlations to predict the permeability coefficient considering

the chain ordering and the temperature effects for gel-phase bilayers comprising pure phospholipid

DPPC. Regression analysis has been performed to obtain correlations for several values of the

exponent n on the molecular weight of the solute appearing in the correlating variable χ (see Table

5). Previous correlations based on fluid-phase are not good predictors for the gel phase.

46
Chapter 5: Intercalation of Cholesterol

In the preceding chapters we have discussed the permeability of various carboxylic acids across

the pure phospholipid bilayers taking into account the chain ordering effect observed. In this

current chapter, we further extend our research in the binary mixtures of the PC bilayers and

cholesterol. As discussed earlier (in Chapter 4), cholesterol induced the chain ordering effect and

reduced the condensation effect observed in pure PC bilayers.

Bilayers membranes are composed of multiple lipids and sterols, where the non-ideal mixing

causes lateral heterogeneity and formation of two or more phase coexistence regions. The presence

of cholesterol would result in the phases of liquid disordered (low-cholesterol) and liquid ordered

(high- cholesterol) at the physiological conditions. Due to the high content of cholesterol present

in the biological membranes (~ 40% mole percent, discussed in Chapter 1), many bilayers exist in

the liquid-ordered/liquid-disordered coexistence region at 370C [78, 83, 111]. The fundamental

role for the study of the phase behavior of the PC and cholesterol mixtures is for the better

understanding of the solute permeation across the lipid bilayers. Researchers like Xiang &

Anderson, have published that there is strong correlation between phase behavior and solute

permeability across the bilayer membranes[34, 95].

5.1 Estimation of Permeability relation in 2-phase

coexistence region

Based on the study of the phase behavior of the binary mixtures of PC and cholesterol

bilayers, we are aware that at very low cholesterol concentrations (~ 0-7 %), the mixture exists in

47
gel phase (liquid-disordered). At these conditions, we assume the permeability to be constant and

takes a value which is observed as in pure PC bilayer. As the cholesterol is increased, due to

conformational changes in the bilayer mixtures, we observe the phase coexistence between the gel

phase and liquid ordered phase. To find out the relation, we use the concept of the lever rule which

determines the weight percentages across each phase. Applying lever rule for cholesterol, the total

weight of cholesterol in gel phase is same as that in the liquid ordered phase.

5.1

5.2

5.3

We obtain a relation for the area of the lipid bilayer across each phase and the molar

concentrations of the cholesterol at the phase boundaries in equation 5.3. To further apply this

relation to relate the permeability across the bilayer, we apply the mass balance across the bilayer

membrane. Applying concentration difference across the bilayer

5.4

Substituting the area terms in cholesterol concentration (from Equation 1.3) and cancelling the

common terms, we obtain the relation for permeability constant across the phase coexistence

region.

48
Rearranging we have,

5.5

Rearranging all the terms, we obtain a linear relation for the permeability and cholesterol

concentrations across the phase coexistence region where the slope is a function of permeability

constants at higher and lower concentrations of the cholesterol at the phase boundaries.

At higher concentrations of cholesterol, we assume a constant permeability relation which

is obtained by averaging the permeability data collected for cholesterol concentration above the

phase transition boundary (i.e. Xc > 0.22). Based on the linear and constant theory for the

permeability coefficient, we obtain the following relation,

5.6

The datum collected from various published resources [17, 33, 34, 38, 60, 95, 98] (extrapolated

from the semi-log plot, detailed in Appendix) and the permeability for acetic acid calculated at

various temperatures below Tm from the above relation (equation 1.6) have been plotted on a semi-

log plot of Pm and Xc. The solid lines represent the permeability coefficients calculated from the

relation (equation 1.6) at various concentrations of cholesterol. The filled circles represent the

permeability data for acetic acid obtained from Xiang & Anderson [95] at T=370C. The filled

diamond represent the permeability data for acetic acid from Xiang & Anderson [95] at T=330C.

The filled triangles represent the permeability data for acetic acid from Xiang & Anderson [95] at

T=300C.

49
 Acetic Acid
-2.50

-3.00

-3.50 t=30 DATA

t=30 CALC
log Pm

t=33 DATA
-4.00 t=33 CALC
t=37 DATA
t=37 CALC
-4.50

-5.00
0 0.1 0.2 0.3 0.4 0.5
XC

Fig 5.1 Semi-log plot of Permeability coefficient of acetic acid and cholesterol at various concentrations

The above procedure is followed for another solute, trimethyl acetic acid to

support the linear relation of the permeability with the cholesterol concentration at the phase

existence. The datum collected from various published resources [17, 33, 34, 38, 60, 95, 98]

(extrapolated from the semi-log plot, detailed in Appendix) and the permeability for trimethyl

acetic acid calculated at various temperatures below Tm from the above relation have been plotted

on a semi-log plot of Pm and Xc. The solid lines represent the permeability coefficients for trimethyl

acetic acid calculated from the relation (equation 16) at various concentrations of cholesterol. The

filled circles represent the permeability data for trimethyl acetic acid obtained from Xiang &

Anderson [95] at T=370C. The filled diamonds represent the permeability data for trimethyl acetic

50
acid from Xiang & Anderson [95] at T=330C. The filled triangles represent the permeability data

for trimethyl acetic acid from Xiang & Anderson [95] at T=300C.

Trimethyl Acetic Acid

-1.50

-2.00

-2.50
t=30 DATA
t=30 CALC
log Pm

-3.00
t=33 DATA
t=33 CALC
-3.50 t=37 DATA
t=37 CALC
-4.00

-4.50
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
XC

Fig 5.2 Semi-log plot of Permeability coefficient for trimethyl acetic acid and cholesterol at various concentrations.

There is disagreement and lot of error is observed between the calculated values from the relation

and the data set collected from various sources [17, 33, 34, 95]. To obtain an optimum value, we

calculate the total squared error between the calculated values and experimental values. We

minimize the error value by varying the constant permeability obtained from the higher phase

boundary of the phase coexistence using the Solver software in Microsoft Excel. For instance, the

error for acetic acid at T=370C was reduced by 41% (from 0.4814 to 0.2838) by varying the

constraint, the permeability coefficient at phase boundary (from -3.18(average value) to -3.00

(minimized value for error)). The detailed error analysis values are provided in the table 7. This

51
error analysis would lead to optimum permeability coefficient values with the cholesterol

concentrations. The following are the semi-log plots of permeability coefficients of acetic acid and

trimethyl acetic acid after the error minimization.

Acetic Acid
-2.50

-3.00

-3.50 t=30 DATA

t=30 CALC
log Pm

t=33 DATA
-4.00 t=33 CALC
t=37 DATA
t=37 CALC
-4.50

-5.00
0 0.1 0.2 0.3 0.4 0.5
XC

Fig 5.3 Semi-log plot of Permeability coefficient of acetic acid and cholesterol at various concentrations minimizing

the errors calculated.

As compared to Fig 5.1, Fig 5.3 is in good agreement for the permeability relation for acetic acid

derived with the experimental data collected [95]. Similarly, Fig 5.4 is in good agreement for

trimethyl acetic acid for the permeability data derived from the relation and the experimental data

collected [95]. Thus, summarizing the analysis, in 1 phase region the permeability coefficient is

assumed to be constant and a linear dependence on the cholesterol concentration in the 2-phase

coexistence region. This analysis has shown consistency with the experimental data also. The

52
analysis has also provided the evidence that the permeability follows a linear increase with

cholesterol in a 2-phase coexistence region irrespective of the solute being considered.

Trimethyl Acetic Acid

-1.50

-2.00

-2.50
t=30 DATA
t=30 CALC
log Pm

-3.00
t=33 DATA
t=33 CALC
-3.50 t=37 DATA
t=37 CALC

-4.00

-4.50
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
XC

Fig 5.4 Semi-log plot of Permeability coefficient of trimethyl acetic acid and cholesterol at various concentrations

minimizing the errors calculated.

5.2 Prediction for fluid phase permeability coefficient at Gel

phase temperatures

Taking the analysis to further step ahead, we now aim in estimating the permeability of the fluid

phase bilayer membranes assuming it to be present in gel phase. For this estimating, we gather the

relations for the area of the phospholipid bilayer and study their behavior in the gel phase. From

the discussion of area of phospholipid bilayer in Chapter 2, we use the areal expansion coefficient

and the exponential behavior of the area with respect to temperature.

53
 5.7

The reference temperature for the fluid phase behavior is at 500C and the value of area of the pure

phospholipid (DPPC) is 63.1Å2, the areal expansion coefficient (α) is 0.003(0C)-1. Using this

relation, we find the area of the pure DPPC bilayers at temperatures below Tm.

The area of the total lipid membrane per phospholipid molecule is given in terms of the partial

molar areas according to the equation 1.8. When cholesterol is intercalated in the DPPC bilayers,

they occupy a certain area of 27 Å2. Researchers like Edhom and Nagle [99] have reviewed and

presented analysis detailed on the total membrane area per phospholipid for binary mixtures based

on the concept of partial molar properties. The assumption made in approaching the partial specific

area model is the cholesterol area is constant. The area per lipid molecule has been derived

and published in term of the condensation effect observed due to cholesterol sgiven in equation

1.9.

5.8

5.9

In the above equation, is the total area of the membrane per lipid molecule, is the area

of the pure DPPC molecule, area of condensation of any DPPC molecule in contact with

cholesterol molecule, is the number of DPPC molecules condensed by a cholesterol molecule,

is the cholesterol area and is the mole fraction of cholesterol.

For binary systems, the partial area can be directly calculated from the following expression using

the solution property as a function of composition at constant T is given by equation 1.10.

5.10

From equation 5.9, differentiating w.r.t x we have

54
 5.11

Using equations 5.9 and 5.11, substituting in equation 1.10 we have

5.12

From equation 5.12, we calculate the area of DPPC molecules in particular. The cholesterol

dependence on the area of the bilayer can be quantified by the ratio of from equation

5.12 and .

5.13

The 40.8 Å2 is the minimum area in the crystalline state[3].

The cholesterol dependence on the lipid bilayers are quantified in term of σrel, which is defined as

5.14

Using the σrel, we estimate the cholesterol effect on the normalized surface density of the pure

phospholipid bilayer. This is applied in of molecular weight) and the void region

in the barrier region (quantified in terms of σ). Using the relation published by Nitsche and Kasting

[3], we estimate the permeability coefficient for the fluid phase bilayer mixture DPPC and

cholesterol at gel phase temperatures.

55
5.3 Prediction of Liquid-ordered phase permeability
coefficient
The estimation of the permeability coefficient in liquid ordered phase was done with the help of

gel phase and fluid phase permeability coefficients. For this, we calculated the geometric mean of

the pure gel and fluid phase permeability coefficients. The best conclusion is the liquid ordered

permeability coefficient is approximately 6.5 times (ranges from 4.5 to 8.5) the permeability

coefficient of the pure gel phase bilayer. This result is based on the characterization of liquid

ordered permeability data for only 2 solutes. This analysis is showed clearly in Table 8.

5.4 Summary
In this chapter, we studied the dependence of permeability coefficient on cholesterol concentration

in a bilayer. In a 1 phase region, the permeability coefficient is constant irrespective of the

cholesterol concentration and in 2-phase region, the permeability coefficient depends linearly with

the cholesterol concentration. Also, we concluded that the liquid ordered phase permeability

coefficient is approximately 6.5 times than the value of the pure gel phase permeability coefficient.

56
6. Conclusions
To summarize, I have demonstrated a generic procedure to calculate the permeability coefficient

for gel phase bilayers. Based on the analysis, the permeability coefficient can be calculated from

the partition coefficient, solvent parameters and the chain ordering factors as shown in equation

6.1.

[6.1]

where

Tough the best value of n was 0.1, the permeability coefficient can be found from either of

the functions provided in table 6.

The permeability coefficient foe the bilayers with the intercalation of cholesterol is also

predicted. As discussed in Chapter 5, in 1 phase region (i.e. gel phase) at low cholesterol regions,

the permeability coefficient takes a constant value as that of the pure gel phase permeability

coefficient. And, in 2-phase region it shows linear dependence on the cholesterol concentration.

The 1 phase region (i.e. liquid-ordered) at higher cholesterol regions, take s value approximately

6.5 times that of the pure gel phase value.

57
Table 1: Table showing the melting temperatures of pure PCs[48].

PC Tm(0C)
DLPC -2
DMPC 24
DPPC 41
DSPC 55
DOPC -17
POPC -2

Table 2: Selected values of σ of pure lipids at different temperatures reported in various papers by
Nagle, Xiang & Anderson and other various resources [34, 56, 57].

Area of
Lipid Temp(0C) σa σb
lipid(Å2)
DMPC 10 47.2[112] 8.64E-01
DMPC 20 8.18E-01[34] 8.18E-01
DPPC 5 46.1[57] 8.85E-01
DPPC 7 46.4[57] 8.79E-01
DPPC 8 46.5[57] 8.77E-01
DPPC 10 46.3[57] 8.81E-01
DPPC 13 45.8[57] 8.91E-01
DPPC 14 46.58[56] 8.76E-01
DPPC 20 47[77] 8.68E-01
DPPC 20 47.9[112] 8.52E-01
DPPC 20 46.92[56] 8.70E-01
DPPC 22 46.6[113] 8.76E-01
DPPC 24 47.9[76] 8.52E-01
DPPC 25 47.3[93] 8.63E-01
DPPC 25 8.95E-01[34] 8.95E-01
DPPC 26.8 47.13[56] 8.66E-01
DPPC 30 8.73E-01[34] 8.73E-01
DPPC 34 8.59E-01[34] 8.59E-01
DPPC 35 8.55E-01[34] 8.55E-01
DPPC 36 47.82[56] 8.53E-01
DPPC 36 8.52E-01[34] 8.52E-01
DPPC 38 8.37E-01[34] 8.37E-01
DPPC 40 8.25E-01[34] 8.25E-01
DSPC 4 46.4[57] 8.79E-01
DSPC 25 46.8[50] 8.72E-01

a Values are obtained from the normalized density data provided by Xiang & Anderson [34].

b Values are calculated from the collected values of A [57, 93, 112] from various sources and by using Equation .

58
Table 3: Permeability coefficient (Plipid|w) of pure phospholipids at different temperatures, reported
in various papers by Xiang & Anderson[3, 34, 36].

Solute Lipid T(0C) MolWt Koct|w Kdecadiene|w|250C[3] Plipid|w

Acetic Acid DMPC 20 60.05 6.76E-01a 1.30E-03c 3.90E-04[34]
Acetic Acid DPPC 25 60.05 6.76E-01a 1.30E-03c 1.70E-05[34]
Acetic Acid DPPC 30 60.05 6.76E-01a 1.30E-03c 2.70E-05[34]
Acetic Acid DPPC 33 60.05 6.76E-01a 1.30E-03c 6.92E-05[33]
Acetic Acid DPPC 34 60.05 6.76E-01a 1.30E-03c 7.80E-05[34]
Acetic Acid DPPC 35 60.05 6.76E-01a 1.30E-03c 2.00E-04[34]
Acetic Acid DPPC 36 60.05 6.76E-01a 1.30E-03c 2.20E-04[34]
Acetic Acid DPPC 37 60.05 6.76E-01a 1.30E-03c 2.58E-04[33]
Acetic Acid DPPC 38 60.05 6.76E-01a 1.30E-03c 3.10E-04[34]
Acetic Acid DPPC 40 60.05 6.76E-01a 1.30E-03c 7.10E-04[34]
Butyric Acid DPPC 24.65 88.18 6.17E+00a 3.90E-02c 4.18E-05[33]
Butyric Acid DPPC 29.88 88.18 6.17E+00a 3.90E-02c 4.76E-05[33]
Butyric Acid DPPC 32.66 88.18 6.17E+00a 3.90E-02c 2.65E-04[33]
Butyric Acid DPPC 34.63 88.18 6.17E+00a 3.90E-02c 5.48E-04[33]
Butyric Acid DPPC 37.7 88.18 6.17E+00a 3.90E-02c 1.21E-03[33]
Formic Acid DPPC 24.47 46.02 2.88E-01b 6.20E-04c 7.01E-05[33]
Formic Acid DPPC 29.88 46.02 2.88E-01b 6.20E-04c 1.46E-04[95]
Formic Acid DPPC 33.6 46.02 2.88E-01b 6.20E-04c 1.45E-04[95]
Formic Acid DPPC 37.6 46.02 2.88E-01b 6.20E-04c 4.09E-04[33]
Isovaleric Acid DPPC 29.88 102.13 1.45E+01a 1.94E-01b 6.60E-05[33]
Isovaleric Acid DPPC 32.47 102.13 1.45E+01a 1.94E-01b 3.40E-04[33]
Isovaleric Acid DPPC 34.35 102.13 1.45E+01a 1.94E-01b 4.92E-04[33]
Isovaleric Acid DPPC 36.45 102.13 1.45E+01a 1.94E-01b 8.50E-04[33]
Isovaleric Acid DPPC 37.41 102.13 1.45E+01a 1.94E-01b 9.03E-04[33]
Proplonic Acid DPPC 29.88 74.07 2.14E+00a 1.16E-02b 2.47E-05[33]
Proplonic Acid DPPC 32.67 74.07 2.14E+00a 1.16E-02b 6.34E-05[33]
Proplonic Acid DPPC 34.73 74.07 2.14E+00a 1.16E-02b 1.30E-04[33]
Proplonic Acid DPPC 36.83 74.07 2.14E+00a 1.16E-02b 3.00E-04[33]
Trimethyl acetic acid DPPC 30 102.13 3.02E+01a 4.82E-01b 8.31E-05[33]
Trimethyl acetic acid DPPC 32.14 102.13 3.02E+01a 4.82E-01b 3.87E-04[33]
Trimethyl acetic acid DPPC 34.54 102.13 3.02E+01a 4.82E-01b 8.75E-04[33]
Trimethyl acetic acid DPPC 35.87 102.13 3.02E+01a 4.82E-01b 1.48E-03[33]
Trimethyl acetic acid DPPC 39.35 102.13 3.02E+01a 4.82E-01b 9.56E-03[33]
Valeric Acid DPPC 24.45 102.13 2.45E+01a 2.88E-01b 1.10E-04[33]
Valeric Acid DPPC 29.49 102.13 2.45E+01a 2.88E-01b 9.89E-04[33]
Valeric Acid DPPC 32.29 102.13 2.45E+01a 2.88E-01b 1.59E-03[33]
Valeric Acid DPPC 34.54 102.13 2.45E+01a 2.88E-01b 1.41E-03[33]
Valeric Acid DPPC 36.26 102.13 2.45E+01a 2.88E-01b 2.67E-03[33]
Valeric Acid DPPC 37.47 102.13 2.45E+01a 2.88E-01b 5.35E-03[33]
DB-67 Lactone Form DSPC 40 477.62 8.51E+04a 1.08E+00b 3.50E-08[35]
aValues are obtained from Estimation Program Interface (EPI) Suite, accessed through ChemSpider
b Values are obtained from ACD/Labs’ logP calculator accessed through the ChemSketch interface.
c Values are collected from a source by Nitsche and Kasting[3]

59
Table 4: Permeability coefficient (Plipid|w) of phospholipids with cholesterol at different
temperatures, reported in various papers by Xiang & Anderson[34],[33, 36, 95]

Solute Lipid T(0C) XC Koct Kdecadiene|w|250C Plipid|w

Acetic Acid DMPC 20 0.10 6.76E-01a 1.30E-03c
Acetic Acid DMPC 21 0.10 6.76E-01a 1.30E-03c 2.70E-03[34]
Acetic Acid DPPC 30 0.03 6.76E-01a 1.30E-03c 3.73E-05[95]
Acetic Acid DPPC 30 0.10 6.76E-01a 1.30E-03c 4.70E-05[95]
Acetic Acid DPPC 30 0.15 6.76E-01a 1.30E-03c 7.48E-05[95]
Acetic Acid DPPC 30 0.17 6.76E-01a 1.30E-03c 8.08E-05[95]
Acetic Acid DPPC 30 0.20 6.76E-01a 1.30E-03c 1.10E-04[95]
Acetic Acid DPPC 30 0.25 6.76E-01a 1.30E-03c 1.28E-04[95]
Acetic Acid DPPC 30 0.30 6.76E-01a 1.30E-03c 1.51E-04[95]
Acetic Acid DPPC 30 0.35 6.76E-01a 1.30E-03c 2.39E-04[95]
Acetic Acid DPPC 30 0.40 6.76E-01a 1.30E-03c 1.51E-04[95]
Acetic Acid DPPC 30 0.45 6.76E-01a 1.30E-03c 1.19E-04[95]
Acetic Acid DPPC 33 0.05 6.76E-01a 1.30E-03c 6.92E-05[95]
Acetic Acid DPPC 33 0.07 6.76E-01a 1.30E-03c 7.48E-05[95]
Acetic Acid DPPC 33 0.10 6.76E-01a 1.30E-03c 1.12E-04[95]
Acetic Acid DPPC 33 0.13 6.76E-01a 1.30E-03c 1.89E-04[95]
Acetic Acid DPPC 33 0.15 6.76E-01a 1.30E-03c 1.89E-04[95]
Acetic Acid DPPC 33 0.17 6.76E-01a 1.30E-03c 2.13E-04[95]
Acetic Acid DPPC 33 0.20 6.76E-01a 1.30E-03c 2.58E-04[95]
Acetic Acid DPPC 33 0.22 6.76E-01a 1.30E-03c 3.26E-04[95]
Acetic Acid DPPC 33 0.25 6.76E-01a 1.30E-03c 3.80E-04[95]
Acetic Acid DPPC 33 0.30 6.76E-01a 1.30E-03c 3.80E-04[95]
Acetic Acid DPPC 33 0.40 6.76E-01a 1.30E-03c 2.79E-04[95]
Acetic Acid DPPC 37 0.01 6.76E-01a 1.30E-03c 2.68E-04[95]
Acetic Acid DPPC 37 0.05 6.76E-01a 1.30E-03c 2.68E-04[95]
Acetic Acid DPPC 37 0.06 6.76E-01a 1.30E-03c 3.01E-04[95]
Acetic Acid DPPC 37 0.10 6.76E-01a 1.30E-03c 5.18E-04[95]
Acetic Acid DPPC 37 0.12 6.76E-01a 1.30E-03c 1.04E-03[95]
Acetic Acid DPPC 37 0.15 6.76E-01a 1.30E-03c 9.62E-04[95]
Acetic Acid DPPC 37 0.16 6.76E-01a 1.30E-03c 7.64E-04[95]
Acetic Acid DPPC 37 0.19 6.76E-01a 1.30E-03c 1.04E-03[95]
Acetic Acid DPPC 37 0.20 6.76E-01a 1.30E-03c 9.62E-04[95]
Acetic Acid DPPC 37 0.25 6.76E-01a 1.30E-03c 8.24E-04[95]
Acetic Acid DPPC 37 0.30 6.76E-01a 1.30E-03c 7.06E-04[95]
Acetic Acid DPPC 37 0.40 6.76E-01a 1.30E-03c 4.80E-04[95]
Trimethylacetic Acid DPPC 30 0.02 3.02E+01a 4.82E-01b 9.64E-05[95]
Trimethylacetic Acid DPPC 30 0.07 3.02E+01a 4.82E-01b 1.30E-04[95]
3.14E-04[95]
Trimethylacetic Acid DPPC 30 0.10 3.02E+01a 4.82E-01b

60
 Solute Lipid T(0C) XC Koct Kdecadiene|w|250C Plipid|w
Trimethylacetic Acid DPPC 30 0.13 3.02E+01a 4.82E-01b 4.66E-04[95]
Trimethylacetic Acid DPPC 30 0.15 3.02E+01a 4.82E-01b 5.15E-04[95]
Trimethylacetic Acid DPPC 30 0.17 3.02E+01a 4.82E-01b 6.27E-04[95]
Trimethylacetic Acid DPPC 30 0.20 3.02E+01a 4.82E-01b 8.40E-04[95]
Trimethylacetic Acid DPPC 30 0.25 3.02E+01a 4.82E-01b 1.50E-03[95]
Trimethylacetic Acid DPPC 30 0.30 3.02E+01a 4.82E-01b 1.76E-03[95]
Trimethylacetic Acid DPPC 30 0.35 3.02E+01a 4.82E-01b 2.04E-03[95]
Trimethylacetic Acid DPPC 30 0.40 3.02E+01a 4.82E-01b 1.85E-03[95]
Trimethylacetic Acid DPPC 30 0.45 3.02E+01a 4.82E-01b 1.85E-03[95]
Trimethylacetic Acid DPPC 33 0.05 3.02E+01a 4.82E-01b 5.14E-04[95]
Trimethylacetic Acid DPPC 33 0.07 3.02E+01a 4.82E-01b 4.66E-04[95]
Trimethylacetic Acid DPPC 33 0.10 3.02E+01a 4.82E-01b 9.29E-04[95]
Trimethylacetic Acid DPPC 33 0.13 3.02E+01a 4.82E-01b 1.38E-03[95]
Trimethylacetic Acid DPPC 33 0.15 3.02E+01a 4.82E-01b 1.52E-03[95]
Trimethylacetic Acid DPPC 33 0.17 3.02E+01a 4.82E-01b 1.85E-03[95]
Trimethylacetic Acid DPPC 33 0.20 3.02E+01a 4.82E-01b 2.49E-03[95]
Trimethylacetic Acid DPPC 33 0.25 3.02E+01a 4.82E-01b 3.34E-03[95]
Trimethylacetic Acid DPPC 33 0.30 3.02E+01a 4.82E-01b 3.34E-03[95]
Trimethylacetic Acid DPPC 33 0.35 3.02E+01a 4.82E-01b 3.34E-03[95]
Trimethylacetic Acid DPPC 33 0.40 3.02E+01a 4.82E-01b 2.74E-03[95]
Trimethylacetic Acid DPPC 33 0.45 3.02E+01a 4.82E-01b 2.74E-03[95]
Trimethylacetic Acid DPPC 37 0.02 3.02E+01a 4.82E-01b 1.20E-03[95]
Trimethylacetic Acid DPPC 37 0.05 3.02E+01a 4.82E-01b 2.04E-03[95]
Trimethylacetic Acid DPPC 37 0.07 3.02E+01a 4.82E-01b 4.90E-03[95]
Trimethylacetic Acid DPPC 37 0.10 3.02E+01a 4.82E-01b 6.60E-03[95]
Trimethylacetic Acid DPPC 37 0.13 3.02E+01a 4.82E-01b 8.95E-03[95]
Trimethylacetic Acid DPPC 37 0.15 3.02E+01a 4.82E-01b 8.95E-03[95]
Trimethylacetic Acid DPPC 37 0.17 3.02E+01a 4.82E-01b 1.08E-02[95]
Trimethylacetic Acid DPPC 37 0.20 3.02E+01a 4.82E-01b 1.08E-02[95]
Trimethylacetic Acid DPPC 37 0.25 3.02E+01a 4.82E-01b 8.10E-03[95]
Trimethylacetic Acid DPPC 37 0.30 3.02E+01a 4.82E-01b 6.60E-03[95]
Trimethylacetic Acid DPPC 37 0.35 3.02E+01a 4.82E-01b 6.24E-03[95]
Trimethylacetic Acid DPPC 37 0.40 3.02E+01a 4.82E-01b 4.90E-03[95]
Trimethylacetic Acid DPPC 37 0.45 3.02E+01a 4.82E-01b 4.90E-03[95]

aValues are obtained from Estimation Program Interface (EPI) Suite, accessed through ChemSpider
b Values are obtained from ACD/Labs’ logP calculator accessed through the ChemSketch interface.
c Values are collected from a source by Nitsche and Kasting[3]

61
Table 5: Permeability coefficient (Plipid|w) of phospholipids along with cholesterol at different temperatures, reported in various papers by Xiang &
Anderson [33, 34, 36, 95]

Solute χa χb χc χd χe fsolventf ln (Pm/(Kdeca*fsolv))g ln (Pm/(Koct*fsolv))

Acetic Acid 9.52 24.42 98.29 222.96 296.97 0.0737 1.40 -4.85
Acetic Acid 9.51 24.39 98.15 222.63 296.54 0.0785 3.28 -2.98
Acetic Acid 9.46 24.26 97.63 221.47 294.99 0.0985 -2.00 -8.25
Acetic Acid 9.45 24.25 97.58 221.34 294.81 0.1009 -2.04 -8.30
Acetic Acid 9.39 24.08 96.92 219.84 292.83 0.1341 -1.89 -8.15
Acetic Acid 9.39 24.07 96.87 219.74 292.68 0.1368 -1.89 -8.14
Acetic Acid 9.39 24.07 96.87 219.74 292.68 0.1368 -1.56 -7.82
Acetic Acid 9.39 24.07 96.87 219.74 292.68 0.1368 -1.33 -7.58
Acetic Acid 9.39 24.07 96.87 219.74 292.68 0.1368 -0.87 -7.12
Acetic Acid 9.39 24.07 96.87 219.74 292.68 0.1368 -0.79 -7.04
Acetic Acid 9.39 24.07 96.87 219.74 292.68 0.1368 -0.48 -6.73
Acetic Acid 9.39 24.07 96.87 219.74 292.68 0.1368 -0.33 -6.58
Acetic Acid 9.39 24.07 96.87 219.74 292.68 0.1368 -0.16 -6.42
Acetic Acid 9.39 24.07 96.87 219.74 292.68 0.1368 0.29 -5.96
Acetic Acid 9.39 24.07 96.87 219.74 292.68 0.1368 -0.16 -6.42
Acetic Acid 9.39 24.07 96.87 219.74 292.68 0.1368 -0.40 -6.66
Acetic Acid 9.34 23.97 96.45 218.79 291.42 0.1634 -1.12 -7.38
Acetic Acid 9.34 23.97 96.45 218.79 291.42 0.1634 -1.12 -7.38
Acetic Acid 9.34 23.97 96.45 218.79 291.42 0.1634 -1.04 -7.30
Acetic Acid 9.34 23.97 96.45 218.79 291.42 0.1634 -0.64 -6.89
Acetic Acid 9.34 23.97 96.45 218.79 291.42 0.1634 -0.12 -6.37
Acetic Acid 9.34 23.97 96.45 218.79 291.42 0.1634 -0.12 -6.37
Acetic Acid 9.34 23.97 96.45 218.79 291.42 0.1634 0.00 -6.25
Acetic Acid 9.34 23.97 96.45 218.79 291.42 0.1634 0.19 -6.06
Acetic Acid 9.34 23.97 96.45 218.79 291.42 0.1634 0.43 -5.83

62
 Solute χa χb χc χd χe fsolventf ln (Pm/(Kdeca*fsolv))g ln (Pm/(Koct*fsolv))
Acetic Acid 9.34 23.97 96.45 218.79 291.42 0.1634 0.58 -5.67
Acetic Acid 9.34 23.97 96.45 218.79 291.42 0.1634 0.58 -5.67
Acetic Acid 9.34 23.97 96.45 218.79 291.42 0.1634 0.27 -5.98
Acetic Acid 9.34 23.96 96.42 218.72 291.33 0.1656 -1.05 -7.30
Acetic Acid 9.33 23.93 96.31 218.47 291.00 0.1733 -1.06 -7.31
Acetic Acid 9.32 23.91 96.23 218.27 290.73 0.1799 -0.21 -6.46
Acetic Acid 9.32 23.90 96.18 218.16 290.58 0.1837 -0.18 -6.43
Acetic Acid 9.31 23.89 96.13 218.06 290.45 0.1871 -0.11 -6.37
Acetic Acid 9.30 23.86 96.04 217.85 290.16 0.1946 -0.14 -6.39
Acetic Acid 9.29 23.83 95.90 217.53 289.75 0.2061 -0.04 -6.29
Acetic Acid 9.29 23.83 95.90 217.53 289.75 0.2061 0.00 -6.25
Acetic Acid 9.29 23.83 95.90 217.53 289.75 0.2061 0.00 -6.25
Acetic Acid 9.29 23.83 95.90 217.53 289.75 0.2061 0.12 -6.14
Acetic Acid 9.29 23.83 95.90 217.53 289.75 0.2061 0.66 -5.59
Acetic Acid 9.29 23.83 95.90 217.53 289.75 0.2061 1.36 -4.90
Acetic Acid 9.29 23.83 95.90 217.53 289.75 0.2061 1.28 -4.98
Acetic Acid 9.29 23.83 95.90 217.53 289.75 0.2061 1.05 -5.21
Acetic Acid 9.29 23.83 95.90 217.53 289.75 0.2061 1.36 -4.90
Acetic Acid 9.29 23.83 95.90 217.53 289.75 0.2061 1.28 -4.98
Acetic Acid 9.29 23.83 95.90 217.53 289.75 0.2061 1.12 -5.13
Acetic Acid 9.29 23.83 95.90 217.53 289.75 0.2061 0.97 -5.28
Acetic Acid 9.29 23.83 95.90 217.53 289.75 0.2061 0.58 -5.67
Acetic Acid 9.28 23.80 95.76 217.22 289.33 0.2182 0.09 -6.16
Acetic Acid 9.27 23.79 95.73 217.15 289.24 0.2209 -0.01 -6.27
Acetic Acid 9.25 23.73 95.49 216.60 288.51 0.2442 0.80 -5.45
Butyric Acid 9.83 27.53 126.21 309.10 422.91 0.0797 -4.31 -9.37
Butyric Acid 9.75 27.32 125.26 306.76 419.71 0.1096 -4.50 -9.56
Butyric Acid 9.71 27.21 124.76 305.53 418.03 0.1293 -2.95 -8.01

63
 Solute χa χb χc χd χe fsolventf ln (Pm/(Kdeca*fsolv))g ln (Pm/(Koct*fsolv))
Butyric Acid 9.69 27.14 124.40 304.67 416.84 0.1450 -2.33 -7.40
Butyric Acid 9.64 27.02 123.86 303.33 415.01 0.1730 -1.72 -6.78
DB- 67 lactone form 11.38 47.04 383.13 1315.71 2026.26 0.0765 -14.67 -25.95
Formic Acid 9.21 22.23 81.71 175.73 229.75 0.1134 0.00 -6.15
Formic Acid 9.14 22.05 81.07 174.36 227.95 0.1577 0.40 -5.74
Formic Acid 9.09 21.93 80.63 173.42 226.73 0.1965 0.17 -5.97
Formic Acid 9.04 21.81 80.17 172.43 225.43 0.2475 0.98 -5.16
Isovaleric Acid 9.90 28.69 138.29 348.85 482.25 0.1009 -2.89 -10.00
Isovaleric Acid 9.86 28.58 137.78 347.54 480.45 0.1177 -4.21 -8.52
Isovaleric Acid 9.84 28.50 137.41 346.60 479.15 0.1314 -3.95 -8.26
Isovaleric Acid 9.81 28.42 136.99 345.56 477.71 0.1483 -3.52 -7.83
Isovaleric Acid 9.79 28.38 136.80 345.09 477.05 0.1567 -3.52 -7.83
Proplonic Acid 9.59 25.80 111.51 263.79 356.56 0.1208 -4.04 -9.25
Proplonic Acid 9.55 25.70 111.07 262.73 355.13 0.1425 -3.26 -8.48
Proplonic Acid 9.52 25.62 110.74 261.95 354.08 0.1608 -2.66 -7.88
Proplonic Acid 9.49 25.55 110.40 261.16 353.01 0.1814 -1.95 -7.16
Trimethyl acetic acid 9.90 28.69 138.33 348.94 482.38 0.0998 -6.08 -10.22
Trimethyl acetic acid 9.87 28.59 137.84 347.71 480.68 0.1154 -4.97 -9.11
Trimethyl acetic acid 9.83 28.50 137.37 346.51 479.02 0.1328 -4.29 -8.43
Trimethyl acetic acid 9.81 28.44 137.11 345.85 478.11 0.1434 -3.84 -7.98
Trimethyl acetic acid 9.77 28.30 136.42 344.13 475.73 0.1749 -2.18 -6.31
Trimethylacetic Acid 9.90 28.68 138.27 348.79 482.17 0.1016 -6.38 -10.52
Trimethylacetic Acid 9.90 28.68 138.27 348.79 482.17 0.1016 -6.23 -10.37
Trimethylacetic Acid 9.90 28.68 138.27 348.79 482.17 0.1016 -5.93 -10.07
Trimethylacetic Acid 9.90 28.68 138.27 348.79 482.17 0.1016 -5.05 -9.19
Trimethylacetic Acid 9.90 28.68 138.27 348.79 482.17 0.1016 -4.66 -8.79
Trimethylacetic Acid 9.90 28.68 138.27 348.79 482.17 0.1016 -4.56 -8.69
Trimethylacetic Acid 9.90 28.68 138.27 348.79 482.17 0.1016 -4.36 -8.50

64
 Solute χa χb χc χd χe fsolventf ln (Pm/(Kdeca*fsolv))g ln (Pm/(Koct*fsolv))
Trimethylacetic Acid 9.90 28.68 138.27 348.79 482.17 0.1016 -4.07 -8.20
Trimethylacetic Acid 9.90 28.68 138.27 348.79 482.17 0.1016 -3.49 -7.62
Trimethylacetic Acid 9.90 28.68 138.27 348.79 482.17 0.1016 -3.33 -7.46
Trimethylacetic Acid 9.90 28.68 138.27 348.79 482.17 0.1016 -3.18 -7.32
Trimethylacetic Acid 9.90 28.68 138.27 348.79 482.17 0.1016 -3.28 -7.41
Trimethylacetic Acid 9.90 28.68 138.27 348.79 482.17 0.1016 -3.28 -7.41
Trimethylacetic Acid 9.85 28.56 137.67 347.28 480.09 0.1214 -4.83 -8.97
Trimethylacetic Acid 9.85 28.56 137.67 347.28 480.09 0.1214 -4.73 -8.87
Trimethylacetic Acid 9.85 28.56 137.67 347.28 480.09 0.1214 -4.83 -8.97
Trimethylacetic Acid 9.85 28.56 137.67 347.28 480.09 0.1214 -4.14 -8.28
Trimethylacetic Acid 9.85 28.56 137.67 347.28 480.09 0.1214 -3.75 -7.89
Trimethylacetic Acid 9.85 28.56 137.67 347.28 480.09 0.1214 -3.65 -7.79
Trimethylacetic Acid 9.85 28.56 137.67 347.28 480.09 0.1214 -3.45 -7.59
Trimethylacetic Acid 9.85 28.56 137.67 347.28 480.09 0.1214 -3.16 -7.30
Trimethylacetic Acid 9.85 28.56 137.67 347.28 480.09 0.1214 -2.86 -7.00
Trimethylacetic Acid 9.85 28.56 137.67 347.28 480.09 0.1214 -2.86 -7.00
Trimethylacetic Acid 9.85 28.56 137.67 347.28 480.09 0.1214 -2.86 -7.00
Trimethylacetic Acid 9.85 28.56 137.67 347.28 480.09 0.1214 -3.06 -7.20
Trimethylacetic Acid 9.85 28.56 137.67 347.28 480.09 0.1214 -3.06 -7.20
Trimethylacetic Acid 9.80 28.39 136.88 345.29 477.33 0.1531 -3.88 -8.02
Trimethylacetic Acid 9.80 28.39 136.88 345.29 477.33 0.1531 -4.12 -8.26
Trimethylacetic Acid 9.80 28.39 136.88 345.29 477.33 0.1531 -3.59 -7.73
Trimethylacetic Acid 9.80 28.39 136.88 345.29 477.33 0.1531 -2.71 -6.85
Trimethylacetic Acid 9.80 28.39 136.88 345.29 477.33 0.1531 -2.41 -6.55
Trimethylacetic Acid 9.80 28.39 136.88 345.29 477.33 0.1531 -2.11 -6.25
Trimethylacetic Acid 9.80 28.39 136.88 345.29 477.33 0.1531 -2.11 -6.25
Trimethylacetic Acid 9.80 28.39 136.88 345.29 477.33 0.1531 -1.92 -6.06
Trimethylacetic Acid 9.80 28.39 136.88 345.29 477.33 0.1531 -1.92 -6.06

65
 Solute χa χb χc χd χe fsolventf ln (Pm/(Kdeca*fsolv))g ln (Pm/(Koct*fsolv))
Trimethylacetic Acid 9.80 28.39 136.88 345.29 477.33 0.1531 -2.21 -6.35
Trimethylacetic Acid 9.80 28.39 136.88 345.29 477.33 0.1531 -2.41 -6.55
Trimethylacetic Acid 9.80 28.39 136.88 345.29 477.33 0.1531 -2.47 -6.61
Trimethylacetic Acid 9.80 28.39 136.88 345.29 477.33 0.1531 -2.71 -6.85
Trimethylacetic Acid 9.80 28.39 136.88 345.29 477.33 0.1531 -2.71 -6.85
Valeric Acid 9.98 28.91 139.39 351.61 486.07 0.0725 -5.25 -9.69
Valeric Acid 9.90 28.70 138.37 349.04 482.53 0.0986 -3.36 -7.80
Valeric Acid 9.86 28.59 137.81 347.63 480.58 0.1164 -3.05 -7.49
Valeric Acid 9.83 28.50 137.37 346.51 479.02 0.1328 -3.30 -7.75
Valeric Acid 9.81 28.42 137.03 345.66 477.84 0.1467 -2.76 -7.21

a χ is the chain ordering effect and the values are obtained using the equation , when n=0.1

b
χ is the chain ordering effect and the values are obtained using the equation , when n=0.33

c χ is the chain ordering effect and the values are obtained using the equation , when n=0.67

d χ is the chain ordering effect and the values are obtained using the equation , when n=0.87

e χ is the chain ordering effect and the values are obtained using the equation , when n=0.94

f is the solvent factor for the diffusion and partition coefficient due to the temperature difference and is obtained using the equation

g the normalized permeability is calculated from the permeability data sets obtained from research papers of Xiang & Anderson [33, 34, 36].

66
Table 6: Table of fits of the chain ordering coefficient and the permeability of the pure PCs.

N r2 (deca) r2 (oct) Equn based on deca F(χ)

0.1 0.8285 0.5746 y = -6.47x + 59.599 7.647x1025 exp(-6.47 χ)
0.33 0.7622 0.4677 y = -0.709x + 16.119 10.00x106 exp(-0.709 χ)
0.67 0.7407 0.4358 y = -0.0793x + 6.7857 885.01 exp(-0.0793 χ)
0.87 0.734 0.4256 y = -0.0256x + 4.738 114.205 exp(-0.0256 χ)
0.94 0.7319 0.4225 y = -0.0174x + 4.2301 68.724 exp(-0.0174 χ)
1 0.7303 0.4200 y = -0.0126x + 3.8522 47.09 exp(-0.0126 χ)

where y is the value of and and x is the value of .

Table 7: Error analysis for acetic acid for DPPC and cholesterol bilayers at various temperatures

XC Temp log Pma log Pmb ERRORc log Pmd ERRORc

0 30 -4.57 -4.57 0.0000 -4.57 0.0000
0.03 30 -4.43 -4.57 0.0197 -4.57 0.0197
0.07 30 -4.57 -4.57
0.08 30 -4.45 -4.46
0.09 30 -4.60 -4.38
0.1 30 -4.33 -4.29 0.0018 -4.31 0.0005
0.15 30 -4.13 -4.03 0.0092 -4.06 0.0041
0.17 30 -4.09 -3.96 0.0178 -3.99 0.0099
0.2 30 -3.96 -3.87 0.0076 -3.91 0.0026
0.25 30 -3.89 -3.82 0.0053 -3.86 0.0012
0.3 30 -3.82 -3.82 0.0000 -3.86 0.0013
0.35 30 -3.62 -3.82 0.0393 -3.86 0.0556
0.4 30 -3.82 -3.82 0.0000 -3.86 0.0013
0.45 30 -3.92 -3.82 0.0109 -3.86 0.0045
Total error 0.1118 0.1006
0 33 -4.16 -4.16 0.0000 -4.16 0.0000
0.05 33 -4.16 -4.16 0.0000 -4.16 0.0000
0.07 33 -4.13 -4.16 0.0011 -4.16 0.0011
0.08 33 -4.06 -4.07
0.09 33 -3.98 -3.99
0.1 33 -3.95 -3.91 0.0017 -3.92 0.0009
0.125 33 -3.72 -3.77 0.0026 -3.79 0.0045
0.15 33 -3.72 -3.67 0.0027 -3.69 0.0011
0.17 33 -3.67 -3.60 0.0047 -3.62 0.0024
0.2 33 -3.59 -3.52 0.0049 -3.54 0.0023
0.22 33 -3.49 -3.47 0.0003 -3.49 0.0000

67
 0.25 33 -3.42 -3.47 0.0025 -3.49 0.0051
0.3 33 -3.42 -3.47 0.0025 -3.49 0.0051
0.4 33 -3.55 -3.47 0.0071 -3.49 0.0039
Total Error 0.0301 0.0265
0 37 -3.59 -3.59 0.0000 -3.59 0.0000
0.01 37 -3.57 -3.59 0.0003 3.59 0.0003
0.05 37 -3.57 -3.59 0.0003 -3.59 0.0003
0.06 37 -3.52 -3.59 0.0045 -3.59 0.0045
0.07 37 -3.59 -3.59
0.08 37 -3.55 -3.51
0.09 37 -3.51 -3.45
0.1 37 -3.29 -3.47 0.0341 -3.39
0.12 37 -2.89 -3.41 0.1793 -3.30 0.0114
0.15 37 -3.02 -3.33 0.0952 -3.19 0.0995
0.16 37 -3.12 -3.30 0.0340 -3.16 0.0289
0.19 37 -2.98 -3.24 0.0643 -3.07 0.0015
0.2 37 -3.02 -3.22 0.0400 -3.05 0.0080
0.22 37 -3.18 -3.00 0.0010
0.25 37 -3.08 -3.18 0.0092 -3.00 0.0066
0.3 37 -3.15 -3.18 0.0008 -3.00 0.0219
0.4 37 -3.32 -3.18 0.0193 -3.00 0.0999
Total Error 0.4814 0.2838

aThe values of permeability are calculated experimentally and are collected from the research papers of Xiang & Anderson [33,
34, 36, 95].

b The values of permeability are calculated based on the theory proposed; i.e permeability coefficient has linear dependence on
cholesterol relation, , where and are the permeability coefficients in gel and liquid

ordered phase

c errors are estimated by the square of the difference in permeability coefficient

d the value of permeability coefficients obtained when the error calculated from c is minimized by varying

68
Table 8: Prediction of permeability in the liquid ordered phase (theory based on Chapter 5).

Solute Temp( 0C ) MW log Pgela log Pfluidb log Ploc Geo Meand log(Plo/Pgel)e Averagef
AA 30 60.05 -4.57 -2.07 -3.858 -3.319 0.710
AA 33 60.05 -4.16 -1.98 -3.492 -3.071 0.670 0.657
AA 37 60.05 -3.59 -1.87 -3.003 -2.730 0.590
TMAA 30 102.13 -3.96 0.18 -2.871 -1.888 1.090
TMAA 33 102.13 -3.41 0.27 -2.533 -1.569 0.880 0.917
TMAA 37 102.13 -2.82 0.39 -2.035 -1.213 0.780

a Value of permeability coefficient is collected from the research paper of Xiang & Anderson[95].

b Value of permeability coefficient in the fluid is calculated based on the correlations provided by Nitsche & Kasting [3], assuming gel phase temperatures.

c Value of permeability coefficient is predicted by the theory proposed in Chapter 5.

d Geometric mean of the permeability coefficients obtained in a and b.

e Difference of the log of permeability coefficients obtained in a and c.

f Average of the log of permeability coefficients values for acetic acid marked in blue and trimethyl acetic acid marked in red.

69
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82
Nomenclature
Partition coefficient of the solute across the lipid bilayer relative to water

Solute diffusion coefficient in the lipid bilayer

Bilayer thickness

Melting Temperature

Area of the phospholipid membrane per phospholipid molecule

Head-to- Head thickness

Hydrocarbon chain thickness

Luzzati Thickness

Volume of the Lipid bilayer

Are of the phospholipid membrane at reference temperature

Normalized phospholipid surface density

Area of the phospholipid bilayer in the perpendicular direction

Chain tilt

Mole fraction of cholesterol in the bilayer mixtures

Relative bilayer surface density, see equation 5.14

Liquid ordered phase

Liquid disordered phase

Permeability coefficient of the solute across the phospholipid bilayer

Free surface area, see equation 4.2

Total bilayer area (intercalated with cholesterol) per phospholipid molecule

Chain ordering factor, ratio of solute size to void space within the barrier region, see equation
4.4

83
Appendix
Estimation of the permeability coefficient
The quantitative estimation of the permeability coefficients that have been tabulated (Table

2,3,4,5,6,7,8 & 9) in the main text is briefed below. The dependence of the surface density on the
28,
partitioning and permeability of a solute, acetic acid has been briefed by Xiang and Anderson
48, 49, 55, 56
. From the data provided, the graph of vs , we estimate the permeability

coefficient at various temperatures can be found using the numerical analysis. From the discrete

set of the permeability points, we use linear interpolation the permeability coefficients at

temperatures below the melting transition temperature, .

Here, and are permeability coefficients of acetic acid at various temperatures and .

Choice of DPPC
One of the most important facts in the biophysics is the diversity of the lipids that occur in

epidermis layer. Various authors have published the lipid compositions in the various layers of the

epidermis, majorly as the phospholipids and cholesterol. Gray & Yardley have proclaimed that

phospholipids & cholesterol constitute around 60-65 wt% of the human dermis; out of which

phospholipid make up for 53 % and cholesterol for about 8-12 % [114, 115]. The characterization

of the lipid bilayers in gel phase at physiological temperatures (~370C) is majorly performed on

DPPC bilayers. We have restricted the calculations on DPPC as our model phospholipid due to its

availability in the gel phase at the physiological conditions, given the of the DPPC at 41.40C.

84