Human Monocyte Response To Andean-Native Starch Nanoparticles
Human Monocyte Response To Andean-Native Starch Nanoparticles
Human Monocyte Response To Andean-Native Starch Nanoparticles
201600105 1
RESEARCH ARTICLE
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.
internal pathological threats. The immune system consists water and stored at 4°C. Nanoparticle yield, calculated as the
of two different lines of control, the innate and the adaptive amount of NPs recovered per 100 g of processed dry starch,
responses, which allow the body to distinguish between self was 19.74% for S. tuberosum subsp. Andigena samples and
and non-self molecules (namely, organisms or materials 14.98% for the S. goniocalyx samples.
from external sources). The innate reaction defines an
immediate non-specific response to alien bodies [20] 2.2 Transmission electron microscopy
mediated by phagocytic cells, such as neutrophils or
monocytes/macrophages, which act by encapsulating, and Transmission electron microscopy (TEM) images were
destroying the foreign substances through specific enzy- recorded using a JEOL JEM 1011 microscope operating at
matic reactions. Resting monocytes travel around the body, an accelerating voltage of 100 kV. The TEM samples were
sensing environmental signals, and migrating to specific prepared by dropping a dilute solution of starch NPs in water
sites of inflammation. They are driven into the inflamed on carbon-coated copper grids (Formvar/Carbon 300 Mesh
tissue by a gradient of specific chemo-attractant cytokines Cu). Nanoparticle size was evaluated by measuring the
(chemokines) released by tissue cells, indicating their average diameter of more than 100 NPs.
pathological condition. Once in the tissue, macrophages
phagocytize pathogens and damaged cells, further regulating 2.3 Cell culture
the growth, and the differentiation of other cells by releasing
specific molecular messengers [21]. In this way, monocytes Human THP-1 monocytic cells (ATCC1 TIB-202TM) were
link innate and adaptive immunity, tailoring the pathogen- purchased from the American Type Culture Collection (ATCC
specific response of lymphocytes. Monocytes/macrophages Manassas, VA, USA). The cells were cultured in RPMI-1640
recognize some of the polymers used for biomedical purpose (Life Technologies, cat. no. A10491-01) supplemented with
as non-self, triggering unforeseen immune reactions [22]. 10% fetal bovine serum (FBS) (Life Technologies, cat. no.
We have recently shown that starch films derived from 10108-165), 1% antibiotics (Penicillin–Streptomycin, Sigma–
different types of potatoes have a specific ability to interact Aldrich, cat. no. P0781), and 0.05 mM 2-mercaptoethanol
with the immune cell surface [23], and the response may be (Sigma–Aldrich cat. no. 63689) at 37°C in 5% CO2, and the
related to the different molecular structures of the various cultures were split every 3 days.
starches. The aim of the present study, was to investigate the
reaction of human monocytes primed with two different 2.4 Cell viability
types of Andean-native starch nanoparticles, focusing on the
release of monocyte-derived pro-inflammatory cytokines, The cells were seeded in a 96-well plate (Sigma–Aldrich,
and their consequences. 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
2 Experimental section activity was determined 24 h after exposure using a standard
WST-8 assay (Sigma–Aldrich, cat. no. 96992). Briefly, 10 mL of
2.1 Starch extraction and starch nanoparticle Cell Counting Reagent WST-8 were added to each well, and the
preparation plate was incubated in a humidified atmosphere of 5% CO2
at 37°C for 3 h. The orange WST-8 formazan product was
The starches used in this study were extracted from the measured on a Flo Star Optima (BMG LABTECH) microplate
potatoes Solanum tuberosum subsp. Andigena (common reader at a wavelength of 460 nm. The data were collected by
name “Negra”) and Solanum goniocalyx (common name the Control Software and further analyzed with the MARS Data
Peruanita), as reported in Torres et al. [24]. The amylose Analysis Software (BDG LABTECH). As a positive control for
content of the starches, as measured by a colorimetric cytotoxicity, the cells were incubated with 5% DMSO (Sigma–
procedure [24], was 25.43% for the S. tuberosum subsp. Aldrich, Cat. no. D8418).
Andigena starch and 24.10% for the S. goniocalyx starch.
Nanoparticles (NPs) were prepared using the method 2.5 Cytokine release
reported by Torres et al. [25], which is based on the method
developed by Angellier et al. [26]. Briefly, 11.75 g of starch Cells were cultured in 12-well plates (Sigma–Aldrich,
were mixed with 80 mL of 3.16 M H2SO4 (Merck–Millipore, Corning1 Costar1 cat. no. CLS3512) at a density of
Billerica, MA, USA cat. no. 100731) at 40°C under magnetic 5 105 cells/mL and incubated with the appropriate suspen-
agitation for 5 days. The final suspension was centrifuged, sion of starch NPs at a concentration of 300 mg/mL for 2, 6,
and the precipitate washed by successive centrifugations and and 24 h. IL-6, IL-1b, TNF-a, CCL2, CCL4, and CCL5 release
re-dispersions in distilled water until it reached neutral pH. from the THP-1 cells were assessed with a Bio-Plex1
Finally, the starch NPs were dispersed in 100 mL of distilled MAGPIXTM Multiplex Reader (Bio-Rad) according to the
Similar to the THP-1 cells treated with 100 ng/mL LPS, the
expression of the immunoglobulin receptor CD16 was
Figure 1. TEM images and measurements of the starch NP sizes.
significantly increased by the 300 mg/mL NEG NP treatment
for 24 h, but not by PER NPs treatment (Fig. 5). In contrast,
although weak production of IL-1b and the chemokine CCL4
no variation in the CD11b levels was observed in the THP-1
was observed at 24 h. The release of inflammatory chemo-
cells after a 24 h treatment with the NEG NPs or PER NPs
kines (CCL2, CCL4, and CCL5) by THP-1 cells in response to
(SI Fig. S4).
NEG NPs, but not the PER NPs, persuaded us to further
investigate its functional impact on the monocytes.
4 Discussion
3.4 Cell migration
The influence of the botanic origin on the characteristics of
The chemotaxis of THP-1 cells was evaluated using the
starch and the starch NPs has been reported in several
culture media collected from the monocytes treated with
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
Figure 2. Cell viability. The columns represent the percentage of a-D-(1-6)-glycosidic linkages. When starch is treated with
metabolic activity of THP-1 cells 24 h after exposure to increasing acid, the glucosidic linkages are disrupted, provoking
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 alterations in its native structure, and properties. In
expressed relative to the untreated control cells (white column). acid hydrolysis, the hydroxonium ion (H3Oþ) attacks the
oxygen atom of the a(1-4) glucosidic bond. The electrons peak temperature. We associated this change with changes in
in the carbon-oxygen bonds move towards the oxygen the starch structure.
atom, generating a carbocation intermediate that reacts The starch granule is formed by growth rings (120–
with water, leading to the regeneration of a hydroxyl 500 nm) composed of blockets (20–50 nm). These blockets
group [33]. are composed of amorphous and crystalline lamellae
We have previously studied the influence of acid containing amylose, and amylopectin molecules [34]. The
hydrolysis on the gelatinization of S. tuberosum subsp. analysis of the hydrolysis kinetics revealed a two-stage
Andigena and S. goniocalyx starches [25] by comparing the process [35, 36]. In the first stage, hydrolysis is rapid and
DSC thermograms of native starch and starch NPs extracted occurs on the more amorphous part of the starch granule.
by acid hydrolysis. The endothermic peak gelatinization During the second stage, the crystalline material is
temperature of starch NPs was higher than the native starch slowly degraded due to the slower penetration of H3Oþ
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