Understanding surface-adsorption of proteins: the Vroman effect

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by  Pol Vilaseca

Understanding surface-adsorption of proteins: the Vroman effect
Pol Vilaseca,1,2 Kenneth A. Dawson,3 and Giancarlo Franzese1
1Departament de F´ısica Fonamental, Facultat de F´ısica,
Universitat de Barcelona, Diagonal 645, 08028, Barcelona, Spain
2School of Mathematics, Trinity College Dublin, Ireland
3Center for BioNano Interactions (CBNI), University College of Dublin, Ireland∗
(Dated: February 20, 2012)
It is now well accepted that cellular responses to materials in a biological medium reflect greatly
the adsorbed biomolecular layer, rather than the material itself. Here, we study by molecular
2 dynamic simulations the competitive protein adsorption on a surface (Vroman-like effect), i.e. the
1 non-monotonic behavior of the amount of protein adsorbed on a surface in contact with plasma as
0 a function of contact time and plasma concentration. We show how the effect can be understood,
2
controlled and inverted.

b
PACSnumbers: 87.10.Tf,81.16.Fg,87.85.J-
e
F

6 When nanoparticlesareincontactwith bloodplasma, (a) (b)
1
or other biological fluids, biomolecules rapidly coat the

bare surface in a relatively selective manner [1]. It is in-
]
t creasingly accepted that the early biological responses
f
o to nanoparticles will be determined by the adsorbed
s biomoleculesratherthanthe pristinesurfacealone[2,3]. (c) (d)
.
t Becauseoftheirsize[2,4]nanoparticlesaretraffickedby
a
m active transport processes throughout the organism, us-
ingtheinformationfromtheproteinsequencesassociated
-
d with the surface of nanoparticles. Unlike the situation
n for flat macroscopicsurfaces sayof medicalimplants, for FIG. 1. Schematic representations of different proteins ad-
o
nanoparticles the protein environment changes in differ- sorbed on the surface. (a) Alb is a globular protein charac-
[c entcompartmentsofcellsandorgans,asthenanoparticle terized by the radius RA. In all the panels the continuous
linerepresentsthesurfaceprofile. IgGorFibarerepresented
traffics. This has lent urgency to the modern interest in
1 asellipsoids thatinteractwith eachotheralong thelongaxis
v understanding the phenomenon at a more fundamental RS,i (b),or along theshort axis Ri (c). In thelast case they
6 level [2]. Still, we can learn a lot from an understanding arepartiallydeformed(denatured)bythesurfaceadsorption,
9 of the process for flat surfaces [5]. Studying the adsorp- representedherebyapartialoverlapoftheellipsoidswiththe
7 tion of Fibrinogen on a surface in contact with blood surface. (d)Atarandommoment,theadsorbedellipsoidscan
3 plasma, Vroman found that the surface concentration of havedifferent arrangements.
.
2 Fibrinogenshowsamaximumatanintermediatecontact
0 time,indicatingthatFibrinogenisreplacedwithtimeby
2 competitive adsorptionhas beenobserved[11]. We show
one or more families of different proteins [6]. The phe-
1 thatthesequenceofadsorptioncanbeexplainedinterms
nomenon is not specific to Fibrinogen, but is a general
:
v effect for many other proteins [7]. The plasma proteins of the relative bulk concentrations, difusivities and sur-
i face affinities of the proteins and that by thermal or
X compete for the occupation of the surface, resulting in a
chemical energy-depletion is possible to control and in-
sequentialcompetitiveadsorption,knownastheVroman
r
a effect. vert the effect.
Alb is a globular protein, with an almost spherical
The effects depends on numerous factors such as the shape. We model Alb-Alb interaction as
plasmadilution,thetemperature,andthespecificsurface
chemistry[8]. Inhighlyconcentratedplasma,thesequen- 2R 24
A
V (r)= (1)
tial adsorption takes place in seconds, but it takes sev- A (cid:18) r (cid:19)
eral minutes when the plasma concentrations has phys-
iological values [9]. The more hydrophobic the surface, where R is the radius of Alb and r the protein-protein
A
the stronger is the protein adsorption, eventually induc- distance (Fig. 1a). Attraction among protein is not in-
ing irreversible adsorption [10]. Here we study the ef- cludedatthislevelofdescription,asitissmallcompared
fect by Molecular Dynamics (MD) of a model protein to protein-surface interaction [11]. Despite this rough
solution in contact with glass. We consider the three zero-order approximation, our results support a posteri-
mostabundantproteinsinhumanblood: Albumin(Alb), orithisassumptionwithintheapproximationsofourap-
Immunoglobulin-γ (IgG)andFibrinogen(Fib),forwhich proach. IgG and Fib in their folded conformation have
2
non-spherical shapes. In particular, the IgG structure 6.08 ǫ . We express all the results in terms of the Alb
A
resembles a γ and the Fib resembles an elongated ellip- units: M∗ ≡ Mi for the mass, R∗ ≡ Ri for lengths
i MA i RA
soid. They both can be approximated by ellipsoids with and ǫ∗ ≡ ǫi for the energies, t∗ = R2AMA 12 for the
two principal axes of rotation along which they interact i ǫA (cid:16) ǫA (cid:17)
witheachother(Fig.1b-d). This isencodedthroughthe time, T∗ = kBT for the temperature, and ∆E/ǫ for
ǫA A
protein-proteinpotential,withinthesameproteinfamily, the energy changes of the buffer. In experiments ∆E is
controlled by adding sodium azide, or other depletion-
24
2Ri 3 energy chemical agents, to the protein solution [12]. In
V (r)= + (2)
i (cid:18) r (cid:19) 1+exp(30(r−2R )/2R ) the following we drop the ∗ for sake of clarity.
S,i A
We perform MD simulations at constant T, constant
wherethe index i=I,F standsfor IgG(I) andFib (F),
volume V and constant number of proteins N , in a par-
i
with a hard-core radius R and a soft-core radius R
i S,i allelepiped with two square faces and four rectangular
[13]. Interaction between pairs of proteins of different
faces. A square face is occupied by the attractive sur-
familiesaregivenbyEq.(2)withparametersRi andRS,i face, the other by a wall interacting with the proteins
equaltotheaveragesofthecorrespondingparametersfor through the repulsive part of the V potential. We
24,12
each family, and with RA =RS,A for Alb. apply periodic boundary conditions (pbc) along the four
The protein-surface interaction is given by rectangularfaces. Thevolumeconcentrationsofproteins
is taken to match the average concentrations of the hu-
σ 24 σ 12
V24,12(r)=4ǫi i − i (3) man plasma, with cA = 4.25 g/dl, cI = 1.25 g/dl and
(cid:18)(cid:16) r (cid:17) (cid:16) r (cid:17) (cid:19) c = 0.325 g/dl, at X = 100% plasma concentration
F P
in blood. When a protein is adsorbed on (released by)
whereǫ istheattractiveenergybetweenthe surfaceand
i
a protein of the family i, and σ = R /21/6, with i = the surface, we keep its bulk concentrations constant by
i i
inserting (deleting) a protein of the same family in a
A,I,F,isthemaximumapproachdistancebetweeneach
randomly-chosenempty (occupied) space of the box.
protein and the surface.
Experiments are usually carriedout for highly diluted
For each family of proteins i, we set the soft-core ra-
plasma,atconcentrationassmallasX =0.1%,toslow
dius R = R the hydrodynamic radius, determined P
S,i H
down the adsorption rate to minutes or hours, allowing
experimentally from the diffusion coefficient D through
the Einstein-Stokes equation D = kBT , where η is the precise measurements. However, such low rates would
6πηRH decreasethestatisticsofourMDsimulations. We,there-
viscosity of the medium, under the assumption that the
fore,performoursimulationsinconditionsthatarecloser
proteins can be approximated by a sphere. The hard-
to those of practical interest, with X as high as 100%,
core radiuses R are set by imposing for each protein P
i
50% and 25%, by considering different sizes of the sim-
that the experimental surface concentrationcorresponds
ulation box while keeping constant the initial number of
to the close packing configuration[11]. These conditions
proteins, their relative proportions, and the size of the
give R = 3.55 nm, R = 4.9 nm, R = 6.58 nm
A I F
adsorption surface.
R = 5.51 nm and R = 11 nm. Protein masses
S,I S,F
For each X we average the results over fourteen in-
M = 67 KDa, M = 150 KDa, M = 340 KDa, neces- P
A I F
dependentruns,startingfromindependentinitialconfig-
sarytodeterminethetimescales,areknownfromexper-
urations that have been equilibrated by applying pbc in
iments [10]. Protein-surface attraction energy ǫ can be
i
any direction. We find that protein surface concentra-
calculatedfromtheadsorptionrateconstants[11]. These
tions CS are non-monotonic in time (Fig. 2). For any
rates are proportional to the probability for a protein i i
considered X , Alb is the first protein that reaches the
to attach to the nearby surface P
surface, inducing an increase of CS. When the second
A
ǫ fastest and second most affine protein, IgG, diffuses to
Pi ∝exp(cid:18)kBiT(cid:19). (4) the surface, it displaces Alb, leading to a decrease of CAS
and an increase of CS. Finally Fib, which is the slowest
I
However,the ǫ inphysicalunits arenot knowna priori. andmostaffineproteintothesurface,takesoverdecreas-
i
Hence, we consider the relative probabilities for differ- ing CS and increasing CS. Each CS saturates toward
I F i
ent proteins Pi ∝exp ǫi−ǫj , from which is possible to an equilibrium value at long times, while the total sur-
Pj (cid:16) kBT (cid:17) faceconcentrationofproteinsissaturatedatearlytimes.
determine the values of the different energies as
This behavior qualitatively reproduces the Vroman ef-
ǫ k T P fect, apart from the behavior of Fib that here is mono-
j =1− B ln A (5)
ǫ ǫ (cid:18)P (cid:19) tonic, while in experiments has a maximum due to the
A A j
competitive adsorption with heavier and more surface-
adopting ǫ for Alb as the energy units. We set ǫ by affine plasma proteins not included in our model [14].
A A
comparing our simulations results with experiments at The only effect of reducing X is a slowing down in the
P
ambient temperature, and get ǫ = 2.79 ǫ and ǫ = dynamicsoftheprocess,asobservedinexperiments[15].
I A F
3
(a)
0.25 0.25 (a) X =100%
P
) 0.2 X =100% ) 0.2
2 2
m P m
g/c 0.15 g/c 0.15
µ µ
( 0.1 ( 0.1
S S
Ci Ci
n 0.05 n 0.05
o o
ati 0 ati 0
r r
nt (b) nt (b)
ce 0.25 ce 0.25 X =25%
n n
o o P
C 0.2 X =25% C 0.2
e P e
c 0.15 c 0.15
a a
f f
r r
u 0.1 u 0.1
S S
0.05 0.05
0 0
0 0.005 0.01 0.015 0 0.005 0.01 0.015
Time t (s) Time t (s)
FIG.2. SimulationsatT =300Kand(a)XP =100%and(b) FIG. 3. Surface concentrations CiS as function of time for
XP = 25% show that surface concentration CAS of Alb (#), T = 120 K at (a) XP = 100% and (b) XP = 25%. At
CS of IgG (2) and CS of Fib (∆) are non-monotonic with long time, CS > CS, with an inversion with respect to the
I F I F
time, while their sum is (∇). At XP =50% (not shown) we standard conditions in Fig. 2 where CFS > CIS. We find the
findthesamebehaviorwithtime-scalesintermediatebetween same qualitative behavior at XP = 50%, not shown. Errors
those in (a) and (b). Bulk concentrations are as indicated in and symbols are as in Fig. 2.
thetext. Errors are smaller than symbol sizes.
2m) 0.2 Albumin
surTfahceemococdueplaatliloonwassuas ctoonsuenqdueernstcaenodf tthhee csoeqmupeentcietioonf µC (g/cSi0.15 T=120K T=300K IFnimbruinnoog γen
between the smaller, but less affine, proteins with the on 0.1
more affine, but bigger, proteins. For example, we test ntrati
that by increasing the Alb affinity, or artificially setting ce0.05
n
o
to the same value all the diffusion constants, the effect C
e
disappears. Therefore,affinity and hydrodynamicradius ac 0
are the relevant protein parameters for the effect. Surf 0 0.01t00.02 0.03 0.04 0.05 0.06
Time t (s)
Next,westudyhowenergydepletionoftheproteinso-
lutionaffectsthesequenceofadsorption. Here,forsakeof
simplicity, we decrease T, reducing the kinetic energy of FIG.4. Thesurfaceconcentrations CiS,asafunction oftime
thesolution,butneglectingpossibleeffectsoftheprotein for XP = 100%, is drastically affected when the system un-
dergoes a sudden change from en energy-depleted condition
stability. We find (i) that, although the surface affinity
to a normal condition. The vertical dashed line marks the
of Fib is stronger than that for IgG, the latter becomes time t0 of the change. We control the energy of the solution
the dominant protein adsorbed on the surface for long by changing the external parameter T from T = 120 K to
time scales; (ii) that, by changing X , the time scale T =300 K. Errors and symbols are as in Fig. 2.
P
of the process becomes longer, but the inversion of the
protein concentration is always present (Fig. 3). Hence,
the energydepletionleads to aninversionofthe Vroman tion of this layer, determining different biomimetics sur-
effect. face properties. This situation could occur, for example,
By comparing the results at different energies, k T, when a medical device is manipulated in a bioenviron-
B
and same X (Fig. 2-3), we observeonly a week energy- mentwhosecompositionisexternallycontrolledduringa
P
dependence of the times at which each CS reaches its surgery[18]. Inparticular,westudythecaseinwhichthe
i
maximum. Hence, the time-scales of the process are system is first equilibrated under energy-depleted condi-
mainlycontrolledby thetotalplasmaconcentrationX . tions and subsequently undergoes a sudden change that
P
Once we have understood that the protein layer cov- reestablishes the normal conditions (Fig. 4).
ering the surface is controlledby the energy depletion of Atshorttimestheenergy-depletedsystemevolvesuntil
the system, it is interesting to ask if a sudden change the equilibrium concentrations are reached. Under these
of external conditions could induce a different composi- conditions, as discussed (Fig. 3), the dominant protein
4
is IgG instead of Fib. At time t we switch to normal on a surface, in which the different families of proteins
0
conditions, forcing the system out of equilibrium. As a occupysequentiallythesurface,replacingeachother,un-
consequence, the system re-enters a transitory situation tilanequilibriumsituationisreached. Bydecreasingthe
in which the concentrations CS evolve until they reach totalconcentrationofproteininthesolution,keepingthe
i
their new equilibrium values at long times. In the spe- relative concentrations fixed, the time scales of the pro-
cificcaseconsideredhere,weobserveafastchangeinthe cessincreaseandthemaximaofsurfaceconcentrationfor
surface concentrations, with CS of Fib overcoming CS each family of proteins occur at longer times.
F I
of IgG, being the first, under normal conditions, more Wefindthattheproteinsurfaceconcentrationsatequi-
stable on the surface than the second. The final equi- librium depend on external control parameters. In par-
librium concentrations are reached at large times. We ticular, we find that energy depletion induces a drastic
observe also a sudden change in CS of Alb, between the change in the composition of the covering protein-layer,
A
twoequilibriumconcentrationscharacteristicsofthe two leading to an inversion of the Vroman effect. Our re-
valuesoftheexternalparametersT. However,CS always sults show that the inversion can be used to quantify
A
equilibrates to a value that is smaller then CS and CS, howstronglyirreversibleistheprocessofsurfaceadsorp-
I F
consistent with its long-time values in Fig. 2-3. By de- tion of the proteins, an information useful in studies of
creasing X , we find the same qualitative behavior for a thromboembolic events [17]. Furthermore, these results
P
sudden energy-change,but with the transientregime ex- suggest the possibility of engineering the composition of
tendingtolongertimes,consistentwithFig.3. Hence,at the protein layer covering a surface in a controlled way,
experimentalvalues of X the switching behavior would a feature particular relevant in biomimetic applications.
P
occur on time scales that are comparable to those char- We thank C. ˚Aberg, F. Baldelli Bombelli, and M. P.
acteristic of the Vroman effect. Monopoli for discussions. We acknowledge the support
We remark that our predictions about inverting the of EU FP7 grantNMP4-SL-2011-266737;PV and GF of
Vroman effect by changing the experimental control pa- SpanishMEC grantFIS2009-10210co-financedFEDER.
rameters should hold only if the protein adsorption on
the surface is reversible. If the adsorption is, instead, ir-
reversible the change of external parameters should not
lead to a new composition of the protein layer. Indeed,
∗
[email protected], [email protected]
under many practical conditions of interest for blood
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