Bioadhesion, nanoparticles, intravascular deliveryThe non-specific adhesion of spherical particles to a cell substrate is analyzed in a parallel plate flow chamber, addressing the effect of the particle size. Differently from other experiments, the total volume of the injected particles has been fixed, rather than the total number of particles, as the diameter d of the particles is changed from 500 nm up to 10 ìm. From the analysis of the experimental data, simple and instructive scaling adhesion laws have been derived showing that (i) the number of particles adherent to the cell layer per unit surface decreases with the size of the particle as d^(-1.7) ; and consequently (ii) the volume of the particles adherent per unit surface increases with the size of the particles as d^(+1.3). These results are of importance in the ‘rational design’ of nanoparticles for drug delivery and biomedical imaging

Decuzzi P.

Ferrari, M.

Gentile F.

Granaldi A.

I-Revues

ENS’06 Paris, France, 14-15 December 2006 
©TIMA Editions/ENS 2006 -page- ISBN:  
 
ON THE ADHESION OF PARTICLES TO A CELL LAYER UNDER FLOW 
 
F. Gentile a, A. Granaldi b, M. Ferrari c, P. Decuzzi a,b,c * 
 
a
 Center of Bio-/Nanotechnology and -/Engineering for Medicine (BioNEM), University of Magna 
Graecia, Catanzaro - ITALY  
b
 Center of Excellence in Computational Mechanics (CEMEC), Politecnico di Bari, Bari - ITALY  
c
 The Univeristy of Texas Health Science Center at Houston, Houston, Texas - USA 
 
 
ABSTRACT 
 
The non-specific adhesion of spherical particles to a cell 
substrate is analyzed in a parallel plate flow chamber, 
addressing the effect of the particle size. Differently from 
other experiments, the total volume of the injected 
particles has been fixed, rather than the total number of 
particles, as the diameter d of the particles is changed 
from 500 nm up to 10 µm. From the analysis of the 
experimental data, simple and instructive scaling adhesion 
laws have been derived showing that (i) the number of 
particles adherent to the cell layer per unit surface 
decreases with the size of the particle as d-1.7; and 
consequently (ii) the volume of the particles adherent per 
unit surface increases with the size of the particles as d1.3. 
These results are of importance in the ‘rational design’ of 
nanoparticles for drug delivery and biomedical imaging. 
 
1. INTRODUCTION 
 
      Advances in nanosciences and micro/nanofabrication 
techniques have led to novel strategies and devices for 
drug delivery and diagnosis in biomedical applications 
[1,2]. In particular, biomimetic artificial carriers are 
emerging as powerful tools for cancer and heart diseases 
treatment and molecular imaging. These are particulates 
carrying therapeutic and/or imaging agents administered 
at the systemic level and delivered towards a specific 
biological target (diseased cell or microenvironment). The 
surface of these particulates is generally coated by ligand 
molecules that can recognize countermolecules (receptors) 
expressed at the biological target. The power of 
intravascularly injectable particles over free molecules 
administration lies in their multifunctionality and 
engineerability [3]. The use of such particles as 
intravascular delivery vectors affords substantial 
advantages including (i) the specific biomolecular 
targeting through one or more conjugated antibodies or 
other recognition molecules; (ii) the ability to carry one or 
more therapeutic agents; (iii) the signal amplification for 
imaging through co-encapsulated contrast agents; (iv) the 
tailoring of the physico-chemical and geometrical 
properties favoring avoidance of the biological and 
physiological barriers and improve the recognition of the 
target cells or microenvironment. 
A broad spectrum of particulates have been presented 
in the literature which can be used both in medical therapy 
and imaging. These have different sizes, ranging from few 
tens of nanometers (dendrimers [4]) to hundreds of 
nanometers (polymer particles and liposome [5,6]) up to 
few microns (silicon and silica based particles [7]); 
different shapes from the classical spherical to spheroidal 
[8] and even more complex shapes [9]; and with different 
compositions and surface chemico-physical properties. 
Differently from freely administered drugs, these particles 
can be engineered [10,11] and it is of fundamental 
importance to understand if, in the great variety of shapes, 
sizes and compositions, there is any 'optimal particle’ with 
the largest selectivity and delivery efficiency. 
In this work, the adhesive performance of the particles 
with a size ranging between 500 nm and 10 µm is assessed 
in terms of the number of particles adhering non-
specifically to a confluent layer of endothelial cells under 
fixed hydrodynamic conditions. Goetz and colleagues [12] 
have already analyzed the effect of the size of spherical 
particles on their adhesive performance. However, since 
they were interested in analyzing the interaction between 
leukocytes and endothelial cells, employed 5−, 10−, 15−, 
and 20 − µm diameter microspheres. Also, the 
experimental analysis was carried out using a biological 
substrate consisting of a glass slide covered with P-
selectin molecules, whereas in this work the biological 
substrate is made up of a confluent layer of endothelial 
cells. Nonetheless, they showed that the adhesive strength 
of the particles, expressed in terms of the critical shear 
stress and rolling velocity, is significantly influenced by 
their diameter.  
 
2. MATERIALS AND METHODS 
 
       A Glycotech (Rockville, MD) parallel plate flow 
chamber has been used as described in [13]. The flow 
 ©TIMA Editions/ENS 2006 -page- ISBN:  
chamber is connected to a Harvard Apparatus syringe 
pump through the inlet hole and a silastic tubing; and to a 
reservoir through the outlet hole and a silastic tubing. The 
channel within the flow chamber is defined by a silicon 
rubber gasket sitting between the flow deck and the 
microscope 35-mm dish. The gasket used in the present 
experiments has a thickness of 0.01 in and a width of 1 
cm. The volumetric flow rate Q has been fixed equal to 50 
µl/min for all the experiments. After assembling the flow 
chamber on the stage of an inverted microscope, a region 
at the center of the channel is chosen with a sufficiently 
large number of confluent cells. This is the region of 
interest (ROI). The microscope objective is focused on 
this region and after a short rinse, the flow with the 
particles enters the chamber.      
Using the fluorescent module of the microscope, 
the flow of the particles in the ROI is recorded during the 
whole experiments. Each acquired frame is then analyzed 
in MatLab® (Matlab6.5) using a self-developed imaging 
analysis code. The number of particles adherent to the 
whole substrate ntot within the ROI and the number of 
particles adherent to the sole cells nc within the ROI are 
measured as a function of the frame number, which is 
easily converted in time by knowing the acquisition rate. 
Human umbilical vein endothelial cells 
(HUVECs), purchased from Cambrex, Inc. (East 
Rutherford, NJ), were plated on a borosilicate glass with a 
0.2 mg/cm² substratum of type A gelatine (Sigma-Aldrich 
Corporation, MO). 
Fluoresbrite® Microspheres from Polysciences 
were used. 
 
3. RESULTS 
 
The number of adherent particles within the region of 
interest (ROI) has been monitored as a function of the size 
during the whole experiment. Figure1 shows, as an 
example, the variation with time of the particles nc 
adhering solely to the cells (lower curve) and the total 
number ntot of adherent particles within the ROI (upper 
curve), for the 1 µm, 750 and 500 nm particles. An image 
of the fluorescent particles adhering to the culture dish 
and to the cells is shown in Figure2, for the 1 µm particle. 
From the analysis of the experimental data, the number of 
particles ntot and nc at the end of each experiment can be 
plotted as a function of the particle diameter d (Figure3). 
As the diameter of the particle reduces the number of 
adherent particles, both ntot and nc, increases; and a non 
linear regression of the experimental data leads to the two 
following approximate expressions 
 
696.196.627 −= dntot  (1) 
143.1754.164 −= dnc   (2) 
 
Differently, in Figure4, the number of particles adherent 
per unit surface is shown as a function of their diameter: 
the total number of adherent particles ntot is normalized by 
the area of the ROI, whereas the number of particles 
adherent to the sole cells nc is normalized by the area 
occupied by the cells within the ROI, which depends on 
the level of cell confluency. In a double logarithm 
diagram, the surface densities of the total number of 
particles totn
~
 and of the particles adherent on the cells  
cn
~ are well aligned along two straight and almost parallel 
lines (Figure4), and the non linear regression of the 
experimental data leads to 
 
696.139.1116~ −= dntot   (3) 
 
746.134.2784~ −= dnc   (4) 
 
 
4. DISCUSSION AND CONCLUSIONS 
 
The non-specific adhesion under flow of spherical 
particles onto a confluent layer of endothelial cells is 
analyzed in-vitro using a parallel plate flow chamber. 
Differently from other experimental analysis, the total 
number of particles injected in the flow chamber has been 
changed to keep fixed the total volume of the particles 
with their size. Since the focus is on drug delivery and 
biomedical imaging, by keeping fixed the total volume of 
the particles, the total volume of therapeutic or imaging 
agents delivered to the cell layer are fixed in the analysis. 
The experimental results, which have been derived under 
fixed hydrodynamic conditions, lead to conclude that (i) 
the surface density of particles adhering to the cells cn
~
 
decreases with the diameter d following the scaling law 
7.1−
≈ d ; (ii) the volume per unit surface of the particles 
adhering to the cells 3( / 6)cV n dpi=ɶ ɶ  increases with the 
diameter d following the scaling law 3.1+≈ d . These 
scaling laws have been also justified by theoretical 
reasonings, as described in details in [14]. 
Since in drug delivery, it is desirable to increase the 
amount of drug that can be released per unit surface on a 
cell layer, the above results would suggest to use the 
largest possible particles in drug delivery. Notice 
however, that the following suggestion is derived by 
experimental observations of non-specific adhesion at 
sufficiently small shear stresses at the wall. In specific 
adhesion, Decuzzi and Ferrari [15] have already shown 
that there is an optimal particle size corresponding to the 
largest strength of adhesion. 
 ©TIMA Editions/ENS 2006 -page- ISBN:  
Differently in biomedical imaging, it is desirable to 
increase the number of particles adherent per unit surface 
to have the largest possible resolution of the cell layer. As 
a consequence, the above results would suggest to use the 
smallest possible particles in biomedical imaging. 
 
 
5. REFERENCES 
 
[1] LaVan DA, McGuire T and Langer R. Nat. 
Biotechnol. 21; 1184 (2003) 
[2] Ferrari M. Nat Rev Cancer ;5:161-171 (2005) 
[3] M. Ferrari, Curr Opin Chem Biol. 9, 343 (2005) 
[4] Choi YS, Thomas T, Kotlyar A, Islam MT, Baker JR, 
Jr., Chemistry & Biology 12, (2005) 
[5] Duncan R. Nat RevDrug Discov. 2; 347 (2003) 
[6] Crommelin DJA, Schreier H. Liposomes. In: Colloidal 
Drug Delivery Systems, J. Kreuter, editor. Marcel Dekker, 
Inc.. New York. (1994) 
[7] Cohen MH, Melnik K, Boiarski AA, Ferrari M, Martin 
FJ. Biomedical Microdevices 5; 253 (2003) 
[8] van Dillen T, van Blaaderen A, Polman A. Materials 
Today; 40 (2003). 
[9] Rolland JP, Maynor BW, Euliss LE, Exner AE, 
Denison GM, DeSimone J., J. Am. Chem. Soc. 127; 
10096 (2005) 
[10] Decuzzi P, Lee S, Decuzzi M, Ferrari M. Annals of 
Biomedical Engineering 33; 179 (2004) 
[11] Decuzzi P, Lee S, Bhushan B, Ferrari M. Annals of 
Biomedical Engineering 33; 179 (2005) 
[12] Vivek R, Patil S, Campbell CJ, Yun YH, Slack SM, 
Goetz DJ. Biophysical Journal. ;80:1733- 1743 (2001) 
[13] David C Brown and Richard S Larson, BMC 
Immunology 2; 9 (2001) 
[14] Decuzzi P. et al., International Journal of 
Nanomedicine ;in print, (2006) 
[15] Decuzzi P and Ferrari M, Biomaterials. 27; 5307 
(2006) 
 
 
*  Corresponding Author: 
 
Paolo Decuzzi, PhD              p.decuzzi@poliba.it 
 
Associate Professor of Mechanical and Biomedical 
Engineering 
 
Center of Bio-/Nanotechnology and -/Engineering for 
Medicine (BioNEM); University of Magna Graecia, Viale 
Europa - Loc. Germaneto; 88100 Catanzaro, ITALY 
and Center of Excellence in Computational Mechanics 
(CEMEC), Politecnico di Bari, Via Re David 200 Bari, 
ITALY 
 
0 100 200 300 400 500 600
t @sD
0
200
400
600
n
AdheredParticles - 1 mm
 
 
 
0 200 400 600 800 1000
t @sD
0
500
1000
1500
2000
2500
n
Adhered Particles - 750 nm
 
 
0 100 200 300 400 500 600 700
t @sD
0
500
1000
1500
2000
2500
3000
3500
n
Adhered Particles - 500 nm
 
 
Figure1. The variation of the number n of adherent 
particles with time for d = 1µ m, d = 750 nm and d = 500 
nm. The upper curve is for the total number of adherent 
particles ntot, whereas the lower curve is for the particles 
adhering solely on cells nc. 
 
 
 
 ©TIMA Editions/ENS 2006 -page- ISBN:  
 
 
Figure2. An image showing red fluorescent 1 µm - 
particles adhering to a layer of HUVECs within the region 
of interest (ROI). 
 
 
0.5 0.6 0.7 1. 1.5 2. 3. 4. 5. 6. 7. 10.
d @mmD
10.
20.
50.
100.
2
5
lO3
2
n
628 d-1.7
165 d-1.14
 
 
Figure3. The variation of the number of particles 
adhering at the end of the experiments within the ROI, on 
the sole cells (black boxes) and on the glass dish (white 
circles) as a function of the particle diameter d. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
0.5 0.6 0.7 1. 1.5 2. 3. 4. 5. 6. 7. 10.
d @mmD
10.
20.
50.
100.
2
5
lO3
2
5
lO4
2
nêmm2 Log Log Plot - normalizednumber of particles
3
4
8
4
41116.39 d-1.696
2784.34 d-1.74589
 
 
Figure4. The variation of the surface density of particles 
adhering at the end of the experiment within the ROI, on 
the sole cells (black boxes) and on the glass dish (white 
circles) as a function of the particle diameter d. 


On the adhesion of particles to a cell layer under flow

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