Preprint Title Silver nanoparticles nucleated in NaOH treated halloysite : a potential antimicrobial material

16 Despite all recent advances in medical treatments, infectious diseases remain dangerous. This 17 scenario has led to intense scientific research on materials with antimicrobial properties. Silver 18 nanoparticles (Ag-NPs) are a well established solution in this area. The present work studied the 19 nucleation of silver in halloysite substrates (HNT) modified by a NaOH chemical treatment. The 20 resulting stabilized Ag-NPs were characterized by X-ray diffraction (XRD), transmission electron 21 microscopy (TEM) and energy dispersive x-ray spectroscopy (EDS). The nucleation was charac22 terized by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Ag-NPs 23 antimicrobial properties were investigated against E. coli and S. aureus. The potential of Ag-NPs 24


Introduction
Deaths from bacterial infections have been significantly reduced with the emergence of antibiotics, 34 but some bacterial diseases still remain amongst the most dangerous to human life. In addition, 35 as antibiotics become more popular, bacteria are evolving to become resistant -a phenomenon 36 known as "antimicrobial resistance", and listed by the World Health Organization as one of the top 37 ten threats to public health [1]. Research on antimicrobial nanomaterials is one of the most promis-38 ing antibiotic free alternative for many applications; among them metallic nanoparticles, which 39 could be potent inorganic antimicrobial agents in due to ion-releasing properties and the capability 40 to rupturing the cellular membrane and disrupting internal cellular components, such as the DNA 41 [2 -6]. 42 Silver nanoparticles (Ag-NPs), in particular, are specially prominent for their antimicrobial prop-43 erties, being one of the most studied inorganic antimicrobial agents [7][8][9]. Early studies sugest 44 that Ag-NPs displays strong growth-inhibitory properties against common microorganisms, and 45 that they may be used as an alternative way to overcome bacterial resistance to antibiotics [10,11]. 46 However, large scale industrial usage of Ag-NPs is still limited, because most synthesis techniques 47 used in lab are unsuitable to commercial use -the main issues being high procedural complexity 48 and elevated costs, which jeopardize scalability efforts fundamental to industrial mass-production 49 of Ag-NPs. 50 there is even evidences suggesting that silver nucleation in HNTs external surface tends to occur 66 in crystalline defects, where aluminol is exposed [22]. Synthesizing Ag-NPs supported by HNT 67 has the advantage of improving stability, thus enhancing antimicrobial properties of the material 68 [21][22][23]26,27]. 69 In this work we treated the HNT surface with NaOH to improve Ag-NP nucleation into the clay 70 substrate. The resulting nanocomposite was evaluated in terms of chemical composition, mor-71 phology and antimicrobial properties, while the nucleation process was characterized in terms of 72 thermal behaviour and structural changes. We also investigated the Ag-NPs potential for industrial 73 application by dispersing them into low density polyethylene (LDPE), providing a way to evaluate 74 chemical compatibility between matrix and additive by testing a dodecanethiol (DIO) coating to 75 improve Ag-NPs dispersion into LDPE. The resulting composites were then evaluated in terms of 76 surface antimicrobial activity.

78
Halloysite (>99%), silver nitrate (AgNO 3( ) , >99%) and dodecanethiol were obtained from Sigma 79 Aldrich; sodium hydroxide (NaOH ( ) , >99%) was purchased from Alpha Quimica; low Density   To prepare the substrates for silver nucleation, halloysite was treated in a NaOH(aq) bath. Three 89 HNT suspensions were prepared sonicating 100 g of HNT powder into 400 mL of deionized water 90 (DI), for 20 min, at 20 kHz. One suspension, labelled HNT-0, was not subjected to the NaOH(aq) 91 treatment, and was used as a control sample. The two remaining suspensions received 200 g of 92 days -resulting in the samples HNT-4 and HNT-8. After the resting periods, the three suspensions 94 were filtered and extensively washed with deionized water to remove residual NaOH and then dried 95 naturally. To characterize the prepared substrates, samples HNT-0, HNT-4 and HNT-8 were sub-96 jected to TEM, while sample HNT-8 was also subjected to XRD (XRD was also performed for 97 HNT-0 and HNT-4 but omitted from this report because it did not show any significant difference 98 from HNT-8).

99
Silver nanoparticle synthesis 100 To synthesize silver nanoparticles, 10 g of HNT (from the three samples previously prepared:  Ag-NPs nucleation analysis 107 In order to study the nucleation of silver nanoparticles, 1 g of Ag/HNT-8 were submited to TGA 108 and DSC analysis, from room temperature up to 600 • C, at constant heating of 20 • C/min.

109
In order to observe the phases identified in the DSC/TGA measurements, four 10 g samples of 110 HNT-8 were loaded with AgNO 3 using the process described in the section "silver nanoparticle    the plastic surface was quantified by comparing the number of viable colonies from the suspensions 160 that were exposed to treated LDPE with the number of viable colonies from the suspensions that 161 were exposed to the control polymer.   The results of HNT's loaded with silver nitrate TGA and DSC analysis are presented in figure 5.

222
Since the sub-products of silver nitrate reduction are oxygen and nitrogen dioxide (both gaseous 223 substances) from reaction: 2AgNO 3 → 2Ag ( ) +O 2 +2NO 2( ) [35], we expect to see mass reduction 224 11 as AgNP nucleates; and since nucleation is an endothermic process, we expect to see endothermic 225 peaks as well. presented in figure 6 and also correlated to the data in table 2.

288
Treating halloysite with NaOH is an efficient way to enhance silver nucleation into the clay's sur-289 face. Substrates treated with this technique were able to anchor the formation of nanosized silver 290 particles, that displayed strong antimicrobial effect against E. coli and S. aureus, as was expected.

291
The resulting antimicrobial effect for treated substrates was also higher than for the untreated hal-292 loysite. TGA and DSC analysis indicated that most reduction of metallic silver from AgNO 3 into 293 halloysite substrates occurs at temperatures above 420 C, while TEM images showed that nucle-294 ation at this temperature does not result in uncontrolled grain growth into microscopic scale. The

295
Ag-NPs produced in this work were also tested for practical applications by dispersing them into 296 antimicrobial effect of LDPE doped with "natural" Ag-NPs was disappointing, it was possible to 298 greatly improve the material's antimicrobial properties by coating the nanoparticles with dode-299 canethiol -a non-polar surfactant agent, highly compatible with LDPE. This shows that the Ag-

300
NPs synthetized is this work are viable as antimicrobial additives for plastics -probably the most 301 important material for large scale industrial applications -while also highlighting the importance 302 of controlling chemical affinity between nanoparticle's surface and matrix, a fact often overlooked 303 in nanoparticle's literature.

304
The synthesis process presented in this work uses only common reactants, thermal reduction 305 and the inexpensive halloysite clay as substrate, being thus a low cost solution for antimicrobial 306 nanoparticle production, that is also scalable to industrial production.