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

Tumor cells

Tumor ECM

Drug Molecules (Doxorubicin)

ATP Adenosine -TriPhosphate

The Challenge

Distance from the Blood vessel

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)

Limited Drug Penetration +

Multi-Drug Resistance

LOSS OF CHEMOTHERAPEUTIC EFFECTIVENESS

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

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

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.

The Effect of Surface Functionalization  and Temperature on Nanoparticle Penetration into Tumor SpheroidsAbhignyan  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

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 tumorspheroids. Biotechnol Bioeng., 101(2) (2008) pp. 388-399.

Acknowledgements : Department of Biomedical Engineering; FIU MBRS BRI Summer Grant

Silica nanoparticles prepared by covalently conjugating Fluorescein Isothiocyanate dye to the silica matrix.

PEG-COOH and m-PEG particles possess similar physical characteristics.

PEGylation improved aqueous stability.

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.

c

b

d

Spheroid cell culture

a

Table 1: Size, distribution and zeta potential properties of the nanoparticles

a

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).

M-PEG PEG-COOH0E+00

1E+09

2E+09

To

tal

Nu

mb

er

of

Na

no

pa

rtic

les

0

10

20

30

0.1 1 10 100 1000 10000

Num

ber

(%)

Size (d.nm)

Size Distribution by Number

Record 486: f itc13.3 Record 489: f itc13.3_PEG_Cooh

Average Diameter

(nm)

PDI Zeta (mV/pH)

Bare 58.8 0.008 -30.2 / 9.0

PEG-COOH 63.4 0.020 -5.81 / 7.4

m-PEG 65.3 0.024 -5.61 / 7.4

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.

Nugent et.al.

@ +43°C Value -ve 95 % CI +ve 95 % CI

ka (m-1 s-1) 1.84E+05 1.78E+05 1.90E+05

kd (s-1) 6.99E-02 6.61E-02 7.37E-02

ke (s-1) 8.43E-03 7.99E-03 8.88E-03

@+37°C Value -ve 95 % CI +ve 95 % CI

ka (m-1 s-1) 9.75E+04 9.70E+04 9.79E+04kd (s-1) 1.97E-02 1.95E-02 2.00E-02ke (s-1) 1.35E-03 1.26E-03 1.45E-03

Table 2: Uptake rate constants estimated using MCMC method at +37ºC, 95 % confidence indicate good agreement with experimental measurements

Table 3: Uptake rate constants estimated using MCMC method at +43ºC, 95 % confidence indicate good agreement with experimental measurements

b c

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.

Method

Results Conclusions and Future work

Results

Results

b

Plasma membrane

a b

c

Bare nanoparticles PEGylated