Subscriber access provided by CARLETON UNIVERSITY Article Synergic Effect of Nanolignin and Metal Oxide Nanoparticles into Poly (L-lactide) Bionanocomposites: Material Properties, Antioxidant Activity and Antibacterial Performance Erlantz Lizundia, Ilaria Armentano, Francesca Luzi, Federico Bertoglio, Elisa Restivo, Livia Visai, Luigi Torre, and Debora Puglia ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.0c00637 • Publication Date (Web): 11 Jul 2020 Downloaded from pubs.acs.org on July 12, 2020 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. 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However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties. Page 1 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Applied Bio Materials Synergic Effect of Nanolignin and Metal Oxide Nanoparticles into Poly (L-lactide) Bionanocomposites: Material Properties, Antioxidant Activity and Antibacterial Performance Erlantz Lizundia†,‖,°, Ilaria Armentano‡, Francesca Luzi§, Federico Bertoglio#1, Elisa Restivo#,&, Livia Visai#,&, Luigi Torre§, Debora Puglia§,+, * †Department of Graphic Design and Engineering Projects. Faculty of Engineering in Bilbao. University of the Basque Country (UPV/EHU), Bilbao 48013, Spain. ‖BCMaterials, Basque Centre for Materials, Applications and Nanostructures, UPV/EHU Science Park, 48940 Leioa, Spain °Laboratory for Multifunctional Materials, Department of Materials, ETH Zürich. VladimirPrelog-Weg 5, 8093 Zürich, Switzerland. ‡Department of Economics, Engineering, Society and Business Organization (DEIM), University of Tuscia, Largo dell’Università snc, 01100 Viterbo, Italy §University of Perugia, Civil and Environmental Engineering Department, UdR INSTM, Strada di Pentima 4, 05100 Terni, Italy. #Molecular Medicine Department, UdR INSTM, University of Pavia, Viale Taramelli 3/B – 27100 Pavia – Italy & Center for Health Technologies University of Pavia, 27100 Pavia – Italy &Department of Occupational Medicine, Toxicology and Environmental Risks, Istituti Clinici Scientifici Maugeri S.p.A Società Benefit, IRCCS, Via S. Boezio, 28, 27100 Pavia, Italy 1 ACS Paragon Plus Environment ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1Present Page 2 of 32 address: Technische Universität Braunschweig, Institute for Biochemistry, Biotechnology and Bioinformatics, Department of Biotechnology, 38106 Braunschweig, Germany. *Corresponding author: Debora Puglia, debora.puglia@unipg.it 2 ACS Paragon Plus Environment Page 3 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Applied Bio Materials ABSTRACT Binary and ternary poly (L-lactide) (PLLA) based nanocomposites, containing nanolignin (1% wt.) and different metal oxide nanoparticles (0.5% wt., Ag2O, TiO2, WO3, Fe2O3 and ZnFe2O4) were realized by solvent casting and their morphological, thermal, surface, optical, antioxidant and antimicrobial characterization was performed. The presence of metal oxide nanoparticles at the selected weight concentration affects the surface microstructure of the PLLA polymer and this outcome is particle-type dependent, accordingly to the shape, morphology and chemical properties of the selected NPs. Analogously, wettability of PLLA based nanocomposites was slightly modified by the presence of hydrophobic lignin nanoparticles and different shaped metal oxides. Results of DSC and XRD tests confirmed that nanoparticles addition confined the mobility of the amorphous phase, increasing at the same time the formation of more numerous but less perfect PLLA crystals. Interestingly, antioxidant activity was also obtained in ternary based nanocomposites, where a synergic effect of lignin and metal oxide nanoparticles was obtained. Antibacterial tests showed manifest activity of TiO2 and Ag2O nanoparticles containing PLLA films, and the time dependence was more evident for S.aureus than for E.coli. Lignin nanoparticles are able to provide protection against UV light while still allowing visible light to pass and even surpassing the UV-protection capacity provided by many inorganic nanoparticles. This make them an attractive renewable additive for the realization of PLLA/metal oxides nanocomposites for the fields of food, drug packaging and biomedical industry, where antibacterial and antioxidant properties are required. Keywords: polylactic acid, nanocomposite, metal oxide, lignin, antioxidant, antibacterial 3 ACS Paragon Plus Environment ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 4 of 32 INTRODUCTION Polylactic acid (PLA) is a high potential polymeric material, owing to its exceptional mechanical, transparency, biocompatibility and biodegradability properties. However, some limitations, such as narrow processing window due to the high melting temperature and poor thermal stability, limited protection towards many biological, chemical, and physical environmental conditions (physical stress, light, microorganisms, oxygen, and moisture) restrict its complete use in some industrial applications, as in the case of food packaging sector.1 In recent times, lignin, as the most abundant natural material after cellulose, has obtained much interest, due to its multifunctionalities: reinforcement effect, UV protection, antimicrobial and antioxidation properties.2 In addition, its use in polylactic acid is considered particularly attractive, being both materials biobased and biodegradable. There are many examples in the literature of PLA/(micro)lignin systems, where hydrolytic degradation, antioxidant activity, and mechanical properties of PLA/lignin systems has been analyzed, showing, in general, large-sized immiscible areas and impairment of mechanical characteristics, due to limited compatibility of lignin with polylactic acid.3 To overcome these limits, different facile and practical routes have been proposed, such as reactive compatibilization,4 copolymer addition,5 filler acetylation.6 Quite recently, the introduction of the lignin at the nanoscale was also verified to enhance the overall performance of PLA matrix,7 some studies already exist offering information about the effect of the inclusion of lignin nanoparticles (LNPs) into PLA by different methods. Melt blending, masterbatch procedure, solvent casting and Pickering emulsion method were considered as suitable methods to incorporate LNPs,8,9 and the authors evidenced how these nanoparticles could effectively work in improving limited or absent properties in PLA, such as flame retardancy, antioxidant activity and UV shielding. Regarding these last specific stuffs, renewable and biodegradable UV-shielding thin systems are demanded to meet the pressing sustainable environmental requests and lignin, as a natural antioxidant and UV blocker, has gained significant consideration.10 In parallel, UV blocking additives based on inorganic metal oxide nanoparticles (TiO2, ZnO, Fe2O3),11,12 have been widely used for these purposes, as their incorporation into different supporting matrices helps to reduce the yellowing of their hosting matrix during UV weathering. Inorganic metal oxide nanoparticles are able to absorb UV light but not visible light, making them especially relevant for transparent packaging development,13 they also demonstrated to have appreciable antioxidant activity.14 4 ACS Paragon Plus Environment Page 5 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Applied Bio Materials Antimicrobial effect of the same nanoparticles has been also found, as reported in the case of LNPs,15 and nanosized metal oxides.16 Furthermore lignin, as a natural three-dimensional network, has shown ability to create uniform composite structure with inorganic nanoparticles,17 so organic–inorganic hybrid materials have similarly received wide-ranging attention. The production of lignin/inorganic nano-systems represents a new methodology for high-value reuse of this material, since the hybridization potentially combines the benefits of all constituents to acquire matching synergic properties. There are few works regarding the realization of these systems, where emphasis is put primarily on the chance of using lignin as a precursor for metal oxide synthesis to be further incorporated in polymeric matrices,18,19 while, to the best of authors’ knowledge, limited researches are available dealing with the incorporation of lignin nanoparticles,20 in presence of other inorganic metal oxide nanoparticles, with the main aim of synergically combine the two nanofillers to enhance UV, antioxidant and antibacterial protection of biodegradable polymer matrix. On the basis of previous results,12 where the authors developed efficient and cost-effective transparent poly (L-lactide) PLLA films containing different nanofillers/particles to be used in packaging industry, here we extended the research to the preparation of binary and ternary PLLA based nanocomposites, realized by using both lignin nanoparticles (fixed content at 1% wt.) and different organic nanofillers (content of 0.5% wt. for Ag2O, TiO2, WO3, Fe2O3 and ZnFe2O4). Although here we use commercial metal oxide nanoparticles, the abundant aliphatic hydroxyl groups of lignin provide reducing surfaces for metallic ions into metal oxide nanofillers, potentially reducing the need of environmentally toxic reagents needed during nanoparticle synthesis. Here we aim to explain how the introduction of organic NPs affects the morphological, thermal and wettability behavior of PLLA mixed with metal oxides. In the meanwhile, functional properties, such as antibacterial and antioxidant response of binary and ternary systems, were also investigated. EXPERIMENTAL Materials Polylactic acid (PLLA) with a polydispersity index of 1.27 and molecular weight (Mw) of 120.000 g mol−1 was obtained by Purac Biochem. Titanium (IV) oxide anatase phase (TiO2) (<25 nm, 637254-50G), tungsten (VI) oxide (WO3) (<100 nm, 550086-25G), iron (III) oxide (Fe2O3) (<50 nm, 544884-5G) and zinc iron oxide (ZnFe2O4) (<100 nm, 633844-10G) were supplied from Sigma. Silver oxide nanoparticles (Ag2O), P203 were purchased from Cima NanoTech (Corporate 5 ACS Paragon Plus Environment ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 6 of 32 Headquarters Saint Paul, MN, USA). Chloroform was used as a solvent to solution-cast PLLA based films. Lignin nanoparticles (LNP) were extracted from alkali lignin (purchased from Sigma Aldrich®) by an hydrochloric acid process based on the methodology previously published.21 Specifically, the treatment is based on the stirring of 4% (m/v) of lignin in ethylene glycol suspension for 2 h at 35 °C. Subsequently, hydrochloric acid (8 mL, 0.25 M) was slightly mixed to the solution at a rate of 3 − 4 drops·min-1, after that the suspension was stirred again for other 2 h. The material was filtered and then dialyzed in deionized water as long as the solution has not reached the neutral pH. Nanoparticles Characterization Metal oxide and lignin nanoparticles were morphological and chemically characterized. The morphology of the nanoparticles was analyzed by Transmission Electron Microscopy (TEM). A small drop of nanoparticle water dispersion was deposited on carbon coated copper grid and analysed with a JEOL 1200EXII electron microscope (JEOL, Peabody, MA USA). Micrographs were captured using a SIS VELETA CCD camera (Olympus, Muenster, Germany) equipped with iTEM software. Fourier Transform Infrared spectroscopy analysis (FT-IR) was done at room temperature (RT) in transmission mode on KBr disks by using JASCO FT-IR 615 spectrometer at a wavenumber range 4000-400 cm-1. Nanocomposite fabrication Binary and ternary nanocomposites were prepared by solution-cast method in chloroform. PLLA (2.5 g) was dispersed in 25 mL of chloroform (CHCl3) and mixed at RT for 4 h. For the realization of PLLA binary films, a specific amount of LNPs (1% wt.) or metal oxide (MO) nanoparticles (0.5 % wt.) was dispersed in CHCl3 by using a tip sonication in an ice bath (VIBRA CELL Sonics mod. VC 750, USA) for 2 min at 30% of amplitude (nanoparticles/CHCl3 ratio was selected at 1% wt/v). Polymeric solution and NP suspensions (with exception of Fe2O3) were mixed at RT under magnetic stirrer for 1 h and then casted. In the case of Fe2O3 based films, polymer and metal oxide dispersions were mixed by using tip sonication for 2 min at 30 % of amplitude. For the preparation of PLLA ternary nanocomposites, LNPs and metal oxide NPs suspension were previously mixed by using tip sonication (2 min at 30 % of amplitude) and added to PLLA 6 ACS Paragon Plus Environment Page 7 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Applied Bio Materials solution, the combination of LNPs and metal oxide NPs was performed following the same procedure reported for the PLLA binary based films. After complete polymer dissolution, the polymeric solution was then cast on glass Petri dish (diameter 15 cm) coated with Teflon® substrate and dried at RT and in air for 24 h. The films (thickness of ca. 250 μm) were removed from the Petri dish after drying. The films were positioned in a vacuum oven for 1 week at 40 °C in order to remove the chloroform still present in the polymeric matrix. The realized formulations are reported in Table 1. Table 1: Formulations for PLLA binary and ternary nanocomposites Material PLLA (wt. %) LNP (wt. %) MO (wt.%) PLLA 100 PLLA/1LNP 99 1 PLLA/0.5ZnFe2O4 99.5 0.5 PLLA/0.5Fe2O3 99.5 0.5 PLLA/0.5WO3 99.5 0.5 PLLA/0.5TiO2 99.5 0.5 PLLA/0.5Ag2O 99.5 0.5 PLLA/1LNP/0.5ZnFe2O4 98.5 1 0.5 PLLA/1LNP/0.5Fe2O3 98.5 1 0.5 PLLA/1LNP/0.5WO3 98.5 1 0.5 PLLA/1LNP/0.5TiO2 98.5 1 0.5 PLLA/1LNP/0.5Ag2O 98.5 1 0.5 Nanocomposite Characterization Surface microstructure of the PLLA, binary and ternary films was analysed by field emission scanning electron microscopy (FESEM, Supra 25-Zeiss, Germany). UV-Visible (UV-Vis) spectroscopy analysis in transmittance mode were performed with a JASCO V-670 instrument in the 200-900 nm range using a film holder. Thermal analysis of PLLA films was performed by using thermogravimetric measurement (TGA, Seiko Exstar 6000) and carried out as follows: 10 mg weight samples, nitrogen flow (250 mL/min), temperature range from 30 to 900 °C, 10°C/min heating rate. The thermal degradation temperatures were mainly evaluated and compared. 7 ACS Paragon Plus Environment ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 8 of 32 X-ray powder diffraction (XRD) patterns were obtained byusing a PANalytical's X'Pert PRO MRD powder diffractometer in reflection mode using Cu Kα radiation and operating at 45 kV and 30 mA Thermal transitions of PLLA films were determined with a Mettler Toledo DSC 822e calorimeter under nitrogen atmosphere (50 mL·min-1). Samples having 8 ± 1 mg were sealed in an aluminum pan and applying an heating scan from -20 to 220 ºC at 10 ºC·min-1. The crystalline fraction Xc (%) of PLLA matrix was calculated as: X c (%)  H f  H cc H f ·Wm 0 ·100 (1) where ΔHf and ΔHc represent the fusion and cold crystallization enthalpies of samples, respectively and Wm is the PLLA weight fraction in the sample. ΔHf0 = 106 J/g was considered as the melting heat of an infinitely thick PLLA crystal.22 The surface characteristics of PLLA based films were determined by static contact angle measurements, by using FTA1000 Analyser (USA) with the sessile drop method in air with HPLCgrade water. The presence of the different nanoparticles and the surface characteristics of the systems on the wettability properties were evaluated. Antioxidant properties Antioxidant activity of neat PLLA and PLLA binary and ternary systems was estimated by using a spectroscopic method,23 In details, PLLA films (0.1 g) were cut into small pieces, added in 2 mL of methanol and maintained for 24 h at RT. Methanol solution (1 mL) was added to 1 mL of DPPH in methanol (50 mg L-1). Methanol solution and extract were maintained at RT in the dark for 60 min. The absorbance was analyzed at 517 nm using a UV spectrometer (Perkin Elmer Lambda 35). The DPPH solution extracted from neat PLLA film was utilized as control. DPPH radical scavenging activity (RSA) was evaluated according to the Eq.2: RSA(%)  AControl  ASample AControl * 100 (2) where Asample is the absorbance of different samples and Acontrol is the absorbance of the control. 8 ACS Paragon Plus Environment Page 9 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Applied Bio Materials Bacterial strains, culture conditions and antibacterial assay The microorganisms used in this study were Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922), kindly supplied by R. Migliavacca (Department of Clinical Surgical, Diagnostic and Pediatric Sciences, University of Pavia, Italy). Both strains were cultured in their appropriate medium: S. aureus in Brain Heart Infusion (BHI) (Difco, Detroit, MI, USA), and E. coli in Luria Bertani Broth (LB) (Difco). They were grown at 37 °C, overnight, under aerobic conditions using a shaker incubator (New Brunswick Scientific Co., Edison, NJ, USA). The bacterial growths were monitored by checking the optical density at 600 nm until reaching a density of 1x1010 cells·mL-1, determined with a standard curve relating OD600 to cell number.24 Before carrying out the antibacterial tests, all types of binary and ternary nanocomposite films were sterilized with 70% Et-OH for 10 min, and successively washed with sterile dH2O. Later, 200 L (5x103) of bacterial suspensions were incubated on films for 6 and 24 h at 37 °C. After the desired incubation time , the viability test was performed through the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; Sigma-Aldrich®, USA) assay. The test was performed on the planktonic cell cultures removed after being in contact with the binary and ternary nanocomposite films. The same aliquot of bacteria was inoculated in Tissue Culture Plate (TCP). Results for binary nanocomposites films were firstly normalized to TCP and then to plain PLLA films set as 100%. Results for ternary nanocomposites films were firstly normalized to TCP, then to plain PLLA films and finally to PLLA_LNPs set as 100%. Furthermore, the data for both binary and ternary nanocomposite films were also presented normalized to TCP. The experiments were performed in duplicate and repeated 3 times. Finally, the results obtained were statistically analyzed using GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA). A Student’s unpaired, two-sided t-test and one-way variance analysis (ANOVA) were performed.Eventually, multiple comparisons were analyzed with Bonferroni post hoc (significance level of p ≤ 0.05). RESULTS AND DISCUSSION Nanoparticle Characterization Particle shape, size and morphology of the selected nanoparticles were investigated by TEM analysis. Figures 1 (a-e) show images of the as prepared nanoparticles selected for the binary and ternary nanocomposites, at different resolution, while in Figure 1f the TEM image of lignin nanoparticles is included. ZnFe2O4 and Fe2O3 nanoparticles have a hexagonal shape with dimensions ranging, respectively, from 20 to 150 nm and 10 to 100 nm, with a broad distribution in dimension. Spheroid shape was observed in WO3 nanoparticles, with a narrow size dimension in 9 ACS Paragon Plus Environment Page 10 of 32 ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 diameter (20-40 nm). Elongated particles are visible in TiO2 sample, with reduced dimension respect to other systems. Ag2O nanoparticles dimension is ranging around 20-80 nm, a nanostructured random morphology with particle agglomeration is visible (Figure 1c). Figure 1. TEM images of metal oxide (a-e) and lignin (f) nanoparticles Lignin NPs showed diameters ranging 50 ± 20 nm.21 Nanoparticles with different morphology and dimension were selected. All images highlight the absence of precursor residual material from synthesis phase, with exception of Ag2O, that showed the presence of surface capping agent used during the production process.25 Chemical nature of the nanoparticles was confirmed by infrared spectroscopy (Figure S1). All spectra show the typical infrared signals of the inorganic nanoparticles in the low wavenumber regions. ZnFe2O4 shows two broad adsorption peaks in the range 380-600 cm-1, that is a common characteristic of spinel ferrite NPs. In the case of Fe2O3, the absorption peaks at 550, 580, 630 and 680 cm-1 are assigned to the stretching vibration mode of Fe-O bonds.26 Tungsten oxide NPs show the vibration of the W-OH and W=O at the wavenumbers of about 848 and 914 cm-1 respectively,27 while TiO2 NPs show a broad absorption peak at 483 cm-1, due to the Ti-O stretching vibration.28 The FTIR spectrum of Ag2O NPs shows abroad band at 3253 cm-1, due to the Ag-O-H stretching, while another peak at 1096 cm-1 is induced by O-Ag-O vibration of Ag2O crystal. The 10 ACS Paragon Plus Environment Page 11 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Applied Bio Materials adsorption bands at 1450 and 1562 are attributed to the bending vibration of –OH (due to the presence of moisture), while the two sharp bands with low intensity at 841 and 663 cm-1 are attributed to the stretching vibration of Ag-O-Ag and Ag-O, respectively.29,30 Nanocomposite Characterization Surface microstructure The cast samples were flexible, smooth and free standing, with different color according to the initial color of selected inorganic NPs, brown in the case of Fe2O3 and ZnFe2O4 NPs, green/blue in the case of WO3 and AgO2 NPs, white in the case of TiO2 NPs binary films (see visual images in Figure 2-top). Figure 2 shows FESEM surface micrographs of binary PLLA films compared at the same resolution with the corresponding film in presence of lignin nanoparticles. The presence of metal oxide nanoparticles at the selected weight concentration (0.5 wt.%) affects the surface microstructure of the PLLA polymer and this effect is particle dependent, accordingly to the shape, morphology and chemical properties of the selected NPs. The images depict that the pristine PLLA film has an even, compacted and smooth surface with evidence of parallel lines, due to the mold surface morphologies, while the nanocomposite films consist of rougher surface morphology. The processing conditions and the nanoparticle presence impact the surface morphology of PLLA and its nanocomposites. Additionally, the solvent effect was clearly evident, since final morphological surface aspect was affected by the different solubility of PLLA in chloroform when mixed with oxide nanoparticles.31 As already reported,32 aggregation of several nanoparticles, evidenced in case of ZnO and TiO2 containing PE films, gave surface morphologies similar to the ones observed here for TiO2 and ZnFe2O4. In particular, the incorporation of 0.5 wt.% of Ag2O changed the PLLA smooth surface in discrete micro-phase aggregation (diameter more than 10 m). Interestingly, it was observed that the addition of LNPs affects the surface properties of the binary nanocomposites. All ternary nanocomposites show a smoother surface, even if the binary corresponding materials showed a quite rough and controlled microstructure. This effect is particular marked in silver based films and also in the case of ZnFe2O4 NPs. Hence the different morphologies obtained in the ternary samples could be ascribed to a different interfacial adhesion between PLLA polymer and metal oxide nanoparticles in presence of lignin NPs. 11 ACS Paragon Plus Environment ACS Applied Bio Materials PLLA P/1LNP P/0.5Fe2O3 P/0.5znFe2O4 P/1LNP/0.5ZznFe2O4 P/0.5TiO2 P/1LNP/0.5TiO2 P/0.5WO3 P/1LNP/0.5WO3 P/0.5Ag2O 2 m P/1LNP/0.5Fe2O3 P/1LNP/0.5Ag2O 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 12 of 32 Figure 2. Visual images (1=PLLA; 2=PLLA/1LNP; 3=PLLA/0.5TiO2; 4=PLLA/1LNP/0.5TiO2; 5= PLLA/0.5Ag2O; 6=PLLA/1LNP/0.5Ag2O; 7=PLLA/0.5WO3; 8=PLLA/1LNP/0.5WO3; 9 = PLLA/0.5Fe2O3; 10=PLLA/1LNP/0.5Fe2O3; 11=PLLA/0.5ZnFe2O4; 12=PLLA/1LNP/0.5ZnFe2O4) and FESEM images depicting the surface microstructure of PLLA ternary films compared with the corresponding PLLA binary films 12 ACS Paragon Plus Environment Page 13 of 32 Furthermore, we can underline that surface microstructure analysis of both binary and ternary films evidenced no physical defects, like scratches, micro-cracks or pin holes, due to solvent evaporation, which suggests successful preparation of the nanocomposites by using the solvent casting method. UV-Vis Spectroscopy Thanks to their electronic property including a filled valence band and an empty conduction band, many semiconductor nanoparticles such as zinc oxide, titanium dioxide or iron (III) oxide present a strong UV-absorption tendency.33 Such inorganic nanoparticles can thus be used as UVblocking additives to develop films which protect against UV light, which is especially interesting for food packaging applications.12 Accordingly, nanocomposites were characterized by UV-Vis spectroscopy measurements in transmittance mode. Figure 3 reports the transmittance mode UVVis spectra in the 200-800 nm range (see Table S1 for the amount of transmitted light (%) in the UV region (at λ= 360 nm) and visible region (540–560 nm). Neat PLLA film shows an optical transparency in the 540 to 560 nm range of 40.2%. Overall, upon nanoparticle addition, such transparency markedly decreases up to a minimum values of 0.6% for the PLLA containing 0.5 wt.% TiO2 and LNPs. It is noteworthy the decreased transmittance in the UV region (λ< 400 nm) provided by the LNPs, which are able to block almost the 100 % of UV light while keeping the optical transparency of the film at 7.5%. 60 50 Transmittance (%) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Applied Bio Materials PLLA PLLA/1LNP PLLA/0.5TiO2 PLLA/0.5WO3 PLLA/1LNP/0.5TiO2 PLLA/1LNP/0.5Fe2 O3 PLLA/0.5Ag2O PLLA/0.5ZnFe2O4 PLLA/1LNP/0.5Ag2O PLLA/1LNP/0.5ZnFe2O4 PLLA/1LNP/0.5WO3 PLLA/0.5Fe2 O3 40 30 20 10 0 200 300 400 500 600 700 800 Wavelength (nm) Figure 3. UV−Vis transmittance spectra of PLLA binary and ternary films 13 ACS Paragon Plus Environment Page 14 of 32 ACS Applied Bio Materials Among inorganic nanoparticles, WO3 shows the better performance for packaging applications, where as low as possible transmittances at wavelengths below 400 nm and optically transparent films are required.34 Overall, these results denote that the capacity of lignin nanoparticles to provide protection against UV light while still allowing visible light (λ> 400 nm) to pass through matches and even surpasses the UV-protection capacity provided by many inorganic nanoparticles, making them an attractive renewable additive to develop materials for food packaging. Interestingly, the UV-screening character of fabricated films may protect the PLLA hosting matrix from photodegradation by absorption of UV radiation and its re-emission in the visible region,35 providing longer-lasting materials. TGA analysis The thermal performance of PLLA nanocomposites has been evaluated by thermogravimetric measurements, in Figure 4 DTG curves of binary (a) and ternary (b) PLLA nanocomposites are reported. 0,40 a) PLLA PLLA/0.5Ag2O 0,35 PLLA/0.5TiO2 PLLA/1LNP/0.5Fe2O3 DTG [g/gi min] PLLA/0.5WO3 PLLA/0.5ZnFe2O4 0,25 b) PLLA PLLA/1LNP PLLA/1LNP/05Ag2O 0,4 PLLA/0.5Fe2O3 0,30 DTG [g/gi min] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 0,20 0,15 0,10 PLLA/1LNP/0.5TiO2 0,3 PLLA/1LNP/0.5WO3 PLLA/1LNP/0.5ZnFe2O4 0,2 0,1 0,05 0,00 0,0 200 250 300 350 400 200 250 Temperature (°C) 300 350 400 Temperature (°C) Figure 4. DTG curves of binary (a) and ternary (b) PLLA based nanocomposites. As suggested by the bell-shaped curve, the thermodegradation process of PLLA proceeds through a single step (via intramolecular transesterification reactions with the formation of cyclic oligomers),36 reaching its maximum rate (Tpeak) at ~362 ºC. Despite to their high thermal stability, the presence of inorganic nanoparticles decreases the thermal stability of the hosting PLLA matrix as shown by the decrease of Tpeak up to a minimum of ~327 ºC for the PLLA/0.5ZnFe2O4 nanocomposite. This catalyzing effect of inorganic nanoparticles can be ascribed to the fact that 14 ACS Paragon Plus Environment Page 15 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Applied Bio Materials nanoparticle surfaces boost depolymerization reactions of adjacent L-lactide units,36 accelerating the overall thermodegradation process of PLLA. It has been reported that lignin decomposes through a complex mechanism involving an initial formation of low molecular weight elements from lignin side chains cleavage followed by a methane release due to the cleavage of the main chain (C-C and β-β scission or aryl-ether cleavage) and final condensation of the aromatic structure to lead a char residue.37 In this sense, it is worth noticing that although the thermal stability of ternary nanocomposites is also lower than that corresponding to pure PLLA,38 it is observed that the addition of lignin is able to counteract up to a certain extent the marked catalyzing effect of inorganic nanoparticles. The thermal stabilizing effect of lignin is related to the addition of complex phenylpropanoid moieties consisting on aromatic phenyl groups.3 XRD analysis Powder X-ray diffraction (XRD) measurements were implemented to verify whether or not the incorporation of different nanoparticles (either inorganic or organic) into PLLA could induce changes on the crystalline morphology. Accordingly, Figure 5 shows the XRD patterns on nanocomposite films in the 2θ range of 10-50°. The diffraction pattern of all samples are characterized by two main reflections at 2θ of 16.8 and 19.2° which are ascribed to the (110)/(200) and (203) planes of α PLLA phase with a 103 chain conformation,39 together with a less intense peak at 2θ=22.4º arising from the (105) plane. No significant changes on the resulting XRD patterns are observed upon nanoparticle incorporation, indicating that their addition does not modify the crystalline form of PLLA. The size of crystalline domains can be extracted from the most intense (110)/(200) diffraction peak in the XRD patterns according to the equation (3):40 𝐾∙𝜆 𝜏 = 𝛽𝜏 ∙ 𝑐𝑜𝑠𝜃 (3) where τ is the average size of the crystals, λ is the wavelength of the incident radiation (λ = 1.5418 Å), θ is the diffraction angle, K is the Scherrer constant (set at 0.9) and βτ is the peak width at half height. As a general trend the size of crystal domains (see Table S2 for further details) slightly decreases, after addition of inorganic nanoparticles, from 17.7 nm for neat PLLA film to values in the range 14.2-17.4 nm depending on the nanoparticle type. Such smaller sizes can be due to the nucleating effect of nanoparticles, which acting as heterogeneous nucleation surfaces for PLLA crystallization, yield more but smaller crystals.41 Additionally, for a given inorganic nanoparticle, the presence of lignin yields crystal size domains comparable to that obtained for neat PLLA, 15 ACS Paragon Plus Environment ACS Applied Bio Materials suggesting that lignin nanoparticles limit to a certain extent the nucleating effect of inorganic nanoparticles. We can consider that lignin nanoparticles, especially in solvent cast polymeric films, have limited propensity to induce crystallization. As already observed in our previous work8, nucleation effect is improved when homogeneous LNPs distribution in PLA matrix is achieved by using melt extrusion process, while the addition of LNPs at the same concentration (1% wt.) has no effect on the crystallization of the PLA matrix for solvent cast films. PLLA PLLA/1LNP PLLA/0.5TiO2 PLLA/1LNP_/0.5TiO2 PLLA/0.5Ag2O PLLA/1LNP/0.5Ag2O PLLA/1LNP/0.5WO3 PLLA/1LNP/0.5WO3 PLLA/0.5Fe2 O3 Intensity (a.u.) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 16 of 32 PLLA/1LNP/0.5Fe2 O3 PLLA_/0.5ZnFe2O4 PLLA /1LNP/0.5ZnFe2O4 10 20 30 40 50 2 Figure 5. XRD patterns of PLLA binary and ternary films DSC analysis Differential scanning calorimetry has been utilized to study the changes induced by nanoparticle addition on the thermal characteristics of PLLA nanocomposites. Accordingly, Figure 6 shows the heating scans of nanocomposite films, while the corresponding main thermal values are reported in Table 2. Neat PLLA is characterized by a smooth enthalpy jump at 34.4 ºC arising from the glass transition (Tg) and a marked endothermic peak centered at 179.2 ºC corresponding to the melting of its crystals (Tm).42 The presence of an exothermic peak at intermediate temperatures between the Tg and the Tm (typically arising from the formation of partial crystallinity during heating scan), not present in the binary nanocomposite formulations, suggests a relative initial large crystalline fraction of neat PLLA film (a Xc value of 24.5 % was obtained for neat PLLA film). Binary (Figure 6a) and ternary (Figure 6b) nanocomposites display similar thermograms, where only the transition temperatures resulted slightly shifted. In this sense, Tg is increased by 3-6 ºC 16 ACS Paragon Plus Environment Page 17 of 32 depending on the nanoparticle type, suggesting that amorphous PLLA chains are confined by the nanoparticle (either inorganic or organic) surfaces, where the mobility of amorphous segments markedly drops as a result of attractive forces between the particle surface and the PLLA chains.43 On the contrary, Tm temperatures are decreased from 179.2 ºC for pure PLLA to values in the range of 172.3-177.8 ºC for the binary and ternary nanocomposites. This behavior may be ascribed to the development of less perfect crystals having thinner lamella, which undergo through melting phenomena at lower temperatures.44 Table 2. Thermal properties extracted from DSC analysis (1st heating scan) for PLLA nanocomposite films. Tg: glass transition temperature; Xc: crystalline fraction; Tm: melting temperature Formulations Tg (°C) Xc (%) Tm (°C) PLLA 34.4 25 179.2 PLLA/1LNP 37.0 39 176.7 PLLA/0.5ZnFe2O4 40.4 42 176.1 PLLA/0.5Fe2O3 39.2 44 173.4 PLLA/0.5WO3 37.3 47 173.5 PLLA/0.5TiO2 36.9 41 172.7 PLLA/0.5Ag2O 36.4 47 173 PLLA/1LNP/0.5ZnFe2O4 38.2 42 173.2 PLLA/1LNP/0.5Fe2O3 38.6 44 172.3 PLLA/1LNP/0.5WO3 40.5 45 173.3 PLLA/1LNP/0.5TiO2 38.3 41 177.8 PLLA/1LNP/0.5Ag2O 38.6 46 173.2 b) Relative heat flow, exo > a) Relative heat flow, exo > 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Applied Bio Materials PLLA PLLA/0.5AgO2 PLLA/0.5Fe2O3 PLLA/0.5TiO2 PLLA/0.5WO3 PLLA/0.5ZnFe2O4 0 50 100 150 200 PLLA PLLA/1LNP PLLA/1LNP/0.5AgO2 PLLA/1LNP/0.5Fe2O3 PLLA/1LNP/0.5TiO2 PLLA/1LNP/0.5WO3 PLLA/1LNP/0.5ZnFe2O4 0 50 100 150 200 Temperature (°C) Temperature (°C) Figure 6. DSC heating curves of PLLA binary (a) and ternary (b) nanocomposites. 17 ACS Paragon Plus Environment ACS Applied Bio Materials Moreover, thanks to the effective nucleating behavior of nanoparticles when homogeneously dispersed into a polymer matrix, all nanocomposites present a marked increase on the crystalline fraction in comparison with neat PLLA (from 24.5 % for neat polymer to values in the 38.5-46.5 % range).45 Overall, nanoparticle addition confines the mobility of the amorphous phase while boosts the formation of more numerous but less perfect PLLA crystals. Wettability Water contact angle is one of the most important and representative parameter to control and study the surface physical characteristics of different films.46 The static water contact angle (WCA) of nanocomposite surfaces has been determined by the sessile drop methodology and obtained values are reported in Figure 7. PLLA Binary Films PLLA Ternary Films 90 80 70 60 WCA (°) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 18 of 32 50 40 30 20 10 0 PL LA L PL P O O 2 O 3 Fe 2O 3 O 4 O 4 O 3 O3 g 2O LN Ag 2 iO 2 .5Ti e2 e2 5 5W .5 A A /1 0 .5 / 0 . 5 T P /0 . 5 F e 2 P / 0 . 5 Z n F 5 Z n F / 0 . 5 W P / 0 . 0 / / P /0 L N A A N LA LN 0. /0 . LN /1 L P L L L A /1 L L A L A / 1 L L A / L N P P L L A / 1 PL L P PL LA P A /1 PL PL PL PL L Figure 7. Water contact angles of PLLA binary and ternary films Neat PLLA film shows a water contact angle of about 68°.47 The wettability of PLLA based nanocomposites is slightly modified with the presence of hydrophobic nanofillers, as in the case of lignin nanoparticles. Specifically, PLLA/1LNP shows increased values of WCA, while a different trend was found for binary systems, according to the different chemical structure, morphology, and dimension of the nanoparticles. The mean value of WCA for PLLA/0.5TiO2 and PLLA/0.5ZnFe2O4 is 68°, almost constant respect to PLLA neat film, this trend being in accordance with the 18 ACS Paragon Plus Environment Page 19 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Applied Bio Materials literature.12 A slight decrease is registered for PLLA/0.5WO3, this behavior is related to the low dimension of WO3 nanoparticles as evaluated by TEM image (Figure 1), that induces a smooth surface in the polymeric matrix (see Figure 2). An increase of 12° for WCA is obtained by the introduction of Ag2O in PLLA, which is caused by the different film surface morphology induced by this formulation (Figure 2). In PLLA/0.5Ag2O binary films, the surface appears with discrete microaggregates, which clearly induces a variation in physical properties. In ternary films, the addition of LNPs induces a variation of water contact angle values in comparison with the same binary films. Generally, it is observed a slight increase of WCA in ternary based films respect to the binary films, with exception of PLLA/1LNP/0.5Ag2O that is characterized by a reduction. This behavior is strongly related to the morphology of the film, since a more smooth and uniform surface (Figure 2) respect to the binary based film was observed. As reported by Moradi et al.48 surface roughness can cause noteworthy changes in the contact angle values as a function of the characteristics of the asperities in quantity and size,49 as developed by Cassie´s and Wenzel´s models, respectively for hydrophobic and hydrophilic surfaces so wettability can be strongly influenced.50 Nanocomposite antioxidant and antibacterial activities Antioxidant Properties Antioxidant characteristicsof PLLA and PLLA/1LNP nanocomposites are determined and expressed as radical scavenging activity (RSA, %). The values of antioxidant activity of migrating extracts in methanol for 24 h for different PLLA based films are summarized in Table 3, while Figure 8 shows the UV monitoring of the absorbance for band at 517 nm (Figure 8a) and color difference of DPPH methanol solution for PLLA/LNP binary and ternary (Figure 8b) systems. LNP are able to trap DPPH radicals, behavior which is confirmed by color change of DPPH solution from deep violet to soft and pale violet color or plate yellow. Metal oxide binary based nanocomposites show antioxidant activity, mainly due to transfer of free electrons from the oxygen atoms present in the nanoparticles to free radicals present at the nitrogen atom of DPPH molecules.51 Many metal oxide nanoparticles act as antioxidants and scavenge free radicals. Das et al.52 studied that the RSA activity is largely due to the high surface to volume ratio of nanofillers. PLLA/0.5ZnFe2O4 film shows the highest value of radical scavenging activity among the different metal oxide binary based nanocomposites (RSA=(7.6±0.1)%).51 The same trend is also detectable for PLLA ternary based nanocomposites. The high values of RSA in ternary based nanocomposites are induced by the presence of natural LNP nanofillers used as antioxidant additive as previously 19 ACS Paragon Plus Environment ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 20 of 32 evaluated in binary based system (RSAPLA/LNP=(43.1±0.1)%).53 The combination of LNP and metal oxide nanoparticles influences positively the RSA response, inducing high values of antioxidant activity (Table 3). Table 3: DPPH Scavenging activity of PLLA binary and ternary nanocomposites Formulations Radical scavenging activity, RSA (%) PLLA - PLLA/1LNP 43.1±0.1 PLLA/0.5ZnFe2O4 7.6±0.1 PLLA/0.5Fe2O3 4.2±0.1 PLLA/0.5WO3 3.3±0.3 PLLA/0.5TiO2 6.8±0.1 PLLA/0.5Ag2O 2.4±0.2 PLLA/1LNP/0.5ZnFe2O4 57.6±1.4 PLLA/1LNP/0.5Fe2O3 44.0±2.0 PLLA/1LNP/0.5WO3 48.0±1.1 PLLA/1LNP/0.5TiO2 51.5±1.2 PLLA/1LNP/0.5Ag2O 45.5±0.4 20 ACS Paragon Plus Environment Page 21 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Applied Bio Materials Figure 8. Antioxidant properties of migrated substances for PLLA based formulations immersed in the methanol solution for 24 h: detection of the absorbance for band at 517 nm (a), and color modifications of the DPPH methanol mixture (b). The highest values of RSA in ternary based systems is registered for PLLA/1LNP/0.5ZnFe2O4 (RSA =(57.6±1.4)%). Also WO3 and Fe2O4 nanoparticles showed good antioxidant properties, nevertheless systematic investigations on antioxidant characteristics of ferrite nanoparticles are not available in literature. The data of our work are promising and offer a lead in the exploration of Fe2O4 and ZnFe2O4 nanoparticles as a new source of antioxidant materials. Antibacterial Properties The antibacterial properties of both binary and ternary nanocomposite films were tested by checking viability of planktonic cell cultures of S. aureus and E. coli after their removal from materials surface to determine the number of live cells (Figure 9). Figure 9 shows the comparison of S. aureus (Figure 9 a,c) and E. coli (Figure 9 b,d) viability on binary (Figure 9 a,b) and ternary (Figure 9 c,d) nanocomposites at 6 and 24 h, respectively. Considering the times of incubation (6h and 24h) and the bacterial strains, different results were obtained for binary and ternary nanocomposites films. 21 ACS Paragon Plus Environment ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 22 of 32 Binary based nanocomposites films showed an interesting antibacterial activity at 6h in comparison to higher incubation times (24h) with some differences. Specifically, at 6h for S. aureus, the antibacterial activity trend starting from the higher to the lower value was: PLLA/0.5Ag2O (50%) > PLLA/0.5WO3 (~42%) > PLLA/0.5TiO2 (~40%) > PLLA/0.5Fe2O3 (~15%). PLLA/0.5ZnFe2O4 and PLLA/1LNP were not effective on the gram positive bacterium (Figure 9a); at 24h for S.aureus the trend was: PLLA/0.5WO3 (~35%) > PLLA/0.5TiO2 (~33%) > PLLA/0.5ZnFe2O4 (~18%) > PLLA/0.5Ag2O (~11%)  PLLA/0.5Fe2O3  PLLA/1LNP even if the antibacterial activity was relatively reduced on all the binary nanocomposites films (Figure 9a). For S. aureus, the antibacterial activity between 6h and 24h was quite similar, with the exception of PLLA/0.5Ag2O which was majorly effective at 6h and PLLA/0.5ZnFe2O4 which had an antibacterial activity only after 24h. At the short incubation time, E. coli showed a different behavior on binary based nanocomposites films: PLLA/0.5TiO2 (20%) > PLLA/0.5ZnFe2O4 (5%)  PLLA/0.5Ag2O whereas PLLA/0.5Fe2O3, PLLA/0.5WO3 and PLLA/1LNP did not show any antibacterial activity (Figure 9a). At 24h for E. coli, the trend was slightly different with PLLA/0.5Fe2O3. PLLA/0.5Ag2O and PLLA/0.5ZnFe2O4 showed the best antibacterial performance maintaining the values observed at 6h (Figure 9b). For E. coli, no significant difference was observed at 6h when compared with results at 24h. 22 ACS Paragon Plus Environment Page 23 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Applied Bio Materials a) c) b) d) Figure 9. Antibacterial properties of PLLA binary and ternary nanocomposites films on Gram positive and negative bacteria. The antibacterial activity was evaluated on the planktonic bacterial cultures removed after being in contact with PLLA and PLLA nanocomposite films containing LNP and/or metal oxide nanoparticles for 6 h and 24 h at 37 °C. Results for both binary (Panels a, b) and ternary (c, d) based nanocomposites films, in S. aureus (a, c) and E. coli (b, d), are expressed as the percentage of bacterial cells grown on PLLA film set equal to 100% (A, B) or on PLLA/1LNP film set as 100% (c, d). Data are presented as the average of three replicates ± standard deviations. *, # P < 0,05. *statistical analysis obtained from Student’s t-test, normalized vs PLLA (a, b) and vs PLLA/1LNP (c, d); #-statistical analysis from ANOVA, followed by Bonferroni post hoc for comparison of nanocomposites between 6 and 24h. Interestingly, on the contrary to our previous studies performed with hydrogels containing the same amount (1% wt.) of lignin nanoparticles (LNPs),54 the presence of LNPs in PLLA nanocomposites films without metal based NPs was slightly more effective against Gram positive (S. aureus) in comparison to Gram negative bacteria (E. coli) strain; in the latter case, no antibacterial activities were observed. The main reason of the difference may be related to the experimental protocol, that was modified to provide a more accurate understanding on the antibacterial performance: in the previous study, test was performed directly on the bacteria attached to the material surfaces, whereas in this study it was detected on the planktonic cell cultures of S. aureus and E. coli after their removal from the different types of nanocomposite films. Again, we cannot exclude that the different effects could be related to the nature of the PLLA 23 ACS Paragon Plus Environment ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 24 of 32 polymer surface induced by the fabrication procedure, as well as LNPs distribution in the PLLA films. As shown by the test performed using binary nanocomposite films made of PLLA enriched with metal oxide based NPs, the antibacterial activity was manifest with PLLA/0.5Ag2O in S. aureus and PLLA/0.5TiO2 for both bacterial strains, even if at a different extent. As previously reported, metal oxide nanoparticles, especially TiO2 and Ag2O nanoparticles, have demonstrated significant antibacterial activity.55,56 Within this study, we showed that TiO2 and Ag2O nanoparticles, immersed in a polymer such as PLLA, seem to retain at some extent their antibacterial effect, indeed, it is supported by the supplementary Figure S2, whose results were normalized only vs. TCP. Interestingly, regarding the other samples tested, PLLA/0.5WO3 showed a better result with S.aureus, whereas PLLA/0.5ZnFe2O4 reduced at some level E.coli viability (Figure 9b) and supported by supplementary figure (Figure S2b). The iron oxide nanoparticles have shown therapeutic activities and have demonstrated to be efficient antimicrobial agents.57 These nanoparticles can be used in the treatment of microbial infections, because they might function as membrane permeability enhancer, either causing damages in the cell wall or generating reactive oxygen species. In our study, the antimicrobial activity of iron oxide nanoparticles was better performed in the presence of zinc ferrite, in particular retaining the antibacterial activity at 6 and 24h in E. coli. As previously reported,58 also zinc nanoparticles have shown to exhibit interesting antimicrobial properties against bacteria, both gram positive and negative,and fungi. To better explain these results, it is firstly important to consider that the main toxicological effect induced by metal oxide based NPs in bacteria occurs by direct contact with the bacteria cell surface. It has been reported that both Gram positive and negative bacteria have a negatively charged surface: the cell wall of a gram positive is composed of a thick layer of peptidoglycan forming a cohesive mesh, additionally enriched with the external protrusion of the negatively charged teichoic acids (with high levels of phosphate groups). Gram negative bacteria instead, have a thin layer of peptidoglycan and anouter membrane with partially phosphorylated lipopolysaccharides. The latter increase the negative charge of the surface.59 Due to electrostatic interactions, positively charged metal-based nanoparticles are attracted by the negatively charged bacterial surface, and their consequent bond causes the disruption of cell walls and the increased permeability. Furthermore, NPs can release metal ions, which can enter the cell to induce the production of reactive oxygen species (ROS) and can interact with cellular structures like proteins, membranes andDNA, ending with the disruption of cell functions.59 In this study we need to consider at least 2 aspects; a. the lignin and the metal-oxide nanoparticles were not free but included in the PLLA films; b. the evaluation was performed on the planktonic cell cultures of both bacterial strains after their removal from the different nanocomposite films at 6h or 24h. It may be possible that bacterial cells that are 24 ACS Paragon Plus Environment Page 25 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Applied Bio Materials in direct contact with material surface during incubation time can reduce their survivability, whereas those cells that are distant cannot interact directly and hence can survive. Our data seems to confirm the retained effect of some of the inorganic metal oxide nanoparticles even when they are incorporated in PLLA. Regarding the ternary nanocomposite films, we evaluated the antibacterial performance of PLLA/1LNP containing metal oxide based nanoparticles to determine in what manner their concurrent presence can effectively reduce bacterial viability. Ternary based nanocomposites films showed a trend in antibacterial activity quite similar to the one exerted by binary nanocomposites films with some differences (Figure 9 c,d). Specifically, at 6h for S.aureus, the antibacterial activity trend starting from the higher to the lower value showed PLLA/1LNP/0.5Ag2O (60%)  PLLA/1LNP/0.5ZnFe2O4 (60%) > PLLA/1LNP/0.5Fe2O3 (40%), whereas no activity was observed on PLLA/1LNP/0.5WO3 and PLLA/1LNP/0.5TiO2 (Figure 9c); at 24h for S.aureus, the trend of the antibacterial activity was relatively different on all the ternary nanocomposites films: PLLA/1LNP/0.5WO3 (56%) > PLLA/1LNP/0.5Ag2O  PLLA/1LNP/0.5Fe2O3(45%)  PLLA/1LNP/0.5ZnFe2O4 (35%). PLLA/1LNP/0.5TiO2 did not show an antibacterial activity (Figure 9c). For S.aureus, the antibacterial activity exerted at 6h was retained at some extent at 24h, showing an improvement in particular for PLLA/1LNP/0.5WO3. At 6h incubation time, the E.coli showed a different behavior: PLLA/1LNP/0.5ZnFe2O4 (35%) > PLLA/1LNP/0.5Ag2O (5%)  PLLA/1LNP/0.5WO3 (5%) whereas PLLA/1LNP/0.5Fe2O3 and PLLA/1LNP/0.5TiO2 did not show any efficient activity against bacteria (Figure 9d). At 24h incubation time for E.coli, the trend was slightly different with PLLA/1LNP/0.5Ag2O and PLLA/1LNP/0.5TiO2 showing the best antibacterial performance even if at quite low value, whereas no activity was observed on the other ternary nanocomposites films (Figure 9d). For E. coli, no great difference was observed at 6h if compared at 24h with a slightly increment in efficacy exerted by both PLLA/1LNP/0.5Ag2O and PLLA/1LNP/0.5TiO2. In addition, PLLA/1LNP/0.5ZnFe2O4 has an inhibitory effect at 6h of incubation. Ternary blends are much more complex multiphase polymeric systems because they can show different microstructures and interactions between polymer, LNPs and metal oxide nanoparticles. The characteristics of the final products may result from the different properties of each component of the blend, whereas the various interfacial features could affect the properties of multiphase systems.60 The interest of ternary nanocomposites is in continuous expansion60 and it might be due to the fact that new functional properties could be obtained from the synergy resulting from interfacial interactions of different phases in the blend thus to be differently influenced. Moreover, 25 ACS Paragon Plus Environment ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 26 of 32 the different localization of nanoparticles could influence the morphology and the surface,60 indeed they can explain the different behavior in bacterial strains, in accordance with literature.61,62 CONCLUSIONS Solvent cast films of PLLA based nanocomposites containing lignin (1% wt.) and different metal oxide nanoparticles (0.5% wt., Ag2O, TiO2, WO3, Fe2O3 and ZnFe2O4) were prepared and tested determining thermal, morphological, surface, optical, antioxidant and antibacterial characteristics. The presence of lignin nanoparticles affected the morphologies of produced ternary films, as confirmed by smoother and more hydrophobic surfaces, with exception of PLLA/1LNP/0.5Ag2O, where the roughness effect was predominant on chemical nanoparticles reactivity. The combined use of metal oxides and lignin demonstrated to be also effective in UV protection capacity, surpassing the results obtainable with inorganic nanoparticles. The decreased thermal stability of PLLA upon inorganic NP addition was partially recovered by the stabilizing effect of lignin, while on the other hand lignin inhibited the nucleating effect of inorganic nanoparticles in the polymer, by confining the mobility of the amorphous phase and boosting the formation of more numerous but less perfect PLLA crystals. Interestingly, the presence of metal oxide NPs also corroborate the antioxidant character of the lignin containing films, that was present in the ternary films, and maximum in ZnFe2O4 based films. Binary nanocomposite films with metal oxide NPs displayed also a significant antibacterial activity against PLLA, more evident for S. aureus than E. coli, which was and maintained only for few ternary nanocomposites films in a time dependence that was more evident for S. aureus than for E. coli. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at https://pubs.acs.org/doi/XXX. Tables showing transmitted light (%), the size of the crystalline domains according to Scherrer equation, FTIR of metal oxide nanoparticles, and the antibacterial properties of binary and ternary nanocomposites. AUTHOR INFORMATION 26 ACS Paragon Plus Environment Page 27 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Applied Bio Materials Corresponding Author E-mail address: debora.puglia@unipg.it ORCID Erlantz Lizundia: 0000-0003-4013-2721 Ilaria Armentano: 0000-0002-8266-4366 Francesca Luzi: 0000-0001-8785-5033 Federico Bertoglio: 0000-0001-6477-1785 Elisa Restivo: 0000-0003-1752-9341 Livia Visai: 0000-0003-1181-3632 Luigi Torre: 0000-0001-7872-5712 Debora Puglia: 0000-0001-8515-7813 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This study was supported with a grant to F.B., E.R. and L.V. of the Italian Ministry of Education, University and Research (MIUR) to the Department of Molecular Medicine of the University of Pavia under the initiative “Dipartimenti di Eccellenza (2018–2022)”. E.L. is grateful to ETH Zurich for financial support and Markus Niederberger for providing laboratory facilities. We thank Stefano Iervese (University of Pavia) for sample sterilizations and preparation of bacterial cultures and Dr Anna Rita Taddei from CGA University of Tuscia, Viterbo Italy, for her valuable technical assistance for microscopic analysis. Data availability All the data used to support the findings of this study are included within the article. 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