AN IN VIVO HUMAN TIME-EXPOSURE INVESTIGATION OF A COMMERCIAL SILVER NANO-PARTICLE SOLUTION.

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Feature Article
In vivo human time-exposure study of orally dosed commercial
silver nanoparticles
Mark A. Munger, PharmD
a,d,
⁎, Przemyslaw Radwanski, PharmD, PhD
b
,
Greg C. Hadlock, PhD
b
, Greg Stoddard, MS
d
, Akram Shaaban, MD
e
, Jonathan Falconer, BS
c
,
David W. Grainger, PhD
c
, Cassandra E. Deering-Rice, PhD
b
a
Department of Pharmacotherapy, University of Utah, Salt Lake City, UT, USA
b
Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT, USA
c
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT, USA
d
Department of Internal Medicine, University of Utah, Salt Lake City, UT, USA
e
Department of Radiology, University of Utah, Salt Lake City, UT, USA
Received 22 April 2013; accepted 22 June 2013
Abstract
Human biodistribution, bioprocessing and possible toxicity of nanoscale silver receive increasing health assessment. We prospectively
studied commercial 10- and 32-ppm nanoscale silver particle solutions in a single-blind, controlled, cross-over, intent-to-treat, design.
Healthy subjects (n = 60) underwent metabolic, blood counts, urinalysis, sputum induction, and chest and abdomen magnetic resonance
imaging. Silver serum and urine content were determined. No clinically important changes in metabolic, hematologic, or urinalysis measures
were identified. No morphological changes were detected in the lungs, heart or abdominal organs. No significant changes were noted in
pulmonary reactive oxygen species or pro-inflammatory cytokine generation. In vivo oral exposure to these commercial nanoscale silver
particle solutions does not prompt clinically important changes in human metabolic, hematologic, urine, physical findings or imaging
morphology. Further study of increasing time exposure and dosing of silver nanoparticulate silver, and observation of additional organ
systems are warranted to assert human toxicity thresholds.
From the Clinical Editor: In this study, the effects of commercially available nanoparticles were studied in healthy volunteers, concluding
no detectable toxicity with the utilized comprehensive assays and tests. As the authors rightfully state, further studies are definitely
warranted. Studies like this are much needed for the more widespread application of nanomedicine.
© 2014 Elsevier Inc. All rights reserved.
Key words: Biological activity nanoparticles; Nanotechnology; Nanotoxicology oral ingestion; Safety research
Nanotechnology is driving potentially the most important
engineering revolution since the industrial age. Currently, over
1,300 manufactured nanotechnology-enabled consumer products
are available in the marketplace.
1
This abundant, increasingly
common consumer accessibility to engineered nanomaterials
included in diverse health, cosmetic, food and agricultural,
recreational equipment and clothing products has afforded
increasing exposure of human tissue and physiology to different
routes of nanomaterial entry into the human body. The con-
sequences of such exposure, both deliberate and inadvertent, to
large populations are currently debated, with little current
consensus on the risks, toxicities, risk management and expo-
sure.
2-5
This scenario has contributed to nanoparticulate silver's
reemergence as a nutraceutical product and possible medical
modality.
6-8
Nanoscale silver makes up approximately a quarter
of the inventory of the present commercially available
nanoproduct inventory. With centuries of silver therapeutic
attributes, silver nanoproducts are claimed to provide unique
physiochemical properties and biological activities broadening
its application as an antibacterial, anti-viral, and anti-inflamma-
tory therapy.
9-12
However, elemental silver in nanoparticulate
CLINICALLY RELEVANT
Nanomedicine: Nanotechnology, Biology, and Medicine
10 (2014) 1 –9
nanomedjournal.com
Trial Registration: Clinical-Trials.gov: https://register.clinicaltrials.gov/
(Identifier: NCT01243320 and NCT01405794).
Funding: This study was funded in part by Award Number
UL1RR025764 from the National Center for Research Resources.
Conflict of Interest: The authors declare no conflicts of interest or
financial interests in any product or service mentioned in this article,
including grants, employment, gifts, stock holdings, honoraria, consultan-
cies, expert testimony, patents, and royalties.
The authors express appreciation to The Utah Lung Health Study Clinic,
and Nicholas Cox, and Angela Wolsey for their technical and clinical
support.
⁎Corresponding author: University of Utah, Salt Lake City, Utah, USA.
E-mail address: mmunger@hsc.utah.edu (M.A. Munger).
1549-9634/$ –see front matter © 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.nano.2013.06.010
Please cite this article as: Munger MA, et al, In vivo human time-exposure study of orally dosed commercial silver nanoparticles. Nanomedicine: NBM
2014;10:1-9, http://dx.doi.org/10.1016/j.nano.2013.06.010

form (i.e., Ag
0
) has distinct physical, chemical and toxicological
properties from long-studied soluble silver ions (Ag
+aq
)made
bioavailable from diverse salts. Increasing systemic exposure to
humans in dermally absorbed, ophthalmologically applied,
ingested, inhaled and possibly parenterally injected silver forms
facilitates vascular transport and penetration of silver nanoparticles
across tissue surfaces and through membranes. Human bioavail-
ability, biodistribution, and possible accumulation of these
nanoparticles have not been reported. Therefore, the potential for
evasion of immune cell-based clearance, leading to systemic acute
or chronic cytotoxicity or illness remains an open question.
8
A growing body of in vitro evidence supports cell toxicity
for silver nanoparticles in concentrations between 5-50 μg/ml.
The lung,
12-14
liver,
10,15-18
brain,
19
vascular system,
20
and
reproductive systems
21,22
may be negatively influenced.
Given the increased introduction of engineered new nanoscale
products into the consumer marketplace, it is important to
understand whether in vitro findings translate to in vivo
human toxicity.
To this end, we studied in an intent-to-treat analysis 60
healthy volunteers through several time-length exposures to orally
dosed commercial silver nanoparticles in a prospective, placebo-
controlled, single-blind, dose-monitored, cross-over design. The
study is the first to quantitate changes in human metabolic,
hemotologic, and sputum morphology, and to monitor for
changes in physical findings and organ imaging after exposure
to a commercially available aqueous silver colloid nanoparticle
oral formulation.
Methods
Study population
Two intent-to-treat studies were conducted at the University
of Utah Lung Health Study Clinic and Center for Clinical and
Translational Sciences at the University of Utah Hospital. Each
subject underwent a screening evaluation to assess enrollment
eligibility. Sixty healthy volunteers, between 18-80 years of age,
were subsequently enrolled (i.e., 10 ppm oral silver particle
dosing [36 subjects] and 32 ppm oral silver particle dosing
[24 subjects]). Females of child-bearing potential, defined as women
physically capable of becoming pregnant, whose career, lifestyle, or
sexual orientation precluded intercourse with a male partner and
women whose partners were using 2 barrier birth control methods or
hormonal contraceptive method were allowed to participate. Subjects
with a history of any heavy metal allergy; asthma, chronic bronchitis
or emphysema; or renal impairment defined by a creatinine clearance
≤30 ml/minute; or significant acute or chronic disease as determined
by the investigators were excluded. Subjects unable to complete the
study were excluded from analysis and replaced.
All patients provided written informed consent. The study
was conducted in accordance with the International Conference
on Harmonisation of Technical Requirements for Registration of
Pharmaceuticals for Human Use Guidelines for Good Clinical
Practice and the Declaration of Helsinki, and received approval
from the University of Utah Institutional Review Board. The
trials are registered with Clinical-Trials.gov (Identifier:
NCT01243320 and NCT01405794).
To minimize risk to study subjects, a dose-time escalation
dosing scheme was employed. Study one used 10 ppm oral silver
particle dosing with 3-, 7-, and 14-day time periods; study two
used 32 ppm for 14 days. After completion of each time period,
an independent Data Safety and Monitoring Board (DSMB)
reviewed every measurement for evidence of toxicity.
Study product
The silver nanoparticle (AgNP) study product was manufactured
by American Silver, LLC. (Alpine, Utah, USA) by a published AC
high voltage (10(3)-10(4)) aqueous electrolysis of de-ionized water
using silver metallic electrodes as detailed previously (US patents
6,214,299 and 7,135,195).
7,23
Silver is in the form of zero-valent
elemental silver particles coated with silver oxide, with manufacturer's
claims to particle size ranging between 5-10 nm (10 ppm; lot
#122810) or claims to a mean of 32.8 nm, range of 25-40 nm
(32 ppm: lot #071511), respectively. The average daily ingestion of
this elemental silver colloid formulation is estimated to be 100 μg/day
for 10 ppm, and 480 μg/day for 32 ppm silver.
Silver nanoparticle characterization
Silver particle sizing. To determine nanoparticle hydrody-
namic diameter, dynamic light scattering (DLS) was performed
on the 32 ppm AgNPs (lot #09280, 32 ppm silver) in Millipore
ASTM grade I water using a commercial instrument (Brookha-
ven Instruments, Holtsville, USA) at a laser wavelength of
677 nm and fixed angle of 90° at 25 °C after instrument
calibration using 100-nm diameter commercial polystyrene
nanosphere size standards (Thermo Scientific, Fremont, Cali-
fornia, USA). Instrument background was tested with a control
sample of 0.2 micron filtered Millipore American Society for
Testing and Materials (ASTM) grade I prior to sampling and no sub-
micron particles were detected. Cuvette temperature was controlled
using a recirculation bath. Optical scattering intensities were
collected at 50% laser intensity to ensure proper particle counts.
Each measurement was taken between 5 and 10 minutes' duration.
Time correlation functions of scattered intensity were calculated by
inverse Laplace transformation (regularized positive exponent sum)
as described previously.
24
Optical spectroscopy and plasmon
absorption analysis were performed on the 32 ppm silver
nanoparticles using a Varian Cary 400 Bio Ultraviolet (UV)–
visible spectrophotometer (Varian Cary, Palo Alto, California).
Two 1-cm quartz cells were used: one cell containing sample and
one reference cell containing ASTM grade I water. Scans were
performed at 5 nm per second from 800 to 200 nm. Samples were
vortexed prior to measurementatambienttemperature.
Silver ion content. The study silver solution (lot #09280,
32 ppm) was centrifuged for 120 minutes at 20,800 RCF × g,
separating N96% of silver nanoparticles N10 nm in diameter to
obtain a supernatant with residual ions.
25
Inductively coupled
plasma mass spectrometry (ICP-MS) was then performed on the
supernatant using a liquid chromatography ICP-MS (LC-ICP-
MS) Agilent 7500ce (Agilent Technologies, Inc., Santa Clara,
California USA). Fresh silver ion standards were prepared prior
to sample analysis using known concentrations (0.028-
0.833 ppm) of silver nitrate (AgNO
3
) (Inorganic Ventures,
Christiansburg, Virginia USA) in ASTM grade I (Millipore
2M.A. Munger et al / Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 1–9

filtered) ultrapure water. All ICP-MS samples (centrifuged
supernatants or uncentrifuged product stock) were oxidized
with nitric acid to solubilize residual silver nanoparticles to ions.
Aliquots of each supernatant or product sample were measured
in triplicate. Detection limit for silver ion in this instrument was
determined to be 0.1 ppb using the standards.
Study design for metabolic panel, silver concentrations, induced
sputum, and MRI
Subjects received silver nanoparticle colloidal solution
diluent (e.g. sterile water [no silver nanoparticles]) followed by
the active silver solution. A 72-hour washout period preceded the
dosing cross-over. Each subject received 15 mL of study
material daily from a pre-mixed oral syringe. Each dose
administration was observed by study personnel to ensure
compliance. Subjects were blinded to the study product received.
At baseline and the end of each time-period, subjects
underwent a medical and drug history, complete physical
examination, comprehensive metabolic panel, blood count with
differential and urinalysis. Blood and urine were collected for
serum and urine silver concentrations at trough concentrations
(≥24 hours post-dose) for the 3- and 7-day time periods at
10 ppm and at peak concentration (≤2 hours post-dose) for the
14-day 10 ppm dose and for the 32 ppm study population. Silver
concentrations in serum and urine were determined by using
ICP-MS (NMS Laboratories, Willow Grove, USA). Calibration
silver samples in dilute nitric acid and controls in human serum
matrix were used in each ICP-MS assay cohort. The ICP-MS
assay dynamic range of silver samples for this study was 0-
40 μg/L. Sputum was collected by induction protocol within
24 hours of the last dose for each time-period, as previously
described.
26
Sputum analysis
Hydrogen peroxide concentrations were determined using a
modification of a previous method.
27
Peroxiredoxin protein
expression was measured as described.
28
Determination of RNA
expression using quantitative real-time polymerase chain
reaction (qPCR) was determined by methods previously
described.
29
Magnetic resonance imaging (MRI) protocols
A cardiac and abdominal MRI was obtained at the end of each
phase of each time period. Patients were examined on a 1.5-T 32-
channel superconducting magnetic resonance system (Magne-
tom Avanto, Siemens Medical Solutions). Cardiac MRI studies
were performed using breath-hold acquisitions prospectively
triggered by the electrocardiogram. Cine steady-state free
precession (SSFP) and true fast imaging magnetic resonance
with steady state precession (TrueFISP) cardiac images were
acquired in multiple standard short-axis and long-axis views,
including specific right ventricular outflow tract (RVOT) and left
ventricular outflow tract (LVOT) orientations (slice thickness 8
mm, echo time 1.2 ms, pixel bandwidth 1.150 hertz (Hz),
repetition time 3.0 ms, temporal resolution about 43 ms, matrix
256 × 202). Abdominal MRI protocol included a transverse
T1-weighted fast gradient-recalled dual-echo sequence (TR/in-
phase TE/out-of-phase TE, 129/4.36/2.0; flip angle, 70°; matrix,
134 × 256; section thickness and intersection gap, 6 and 0.6 mm;
signal average, 1; field of view, 220-340 mm [depending on
body habitus]) and a transverse T2-weighted Half Fourier
Acquisition Single Shot Turbo Spin Echo (HASTE) (time to
repetition (TR)/echo time (TE), 1,000/89; refocusing angle,
180°; slices, 20; slice thickness, 6 mm with a 10% gap; matrix,
168-192 × 256; field of view, 220-340 mm [depending on body
habitus]). A dynamic contrast-enhanced 3 dimensional (D)
gradient-echo volumetric interpolated breath-hold examination
(VIBE) sequence was performed in the arterial, venous, and
delayed phases, after the injection of 0.1 mmol/kg of body
weight of Gadopentetate dimeglumine (Magnevist; Bayer
HealthCare Pharmaceuticals, Berlin, Germany) at a rate of
2 mL/s using a pressure injector. This was followed by delayed
contrast‐enhanced cardiac study using a segmented inversion–
recovery sequence in the same views used for cine cardiac MRI
10-20 min after contrast administration. All images were
deidentified and transferred to a 3D postprocessing workstation
(Leonardo, Siemens Healthcare). LV function and volumes were
calculated by planimetry of the endocardial and epicardial
borders from the serial short‐axis views (usually 8-14) with no
gap between the slices. Ejection fraction, end diastolic volume
and end systolic volume were analyzed.
Statistical analysis
Average effect analysis was employed to assess toxicity. A ±
4 SD range was applied as the statistical rule based on the
concept that a normal distribution contains nearly all individual
observations within a ±3 SD interval. A mixed effects linear
regression model with repeated measurements from the cross-
over periods nested within subjects was used to determine effect
differences. In this model, the baseline value was included as a
covariate. The crossover period was the primary predictor
variable. To test for a linear trend across exposure time, the 3-,
7-, and 14-days a mixed effects model using exposure time as the
predictor was fitted. The Pvalue for the time variable in such a
model represents a linear dose–response significance test. All
reported Pvalues were from a two-sided comparison.
The power of the study to detect toxicity was sufficient for
individual observation analysis and the usual mean difference
analysis. Combining the 10 ppm and 32 ppm silver colloidal
solutions, for a total sample size of n = 60, based on a
binomial probability, using 1-Prob (observed incidence = 0|
true incidence = 0.027), the total sample size of n = 60
provided 80% probability of observing toxicity in at least
one study subject if the true toxicity incidence is 2.7%. For the
analysis examining paired sample mean differences between
placebo (diluent only) and active silver solution, the n = 24
subjects with maximum exposure of 32 ppm silver colloidal
solution for 14 days provided 80% power to detect a mean
difference of 0.48 standard deviations (SD), using a two-sided
alpha 0.05 comparison and assuming a correlation of r = 0.70
between the placebo and active phases. Given the reference
range is ± 2 SDs, the effect sizes of 0.48 SD and 0.30 SD
represented detectable effects well within normal laboratory
reference ranges.
3M.A. Munger et al / Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 1–9

Results
A total of 62 subjects were enrolled in the study. Sixty
subjects completed the study. Two were discontinued, one due to
inability to draw blood (subject never received study product)
and one due to hospitalization for pulmonary embolism (subject
received 12 days of placebo diluent only) (Table 1).
Silver nanoparticles used here were commercially prepared
by alternating current high voltage (10
3
volts) aqueous
electrolysis of silver salt solutions as detailed previously.
7,23
These previous studies on the same particles of 32 ppm product
showed that the process produces nanoparticles 20-40 nm in
diameter, with the dominant crystalline solid phase being
metallic silver as asserted by scanning electron microscope,
transmission electron microscope, Raman and UV/Visible
spectrometry analysis.
7,23
The metallic nanoparticle silver has
some oxygen, proposed as an oxide or ‘oxyhydroxide’overlayer
in the sol, consistent with minute amounts of crystalline silver
oxide under TEM studies.
7,23
Many 20-30 nanometer (nm) silver
nanoparticles comprise a substructure, with 5-7 nm particles held
together by relatively weak bonds.
7,23
Optical spectroscopic analysis of silver nanoparticles
The visible appearance of the as-supplied commercial silver
solution from the stock bottle was yellow, consistent with the
presence of typical silver nanoparticles with plasmon behavior.
30
Optical absorption of silver colloids (see Figure 1) also supports the
presence of silver nanoparticles. The molar extinction coefficient
for silver particles is considerable (3 × 10
11
M
−1
cm
−1
), providing
a strong, distinct absorption band at low concentrations. The
peak and width of the spectrum in Figure 1 follow theoretical
Mie optical plasmon predictions consistent with previously
reported results for similar silver nanoparticle systems.
31
Full
width at half maximum absorbance was found to be 150 nm
and λ
max
was determined to be 448 nm. Although the observed
peak position of the surface plasmon band is slightly larger than
these previously reported, its position is still consistent with Mie
theory predictions. This optical absorption strongly depends on
particle size, extent of aggregation,
32
dielectric, and chemical
properties of the milieu.
33
Small silver spherical nanoparticles (i.e.,
b20 nm diameter) exhibit a single, narrow surface plasmon band
inconsistent with experimental observations here.
34
Previous data
suggested that for a full width at half maximum (FWHM) of 229
and a λ
max
of 400 nm, silver nanoparticle size was determined to
be 63 ± 19 nm.
35
Direct size predictions from particle optical
absorption are difficult because of many influences on the system,
but the red-shifting of the surface plasmon absorption band
maximum to 448 nm and FWHM bandwidth broadening both
indicate some alteration of ideal plasmonics compared to
theoretical predictions calculated for silver nanoparticles.
36
Specifically, the spectrum supports a broad polydispersity of
these commercial silver nanoparticles, prepared in water by high
voltage electrolysis.
7,23
This method also seems to produce
particles that experience aggregation or size dispersity to produce
the spectral shift and broadeningobserved.
36
Previous data provide
evidence that exposure of silver nanoparticles to low-level UV
light (including ambient laboratory lighting) can convert spherical
AgNPs to nanoprisms with a corresponding red-shift in their UV
absorbance profile.
37
However, the silver nanoparticles are
shipped and stored from the commercial supplier in light-protected
(opaque) plastic stock bottles in DI water and transferred to sterile,
but transparent plastic dispensing syringes in the clinical trials
dispensing room prior to each patient's oral dosing/administration.
Light effects changing silver particle sizes are therefore considered
unlikely. Finally, the observed spectrum minimum at ~ 320 nm
corresponds to the wavelength at which both real and imaginary
parts of the silver particle's dielectric function diminish drastically,
consistent with other similar observations for silver NP systems.
34
Hence, the collective UV/Vis optical data support the presence of
silver NPs with broad sizing polydispersity and classic plasmonic
properties of particles with sizes below 100 nm.
Silver nanoparticle DLS hydrodynamic diameter determinations
Figure 2 shows that the average silver nanoparticle
hydrodynamic diameter was determined by dynamic light
scattering (DLS) to be 59.8 nm ± 20 nm. This particle size is
consistent with that expected for the observed absorption
maximum of λ
max
~448 nm from Figure 1, given the broad
particle polydispersity.
Table 1
Study population demographics.
Demographic/Clinical
Variable
10 ppm
(n = 36)
32 ppm
(n = 25)
Total Sample
(n = 61)
Age, years,
mean ± SD min, max
52 ± 11
26-76
41 ± 15
20-67
47 ± 14
20-76
Gender, n M/F (%) 17/19 (47/53) 18/6 (75/25) 35/25 (58/42)
BMI (kg/m
2
)
mean ± SD (min, max)
29 ± 6
20-45
29 ± 6
21-43
29 ± 6
20-45
SBP (mmHg)
mean ± SD (min, max)
127 ± 19
84-176
127 ± 13
102-150
127 ± 17
84-176
DBP (mmHg)
mean ± SD (min, max)
83 ± 11
55-106
81 ± 11
62-107
82 ± 11
55-107
Heart Rate (bpm)
mean ± SD (min, max)
69 ± 9
42-84
65 ± 7
52-79
68 ± 8
42-84 Figure 1. Optical absorbance spectrum of 32 ppm commercial AgNPs
measured on samples taken directly from the study stock bottle used in
human oral ingestion and exposures (lot # 09280).
4M.A. Munger et al / Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 1–9

Silver ion content
ICP-MS detected quantitative recovery of total silver from the
(as supplied) nitric acid-digested 32 ppm product stock sample
(average control silver content of 31.5 ± 0.2 ppm). Importantly,
significant amounts of silver ion were recovered in the
supernatant of all centrifuged samples analyzed (26.8 ±
0.6 ppm silver ion), corresponding to ionic silver comprising
some 84.3% of the total silver content in the product
administered orally to patients.
Clinical findings
For all outcomes measured in the subjects administered the
10 ppm dose at 3-, 7-, and 14-day exposure, a linear trend across
exposure times was tested in a mixed effects model without a
significant trend in any outcome (all Pb0.20). Thus, combining
the various exposure groups into one large, a 10 ppm, group,
provided greater statistical power for the 10 ppm versus 32 ppm
dose comparisons. Changes in subject hemodynamics are listed
in Table 2. No clinically important changes in weight, BMI,
systolic or diastolic blood pressure or heart rate were noted.
However, heart rate significantly declined by 2.3 beats per
minute for the total group.
Results of the complete metabolic panel are listed in Table 3.
There were no significant or clinically important changes
observed in laboratory finding across the total population.
Blood urea nitrogen and alanine aminotransferase tests from the
10 ppm dose were analyzed to be statistically significant, but
nothing in the 32 ppm dose cohort was noted. However, when
the 95% CI limits of these significant tests are added to the mean
value of the active period, representing a statistical comparison to
the normal reference range limits, all values remain within the
normal reference range. There was no significance in any
metabolic test, suggesting that increasing dosing from 10 to
32 ppm does not elicit silver toxicity.
The results of the complete blood count with differential are
displayed in Table 3. Comparison of the red blood cell count
(RBC) between active vs. placebo solutions was significant at the
10 ppm dose, but not at the 32 ppm dose. There were no
clinically important changes in any blood count value including
erythrocytes, granulocytes, or agranulocyte counts. Exposure
time was not associated with changes in blood counts.
No significant or clinically important changes were evident in
the complete urinalysis. Although there were individual subject
positive tests for urine ions, proteins, blood cells, and some other
solutes, these changes remained unchanged in comparison
between the active and placebo solutions.
Serum silver and urine findings
Serum and urine silver concentrations were determined at
different time variables. There was no detection of serum silver
Table 2
Study population changes in hemodynamics.
Hemodynamic Variable 10 ppm
Mean Change
[95% CI] (Pvalue)
32 ppm
Mean Change
[95% CI] (Pvalue)
Total Sample
Mean Change
[95% CI] (Pvalue)
Weight (kg) −1.1 [−2.6, 0.4] (0.17) −0.4 [−0.1, 0.8] (0.13) −0.5 [−1.4, 0.4] (0.30)
BMI (kg/m
2
)−0.4 [−0.9, 0.1] (0.15) −0.1 [−0.04, 0.3] (0.14) −0.2 [−0.5, 0.1] (0.26)
SBP (mmHg) 1.3 [−1.7, 4.3] (0.40) −1.3 [−3.0, 5.6] (0.54) 1.3 [−1.2, 3.8] (0.30)
DBP (mmHg) −2.4 [−5.5, 0.7] (0.13) −0.7 [−2.5, 3.8] (0.67) −1.2 [−3.4, 1.1] (0.31)
HR (bpm) −1.9 [−5.0, 1.3] (0.25) −3.1 [−6.4, 0.3] (0.07) −2.3 [−4.6, −0.93] (0.05)
BMI: Body Mass Index; SBP: Systolic blood pressure; DBP: Diastolic Blood Pressure; HR: Heart Rate; CI; Confidence Interval.
Figure 2. Dynamic Light Scattering (DLS) particle size characterization of 32 ppm commercial AgNPs as taken directly from commercial silver nanoparticle
sample (lot # 09280).
5M.A. Munger et al / Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 1–9

from subjects at trough concentrations throughout the 3- and 7-
day time periods at 10 ppm. Peak serum silver concentration was
detected in 42% of subjects in the 14-day 10 ppm dosing
showing a mean of 1.6 ± 0.4 mcg/L. The 32 ppm dose mean
concentration was detected in 92% of subjects at 6.8 ± 4.5 mcg/
L. No silver was detected in the urine, independent of dose or
time period.
Sputum reactive oxygen species (hydrogen peroxide) and
pro-inflammatory cytokine RNA findings
Quality paired samples allowing determination of ROS
concentrations and pro-inflammatory cytokine RNA expression
were analyzed in 72% and 83% of 10 ppm and 32 ppm study
samples, respectively (Table 4). No statistically significant
change in markers of hydrogen peroxide production or
peroxiredoxin protein expression was detected. Analysis of IL-
8, IL-1α, IL-1β, MCP1 and NQO1 also showed no statistical
difference between the active silver and placebo solutions.
MRI findings
Eighteen 10 ppm and eleven 32 ppm subjects underwent a
post 3-14 day, respective active and placebo solution cardiac and
abdominal MRIs. No morphological or structural changes were
noted between active and placebo solutions.
Discussion
Nanoscale colloidal elemental silver is widely found in
diverse consumer products, medical devices, and pharma-
ceuticals.
1
Therefore, study of human processing and possible
toxicological response to this nanomaterial is critically important
to understanding potential human exposure risks and benefits. To
assess the human risk of oral ingested exposure to a commercial
nanoscale silver product, we conducted a prospective, controlled,
parallel design systematic in vivo study with two doses of a
commercial silver nano-particle solution over a 3-14 day
monitored exposure. Findings show that this colloidal elemental
silver particulate formulation produces detectable silver (pre-
sumed to be Ag
+(aq)
ion) in human serum but do not demonstrate
clinically significant changes in metabolic, hematologic, urine,
physical findings, sputum morphology or imaging changes. To
our knowledge, this is the first systematic in vivo human study of
a systemic ingested nanoscale product.
Oral administration of the silver aqueous product delivers
both ionic silver and silver nanoparticles. Given the substantial
Table 3
Study population comprehensive metabolic panel and complete blood count with differential (10 ppm [n = 36], 32 ppm [n = 24], Total Sample [n = 60]).
Comprehensive Metabolic Panel and
Complete Blood Cell Count
with Differential
10 ppm
Mean Change
[95% CI: min, max] (Pvalue)
32 ppm
Mean Change
[95% CI: min, max] (Pvalue)
Total Sample
Mean Change
[95% CI: min, max] (Pvalue)
Sodium [mmol/L] −0.1 [−0.7, 0.6] (0.87) 0.2 [−0.7, 1.0] (0.71) 0.03 [−0.5, 0.6] (0.90)
Potassium [mmol/L] −0.1 [−0.3, 0.02] (0.10) −0.03 [−0.2, 0.1] (0.74) −0.08 [−0.2, 0.03] (0.13)
Chloride [mmol/L] −0.4 [−1.2, 0.3] (0.23) 0.04 [−1.1, 1.2] (0.94) −0.3 [−0.9, 0.4] (0.44)
Carbon Dioxide [mmol/L] 0.5 [−0.5, 1.6] (0.33) −0.04 [−1.1, 1.0] (0.94) 0.3 [−0.5, 1.1] (0.44)
BUN [mg/dL] −0.9 [−1.7, −0.1] (0.03)⁎0.5 [−0.08, 1.8] (0.41) −0.3 [−1.1, 0.4] (0.37)
Creatinine [mg/dL] 0.01 [−0.02, 0.03] (0.60) −0.02 [−0.04, 0.01] (0.21) −0.003 [−0.02, 0.01] (0.74)
Glucose [mg/dL] 3.6 [−1.6, 8.7] (0.17) −0.7 [−4.2, 2.9] (0.71) 1.9 [−1.5, 5.3] (0.28)
ALP [U/L] −1.4 [−3.9, 1.1] (0.28) 2.0 [−1.0, 5.0] (0.18) −0.03 [−2.0, 1.9] (0.97)
AST [U/L] −0.44 [−2.1, 1.2] (0.60) 2.0 [−2.6, 6.5] (0.40) 0.5 [−1.5, 2.5] (0.61)
ALT [U/L] −2.6 [−4.8, −0.3] (0.03)⁎2.3 [−1.6, 6.3] (0.25) −0.6 [−2.7, 1.5] (0.58)
Total Protein [g/dL] −0.02 [−0.3, 0.2] (0.87) 0.2 [−0.001, 0.4] (0.051) 0.1 [−0.1, 0.3] (0.45)
Total Bilirubin [mg/dL] −0.02 [−0.08, 0.3] (0.43) −0.03 [−0.11, 0.05] (0.49) −0.02 [−0.07, 0.02] (0.29)
Albumin [g/dL] −0.07 [−0.1, 0.0004) (0.06) 0.11 [−0.01, 0.23] (0.09) 0.002 [−0.07, 0.07] (0.96)
Calcium [mg/dL] −0.1 [−0.2, 0.04] (0.20) 0.1 [−0.003, 0.2] (0.06) 0.0 [−0.1, 0.1] (0.99)
WBC Count [k/μL] −0.17 [−0.53, 0.19] (0.35) −0.09 [−0.68, 0.49] (0.75) −0.14 [−0.46, 0.17] (0.38)
RBC Count [M/μL] −0.08 [−0.17, −0.001] (0.047)⁎0.06 [−0.03, 0.14] (0.23) −0.03 [−0.09, 0.03] (0.37)
Hemoglobin [gm/dL] −0.2 [−0.4, 0.03] (0.08) 0.1 [−0.2, 0.4] (0.41) −0.1 [−0.3, 0.1] (0.41)
Hematocrit [%] −0.7 [−1.5, 0.07] (0.075) 0.8 [−0.1, 1.7] (0.095) −0.1 [−0.8, 0.5] (0.68)
MCV [fL] 0.1 [−0.6, 0.8] (0.80) 0.5 [−0.1, 1.2] (0.12) 0.3 [−0.2, 0.7] (0.29)
MCH [pg] 0.1 [−0.1, 0.3] (0.48) −0.1 [−0.4, 0.2] (0.38) −0.01 [−0.2, 0.2] (0.96)
MCHC [gm/dL] 0.1 [−0.3, 0.4] (0.72) −0.3 [−0.7, 0.1] (0.12) −0.1 [−0.3, 0.2] (0.53)
Platelets [k/μL] 5 [−6, 15] (0.39) −4[−16, 8] (0.53) 1.3 [−6.8, 9.4] (0.76)
Granulocytes [%] −0.9 [−3.0, 1.1] (0.36) −0.7 [−4.0, 2.7] (0.70) −0.9 [−2.8, 1.0] (0.36)
Lymphocytes [%] 0.7 [−1.2, 2.6] (0.46) 1.3 [−1.5, 4.1] (0.36) 1.0 [−0.8, 2.7] (0.27)
Monocytes [%] −0.1 [−0.6, 0.4] (0.70) −0.3 [−0.8, 0.3] (0.34) −0.2 [−0.5, 0.2] (0.38)
Basophils [%] 0.04 [−0.1, 0.2] (0.58) 0.001 [−0.1, 0.1] (0.99) −0.02 [−0.1, 0.1] (0.61)
Eosinophils [%] −0.06 [−0.3, 0.2] (0.69) −0.2 [−0.7, 0.2] (0.36) −0.1 [−0.4, 0.1] (0.35)
BUN: Blood Urea Nitrogen; ALP: Alkaline Phosphatase; AST: Aspartate Aminotransferase; ALT: Alanine Aminotransferase; WBC: White Blood Cells; RBC:
Red Blood Cells; MCV: Mean Corpuscular Volume; MCH: Mean Corpuscular Hemoglobin; MCHC: Mean Corpuscular Hemoglobin Concentration.
⁎P≤0.05, comparison of 10 ppm or 32 ppm active solution vs. placebo solution, controlling for baseline value, in a mixed effects linear regression model.
6M.A. Munger et al / Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 1–9

fraction of ionic silver in the product detected by ICP-MS, and
very limited GI absorption of metallic nanoparticles of similar
size in other studies in rodents, we assert that most silver detected
in patient blood samples is ionic, with no evidence that intact
silver nanoparticles are either absorbed into circulation through
the human digestive tract, or attached to blood components (e.g.,
proteins, platelets and cells).
38
Recent reports detail the potential for human toxicity from
nanoparticles of various natural and engineered forms, material
chemistries and morphologies, and exposure routes. Suggested
and defined target organ systems susceptible to nanoparticle
adverse health effects include the pulmonary, cardiovascular,
neurologic, reticuloendotheilal, renal, and reproductive sys-
tems.
5,39-41
To this end, the literature has called for new
toxicological study designs to establish procedures capable of
reliable predictive assay of nanoparticles in model in vitro
systems, as well as the critical need for validating in vivo
studies.
5,42,43
Our study begins to address existent human
toxicity to silver oral nanoparticle exposure in a systematic way.
Abdominal organ system toxicity has been shown to occur
from exposure to silver nanoparticles. The liver, in particular, has
been noted as a toxicity target, possibly due to oxidative stress.
15
Non-cytotoxic doses of silver nanoparticles reduced cell
mitochondrial function, cell proliferation, and induced apoptosis
in rat and human liver, and human mesenchymal cell lines in
vitro, respectively.
15,16,44
We have shown in MDR1.C and Hep
G2 cell lines that after 24 hour exposure of a commercial 32 ppm
silver nanoparticle solution where cell viability was maintained
that nanoscale colloidal silver may be a potential source of drug-
drug interactions.
45
Potential interactions may occur through
reductions in NADPH cytochrome c reductase activity.
46
In
contrast, a prospective, controlled 90-day exposure of 56 nm
silver nanoparticles at 30 mg/kg in vivo in rats did not show any
clinical chemistry, hematological, body weight, food consump-
tion, or water intake changes.
47
Our human clinical findings
correlate closely with these results. Hence, potential in vitro
hepatocellular toxicity could occur from non-cytotoxic doses of
nanoscale silver but these results do not correlate with in vivo rat
or our human findings. Reasons for this disconnect, while often
observed in nanomaterials toxicity exposure studies, may result
from differences using in vitro direct nanoparticle-cell exposure,
non-comparable dosing to cultured cells versus in vivo systemic
exposure, or incomplete understanding of nanoscale silver
particle in vivo bioavailability, biodistribution, or liver blood
flow dynamics and processing. MRI results from this study were
not able to differentiate any abdominal changes from exposure to
nanoscale silver.
The lung is another major target of silver nanoparticle
exposure, particularly through inhalation. Silver nanoparticles
(15-18 nm) may bind to lung epithelial cells and alveolar
macrophages, producing reactive oxygen species, potentially
limiting function of cells
10,12,13,47
Histopathological examina-
tion from inhaled 18 nm silver nanoparticles for 90 days in
Sprague–Dawley rats shows dose-dependent alveolar infiltra-
tion, thickened alveolar walls and small granulomatous
lesions.
13
These histology changes were associated with re-
ductions in tidal and minute volumes. Intratracheally instilled
silver nanopowders enhanced systemic platelet aggregation in
rats.
48
Silver nanoparticles enhance thrombus formation through
increased platelet aggregation and procoagulant activity.
49
Congruence of results from multiple in vitro and a single in
vivo animal models supports cellular and functional toxicity
from inhaled nanoscale silver particles. However, orally dosed
nanoscale silver from our study failed to induce detectable
changes in reactive oxygen species or pro-inflammatory cytokine
RNA from induced sputum samples. This attributed to poor
absorption of silver nanoparticles in the gut and poor
translocation of these particles to the respiratory system from
the gastrointestinal route.
38,49
Further studies are necessary to better understand possible
silver nanoscale particle toxicity risks on the human reproductive
system, systemic bioavailability and toxicity from subcutaneous
delivery systems or from leaching of embedded silver in
catheter-based medical devices, and to the central nervous
system from different delivery systems. Our limited study did not
assess these other physiologic systems or other delivery systems.
Our study timeframe of 14 days, although one dosing interval,
should be extended to better determine whether longer particle
oral exposure leads to higher silver systemic bioavailability and
subsequent accumulation in human lipid compartments, a
possible source of chronic toxicity. Another important issue
regards the form of silver detected in serum in the patients in this
study: ICP-MS detects silver in ionic form, and our analytical
methods cannot discriminate ionic silver from nanoparticulate
silver in physiological fluids. However, the ICP-MS product
analysis indicates that oral administration of the silver aqueous
product delivers both ionic silver and silver nanoparticles to
humans. Given the substantial fraction of ionic silver (Ag
+(aq)
)
Table 4
Sputum reactive oxygen species and pro-inflammatory cytokine analyses.
ROS or Cytokine Parameter 10 ppm
Mean Change
[95% CI] (Pvalue)
32 ppm
Mean Change
[95% CI] (Pvalue)
Total Sample
Mean Change
[95% CI] (Pvalue)
ROS μM 0.89 [−0.6, 2.38] (0.24) −0.44 [−1.23, 0.35] (0.28) 0.52 [−0.56, 1.60] (0.34)
IL-8 (copies/1000 B2M) 2.19 [−1.53, 5.91] (0.25) 6.39 [−5.83, 18.60] (0.31) 4.52 [−2.47, 11.51] (0.21)
IL-1α(copies/1000 B2M) −0.0005 [−0.0007, 0.0006] (0.88) 0.0197 [−0.0014, 0.0408] (0.07) 0.0128 [−0.0014, 0.0269] (0.08)
IL-1β(copies/1000 B2M) 0.017 [−0.011, 0.044] (0.24) 0.027 [−0.058, 0.112] (0.53) 0.022 [−0.027, 0.072] (0.38)
MCP1 (copies/1000 B2M) −0.028 [−0.084, 0.028] (0.34) −0.004 [−0.026, 0.017] (0.69) −0.015 [−0.046, 0.015] (0.33)
NQO1 (copies/1000 B2M) −0.0043 [−0.0115, 0.029] (0.24) −0.0279 [−0.4671, 0.4114) (0.90) −0.0182 [−0.2850, 0.2487] (0.89)
ROS: Reactive Oxygen Species; B2M: Beta-2 microglobulin; MCP1: Monocyte chemoattractant protein-1; NQO1: NADH quinine oxioreducatase-1.
7M.A. Munger et al / Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 1–9

resident in the as-supplied product as detected by ICP-MS, and
very limited GI absorption of metallic nanoparticles of similar size
in other studies in rodents
38
we assert that most silver detected in
patient blood samples is ionic, with no evidence that intact silver
nanoparticles are either absorbed into circulation through the
human digestive tract, or attached to blood components (e.g.,
proteins, platelets and cells). Previous studies on nanoparticle
colloidal gold oral bioavailability showed that miniscule (b1%)
amounts of 10-nm gold nanoparticles permeate across the gut to
enter systemic vascular circulation from the intestine in rodents.
50
We assert that silver metallic particle absorption is similar, and that
the silver detected in serum in human subjects has sources from
both the soluble ionic fraction in the formulation, and fromthat first
solubilized from ingested colloidal form in the upper gastrointes-
tinal tract and absorbed into blood in ionic form.
In summary, nanoscale colloidal silver is an increasingly
deployed engineered nanomaterial, with potential nutraceutical
and therapeutic properties, increasingly found in consumer
products and medical devices. We have demonstrated that 14-
day monitored human oral dosing of a commercial oral
nanoparticle silver colloidal product does not produce observ-
able clinically important toxicity markers. Further study of
nanomaterials over longer human exposures is clearly warranted
to determine risks.
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