Copper nanoparticles have negligible direct antibacterial impact

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Bastos, C., Faria, N., Wills, J. et al (2020). Copper nanoparticles have negligible direct antibacterial
impact. NanoImpact, 17. http://dx.doi.org/10.1016/j.impact.2019.100192

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 NanoImpact 17 (2020) 100192

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Research paper

Copper nanoparticles have negligible direct antibacterial impact T

a,⁎ a a b c d,e
Carlos A.P. Bastos , Nuno Faria , John Wills , Per Malmberg , Nathalie Scheers , Paul Rees ,
Jonathan J. Powella,
⁎

a
Biomineral Research Group, Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK
b
Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, Sweden
c
Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
d
Centre for Nanohealth, Swansea University College of Engineering, Fabian Way, Crymlyn Burrows, Swansea SA1 8EN, UK
e
Broad Institute of MIT and Harvard, 415 Main Street, Cambridge, MA 02142, United States

ARTICLE INFO ABSTRACT

Editor: Jamie Lead Introduction: Soluble copper that can be acquired by bacteria is toxic and therefore antimicrobial. Whether
Keywords: nanostructured copper materials, in either disperse or agglomerated form, have antimicrobial impact, aside from
Copper that of their dissolution products, is not clear and was herein addressed.
Antimicrobial Methods: We took five nanostructured copper materials, two metallic, and three oxo-hydroxides with one of
Nanoparticle these being silicate-substituted. Four agglomerated in the bacterial growth media whilst the silicate-substituted
Nano material remained disperse and small (6.5 nm diameter). Antibacterial activity against E. coli was assessed with
Antibacterial copper phase distribution measured over time. Using the dose of soluble copper, and benchmark dose non-linear
regression modelling, we determined how well this phase predicted antimicrobial activity. Finally, we used
Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) analysis to investigate whether membrane adhe-
sion effects by copper were plausible or if intracellular uptake most likely explained the bacterial impact of
copper.
Results: Comparison over time of antimicrobial activity against particulate or soluble phases of the aquated
materials clearly demonstrated that soluble copper but not particulate forms were associated with inhibition of
bacterial growth. Indeed, the benchmark dose modelling showed the soluble dose required to cause a 50%
reduction in E. coli growth was strongly clustered – for all particle formulations – at 14.5 mg/L (10–19 mg/L 90%
confidence interval). By comparison, total copper levels associated with the same reduction in viability varied
widely (45–549 mg/L). Finally, in favour of this soluble product dominance in terms of antimicrobial activity,
copper had low association with bacterial membrane (something both soluble and particulate materials could
do) but showed high intra-bacterial levels (something only soluble copper could do).
Conclusion: Taken together our data show that it is the uptake of soluble but not particulate copper, and the
intracellular loading not just contact and membrane association, that drives copper toxicity to bacteria.
Therapeutic strategies for novel antimicrobial copper compounds should consider these findings.

1. Introduction animals and the environment (Chen et al., 2017; McClements and Xiao,
2017).
Nanostructuring of materials may lead to changes in physicochem- Copper has broad-spectrum antimicrobial properties (Ingle et al.,
ical properties, beyond size differences, when compared to the bulk 2014; Lemire et al., 2013). This could be exploited for therapeutic
material counterparts (Pfeiffer et al., 2014; Santos et al., 2018). Whilst benefit but, equally, exposures could yield undesirable activity against
this can create novel applications and commercial opportunities, it has symbiotic bacteria in the environment, the skin or the intestine (i.e. the
also brought about concerns over exposure and safety for humans, other microbiome). So understanding copper-bacteria interaction is

Abbreviations: BMD, benchmark dose; CuNPs, elemental copper nanoparticles; CuO NPs, copper oxide nanoparticles; CuSi NPs, silicate-stabilised copper hydroxide
nanoparticles; DLS, Dynamic Light Scattering; E. coli, Escherichia coli; EFSA, European Food Safety Authority; FT-IR, Fourier Transform – Infra Red; HMM, Heavy
Metal MOPS medium; ICP-OES, Inductively Coupled Plasma – Optical Emission Spectrometry; MOPS, 3-(N-morpholino)propanesulfonic acid; OD, optical density;
SLS, static light scattering; TBS, Tryptic Soy Broth medium; ToF-SIMS, Time-of-Flight Secondary Ion Mass Spectrometry; UHP, ultra high purity
⁎
Corresponding authors.
E-mail addresses: [email protected] (C.A.P. Bastos), [email protected] (J.J. Powell).

https://doi.org/10.1016/j.impact.2019.100192
Received 9 August 2019; Received in revised form 24 October 2019; Accepted 4 November 2019
Available online 20 November 2019
2452-0748/ © 2019 Elsevier B.V. All rights reserved.
C.A.P. Bastos, et al. NanoImpact 17 (2020) 100192

important (Lemire et al., 2013). How much the nanostructuring of 18.2 ΩM/cm). Dispersions of copper oxide nanoparticles (CuO NPs A)
copper-based materials governs their antimicrobial properties is not were prepared from commercial powders (Sigma 544868) that were
fully understood. Certainly this nanostructuring strategy may be used to reported as free of impurities and with a primary particle size of 34 nm
tailor ‘slow release’ properties for copper ions or to allow stable aqu- (Moschini et al., 2013). These have been previously investigated as
eous formulations of copper at high concentrations without issues with antimicrobial agents (Aruoja et al., 2009; Bastos et al., 2018). The
hydrolysis and precipitation (Bastos et al., 2018; Herrmann and stocks were prepared at 20 mM copper by dispersing the powder in
Bucksch, 2015; Paulson and Kester, 1980). However, this does not ad- water with vigorous agitation. The same protocol was repeated for
dress whether particles themselves have a microbial impact beyond or copper oxide nanoparticles Alfa Aesar 44663 (termed here as CuO NPs
in addition to released soluble copper. On the one hand, by comparison B). Elemental copper nanoparticles, Sigma 774081 (termed here as
to silver (Xiu et al., 2012), one might assume that it is only soluble CuNPs A) and Sigma 774111 (termed here as CuNPs B), were main-
copper, not particles, that are antimicrobial. On the other hand, in- tained in an oxygen-free environment before use and were dispersed in
creased activities of copper-based nanoparticles, above and beyond just UHP water (20 mM copper) that had been flushed with nitrogen gas to
copper ion content, have been indicated (Chen et al., 2019; Raffi et al., reduce oxygen exposure. Their purity was confirmed via Fourier
2010; Sarkar et al., 2012). Indeed, the issue of whether nanoparticles Transform – Infrared (FT-IR) spectroscopy and by determining that the
per se have specific activity against bacteria, remains unclear but im- particles were 97% copper (Appendix A). Finally, silicate-stabilised
portant. For example, the European Food Safety Authority (EFSA) re- copper hydroxide nanoparticles (CuSi NPs) were synthesised by mixing
quests that, for new oral nanoparticles that are relevant to food, tests equal volumes of 400 mM sodium silicate (ca. pH 12) and 40 mM
are carried out in terms of microbiome effects for safety assessment copper chloride, which resulted in formation of blue-coloured ag-
(EFSA, 2018). gregates. 5 M NaOH was then added, under agitation to adjust the
In trying to determine whether copper-based particles, or the copper suspension to pH 12 ± 0.2 and the mixture was kept under stirring
ions that they release in an aqueous environment, are responsible for until full colloidal dispersion was observed (ca. after 24 h).
antimicrobial activity, a number of aspects must be carefully con-
sidered. Firstly, in standard bacterial assays, total exposure to particu- 2.2. Particle characterisation in liquid suspensions
late or soluble (solubilised) copper will be determined by an interaction
between time and rates of particle dissolution. Secondly, the chemistry 2.2.1. Particle sizing measurements
of copper is complicated. At typical biological (near–neutral) pHs, Following Initial inspection of commercial copper-based particles
particles may form when ‘soluble’ salts are added to a solution phase, dispersed in bacterial medium (Heavy Metals MOPS medium here
due to oxo-hydroxide formation, polymerisation, cross-linking and termed as HMM; full description in Appendix B), fast sedimentation
precipitation (Paulson and Kester, 1980; Zhang and Richardson, 2016). indicated that these were not nano-sized when suspended in aqueous
Finally, particles, whether inadvertently formed or specifically added to media. Therefore, static light scattering (SLS) was used to formally
a fluid phase, may agglomerate and/or aggregate. As such, studies in- determine the de facto particle size distribution of commercial copper
vestigating exactly what bacteria experience in terms of exposure to nanoparticles using a Mastersizer 2000 fitted with a Hydro 2000 μP
copper-based particles, and thus what chemical form is active, are Micro Precision sample dispersion unit (Malvern Instruments Ltd., UK).
lacking (Ingle et al., 2014). For each measurement, ca. 787 μL of each 20 mM copper stock was
Here, we took advantage of the differential solution behaviour of added directly to the 20 mL dispersion cell unit containing HMM. The
various formulations of copper-based particles and we measured their resulting dispersion (50 mg/L copper in HMM) was run at 1000 rpm
low molecular weight (soluble), nano and agglomerated fractions over and three consecutive measurement cycles were carried out for each
time in bacterial culture medium alongside their antimicrobial impact material with the settings described in Appendix C.
against Escherichia coli, a laboratory standard for clinically relevant Dynamic Light Scattering (DLS) was used to determine the hydro-
gram-negative bacteria which are increasingly associated with anti- dynamic particle size distribution by of the CuSi NPs suspension
biotic resistant infections (Cassini et al., 2019). We then used bench- (20 mM copper). The measurements were performed on a Zetasizer
mark dose modelling to determine the variability in effector activity of NanoZS (Malvern Instruments Ltd., UK) using a 1 mL disposable cuvette
the different phases of each particle type. Finally, we used ToF SIMS and a temperature of 25 ± 2 °C (n = 3). Detailed settings for the
analysis to investigate whether membrane adhesion effects by copper analysis can be found in Appendix C.
were plausible, or, if intracellular uptake most likely explained the
bacterial impact of copper. 2.2.2. Size categorisation in bacterial medium
To ensure that the light scatter signal from aquated microparticles,
2. Materials and methods above, was not masking a significant nano-fraction (Malvern
Instruments, 2015), we also carried out size categorisation by ultra-
2.1. Preparation of copper stocks filtration and centrifugation followed by elemental analysis. Copper-
based particle stocks with starting concentration of 20 mM copper were
All reagents were purchased from Sigma Aldrich and used as re- diluted in bacterial medium – Heavy Metal MOPS medium (HMM) – to
ceived, unless otherwise detailed. Copper nanopowders were purchased 50 mg/L (ca. 0.8 mM). Three aliquots of each stock were collected to
from Sigma Aldrich or Alfa Aesar (Table 1). Stock solutions were pre- determine: a) the total copper content (CuTotal), b) copper in the su-
pared using ultra high purity (UHP) water (reverse osmosis purification; pernatant after centrifugation at 16000g for 5 min (CuSupernatant), and c)

Table 1
Properties of commercial copper nanopowders as described by suppliers.
Reference Label Composition Molecular weight Primary particle size

Sigma 544868 CuO NPs A Copper oxide (CuO) 79.545 < 50 nm

10–50 nm
(Moschini et al., 2013)
Alfa Aesar 44663 CuO NPs B Copper oxide (CuO) 79.545 30–50 nm
Sigma 774081 Cu NPs A Elemental Cu (Cu0) 63.546 25 nm
Sigma 774111 Cu NPs B Elemental Cu (Cu0) 63.546 40–60 nm

2
C.A.P. Bastos, et al. NanoImpact 17 (2020) 100192

A B 20
12
CuO NPs A
10
CuO NPs B 15

Volume %
Volume %

8 CuNPs A
CuNPs B 10
6

4
5
2

0 0
0.1 1 10 100 1000 10000 1 10 100 1000
Size (nm)
Size (µm)

C D 40
30
Cu Phase Distribution %

Zeta Potential (mV)

100
20
80 10
Soluble %
60 Nanoparticulate % 0

Agglomerated % -10
40
-20
20
-30
0 -40
Ps
A
Ps
B sA sB Ps A B sA sB NP
s
ON ON NP NP Si N Ps Ps NP NP Si
Cu Cu Cu Cu Cu ON ON Cu Cu Cu
Cu Cu

Fig. 1. Particle characterisation in stock solutions and bacterial medium. (A) Particle size distribution of commercial copper nanoparticles in bacterial medium
(HMM) at 50 mg/L copper obtained by static light scattering. (B) Particle size distribution of CuSi NPs in its original stock at 20 mM in water. (C) Copper phase
distribution of nanoparticle in bacterial medium (HMM) at 50 mg/L Cu. (D) Zeta potential of copper nanoparticles in their corresponding stocks at 20 mM copper.

soluble copper content, after filtration with a 3KDa centrifuge filter syringe to avoid air bubbles, and analysis carried out as per standard
(Sartorius Vivaspin 500 VS0192) for 5 min at 16000 g (Cu3KDa). Fol- guidelines of the instrument manufacturer.
lowing size differentiation, aliquots were diluted down to 20 mg/L in
5% HNO3 (v/v) or lower and kept for at least 24 h prior to analysis to
ensure full dissolution. Copper concentration was then determined by 2.3. Antibacterial activity of copper materials
inductively coupled plasma-optical emission spectroscopy at
324.754 nm (ICP-OES; Jobin Yvon 2000, Horiba, UK) using matrix- Antibacterial activity of copper materials was assessed using a broth
matched calibration standards (0.1 to 20 mg/L). Finally, the different microdilution assay which is a standard approach for antimicrobial
fractions were calculated as per equations below: susceptibility testing in solution (ESCMID, 2003; Reller et al., 2009).
The assays were carried out using starting bacterial loads of ca.
CuTotal Cusupernatant
Agglomerated (%) = × 100 106 CFU/mL, which is the minimum quantifiable by optical density
CuTotal (OD595nm = 0.05–0.1) allowing bacterial growth to be followed over
Cu3Kda time and, therefore, copper effects to be assayed quantitatively. Copper
Soluble (%) = × 100 speciation and bacterial growth were followed over time. For this, Es-
CuTotal
cherichia coli (NCTC11100) cultures were grown in HMM (full compo-
Nanoparticulate (%) = 100 (Soluble (%) + Agglomerated (%)) sition in Appendix B), a defined media with low metal chelation ability
(LaRossa et al., 1995), which was supplemented with 0.4% glucose and
0.1% casein acid hydrolysate, and pH adjusted to 7.2 ± 0.2. Overnight
2.2.3. Soluble and particulate content in bacterial medium bacterial cultures were prepared at 30 °C in HMM under constant agi-
20 mM coper stocks were diluted in HMM to 12.5, 25 and 50 mg/L tation (80 rpm) in an Infors HT Minitron incubator. A turbidimetric
copper and incubated overtime at 30 °C under constant agitation assay was used to follow bacterial growth over time by measuring op-
(80 rpm) in an Infors HT Minitron incubator. Samples were collected tical density (OD) at 595 nm using a Multiskan RC 351 Labsystem plate
overtime for elemental analysis as described in 2.2.2. Soluble copper reader. 20 mM (ca. 1.27 g/L) copper particle stocks were pre-diluted in
content was defined as low molecular weight copper that passed HMM and then mixed with E. coli suspensions that, as noted above,
through a 3KDa filter (Cu3kDa), whilst the remaining copper was con- were diluted to an optical density of 0.05–0.1 OD reading (ca. 106 CFU/
sidered particulate (CuTotal − Cu3kDa). mL). Final copper concentrations were 0, 12.5, 25 and 50 mg/L copper.
These were chosen to be measurably toxic to bacteria whilst allowing
2.2.4. Zeta Potential Measurements for observations on how variance in particulate and soluble copper
The zeta potential of copper particles stocks was determined by concentrations impacted growth inhibition. Bacterial cells and particles
Laser Doppler Micro-electrophoresis (Zetasizer NanoZS, Malvern were incubated for 8 h at 30 °C under agitation (80 rpm) and OD
Instruments Ltd., UK) using disposable folded capillary cells (DTS1070) measurements were obtained at different time points. Background, i.e.
and assuming a dielectric constant of 78.5 and a viscosity of 0.89 cP. OD of bacteria-free media, was subtracted for both control (OD for
Cells were carefully filled with copper particle suspensions using a media only) and for copper (OD for media plus copper material) and

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C.A.P. Bastos, et al. NanoImpact 17 (2020) 100192

A) E. coli Growth inhibition B) Particulate Copper C) Soluble Copper

50 50
40

Particulate Cu, mg/L

100
E.coli Growth Inhibtion %
40 30

Soluble Cu, mg/L

25
80
30 20
60
12.5 20 15
40
mg/L 10
20 10
5
0 0 0
2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10
-20 Time (h) Time (h) Time (h)

50 50
40

Particulate Cu, mg/L

100 30
E.coli Growth Inhibtion %

Soluble Cu, mg/L

40
25
80
30 20
60
15
25 40
20
10
mg/L 20 10
5
0 0 0
2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10
-20 Time (h) Time (h)
Time (h)

50 50
100 40
Particulate Cu, mg/L
E.coli Growth Inhibtion %

45 30

Soluble Cu, mg/L

80 25
40 20
60
50 35
15
40
mg/L 10
20 30
5
0
2 4 6 8 10 25 0
-20 0 2 4 6 8 10 0 2 4 6 8 10
Time (h)
Time (h) Time (h)

Legend: CuSi NPs CuO NPs A CuO NPs B CuNPs A CuNPs B

Fig. 2. Antimicrobial activity and phase distribution of the materials in bacterial medium.
(A) E. coli growth inhibition (%) as a function of time upon incubation with copper nanoparticles at 12.5 mg/L (top), 25 mg/L (middle) and 50 mg/L (bottom). (B)
Particulate copper in bacterial medium overtime and (C) soluble copper released in bacterial medium over time upon incubation with copper nanoparticles at
12.5 mg/L (top), 25 mg/L (middle) and 50 mg/L (bottom).

Bacterial growth inhibition over time was determined by comparing that could be expected to cause the benchmark response (set here at a 50%
with OD readings for copper treated against control, as per equation reduction in bacterial viability) for each of the particle formulations.
below:
2.5. TOF-SIMS analysis
OD Control OD Copper Treated
Growth Inhibition% = × 100
OD Control A 40 mM copper chloride solution was diluted to 500 mg/L (ca.
7.9 mM) in HMM and this stock was then further diluted to 12.5 mg/L
copper in an E. coli (DH5 alpha) culture in the stationary phase (pre-
2.4. Benchmark response modelling cultured in Tryptic Soy Broth medium (TBS) and added in the ex-
ponential phase to HMM overnight cultures (37 °C, 200 rpm; 1.9 cm
Benchmark dose (BMD), i.e. non-linear regression analysis, was con- orbit; Max 4000 Barnstead Lab-line)). Copper and bacteria were in-
ducted using the combined BMD-covariate approach described extensively cubated at room temperature for 2 h with no agitation. This culture was
in previous work (Wills et al., 2015, 2016, 2017). Analyses were carried out subsequently centrifuged at 1500 rpm (250g; Heraeus multifuge) for
the R computing environment using the freely available PROAST package 5 min and the supernatant containing the medium and free copper was
(http://www.proast.nl). The bacterial viability dose-response data were discarded. The remaining cell pellet was resuspended to the original
analysed using a nested family of four-parameter exponential models re- volume in UHP water. A sample droplet was mounted on a silicon
commended for the analysis of continuous toxicity data by the EFSA (EFSA, wafer, heat-dried, and analysed by TOF-SIMS.
2009, 2017). In each analysis, combined dose response data for either the ToF-SIMS imaging and depth profiling was performed using a TOF.SIMS
total dose of copper (i.e., the dose-time product) or the released dose of V (ION-TOF, GmbH) using a 25 keV bismuth liquid metal ion gun as a
soluble copper were analysed using the factor discriminating the subgroups primary ion source and a 10 KeV C60 ion source as a sputter source. The
(i.e., copper-based nanoparticle type) as a covariate. Models were iteratively delayed extraction mode was used for imaging with an approximate lateral
fitted to the data with more complex models with increasing numbers of resolution of 400 nm at a mass resolution of approx. 3500 at m/z 58. The
covariate-dependent parameters only accepted if the difference in log-like- pulsed primary ion current of the Bi3+ ion used was 0.2 pA. The total
lihood exceeded P < 0.05. This approach allowed determination of which primary ion dose density was 1 × 1011 ions/cm2 and therefore below the
model parameters needed to be estimated for each subgroup, and which static limit. The SurfaceLab 6 software (v. 6.6, ION-TOF GmbH) was used
parameters could be considered constants across the combined data. Inter- for all processing of images and depth profiles. The mass spectra were ca-
polation from the fitted model allowed estimation of the dose (i.e., the BMD) librated internally to signals of [CH3]+, [C5H12N]+, and [C5H15PNO4]+.

4
C.A.P. Bastos, et al. NanoImpact 17 (2020) 100192

oxides were mostly 1–10 μm in diameter and the copper metal particles
A were larger (10–1000 μm). In contrast, the in-house-synthesised, sili-
cate-substituted copper oxo-hydroxides (termed CuSi NPs) had a modal
diameter of 6.5 nm and almost all were 4–20 nm in size (Fig. 1B). Size
differentiation experiments, based on filtration and centrifugation,
confirmed these findings with the aquated commercial particles domi-
nated by agglomerated material and the in-house-synthesised particles
Log10 Bacterial Growth (OD595nm)

dominated by a small nano fraction (Fig. 1C). As expected, the copper

metal particles were positively-charged in the HMM medium and the
ratio: Treated:Untreated

copper oxides were negatively charged (Fig. 1D) with the aquated na-
noparticles (i.e. the CuSi NPs) showing a large enough negative zeta
potential (−27 ± 1 mV; Fig. 1D) for stable suspension in aqueous
media (Bhattacharjee, 2016).
When the particles were aquated in the E. coli-containing bacterial
medium, it was apparent that, except at the highest levels of exposure
(50 mg/L copper), the aquated nanoparticulate material had much
greater antimicrobial activity than the large particle material, during
the 8 h of experimentation (Fig. 2A). Moreover, the particulate fraction,
whether nano (CuSi NPs) or larger (other materials) or whether posi-
tively or negatively charged, appeared inversely related to bacterial
growth inhibition (Fig. 2B). Indeed, as therefore expected, the soluble
fraction of copper appeared clearly related to inhibition of E. coli
growth, over time, for all materials (Fig. 2C). Notwithstanding, the
interaction between time and solubility did not facilitate intuitive un-
derstanding of just how well soluble copper concentration might ex-
plain antimicrobial activity. To enable this, we modelled the effects on
Log10 of BMD50 : (Dose of soluble copper, mg.L-1.h-1) bacterial viability over time using benchmark dose non-linear regres-
sion modelling. Firstly, we confirmed that exposure to total copper in
suspension was a very poor predictor of antimicrobial potency as would
B 10 19 be expected for combined soluble and particulate copper (Appendix D).
CuNPs A In contrast, the results for soluble copper converged across all for-
mulations tested to very similar benchmark dose levels (Fig. 3). Indeed,
CuNPs B when the dose required to cause a 50% reduction in E. coli viability was
compared, the total copper data showed an order of magnitude var-
iance (45–549 mg/L Cu) between particle types (Appendix D). Con-
CuO NPs A

trastingly, the dose of soluble copper required was tightly clustered for
CuO NPs B
all formulations around 14.5 mg/L with the 90% confidence interval
across all particle types spanning a range of just 10–19 mg/L (Fig. 3).
CuSi NPs
Collectively, these data strongly imply that soluble, but not particulate
0 5 10 15 20 25 30 copper levels whether nano or larger, achieved by hydrated copper-con-
50% Growth inhibition taining particles, determine a material's antimicrobial activity. Finally,
(Copper dose, mg.L-1.h-1) therefore, to confirm this we considered the intracellular (i.e. intra-bacterial)
levels of copper versus that of the outer membrane following soluble copper
Fig. 3. Correlation between bacterial growth inhibition and total soluble exposure. We reasoned that a non-contact effect would favour intracellular
copper released from the particles. (A) Benchmark dose non-linear regression copper localisation rather than membrane localisation, because the latter
modelling to compare the effect of released soluble copper on bacterial growth could have been achieved in the studies above with particles, whereas the
inhibition. (B) 90% confidence intervals from each analysis, showing for each former could not have been achieved with particle surfaces since bacteria
particle formulation the dose required to elicit a 50% reduction in bacterial have rigid membranes and no ability to internalise particles (Santos et al.,
growth. The intervals for soluble copper converge into a narrow range, unlike 2018). We did not use intracellular copper sensors as we have previously
for total copper that shows poor correlation across formulations, suggesting ion
(Bastos et al., 2018; Kasemets et al., 2009) because that approach does not
release drives the inhibition effect observed.
enable membrane localisation to be assessed. Instead we used ToF-SIMS,
where individual bacterial cells were first identified using K+ mapping and
Depth profiling was performed in the non-interlaced mode (2 shots copper was directly analysed with a combined 63Cu + 65Cu signal
per pixel, 1 frame per sputter cycle of 1 s with a 0.5 s pause) using (Blockhuys et al., 2018). Following exposure to 12.5 mg/L copper, i.e. non-
C60++ ions at a current of 0.15 nA. Craters were sputtered ranging precipitating conditions in the bacterial medium, the copper signal was low
from 150 × 150 μm2 to 300 × 300 μm2 whilst analysing areas of at the bacterial cell membrane, the site of which was determined by
50 × 50 μm2 to 150 × 150 μm2 using the primary ions. The ratio C5H15PNO4+ as before (Nygren et al., 2007), and, was high intracellularly
between the analysed area and the sputtered area was > 1.5. The total (Fig. 4). Collectively, our findings show that regardless of size, charge and
C60 dose in the experiments ranged from 4.9 × 1013 ions/cm2 to composition of copper-based particles, it is the release of copper ions into
5.8 × 1014 ions/cm2. solution that primarily determines their antimicrobial activity when
aquated.
3. Results
4. Discussion
In the HMM bacterial medium, the commercially available nano-
particles were chiefly polydisperse micron-sized agglomerates as de- It is well recognised that soluble forms of copper have antibacterial
termined by static light scattering measurements (Fig. 1A). The copper activity. However, whether particulate copper in suspension also has a

5
C.A.P. Bastos, et al. NanoImpact 17 (2020) 100192

A B

Intensity (counts)- Membrane

25 25 10

Intensity (counts) - Cu
20 20 8 Cu
Intensity Counts

12.5 mg/L Cu Membrane

15 0 mg/L Cu 15 6

10 10 4

5 5 2

0 0 0
0 20 40 60 80 100 0 20 40 60 80 100
Sputter Time (s) Sputter Time (s)

Fig. 4. Copper mapping in bacteria using Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). (A) Depth profile for combined 63Cu+, 65Cu+ signal as a
function of sputter time in samples where bacteria were exposed to 0 or 12.5 mg/L copper chloride; (B) and comparison between copper (combined 63Cu+, 65Cu+)
and a membrane marker (C5H15PNO4+).

direct impact has not been established. Plausibility for this is posited on Declaration of competing interest
the fact that copper surfaces can be used to facilitate environmental
infection control. Moreover, particles in suspension are, generally, at- The authors declare no conflict of interest but C.A.P.B., N.F., and
tracted to cell membranes due to entropic and, often, electrostatic J.J.P. wish to note that they are inventors on a patent detailing use of
forces. Indeed, attractive forces could be enhanced using particle sur- copper oxo-hydroxide structures as antimicrobials.
face coatings although these then increase particle size, which is
especially noticeable for the ultra small particle size range. On this Acknowledgements
point, it is unlikely that bacteria acquire and internalise ultra small (or
larger) nanoparticles as the rigidity of the bacterial cell wall does not This work was supported by the University of Cambridge (UK) and
support endocytic mechanisms (Santos et al., 2018). Particle penetra- funded by the UK Medical Research Council grant number MR/
tion could occur if the cell membrane was degraded by a specific par- R005699/1.
ticle coating or carrier (e.g. surfactants), but this would not be a direct
nanoparticle interaction (Li et al., 2015; Xiu et al., 2012). Appendices. Supplementary Information
Here we show that, in the absence of a permeabilising agent, neither
large nor small particles of copper-based materials have a measurable im- Supplementary data to this article can be found online at https://
pact on bacterial growth above and beyond the effect through loss of copper doi.org/10.1016/j.impact.2019.100192.
ions into the aqueous medium. In other words, it is the dissolution of
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