Starch/Stärke 2016, 68, 1–8 DOI 10.1002/star.

201600105 1

RESEARCH ARTICLE

Human monocyte response to Andean-native starch

nanoparticles
Francesca Gatto 1,2, Omar P Troncoso 3, Virgilio Brunetti 4, Maria Ada Malvindi 4, Pier Paolo Pompa 1,
Fernando G Torres 3 and Giuseppe Bardi 1

1
Istituto Italiano di Tecnologia (IIT), Genova, Italy
2
Department Of Engineering for Innovation, University of Salento, Lecce, Italy
3
Department of Mechanical Engineering, Pontificia Universidad Catolica del Peru, Lima, Peru
4
Istituto Italiano di Tecnologia (IIT), Center for Bio-Molecular Nanotechnology@UniLe, Arnesano, Lecce, Italy

We used starches from two different Andean-native tubers to prepare nano-sized particles, and Received: March 24, 2016
tested their ability to stimulate inflammatory reactions in human monocytes. Our data show Revised: May 16, 2016
that the release of inflammatory cytokines by monocytes can be differentially modulated by Accepted: May 20, 2016
the administration of non-toxic doses of nanoparticles synthesized from the starch of the
Andean sub-species Solanum tuberosum and Solanum goniocalyx. Furthermore, we observed a
starch-nanoparticle-specific increase in inflammatory chemokine-dependent migration, and an
up-regulation of immunoglobulin receptor CD16. Based on this preliminary study, we conclude
that different potato starch nanoparticles possess specific properties that can induce immune
responses and may be employed as immune modulators in the future.

Keywords:
Inflammation / Monocytes / Nanoparticles / Starch

: Additional supporting information may be found in the online version of this article at the publisher’s web-site.

1 Introduction characterized by several alpha-1,6-linked branch points [2].

Natural polymers play a key role in materials science and are
widely used for biomedical applications. These polymers are
derived from several vegetable sources, such as roots, seeds,
stems or tubers, and they are considered attractive options
because of their biodegradability and similarity with other
polymers found in the human body [1]. Starch is a highly
biocompatible and biodegradable carbohydrate composed of
two different polymers: amylose and amylopectin. The
former shows a linear structure of glucose units linked by
alpha-1,4-bonds, whereas, amylopectin is a branched
polymer with the same backbone structure of amylose
Correspondence: Dr. Giuseppe Bardi, Istituto Italiano di Tecnologia
(IIT), Via Morego 30, Genova 16163, Italy
E-mail: giuseppe.bardi@iit.it
Fax: þ39 010 71781230
Abbreviations: TEM, transmission electron microscopy; FSC,
forward scattering; SSC, side scattering; NPs, nanoparticles
Currently, starch-based materials are widely used for
biomedical purposes, including topical release in skin [3],
drug delivery [4–9], tissue engineering [10–13], cell seed-
ing [14], and bone replacement [15–17]. However, starch may
exhibit immunogenic properties by modulating the biologi-
cal response of human immune cells [1]. Actually, carbohy-
drate-based materials are also currently investigated as
immune modulators, due to the prominent role of
carbohydrates in immune recognition and response [18];
other classes of natural polymers are also being investigated.
For instance, attempts to produce adjuvants for vaccines have
used bacterial walls, relying on the glucans, and mannans
present on their surface [19]. However, bacteria-derived
materials may raise some concerns regarding their potential
pathogenicity. One of the major issues it is the undesired
activation of the immune system, which is an extremely
complex and coordinated network composed of several cell
types, and cellular mediators. All immune system compo-
nents interact in a concerted fashion to establish an
appropriate and balanced response against external, and

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2 F. Gatto et al.
internal pathological threats. The immune system consists
of two different lines of control, the innate and the adaptive
responses, which allow the body to distinguish between self
and non-self molecules (namely, organisms or materials
from external sources). The innate reaction defines an
immediate non-specific response to alien bodies [20]
mediated by phagocytic cells, such as neutrophils or
monocytes/macrophages, which act by encapsulating, and
destroying the foreign substances through specific enzy-
matic reactions. Resting monocytes travel around the body,
sensing environmental signals, and migrating to specific
sites of inflammation. They are driven into the inflamed
tissue by a gradient of specific chemo-attractant cytokines
(chemokines) released by tissue cells, indicating their
pathological condition. Once in the tissue, macrophages
phagocytize pathogens and damaged cells, further regulating
the growth, and the differentiation of other cells by releasing
specific molecular messengers [21]. In this way, monocytes
link innate and adaptive immunity, tailoring the pathogen-
specific response of lymphocytes. Monocytes/macrophages
recognize some of the polymers used for biomedical purpose
as non-self, triggering unforeseen immune reactions [22].
We have recently shown that starch films derived from
different types of potatoes have a specific ability to interact
with the immune cell surface [23], and the response may be
related to the different molecular structures of the various
starches. The aim of the present study, was to investigate the
reaction of human monocytes primed with two different
types of Andean-native starch nanoparticles, focusing on the
release of monocyte-derived pro-inflammatory cytokines,
and their consequences.
2 Experimental section
2.1 Starch extraction and starch nanoparticle
preparation
The starches used in this study were extracted from the
potatoes Solanum tuberosum subsp. Andigena (common
name “Negra”) and Solanum goniocalyx (common name
Peruanita), as reported in Torres et al. [24]. The amylose
content of the starches, as measured by a colorimetric
procedure [24], was 25.43% for the S. tuberosum subsp.
Andigena starch and 24.10% for the S. goniocalyx starch.
Nanoparticles (NPs) were prepared using the method
reported by Torres et al. [25], which is based on the method
developed by Angellier et al. [26]. Briefly, 11.75 g of starch
were mixed with 80 mL of 3.16 M H2SO4 (Merck–Millipore,
Billerica, MA, USA cat. no. 100731) at 40°C under magnetic
agitation for 5 days. The final suspension was centrifuged,
and the precipitate washed by successive centrifugations and
re-dispersions in distilled water until it reached neutral pH.
Finally, the starch NPs were dispersed in 100 mL of distilled
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Starch/Stärke 2016, 68, 1–8
water and stored at 4°C. Nanoparticle yield, calculated as the
amount of NPs recovered per 100 g of processed dry starch,
was 19.74% for S. tuberosum subsp. Andigena samples and
14.98% for the S. goniocalyx samples.
2.2 Transmission electron microscopy
Transmission electron microscopy (TEM) images were
recorded using a JEOL JEM 1011 microscope operating at
an accelerating voltage of 100 kV. The TEM samples were
prepared by dropping a dilute solution of starch NPs in water
on carbon-coated copper grids (Formvar/Carbon 300 Mesh
Cu). Nanoparticle size was evaluated by measuring the
average diameter of more than 100 NPs.
2.3 Cell culture
Human THP-1 monocytic cells (ATCC1 TIB-202TM) were
purchased from the American Type Culture Collection (ATCC
Manassas, VA, USA). The cells were cultured in RPMI-1640
(Life Technologies, cat. no. A10491-01) supplemented with
10% fetal bovine serum (FBS) (Life Technologies, cat. no.
10108-165), 1% antibiotics (Penicillin–Streptomycin, Sigma–
Aldrich, cat. no. P0781), and 0.05 mM 2-mercaptoethanol
(Sigma–Aldrich cat. no. 63689) at 37°C in 5% CO2, and the
cultures were split every 3 days.
2.4 Cell viability
The cells were seeded in a 96-well plate (Sigma–Aldrich,
Corning1 cat. no. CLS3595) at a density of 5,000 cells/50 mL.
Starch NPs were added to obtain final concentrations of 30, 60,
and 300 mg/mL in a final volume of 100 mL per well. Metabolic
activity was determined 24 h after exposure using a standard
WST-8 assay (Sigma–Aldrich, cat. no. 96992). Briefly, 10 mL of
Cell Counting Reagent WST-8 were added to each well, and the
plate was incubated in a humidified atmosphere of 5% CO2
at 37°C for 3 h. The orange WST-8 formazan product was
measured on a Flo Star Optima (BMG LABTECH) microplate
reader at a wavelength of 460 nm. The data were collected by
the Control Software and further analyzed with the MARS Data
Analysis Software (BDG LABTECH). As a positive control for
cytotoxicity, the cells were incubated with 5% DMSO (Sigma–
Aldrich, Cat. no. D8418).
2.5 Cytokine release
Cells were cultured in 12-well plates (Sigma–Aldrich,
Corning1 Costar1 cat. no. CLS3512) at a density of
5  105 cells/mL and incubated with the appropriate suspen-
sion of starch NPs at a concentration of 300 mg/mL for 2, 6,
and 24 h. IL-6, IL-1b, TNF-a, CCL2, CCL4, and CCL5 release
from the THP-1 cells were assessed with a Bio-Plex1
MAGPIXTM Multiplex Reader (Bio-Rad) according to the
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Starch/Stärke 2016, 68, 1–8 Andean-native potato starch nanoparticles induce dissimilar cytokine release
manufacturer’s instructions. The cells were stimulated
with 100 ng/mL lipopolysaccharide (LPS, from Escherichia
coli 0111-B4, Sigma–Aldrich, cat. no. L4391) as a positive
control.
2.6 Cell migration assay
Chemotaxis was assessed in a 24-well plate (Sigma–Aldrich,
Corning1 Costar1 cat. no. CLS3987). After the administration
of starch nanoparticle suspensions (300 mg/mL for 24 h), the
supernatants were collected and 650 mL were poured in the
bottom of the wells. Hanging polyethylene terephthalate (PET)
cell culture inserts with a pore size of 5 mm (Millipore, cat. no.
PIMP12R48) were placed over the wells. The cells were serum
starved in RPMI 1640 (Life Technologies, cat. no. A10491-01)
supplemented with 1% antibiotics (Penicillin–Streptomycin,
Sigma–Aldrich, cat. no. P0781), 0.05 mM 2-mercaptoethanol
(Sigma–Aldrich, cat. no. 63689), and 0.1% BSA (Miltenyi
Biotec cat. no. 130-091-376) for 30 min and plated onto the filter
wells at a density of 2  105 cells/200 mL for 2 h at 37°C in cell
culture incubator. Finally, the filters were removed and 450 mL
of medium from the bottom of the wells were analyzed by
flow cytometry (MACSQuant1 Analyzer, Miltenyi Biotec,
Bergish, Germany). The cell number was counted using the
MACSQuantifyTM Software (Miltenyi Biotec).
2.7 Cell membrane receptor expression and flow
cytometry
THP-1 cells were cultured in 12-well plates (Sigma–Aldrich,
Corning1 Costar1 cat. no. CLS3512) at a density of 5  105
cells/mL and incubated with 300 mg/mL starch NPs for 24 h.
After incubation, the cells were washed with RPMI (Life
Technologies, cat. no. A10491-01) at 300g for 5 min,
resuspended in RPMI/0.5% BSA (Miltenyi Biotec, cat. no.
130-091-376), and incubated with fluorescently labeled
antibodies (APC-conjugated mouse anti-human CD16,
Miltenyi Biotec, cat. no. 130-091-246; PE-conjugated mouse
anti-human CD11b, Miltenyi Biotec, cat. no. 130-097-336) at
the manufacturer’s recommended concentration for 30 min
on ice in the dark. The cells were washed twice in ice-cold
RPMI and samples prepared in the appropriate volume. Cell-
associated fluorescence was analyzed by flow cytometry with
MACSQuant1 Analyzer (Miltenyi Biotec), and 50 000 events
per sample were acquired. The live cells used for the analysis
were gated based on light forward scattering (FSC) and
side scattering (SSC), and further analyzed using MACS-
QuantifyTM software (Miltenyi Biotec).
2.8 Endotoxin assay (LAL assay)
The presence of endotoxin was assessed according to
established protocols with standardized Limulus Amebocyte
Lysate (LAL) reagents QCL-1000TM (Lonza, cat. no. 50-648U).
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2.9 Statistical analysis
All the experiments were performed in triplicate. Statistically
significant differences were determined by one-way ANOVA
analysis followed by Bonferroni’s post hoc test. A p value of
<0.05 was considered significant.
3 Results
3.1 Starch nanoparticle size
NPs were prepared from two different Andean-native potato
starches, S. tuberosum subsp. Andigena (common name
“Negra”) and S. goniocalyx (common name “Peruanita”)
using the method described in Torres et al. [25]. Nanoparticle
size ranged from 40 to 100 nm for at least 90% of the
observed starch particles, as measured by TEM (Fig. 1).
3.2 Cell viability
THP-1 monocytes were treated with starch NPs from “Negra”
(NEG NPs) and “Peruanita” (PER NPs) potatoes at three
different concentrations, from 30 to 300 mg/mL, for 24 h. The
results of the WST-8 assay showing the metabolic activity of
the THP-1 cells in the presence or absences of NPs are reported
in Fig. 2. No toxicity was observed in our samples within 24 h,
and the cell morphology of the treated cells was not dissimilar
from the control cells (data not shown).
3.3 Cytokine and chemokine release
The release of inflammatory cytokines and chemokines was
measured to evaluate the possible immune reaction of THP-
1 monocytes to the starch NPs. To avoid false positive results,
LAL test for endotoxins was performed on nanoparticle
suspensions before they were administered to the cells. As
reported in SI Fig. S1, both NP suspensions produced
negative results. The THP-1 cells were treated with 300 mg/
mL NEG NPs or PER NPs for 24 h, and the cytokine
concentrations were evaluated at three different time points
(2, 6, and 24 h). The THP-1 monocytes were stimulated with
100 ng/mL LPS as a positive control for cytokine release by
monocytes (SI Fig. S2). A dissimilar cytokine and chemokine
release pattern appeared when the cells were treated with the
NPs from the two different starches. The administration of
NEG NPs to the THP-1 cells induced a significant release of
all the probed inflammatory cytokines, namely IL-6, IL-1b,
TNF-a, CCL2, CCL4, and CCL5 (Fig. 3). Monocytes released
these cytokines with a specific temporal pattern, as shown by
their time curves. The maximal concentration of TNF-a was
detected after 6 h of NEG NPs administration, whereas, the
release of the other cytokines peaked at 24 h. In contrast, the
PER NPs did not induce any significant cytokine release,
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4 F. Gatto et al.
Figure 1. TEM images and measurements of the starch NP sizes.
although weak production of IL-1b and the chemokine CCL4
was observed at 24 h. The release of inflammatory chemo-
kines (CCL2, CCL4, and CCL5) by THP-1 cells in response to
NEG NPs, but not the PER NPs, persuaded us to further
investigate its functional impact on the monocytes.
3.4 Cell migration
The chemotaxis of THP-1 cells was evaluated using the
culture media collected from the monocytes treated with
Figure 2. Cell viability. The columns represent the percentage of
metabolic activity of THP-1 cells 24 h after exposure to increasing
doses (30, 60, 300 mg/mL) of starch NPs, as evaluated by the
WST-8 assay. The metabolic activity of the NP-treated cells is
expressed relative to the untreated control cells (white column).
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Starch/Stärke 2016, 68, 1–8
starch NPs as the chemo-attractants. The experimental
rationale was to evaluate whether the CCL2, CCL4, and CCL5
concentrations in the NEG NP-treated cell-culture medium
were sufficient to provoke the chemotaxis of resting THP-1
cells that constitutively express their cognate receptors CCR2
and CCR5 [27]. As expected, more THP-1 cells migrated
towards the medium from the NEG NP-treated monocytes
compared to the medium from the PER NP-treated
monocytes (Fig. 4, SI Fig. S3), although a slight migration
was also detected in the latter case. Medium from cells
treated with 100 ng/mL LPS was used as chemo-attractant
positive control, relying on the paradoxical release of
chemokines induced by LPS stimulation of THP-1 cells
(SI Fig. S2).
3.5 CD16 expression
Similar to the THP-1 cells treated with 100 ng/mL LPS, the
expression of the immunoglobulin receptor CD16 was
significantly increased by the 300 mg/mL NEG NP treatment
for 24 h, but not by PER NPs treatment (Fig. 5). In contrast,
no variation in the CD11b levels was observed in the THP-1
cells after a 24 h treatment with the NEG NPs or PER NPs
(SI Fig. S4).
4 Discussion
The influence of the botanic origin on the characteristics of
starch and the starch NPs has been reported in several
studies [25, 28, 29]. The size of the starch granules extracted
from S. tuberosum subsp. Andigena reported by Torres
et al. [24] was 42.5 mm, whereas, the size of the S. goniocalyx
starch granules was 30.5 mm. In contrast, the amylose
content measured for both starches was similar (25%).
X-ray diffraction tests performed on potato starch [30]
showed the typical B-pattern of crystalline structure (2u ¼ 5,
15, 17, 20, 22, and 24°), in agreement with other published
reports on potato starch [31, 32].
In our previous report [25], we characterized the nano-
particles extracted by acid hydrolysis from S. tuberosum
subsp. Andigena and S. goniocalyx starches. We found that
the granule size, amylose content, and nanoparticle size were
different for each starch source.
Acid hydrolysis modifies the structure of starch. Starch is
mainly composed of two glucosidic macromolecules:
amylose and amylopectin. Amylose is a linear polymer of
glucose units linked by (1-4) a-D-glycoside bonds, whereas,
amylopectin is highly branched, consisting of short
branches of a-D-(1-4) glycopyranose interlinked with
a-D-(1-6)-glycosidic linkages. When starch is treated with
acid, the glucosidic linkages are disrupted, provoking
alterations in its native structure, and properties. In
acid hydrolysis, the hydroxonium ion (H3Oþ) attacks the
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Starch/Stärke 2016, 68, 1–8 Andean-native potato starch nanoparticles induce dissimilar cytokine release
oxygen atom of the a(1-4) glucosidic bond. The electrons
in the carbon-oxygen bonds move towards the oxygen
atom, generating a carbocation intermediate that reacts
with water, leading to the regeneration of a hydroxyl
group [33].
We have previously studied the influence of acid
hydrolysis on the gelatinization of S. tuberosum subsp.
Andigena and S. goniocalyx starches [25] by comparing the
DSC thermograms of native starch and starch NPs extracted
by acid hydrolysis. The endothermic peak gelatinization
temperature of starch NPs was higher than the native starch
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Figure 3. Cytokine release by the
starch NP-treated THP-1 cells. IL-
6, IL-1b, TNF-a, CCL2, CCL4, and
CCL5 release from THP-1 cells
after 2, 6, and 24 h of exposure to
starch NPs. The control columns
represent the untreated cells. The
data are expressed as the means
 SD of at least three independent
experiments. One-way ANOVA
analysis followed by Bonferroni’s
post hoc test were performed.
( represents a statistically signifi-
cant different between NEG and
the control,  p < 0.05,  p
< 0.01,  p < 0.0001; ^ statisti-
cally significant difference be-
tween NEG and PER, ^p < 0.05,
^^p < 0.01, ^^^^p < 0.0001).
peak temperature. We associated this change with changes in
the starch structure.
The starch granule is formed by growth rings (120–
500 nm) composed of blockets (20–50 nm). These blockets
are composed of amorphous and crystalline lamellae
containing amylose, and amylopectin molecules [34]. The
analysis of the hydrolysis kinetics revealed a two-stage
process [35, 36]. In the first stage, hydrolysis is rapid and
occurs on the more amorphous part of the starch granule.
During the second stage, the crystalline material is
slowly degraded due to the slower penetration of H3Oþ
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6 F. Gatto et al.
Figure 4. Chemotaxis. The bar graph shows the number of THP-1
cells (untreated) that migrated towards the supernatants from
starch NEG NP-treated (dark gray column), or PER NPs-treated
(light gray column) cells. The supernatant from the 100 ng/mL LPS-
treated cells was used as a positive control (black column). The
data are expressed as fold increases for the treatments versus
controls. One-way ANOVA and Bonferroni’s post hoc test were
performed ( p < 0.01,  p < 0.0001).
within the densely packed starch chains in the crystallites.
We suggested that the increase in the peak temperature of
starch NPs compared to starch granules could be due to an
increase in crystallinity, or the presence of more regular
starch crystals [25]. Because acid hydrolysis attacks the
amorphous regions in the granule, the crystallites are
decoupled from the amorphous parts, inducing starch NP
melting at a higher temperature.
Figure 5. CD16 receptor expression. Relative mean fluorescence
intensity (RMFI) of CD16 receptors 24 h after the THP-1 cells were
treatewith the starch NPs. The control represents the untreated
cells. The data are expressed as the means  SD of three
experiments. One-way ANOVA followed by Bonferroni’s post
hoc test were performed ( p < 0.05,  p < 0.01).
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Starch/Stärke 2016, 68, 1–8
However, according to LeCorre et al. [34], acid hydrolyzed
starches are not 100% crystalline, but rather 45% crystalline,
with variations depending on the botanic origin. The acid
used for hydrolysis also changes the characteristics of the
process. Angellier et al. [26] obtained a lower yield of
hydrolysis with H2SO4 compared to HCl, but showed that
the final suspensions were more stable following H2SO4
hydrolysis due to the presence of sulfate groups at the
surface.
Our results show that starch nanoparticles from different
sources induce dissimilar immune responses. The non-
cytotoxicity of starch materials is an expected characteristic
shared by many other vegetable products. Nevertheless,
viability tests are not sufficient to validate biocompatibility.
The application of alien materials to the human body involves a
defense system of specialized cells, and molecules that are
finely regulated in a complex network. The precise tuning of
the immune system can be disrupted by non-toxic and even
biodegradable nanomaterials. For instance, the altered
inflammatory cytokine expression induced by the administra-
tion of NEG NPs revealed in our experiments (Fig. 3) could
push the innate immune system to over-react, and lead to
abnormal chemokine-mediated migration of monocytes/
macrophages (Fig. 4) to an existing site of inflammation.
Furthermore, the differentiation of resting monocytes
towards active cell types is also represented by the up- or
down-regulation of cell membrane immune receptors. The
immunoglobulin receptor CD16 mediates the phagocytosis
of opsonized microbes and is often overexpressed in
activated monocytes that are differentiating into tissue
macrophages. As shown in Fig. 5, CD16 up-regulation
could facilitate the internalization of opsonized NPs, and
increase the inflammatory state by triggering intracellular
inflammasomes [37].
Because we prepared both starch NP suspensions in the
same way and the size range of the particles was similar, we
hypothesize that the differences in the starch material could
be responsible for the dissimilar results. This hypothesis
would agree with our previous results obtained with starch
films from diverse Andean-native sources [23], indicating
that their immunological properties were not directly
correlated with film roughness, or stiffness.
The information regarding the immune response to
starch nanoparticles stemming from our observations may
lead to forecast-specific application for starch-based materi-
als from diverse sources. Although the nanoparticles
prepared with starch obtained from S. goniocalyx (PER)
display a minimal innate immune response, and could be
studied and engineered for the delivery of bio-active
molecules, such as drugs or genes, the immunogenicity of
the starch nanoparticles from S. tuberosum subsp. Andigena
(NEG) provides a warning about potential reactions that
could be harmful to potential delivery. On the other hand, the
inflammatory cytokine release induced by the NEG NPs
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Starch/Stärke 2016, 68, 1–8 Andean-native potato starch nanoparticles induce dissimilar cytokine release
would suggest a feasible employment of these particles to
prepare biodegradable vaccine adjuvants. At the present
stage, the employment of nanomaterials obtained from
various Andean-native potato species in biomedicine is in its
infancy due to synthesis limitations. The preparation of
homogeneous starch NPs is limited to research labs, with
very low commercial availability. NP size heterogeneity and
diverse NP shapes are common features of suspensions of
bio-derived materials. We cannot fairly exclude that these or
other physical characteristics of the starch NPs might also
have roles in immune system activation. However, the
continuous investigation of the immunological properties of
Andean-native starch nanomaterials will certainly help to
identify future biomedical applications of these, and other
low-cost biological resources.
5 Conclusions
In the present manuscript, we present data on human
monocyte reactions to two different Andean-native potato
starch nanoparticles. Our observations show that these
nanoparticles produce different cytokine release and cell
receptor expression profiles. Moreover, these nanoparticles
may differentially stimulate some related functions of
activated monocytes as an indirect consequence; specifically,
the monocyte migration towards inflammatory chemokines,
which were themselves released by cells that had been
previously stimulated with starch nanoparticles from S.
tuberosum subsp. Andigena. Our results emphasize the roles
of biodegradable materials from natural sources in nano-
technology and biomedical applications.
The authors would like to thank the Peruvian Council of
Science and Technology (Concytec-162-2015-FONDECYT) and
the Vice-Rectorate for Research of the Pontificia Universidad
Catolica del Peru (VRI-PUCP).
The authors do not have any conflicts of interest. The present
manuscript reports for the first time experimental results of
THP-1 cell immune responses to nanoparticles synthesized
from the starch of the Andean sub-species S. tuberosum and
S. goniocalyx.
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