Antimicrobial Activity and Cytotoxicity to Tumor ... - ACS Publications

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Antimicrobial activity and cytotoxicity to tumor cells of nitric oxide donor and silver nanoparticles containing PVA/PEG films for topical applications Wallace R Rolim, Joana C Pieretti, Débora L S Renó, Bruna A Lima, Mônica H. M. Nascimento, Felipe N. Ambrósio, Christiane B. Lombello, Marcelo Brocchi, Ana Carolina Santos de Souza, and Amedea B Seabra ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19021 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019

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ACS Applied Materials & Interfaces

Antimicrobial Activity and Cytotoxicity to Tumor Cells of Nitric Oxide Donor and Silver Nanoparticles Containing PVA/PEG Films for Topical Applications

Wallace R. Rolim,1,2 Joana C. Pieretti,1,2 Débora L. S. Renó,1 Bruna A. Lima,3 Mônica H. M. Nascimento1,2, Felipe N. Ambrosio4, Christiane B. Lombello2,4, Marcelo Brocchi,3 Ana Carolina S. de Souza,1 Amedea B. Seabra1,2*

1

Center for Natural and Human Sciences (CCNH), Federal University of ABC

(UFABC), Santo André, SP, 09210-580, Brazil 2

Nanomedicine Research Unit (NANOMED), Federal University of ABC (UFABC),

Santo André, SP, 09210-580, Brazil 3

Tropical Disease Laboratory, Department of Genetics, Evolution, Microbiology and

Immunology, Institute of Biology, University of Campinas (UNICAMP), Campinas, SP, 13083-862, Brazil 4

Center for Engineering, Modeling and Applied Social Science, Federal University of

ABC (UFABC), Santo André, SP, 09210-580, Brazil

*Corresponding author Amedea B. Seabra Center for Natural and Human Sciences (CCNH), Federal University of ABC (UFABC) Av. dos Estados 5001, CEP 09210-580, Santo André, SP, Brazil Email: [email protected] Keywords: Nitric oxide, S-nitrosoglutathione, silver nanoparticles, polymeric films, antibacterial, cytotoxicity.

1 ACS Paragon Plus Environment

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Abstract Due to their antibacterial activity, silver nanoparticles (AgNPs) have been explored in biomedical applications. Similarly, nitric oxide (NO) is an important endogenous free radical with an antimicrobial effect and toxicity towards cancer cells that plays pivotal roles in several processes. In this work, biogenic AgNPs were prepared using green tea extract and the principles of green chemistry, and the NO donor S-nitrosoglutathione (GSNO) was prepared by the nitrosation of glutathione. To enhance the potentialities of GSNO and AgNPs in biomedical applications, the NO donor and metallic nanoparticles were individually or simultaneously incorporated into polymeric solid films of poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG). The resulting solid nanocomposites were characterized by several techniques, and the diffusion profiles of GSNO and AgNPs were investigated. The results demonstrated the formation of homogenous PVA/PEG solid films containing GSNO and nanoscale AgNPs distributed in the polymeric matrix. PVA/PEG films containing AgNPs demonstrated potent antibacterial effect against gram-positive and gram-negative bacterial strains. GSNOcontaining PVA/PEG films demonstrated toxicity towards human cervical carcinoma cell and human prostate cancer cell lines. Interestingly, the incorporation of AgNPs in PVA/PEG/GSNO films had a superior effect on the decrease of cell viability of both cancer cell lines, compared with cells treated with the films containing GSNO or AgNPs individually. To our best knowledge, this is the first report to describe the preparation of PVA/PEG solid films containing GSNO and/or biogenically synthesized AgNPs. These polymeric films might find important biomedical applications as a solid material with antimicrobial and antitumorigenic properties.


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1.

Introduction

Silver nanoparticles (AgNPs) are important antibacterial agent, with a broadspectrum of antibacterial, antifungal, and antiviral effects.1–3 In addition, AgNPs have antiproliferative effects against different tumor cell lines.4 The anticancer effect of AgNPs is due to the ability of these nanoparticles to impair the mitochondrial respiratory chain, inducing the formation of ATP and reactive oxygen species (ROS), leading to DNA damage.5,6 Thus, AgNPs are extensively used in different applications and are present in several commercial products.3,7 Interestingly, the development of resistance against AgNPs is difficult for bacteria.8 Indeed, AgNPs can limit the growth of resistant bacteria strains, at low concentrations with minimum toxicity toward mammalian cells.3,9,10 Similar to AgNPs, nitric oxide (NO) has antimicrobial and anticancer effects.11– 13

NO is an endogenous found molecule that mediates different pathophysiological and

physiological processes.13 NO promotes the increase of blood flow,14 the regulation of platelet adhesion and aggregation,15 and the promotion and acceleration of tissue repair.16 In addition, depending on its concentration, NO has antimicrobial17,18 and anticancer effects.13,19 As NO is a free radical, different NO donor molecules have been synthesized and used in several biomedical applications.13 S-nitrosothiols (RSNOs) are NO donors able to spontaneous produce NO.20 S-nitrosoglutathione (GSNO) is an RSNO found in vivo. GSNO undergoes spontaneous decomposition yielding NO and oxidized glutathione (GSSG) (Eq. 1). GSNO has antimicrobial, antioxidant and anticancer effects.13

2 GSNO  2 NO + GSSG

(1)



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In this scenario, there is a potential interest in the uses of AgNPs and NO donors, such as GSNO, in several applications, including biomedical applications.3,19,21 However, the sustained and localized release of NO donors or AgNPs is a major issue that needs to be addressed. Entrapment of NO donors or AgNPs in non-toxic matrices might contribute to the sustained and localized release of therapeutic amounts of AgNPs or NO directly to the desired application site.9,20,22 To use NO donors or AgNPs potentialities in biomedical applications, they should be trapped in a matrix that can form thin or scaffold films. Polymers are interesting materials due to their flexible features and the presence of suitable functional groups that allow the entrapment of GSNO or AgNPs.9,23,24 The preparation of polymeric flexible solid films containing GSNO able to locally release NO in topical applications was reported to promote the increase of skin blood flow and to promote cutaneous wound healing.24,25 In a similar manner, the incorporation of AgNPs in polymeric solid films produces nanocomposites with improved functional and structural properties.26 Since solid polymer films containing AgNPs are expected to have antimicrobial properties, these films can be used in biomedical applications or in food packaging.27 Polymer nanocomposites contains the antimicrobial properties of AgNPs and the desired aspects of host polymers.9,28 Among the different polymers that can be used to host NO donors or AgNPs in biomedical applications, using poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG) as film-forming polymers are an appropriate choice. PVA is non-toxic, soluble in water, non-carcinogenic, hydrophilic, synthetic polymer able to produce film, and therefore, it is extensively used in biomedical applications, including wound dressings.9,26,27,29 PVA has high surface stabilization and chelation properties,9,30 and the use of PVA in many medical devices, including contact lenses, orthopedic devices,


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and drug delivery systems, is approved by the Food and Drug Administration.31 Several papers described the incorporation of AgNPs into PVA films as an antimicrobial surface.9,26–28 Similarly, GSNO was incorporated into PVA based films to promote sustained NO release, increasing dermal blood flow and promoting cutaneous wound repair.20,24,25 Likewise, PEG is soluble in water, non-toxic polymer that has been extensively applied in pharmacological applications.32 A PEG matrix was used to incorporate GSNO,33 to coat metallic nanoparticles including AgNPs,34 and PEG hydrogels have been used in wound healing and tissue engineering.35 Polymer blends often exhibit superior properties compared to those of each individual component polymer.36 The polymer blend of PVA and PEG is important due to its enhanced properties, including superior mechanical properties, non-toxicity, noncarcinogenic effect, and bioadhesive properties.37 Semi-crystalline PVA has important applications due to its OH groups and hydrogen bonds.36 PEG is known to enhance the physical properties of PVA films.36 PEG and PVA form strong interactions due to the formation of hydrogen bonds.36,37 In this work, blended solid polymeric films of PVA/PEG with monodispersed and well distributed GSNO and/or AgNPs were successfully prepared. GSNO was prepared by the nitrosation of glutathione,14,33 while AgNPs were biogenically synthesized using plant extract, green tea (Camellia sinensis).10 According to the principles of green synthesis, the use of plant extract to synthesize AgNPs is based on the use of aqueous solvents in the place of organic solvents, nontoxic chemicals, and renewable materials.38 Solid PVA/PEG films containing GSNO and/or green tea synthesized AgNPs were prepared by solvent cast evaporation. The films were characterized by several techniques. The NO release from the polymeric films and the diffusion of GSNO and AgNPs from the blended films were monitored. PVA/PEG films



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containing AgNPs demonstrated potent antibacterial activity against different bacterial strains. GSNO-containing PVA/PEG films demonstrated toxicity towards human cervical carcinoma cell (HeLa) and human prostate cancer cell (PC3). Interestingly, the incorporation of AgNPs in PVA/PEG/GSNO films had a superior effect on the decrease of cell viability of both cancer cell lines, compared with cells treated with either PVA/PEG films containing only GSNO or PVA/PEG films containing only AgNPs. To the best of our knowledge, this is the first report to describe the preparation, characterization, antibacterial effect, and cytotoxicity towards cancer cell lines of PVA/PEG solid films containing GSNO and/or biogenic synthesized AgNPs. These polymeric films might find important biomedical applications as a solid material with antimicrobial and antitumorigenic properties, including wound dressing in tissue engineering.

2. Materials and Methods

2.1. Materials

Camellia Sinensis was purchased from Sumioka Shokuhin Kabushikikaisha, Hiraguti, Japan. Silver nitrate (AgNO3), poly(vinyl alcohol) (MW 85,000-124,000; 99% hydrolyzed, PVA), poly(ethylene glycol) (MW 200, PEG), glutathione (GSH), sodium nitrite (NaNO2), and phosphate buffer saline (PBS, pH 7.4) were obtained from SigmaAldrich (St. Louis, MO, USA). Sodium hydroxide (NaOH), acetone, hydrochloric acid (HCl), and acetic acid were obtained from Labsynth (Diadema, SP, Brazil). All the experiments were performed with analytical grade water from a Millipore Milli-Q Gradient filtration system. Dulbecco’s modified Eagle’s medium (DMEM), HAM F-10,


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RPMI media, and MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide] were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) and αMEM medium (without ascorbic acid) were purchased from Gibco (Grand Island, NY, USA). Mueller-Hinton media (broth and agar) were purchased from Difco (Franklin Lakes, USA). Sodium chloride (NaCl, P.A. grade), used to obtain an 0.85% w/v saline solution, was purchased from J.T. Baker (Ciudad del Mexico, Mexico). Saline and culture solutions were sterilized at 120 ºC.

2.2. Synthesis of GSNO

GSNO was prepared by the nitrosation of GSH, as previously described.18,39 Briefly, GSH was mixed with NaNO2, in acid medium for 40 min, protected from light in an ice bath. The obtained GSNO was precipitated with acetone and cold water, filtered, washed with cold water, and lyophilized for 24 h. The resulting pink GSNO precipitate was stored at -20 °C.

2.3. Synthesis of AgNPs by green tea extract

AgNPs were synthesized as previously described.10 The mass of 2 g of powdered commercial green tea was dissolved in water (100 mL), stirred and heated at 60 °C, and filtered. Aqueous solution of AgNO3 (0.1 mol L-1) was mixed with aqueous suspension of green tea extract at room temperature, with magnetic stirring, and the pH of the final mixture was adjusted to 10.5. The final suspension was centrifuged, and the precipitate of AgNPs was washed and freeze-dried.



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2.4. Dynamic light scattering (DLS)

The determination of the hydrodynamic size, polydispersity index (PDI), and zeta potential values of green tea synthesized AgNPs were analyzed by DLS (Zetasizer Nano ZS, Malvern Instruments Co, UK), as previous described.10

2.5. Solvent cast film preparation

The preparation of PVA/PEG solid blended films with a PVA:PEG ratio of 1:2 (wt/wt) were obtained using the adapted protocols reported in our previous works.20,22 The PVA:PEG ratio of 1:2 wt/wt was used in all the blended PVA/PEG films prepared. GSNO and/or AgNPs were embedded in PVA/PEG solid films. We prepared 4 groups of solid films: (i) PVA/PEG, (ii) PVA/PEG containing GSNO (2.5 wt%), (iii) PVA/PEG containing AgNPs (2.5 wt%), and (iv) PVA/PEG containing GSNO (2.5 wt%) and AgNPs (2.5 wt%). All groups were prepared by solvent cast evaporation, as described below. Group (i): Solid PVA/PEG films were prepared by dissolving 0.56 g of PVA in 7.0 mL of deionized water using magnetic stirring at 80.0 °C. After the total dissolution of PVA, 1.124 g of PEG was added to the PVA solution, and the final solution was further homogenized using magnetic stirring at room temperature. Group (ii): Solid PVA/PEG films containing GSNO were prepared by dissolving 0.56 g of PVA in 6.8 mL of deionized water using magnetic stirring at 80.0 °C. After the total dissolution of PVA, 1.124 g of PEG was added to the PVA solution, and the final solution was further homogenized using magnetic stirring at room temperature. 0.2


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mL of aqueous GSNO (0.21 mol L-1) was added to the PVA/PEG solution, and the final solution was further homogenized using magnetic stirring at room temperature. Group (iii): Solid PVA/PEG films containing AgNPs were obtained by dissolving 0.56 g of PVA in 6.0 mL of deionized water using magnetic stirring at 80.0 °C. After the total dissolution of PVA, 1.124 g of PEG was added to the PVA solution, and the final solution was further homogenized using magnetic stirring at room temperature. 1.0 mL of aqueous a suspension of AgNPs (14 mg mL-1) was added to the PVA/PEG solution, and the final solution was further homogenized using magnetic stirring at room temperature. Group (iv): Solid PVA/PEG films containing GSNO and AgNPs were obtained as described for groups (iii) and (iv); 0.8 mL of aqueous suspension of AgNPs (17.5 mg mL-1) and 0.2 mL of aqueous GSNO (0.21 mol L-1) were added to an aqueous solution of PVA/PEG using magnetic stirring at room temperature. All aqueous polymeric solutions (groups i-iv) were individually cast by solvent evaporation. A volume of 300 µL of each of the polymeric solutions was added to the cavity of a quartz cell with a detachable window (Hellma 106-QS optical path 0.1 mm) and allowed to cast the films.20 Alternatively, each of the polymeric solutions was cast in a 3.5 cm diameter silicone mold for 8 h. After drying, the solid films were removed from the silicone mold. These processes led to the formation of solid films of PVA/PEG (group i, 1:2 wt/wt PVA:PEG ratio). In the case of groups ii, iii and iv, these processes led to the formation of solid PVA/PEG films containing 2.5 wt% GSNO (group ii), 2.5 wt% AgNPs (group ii), 2.5 wt% GSNO and 2.5 wt% AgNPs (group iv). Figure 1 illustrates the preparation of the solid films.

2.6. Characterization of the prepared polymeric films



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The polymeric films were characterized by different techniques, as described below.

2.6.1. UV-Visible spectroscopy analysis

Solid films of PVA/PEG, PVA/PEG containing GSNO, PVA/PEG containing AgNPs, and PVA/PEG containing GSNO and AgNPs, cast onto the cavity of a detachable quartz cell, were characterized by UV-Visible spectroscopy analysis using a UV–Vis spectrophotometer (Agilent, model 8453, Palo Alto, CA, USA).

2.6.2. X-ray diffraction (XRD)

Solid films of PVA/PEG and PVA/PEG containing GSNO and AgNPs were characterized by XRD (STADI-P powder diffractometer (Stoe®, Darmstadt, Germany) in transmission geometry by using a MoKα1 wavelength, from 2.0° to 60.485°).10 To this end, 3.0 mm diameter pieces of PVA/PEG or PVA/PEG containing GSNO and AgNPs were placed in a sample holder during data collection.

2.6.3. Fourier transformed infrared spectroscopy (FTIR)

FTIR analysis was used to identify the functional groups on solid films of PVA/PEG and PVA/PEG containing GSNO and AgNPs. To this end, 6 mm pieces of PVA/PEG or PVA/PEG containing GSNO and AgNPs were placed in the sample holder of a Spectrum Two FT-IR Spectrometer (PerkinElmer, USA). The measurements were


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performed using an attenuated total reflectance (ATR) accessory, the spectra were recorded in the range of 400–4000 cm-1, with a resolution of 2 cm-1, and 32 scans were carried out.

2.6.4. Scanning electron microscopy (SEM) and energy dispersive X-ray fluorescence spectrometry (EDS)

The morphology of a solid PVA/PEG film containing GSNO and AgNPs was examined by SEM (SEM, Quanta 250 SEM-FEI, FEI/ Thermo Fisher Scientific, OR, USA), with an acceleration voltage of 2.0 and 5.0 kV. The film sample was mounted on aluminum stubs, sputter coated for 2 min in a high-vacuum gold sputter coating unit (Balt-Tec MED 020, Liechtenstein). The elemental composition of the films was obtained by EDS analysis (EDS, JEOL, JSM-6010LA). The EDS technique was used to map the distribution of carbon, sulfur, oxygen and silver atoms on the PVA/PEG containing GSNO and AgNPs film.

2.7. Kinetics of GSNO decomposition with NO release in PVA/PEG films containing GSNO

The kinetics of GSNO decomposition in solid PVA/PEG film containing GSNO (2.5 wt%) were recorded from the absorption changes at 336 nm, at 25, 32.5, and 37 °C, for 24 h in 30 min intervals, using a UV–vis spectrophotometer (Agilent, model 8453, Palo Alto, CA, USA), with a temperature-controlled cuvet holder.20,22 The GSNO decomposition was converted to NO released. This calculation was based on the decay of the absorption band of GSNO at 336 nm ( = 922 mol-1 L cm-1) being solely



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associated with the cleavage of the S-N. The Lambert-Beer Law was used to calculate the concentration of NO released (Eq. 2).20,22

[𝑁𝑂]𝑡 = [𝐺𝑆𝑁𝑂]0 − [𝐺𝑆𝑁𝑂]𝑡 = (𝐴0 𝑏/ε𝐺𝑆𝑁𝑂 ) − (𝐴𝑡 𝑏/ε𝐺𝑆𝑁𝑂 )

(Eq. 2)

where A0 is the initial absorbance, At is the absorbance at a given time, b is the optical path, and ε is the molar absorptivity of GSNO at 336 nm (980 mol L-1 cm-1). Initial rates (IR) of NO release from PVA/PEG film containing GSNO at different temperatures, were calculated by linear regression from the slopes, as previously described.22 Each point of the kinetic curve was reported as the mean ± standard deviation (SD) of the three independent experiments with error bar values set at the standard error of the mean.

2.8. In vitro diffusion of AgNPs and GSNO from PVA/PEG films containing GSNO and AgNPs

The in vitro kinetics diffusion of intact GSNO and AgNPs from PVA/PEG containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) were performed with a vertical Franz diffusion cell (Hanson Research Corporation).40 The cell consists of donor and receptor chambers that were separated by a hydrophilic nitrocellulose membrane with a 50 nm porosity and 25 mm diameter (Merck Millipore Ltd.). Solid PVA/PEG films containing GSNO and AgNPs were cut in a spherical shape with a 3.5 cm diameter and mounted in the donor compartment. A volume of 7 mL of PBS at 32.5 °C (human skin temperature) was added in the receptor chamber, with constant stirring. Ever 30 min, a volume of 500 μL was withdrawn from the receptor chamber and replaced by the same volume of fresh PBS solution. For the detection of GSNO diffusion from the films, the


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withdrawn samples were analyzed by UV-Visible spectrophotometry, as previous reported.40 The amount of total silver diffusion through the membrane from the polymeric film was performed using inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7900, Hachioji, Japan). For the ICP-MS analysis, 50 µL aliquots of each sample were diluted in 1.0 mL of an aqueous solution of 2% (v/v) nitric acid (HNO3).

2.8.1. Mathematical models

To investigate the mechanisms of GSNO and AgNPs diffusion from the solid films, the kinetic curves obtained in Section 2.8 were adjusted for two mathematical models: Higuchi and Korsmeyer-Peppas. Higuchi model:40,41

𝑄 = 𝐾𝐻 𝑡 0.5

(Eq. 3)

Korsmeyer-Peppas model:40

𝑀𝑡 𝑀∞

= 𝐾𝐾𝑃 𝑡 𝑛

(Eq. 4)

2.9. Antibacterial activities of polymeric films (disk diffusion method)

The antibacterial effects of solid films were investigated through testing the inhibition zone according to the disk-diffusion method, with CLSI recommendations (M02-A12, 2015 with adaptations,) against the gram-positive strain Staphylococcus 13 ACS Paragon Plus Environment


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aureus American Type Culture Collection (ATCC) 29213 (CLSI standard) and the gram-negative bacterial strains (all CLSI standards) Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Klebsiella pneumoniae ATCC 700603. The disk was impregnated with 6 mm diameter pieces of the films with (i) PVA/PEG, (ii) PVA/PEG containing AgNPs (0.5 wt%), (iii) PVA/PEG containing AgNPs (2.5 wt%), or (iv) PVA/PEG containing AgNPs (2.5 wt%) and GSNO (2.5 wt%). The films were put on Mueller-Hinton agar media previously inoculated with bacterial strains in Mueller-Hinton broth after appropriate dilution in saline solution. As the substance diffuses from the films into the agar, the concentration decreases as a function of the square of the distance of diffusion. In order to validate the method, as positive controls, commercial paper filter disks of purified antibiotics: Kanamycin (30 µg), Gentamicin (10 µg), Meropenem (10 µg) and Cefoxitin (30 µg) were tested against E. coli, K. pneumoniae, P. aeruginosa and S. aureus strains, respectively. These compounds and concentrations tested were selected according to the recommendations of the ATCC. As negative control, pure PVA/PEG films were used.

2.10. Cell culture, cell treatment, and viability assay

The human cervical carcinoma cell line HeLa and human prostate cancer cell line PC3 were provided by Dr. Marcelo Bispo de Jesus (State University of Campinas, Brazil). The human foreskin fibroblast (HFF-1) cell line was provided by Dr. Giselle Zenker Justo (Federal University of São Paulo, Brazil). The cells were cultured at 37 °C in RPMI 1640 medium or Dulbecco’s DMEM with high glucose, for PC3 and HeLa respectively, supplemented with 10% (v/v) FBS, 100 U/mL of penicillin and 100 μg/mL of streptomycin. For HFF-1 cells, Dulbecco’s DMEM with high glucose was


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supplemented with 15% (v/v) FBS and 1 mmol/L sodium pyruvate. The Vero cells (CCIAL-57, Adolfo Lutz Institute, São Paulo, Brazil), epithelial cells from African Green monkey (Cercopithecus aethiops), and pre-osteoblastic MC3T3-E1 cells (BCRJ:0285, Rio de Janeiro Cell Bank, Brazil), obtained from Mus musculus calvaria, were cultured in HAM F-10 and alfa-MEM medium without ascorbic acid, respectively, supplemented with 10% (v/v) FBS, 100 U/mL of penicillin and 100 μg/mL of streptomycin. Cultures were maintained at 37 °C in a humidified incubator with a 5% CO2 atmosphere. Cells were passaged every 2–3 days, and viability and cell density were periodically checked by the trypan blue dye exclusion test. The effects of the polymeric films on the viability of cancer cell lines (HeLa and PC3), and non-cancerous cell lines (HFF-1, Vero and MC3T3) were assessed by MTT reduction assay.42 The analyzed solid films were: (i) PVA/PEG, (ii) PVA/PEG containing GSNO (2.5 wt%), (iii) PVA/PEG containing AgNPs (2.5 wt%), and (iv) PVA/PEG containing GSNO (2.5wt%) and AgNPs (2.5 wt%). Briefly, the cells were seeded in a 24-well plate at a density of 3 x 104 cell/well (HFF-1) or 6 x 104 cells/well and incubated for 24 h at 37 °C for attachment. At the end of the incubation period, the medium was replaced with 900 µL of fresh medium and 14 mm2 pieces of each solid polymeric film were added to each well. The cells were then incubated with the polymeric films for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. The polymers and medium were removed from the wells and the cells were washed three times with PBS. Then 600 µL of MTT solution (0.3 mg/mL) prepared in the FBS-free medium was added to each well and the plates were incubated for 50 min (HeLa) or 2 h (PC3, HFF-1, Vero, MC3T3) at 37 °C. Finally, the MTT solution was removed from the wells and the formazan crystals were dissolved in 500 µL of dimethyl sulfoxide (DMSO). The plate was gently shaken at room temperature for 20 min and the absorbance was measured at 570 nm using a plate



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reader (Asys Expert Plus Microplate Reader, Biochrom, USA). Cells without treatment were used as a control for 100% cell viability. Each group included quadruplicate assays to confirm the results. All data were expressed as the mean ± SD. Statistical differences between groups were evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. A value of P < 0.05 was considered statistically significant.

2.11. Morphological evaluation of cells after treatment with the polymeric solid films

The morphology of HeLa, PC3, Vero and MC3T3 cells after 24 h incubation with (i) PVA/PEG, (ii) PVA/PEG containing GSNO (2.5 wt%), (iii) PVA/PEG containing AgNPs (2.5 wt%), and (iv) PVA/PEG containing GSNO (2.5wt%) and AgNPs (2.5 wt%) was observed with an inverted microscope (AxioVert A1, Zeiss, Jena, Germany) with phase contrast.18

3. Results and Discussion

3.1. Synthesis and characterization of PVA/PEG films containing GSNO and/or biogenic AgNPs

In this work, GSNO was synthesized and incorporated in PVA/PEG films, in the presence and absence of AgNPs. GSNO releases NO through homolytic S-N bond cleavage with free NO release (Eq. 1). To enhance the stability of GSNO, the NO donor was incorporated in solid PVA/PEG films, leading to the formation of transparent films. The incorporation of GSNO in PVA/PEG blended films allows a localized NO delivery


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suitable for biomedical applications, mainly topical NO delivery in dermatological applications.24,25 In addition, biogenic AgNPs were synthesized using green tea (Camellia sinensis) extract in an environmentally friendly protocol, as recently described.10 Traditional methods to synthesize AgNPs, such as the chemical reduction of silver ions, are generally expensive and involve the presence of hazardous chemicals imposing biological and environmental risks.34,38 In the light of green chemistry, in this work, AgNPs were biogenically synthesized using plant extracts. We recently reported the successful preparation of AgNPs using commercial green tea extract.10 In our previous publication,10 X-ray powder diffraction patterns (XDR) and X-ray photoelectron spectroscopy (XPS) were used to characterize the green tea synthesized AgNPs. The XRD patterns demonstrated the formation of Ag0 (AgNPs) face centered cubic planes, of single phase cubic AgNPs. Furthermore, the crystallite size of the AgNPs was found to be 2.17 nm.10 XPS was used to investigate the composition of the surface of green tea synthesized AgNPs.10 As expected, the chemical elements silver, oxygen and carbon were found on the surface of the nanoparticles. In addition, highresolution spectrum of the Ag indicated the formation of AgNPs.10 The obtained nanoparticles showed potent antimicrobial effects at concentrations that did not cause toxicity to mammalian cells.10 Green tea is rich in polyphenols, such as catechin, which act as reducing agents for silver ions yielding AgNPs.38 In addition, the presence of phytochemicals on the surface of AgNPs prevents nanoparticle agglomeration and oxidation.38 The formation of green tea synthesized AgNPs and their detailed characterization have been recently reported by our group.10 DLS measurements showed the formation of AgNPs with an average hydrodynamic size of 34.68 ± 4.95 nm, a PDI value of 0.28 ± 0.01, and negative zeta



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potential of - 35.5 ± 3.32 mV. The negative zeta potential value is due to the presence of polyphenols on the surface of green tea synthesized AgNPs.10 The NO donor, GSNO, and green tea synthesized AgNPs were individually or simultaneously incorporated in blended PVA/PEG films, suitable for biomedical applications, by solvent casting. Figure 2 shows the UV-visible absorption spectra of solid films of (i) PVA/PEG, (ii) PVA/PEG containing GSNO (2.5 wt%), (iii) PVA/PEG containing AgNPs (2.5 wt%), and (iv) PVA/PEG containing GSNO (2.5 wt%) and AgNPs (2.5 wt%), as indicated in the Figure. The addition of GSNO in PVA/PEG films (curves ii, and iv) causes the presence of a characteristic absorption band at 336 nm due to the π  π* transitions of the S-N bond group of GSNO, confirming the incorporation of the NO donor in the films.20,22,24 The presence of the absorption peak of GSNO at 336 nm in GSNO-containing PVA/PEG film was used to monitor the kinetics of GSNO decomposition and NO release in the film. For AgNPs-containing PVA/PEG films (curves iii and iv), the presence of the surface plasmon resonance (SPR) absorption band of AgNPs at about 405 nm, confirms the presence of AgNPs. The SPR absorption band is due to the interactions of incident light with the electrons on the surface of AgNPs.26 The presence of a sharp characteristic SPR absorption band at 405 nm indicated the presence of nanometer-sized AgNPs in the solid matrix of PVA/PEG films.9,10 Thus, Figure 2 confirms the incorporation of GSNO and nanosized AgNPs in PVA/PEG films. Figure 3A shows the XRD patterns of the PVA/PEG film (black line) and PVA/PEG films containing-AgNPs and GSNO (red line). Figure 3B shows detailed XRD spectra of both films. The XRD spectrum of solid a PVA/PEG film exhibits a broad diffraction peak at 2  8.71º due to the (101) reflection plane of semicrystalline


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PVA.26,27,43–45 This peak is due to the semi-crystalline nature of PVA due to the presence of hydrogen bonding between PVA molecule chains.29,45 The peak observed at 2  10.51º corresponds to the presence of PEG.36 It should be noted that the presence of PEG in PVA/PEG films favors the formation of hydrogen bonding between PEG and PVA chains, decreasing the semi-crystalline nature of PVA.46 The presence of AgNPs in PVA/PEG films led to the appearance of diffraction peaks at 2 of º28.40º and 33.24º, which correspond to the XRD pattern of the indexed (111), (220), and (311) planes of face-centered cubic (FCC) AgNPs. Similar results were reported elsewhere.7,27,28,47 The broadening of the XRD peaks is related to the small grain size of AgNPs.7 These results demonstrated the successful incorporation of well dispersed AgNPs in the amorphous PVA/PEG matrix.26 Figure 4 shows the FTIR spectra of PVA/PEG film (black line) and PVA/PEG film containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) (red line). The broad band at 3325 cm-1 is assigned to the stretching mode of the O-H group of the polymeric chains involved in H-bonding.7,28 Changes in the intensity and position of this band (by comparing black and red lines in Figure 4) indicate the -OH group interactions between PVA/PEG and the GSNO and AgNPs.28 In the PVA/PEG film (black line), the band at 2954 cm-1 is attributed to the presence of asymmetric stretching vibrations of C-H groups.28,48,49 However, in PVA/PEG films containing GSNO and AgNPs (red line), this band was shifted from 2954 cm-1 to 2931 cm-1 due to interactions of AgNPs, GSNO and the PVA/PEG matrix.28 The band at 1438 cm−1 is attributed to the in-plane swaying vibration of C-H groups.7 The band at 1095 cm-1 (black curve) is attributed to C-O stretching, present in polymeric chains.28,48 This band was shifted to 1067 cm-1 for PVA/PEG films containing GSNO and AgNPs (red curve). This band increased its intensity and shifted to lower wavenumber values compared to pure PVA/PEG film.



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This might be a result of molecular interactions, such as H-bonding between O-H groups of PVA/PEG chains with GSNO, in addition to AgNPs, as previously reported.23,28 The band at 1431 cm-1 is attributed to the in-plane swaying vibration of C-H groups.7 Characteristic vibration bands of carbonyl function (originally in the PVA moieties due to incomplete hydrolysis) are found at 1644 cm−1, as previously reported.48 Overall, FTIR measurements indicate the presence and interactions of GSNO and AgNPs with the polymeric matrix. Figure 5 shows the morphology of the solid PVA/PEG film (Fig. 5A) and PVA/PEG film containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) (Fig. 5B), using SEM micrographs. As expected, PVA/PEG films have a homogenous surface morphology, a smooth texture with an absence of pores, due to the positive interactions of PVA and PEG chains, and no significant formation of aggregates and phase separation (Fig. 5A). Similar results were previously reported for solid PVA films.20,24,28 In contrast, the SEM micrograph of a PVA/PEG film containing GSNO and AgNPs obviously shows a number of homogeneously distributed grains with smaller sizes (at nanoscale) attributed to quasi-spherical shaped AgNPs (Fig. 5B). During solvent cast evaporation, a degree of agglomeration of smaller AgNPs through the less viscous PVA/PEG solution might lead to the formation of nanoparticle aggregates.9 Similar results have been reported for PVA-based films containing AgNPs.9,27,29,45 EDS microanalysis is a technique used for identification of the elemental composition of a material. In addition, EDS microanalysis can generate a map of one or more selected chemical elements of interest in a sample. Analysis of a PVA/PEG film containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) through an EDS spectrometer confirmed the presence of elemental carbon, oxygen, sulfur, and silver signals. As expected, carbon and oxygen in the examined film are attributed to the polymeric matrix


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of PVA/PEG, and GSNO. Sulfur is due to the presence of the NO donor, GSNO; while silver is due to the presence of AgNPs in the films. Figure 6A shows the SEM image of a PVA/PEG film containing GSNO (2.5 wt%) and AgNPs (2.5 wt%). This image was selected to map carbon (Fig.6B), oxygen (Fig.6C), sulfur (Fig. 6D) and silver (Fig. 6E) atoms. The colored dots in Figures 6(B–E) represent the EDS elemental mapping of carbon, oxygen, sulfur and silver atoms, respectively. In all cases, a homogeneous distribution of dots can be observed in the mapped area. Our results are in accordance with previous reports for AgNPs containing PVA-based films.7,27,45

3.2. Kinetics of GSNO decomposition with NO release in PVA/PEG films containing GSNO

Figure 7 shows the kinetic curves of NO release from GSNO (2.5 wt%), incorporated in the solid PVA/PEG film in the dry condition. These kinetic curves correspond to the release of NO from GSNO (Eq. 1).20,22 The kinetic curves were monitored at 25 °C (room temperature), at 32.5 °C (skin temperature), and at 37 °C (physiological temperature) for 24 h, in the dark. In all temperatures, a sustained NO release was observed for at least 24 h. The viscous polymeric matrix increases the thermal stabilization of the NO donor.20 The initial rates IR of NO release from GSNOcontaining PVA/PEG films were found to be 0.44 µmol cm-2 h-1, 0.59 µmol cm-2 h-1 and 0.92 µmol cm-2 h-1 at 25 ºC, 32.5 ºC and 37 °C, respectively. As expected, the initial rates of NO release from GSNO increase with the increase of the temperature.18,20,33 As shown in Figure 7, the amount of NO release from the polymeric films is in the range of 4–7 µmol of NO per cm2 of the polymeric film. At this range of concentration, NO is expected to have biological effects, such as cytotoxicity towards



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tumorigenic cells and antibacterial effects.13 For topical applications, the amount of NO release from other formulations is in the range of low µmol of NO per 100 gram of the matrix, allowing a transdermal NO delivery.14 In addition, the preparation of modified solid PVA films containing RSNO for topical NO release has been reported.24 The authors reported that these films, which release 0.3 µmol of NO per cm2, were able to increase dermal vasodilation more than 5-fold in human subjects.24 In a similar approach, NO-release from PVA membrane (64–101 nmol of NO released per cm2) was observed to produced dermal vasodilation.50 Moreover, Seabra et al. reported the preparation of polymeric solid films of polynitrosated polyester blended with poly(methyl methacrylate) for topical NO delivery. The obtained film released 4.6 nmol of NO per cm2 per hour. At this concentration, the polymeric film displayed potent antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa strains.51 Therefore, GSNO-containing PVA/PEG films might have important biomedical applications due to the ability to release therapeutic amounts of NO. 3.3. In vitro diffusion of AgNPs and GSNO from PVA/PEG films containing GSNO and AgNPs

The diffusion of intact GSNO and AgNPs from PVA/PEG films containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) was monitored by Franz diffusion cell.40 The donor-receptor compartment was monitored for intact free GSNO and AgNPs diffusion for 4 h. The amount of GSNO diffused from the film through the membrane to the receptor compartment was assayed by the detection of the absorption band of GSNO at 336 nm (ε = 980 mol−1 L cm−1), while the diffusion of AgNPs was assayed by the detection of total silver using ICP-MS. Figure 8 shows the diffusion profile of GSNO and AgNPs from the polymeric films. An initial burst of 32% of the GSNO and 37% of


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the AgNPs diffused from the films after 4 h of monitoring. Moreover, after 24 h of monitoring the amounts of GSNO and AgNPs diffused from the films were 38% and 41%, respectively, (data not shown). As can be seen in Figure 8, there is an initial burst in GSNO and AgNPs diffusion, during the first 2 hours of monitoring and a sustained diffusion that continued for at least 24 h (data not shown). Similar results were reported for GSNO-diffusion from polymeric hydrogels.40 Thus, these results indicate that PVA/PEG films can promote a sustained diffusion of GSNO and AgNPs. It should be noted that ICP-MS detects the total amount of silver released from the polymeric films, which might contain a contribution of AgNPs and silver ions (Ag+). AgNPs and Ag+ are reported to have cytotoxic and antimicrobial effects.3,34 To investigate the diffusion mechanism of GSNO and AgNPs from PVA/PEG films, two mathematical models were used to the results (data recorded in Figure 8): Higuchi and Korsmeyer-Peppas. Table 1 shows the correlation coefficients (R2) and the release constants for the two models applied to the kinetic curves obtained in Figure 8. Both mathematical models exhibit a good coefficient of linearity. The Higuchi model presented a linearity of 0.941 for GSNO and 0.951 for AgNPs, indicating fickian diffusion.52 The Korsmeyer-Peppas model demonstrated suitable coefficients of linearity of 0.932 for GSNO and 0.947 for AgNPs. The Korsmeyer-Peppas model suggests classic fickian diffusion with n values of 0.2 for GSNO and 0.309 for AgNPs.53,54 Taken together, these two mathematical models indicate the fickian diffusion mechanism for both GSNO and AgNPs from PVA/PEG films.

3.4. Antibacterial activities of PVA/PEG films containing GSNO and/or AgNPs (disk-diffusion method)



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The disk-diffusion method was employed to characterize the antibacterial effect of the following polymeric films: PVA/PEG containing AgNPs (0.5 wt%); PVA/PEG films containing AgNPs (2.5 wt%), and PVA/PEG films containing GSNO (2.5 wt%) and AgNPs (2.5 wt%). The antibacterial activity was assayed against E. coli, K. pneumoniae, P. aeruginosa, and S. aureus ATCC strains. In the agar disk-diffusion assay, the formation of a zone of inhibition indicates an antibacterial effect. For all tested groups, a noticeable zone of inhibition was observed. Figure 9 shows the mean diameter of the bacterial zone of inhibition for the tested polymeric films, as indicated in the Figure. The experiments showed no zone of inhibition for a pure PVA/PEG film, which was used as negative control. The antibacterial activity of Ag/PVA film is supported by the literature.9,29 As expected, by increasing the amount of AgNPs in PVA/PEG films (from 0.5 to 2.5 wt%), an increase in the antibacterial effect was observed (Figure 9). This effect was most significant for P. aeruginosa. The combination of GSNO and AgNPs in the polymeric film (PVA/PEG films containing GSNO (2.5 wt%) and AgNPs (2.5 wt%)) slightly increased the antibacterial effect of the film in comparison to the film without the NO donor. This effect was more evident for E. coli. Thus, our results demonstrate that the polymeric films containing AgNPs have an antibacterial effect against different bacteria strains, and this effect can be enhanced by increasing the amount of AgNPs in the film or by adding GSNO in the film. Polymer films of PVA/PEG containing only GSNO (2.5 wt%) had no antibacterial effect (data not shown). Fatema et al. reported antibacterial effects of Ag/PVA nanocomposite films against E. coli and S. aureus.9 Similarly, a PVA-based film containing AgNPs showed an antibacterial effect against E. coli and S. aureus, as assayed by the disk-diffusion method (diameters the inhibition zones reported to be 13 and 12 mm for E. coli and S.


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aureus, respectively).29 The antibacterial activity of PVA-based films containing AgNPs has also been demonstrated against foodborne pathogens Salmonella paratyphi and S. aureus.28 The antibacterial activity of PVA-based film containing different concentrations of AgNPs against S. aureus (MRSA) and E. coli (DH5-alpha), with similar diameters of inhibition zones, was also reported.27 The antibacterial mechanisms of AgNPs are not completely understood. However, it is thought that AgNPs might interact with the bacterial cell membrane, changing the outer membrane, resulting in degradation and eventually causing bacteria death.3 In addition, AgNPs might release silver ions (Ag+), which are involved in the generation of reactive oxygen species (ROS) (i.e. hydrogen peroxide, hydroxyl radical, superoxide), and singlet oxygen leading to oxidative stress and bacteria death.55 Moreover, AgNPs can diffuse through the bacterial membrane, inhibiting of cell proliferation and causing bacteria death.3,56,57 Although PVA/PEG films containing GSNO (2.5 wt%) did not cause antibacterial effects against the tested bacteria strains, the combination of GSNO (2.5 wt%) and AgNPs (2.5 wt%) slightly increased the antibacterial effect of the film. This effect was more evident for E. coli and absent for S. aureus (Figure 9). Depending on its concentration, NO donors can have antibacterial effects.13 Since it is relatively lipophilic, NO can easily cross cell membranes. NO can have antibacterial effects by reacting with superoxide (a product from bacteria respiration) leading to the formation of peroxynitrite, a harmful and powerful oxidant molecule. Moreover, NO can react with oxygen, leading to trioxide dinitrogen, which can modify intracellular and membrane proteins.13,58–60 In practice, PVA/PEG films containing GSNO and/or AgNPs can be used as antibacterial coating either directly to the infected wounds or to coat medical devices.



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In our study, commercial antibiotics were used to validate the disk-diffusion method employed. Different antibiotics with different concentrations are recommended to validate the method, according to the ATCC and CLSI guidelines. The antimicrobial compounds Kanamycin (at a concentration of 30 µg), Gentamicin (at a concentration of 10 µg), Meropenem (at a concentration of 10 µg), and Cefoxitin (at a concentration of 30 µg) were used as positive controls for E.coli, K. pneumoniae, P. aeruginosa and S. aureus strains, respectively. In all cases, we observed the formation of the inhibition zone, indicating that the technique is validated. However, a direct comparison between the diameters of the inhibition zone of the bacteria strains treated with recommed antibiotics and PVA/PEG films containing AgNPs and/or GSNO is not appropriate since: (i) each bacteria strain has a specific recommended positive control (in contrast, we used the same PVA/PEG film containing AgNPs for all bacteria strains), (ii) the positive controls have a high purity and high concentration, (iii) the positive controls are recommend for systemic application in vivo. In contrast, PVA/PEG films containing AgNPs and/or GSNO are designed for topical/local applications. A direct comparison with the antimicrobials used as positve controls is not appropriated since these drugs are for systemic employment. Thus, the positive control groups can be used to validate the technique.

3.5. Cell viability assay

The cell viability of two carcinogenic cell lines, human cervical carcinoma cell (HeLa) and human prostate cancer cell (PC3) was evaluated upon treatment with: PVA/PEG films, PVA/PEG films containing GSNO (2.5 wt%), PVA/PEG films containing AgNPs (2.5 wt%), and PVA/PEG films containing GSNO (2.5 wt%) and


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AgNPs (2.5 wt%), as indicated in Figure 10. Cells without treatment were used as a control group for 100% cell viability. As can be seen in Figure 10, for both cell lines, PVA/PEG solid films did not decrease cell viability, as expected. In addition, the presence of GSNO (2.5 wt%) in the polymeric films slight decreases cell viability for both cell lines. The viability of HeLa cells was not affected upon treatment with PVA/PEG films containing AgNPs (2.5 wt%) (Figure 10A), while PC3 cells were significantly affected by the same treatment (Figure 10B). These results indicate a selective sensitivity of AgNPs-containing polymeric films to PC3 cells. Interestingly, the combination of GSNO and AgNPs in the PVA/PEG films significantly reduced the viability of both cancer cell lines (Figure 10). These results suggest a synergistic effect of GSNO and AgNPs towards these two cancer cell lines. To the best of our knowledge, this is the first report to show the synergist effect of GSNO and AgNPs towards tumorigenic cell lines. In addition, the cytotoxicity of PVA/PEG films, PVA/PEG films containing GSNO (2.5 wt%), PVA/PEG films containing AgNPs (2.5 wt%), and PVA/PEG films containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) was evaluated against noncancerous cell lines: human foreskin fibroblast (HFF-1) (Figure 11A), epithelial cells from African Green monkey (Vero) (Figure 11B), and pre-osteoblastic (MC3T3) (Figure 11C). As can be observed in Figure 11, PVA/PEG and PVA/PEG containing GSNO did not significantly decreased cell viability for all tested cells. Although a slight decrease of Vero cells viability was decrease upon treatment with PVA/PEG containing GSNO (Figure 11 B). A significant decrease in HFF-1 cell viability was observed for cells treated with PVA/PEG films containing AgNPs and for PVA/PEG films containing GSNO and AgNPs. These results suggested that the observed decrease of HFF-1 viability might be associated with the presence of AgNPs in the polymeric films.



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However, for Vero and MC3T3 cells, PVA/PEG films containing AgNPs did not decrease cell viability (Figure 11B, C). Differently from the cancer cell lines (Figure 10), a synergistic effect of GSNO and AgNPs were not observed for HFF-1 and MC3T3 cell lines (Figure 11A, C). However, a significant decrease in Vero cell viability was observed for treatment with PVA/PEG films containing both GSNO and AgNPs (Figure 11B). These results indicate that the effects of these polymeric films depend on the cell line. It should be highlighted that these films were designed for topical applications (localized release of AgNPs and/or NO to the target site of application) directly to cancer tissue/organ in order to avoid undesired toxicity to non-cancerous cells. Taken together, these results suggested that the cytotoxicity of these films is depended on the target cell, and more studies are required to verify the toxicity of these films towards different noncancerous and cancerous cell lines. Results reported in Figure 10 (cytotoxicity of polymeric films to cancer cell lines) were also confirmed using a qualitative morphological characterization. The morphology of HeLa and PC3 cancer cell lines incubated with PVA/PEG films, PVA/PEG films containing GSNO (2.5 wt%), PVA/PEG films containing AgNPs (2.5 wt%), and PVA/PEG films containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) for 24 h was investigated (Figures 12 and 13, respectively). For both cell lines, it can be observed the presence of confluent monolayer cells for the negative control (non-cytotoxic), typically epithelial like, as expected for the growth pattern of HeLa and PC3 cells. 61-63 HeLa cells present a polygonal shape, with regular dimensions and discrete separation from each other (Figure 12A).61,62 PC3 cells present a polygonal morphology but more scattered and elongated (Figure 13A).63 For both cell lines, it was not possible to observe signals of cell degradation or cellular debris (Figures 12A and 13A). The presence of PVA/PEG solid films did not decrease cell viability, as can be seen in Figures 12B and


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13B. Both cell lines showed a growth pattern similar to the negative control group, with no changes in cell morphology. For PC3 cell line, it was also observed through the images the core presenting one or more nucleoli and vacuoles (Figure 13B2). For HeLa cells, these features were more difficult to observe, due the high cellular density (Figure 12B2). In quantitaive assays (MTT) (Figure 10) the presence of GSNO in the polymer film did not significant decrease the cell viability for both cell lines, presenting a cell viability above 75% (Figures 12C and 13C). This decrease was not noticed in morphological analyzes (Figures 12C2 and 13C). The cells maintain the typical characteristics of viability, according to the growth pattern of HeLa and PC3 cells, as well as observed for negative control (Figure 12A and Figure 13A). In addition, with MTT results, the viability of HeLa cells was not affected upon treatment with PVA/PEG films containing AgNPs (2.5 wt%), while PC3 cells were significantly affected by the same treatment (Figure 10). On the other hand, the morphological assays showed that the presence of AgNPs caused a cytotoxicity for HeLa cells (Figure 12D), although less prominently than that observed for PC3 cells (Figure 13D). In both cases, it was possible to observe the presence of remnant AgNPs particles 12D1 and 13D1) and the cells presented non-attached rounded loose morphology, lifted from the culture plates, and indications of cell death (Figure 12D2 and 13D2). The synergistic effect of GSNO and AgNPs in the PVA/PEG films was also observed and confirmed by morphological assays (Figures 12E and 13E). Significant reduction of cell viability was noticed for both cancer cell lines upon treatment with PVA/PEG films containing GSNO and AgNPs. In this case, the presence of a lot of cells in suspension, rounded cells and debris, indicate typical phenotype of cytotoxicity (Figures 12E1 and 13E2). The morphological characterization of non-cancerous cell lines (Vero and MC3T3) upon treatment with PVA/PEG films containing GSNO (2.5 wt%), PVA/PEG



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films containing AgNPs (2.5 wt%), and PVA/PEG films containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) are presented and discussed in the Supporting information, Figures S1 and S2). Overall, the MTT analyses of cell viability of non-cancerous cell lines are in accordance with cell morphological evaluation (Supporting Information). The anticancer activity of AgNPs has been reported on several different cell lines such as, skin cancer cell line, human and murine alveolar macrophage cell line, human breast adenocarcinoma cell line, human peripheral blood mononuclear cells, epithelioma and lung , human alveolar epithelial cell line, among others.6,64 The cytotoxicity of AgNPs to cancer cell lines is attributed to the cellular apoptosis.65 Indeed, cellular uptake of AgNPs is known to cause cell shrinkage, fragmentation, damage to the plasma cell membrane, externalization, nuclear condensation, disruption in the mitochondrial respiratory chain, generation of ROS, and cleavage of substrates responsible for the repair of DNA. These alterations are related to the increase of Bax (pro-apoptotic protein) and a decrease of Bcl-2 (anti-apoptotic protein).5,64,66,67 The cytotoxicity of AgNPs towards cancer cells is known to depend on the cancer cell line, nanoparticle size distribution, chemical surface, surface charge, aggregation state, and concentration, among others.5,66,67 To the best of our knowledge, this is the first report to show the in vitro toxicity of plant-mediated synthesis AgNPs incorporated in a polymeric matrix. NO is important in cancer biology and NO donors have been extensively used for anticancer activities.13,19 The cytotoxicity of NO donors to cancer cells is due to the cancer cell line, the nature of the NO donor and its concentration.13,66 There are many proposed mechanisms of cytotoxicity, or proliferation arrest, due to NO donors and a cellular specific response.68,69 NO affects the mitochondrial physiology of cancer cells, exerting concentration-dependent effects on mitochondrial respiration, ATP formation,


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cytochrome c release and the generation of ROS.13,68 The toxicity of NO might also involve the inhibition of ATP and ribonucleotide reductase, NF-κB (multifunctional transcription factor), mediators of protein expression, or cell membrane damage.70-72 The cellular apoptosis is frequently the final cytotoxic effect of these compounds.70 Interestingly, Figures 10, 12E and 13E shows that the combination of GSNO and AgNPs has a superior effect on the decrease of cell viability of both cell lines, compared with cells treated individually with GSNO or AgNPs. Currently, NO-mediated anticancer therapy might be achieved by chemosensitization of tumor cells.67 Thus, this observation has motivated and inspired researchers to combine NO donors allied with cytotoxic nanomaterials for the improvement of chemotherapy. Indeed, several papers described the successful combination of NO donors with traditional chemotherapeutic drugs (such as doxorubicin and cisplatin) in cancer treatment.69,70,74 As AgNPs are known to have toxicity towards different cancer cell lines, this observation has motivated us to combine NO donor (GSNO) with AgNPs in a single polymeric matrix to potentiate the cytotoxicity of the obtained nanocomposite. As far as we know, this is the first report to demonstrate the potential synergistic effect of GSNO and AgNPs, in a polymer matrix, against tumorigenic cell lines. Further studies are required to better understand the biological effects of PVA/PEG films containing GSNO and/or AgNPs. For instance, the mechanisms of toxicity of PVA/PEG films containing GSNO and AgNPs are under investigation, as well as the impact of these films on different cancer cell lines, compared to non-cancerous cells. Moreover, as NO donors are known to regulate cancer cell sensitivity, the combination of PVA/PEG films containing GSNO and AgNPs with to traditional chemotherapeutic drugs (such as cisplatin) represents a promising topic to be explored.



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4. Conclusions

In this work, polymeric blended films of PVA/PEG containing GSNO and/or AgNPs were successfully prepared by a solvent casting method, leading to the formation of homogeneous solid films containing well-dispersed AgNPs. Using green chemistry, AgNPs were biogenically synthesized with green tea extract and incorporated in the polymeric matrix. The NO donor, GSNO, was synthesized by nitrosation of glutathione. The obtained biomaterial demonstrated suitability for biomedical applications, highlighting the possibility of a direct application of NO and/or AgNPs to the target location, where a sustained release of GSNO and AgNPs was demonstrated. GSNO and/or AgNPs incorporated in PVA/PEG films were characterized by different techniques, which demonstrated the successful formation of the nanocomposite. In addition, the films demonstrated a potent antibacterial activity against Gram-negative E. coli, K. pneumoniae, and P. aeruginosa and gram-positive S. aureus. In addition to the antibacterial activity, the cytotoxicity of the PVA/PEG polymeric films containing GSNO and/or AgNPs was observed against HeLa and PC3 tumor cell lines. Interestingly, cell viability assays showed a significant decrease of tumor cell viability when combining GSNO and AgNPs into PVA/PEG films, suggesting a potent synergic effect. Thus, the PVA/PEG films containing GSNO and/or AgNPs films demonstrated a potent antibacterial activity and cytotoxic effect against tumor cell lines. These films might find important biomedical applications in tissue engineering for the topical and sustained release of NO and AgNPs. In clinical setting these films can be used as wound dressing (in particular to treat infected wounds), in direct applications of the films on solid tumors, where NO and AgNPs can be released and have localized cytotoxicity effects, and to coat medical devices creating an antibacterial coating to avoid the growth


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of bacteria. It should be noted that before clinical applications, the in vivo toxicity of these films must be carefully evaluated.

Acknowledgments: We have appreciated the support from CNPq, FAPESP (2016/10347-6, 2018/02832-7). We would like to thank Prof. Bruno Lemos Batista for his assistance with ICP-MS measurements. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) Finance Code 001.

Figure Captions

Figure 1. Schematic representation of the preparation of solid films of (i) PVA/PEG, (ii) PVA/PEG containing GSNO (2.5 wt%), (iii) PVA/PEG containing AgNPs (2.5 wt%), and (iv) PVA/PEG containing GSNO (2.5 wt%) and AgNPs (2.5 wt%). All groups were prepared by solvent cast evaporation.

Figure 2. Uv-visible absorption spectra of solid films of (i) PVA/PEG, (ii) PVA/PEG containing-GSNO (2.5 wt%), (iii) PVA/PEG-containing AgNPs (2.5 wt%), and (iv) PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%), as indicated in the Figure. All solid films were cast by solvent evaporation onto the cavity of a detachable quartz cell.

Figure 3. XRD patterns of the PVA/PEG film (black line) and PVA/PEG films containing-GSNO (2.5 wt%) and AgNPs (2.5 wt%) (red line) (A). Detailed XRD spectra of both films (B).



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Figure 4. FTIR spectra of PVA/PEG film (black line) and PVA/PEG film containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) (red line).

Figure 5. Morphology of PVA/PEG solid film (A) and PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) (B), evaluated by SEM.

Figure 6. SEM image of PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) (A). This image was selected to map carbon (B), oxygen (C), sulfur (D) and silver (E) atoms. The colored dots in Figures (B-E) represent the EDS elemental mapping of carbon, oxygen, sulfur and silver atoms, respectively.

Figure 7. Kinetic curves of NO release from GSNO (2.5 wt%), incorporated in solid film of PVA/PEG, in dry condition, at (i) 25°C (room temperature), (ii) 32.5°C (skin temperature), (iii) 37°C (physiological temperature) for 24 h, in the dark. The films were cast by solvent evaporation onto the cavity of a detachable quartz cell.

Figure 8. The diffusion profile of intact GSNO (curve i) and AgNPs (curve ii) from PVA/PEG films-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%), monitored through an artificial membrane in a vertical Franz diffusion cell.

Figure 9. Mean of diameters of the bacteria zone of inhibition for: PVA/PEG-containing AgNPs (0.5 wt%) (black column), PVA/PEG-containing AgNPs (2.5 wt%) (red column), and PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) (blue


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column). The antibacterial activity was assayed against E. coli ATCC 25922, K. pneumoniae ATCC 700603, P. aeruginosa ATCC 27853, and S. aureus ATCC 29213. Inhibition zones were not observed for free PVA/PEG films used as negative controls.

Figure 10. Cell viability of human cervical carcinoma cell (HeLa) (A) and human prostate cancer cell (PC3) (B), evaluated upon treatment with: PVA/PEG solid film (red column), PVA/PEG-containing GSNO (2.5 wt%) (blue column), PVA/PEG-containing AgNPs (2.5 wt%) (green column), PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) (white column). Cells without treatment were used as a control group for 100% cellular viability, as a negative control group (black column).

Figure 11. Cell viability of non-cancerous cell lines: human foreskin fibroblast (HFF1) (A), fibroblast-like epithelial Vero cell (B), and pre-osteoblastic MC3T3 cell (C) evaluated upon treatment with: PVA/PEG solid film (red column), PVA/PEGcontaining GSNO (2.5 wt%) (blue column), PVA/PEG-containing AgNPs (2.5 wt%) (green column), PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) (white column). Cells without treatment were used as a control group for 100% cellular viability, as a negative control group (black column).

Figure 12. Cell viability of human cervical carcinoma cell (HeLa), morphological characterization, evaluated upon treatment with: (A1, A2) Cells without treatment, negative control group, (B1, B2) PVA/PEG solid film, (C1, C2) PVA/PEG-containing GSNO (2.5 wt%), (D1, D2) PVA/PEG-containing AgNPs (2.5 wt%) and (E1, E2) PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%). Groups A1-E1: 10x and Groups A2-E2: 20x. 35 ACS Paragon Plus Environment


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Figure 13. Cell viability of human prostate cancer cell (PC3), morphological characterization, evaluated upon treatment with: (A1, A2) Cells without treatment, negative control group, (B1, B2) PVA/PEG solid film, (C1, C2) PVA/PEG-containing GSNO (2.5 wt%), (D1, D2) PVA/PEG-containing AgNPs (2.5 wt%) and (E1, E2) PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%). Groups A1-E1: 10x and Groups A2-E2: 20x.

Table 1. Release parameters for GSNO and AgNPs diffusion from PVA/PEG films accordingly to the Higuchi and Kosmeyer-Peppas mathematical models.

Mathematical model Higuchi

Korsmeyer-Peppas

r2

KH (%.h-1/2)

r2

n

Kk (%.h-n)

GSNO

0.941

5.512

0.932

0.2

0.258

AgNPs

0.951

21.142

0.947

0.309

1.264

ASSOCIATED CONTENT Supporting Information available: Morphological evaluation of non-cancerous cells (Vero and MC3T3) after treatment with the polymeric solid films - cell viability assay.

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Antibacterial and cytotoxicity activity to tumor cells of polymeric solid film containing biogenic AgNPs and nitric oxide donor for topical applications 421x189mm (150 x 150 DPI)

ACS Paragon Plus Environment


Figure 1. Schematic representation of the preparation of solid films of (i) PVA/PEG, (ii) PVA/PEG containing GSNO (2.5 wt%), (iii) PVA/PEG containing AgNPs (2.5 wt%), and (iv) PVA/PEG containing GSNO (2.5 wt%) and AgNPs (2.5 wt%). All groups were prepared by solvent cast evaporation. 367x196mm (150 x 150 DPI)


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Figure 2. Uv-visible absorption spectra of solid films of (i) PVA/PEG, (ii) PVA/PEG containing-GSNO (2.5 wt%), (iii) PVA/PEG-containing AgNPs (2.5 wt%), and (iv) PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%), as indicated in the Figure. All solid films were cast by solvent evaporation onto the cavity of a detachable quartz cell. 254x190mm (96 x 96 DPI)



Figure 3. XRD patterns of the PVA/PEG film (black line) and PVA/PEG films containing-GSNO (2.5 wt%) and AgNPs (2.5 wt%) (red line) (A). Detailed XRD spectra of both films (B). 318x140mm (150 x 150 DPI)


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Figure 4. FTIR spectra of PVA/PEG film (black line) and PVA/PEG film containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) (red line). 254x190mm (96 x 96 DPI)



Figure 5. Morphology of PVA/PEG solid film (A) and PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) (B), evaluated by SEM. 204x92mm (150 x 150 DPI)


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Figure 6. SEM image of PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) (A). This image was selected to map carbon (B), oxygen (C), sulfur (D) and silver (E) atoms. The colored dots in Figures (B-E) represent the EDS elemental mapping of carbon, oxygen, sulfur and silver atoms, respectively. 329x141mm (150 x 150 DPI)



Kinetic curves of NO release from GSNO (2.5 wt%), incorporated in solid film of PVA/PEG, in dry condition, at (i) 25°C (room temperature), (ii) 32.5°C (skin temperature), (iii) 37°C (physiological temperature) for 24 h, in the dark. The films were cast by solvent evaporation onto the cavity of a detachable quartz cell. 254x190mm (96 x 96 DPI)


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Figure 8. The diffusion profile of intact GSNO (curve i) and AgNPs (curve ii) from PVA/PEG films-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%), monitored through an artificial membrane in a vertical Franz diffusion cell. 254x190mm (96 x 96 DPI)



Figure 9. Mean of diameters of the bacteria zone of inhibition for: PVA/PEG-containing AgNPs (0.5 wt%) (black column), PVA/PEG-containing AgNPs (2.5 wt%) (red column), and PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) (blue column). The antibacterial activity was assayed against E. coli ATCC 25922, K. pneumoniae ATCC 700603, P. aeruginosa ATCC 27853, and S. aureus ATCC 29213. Inhibition zones were not observed for free PVA/PEG films used as negative controls. 163x136mm (150 x 150 DPI)


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Figure 10. Cell viability of human cervical carcinoma cell (HeLa) (A) and human prostate cancer cell (PC3) (B), evaluated upon treatment with: PVA/PEG solid film (red column), PVA/PEG-containing GSNO (2.5 wt%) (blue column), PVA/PEG-containing AgNPs (2.5 wt%) (green column), PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) (white column). Cells without treatment were used as a control group for 100% cellular viability, as a negative control group (black column). 278x127mm (150 x 150 DPI)



Figure 11. Cell viability of non-cancerous cell lines: human foreskin fibroblast (HFF-1) (A), fibroblast-like epithelial Vero cell (B), and pre-osteoblastic MC3T3 cell (C) evaluated upon treatment with: PVA/PEG solid film (red column), PVA/PEG-containing GSNO (2.5 wt%) (blue column), PVA/PEG-containing AgNPs (2.5 wt%) (green column), PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%) (white column). Cells without treatment were used as a control group for 100% cellular viability, as a negative control group (black column). 440x147mm (150 x 150 DPI)


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Figure 12. Cell viability of human cervical carcinoma cell (HeLa), morphological characterization, evaluated upon treatment with: (A1, A2) Cells without treatment, negative control group, (B1, B2) PVA/PEG solid film, (C1, C2) PVA/PEG-containing GSNO (2.5 wt%), (D1, D2) PVA/PEG-containing AgNPs (2.5 wt%) and (E1, E2) PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%). Groups A1-E1: 10x and Groups A2-E2: 20x. 190x338mm (96 x 96 DPI)



Figure 13. Cell viability of human prostate cancer cell (PC3), morphological characterization, evaluated upon treatment with: (A1, A2) Cells without treatment, negative control group, (B1, B2) PVA/PEG solid film, (C1, C2) PVA/PEG-containing GSNO (2.5 wt%), (D1, D2) PVA/PEG-containing AgNPs (2.5 wt%) and (E1, E2) PVA/PEG-containing GSNO (2.5 wt%) and AgNPs (2.5 wt%). Groups A1-E1: 10x and Groups A2-E2: 20x. 190x254mm (96 x 96 DPI)


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Antimicrobial Activity and Cytotoxicity to Tumor ... - ACS Publications

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