Download The Effect of Surface Functionalization and Temperature

The Effect of Surface Functionalization and Temperature on Nanoparticle Penetration into Tumor Spheroids
Abhignyan Nagesettia, Diego Estumano b, Helcio R. B. Orlandeb, Marcelo J. Colaçob, George S. Dulikravicha & Anthony McGoronb
a
Florida International University, Miami, FL, U.S.A.
b Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
The Challenge
Results
Spheroid cell culture
Tumor cells
b
a
Method
 Conventional cell cultures (monolayers) do not have the functional (hypoxic core etc.)
or anatomical (ECM, stratified organization) attributes of tumor tissue.
 Tumor spheroids are representative in-vitro models of in-vivo avascular tumors
(micrometastases, early stage tumors) .
a
Tumor ECM
b
Bare nanoparticles
Distance from the Blood vessel
Plasma membrane
c
c
d
Figure 2: Optical microscope (4X) image of (a) 5 day old SKOV-3 spheroid, tight
aggregation of cells is evident. (b) SEM image of Dx-5 spheroid, (c) dense
aggregation of cells can be seen in SEM image (scale bar 1μm). (d) Maximal
intensity projection of a confocal image shows 3D shape of the spheroid
Adenosine -TriPhosphate
Total Number of
Nanoparticles
a
2.E+09
2.E+09
1.E+09
1.E+09
1.E+09
8.E+08
6.E+08
4.E+08
2.E+08
0.E+00
Zeta
(mV/pH)
0.008
0.020
0.024
-30.2 / 9.0
-5.81 / 7.4
-5.61 / 7.4
Table 1: Size, distribution
and zeta potential properties
of the nanoparticles
b
 PEG-COOH and m-PEG particles possess similar physical characteristics.
 PEGylation improved aqueous stability.
Conclusions and Future work
Results
Results
b
PDI
 Silica nanoparticles prepared by covalently conjugating Fluorescein Isothiocyanate
dye to the silica matrix.
LOSS OF CHEMOTHERAPEUTIC
EFFECTIVENESS
a
Average
Diameter
(nm)
58.8
63.4
65.3
Bare
PEG-COOH
m-PEG
Limited Drug Penetration
+
Multi-Drug Resistance
Drug Molecules (Doxorubicin)
ATP
Figure 3: Dynamic light scattering results of silica particles. Size results: bare
(red): 58 ± 8 nm (n=3) PDI: 0.070, PEGylated (green) 68 ± 7 nm (n= 3), PDI:
0.118.
Figure1: (a) Trastuzumab (green) is unable to
penetrate to the hypoxic regions of the tumor
(blue) from the blood vessels (red). (b) Tumor
Extracellular Matrix (ECM) is a barrier to
delivery of drugs, nanoparticles and nutrients.
(c) P-glycoprotein: Actively (requiring ATP)
pumps out drugs from cell interior, reducing
intracellular drug concentration and causing
Multi-Drug Resistance (MDR)
Nugent et.al.
PEGylated
c
@+37°C
Value
-ve 95 % CI
+ve 95 % CI
ka (m-1 s-1)
kd (s-1)
ke (s-1)
9.75E+04
1.97E-02
1.35E-03
9.70E+04
1.95E-02
1.26E-03
9.79E+04
2.00E-02
1.45E-03
Table 2: Uptake rate constants estimated using MCMC method at +37ºC, 95 %
confidence indicate good agreement with experimental measurements
M-PEG
PEG-COOH
Figure 4: (a) Silica particles internalized and fluorescent at 488 nm excitation.
Punctate formations indicate clathirin mediated endocytosis. (b) Uptake of PEG-COOH
and m-PEG particles in Skov-3 cells (incubation time 2 hours).
 Surface functionalization affects the uptake of nanoparticles in Skov-3 cells.
 Methoxy groups are inert and do not interact with plasma proteins. Carboxyl
groups interact with plasma proteins and hence are internalized by the cells.
Figure 5: (a) Confocal section of m-PEG particle distribution (green) in a tumor
spheroid (nuclei stained blue) (40x magnification) (b) Confocal section of PEG-COOH
particle distribution (green) (20x magnification) (c) Results from MCMC simulations
overlaid on experimental data
 m-PEG particles showed a disperse distribution in tumor spheroids.
 PEG-COOH particles showed a homogeneous distribution at the periphery of the
spheroid.
 Markov Chain Monte Carlo (MCMC) simulations were used to estimate the rate
constants of uptake at different temperatures.
 Uptake experiments done in monolayers at 90 % confluency.
@ +43°C
Value
-ve 95 % CI
+ve 95 % CI
ka (m-1 s-1)
kd (s-1)
ke (s-1)
1.84E+05
6.99E-02
8.43E-03
1.78E+05
6.61E-02
7.99E-03
1.90E+05
7.37E-02
8.88E-03
Table 3: Uptake rate constants estimated using MCMC method at +43ºC, 95 %
confidence indicate good agreement with experimental measurements
 Surface functionalization plays a major role in particle uptake by the cells and their
penetration into the spheroids.
 Temperature dependence of rate constants has been successfully modeled by
MCMC simulations.
 Parameters obtained from 2D experiments will be used in a diffusion - reaction
model to study the effect of hyperthermia on nanoparticle penetration in spheroids.
References : 1. L.J. Nugent, R.K. Jain Extravascular diffusion in normal and neoplastic tissues. Cancer Res.,
44 (1) (1984), pp. 238–244.
2. Goodman, T.T, Spatio-temporal modeling of nanoparticle delivery to multicellular tumor
spheroids. Biotechnol Bioeng., 101(2) (2008) pp. 388-399.
Acknowledgements : Department of Biomedical Engineering; FIU MBRS BRI Summer Grant