1. 978-1-4799-8360-5/15/$31.00 ©2015 IEEE
Characterizing the Effects of Metal Oxide
Nanoparticle Ingestion on Intestinal Function
Nicole Martucci1
, Rajesh Burela2
, and Gretchen Mahler1
1
Department of Biomedical Engineering
2
Department of Neuroscience
Binghamton University, State University of New York
Binghamton, New York
ABSTRACT
Silicon Dioxide (SiO2) and Titanium Dioxide (TiO2) are
engineered nanoparticles often ingested from food and food
packaging. SiO2 is a common additive, where it is used as a flow
agent or to absorb extra water [1]. TiO2 is commonly used as a
pigment for foods including candy and medications, and is often
found in cosmetic skin care products [2]. The effects of ingesting
these materials, however, is not yet fully understood. The
purpose of this study is to characterize the size and surface
chemistry of 30 nm SiO2 and TiO2 nanoparticles following
digestion and to determine how nanoparticle ingestion affects
small intestinal alkaline phosphatase activity.
MATERIALS AND METHODS
Chemicals, enzymes, and hormones
All chemicals, enzymes, and hormones were purchased
from Sigma Chemical Company (St. Louis, MO) unless
otherwise stated.
Cell Culture
The human colon carcinoma Caco-2 cell line was obtained
from the American Type Culture Collection (Manassas, VA).
Caco-2 cells were grown in Dulbecco's Modified Eagle
Medium (DMEM, Life Technologies, Grand Island, NY)
containing 4.5 g/L glucose and 10% fetal bovine serum
(Invitrogen). The cells were maintained at 37°C in 5% CO2
and culture medium was changed every 2 days. For
experimental studies, Caco-2 were seeded at a density of
90,000 cells/cm2
onto 24 well plates (Corning Life Sciences,
Acton, MA) coated with rat tail Type I collagen (BD
Biosciences, San Jose, CA) at 8 μg/cm2
. Experiments were
performed 14 days post-seeding.
Nanoparticle Dose
A mid and high concentration of 30 nm TiO2 and SiO2 (US
Research Nanomaterials, Inc, Houston, TX) were used. The
TiO2 high concentration was 1.4 x 10-4
mg/mL and the mid
concentration was 1.4 x 10-6
mg/mL. The SiO2 high
concentration was 2.0 x 10-4
mg/mL and the mid-concentration
was 2.0 x 10-6
mg/mL. These doses of nanoparticles are
relevant to potential real-life exposure [2, 3].
In Vitro Digestion
A detailed in vitro digestion method has been described by
Glahn et al [4]. Briefly, the gastric digestion phase was
initiated by adding 10 mL of 140 mM NaCl, 5 mL KCl, pH 2
solution to each nanoparticle sample and re-adjusting each
sample to pH 2 with 1 M HCl. An aliquot of 0.5 mL porcine
pepsin solution in 0.1 M HCl was added to each tube and the
samples were rocked on a rocking platform for 1 hour at 37o
C.
After the 1 hour gastric incubation, the pH of the samples was
raised to 5.5-6.0 with 1 M NaHCO3 and 2.5 mL of porcine
pancreatin/bile solution was added. The pH was then adjusted
to 7.0 with 1 M NaHCO3 and the volume of each tube brought
to 15 mL by weight with 140 mM NaCl, 5 mM KCl, and pH
6.7. After this point, the samples were referred to as digests.
Zeta Potential and Size Measurements
Zeta potential and size of particles was measured by
dynamic light scattering with a Malvern Zetasizer Nano-ZS
(Malvern Instruments Inc, Southborough, MA) at 25o
C. The
zeta potential of 30 nm particles was measured in 18 MΩ
water, phosphate buffered saline (PBS), DMEM, and digest;
each nanoparticle dispersion was measured three times.
The colloidal solution is regarded as stable when the zeta
potential is below -30 millivolts (mV) or above +30 mV, and
instable when it lies between -30 mV and +30 mV.
Alkaline Phosphatase Activity Assay
Half of Caco-2 cells were exposed to a mid-concentration
(1.4 x 10-6
mg/mL) of 30 nm TiO2 for 4 hours; the remaining
cells were not exposed to nanoparticles. The cells were then
washed with 0.5 mL of PBS and suspended in 0.2 mL of 18
MΩ water. They were then sonicated for 5 minutes at room
temperature. To obtain the cell lysate, each well was scraped
into individual Eppendorf tubes.
The Alkaline Phosphatase Assay (ALP) detects the
presence of alkaline phosphatase activity by using p-
nitrophenyl phosphate (pNPP) as the substrate. The pNPP
solution was created by dissolving one Tris Buffer tablet and
one pNPP tablet (product code: N2770 SIGMA) in 5mL of
water. Alkaline phosphatase hydrolyzes pNPP to p-
nitrophenol. 25 µL of lysate solution from each Eppendorf
tube was added to each well of a 96-well plate. 85 µL of the
pNPP solution was then added to the wells, bringing the total
volume to 110 µL per well. The plate was then incubated at
room temperature for 1 hour. The absorbance was then read at
405 nm to measure to concentration of p-nitrophenol.
Next the Bradford assay was used to determine the total cell
protein concentration. 5 µL of cell lysate was added to a 96
well plate. 250 µL of Bradford Reagent was then added to
each well. After incubating for 15 minutes at room
temperature, absorbance was read at 595 nm. For each assay, a
standard curve was created with p-nitrophenol or bovine

2. serum albumin (BSA) to calculate the unknown concentrations
of p-nitrophenol or protein.
Statistical Analysis
A two tailed student’s t-test was performed to determine
statistical significance, using the programming language R.
The results were plotted using Graph Pad and samples were
considered significant with a p value ≤ 0.05.
RESULTS AND DISCUSSION
The results of nanoparticle surface chemistry
characterization are summarized in Tables 1-4. The particle
size measurements showed a dramatic decrease in nanoparticle
size following in vitro digestion (Tables 1 and 2). The
measured zeta potential showed a minimal change in
magnitude; both the SiO2 and TiO2 nanoparticles measured
around -25 mV before and after the digestion process. These
results are shown below in Tables 3 and 4.
TABLE 1 - THE PARTICLE SIZE OF SiO2 BEFORE AND AFTER IN VITRO DIGESTION
TABLE 2 - THE PARTICLE SIZE OF TiO2 BEFORE AND AFTER IN VITRO DIGESTION
TABLE 3 - THE RESULTS OF THE ZETA POTENTIAL FOR SiO2 BEFORE AND AFTER IN
VITRO DIGESTION
TABLE 4 - THE RESULTS OF THE ZETA POTENTIAL FOR TiO2 BEFORE AND AFTER IN
VITRO DIGESTION
The purpose of the Alkaline Phosphatase Activity Assay
was to determine how nanoparticle ingestion affects the small
intestinal alkaline phosphatase activity. Alkaline phosphatase
is an enzyme that is responsible for the proper breakdown and
absorption of nutrients, such as proteins, lipids, and
carbohydrates, in the body [5]. Dividing the results of the ALP
assay by the Bradford assay yields milligrams of p-nitrophenol
per milligram of protein. For the control group a value of 6.76
mg of p-nitrophenol per mg of protein was obtained. For the
cells exposed to TiO2, an increased value of 7.9 mg of p-
nitrophenol per mg of protein was obtained. This data was
plotted in a bar chart and can be seen below in Figure 1. A p-
value of 0.05 was obtained after running a t-test to statistically
compare the results using the programming language R. Thus,
results showed a slight statistical difference in the mg of p-
nitrophenol per mg of protein, and a minimal effect on the
alkaline phosphatase activity.
Figure 1- The results of the Alkaline Phosphatase Assay and Bradford Assays
to measure alkaline phosphatase activity. Results show average ± SEM, n = 8.
CONCLUSION
It has been shown that with digestion, nanoparticle
aggregates decrease dramatically in size. Zeta potential
showed relatively no change after the nanoparticles were put
through the in vitro digestion process. It was also shown that
cell exposure to nanoparticles minimally increases the amount
of alkaline phosphatase activity, which could indicate an
alteration of intestinal function due to nanoparticle exposure.
Future work includes testing the effects of SiO2 on ALP
activity, and subjecting the nanoparticles to in vitro digestion
before exposing them to cells.
ACKNOWLEDGEMENTS
Funding for this work was provided by the S3IP
Undergraduate Research Initiatives Award and the National
Institutes of Health (1R15ES022828)
REFERENCES
[1] Chaudhry, Q et al. “Applications and implications of nanotechnologies for
the food sector,” Trends Biotechnol., pp. 7,27,82. 2008
[2] A. Weir, P. Westerhoff, L. Fabricius, N. von Goetz, “Titanium Dioxide
Nanoparticles in Food and Personal Care Products,” Environ Sci Technology,
2012 February 21; 46(4): 2242-2250
[3] Lomer, MCE, Thompson, RPH & Powell, JJ. “Fine and ultrafine particles
of the diet: Influence on the mucosal immune response and association with
Crohn's disease.” P Nutr Soc 61, 123-130, (2002).
[4] Glahn RP, Lee OA, Yeung A, Goldman MI, Miller DD. “Caco-2 cell
ferritin formation predicts nonradiolabeled food iron availability in an in vitro
digestion Caco-2 cell culture model.” Journal of Nutrition, pp. 128(9):1555-
1561, 1998
[5] Jean-Paul Lalles, “Intestinal alkaline phosphatase: multiple biological
roles in maintenance of intestinal homeostasis and modulation by diet,”
Nutrition Reviews, Vol. 68(6):323-332, 2010.
SiO2 Size Before Digestion (nm) After Digestion (nm)
Concentration H20 PBS DMEM Average ± SEM
High 4016 4410 4916 604.65 ± 11.6
Mid 1025 1389 1360 615.45 ± 15.5
TiO2 Size Before Digestion (nm) After Digestion (nm)
Concentration H20 PBS DMEM Average ± SEM
High 706.7 2624 146 625.8 ± 19.7
Mid 435.1 686.5 977.53 644.95 ± 24.5
SiO2 Zeta Potential Before
Digestion (mV)
After Digestion (mV)
Concentration H2O PBS DMEM Average ± SEM
High -45.2 -20.7 -33.9 -26.9 ± 1.1
Mid -35.1 -27.6 -19.4 -28.7 ± 3.7
TiO2 Zeta Potential Before
Digestion (mV)
After Digestion (mV)
Concentration H2O PBS DMEM Average ± SEM
High 38.8 -28.6 -7.56 -27.45 ± 1.75
Mid 28 -20.3 -14.8 -29.75 ± 0.15