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Albumin binding onto synthetic vesicles

Carlotta Pucci, Anita Scipioni and Camillo La Mesa*

Received 20th January 2012, Accepted 4th July 2012

DOI: 10.1039/c2sm26260f

Vesicular entities were obtained by mixing didodecyldimethylammonium bromide and sodium

dodecylsulfate in non-stoichiometric ratios. The vesicles bear a positive surface charge, due to the

cationic species being in excess, and adsorb significant amounts of protein, presumably by electrostatic

interactions. We modulated the net charge of bovine serum albumin by pH and observed its binding

onto the above vesicles. Binding is controlled by the net charge of vesicles and albumin: it is substantial

when albumin has negative charges in excess and is negligible, or non-existent, below its iso-electric

point. For pH values >6.0, the binding efficiency increases in proportion to protein charge. Surface

coverage changes in proportion to pH when the number of charges neutralized upon binding remains

the same. The size of protein–vesicle lipo-plexes was inferred by dynamic light scattering and their

charge by z-potential. The structure of albumin was evaluated by circular dichroism spectroscopy and

estimates of a-helix, b-strand and random coil contents were achieved. Increasing the b-strand and

random coil contents subsequent to binding suggests a significant interaction between vesicles and

albumin. Attempts to determine the binding efficiency were made by elaborating z-potential values.

The results were interpreted in terms of a Gibbs adsorption isotherm. Accordingly, it is possible to

estimate the binding energy under different pH conditions.

1. Introduction

Efforts were devoted to mimic the interaction processes occur-

ring between biopolymers and cells in vivo, and relevant points

were clarified in this regard.1–5 It is accepted nowadays that

biopolymers adsorb onto cells, or vesicular analogues, by elec-

trostatic, depletion, osmotic, hydrophobic effects and combina-

tions thereof. However, the relative weight of the above

contributions is both membrane- and biopolymer-dependent.

Generally, fibrous proteins enter vesicle bi-layers to minimize

hydrophobic interactions with water.1,6 Small globular proteins

or long, highly charged DNA, conversely, hardly enter the

membranes and adsorb on them by electrostatic interactions. For

instance, surface adsorption holds in lysozyme7 and DNA8–10

binding to synthetic vesicles has been observed. Some aspects of

these processes, however, were not investigated in detail.

Controlling pH, ionic strength and/or temperature may help

clarify the details of the binding process. In this paper results on

the pH-dependent binding of bovine serum albumin onto

synthetic cat-anionic vesicles11–13 are reported. Under the inves-

tigated pH conditions the protein charge and conformation

change while the vesicle state is practically unmodified.

The interactions between proteins and phospholipids-based

vesicles are hardly determined, since the charge of both entities is

pH-dependent.14 This fact has unfavourable consequences in

Dept. of Chemistry, La Sapienza University, Cannizzaro Building, P.le A.Moro 5, I-00185 Rome, Italy. E-mail: camillo.lamesa@uniroma1.it; Fax:+39-06-490631; Tel: +39-06-49913707

This journal is ª The Royal Society of Chemistry 2012

determining protein adsorption onto lipid-based vesicular enti-

ties. In addition, the sizes of the latter vesicles are much higher

than those pertinent to cat-anionic systems,14,15 on which we

focus our attention. Such drawbacks hardly allow the use of

dynamic light scattering and z-potential analyses, on the results

of which this contribution relies.

The system reported here does not have the above disadvan-

tages, since pH affects only the protein charge. The vesicle state of

charge is immaterial, since the vesicles are made from strong elec-

trolytes. Minor effects, due to changes in the electrical double-layer

thickness around them, are present, but the role played by such

contribution can be quantified.16,17 These facts offer the opportu-

nity to mimic protein–vesicle interactions in conditions where only

the state of charge of the adsorbing species is modified.Modulating

the electrostatic terms due the binding species allows determination

of whether hydrophobic, osmotic, depletion and other contribu-

tions are relevant in the process. Support comes from ionic

conductivity, dynamic light scattering, DLS, and z-potential

measurements. It is strengthened by circular dichroism, CD, which

gives an indication of the protein conformation.

2. Experimental section

2.1. Materials

Bovine serum albumin, BSA, Sigma-Aldrich, was dialyzed and

lyophilized. Its solution properties were checked by measure-

ments of ionic conductivity, z-potential, density, viscosity and

dielectric relaxation; they are equivalent to previous data.18–20

Soft Matter, 2012, 8, 9669–9675 | 9669

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Sodium dodecylsulfate, SDS, and didodecyldimethylammo-

nium bromide, DDAB, were from Sigma Aldrich. The former

was dissolved in hot ethanol, filtered, precipitated by cold

acetone, and vacuum dried at 70 �C. Its purity was checked by

ionic conductivity and surface tension. Its critical micellar

concentration, CMC, is close to that of previous data.21 DDAB

was purified by the same procedure, vacuum dried and used as

such. Purity was confirmed by determining its CMC.22

Salts and solvents were from Sigma and were used as such.

Water was distilled over alkaline KMnO4 and bubbled by N2 to

reduce the presence of CO2 in the medium. Its ionic conductance,

c, is less than 1 � 10�7 U�1 cm�1 at 25 �C.

Fig. 1 Electrical conductance of the DDAB/SDS vesicular dispersion, in

mS cm�1, as a function of added albumin, in mmol kg�1, (squares) and

conductance of BSA solutions (circles) at different concentrations. Data

are in mmol kg�1, at 25 �C and pH 6.8.

2.2. Vesicle preparation

Cat-anionic vesicles were prepared by mixing aqueous solutions

of SDS and DDAB, both containing 4.05 mmol kg�1 of the

surfactant. The reason for using two species in different amounts

is due to the significant vesicle stability and the fine modulation

of both size23 and z-potential24 that is obtained. Vesicles made by

DDAB are unstable and reform the original lamellar dispersions

with aging. Conversely, the 3.80/1.0 [DDAB/SDS] mixing ratio

gives stable vesicular dispersions. The final dispersion is, thus,

made of charged vesicles dispersed in 2.4 mmol kg�1 NaBr

solution. The free salt is obtained by metathesis between SDS

and DDAB. The metathesis is partial because the surfactants are

mixed in non-stoichiometric ratios.

Care was taken to ensure that the composition was in the

region where vesicles occur.25 The dispersions were extruded,

heated at 50 �C, and filtered. They were thermally equilibrated at

25–30 �C. The vesicle properties were controlled by DLS and

z-potential. The size distribution plots give average vesicle

diameters, h2RHi, in the range 250–300 nm. The corresponding

z-potential isz+70 mV. The DDAB/SDS vesicles are stable and

retain their charge and size for months, provided they are kept at

room temperature.

The quaternary, or five component systems give lipo-plexes

(vesicle–protein complexes) when prepared by dissolving BSA in

the vesicular dispersion and diluting them with the vesicular

pseudo-solvent, if required. Mixing was optimized by stirring

and controlled by DLS. The pH of the dispersions was modified

by adding due amounts of 100 mmol kg�1 HBr, or 100 mmol

kg�1 NaOH, under potentiometric control. Acid or basic solu-

tions were freshly prepared: their analytical concentration was

determined by electrical conductivity, at 25 �C. No buffers were

used, to avoid interference from ions other than H3O+, OH�,

Na+, or Br�. The final dispersions contain ionized BSA, charged

vesicles and interchangeable ions.

2.3. Methods

2.3.1. Ionic conductance. Ionic conductance was measured by

a 1540 Wayne-Kerr bridge, working at 1.00 kHz with a Daggett–

Krauss cell immersed in an oil bath. Measurements were run at

25.00� 0.01 �C. Experiments were performed on the protein and

on vesicle–protein systems; data are reported in Fig. 1.

2.3.2. Light scattering. Measurements were run by using a

Malvern Zeta Nanosizer unit, working at 632.8 nm in

9670 | Soft Matter, 2012, 8, 9669–9675

backscattering mode (at 173�), at 25.0 � 0.1 �C. A Malvern

Autosizer 4700c PS/MV unit, operating at 488 nm, was used at

variable angle conditions. Dispersions were passed through

0.80 mm Waxman filters before entering the cells. The hydrody-

namic diameters of vesicles are reproducible within the experi-

mental data accuracy (�10 nm). CONTIN algorithms analyzed

the auto-correlation functions. Accordingly, g2(s) values were

derived by26,27

g2ðsÞ ¼"hIðtÞIðtþ sÞi

hIðtÞi2#¼ 1þ Bjg1ðtÞj2 (1)

where s is the delay time (in ms), and B an instrumental constant.

An expansion of eqn (1) analyzed g1(s) according to

lnQg1ðsÞS ¼ �G1sþ�G2

2

�s2 (2)

where G1 and G2 are the first and second cumulants, respectively.

Plots of G1 vs. q2 determined the correlation decay. Processes are

diffusion-controlled, and Dapp (¼ G1/q2) is related to h2RHi

through the Stokes–Einstein equation [Dapp ¼ kBT/6phhRHi].An intensity number average distribution plot for vesicles, In.a., is

drawn in Fig. 2.

2.3.3. Electrophoretic mobility. Electrophoretic mobility was

determined by a laser Doppler facility available in the DLS unit,

on cells equipped with gold electrodes, at 25.0 � 0.1 �C. Themeasured electrophoretic mobility, m, allowed calculation of

z-potentials according to28

z ¼ m

�4ph

30

�(3)

In eqn (3) h is the medium viscosity and 30 the static dielectricpermittivity. z-Potentials are directly determined by the

measuring unit; the related accuracy is �1.0 mV. Selected values

This journal is ª The Royal Society of Chemistry 2012

Fig. 2 Intensity-based distribution plot of vesicle sizes before, large

figure, and after filtration (inset at the top). Note the disappearance of the

peak at large h2RHi values. Data refer to compositions and mole ratios

indicated in the text, at 25 �C.

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are reported in Fig. 3. z-Potentials can be related to the surface

charge density of vesicles and lipo-plexes, s, according to7

0�30z

4p

�¼�s

k

�(3)

where 1/k is the Debye screening length (1/k ¼ d). d depends on

the medium ionic strength and, to a lesser extent, on pH.

z-Potentials can be related to electric moment calculated as the

product of average distance between charges on the vesicle and

adsorbing species per unit area.

To achieve estimates of the medium ionic strength and its role

in determining the double layer thickness, a Br� ion selective

electrode was used. According to experiments, the amount of

bromide ions is in the range 3.1 � 0.2 mmol kg�1. The double

layer thickness around vesicles, d, is controlled by ionic strength,

and, apparently, does not depend on the BSA content. Estimates

based on dielectric relaxation indicate d to be in the 2.0–5.0 nm

size range.29 Accordingly, d is about two orders of magnitude

Fig. 3 z-Potential values, in mV, as a function of added protein, in mmol

kg�1, under different pH conditions, at 25 �C. Note the inflection points

in the curves relative to pH 6.8 and 10.0, respectively.

This journal is ª The Royal Society of Chemistry 2012

lower than vesicle size (250–300 nm), and Smoluchowski’s

approximation30 holds (see below for details).

2.3.4. Circular dichroism. Circular dichroism spectra were

obtained on a Jasco J-715 spectropolarimeter, equipped with a

Xenon lamp, operating at 1.0 nm resolution. Accurately cleaned

quartz cells of 0.100 or 0.010 cm path length were used. Spectra

are the average of at least four runs, performed in the range 190–

250 nm. They are expressed as mean residue ellipticity, [q], in deg

cm2 dmol�1. Estimates of a-helix, b-strand and random coil

contents were performed by the program K2D3,31 using the 240–

190 nm wavelength range as data input.

3. Results

The system was investigated at pH 4.0,6.8, and 10.0, respectively,

at 25 �C. The BSA charge varies with pH: at pH 10.0 the nominal

number of charges is�18, at 6.8z�8; at 4.0 is about +2.32 If the

interactions are modulated by the protein state of charge and

hydrophobic contributions, it is possible to determine their

relative weights by changing the pH.

At pH 4.0 the protein is slightly, but positively, charged.

According to DLS data, formation of large lipo-plexes therein is

speculative, although BSA may eventually bind. Apparently, no

significant interactions occur. At such pH values, the size of

colloid entities in the bulk is constant and does not depend on

added albumin. The above statement is supported by visual

observation, turbidity and DLS. The dependence of h2RHi valueson added BSA, under different pH conditions, is reported in

Fig. 4. At pH 4.0, h2RHi values relative to vesicles or lipo-plexes

are close to 300 nm and do not substantially change with albumin

content. This fact indicates moderate vesicle–protein

interactions.

Fig. 4 Dependence of average lipo-plex size, h2RHi (in nm), on the

concentration of added albumin, in mmol kg�1, at 25 �C. The pH

conditions are indicated in the figure. The black and grey vertical bars

indicate the range in which the dispersions are turbid and DLS

measurements could not be performed. The onset of such regions is

considered the surface saturation threshold for vesicles. It is anticipated

at pH 10.0 because of a substantially higher negative charge on the

protein.

Soft Matter, 2012, 8, 9669–9675 | 9671

Table 1 Percentage of a, b and random coil contents for BSA indifferent systems, at pH 6.8 and 25 �C. Evaluation of the secondarystructure from CD was made by the K2D3 program [ref. 31]

Albumin content(in mmol kg�1)

a-Helix(%)

b-Helix(%)

Random coil(%)

0.01 43 13 440.01 + Vesicles 25 23 520.02 34 17 490.02 + Vesicles 14 29 570.0015 50 11 39

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The aggregation processes occurring at high pH, Fig. 4, indi-

cate the electrostatic nature of the interactions taking place

between vesicles and charge-modulated BSA. The higher the

nominal number of negative charges on the protein, the lower the

saturation threshold. Accordingly, the amount of BSA required

for surface saturation and aggregation into large entities is

inversely related to its nominal charge.

The size of lipo-plexes is high in the proximity of the turbid

region and indicates the formation of large objects. The elec-

trostatic repulsion between lipo-plexes is substantially reduced

compared to bare vesicles, and bridging between coated vesicles

and/or lipo-plexes is possible. The combination of two such

effects controls aggregation. The lower limits of turbid areas in

Fig. 4 are related to surface saturation of lipo-plexes. Support to

this statement comes from the nearly constant z-potential values

above that limit, as indicated in Fig. 3.

Similar results were inferred from ionic conductivity

measurements. Note that the lower limit of the turbid area in

Fig. 4 is equivalent to the one observed in conductivity

measurements, Fig. 1. In addition, the slope of the conductivity

curve for albumin is the same as that above saturation.

That is, adsorption is limited and no binding takes place above

saturation, because there is no more room for adsorption. This is

a consequence of the electrostatic and steric repulsions between

protein molecules adsorbing onto vesicles.

Further addition of BSA results in a significant reduction in

the size of the lipo-plexes. When the protein content is higher

than the value required for surface coverage, there is no more

room for binding. In such conditions, presumably, lipo-plexes

are destroyed by the action of osmotic gradients and electrostatic

repulsions.

Very presumably, the BSA in solution is partially self-associ-

ated. At pH 10.0 its conformation is more extended than under

spontaneous pH conditions.32 The binding efficiency is higher

compared to pH 6.8, and albumin occupies a large surface area,

Fig. 4. The compromise between such effects is responsible for

the observed behaviour.

Fig. 5 Normalized CD spectra of bovine serum albumin in water and in

the vesicular pseudo-solvent, at pH 6.8 and 25 �C. The concentrations

indicated in the figure are in mmol L�1. At higher protein contents the

quality of spectra becomes poorer.

9672 | Soft Matter, 2012, 8, 9669–9675

Evaluation of the conformational behaviour of BSA in solu-

tion was obtained by CD spectroscopy at different concentra-

tions, and in vesicular dispersions as well. CD spectra obtained at

pH 6.8 are shown in Fig. 5; the relative contents of the a-helix,

b-strand and random coil were estimated by the programK2D331

and are reported in Table 1.

The percentage of BSA secondary structure in very dilute

regimes is also reported for comparison. The b-strand and

random coil contents increased with protein concentration,

probably because of self-association. Such behaviour is empha-

sized in the vesicular dispersion. Presumably, vesicles bind to the

polypeptide chain, partially destroying the hydrogen bond

network. The contemporary presence of BSA aggregates and

BSA–vesicle association complexes cannot be ruled out ‘‘a pri-

ori’’. At pH 10.0, the effect is similar to that formerly reported,

although the signal intensity is lower.

4. Discussion

In this investigation we chose a system made of oppositely

charged surfactants, namely SDS and DDAB, forming cat-

anionic vesicles upon mixing.11–13 Such vesicles are dispersed in

aqueous NaBr, obtained by partial metathesis of sodium with

didodecyldimethylammonium ion, and of bromide with dode-

cylsulfate, respectively. The reasons for using cat-anionic species

arise from the fact that their state is not pH-dependent, being

vesicles formed by strong electrolytes. Cat-anionic vesicular

matrices, thus, circumvent most drawbacks inherent to phos-

pholipids-based vesicles in studying protein binding.14,15

In addition, in cat-anionic systems, the affinity of cationic, or

anionic, surfactants towards proteins is much lower than in the

reciprocal ones. Wennerstr€om et al.33 stated that ‘‘the most

favourable interaction is between oppositely charged surfactant

ions, despite the strong binding known to lysozyme, in case of

dodecylsulfate’’. In the above system dodecyltrimethy-

lammonium extracted the anionic species from poorly soluble

lysozyme–dodecylsulfate precipitates. The two surfactants

formed cat-anionic precipitates when the mole ratio was close to

unity, and, in sequence, cat-anionic vesicles and mixed micelles.

According to these results, the interaction strength between

surfactants in the vesicles and proteins is much lower than that

between amphiphilic species in the vesicles. Such statements are

supported by independent studies.34 Thus, the uptake of surfac-

tants from vesicles and the formation of protein–surfactant

complexes in the bulk are hardly conceivable.

Albumin charge plays a substantial role in binding efficiency.

In double-layer theories, ion binding and distribution around

This journal is ª The Royal Society of Chemistry 2012

Fig. 6 Surface coverage, w, as a function of added albumin, CBSA (in

mmol kg�1). Data refer to pH 6.8, and 25.0 �C. The upper limit of surface

coverage is indicated by a horizontal black line.

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particles are controlled by an exponential term in the Poisson–

Boltzmann equation.35–38 To quantify binding efficiency, a model

based on a Gibbs adsorption isotherm was introduced. The

reason for replacing Langmuir’s relation with the Gibbs-based

one arises from the need to account for excluded volume effects,39

more precisely for ‘‘excluded surface’’ terms. When BSA binds,

further adsorption reduces, because of surface saturation and

repulsive interactions between molecules adsorbing onto vesicles.

The overall behaviour is controlled by the overlapping of such

effects. It is accounted for by introducing a Boltzmann distri-

bution in Langmuir’s isotherm.

Usually, Langmuir’s adsorption equation is expressed as40

K0ð½L�Þ ¼�

w

1� w

�(4)

where K0 is the binding constant, [L] the concentration of

adsorbing species in the bulk and w the fraction of occupied

surface sites. Eqn (4) applies to point-like entities, or for low

binding degrees onto fixed, identical, and independent loci. For

large and highly charged entities lateral interactions between the

adsorbing species must be accounted for. For this purpose,

Chatelier and Minton41 and, more recently, Zuckermann and

Heimburg2 rewrote Langmuir’s equation for protein adsorption

onto planar surfaces according to2

K0ð½L�Þ ¼�

w

1� w

�exp

w

1� w� 3þ 3

ð1� wÞ2!

(5)

where w is as before and the exponential modulates the surface

saturation terms. The value of 3 in eqn (5) is related to the shape

of particles being adsorbed.2,41 We imposed it to be 1.0, as

assumed for planar projections of identical, spherical objects.

Binding processes were determined when the size of lipo-plexes

did not substantially change, that is in the pre-saturation region

in Fig. 4. In such regimes, the area available for binding is

constant, with a good approximation. The same holds for the

double layer thickness.

To clarify the latter point in more detail, some considerations

are required. The bulk contains about 2.4 mmol kg�1 NaBr,

obtained by ionic metathesis, albumin (and its counter-ions),

plus molecular dodecylsulfate and didodecyldimethylammonium

ions. Estimates of the CMC of cat-anionic systems42 indicate

their concentration in the molecular form to be�0.1 mmol kg�1.

That is why we shall not consider such contributions in the

following. The contribution due to the protein, however, should

be accounted for. At pH 6.8, when BSA has 8 negative charges in

excess, addition of 0.1 mmol kg�1 of protein may imply an

increase in the ionic strength up to 6–7 mmol kg�1. The effective

contribution that BSA plays, perhaps, is hardly quantified.

d values were calculated by defining the ionic strength, I, as the

sum of two contributions

I ¼�1

2

�XciZi

2z

�1

2

�hcNaBr

�ZNa

2 þ ZBr2�

þcBSA

�ZBSA

2 þ kZBSA;count2i

(6)

where k is the number of albumin counter-ions at the given pH. d

values calculated according to eqn (6) are in the 2 nm size range.

This journal is ª The Royal Society of Chemistry 2012

They are lower than those due to NaBr only and the ones inferred

by dielectric relaxation30 and by independent estimates relative to

similar systems.43 It is inferred, thus, that albumin only partially

contributes to the medium ionic strength. The reasons for this are

many: in turn, BSA is partitioned onto vesicles, may associate, or

counter-ions condense onto it. At present, the role of each

contribution is undetermined.

In the following, we assume that the surface charge density of

lipo-plexes, s, is proportional to coverage and is expressed as

s ¼�1� Zw

N

�e

A0

� �(7)

where Z is protein charge at the given pH, N the number of

binding sites onto vesicles, w the surface coverage, e the unit

charge and A0 the area per binding site. N is the ratio of vesicle/

protein areas: the uncertainty of such values can be as high as 5%.

The quantity (e/A0) in eqn (7) is the surface charge density per

binding site (the area is equivalent to that of a BSA molecule, at

different pH values) and is rewritten as s0. Rearrangement leads

to

0w ¼�1� s

s0

��N

Z

�(7)

Data elaboration based on eqn (70) provides the surface

coverage as a function of albumin concentration, Fig. 6. The

vesicle area is known as well as the protein area and charge,

under different pH conditions. Note that the number of N values

in eqn (7) and (70) depends on pH, since the area of BSA changes

accordingly.

Values below the saturation threshold, inferred by DLS, were

considered. Under such conditions, w increases in proportion to

added BSA, and levels off at high protein contents. That is,

surface saturation holds. The effect is controlled by pH; at pH

10.0, w is considerably lower than at pH 6.8, because the area

covered by a single protein is larger. At pH 6.8, Fig. 6, the surface

coverage is about 0.60 and the intrinsic binding constant per site

can be expressed as

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K0 ¼ 0:5½X���1� Zw

N

��2Z

(8)

where [X�] is the concentration of counter-ions released from

vesicles and other symbols are as before. Combination of eqn (8)

and (7) allows us to estimate the Gibbs energy associated with the

process, since DG� ¼ �RTln K0. Estimates based on data relative

to pH 6.8 (Z ¼ �8) and 0.1 surface coverage indicate that the

Gibbs energy is in the range –(15 � 3) kJ per binding site.

Vesicle or lipo-plex sizes by DLS are significantly higher than

in all other species, and it is possible to consider the observed

behaviour in terms of binding of small poly-ions onto large,

oppositely charged surfaces. Albumin charge depends on pH and

its interactions are effective, provided measurements are run

above its iso-electric point. In such cases there is a clear-cut

relation between pH and net protein charge. We do not consider,

in this context, the possibility of vesicle growth. The observed

growth in size, thus, is related to protein-assisted aggregation.

Classical electrostatics holds for protein distribution in the

diffuse double-layer around vesicles. Presumably, the effective

protein charge is considerably lower than its nominal value,

because of ion condensation.44 The albumin distribution around

vesicles was evaluated in terms of classical theories,45,46 to fore-

cast its average distance from the vesicle surface charges. The

distribution around vesicles of radius r was calculated by an

approximation estimating ion location, according to28

jðrÞ ¼�Ze

30r

�exp�kr (9)

where j(r) is the r-dependent potential around vesicles, and other

symbols are as before. From the second derivative of j with

respect to r we get

0V2jðrÞ ¼ k2 sinh

�Zej

kBT

�(9)

Estimates based on eqn (90) determined albumin location in the

double layer. They are based on the approximation of point-like

charged entities binding onto large surfaces.28 Eqn (90) indicatesthat 1/k substantially reduces from 2.0 to 1.4 nm (about 30%)

when pH rises from 6.8 to 10.0. We do not know what the

numerical accuracy is; however, data are self-consistent and in

agreement with experiments. Accordingly, the higher the net

charge of albumin, the closer its location on the vesicle surface.

This hypothesis is reasonable, and is consistent with the decrease

in the z-potential values formerly reported in Fig. 3.

5. Conclusion

The purpose of this research was to determine the role played by

protein charge in binding onto synthetic vesicles. We applied a

pre-existing model41 to account for binding and repulsive inter-

actions between species adsorbing onto charged vesicles. The

results indicate that binding is effective, depends on pH, and that

the area covered by protein molecules is controlled by the state of

charge. At pH 10.0, the area covered by albumin is two–three

times higher than at pH 6.8. That is, the number of neutralized

surface sites scales with protein charge. This effect is consistent

with the essentially electrostatic nature of the interactions

9674 | Soft Matter, 2012, 8, 9669–9675

between albumin and vesicles. Such conclusions are consistent

with information from electrophoretic mobility, CD and DLS.

The biomimetic of this system is noteworthy. It indicates the

substantial role played by the state of charge, charge distribution

and lipo-plex sizes in the observed behaviour. An extension to

lipid-based vesicles is possible. The physical meaning of the

present approach is relevant, given its simplicity; it can be applied

to protein–lipid systems and may be relevant for further

advances in the field.

Acknowledgements

Thanks to F. Tardani (Dept. of Chemistry, La Sapienza) and G.

Risuleo (Dept. of Biology & Biotechnol., La Sapienza) for dis-

cussing some aspects of the manuscript. Thanks to A. Bonin-

contro (Dept. of Physics, La Sapienza) for agreeing to give

preliminary information on d values inferred from dielectric

relaxation. Financial support comes from La Sapienza Univer-

sity funding grants.

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