Roma, SS 2006
Pier Luigi Luisi
Aggregati macromolecolari di
tensioattivi ; self-assembly e
loro applicazioni
La importanza delle vescicole e
liposomi
come modelli per le cellule
biologiche
1
amphiphilic molecules
hydrophilic
H2O
oil
hydrophobic
hexanol
90
10
70
30
50
50
30
70
90
water 10
10
30
50
70
90
CTAB
2
Industrial uses dominate surfactant demand
Personal care
Soaps 10%
Shampoos 6%
Cosmetics & toiletries 1%
Household
Laundry 16%
Dishwashing 7%
Other 6%
54%
Total 1988 demand =
7.59 billion Ib
Source: Freedonia Group
17%
29%
Industrial
Industrial processing 43%
Cleaning products 6%
Food processing 5%
3
Anioics comprise almost two thirds of U.S. surfactant
production while nonionics grab majority of sales
Amphoteric
Total 1988 production =
7.32 billion Ib
Cationic
Nonionic
Anionic
Total 1988 salesa =
$2.30 billion
a Sales of 4.26 billion Ib. Source: U.S. International Trade Commission
4
d = 5 cm
Organic phase
Interface surface
A = 19.6 cm2
Water phase
Micellar surface
A = 19.6  10-14 cm2
r = 12.5 Å
Spherical Micelle of caprylate ions
1.7  10-10
8.5  10-8
8.5  10-5
1.0  10-3
Micellar surface in a
litre of solution
19.6 cm2
1.0 m2
103 m2
0.012 Km2
equals a surface of a
passport
photo
desk
swimming
pool
stadium
[Micelles](mol/lit.)
5
two personal reasons of
fascination
1. self-organization: spontaneous formation of ordered structures
(…evolution, origin of life…)
cmc
2. compartmentation (microheterogenous reactions...)
B
A
A+B
?
6
Surfactat molecule
lipophilic chain
CH3
CH2
polar head
aqueous micelles
CH2
COOCH2
CH
CH2
CH2
CH2
CH3
CH
COOCH2
CH
CH2
CH2
CH2
CH3
SO3
Na+
CH2
CH3
Aerosol-OT (AOT)
bis(2-ethyl-hexyl)sodium sulfosuccinate
reverse micelles
typical conditions:
water
pool
hydrocarbon
Isooctane
25 - 100 mM AOT
0.5 - 2 % water
W0 =
[H2O ]
[AOT ]
Reverse micelles are fairly monodisperse, dynamic
aggregates which can solubilize relatively large (~10%)
amount of water (microemulsions)
7
Micelles from sodium laurylsulfate (SDS)
Average radius of a micelle (RH)
Average aggregation number
Approximate relative mass of a micelle (Mr)
Average half-time of a SDS molecule
in the micelle
CMC (25°C, H2O)
i.e.: monomer concentration by
10g SDS/l (35mM)
2.2 nm
62
1.8  104
0.1 ms
8.1  10-3 M
2.3 g/l
8
MOLECULAR ARCHITECTURE of the animal-cell membrane is determined
primarily by the interactions of phospholipid molecules in water. Phospholipids can
minimize their energy in water by forming a bilayer about 40 angstrom units thick.
The hydrophobic tails of the molecules sequester themselves on the inside of the
bilayer and the hydrophilic heads (blue) face the water on both sides of the
bilayer. If any edge of the bilayer were open to the water, hydrophobic tails along
the edge would be exposed; hence the bilayer closes to form a vesicle, effectively
segregating fluid inside the vesicle from fluid surrounding it.
9
Liposomes (SUV) from egg Lecithin
External radius of a liposome (RH)
50 nm
Approximate aggregation number of
lecithin molecules per liposome
81`900
Approximate relative mass of the
liposome-shell (Mr)
Average residence life of a lecithin
molecule in the liposome
CAC
i.e.: monomer concentration by 10 g lecithin/l (13 mM)
6.6  107
3h
5.0  10-10M
10
self assembly may be described in terms of the curvature which
exists at the hydrocarbon-water interface
the surface packing parameter
v/AL
(*)
A
A: head group area
V
L
L: lengh (fully extend.)
V: volume of the hydrocarbon chain(s)
(*) Mitchell & Ninham, J. Chem. Soc. Farad. Soc. 11 (1981) 77, 601 11
Mean (dynamic) packing shapes of lipids and the stuctures they form
Lipid
Single-chained lipids (surfactants)
with large head-group areas:
SDS in low salt
Single-chained lipids with small headgroup areas:
SDS and CTAB in high salt,
nonionic lipids
Double-chained lipids with large headgroup areas, fluid chains:
Phosphatidyl choline (lecithin),
phosphatidyl serine, phosphatidyl
glycerol, phosphatidyl inositol,
phosphatidic acid, sphingomyelin,
DGDG a, dihexadecyl phosphate,
dialkyl dimethyl ammonium salts
Double-chained lipids with small headgroup areas, anionic lipids in high salt,
saturated frozen chains:
phosphatidyl ethanolamine, phosphatidyl
serine +Ca2+
Double-chained lipids with small headgroup areas, nonionic lipids, poly (cis)
unsaturated chains, high T:
unsat. phosphatidyl ethanolamine,
cardiolipin +Ca2+ phosphatidic acid
+Ca2+ cholesterol, MGDG b
a
Critical packing
parameter v/aolc
Critical
packing shape
Cone
< 1`3
v
a0
lc
Structures
formed
Spherical
micelles
Truncated
cone
1/3-1/2
Truncated
cone
1/2-1
Flexible bilayers, vesicles
Cylinder
Planar
bilayers
~1
Inverted
truncated
cone
or wedge
>1
Inverted
micelles
12
b
DGDG, digalactosyl diglyceride, diglucosyl diglyceride; MGDG, monogalactosyl diglyceride, monoglucosyl diglyceride.
hydrophobic forces as the main factors for the association
of surfactant molecules
water
oil
"free" water
oil
H 2O
H2
O
13
Two main ordering processes
polymerization
 S0 < 0
self-assembly
 S0 > 0
 G 0 =  H0 - T  S < 0
14
hydrophobic forces:
attractive intereactions between
apolar compounds in water
important in membranes
protein folding
DNA duplex
all aggregation forms
G° < 0 !
INCEASE IN ENTROPY
water
made "free"
15
reaction features of
surfactant aggregates
4. The local concentration effect
(micelles as scavengers....)
Oil
+
H 2O
-
reverse micelles: all water-soluble
compounds will be forced in
the water pool
-
H2O
H2O
aqueous micelles: the lipophilic
compounds will be uptaken
by the oil-droplet (soap effect)
....or by the lipophilic
bilayer membrane
16
reaction features of surfactant aggregates
5. Catalysis
hydrophobic
OHOH-
OH- in a
lipidic environment
-more powerful nucleophile
H+
micellar catalysis
H+
H2O
OH-
H 2O
see hydrolysis of
esters & anhydrides....
17
Aggregate
N, XN, °N
kl
Monomer
kN
N=1
Xl
°l
association of N monomers in a micelle
for a thermodynamic treatment see:
J.N. Israelechvili
“Intermolecular & Surface Forces”
N.Y., Acad. Press 1985
18
For a system of micelles with aggregation number i we can write for
Gibb`s free energy:
n
G =  Ni i
1
Ni = number of micelles i with chem. pot. i
For an ideal solution is also
i = oi + kT In i
Xi = molar fraction of micelles with aggregation number i
By minimizing G with constant numbers of monomers
i = i 1
i = chem. pot. of a monomer in solution on
It is thus possible to obtain the fundamental equation for describing the size
distribution of the micelles:
n = n1· exp
on- no1
RT
from mixing entropy.probability that n monomers are together in one
aggregate. 1  10-5, very small!
19
so that, in first approximation,
on- no1
A very useful parameter, A
A = exp

for no < n
ono- no1 1
no – 1
RT
because it expresses the relative concentrations , 1 and no
A = critical micelle concentration, c.m.c.
with a sharp change in the concentration of the monomers and/or the
micelles.
Physical meaning:
(on- no1) / (no – 1)
is the gain of the chemical potential for a monomer, present in the
micelle, with respect to the monomer in solution.
20
n = n1· exp
from
mixing entropy
contribution; probability that n
monomers are together in an
aggregate.
Very small  10-5.
The formation of micelles is
entropically unfavorable.
on- no1
RT
“Boltzmann factor”
energetically favorable intereaction
of the monomers in the micelle,
with respect to the intereaction
with the solvent.
only for on< n·o1 can we have aggregates; till a minimal
micelle size no the formation of micelles will be unfavorable
21
cmc is generally given in molarity
several experimental methods:
Physical property
molecular weight (average)
scattering
conducibility
spectroscopy
½
cmc
[surf.]
Typical values are in the millimolar range 10-3 M;
but there are cases 10-5 M or smaller
22
The determination of the cmc for soap molecules by using pinacyanol chloride
(solubilisation of dye molecules by the micelles, e.g. [30-33])
“The absorption spectrum of pinacyanol chloride in aqueous solution of anionic soaps changes sharply to that
characteristic of its solutions in organic solvents over a short range of soap concentration ( max ~ 610nm). This
effect is attributed to the formation of micelles, in whose hydrocarbon-like layers or cores the dye is solubilized.
The concentration of soap at which this spectral change occurs is taken as `the critical concentration for the
formation of micelles`. …”
CH3
CH3
N
+
Cl
N
pinayanol chloride
The cmc of sodium laurate (=sodium dodecanoate) at 50 °C in water, [33]:
CH3(Ch2)10COO-Na+
“…Each of the laurate solutions was equilibrated in a cuvette at 50 °C
inside a spectrophotometer and the absorbance at 610 nm was adjusted to
zero. A methanolic solution of pinacyanol chloride was added to obtain a
dye concentration of 10.5 M. An absorbance reading was then taken. A
plot of absorbance vs. laurate concentration shows a striking change at
9 mM (cmc). A spectrophotometer is in fact unnecessary for the cmc
determination: above the cmc the solutions are bright blue, while below it
they are a light shade of pink. …”
The absorbance of 1.05 X 10-8 M pinacyanol
chloride at 610.0 m in pH 9.59 sodium
borate buffer (l = 0.1) at 50.0° vs. laurate
concentration.
The use of dyes for the determination of cmc-values may lead to micelle
formation at a concentration below the “true” cmc. ”…In practice, the
method gives only a rough approximation of the cmc. …”
23
An overview on vesicles
and liposomes
(liposomes: vesicles made out of lipids)
24
25
PHOSPHOLIPID MOLECULE is
the primary structural element in
all cell membranes. Four main
kinds of phospholipid are found in
animal-cell-membranes. The one
shown at the left in the diagram is
phosphatidylcholine, but the other
tree differ from it and from one
another only in the chemical
structure of their head groups,
which are diagrammed here as
colored spheres. The electric
charge in each head group makes
the group hydrophilic. The head
group is connected to a glycerol
group, and two hydrocarbon
chains are attached in turn o
glycerol. The hydrocarbon chains
are oily and therefore
hydrophobic.
26
O
O
O P
O
O
H
N
O
O-
+
POPC
O
hydrophobic
(lipophilic)
hydrophilic
(lipophobic)
ONa
O
+
sodium oleate
+ oleic acid
OHO
27
COO -
caprylate
or
oleate
CH 3(CH 2)7 -CH = CH-(CH 2)7COO -
form micelles at alkaline pH
COO (Deamer, 1976)
vesicles at pH=7-8
_ pk)
(pH ~
COO -
HOOC
precursors ( water insoluble! )
R
OH -
CO
micelles or
O
R CO
or
R
COOR'
R COO OH -
vesicles
28
Soaps self-assemble into
micelles as soon as the
cmc is reached.
12
11
Fatty acids are almost
insoluble in water.
pH
10
Mixtures of fatty acids
and the corresponding
soaps assemble into
vesicles, at
concentrations above the
cvc.
9
8
vesicles
micelles
7
-0.2
0.0
0.2
0.4
0.6
0.8
HCl (equivalent)
Equilibrium titration curve of
sodium oleate at 25 °C
29
Cross-sectional views of the three structures that can be formed by
mechanically dispersing a suspension of phospholipids in aqueous solution
Micelle
Liposome
Bilayer sheet
The red circles depict the hydrophilic heads of phospholipids, and the
squiggly lines (in the yellow region) the hydrophobic tails.
30
Liposomes, as closed spherical
bilayers, are considered the most
likely precursors of early living cells
(protocells)
LIPOSOMES ARE JUST TINY SOAP BUBBLES,
50-500 nm radius
31
cac
surfactant
ionic
amphiphilic
hydrophilic
32
A realistic scenario of the emergence of
life can be based on a gradual transition
from random mixtures of simple organic
molecules to spatially ordered assemblies
displaying primitive forms of cellular compartimentation,
self-reproduction and catalysis
33
Liposom / Vesikel
ao
v
lc
v
½<
<1
aolc
J.N. Israelachvili, D.J. Mitchell, B.W. Ninham (1976).
34
500 nm
= 0.5 m
1-10 M
MLV
400 nm
200 nm
20 nm
SUV
LUV
100 nm
35
dynamic of a liposome-membrane
FlipFlop
lateral
diffusion
rotation
diffusion
vertical “vibration”
(amplitude 0.3 nm)
DPPC (T>Tm)
vertical “vibrations”, jump time
 10-10 s
rotation correlation time (c)
 10-9 s
lateral diffusion coefficient
Flip-Flop time
E. Sackmann (1978, 1991)
 7·10-8 cm2 s-1
(wandered  4 m per second)
 8 hours
36
temperature-sensitive phase transmutation of a
liposome-membrane
Tv
L´
crystalline
phase
Tm
P
“Ripple”-phase
(“quasi-crystalline”)
L
fluid phase
(“fluid-crystalline”)
described as gel-phase too
all-trans- (anti-)
confirmations
trans- and gaucheconfirmations
E. Sackmann (1978);
R.R.C. New (1990)
37
permeability of the liposome-membrane
lecithine (T>Tm, pH – 7)
generally:
The permeability for polar, charged molecules and for molecules
with a high molecular weight is small.
Maximal permeability: T=Tm
water
glycerin
urea
tryptophan
glucose
ClLysin
Na+
permeability
coefficient (cm·s-1)
4·10 -3
5·10 -6
4·10 -6
4·10 -10
 10-11
7·10-12
5·10-12
1·10-12
Brunner, D.E. Graham, H. Hauser, G. Semenza (1980); G. Cevc,
Marsh (1987); A.C. Chakrabarti, D.W. Deamer (1992)
38
Are aqueous micelles chemical
equilibrium systems?
How can you demonstrate this?
..the
case of micelles is straightforward
39
You have two preparations of liposomes,
Extruded to 50 nm, resp. 200 nm.
You mix the two solutions.
What happens ?
Does the system reach a mixed state having the
Energy minimum?
O0r: do the two populations remain in solutions
As they are initially?
40
41
what is then the general picture that emerges in the case of oleate
vesicles, a system which is a mixed situation-partly equilibrium, partly not?
nM
Mn
irreversible
nM
yes (Knappl)
irreversible
M`n
Mn
G°
no
Rk
42
to make liposomes is easy
stock surfactant
in water
or methanol
H2O
size distribution
extrusion
through filters
H2O
"film" of
surfactant
relatively monodispers
43
liposomes = vesicles from lipids
REVERSE PHASE EVAPORATION
monomers in
organic solvent
reverse phase
org.
solvent
water
evaporation
hydration
formation of
vesicles
44
Freeze / thaw entrapment method
5x freeze / thaw
Extrusion
45
Liposomes prepared by the “extrusion method“
“LiposoFast“, a small-volume extrusion apparatus
46
47
Size distribution of POPC liposomes prepared by injecting
50 µL of alcoholic solution of 25 mM POPC
into a 0.1 M borate buffer solution, pH 8.5.
[POPC]final 0.5 mM, 2% (v/v) alcohol; measuring angle 90°.
(1) 2,3-butanediol
(2) ethanol
(3) 1,2-propanediol
(4) PEG 200
(5) methanol
48
Spontaneous vesiculation and self-replication
0.40
O
OH
oleic acid
buffer
OD 500 nm
neat surfactant
0.30
0.20
0.10
0.00
0
50
100
150
time (min)
200
increased
solubilisation
spontaneou
s
vesiculation
autocatalytic
population
increase
49
OCH3 O
O
P
+K - O
O (CH2)9CH3
O
O2N
O
KOH 0.2 M
h
P
O (CH2)9CH3
1
+
O
(CH2)9CH3
(CH2)9CH3
O K+
OCH3
2
O2N
3
spontaneous
formation of
vesicles
J. Phys. Chem. B 1998, 102, 7078-7080
50
PHOSPHOLIPONUCLEOSIDES
INVESTIGATED
O
NH2
N
N
O
OH
NH
N
N
N
O
O
OH
OH
Adenosine
OH
Uridine
O
O
O
O P
O
O
O
O_
NH4+
51
O
OO
O
O
P
O
+
N
O
NH2
N
O
HO
+
CHCl3 - H2O, pH 4.5
45 °C, 6 h
HO
O
N
O
OH
Phospholipase D aus
Streptomyces sp. AA 586
NH2
O
N
OO
O
O
P
O
O
N
O
O
HO
O
+
HO
OH
+
N
52
How do you entrap drugs or biochemicals
inside liposomes?
53
Operational
ENZYME
OD
ENZYME
SOLUTION
E
E
oleate film
E
E
make liposomes
(vortex ca. 30”)
E
fractions
E
E
chromat.
Sepharose 4B
-Enzyme free
E
E
oleate film
poly (A)
E
EE
no free
enzyme
outside
E
time
E
EE
chromat.
Sepharose 4B
- ADP free
E
E
ADP
sampling
for poly (A)
E
no external ADP
EE
incubation with ADP
54
Dehydration / Rehydration method for
solute entrapment
Dehydration
Fusion
Rehydration
Extrusion
55
Dispersion of a thin film of POPC in H2O
A
B
C
56
Injection of an ethanolic solution of POPC into H2O
(“ethanol injection method”)
water
EtOH
POPC
Removal of EtOH
by dialysis or
gel permeation
Chromatography
57
3933-3935
The hydrophobicity of the lipid bilayer
Is the main driving force for
The activity/reactivity and applications
Of liposomes
58
Cross-sectional views of the three structures that can be formed by
mechanically dispersing a suspension of phospholipids in aqueous solution
Micelle
Liposome
Bilayer sheet
The red circles depict the hydrophilic heads of phospholipids, and the
squiggly lines (in the yellow region) the hydrophobic tails.
59
LIPOSOMES AS DRUG-DELIVERY AGENTS
CELL
D
D
DD
ENDOCYTOSIS
D
D
DD
60
The hydrophobic effect of the membrane as a further
ordering principle:
It selects out of the bulk solution the most hydrophobic
compounds, forming stable complexes
61
This can give rise to selfreproduction processes
Additionally, these ordered
structures are able to pick up
and order hydrophobic
di- and tripeptides
62
Gene transfer
Is usually done with
Positively charged liposomes
e.g., DDAB
63
.
DNA / LIPOSOMES
+
+
+
+
POPC
+
+
+
+
DNA
POPC / DDAB
DNA
64
65
reaction features of
surfactant aggregates
3. forced compartmentation
of reagents
e.g.
and other charged species
cannot go through
HPO4
O
HPO4
....but uncharged
molecules go through
HPO4
HO
CN
CN
CH2OPO3
and the (charged)
reaction product is
blocked inside !
66
reaction features of
surfactant aggregates
2. The concentration gradient
e.g.
POPC liposomes
HPO4
0.5 M
HPO4 = 10-3 M
=
( OSMOTIC BALANCE is
HOWEVER NECESSARY )
IN
OUT
POPC liposomes
pH = 9.0
these gradients can
be kept for days
or weeks....
pH = 5.0
IN
67
OUT
EXAMPLE OF THE BILAYER HYDROPHOBIC
EFFECT IN CATALYSIS
POPC LIPOSOMES, OR OLEATE / OLEIC ACID VESICLES
BINDING THE WEAKLY CATALYTICALLY
ACTIVE TRIPEPTIDE
H-Phe-His-Leu-OH
AND THE POORLY WATER SOLUBLE
SUBSTRATE
HEXADECYL-P-NITRO-PHENYL ESTER
DUE TO THE SOLUBILIZATION EFFECT,
A HIGH LOCAL CONCENTRATION AND A
HIGH PROXIMITY IS OBTAINED ON THE
LIPIDIC MATRIX
68
Dependency of the initial hydrolysis rate (vin) for the hydrolysis of C16-ONp on
the substrate concentration in spontaneously formed oleic acid/oleate vesicles
([oleic acid] + [oleate] = 20 mM) at 25 °C. The vesicles were prepared in a 0.1 M
borate solution (pH 8.5), either in the absence (filled circles) or in the presence
(open squares) of 1 mM Z-Phe-His-Leu-OH.
69
70
71
reaction features of
surfactant aggregates
.
5. Catalysis
OH-
OH-
OH-
in a
{ hydrophobic
lipidic environment
- more powerful nucleophile
micellar catalysis
H+
H+
OH-
H2O
O CO
CO
see hydrolysis of
esters & anhydrides....
H2O
72
CTP
GTP
ATP
TTP
a
DNA
Plasmid
E
CTP
GTP
ATP
UTP
b
E
RNA
E
RNA
t-RNAPhe
c
Phe
Poly U
(Phe)n
t-RNAPhe
Rib
Ei
Rib
Ei
73
THE IMPORTANCE OF HAVING A BOUNDARY
particular
chemophysical
properties of the
inside (D, µ, 
OUT
IN
H2O
C
A
A
B
B
C A
binding of
hydrophobic
substances
catalysis
selective
permeability
no leakage
gradient
physical
protection
inside different
first step towards
from outside
: the definition of self
... just imagine that cells would all open up:
where would life go?
entrapment & vicinity
or reagents
(high local concentration)
74