1

Roma, SS 2006Pier Luigi Luisi

Aggregati macromolecolari di tensioattivi ; self-assembly e loro applicazioniLa importanza delle vescicole e liposomicome modelli per le cellule biologiche

2

amphiphilic molecules

H2O

oil

hydrophilic

hydrophobic

10

30

50

70

90 10

30

50

70

90

10 30 50 70 90 CTAB

hexanol

water

3

54% 29%17%

Industrial uses dominate surfactant demand

HouseholdLaundry 16%Dishwashing 7%Other 6%

Personal careSoaps 10%Shampoos 6%Cosmetics & toiletries 1%

IndustrialIndustrial processing 43%Cleaning products 6%Food processing 5%

Total 1988 demand =7.59 billion Ib

Source: Freedonia Group

4

Amphoteric

Cationic

Nonionic

Anionic

Anioics comprise almost two thirds of U.S. surfactantproduction while nonionics grab majority of sales

Total 1988 production = 7.32 billion Ib

Total 1988 salesa =$2.30 billion

a Sales of 4.26 billion Ib. Source: U.S. International Trade Commission

5

d = 5 cm

Organic phase

Interface surfaceA = 19.6 cm2

Water phase

Micellar surfaceA = 19.6 10-14 cm2

[Micelles](mol/lit.) 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

Spherical Micelle of caprylate ions

r = 12.5 Å

6

two personal reasons of fascination

1. self-organization: spontaneous formation of ordered structures (…evolution, origin of life…)

cmc

2. compartmentation (microheterogenous reactions...)

AB

A+B ?

7

Surfactat moleculeSurfactat molecule

polar headpolar head

lipophilic chainlipophilic chain

aqueous micellesaqueous micelles

reverse micellesreverse micelles

waterwaterpoolpool

hydrocarbonhydrocarbon

CCHH22 CCHHCCOOOOCCHH22 CCHH33CCHH22 CCHH22 CCHH22

SSOO33 CCHH22

CCHH33

CCHH CCHHCCOOOOCCHH22 CCHH33CCHH22 CCHH22 CCHH22

NNaa++

CCHH22

CCHH33

Aerosol-OT (AOT)Aerosol-OT (AOT)

bis(2-ethyl-hexyl)sodium sulfosuccinatebis(2-ethyl-hexyl)sodium sulfosuccinate

typical conditions:typical conditions: IsooctaneIsooctane25 - 100 mM AOT25 - 100 mM AOT0.5 - 2 % water0.5 - 2 % water WW00 = =

HH22OO[[ ]]

AOTAOT[[ ]]

Reverse micelles are fairly monodisperse, dynamicReverse micelles are fairly monodisperse, dynamicaggregates which can solubilize relatively large (~10%)aggregates which can solubilize relatively large (~10%)amount of water (microemulsions)amount of water (microemulsions)

8

Micelles from sodium laurylsulfate (SDS)

Average radius of a micelle (RH)Average aggregation numberApproximate relative mass of a micelle (Mr)Average half-time of a SDS moleculein the micelleCMC (25°C, H2O)i.e.: monomer concentration by 10g SDS/l (35mM)

2.2 nm62

1.8 104

0.1 ms

8.1 10-3 M2.3 g/l

9

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.

10

Liposomes (SUV) from egg Lecithin

External radius of a liposome (RH)

Approximate aggregation number oflecithin molecules per liposome

Approximate relative mass of the liposome-shell (Mr)

Average residence life of a lecithin molecule in the liposome

CACi.e.: monomer concentration by 10 g lecithin/l (13 mM)

50 nm

81`900

6.6 107

3 h

5.0 10-10M

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self assembly may be described in terms of the curvature whichexists at the hydrocarbon-water interface

the surface packing parameter

v / A L (*)

A

V L

A: head group area

L: lengh (fully extend.)

V: volume of the hydrocarbon chain(s)

(*) Mitchell & Ninham, J. Chem. Soc. Farad. Soc. 11 (1981) 77, 601

12

Lipid Critical packing

parameter v/aolc

Critical

packing shape

Structures

formed

Single-chained lipids (surfactants)

with large head-group areas:

SDS in low salt

< 1`3

Single-chained lipids with small head-group areas:

SDS and CTAB in high salt,

nonionic lipids1/3-1/2

Double-chained lipids with large head-group areas, fluid chains:

Phosphatidyl choline (lecithin), phosphatidyl serine, phosphatidyl

glycerol, phosphatidyl inositol, phosphatidic acid, sphingomyelin,

DGDG a, dihexadecyl phosphate,

dialkyl dimethyl ammonium salts

1/2-1

Double-chained lipids with small head-group areas, anionic lipids in high salt,

saturated frozen chains:

phosphatidyl ethanolamine, phosphatidyl serine +Ca2+

~1

Double-chained lipids with small head-group areas, nonionic lipids, poly (cis)

unsaturated chains, high T:

unsat. phosphatidyl ethanolamine, cardiolipin +Ca2+ phosphatidic acid +Ca2+

cholesterol, MGDG b

>1

a DGDG, digalactosyl diglyceride, diglucosyl diglyceride; b MGDG, monogalactosyl diglyceride, monoglucosyl diglyceride.

Cone a0

v lc

Truncated cone

Truncated cone

Cylinder

Invertedtruncatedconeor wedge

Sphericalmicelles

Flexible bilayers, vesicles

Planar bilayers

Inverted micelles

Mean (dynamic) packing shapes of lipids and the stuctures they form

13

hydrophobic forces as the main factors for the associationof surfactant molecules

water "free" water

H2O H2O

oil oil

14

Two main ordering processesTwo main ordering processes

polymerizationpolymerization

self-assemblyself-assembly

S0 > 0

S0 < 0

G0 = H0 - T S < 0

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hydrophobic forces:attractive intereactions betweenapolar compounds in water

important in membranes protein folding DNA duplex all aggregation forms

watermade "free"

G° < 0 ! INCEASE IN ENTROPY

16

reaction features ofsurfactant aggregates

4. The local concentration effect (micelles as scavengers....)

reverse micelles: all water-soluble compounds will be forced in the water pool

aqueous micelles: the lipophilic compounds will be uptaken by the oil-droplet (soap effect)

....or by the lipophilic bilayer membrane

H2O

-

+

--

Oil

H2O

H2O

17

reaction features of surfactant aggregates

5. Catalysis

OH-

OH-

H+H+

OH-O

CO

CO

H2O

H2O

hydrophobicOH- in a lipidic environment

-more powerful nucleophile

micellar catalysis

see hydrolysis ofesters & anhydrides....

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AggregateN, XN, °N

kl

kN

Monomer

N=1Xl

°l

association of N monomers in a micelle

for a thermodynamic treatment see: J.N. Israelechvili “Intermolecular & Surface Forces” N.Y., Acad. Press 1985

19

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 iBy 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

from mixing entropy.probability that n monomers are together in one

aggregate. 1 10-5, very small!

on- no

1

RT

20

so that, in first approximation,

on- no

1 for no < n

A very useful parameter, A

ono- no

1 1

A = exp 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 themicelles.

Physical meaning: (o

n- no1) / (no – 1)

is the gain of the chemical potential for a monomer, present in the micelle, with respect to the monomer in solution.

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on- no

1

RTn = n

1· exp

from mixing entropycontribution; probability that nmonomers are together in anaggregate.Very small 10-5.The formation of micelles is entropically unfavorable.

“Boltzmann factor”energetically favorable intereactionof the monomers in the micelle,with respect to the intereactionwith the solvent.

only for on< n·o

1 can we have aggregates; till a minimal

micelle size no the formation of micelles will be unfavorable

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cmc is generally given in molarity

several experimental methods:

Physical property

molecular weight (average)scatteringconducibilityspectroscopy

½

cmc [surf.]

Typical values are in the millimolar range 10-3 M;but there are cases 10-5 M or smaller

23

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`. …”

N

CH3

N+

CH3

Cl

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 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. …”

The absorbance of 1.05 X 10-8 M pinacyanolchloride at 610.0 m in pH 9.59 sodium borate buffer (l = 0.1) at 50.0° vs. laurate concentration.

24

An overview on vesicles and liposomes

(liposomes: vesicles made out of lipids)

25

26

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.

27

POPC

sodium oleate + oleic acid

hydrophilic(lipophobic)

hydrophobic(lipophilic)

O

O

O P

O

O

O

O

ON

+

H -

O

O -

Na+

O

OH-

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caprylatecaprylate COOCOO --

oleateoleate

oror

CHCH33(CH(CH 22))77 -CH = CH-(CH -CH = CH-(CH 22))77COOCOO --

form micelles at alkaline pHform micelles at alkaline pH

COOCOO --

(Deamer, 1976)(Deamer, 1976)

vesicles at pH=7-8vesicles at pH=7-8

(pH(pH pk)pk)~~__COOCOO -- HOOCHOOC

precursors ( water insoluble! )precursors ( water insoluble! )

RR COCO

RR COCOOO

OHOH--

OHOH--

RR CCOOOORR''

oror

micellesmicelles oror

R COOR COO --

vesiclesvesicles

29

Soaps self-assemble into micelles as soon as the

cmc is reached.

Fatty acids are almost insoluble in water.

Mixtures of fatty acids and the corresponding soaps assemble into

vesicles, at concentrations above the

cvc. -0.2 0.0 0.2 0.4 0.6 0.8

7

8

9

10

11

12

micelles vesicles

pH

HCl (equivalent)

Equilibrium titration curve ofsodium oleate at 25 °C

30

Micelle

Liposome

Bilayer sheet

Cross-sectional views of the three structures that can be formed by mechanically dispersing a suspension of phospholipids in aqueous solution

The red circles depict the hydrophilic heads of phospholipids, and the squiggly lines (in the yellow region) the hydrophobic tails.

31

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

32

surfactant

amphiphilic

hydrophilic

cac

ionic

33

A realistic scenario of the emergence of life can be based on a gradual transitionfrom random mixtures of simple organic

molecules to spatially ordered assemblies

displaying primitive forms of cellular compartimentation, self-reproduction and catalysis

A realistic scenario of the emergence of life can be based on a gradual transitionfrom random mixtures of simple organic

molecules to spatially ordered assemblies

displaying primitive forms of cellular compartimentation, self-reproduction and catalysis

34

Liposom / Vesikel

ao

lcv

v ½ < <1 aolc

J.N. Israelachvili, D.J. Mitchell, B.W. Ninham (1976).

35

1-10 M MLV

500 nm= 0.5 m

400 nm

LUV200 nm

20 nm SUV

100 nm

36

dynamic of a liposome-membrane

Flip-Flop

rotationdiffusion

vertical “vibration”(amplitude 0.3 nm)lateral

diffusion

DPPC (T>Tm)

vertical “vibrations”, jump time 10-10 s

rotation correlation time (c) 10-9 s

lateral diffusion coefficient 7·10-8 cm2 s-1

(wandered 4 m per second)Flip-Flop time 8 hours

E. Sackmann (1978, 1991)

37

temperature-sensitive phase transmutation of aliposome-membrane

Tv Tm

L´ P L

crystalline “Ripple”-phase fluid phasephase (“quasi-crystalline”) (“fluid-crystalline”)

described as gel-phase too

trans- and gauche-confirmations

all-trans- (anti-)confirmations

E. Sackmann (1978); R.R.C. New (1990)

38

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

permeability coefficient (cm·s-1)water 4·10 -3

glycerin 5·10 -6

urea 4·10 -6

tryptophan 4·10 -10

glucose 10-11

Cl- 7·10 -12 Lysin 5·10 -12

Na+ 1·10 -12

Brunner, D.E. Graham, H. Hauser, G. Semenza (1980); G. Cevc,Marsh (1987); A.C. Chakrabarti, D.W. Deamer (1992)

39

Are aqueous micelles chemicalequilibrium systems?

How can you demonstrate this?

..the case of micelles is straightforward

40

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 solutionsAs they are initially?

41

42

what is then the general picture that emerges in the case of oleatevesicles, a system which is a mixed situation-partly equilibrium, partly not?

nM Mn

M`n

irreversible

irreversiblenM

Mn

yes (Knappl)

noG°

Rk

43

to make liposomes is easy

stock surfactantin wateror methanol

"film" ofsurfactant

size distribution

extrusionthrough filters

relatively monodispers

liposomes = vesicles from lipids

H2O

H2O

44

REVERSE PHASE EVAPORATIONREVERSE PHASE EVAPORATIONmonomers inmonomers inorganic solventorganic solvent reverse phasereverse phase

evaporationevaporation hydrationhydration

water

org.solvent

formation offormation ofvesiclesvesicles

45

Freeze / thaw entrapment methodFreeze / thaw entrapment method

5x freeze / thaw5x freeze / thaw

ExtrusionExtrusion

46

Liposomes prepared by the “extrusion method“Liposomes prepared by the “extrusion method“

“LiposoFast“, a small-volume extrusion apparatus

47

48

(1) 2,3-butanediol(2) ethanol(3) 1,2-propanediol(4) PEG 200(5) methanol

Size distribution of POPC liposomes prepared by injecting50 µL of alcoholic solution of 25 mM POPCinto a 0.1 M borate buffer solution, pH 8.5.[POPC]final 0.5 mM, 2% (v/v) alcohol; measuring angle 90°.

49

Spontaneous vesiculation and self-replicationSpontaneous vesiculation and self-replication

spontaneouspontaneoussvesiculationvesiculation

increasedincreased solubilisationsolubilisation

autocatalyticautocatalyticpopulationpopulationincreaseincrease

0.000.00

0.100.10

0.200.20

0.300.30

0.400.40

00 5050 100100 150150 200200time (min)time (min)

OD

500

nm

OD

500

nm

buffer

neat surfactantneat surfactant

OO

OOHH

oleic acidoleic acid

50J. Phys. Chem. B J. Phys. Chem. B 1998, 1998, 102, 102, 7078-70807078-7080

O2N

OCH3

O P O

O

O

(CH2)9CH3

(CH2)9CH3 O P O

O

O

(CH2)9CH3

(CH2)9CH3-+K

O2N

OCH3

O K+-

spontaneousspontaneousformation offormation of

vesiclesvesicles

11 22 33

KOH 0.2 MKOH 0.2 M

hh++

51

NH2

N

N

N

OH

NO

OH

AdenosineOH

O

OH

N

NH

O

O

Uridine

O O

O

P

O

O

O_O

NH4+

O

PHOSPHOLIPONUCLEOSIDESINVESTIGATED

52

CHCl3 - H2O, pH 4.5

45 °C, 6 h

N+

O

O

OO

O

P O

O

O-

N+HO

O

O

OO

O

P O

O

O- N

N O

NH2

O

HO OH

N

N O

NH2

OHO

HO OH

+

+

Phospholipase D ausStreptomyces sp. AA 586

53

How do you entrap drugs or biochemicals

inside liposomes?

54

E

E E

E

oleate film

oleate film

ENZYMESOLUTION

make liposomes(vortex ca. 30”)

E

EE

EE

fractions

OD

ENZYME

chromat.Sepharose 4B -Enzyme free

E

EEE

no freeenzymeoutside

ADPE

E

E

E

incubation with ADP

chromat.Sepharose 4B

- ADP free

E

E

E

E

no external ADP

samplingfor poly (A)

time

poly (A)

Operational

55

DehydrationDehydration

RehydrationRehydration

FusionFusion

ExtrusionExtrusion

Dehydration / Rehydration method forDehydration / Rehydration method forsolute entrapmentsolute entrapment

56

Dispersion of a thin film of POPC in H2O

A B C

57

Injection of an ethanolic solution of POPC into H2O(“ethanol injection method”)

EtOHPOPC

Removal of EtOHby dialysis or gel permeationChromatography

water

3933-3935

58

The hydrophobicity of the lipid bilayer

Is the main driving force for

The activity/reactivity and applications

Of liposomes

59

Micelle

Liposome

Bilayer sheet

Cross-sectional views of the three structures that can be formed by mechanically dispersing a suspension of phospholipids in aqueous solution

The red circles depict the hydrophilic heads of phospholipids, and the squiggly lines (in the yellow region) the hydrophobic tails.

60

DDD

DD

D D

D

CELL

ENDOCYTOSIS

LIPOSOMES AS DRUG-DELIVERY AGENTS

61

The hydrophobic effect of the membrane as a furtherordering principle:

It selects out of the bulk solution the most hydrophobiccompounds, forming stable complexes

62

This can give rise to self-reproduction processes

Additionally, these orderedstructures are able to pick upand order hydrophobicdi- and tripeptides

63

Gene transferIs usually done with

Positively charged liposomes

e.g., DDAB

64

.

++POPCPOPC POPC / POPC / DDAB

DNA / LIPOSOMESDNA / LIPOSOMESDNA / LIPOSOMESDNA / LIPOSOMES

DNADNA

DNADNA

DNADNA

DNADNA

++

++

++++

++

++

++

65

Plasmid DNA(theoretical length of linearizedDNA 3400 bp 1100 nm)

~180nm

66

reaction features ofsurfactant aggregates

3. forced compartmentation of reagents

and other charged speciescannot go through

....but uncharged molecules go through

and the (charged)reaction product isblocked inside !

HPO4

HO CN

CH2OPO3

HPO4

O CN

HPO4

e.g.

67

reaction features ofsurfactant aggregates

2. The concentration gradient

HPO4 = 10-3 M

( OSMOTIC BALANCE is HOWEVER NECESSARY )

POPC liposomes

HPO4 =

0.5 M

OUT

OUT

IN

IN

these gradients canbe kept for daysor weeks....

pH = 9.0

pH = 5.0

POPC liposomes

e.g.

68

AND THE POORLY WATER SOLUBLEAND THE POORLY WATER SOLUBLESUBSTRATESUBSTRATEHEXADECYL-P-NITRO-PHENYL ESTER

BINDING THE WEAKLY CATALYTICALLYBINDING THE WEAKLY CATALYTICALLYACTIVE TRIPEPTIDEACTIVE TRIPEPTIDE

H-Phe-His-Leu-OH

EXAMPLE OF THE BILAYER HYDROPHOBICEXAMPLE OF THE BILAYER HYDROPHOBICEFFECT IN CATALYSISEFFECT IN CATALYSIS

EXAMPLE OF THE BILAYER HYDROPHOBICEXAMPLE OF THE BILAYER HYDROPHOBICEFFECT IN CATALYSISEFFECT IN CATALYSIS

POPC LIPOSOMESPOPC LIPOSOMES, OR , OR OLEATE / OLEIC ACID VESICLESOLEATE / OLEIC ACID VESICLES

DUE TO THE SOLUBILIZATION EFFECT,DUE TO THE SOLUBILIZATION EFFECT,A HIGH LOCAL CONCENTRATION AND AA HIGH LOCAL CONCENTRATION AND AHIGH PROXIMITY IS OBTAINED ON THEHIGH PROXIMITY IS OBTAINED ON THELIPIDIC MATRIXLIPIDIC MATRIX

69

Dependency of the initial hydrolysis rate (vin) for the hydrolysis of C16-ONp onthe 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 Mborate solution (pH 8.5), either in the absence (filled circles) or in the presence(open squares) of 1 mM Z-Phe-His-Leu-OH.

70

71

72

.

reaction features ofsurfactant aggregates

5. Catalysis

OH-

OH-

OH-

H+H+

{OH- in a

- more powerful nucleophile

micellar catalysis

see hydrolysis ofesters & anhydrides....

hydrophobiclipidic environment

H2O

H2O

73

Plasmid

Rib

Poly U

Ei

E

RNA

DNA

Rib

t-RNAPhe

(Phe)n

Ei

ERNA

CTPGTPATPTTP

CTPGTPATPUTP

t-RNAPhe

Phe

E

a

b

c

74

IINN

OOUUTT

insideinside different different first step towards first step towardsfrom from outsideoutside : the definition of self : the definition of self

... just imagine that cells would all open up:... just imagine that cells would all open up:where would life go?where would life go?

THE IMPORTANCE OF HAVING A BOUNDARYTHE IMPORTANCE OF HAVING A BOUNDARY

H2OC

A AB B

C A

particularparticularchemophysicalchemophysicalproperties of theproperties of theinside inside ((D, µ, D, µ,

physicalphysicalprotectionprotection

entrapment & vicinityentrapment & vicinityor reagentsor reagents(high local concentration)(high local concentration)

no leakageno leakagegradientgradient

selectiveselectivepermeabilitypermeability

catalysiscatalysis

binding ofbinding ofhydrophobichydrophobicsubstancessubstances