Colloids and Surfaces A: Physicochem. Eng. Aspects 457 (2014) 263–274
`
`Contents lists available at ScienceDirect
`Colloids and Surfaces A: Physicochemical and
`Engineering Aspects
`j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / c o l s u r f a
`
`The contribution of zinc ions to the antimicrobial activity of zinc oxide
`Julia Pasquet a,b, Yves Chevalier b,∗, Jocelyne Pelletier b, Emmanuelle Couval a,
`Dominique Bouvier a, Marie-Alexandrine Bolzinger b
`a Strand Cosmetics Europe, 124 route du Charpenay, 69210 Lentilly, France
`b Université Claude Bernard Lyon 1, Laboratoire d’Automatique et de Génie des Procédés (LAGEP), CNRS UMR 5007, 43 bd 11 Novembre,
`69622 Villeurbanne, France
`
`g r a p h i c a l
`
`a b s t r a c t
`
`h i g h l i g h t s
`• Proteins contained in a broth medium
`increase the solubility of ZnO parti-
`cles.
`• The respective activities of ZnO and
`Zn2+ show specificity with respect to
`the microorganisms.
`• The contribution of Zn2+
`the
`to
`antimicrobial activity of ZnO depends
`on the strain.
`• The dissolution process depends on
`time, ZnO concentration, and type of
`ZnO powder.
`• The combination of the three antimi-
`crobial mechanisms of ZnO is benefi-
`cial.
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Article history:
`Received 13 March 2014
`Received in revised form 23 May 2014
`Accepted 24 May 2014
`Available online 2 June 2014
`
`Keywords:
`Zinc oxide
`Zinc ions
`Antimicrobial activity
`Dissolution
`Topical formulations
`
`Zinc ions (Zn2+) exhibit antimicrobial activity against various bacterial and fungal strains. The partial
`dissolution of zinc oxide (ZnO) particles releases Zn2+ ions in aqueous suspension that contributes to
`the antimicrobial activity of ZnO. In addition to the activity of the soluble zinc species that is common
`with water-soluble zinc salts, ZnO combines two additional mechanisms of antimicrobial activity that
`supplement its activity as preservative in topical formulations: generation of reactive oxygen species and
`by direct contact to the cells walls. The present study aims at the evaluation of the contribution of the
`soluble zinc species to the antimicrobial activity of ZnO on microbial cultures in broth medium and the
`investigation of the dissolution of zinc from ZnO suspensions. The antimicrobial activities against the
`five microorganisms of the Challenge Tests were measured for suspensions of three ZnO grades in broth,
`and for the isolated liquid phase of the suspensions containing soluble zinc species. The Zn2+ released
`in the broth brought about a significant contribution to the overall antimicrobial activity of ZnO. The
`complexation of Zn2+ ions by the components of the broth increased the solubility of the zinc in the
`liquid medium. The respective activities of the soluble zinc species and ZnO particles showed specificity
`with respect to the microbial strains. Dissolution was faster for high concentrations of ZnO and for ZnO
`powders of larger specific area. Such conditions led to a better antimicrobial efficacy of ZnO powders.
`ZnO appears an advantageous alternative to soluble zinc salts such as zinc gluconate.
`© 2014 Elsevier B.V. All rights reserved.
`
`∗ Corresponding author. Tel.: +33 4 72 43 18 77; fax: +33 4 72 43 16 82.
`jocelyne.pelletier@univ-lyon1.fr (J. Pelletier),
`jpasquet@strandcosmeticseurope.com (J. Pasquet), chevalier@lagep.univ-lyon1.fr (Y. Chevalier),
`E-mail addresses:
`ecouval@strandcosmeticseurope.com (E. Couval), dbouvier@strandcosmeticseurope.com (D. Bouvier), bolzinger@lagep.univ-lyon1.fr (M.-A. Bolzinger).
`
`http://dx.doi.org/10.1016/j.colsurfa.2014.05.057
`0927-7757/© 2014 Elsevier B.V. All rights reserved.
`
`Petitioner Dr. Squatch
` Ex. 1029
`
`

`

`264
`
`J. Pasquet et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 457 (2014) 263–274
`
`1. Introduction
`
`Zinc oxide (ZnO) is an efficient antimicrobial agent that acts by
`means of several mechanisms involving different chemical species.
`Three distinct mechanisms of action have been put forwards in
`the literature: (i) the production of reactive oxygen species (ROS)
`because of the semiconductive properties of ZnO [1,2,3], (ii) the
`destabilization of microbial membranes upon direct contact of ZnO
`particles to the cell walls [4–6], and (iii) the intrinsic antimicro-
`bial properties of Zn2+ ions released by ZnO in aqueous medium
`[7–9]. The present study aims at investigating the contribution of
`the soluble species released by ZnO, especially Zn2+.
`Zinc is an essential element for microorganisms and higher
`organisms because it is involved in many vital cellular reactions
`at its low endogenous concentrations [10–13]. The concentration
`of zinc is 10−4 M in blood [14]. Optimal rates of this cofactor are
`needed for catalytic and structural activities [14,15]. Zinc con-
`centration is regulated under physiological conditions by several
`transporters [15–17], so that Zn2+ ions are essentially nontoxic to
`higher organisms [3]. Homeostasis regulates zinc uptake by cells,
`but it does not control zinc adsorption to cell membranes how-
`ever. Increase of Zn2+ concentrationsabove optimal levels (typically
`between 10−7 M and 10−5 M depending on the microbial strain
`[18]) perturbs Zn2+ homeostasis and allows entry of Zn2+ inside
`cells, so that zinc starts being cytotoxic to prokaryotes above a con-
`∼10−4 M [18,19]. Therefore, Zn2+ displays an antimi-
`centration of
`crobial activity and could act as either antibacterial or antifungal
`agent. The antimicrobial properties of Zn2+ have been known since
`a long time, both against bacterial [20–22] and fungal strains [23].
`According to several reports on water-soluble zinc salts, the
`antimicrobial activity of Zn2+ depends on its concentration and
`contact duration. Zinc chloride acts in a dose-dependent manner
`against Escherichia coli [21,22]. Zinc acetate exhibits an antibacte-
`rial activity on Pseudomonas aeruginosa and Staphylococcus aureus
`for zinc concentrations above 11 mmol L−1 [20]. Moreover, a pro-
`␮g g−1) to Aspergillus brasiliensis
`longed contact of zinc (at 100
`spores inhibits their germination by 25% [23]. These antimicrobial
`activities were explained by two mechanisms, both leading to cell
`death: (i) a direct interaction with microbial membranes leading to
`membrane destabilization and enhanced permeability [24]; (ii) an
`interaction with nucleic acids and deactivation of enzymes of the
`respiratory system [25].
`In dermatological products, zinc ions are interesting biocides
`and/or antimicrobial preservatives provided that high enough con-
`centrations of Zn2+ are generated. The previously mentioned zinc
`salts can be simply dissolved in the aqueous medium. An alternative
`is solid powder such as ZnO particles that release Zn2+ in the aque-
`ous medium. It is indeed recognized that part of the antimicrobial
`activity of ZnO particles originates from their ability to partially dis-
`solve in aqueous media [26,27]. Release of Zn2+ would contribute to
`the global antimicrobial properties of this inorganic powder [7,29].
`Nevertheless, the contribution of Zn2+ to the antimicrobial activ-
`ity of ZnO is still unclear. Since ZnO particles exhibit two additional
`antimicrobial mechanisms with respect to zinc salts (ROS and direct
`contact) the combination of these three types of action broadens
`the antimicrobial spectrum of ZnO compared to zinc salts.
`The present study has been focused on the contribution of zinc
`cations generated from the partial dissolution of ZnO particles in
`aqueous media to the global antimicrobial action. Even though the
`antimicrobial mechanism of Zn2+ has been disclosed, the antimicro-
`bial activity of ZnO via a contribution of Zn2+ is still under debate
`because of the complexity of the underlying phenomena. Accord-
`ing to Sawai [28] and Jiang et al. [6], the contribution of Zn2+ to
`the antimicrobial efficacy of ZnO particles would be minor because
`too low concentrations of soluble zinc species are released from
`the dissolution of ZnO particles. In other instances reported in the
`
`field of dentistry applications, the contribution of Zn2+ is predom-
`inant [30,31]. The aim of the present work was the assessment of
`the contribution of soluble ionic species of zinc to the antimicro-
`bial efficacy of ZnO powders, as well as the factors that influence
`the dissolution of the particles. Indeed, it has been reported that the
`dissolution phenomenon was influenced by numerous parameters
`sorted into two types:
`
`- the chemistry of the environmental media such as the pH [32,33],
`the duration of exposure [34–36], UV irradiation [32,37], the pres-
`ence of other substances [33,38] or microorganisms [39–41];
`- the physicochemical properties of the particles such as the ele-
`mentary particle size [35,42,43], their porosity [44], their shape
`[35], their concentration [45].
`
`The impact of all these parameters is not fully understood.
`In order to use ZnO particles as an efficient antimicrobial preser-
`vative in cosmetic and dermopharmaceutical products, it was
`firstly aimed at discriminating the contribution of zinc cations gen-
`erated by ZnO particles from the overall antimicrobial activity, and
`secondly identifying the parameters which directly impact the dis-
`solution phenomenon and would enhance this mechanism. The
`study was performed on the five microbial strains used for Chal-
`lenge Tests for checking the safety of pharmaceutical and cosmetic
`products. Microbiological tests on solid agar plate and in broth
`culture were performed to evaluate the antimicrobial efficacy of
`both ZnO particles and Zn2+. This work was performed taking into
`consideration the dissolution of ZnO particles in aqueous media
`depending on the environmental conditions and on the physico-
`chemical characteristics of ZnO powders using three different ZnO
`grades.
`
`2. Materials and methods
`
`2.1. Materials
`
`The following ZnO powders of pharmaceutical grade were stud-
`ied: ZnO-1 from Rockwood Pigments (Beltsville, US); ZnO-2 from
`SILOX (Engis, Belgium); ZnO-3 from Zinc Corporation of America
`(Pittsburgh, US). Zinc gluconate (ZnG) was supplied by Seppic (Cas-
`tres, France).
`
`2.2. ZnO particles characterization studies
`
`The physicochemical properties of the three ZnO grades were
`investigated as previously reported [46]. The characteristics of
`the powders were assessed in a dry state by determining their
`specific area and the porosity by nitrogen adsorption measure-
`ments using a Tristar 3000 Micromeritics BET instrument. The
`specific area was determined by the Brunauer–Emmett–Teller
`(BET) multipoint method and the pore volume was analyzed by
`the Barrett–Joyner–Halenda (BJH) method. The crystal structures
`were established by X-ray diffraction measurements performed at
`the ‘Centre Henri Longchambon’ facility (University of Lyon) using
`a Bruker AXS D8 Advance X-ray diffractometer operating with the
`Cu K␣1 line at 1.54 ˚A wavelength. The crystallite size was esti-
`mated from the width at half height of the Bragg peaks using the
`Debye–Scherer equation. The apparent density was studied follow-
`ing an adapted protocol of the European Pharmacopeia. Size and
`shape of the elementary particles were studied by transmission
`electron microscopy (TEM) performed at the ‘Centre Technologique
`des Microstructures’ facility (University of Lyon) on a Philips CM120
`microscope operating at 80 kV acceleration. A dilute aqueous sus-
`pension (0.1%) was spread on Formvar/carbon grids and dried
`before observation.
`
`

`

`J. Pasquet et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 457 (2014) 263–274
`
`265
`
`for incubation in the dark (24 h for bacterial strains, 48 h for C.
`albicans and 72 h for A. brasiliensis) and counting the colonies. A
`‘positive control’ experiment consisted of inoculation of the micro-
`bial strains in pure zinc-free MH broth.
`Additional experiments on ZnO suspensions and soluble zinc
`species were conducted in water in place of MH broth in order
`to assess the influence of zinc solubilization by MH broth. These
`experiments were conducted according to the same method as for
`those in MH broth.
`
`2.4. ZnO dissolution experiments
`
`The dissolution of ZnO particles under different experimen-
`tal conditions was examined trough separate experiments, each
`performed
`in triplicate. ZnO suspensions were prepared and
`homogenized in hemolysis tubes of 5 mL. For each sample, ZnO sus-
`pensions were vortexed for 10 s and a 10 min centrifugation step
`at 5000 rpm was applied. The supernatant was collected, filtered
`␮m regenerated cellulose syringe-filter (Uptidisc®,
`through a 0.20
`Interchim, Montluc¸ on, France) and introduced in a polypropylene
`1 mL vial for analysis of zinc. Every chemical was of analytical
`grade and solutions/suspensions were prepared with water for
`
`HPLC (resistivity of 18 M(cid:4) cm).
`
`2.5. Zinc ions analysis
`
`Zinc content released from the dissolution of ZnO particles
`was quantified using a high-performance liquid chromatography
`(Waters) coupled to a detection using a post-column reaction with
`the 4-(2-pyridylazo)resorcinol reagent [47]. This method allows a
`chromatographic separation of ions that provides the selectivity
`of the analysis and a sensitive detection through the post-column
`formation of a colored complex [48]; it has already been applied
`to the analysis of Zn2+ ions in protein media [47]. Firstly, zinc
`ions in the supernatant were separated on a non-polar station-
`␮m-3.9
`150 mm, Waters)
`ary phase (column X Terra MSC18-5
`with an acid mobile phase composed of sodium octane sulfonate
`(0.2 mM), tartaric acid (25 mM) and acetonitrile (2%). The pH of the
`mobile phase was adjusted at 3.5 adding sodium hydroxide. Then,
`a post-column reaction took place at 50 ◦C with 4-(2-pyridylazo)
`resorcinol (PAR reagent, Sigma-Aldrich) in ammonia solution. Com-
`plexation of Zn2+ by the PAR reagent yielded colored complex
`species that were detected by means of their absorbance in the vis-
`ible domain. The detection of the complex PAR-Zn2+ was realized
`with a diode array detector at a detection wavelength of 505 nm
`corresponding to the maximum of the absorption spectra of the
`red–orange complex. Acquisition and data processing were done
`with the software Empower Photodiode Array (PDA, Waters). The
`quantitative analysis of Zn2+ concentration was performed with
`help of an external calibration with zinc gluconate solution in acidic
`conditions ensuring a total dissociation of the zinc salt as free zinc
`ions.
`
`×
`
`3. Results and discussion
`
`3.1. Antimicrobial activity of ZnO and Zn2+
`
`Preliminary experiments performed in agar medium revealed
`inhibition zones around ZnO impregnated discs (Fig. 1). Since
`ZnO particles were unable to diffuse out of the cellulose discs,
`antimicrobial species have necessarily been released from the ZnO
`impregnated discs to the agar medium. This indicated that ZnO par-
`ticles were able to exhibit an antimicrobial effect by diffusion of
`soluble species into the agar medium. This test gave a first qualita-
`tive estimate of the sensitivity of microbial strains to ZnO particles
`through the contribution of released Zn2+. E. coli and S. aureus were
`
`The behavior of the aggregates of ZnO particles was also
`evaluated in liquid media by measuring the isoelectric point by
`electrophoretic light scattering measurements using a Zetasizer
`Nano ZS (Malvern Instruments, UK). The sedimentation phe-
`nomenon was followed measuring the turbidity of the samples
`using a Carry 50 UV–vis absorption spectrophotometer (Varian).
`(cm−1) was deduced from the absorbance A(cid:3) mea-
`The turbidity
`sured at a wavelength of 380 nm in a cell of optical path l = 1 cm
`according to the following equation:
`= 2.303 A(cid:3)/l. Finally, the
`diameters of ZnO aggregates in water and in nutrient broth used
`in antimicrobial tests were measured by low-angle laser light scat-
`tering using a Malvern Mastersizer 2000 instrument. Agglomerate
`size distributions were calculated according to Mie theory using the
`refractive indices of water (1.33) and ZnO (2.008). The refractive
`index of the Mueller Hinton culture broth used for antimicrobial
`tests measured using a PAL-1 refractometer (Atago, France) was
`1.336. The full granulometric distribution was retained as a char-
`acteristic parameter.
`
`(cid:2)
`
`(cid:2)
`
`2.3. Evaluation of antimicrobial activity
`
`The microbiological experiments were carried out in tripli-
`cate on the five microbial strains used for Challenge Tests of the
`European 7.0 and US Pharmacopeias 35 supplied by the culture col-
`lection of the Institut Pasteur (France): Escherichia coli CIP® 53.126
`(equivalent strain ATCC® 8739); Staphylococcus aureus CIP® 4.83
`(ATCC® 6538); Pseudomonas aeruginosa CIP® 82.118 (ATCC® 9027);
`Candida albicans IP 48.72 (ATCC® 10231); Aspergillus brasiliensis
`IP 1431.83 (previously named as Aspergillus niger ATCC® 16404).
`Cell cultures and incubations were performed in a thermostated
`chamber in the dark.
`
`2.3.1. Preliminary experiments by the disc diffusion test
`Disc diffusion susceptibility tests on Mueller-Hinton (MH2)
`solid agar plate (BioMérieux, France) were performed. Sterile
`standard discs of cellulose of 6 mm diameter (BioMérieux, France)
`were impregnated with antimicrobial materials and deposited on
`a microbial culture in agar medium. This standard method has
`been used for solutions of ZnG and was adapted to the particu-
`late nature of the present materials as follows. The cellulose discs
`were impregnated with sterile aqueous suspensions of ZnO at 10,
`50 and 100 mg g−1 concentrations by soaking the discs into the sus-
`pensions for 5 s. The impregnated discs were placed onto the agar
`surface previously inoculated by spreading the microbial suspen-
`sion on the surface of the agar. After overnight incubation in the
`dark at 32.5 ◦C for the bacterial strains and 48 h at 22.5 ◦C for the
`fungal strains, zones of inhibition around each disc were observed.
`
`2.3.2. Measurements of antimicrobial activity in liquid broth
`The antimicrobial efficacy of either ZnO particles or soluble
`zinc species was evaluated in Mueller Hinton (MH) broth (AES
`Chemunex, France), a sterile medium intended for microorganisms
`growth. Solutions of soluble zinc species consisted of either solu-
`tions of ZnG in MH broth, or the supernatant of ZnO suspensions in
`MH broth obtained by centrifugation for 10 min at 5000 rpm. Sus-
`pensions of ZnO of various concentrations (from 1.25 to 80 mg g−1)
`were prepared by dispersing the ZnO powder in MH broth for 1 h
`in an ultrasonic bath. A preliminary study has validated the con-
`ditions of this dispersion step [46]; in particular it has shown that
`dispersion by ultrasounds did not alter the intrinsic antimicrobial
`properties of ZnO. Each sample was inoculated with the microbial
`strains and incubated at the appropriate temperature (32.5 ◦C for
`bacterial strains; 22.5 ◦C for fungal strains). Aliquots were collected
`at different incubation times (24 h for bacterial strains and 48 h
`for fungal strains), diluted in tryptone salt broth (AES Chemunex)
`before seeding the suitable dilution on TSA agar plate (BioMérieux)
`
`

`

`266
`
`J. Pasquet et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 457 (2014) 263–274
`
`Fig. 1. Disc diffusion tests for the evaluation of antimicrobial activity of ZnO-1 (10, 50 and 100 mg g−1) (top) and zinc gluconate (10, 50 and 100 mg g−1) (bottom). (A) E. coli;
`(B) S. aureus; (C) P. aeruginosa; (D) C. albicans; (E) A. brasiliensis.
`
`sensitive to soluble species since clear inhibition zones of the bacte-
`rial growth around the discs could be observed. These two bacteria
`were more sensitive than P. aeruginosa for which only a large pale
`green colored halo was observed. Similar results were obtained
`with the soluble ZnG, showing that the antimicrobial activities of
`the soluble zinc species were indeed at the origin of the inhibition
`zones. The disc diffusion test did not reveal any growth inhibition
`of the yeast C. albicans, neither by ZnO nor by ZnG. Conversely, the
`germination of the spores of A. brasiliensis was inhibited by ZnO and
`ZnG, the activity of ZnG being much higher. For the microbial strains
`on which an efficacy was proven with this test, a concentration-
`dependence has been noticed: the inhibition zones were larger for
`the highest concentrations of ZnG and ZnO. This latter result points
`out the impact of the dissolution phenomenon of ZnO particles into
`the culture broth medium.
`Quantitative assessment of antimicrobial activities was done
`by experiments in liquid broth medium and in pure water. Var-
`ious efficacy indexes were determined: the Minimum Inhibitory
`Concentration (MIC) and the Minimum Bactericidal Concentration
`(MBC) evaluated the efficacy on bacteria; an ‘inhibition rate’ was
`defined as the ratio of the population densities of the sample for
`80 mg g−1 of ZnO to that of the positive control measured 48 h after
`inoculation to quantify the activity on C. albicans; and a visual MIC
`estimated as the lowest concentration of ZnO that prevented the
`
`development of mycelium 48 h after inoculation was used to mea-
`sure the efficacy of ZnO on A. brasiliensis. Thanks to these values,
`ZnO grades were sorted with respect to their efficacy (Table 1):
`ZnO-1 exhibited the highest efficacy, whereas ZnO-3 was the less
`effective and ZnO-2 displayed an intermediate activity closer to
`ZnO-1 than ZnO-3 [46].
`The antimicrobial effects of the three types of Zn-containing
`samples were comparatively tested in broth medium: ZnO sus-
`pensions, their supernatants and ZnG solutions. ZnG was used as
`a reference for soluble zinc species although zinc is not present as
`totally free Zn2+ in the solution. The use of ZnG allowed the study
`of high concentrations of soluble zinc that fully dissociated zinc
`salts such as zinc chloride or zinc acetate would not reach because
`of their instantaneous precipitation in the neutral or basic media.
`Zinc ions of ZnG are partially complexed by gluconate and complex-
`ing species contained in the MH broth. High enough concentrations
`of free Zn2+ would require an acidic pH, but the slightly basic pH
`of the culture broth must be retained for an optimal microbial
`growth. The results reported in Fig. 2 for ZnO-1 show a reduction
`of the microbial populations in the presence of ZnO-1suspensions,
`ZnO-1 supernatant, or ZnG. The comparison between ZnO suspen-
`sions and supernatant revealed that the antibacterial activity was
`significantly less for the soluble species alone than for ZnO-1 sus-
`pensions. The difference between the two gave the contributions
`
`

`

`J. Pasquet et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 457 (2014) 263–274
`
`267
`
`Table 1
`Characteristics of the antimicrobial efficacy of ZnO particles and zinc gluconate on the five microorganisms.
`
`Microbial strain
`
`E. coli
`
`S. aureus
`
`P. aeruginosa
`
`C. albicans
`A. brasiliensis
`
`Raw material
`MIC (mg g−1)
`MBC (mg g−1)
`MIC (mg g−1)
`MBC (mg g−1)
`MIC (mg g−1)
`MBC (mg g−1)
`Inhibition rate (%)
`Visual MIC (mg g−1)
`
`ZnO-1
`
`ZnO-2
`
`ZnO-3
`
`Zinc gluconate
`
`1.2
`14.4
`1.8
`15.6
`23.0
`64.0
`33.6
`10
`
`2.5
`17.2
`3.0
`31.8
`34.0
`68.1
`35.9
`20
`
`12.8
`18.1
`46.8
`75.2
`57.0
`>80
`24.7
`80
`
`1.7
`8.4
`4.6
`>80
`33.6
`>80
`47.3
`<10
`
`of the mechanisms that required the presence of ZnO particles
`in situ. The antimicrobial activity by direct contact of ZnO particles
`to the cell walls was absent in the experiments with the super-
`natant. There might be ROS present in the supernatant because
`they have been generated before its separation by centrifugation
`and remained in the state of the stable hydrogen peroxide species
`(H2O2). However, in situ photo-generation of the most active ROS
`by ZnO particles is not operative in the supernatant. As a whole,
`the antimicrobial activity of the supernatant mainly comes from
`the soluble zinc species; a slight supplementary activity may arise
`from residual ROS. The contribution of ROS to the antimicrobial
`activity of ZnO is a complex topic that deserves a full paper to
`be correctly addressed. A specific study will be published soon on
`this subject. Beyond the suppression of the direct action by close
`contact, the removal of ZnO powder from the media stopped any
`further dissolution of soluble zinc species. This part of the study
`demonstrated that soluble Zn2+ released by partial dissolution of
`ZnO had a definite contribution to the antimicrobial activity of ZnO.
`The quantification of the contribution of the soluble zinc species
`is difficult because the shape of the plots of Log(CFU mL−1) with
`respect to [ZnO] (Fig. 2) are not similar for the full ZnO suspen-
`sions and their supernatants. A rough estimate was made from the
`slopes of the curves d(Log(CFU mL−1))/d[ZnO]. For E. coli, the con-
`tribution of soluble zinc species was approximately 15% of the full
`antimicrobial activity of ZnO; it was 71% for S. aureus; and it finally
`
`reached almost 100% for C. albicans (the soluble zinc species were
`responsible for the full activity of ZnO). There was a definite speci-
`ficity of the antimicrobial activity of the soluble zinc species with
`regards to the microbial strains. The behavior was more complex for
`P. aeruginosa: almost 100% of the antibacterial activity was caused
`by the soluble zinc species for [ZnO] < 10 mg g−1 and the contri-
`bution of soluble zinc species turned small for [ZnO] > 10 mg g−1,
`because the antibacterial activity of ZnO became high above this
`ZnO concentration whereas the activity of the soluble zinc species
`did not increase significantly.
`Zinc concentration has been measured
`in the supernatant
`separated by centrifugation, thus allowing the comparison of
`antimicrobial activities of the soluble zinc species released by ZnO
`particles in the MH broth and that of ZnG. Such comparison is pre-
`sented in Fig. 3. The antimicrobial activity of soluble zinc species
`and ZnG appeared of comparable magnitude for most strains except
`E. coli. A noticeable feature of the antimicrobial activity of solu-
`ble zinc species is the weak activity at high concentrations. There
`were two regimes (Fig. 3): the microbial populations decreased sig-
`nificantly with respect to the concentration of zinc for low Zn2+
`concentrations, and remained approximately at the same level
`whatever the concentration of Zn2+ above 1 mg g−1. ZnG had a
`higher antimicrobial activity against E. coli than a similar Zn2+ con-
`centration released from ZnO in the broth. Such an enhancement
`of antimicrobial activity by the gluconate suggests that E. coli is
`
`Fig. 2. Decrease of microbial populations with respect to zinc concentration for ZnO-1 suspension (red squares), its supernatant (blue diamonds), and zinc gluconate solution
`(green triangles). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
`
`

`

`268
`
`J. Pasquet et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 457 (2014) 263–274
`
`Fig. 3. Microbial populations as a function of zinc ions concentration as analyzed in the supernatant of ZnO suspensions (blue diamonds) and in zinc gluconate solutions in
`MH broth (green triangles). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
`
`also sensitive to the ZnG complex. On the contrary, the antimi-
`crobial activity of ZnG against S. aureus and P. aeruginosa was less
`than for the supernatant of ZnO suspensions. The type of bacterial
`membrane does not allow sorting antibacterial activities since the
`two gram negative strains E. coli and P. aeruginosa showed different
`sensitivities to ZnG and soluble zinc species from ZnO. The antimi-
`crobial activities of soluble zinc species appeared strain-specific.
`Conversely, the effect of ZnO-1 particles and ZnG are almost simi-
`lar for the bacterial strain E. coli; their MIC and MBC were of similar
`magnitude, showing that the contributions of the presence of ZnO
`particles in situ compensated the difference of activities between
`ZnG and the ZnO supernatants. This result proved that the combina-
`tion of the three antimicrobial mechanisms is of major importance
`to exert a significant effect on these microorganisms compared to
`a zinc salt.
`The antifungal activity against C. albicans was the same for the
`ZnO suspension and the supernatant. Such a remarkable behav-
`ior indicated that the antifungal activity of ZnO was almost fully
`caused by the soluble zinc species. The mechanism by which the
`microbial membrane was altered either upon contact with ZnO par-
`ticles, or by oxidation by ROS did not operate to a significant extent.
`This behavior is probably the result of a stronger resistance of the
`yeast membrane, compared to that of bacteria. Antifungal activity
`requires that antifungal agents enter the cells and disturb physio-
`logical events of vital importance inside them. ZnG appeared more
`effective against C. albicans than ZnO particles and soluble species
`from ZnO dissolution. Indeed, a significant reduction of the initial
`population was observed after 48 h of contact with ZnG compared
`to ZnO suspensions and supernatant for which yeast populations
`
`remained at the same concentration with respect to contact time
`after inoculation. These results showed that ZnG is more efficient
`than the supernatant of ZnO-1 suspensions, suggesting that higher
`zinc concentrations would exhibit stronger antimicrobial activity.
`Therefore a better antifungal activity should be reached if Zn2+
`could be generated from ZnO particles. The fungistatic behavior in
`MH broth is different of the behavior in the agar medium of the disc
`diffusion test where no inhibition of yeast growth was observed.
`Such a difference probably comes from a lesser Zn2+ solubilization
`capacity or from slower Zn2+ solubilization kinetics of agar than
`MH broth that caused a lesser release of soluble zinc species and a
`lower antimicrobial activity.
`Finally, ZnO particles, soluble species and ZnG inhibited the ger-
`mination of spores of the mold strain A. brasiliensis on agar plate and
`in liquid broth. ZnG was the most efficient between 10 mg g−1 and
`80 mg g−1. No development of mycelium was observed whereas
`germination of spores was observed at [ZnO-1] < 10 mg g−1 and at
`[ZnO-1] < 20 mg g−1 for their supernatant. These results confirmed
`that Zn2+ exhibited an activity on the spore germination, in accor-
`dance with the conclusions reached by Miller and McCallan [23].
`The contribution of Zn2+ to the global antimicrobial activity of
`ZnO powders was definitely significant, especially against fungal
`strains. The direct contact between ZnO particles and microbial
`cells claimed by many authors as the main mechanism operat-
`ing on bacteria is indeed probably the predominant contribution
`to the antibacterial activity. Owing to the combination of three
`antimicrobial mechanisms, the spectrum of antimicrobial activ-
`ity is broad and ZnO appears as a suitable preservative of topical
`products compared to ZnG. Since the antimicrobial activity of Zn2+
`
`

`

`J. Pasquet et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 457 (2014) 263–274
`
`269
`
`increased with respect to their concentration, the dissolution of
`ZnO particles should be enhanced again so as to reinforce the overall
`antimicrobial activity of ZnO.
`
`3.2. Dissolution of ZnO
`
`Our previous study [46] revealed that the antimicrobial efficacy
`of ZnO particles was dependent on the physicochemical parame-
`ters of these powders and the present study shows an important
`contribution of the released zinc ions to the antimicrobial activ-
`ity. The present section reports studies aimed at identifying the
`most relevant parameters that influenced the dissolution process of
`ZnO in aqueous suspensions. Then, in order to optimize and main-
`tain this mechanism over time, the influence of the environmental
`conditions was investigated.
`The dissolution process was depending on whether the solvent
`was pure water or MH broth. The dissolution process of ZnO in these
`two media depended on time, ZnO concentration, and type of ZnO
`powder. The aqueous phases of ZnO suspensions prepared between
`1.25 and 80 mg g−1 in distilled water and MH broth were analyzed
`for their concentration of Zn2+ in order to assess the dissolution of
`ZnO that took place during the microbiological tests.
`The released Zn2+ concentrations immediately after immersion
`of the ZnO-1 powder in the liquid medium and after 24 h dissolution
`time under stirring were quite surprising (Fig. 4). The concentration
`of Zn2+ was strongly dependent