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The nanotechnology revolution has allowed us to speculate on the possibility of hybridising nanoscale materials with live substrates, yet significant doubt still remains pertaining to the effects of nanomaterials on biological matter. In this investigation, we cultivate the ciliated protistic pond-dwelling microorganism Paramecium caudatum in the presence of excessive quantities of magnetite nanoparticles in order to deduce potential beneficial applications for this technique, as well as observe any deleterious effects on the organisms’ health. Our findings indicate that this variety of nanoparticle is well-tolerated by P. caudatum cells, who were observed to consume them in quantities exceeding 5–12% of their body volume: cultivation in the presence of magnetite nanoparticles does not alter P. caudatum cell volume, swimming speed, growth rate or peak colony density and cultures may persist in nanoparticle-contaminated media for many weeks. We demonstrate that P. caudatum cells ingest starch-coated magnetite nanoparticles which facilitates their being magnetically immobilised whilst maintaining apparently normal ciliary dynamics, thus demonstrating that nanoparticle biohybridisation is a viable alternative to conventional forms of ciliate quieting. Ingested magnetite nanoparticle deposits appear to aggregate, suggesting that (a) the process of being internalised concentrates and may therefore detoxify (i.e. render less reactive) nanomaterial suspensions in aquatic environments, and (b) P. caudatum is a candidate organism for programmable nanomaterial manipulation and delivery.
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BioNanoSci. (2018) 8:90–94
DOI 10.1007/s12668-017-0425-z
Toxicity and Applications of Internalised Magnetite
Nanoparticles Within Live Paramecium caudatum Cells
Richard Mayne1·James Whiting1·Andrew Adamatzky1
Published online: 22 June 2017
© The Author(s) 2017. This article is an open access publication
Abstract The nanotechnology revolution has allowed us
to speculate on the possibility of hybridising nanoscale
materials with live substrates, yet significant doubt still
remains pertaining to the effects of nanomaterials on bio-
logical matter. In this investigation, we cultivate the cil-
iated protistic pond-dwelling microorganism Paramecium
caudatum in the presence of excessive quantities of mag-
netite nanoparticles in order to deduce potential beneficial
applications for this technique, as well as observe any
deleterious effects on the organisms’ health. Our findings
indicate that this variety of nanoparticle is well-tolerated
by P. caudatum cells, who were observed to consume
them in quantities exceeding 5–12% of their body vol-
ume: cultivation in the presence of magnetite nanoparticles
does not alter P. caudatum cell volume, swimming speed,
growth rate or peak colony density and cultures may per-
sist in nanoparticle-contaminated media for many weeks.
We demonstrate that P. caudatum cells ingest starch-coated
magnetite nanoparticles which facilitates their being mag-
netically immobilised whilst maintaining apparently normal
ciliary dynamics, thus demonstrating that nanoparticle bio-
hybridisation is a viable alternative to conventional forms
of ciliate quieting. Ingested magnetite nanoparticle deposits
appear to aggregate, suggesting that (a) the process of being
Richard Mayne
Richard.Mayne@uwe.ac.uk
James Whiting
James.Whiting@uwe.ac.uk
Andrew Adamatzky
Andrew.Adamatzky@uwe.ac.uk
1Unconventional Computing Laboratory, University
of the West of England, Bristol, UK
internalised concentrates and may therefore detoxify (i.e.
render less reactive) nanomaterial suspensions in aquatic
environments, and (b) P. caudatum is a candidate organism
for programmable nanomaterial manipulation and delivery.
Keywords Nanotoxicology ·SPION ·Quieting ·
Paramecia ·Biohybridisation
1 Introduction
There are two major justifications for research into the
hybridisation of nanoscale materials with biological matter.
Firstly, despite their widespread use in recent years, signif-
icant doubt remains as to the potential deleterious effects
of nanoparticles and nanomaterials on biological matter as
even inert materials may be rendered reactive, immunogenic
or otherwise harmful to life when fabricated in nanoscale
quantities [1]. Secondly, nanomaterials may be fabricated
to exhibit highly desirable characteristics (specific electrical
properties, magnetism, high tensile strength etc.), the con-
ferral of which to live cells would potentially lead to next-
generation technologies such as bio-computer interfaces for
restorative and/or augmentative medical applications.
Magnetite (iron II/III oxide) nanoparticles (MNPs) (a
variety of superparamagnetic iron oxide nanoparticles,
SPIONs) are one such nanomaterial possessing desirable
properties for applications involving live cells. Exhibiting
superparamagnetism and apparently low toxicity, SPIONs
are now routinely used as a contrast medium in in vivo
magnetic resonance imaging and are a proposed drug-deli-
very (or otherwise therapeutic) agent for cancer treatment
[2,3]. With regards to their effects on protists, the same
class of nanoparticle may be hybridised with the plasmo-
dium of slime mould Physarum polycephalum towards
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BioNanoSci. (2018) 8:90–94 91
enhancing its value in bio-computer interfaces and uncon-
ventional computing devices [47]. Recent evidence has
suggested that MNPs are not as biocompatible as once
thought, however, due to their potential for bioaccumula-
tion and generation of reactive oxygen species within host
cells [8,9]. Furthermore, little is known of the ecotoxi-
cological significance of SPIONs released into the envi-
ronment, but recent studies on water-dwelling eukaryotes
such as Daphnia spp. and various forms of plant life
have demonstrated that they may potentially disrupt aquatic
ecosystems [10].
This study examines the ciliated protistic pond organ-
ism Paramecium caudatum being cultivated in the presence
of excessive quantities of MNPs in order assess the poten-
tial uses of nanohybridised paramecia whilst concurrently
observing for any deleterious effects on the organisms’
health. We conclude by discussing the apparent effects of
this treatment on P. caudatum cells and the ecotoxicologi-
cal significance of these results, and present several novel
applications for hybridised P. caudatum cells containing
MNPs.
2 Materials and Methods
P. caudatum cultures were cultivated in Chalkley’s medium
enriched with 10 g of alfalfa (Sciento, UK) and 40 wheat
grains (Tesco, UK) per litre. Cultures were exposed to a
day/night cycle but were kept out of direct sunlight; ambient
temperatures ranged from 19–24 oC.
In experiments where P. caudatum cells were exposed
to MNPs, suspensions of 200 nm (hydrodynamic diameter)
starch matrix-coated multi-core MNPs (Chemicell GmBH,
Germany) were added to fresh culture medium at a concen-
tration of 0.25 mg ml1(approximately 2.2×1012 particles
per ml). This concentration was chosen as a comparable
quantity of nanoparticles per unit biomass to our previ-
ous studies with other single-celled organisms [5]. Stock
cultures at a concentration of approximately 1000 cells
ml1were harvested in log growth phase and added to the
nanoparticle-infused culture medium, in which they were
incubated for periods in excess of two months.
The following microscopical measurements were made
on a regular (daily) basis (n= 3 per culture per day for each):
average cell count, average swimming speed, total cell cross
sectional area and percentage of cross-sectional cell area
occupied by cytoplasmic/vesicular inclusions whose colour
was suggestive of MNP deposits. Cells were observed in
glass microscope well slides. Cells were chemically fixed
in order to record photomicrographs. Fixation was achieved
by adding 10 μl of 4% paraformaldehyde (Agar Scien-
tific, UK) in pH 7.0 phosphate buffered saline solution
(Sigma, Germany) to each slide well after swimming speed
measurements had been made.
Observations were made with a Zeiss Axiovert 200M
inverted microscope and photo/videomicrographs were cap-
tured with an Olympus SC50 digital camera via CellSens
software. Electron microscopic observations were made
with an FEI Quanta FEG-SEM in high-vacuum mode.
For cell volume measurements, each image was anal-
ysed to extract both the cells’ cross-sectional area in squared
micrometers, but also to estimate the percentage of inter-
nalised MNPs. Each image was imported into Matlab
(Mathworks, USA) and processed in the following man-
ner: the image was converted into greyscale, and a threshold
was applied to extract all material darker than the back-
ground media. Each threshold was visually checked in order
to ascertain that only organisms were isolated in each image.
The number of pixels isolated by the threshold was then
summed to give the cells’ total cross-sectional area. This
process was also performed using a different threshold to
determine the number of pixels whose colour value corre-
sponded to dark cytoplasmic inclusions in order to identify
any internalised MNPs. As the threshold values had to be
determined visually for each image separately, this process
was performed ‘blind’, i.e. without the operator knowing
whether each image were a control or test measurement. The
number of pixels isolated by this method were then com-
pared with the cells’ total volume to give a percentage value.
Video analysis for measurement of organism swimming
speed was also performed using Matlab. RGB images were
imported from the video frame-by-frame for sequential
analysis and organism positioning. For each video set, the
organisms were isolated from the RGB image by colour,
whereupon the data for each frame was converted to a
JPEG image file for further analysis. To detect the position
of the organisms, a Laplace template of a Gaussian filter
was defined before being convolved over the image; the
size of the filter was iteratively determined by visual feed-
back of the user. After organism detection on every frame
had occurred, the positional data was passed to a bespoke
Kalman filter which accurately estimates the position of
the particle across each frame using the data from the full
time-series of particle positions to predict and confirm the
movement of each organism. From this it is possible to
measure the speed of each organism in a noisy video, cre-
ating a dataset of organism speed and momentary position.
While the script ran, frame-by-frame images were shown
on screen allowing visual validation of positional track-
ing by the authors. Average speed was calculated for each
organism.
All numerical data were subject to statistical analysis in
Matlab: two-tailed ttests and Mann-Whitney U tests were
used.
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92 BioNanoSci. (2018) 8:90–94
3 Results & Discussion
All measurement data are shown in Table 1. The micro-
scopical appearance (morphology and swimming patterns)
Fig. 1 Photomicrographs to show appearance of P. caudatum cells
(unfixed): (a) Following exposure to MNPs. Multiple rust-coloured
deposits (arrowed) are visible in the cytoplasm. (b) Control, demon-
strating a lack of rust-coloured deposits. Scale bar 100 μm
Ta b l e 1 Table to show mean (¯x) and standard deviation (σ)values
for measurements of total cell cross-sectional area (xa,inμm2),
cell content comprising dark (including rust-coloured) objects (doc,in
percentage), swimming speed (ss,inμm s1), growth rate (μ,inh
1)
and peak colony density (pcd, in cells ml1)(ttest and Mann-Whitney
Utest)
xa ¯xxaσdoc¯xdocσss¯xssσμ¯σpcd¯xpcdσ
Control 12715.66 5994.59 7.33 1.67 132.54 42.79 0.020 0.009 309 120
Test 13381.09 4700.77 12.68* 3.16 144.94 31.64 0.021 0.009 317 123
Asterisks indicate a statistically significant difference in means to controls at p<0.0001
of P. caudatum cells treated with MNPs was not notice-
ably altered aside from the inclusion of rust-coloured
deposits and more dark objects within intracellular vesi-
cles and the cells’ cytoplasm (Fig. 1). Organisms treated
with MNPs contained approximately 5% more dark intra-
cellular inclusions (including rust-coloured inclusions) than
controls. No statistical difference was observed in total
cell volume or swimming speed between controls and test
organisms.
The microscopical appearance of the rust-coloured
deposits within the organisms treated with MNPs was sim-
ilar to that of suspensions of MNPs in distilled water
(Fig. 2a). The electron microscopic appearance of the MNPs
was consistent with their description as aggregative multi-
core objects (Fig. 2b).
Furthermore, no statistical difference in growth rates or
peak colony density were observed between controls and
organisms treated with MNPs. All cultures (test and control)
persisted over the duration of the experiment (14 days).
Our results indicate that the P. caudatum cell is highly
tolerant to being cultured in the presence of large quantities
of MNPs, as indicated by our not observing any delete-
rious effects on the health of the organisms with regards
to their size, morphology, motility, growth rate or colony
density. This indicates that, despite recent evidence sug-
gesting that this variety of nanoparticle may be harmful
to aquatic microorganisms, they do not appear to induce
any readily-observable toxicological effects in our ciliated
model organism.
Intriguingly, the observations of MNPs within the organ-
isms suggests that they were internalised in the manner of
a nutrient source, noting that the size of the individual par-
ticle cores, 10 nm, were too large to enter the cell via any
other route such as through membrane pores. This apparent
uptake of MNPs was likely a result of their starch coat-
ing. Interactions between P. caudatum and MNPs with other
coatings/no coating remain a topic for further study.
Although no measurements of MNP intracellular reac-
tivity (e.g. generation of reactive oxygen species) or the
longevity of individual cells were made, the longevity of
cultures treated with MNPs was not significantly different
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BioNanoSci. (2018) 8:90–94 93
from that of controls as both varieties were kept in culture
for periods exceeding two months (data not shown).
We propose that P. caudatum may detoxify certain
environmentally dispersed nanomaterials: that dark/rust-
coloured deposits could be easily identified in P. caudatum
cells indicates that they are aggregated in vivo into non-
nanoscale objects. This reduction in surface area to volume
ratio likely renders the deposits less reactive and therefore
less harmful.
This apparent lack of toxicological effects incident of
internalising quantities of MNPs allows us to speculate
on the potential applications of this process of biological–
artificial hybridisation. In further experiments, we exposed
10 μl droplets on glass microscope well slides contain-
ing approximately 5 P. caudatum cells treated with MNPs
to a 1.28 T 25x40x4.0 mm neodymium magnet. Holding
the magnet in close proximity to the margins of the well
caused the organisms to be drawn to the edge of the droplet
where they were held immobile (Fig. 3). By increasing the
distance between the magnet and margins of the well, the
organisms were able to move but at a significantly reduced
speed.
Fig. 2 Microscopic appearance
of MNPs. aLight micrograph
of MNP suspension in distilled
water (25 mg ml1). The
suspension is ‘rust-coloured’
and is similar in appearance to
cytoplasmic inclusions in MNP-
treated P. caudatum cells. (b)
Scanning electron micrograph of
nanoparticle suspension dried
onto a carbon tab
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94 BioNanoSci. (2018) 8:90–94
Fig. 3 Stereomicrograph showing MNP-treated P. caudatum cells
(arrowed) being attracted towards a permanent magnet (black object).
Scale bar 500 μm
4 Conclusions
Magnetic restraint of ciliates via their hybridisation with
biocompatible magnetic nanomaterials would appear to
be an attractive alternative the established methods of
microorganism immobilisation/quieting, such as: replace-
ment of media with inert viscous fluids, induction of
hypoxia through hermetically sealing the observation envi-
ronment [11,12], addition of low-concentration toxins
(e.g. aliphatic alcohols [13], anaesthetic compounds [14]),
ultraviolet light irradiation [15], establishment of fluid
pressure gradients/microfluidic compression [16] and adhe-
sion to solid surfaces via positively-charged proteins (3-
aminopropyltriethoxysilane, protamine sulphate) [17].
The advantage of magnetic restraint over these other
quieting methods is that it does not necessitate inducing
deleterious health effects on the organism, maintains the
chemical composition and hence physical characteristics of
the fluid medium (thus minimising interference with natural
ciliary beating processes) and allows for momentary adjust-
ment of the strength of attraction (i.e. by moving the magnet
or using magnets of different strengths). Although magnetic
restraint of Paramecium spp. has been previously described
via internalised magnetite (particles of ca. 3 μm diameter),
the authors did not describe the use of microparticles in
this context as being a method for fully immobilising the
organisms [18].
Finally, this ciliated model organism’s capacity for gath-
ering, internalising and concentrating nanomaterials holds
exciting possibilities for the prospect of orchestrated bio-
logical manipulation and delivery (guided by gradients of
attractants, repellents or magnetic fields) of nano and micro-
scale compounds of interest, although further research is
required in this area before practical applications can be
realised.
Acknowledgments The authors extend their thanks to Dr. David
Patton for his electron microscopy expertise.
Compliance with Ethical Standards
Conflict of Interest The authors declare no competing financial interest.
Funding This work was funded by the Leverhulme Trust (grant no.
RPG-2013-345).
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were made.
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... Many kinds of nanoparticles being developed by researchers ranging from silica [1-5], cobalt [6-10], ZnO [11][12][13], and so forth. One of the nanoparticles that have low toxicity [14][15][16] and biocompatible [17][18][19] is magnetite so that it can be used in the biomedical field [20][21][22][23]. Practically, magnetite can be utilized as a filler material in the magnetic hydrogel to be subsequently used as actuator [24,25] or as artificial [26][27][28]. ...
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