Sonication Assisted Hierarchical Decoration of Ag-NP on Zinc

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Sonication assisted Hierarchical decoration of Ag-NP on Zinc Oxide Nanoflowers impregnated Eggshell Membrane: Evaluation of Antibacterial Activity and in vitro Cytocompatibility Preetam Guha Ray, Shreya Biswas, Trina Roy, Saptarshi Ghosh, Deblina Majumder, Piyali Basak, Somenath Roy, and Santanu Dhara ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.9b01185 • Publication Date (Web): 15 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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ACS Sustainable Chemistry & Engineering

Sonication assisted Hierarchical decoration of Ag-NP on Zinc Oxide Nanoflowers impregnated Eggshell Membrane: Evaluation of Antibacterial Activity and in vitro Cytocompatibility Preetam Guha Ray1,2, Shreya Biswas2, Trina Roy1, Saptarshi Ghosh3, Deblina Majumder4, Piyali Basak2, Somenath Roy5*, Santanu Dhara1* 1

Biomaterials and Tissue Engineering Laboratory, School of Medical Science and Technology (SMST), Indian Institute of Technology Kharagpur, Kharagpur 721302, India 2

School of Bioscience and Engineering, Jadavpur University, Kolkata-700032, India

3

Faculty of Science, Holon Institute of Technology, Holon 58102, Israel

4

Functional Materials & Devices Division, CSIR - Central Glass and Ceramic Research Institute, Kolkata – 700032, India 5

Central Glass and Ceramic Research Institute, Khurja Center, Khurja-203131

*corresponding authors Dr. Santanu Dhara

Dr. Somenath Roy

E-mail: [email protected]

E-mail: [email protected]

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Abstract Metal/ metal oxide nanoparticles have long been used as an antibacterial substitute; but fabrication of an effective carrier or delivery matrix for achieving a sustain release profile with high bactericidal efficacy alongwith good cytocompatibility is still an unresolved challenge. Herein, the study demonstrates a facile and unique route to fabricate a hierarchical nanobiocomposite with effective loading of ZnO/ silver nanoparticles (Ag–NPs) in order to attain excellent bactericidal efficacy with good and sustainable release profile. Surface functionalized eggshell membranes (ESM) were deployed as three dimensional loading matrices for efficient loading of ZnO/ Ag– NPs. A simple sonochemical guided approach was adopted to synthesize ZnO nanoflakes in-situ onto the microfibrous ESM and decorate it with Ag–NPs to fabricate a nanobiocomposite. Microstructural analysis confirms successful anchorage of ZnO nanoflakes and Ag–NPs on microfibrous

eggshell

membrane

thus

reinstating

hierarchical

morphology

of

the

nanobiocomposites. FT-IR spectra confirms the biochemical composition whereas XPS analysis ratifies the interaction between ZnO and Ag–NPs further substantiating metallic state of Ag. ICPMS studies affirms excellent and sustainable release profile of nanoparticles from the nanobiocomposites. Owing to the synergistic activity of ZnO/ Ag–NPs, the nanobiocomposites demonstrated exceptional bactericidal activity against Gram-negative, E. coli or P. aeruginosa and Gram-positive, S. aureus or B. subtilis bacterial cells. Moreover, inherent antibacterial property of microfibrous natural ESM contributes positively towards the overall bactericidal activity. Further, a direct exposure of nanobiocomposites with NIH 3T3 cells revealed the biocompatible nature of developed matrices. Prolonged exposure also indicated that the 3T3 cells tend to adhere onto the microfibrous nanobiocomposite without any observable deformation in cellular morphology. The architectural tribology and excellent bactericidal performance of the nanobiocomposites along


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with its cytocompatible nature manifests its application as an alternate platform for varying biomedical applications. Keywords: Eggshell Membrane, ZnO/ Ag – NPs nanoparticle, Nanobiocomposite, Antibacterial Activity, Biocompatibility Graphical Abstract:



Introduction Global demography gradually recognizes the obligation of developing novel bactericidal agents with wide spectrum antibacterial applications towards improving the quality of health and hygiene as microbial strains are consistently developing resistance towards existing antibiotics. This has prompted researchers to strive for an effective yet affable means for neutralizing the threats. Vested research has led to development of different antimicrobial materials amongst which metal/ metal oxides nanoparticles enjoy a distinct position.1-2 Further review of the studies performed would reveal zinc oxide (ZnO) and silver as the preferred choices for bactericidal activities.3 The prospect of producing hierarchical nanoarchitectures with conviction on environmentally benign approaches has inspired researchers to utilize the nanoscale properties of ZnO in multiple applications like photocatalysis 4, spintronics5 or as fast switching materials6. However, the field of research involving the antimicrobial activities of ZnO nanoparticles is on a steadfast growth.7-8 The potent anticidal activity of ZnO nanoparticles is further augmented either through rendition of defined and controllable morphologies9-10 or by decorating the surface of these hierarchical architectures with metal nanoclusters with inherent antimicrobial properties.11 In particular, silver nanoparticles anchored ZnO nanoparticles have demonstrated improved antimicrobial activities when compared to their pristine counterparts.12 However, the focus is steadily shifting towards development of a bionanocomposite alternative with biopolymer acting as a platform on which the nanoparticles are anchored.7 The biodegradable nature of such composite is expected to resolve the pertinent issues of food packaging industries in improving the shelf-life and preserve the integrity of the packaged product.13 For example, a recent work delineating the importance of ZnO morphologies on the 4 ACS Paragon Plus Environment

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bactericidal effect highlighted the antibacterial properties of ZnO to be restricted towards grampositive bacteria. Only when the ZnO microstructures were reinforced on an ultra-high molecular weight polyethylene (UHMWP) matrix did the composite showed potency against the gramnegative strain. The affable biopolymer laden with the bactericidal crystals allowed selective proliferation of the gram-negative strains on their surface before targeted killing by ZnO nanoparticles.14 A relevant study reported the use of alginate as a biopolymer matrix for embedding ZnO particles where the Alginate/ZnO composite elicited considerable antimicrobial activities.15 The antibacterial effect for these biocomposites can be entirely ascribed to the ZnO crystals where the polymers simply facilitated embedding of the nanoparticles. This incubated the idea of utilizing a biopolymer with its own antimicrobial properties in conjunction to the zinc oxide nanoparticles. This would inevitably amplify the bionanocomposite’s efficiency towards different strains of bacteria. Amidst which chitosan is of particular interest whose presence either in pristine form16 or in composites17 has been established to be effective against bacterial growths. Logically, in a recent development chitosan/ZnO composites have made their way as flexible antimicrobial pouches for increasing the shelf life in raw meat industries.18 However, utilization of such bionanocomposites often involves precise techniques like electrospinning,19 3D scaffold formation with lyophillization and eventual cross linking through UV radiation20 and strip formation through 3D printing21. The sheer instrumental amendments required in preparation of bionanocomposites shifted the focus to another biopolymer with similar antimicrobial attributes but one that can be prepared in a convenient, time resolute and cost effective fashion. Avian eggs are cheap, environmentally benign and naturally available which do not require any form of pre-processing. Off late, egg shell membranes (ESM) have been recognized as potential platform for DNA sensing,22 substrate for



holding catalytic metal nanoparticle23 and in regenerative medicine24-26. Yet another fascinating statistic about ESM is its proven expertise as structure directing biotemplates.27 But the key attribute which is inspirational to researchers is their antibacterial properties which has been exploited for wound-healing and food packaging.28-29 Not only is the eggshell membrane proficient in external wound healing30 but it has also been utilized for ameliorating inflammation of internal organs.31 Thus the conjugation of an ancandidal membrane as the anchoring platform shall only amplify the antibacterial effect of the nanobiocomposite. This was precisely the motivation behind the work reported by Lee et al. where TiO2 and ZnO crystals loaded eggshell membranes demonstrated enhanced antimicrobial properties.32 The work showed effective antimicrobial properties of the nanocomposite towards E. coli, although the effect towards gram-negative strain was not reported. In a separate report, the featured property for the ESM was promoted through strategic decoration of silver nanoparticles on their surface.30 Nanorange silver particles are known for their inherent antimicrobial capabilities33 and embedding them in ESM-ZnO matrix will only accelerate similar properties for the biopolymer. Such biopolymer – nanocomposite matrix reinforces the overall biocidal efficacy by providing a three dimensional morphology for better exposure. However, the vested effort in this field of research has been limited and definitely deserves further extensive studies. In the present study, a simple yet effective method was adopted for hierarchical decoration of ESM with Ag-ZnO nanocomposites in order to form a nanobiocomposite with enhanced antibacterial activity. Further, to the best of our knowledge, the current report is first to narrate fabrication of such nanobiocomposites comprising of ESM along with Ag-ZnO nanoparticles, without disturbing the 3D architecture and chemical composition of ESM. The very inception of a bionanocomposite synthesized through implantation of distinct nanostructures of antibacterial zinc


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oxide on the polymer matrix of ESM with biocidal activities of their own motivated the present investigation. Decoration of the bionanocomposite with another biocide in the form of silver nanoparticles further enhanced the antimicrobial properties. The implantation and the decoration were performed in-situ through an environmentally benign ultrasonication technique which not only resulted in the bionanocomposite but simultaneously ensued the desired morphology of the ZnO nanoflowers. Experimental Section Preparation of Ag nanoparticles decorated ESM ZnO Nanobiocomposites Synthesis of eggshell membrane based nanobiocomposite involved multiple segregated steps ultimately culminating into an in situ sonochemical process. Such an initial step required preparation of a colloidal solution of silver (Ag) nanoparticles which would later be dispersed on the ZnO surface. The preparation of Ag-NP was inspired from an existing literature wherein, 0.1g ascorbic acid and 200 µl of 50 mM AgNO3 were homogenized in a 20 ml aqueous solution containing 0.15g of tri – sodium citrate. 34 Subsequent heating for 75 mins of the reaction mixture at 90º C under constant stirring resulted in formation of the Ag nanoparticles. The solution was then stabilized at room temperature for 2-3 h before further use. In another exclusive process, eggshell membrane was extracted from disposed eggs with acetic acid, the details of which have been described.22 In brief, eggshell containing the membrane was separated from egg yolk followed by thorough rinsing in DI water to remove any impurity. The eggshell with membrane was incubated in 2% acetic acid for 30 minutes followed by mechanical extraction of the membrane. The extracted membrane was thoroughly washed in DI water to remove any trace amount of unreacted acetic acid followed by air drying the extracted membranes in room temperature prior to use. 7 ACS Paragon Plus Environment


Finally, the nanobiocomposite was prepared through assembly of existing byproducts in an in situ sonochemical technique. Within the same facile process, ZnO nanoflowers were grown on the extracted egg shell membrane and the composite was further decorated with as prepared silver nanoparticles. In a typical synthesis, 0.5g zinc nitrate hexahydrate (Zn (NO3)2.6H2O) (Sigma Aldrich, USA) was added to 150 mL deionized (DI) water, followed by addition of 1g Cetyl trimethylammonium Bromide (CTAB) (Merck, India) under vigorous stirring. Subsequently, 90 µM KOH (Merck) solution and varying volumes from 1 ml to 4 ml with an increment of 1 ml of the already prepared and stabilized AgNP colloidal solution were added to the mixture to fabricate 1-Eaz, 2-Eaz, 3-Eaz and 4-Eaz samples respectively. The solution was then homogenized using a probe ultrasonicator (Hielscher UP200S, 200 W, 24 KHz) for 3 minutes before addition of 6 x 6 cm2 patches cut from the extracted ESM. The dried patches of eggshell membranes were immersed in the reaction mixture and the entire concoction was sonicated for 15 minutes. The output energy for sonication process was fixed at 60% of maximum power and the intermittent pulse duration was preset at 3 min followed by 5 min of interval. After sonication, the as prepared samples were washed in DI water to remove any excess reactant from surface and dried at room temperature. As a control experiment, ZnO nanoflakes implanted ESM membranes were also synthesized by the same procedure without adding silver nanoparticles and hereafter termed as Ez. The microstructural, physicochemical antibacterial and cytocompatiblity activities of prepared samples were then extensively studied. The particulars of experimental methodology implemented for microstructural evaluation, surface area analysis, biochemical analysis, and physico-mechanical characterization of as fabricated nanobiocomposites are detailed in Supporting Information.


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Antibacterial Activity of Nanobiocomposites Antibacterial activity of the as prepared nanobiocomposites was evaluated against gram positive bacteria, S. aureus (ATCC 25923) or B. subtilis (ATCC 23857) and gram negative bacteria, E. coli (ATCC 25922) or P. aeruginosa (ATCC 27853). In order to prepare the bacterial stock solution, a 10 ml nutrient broth containing 3.1% yeast-dextrose was prepared at pH 6.8 to incubate separate cultures of each bacteria at 37˚C until it reaches the exponential growth phase. Following which, broths containing the bacterial cells was centrifuged at 4000 rpm for 12 minutes. Subsequently, the supernatant was discarded and pellets containing bacterial cells were washed in sterile phosphate buffered solution (PBS) thrice. Finally, the bacterial cells were suspended in standard Mueller Hington (MH) broth (Himedia) with a final density of 106 CFU/ml to prepare two stock solution of for each bacterial culture. Disc Diffusion Method. The antibacterial activity of natural ESM and the prepared nanobiocomposites against S. aureus, B. subtilis, E. coli and P. aeruginosa were evaluated using an experimental design similar to Kirby−Bauer disc diffusion method. A solution mixture containing 10 grams of Agar-agar (Merck Millipore) and 6.5 grams of Mueller Hington broth (Himedia) in 500 ml deionized water was prepared and autoclaved before use. The mixture was cooled followed by addition of 1 mL of stock bacterial solution. The whole mixture containing the microbes were mixed evenly and subsequently transferred to sterile petridishes for solidification. As prepared Ez and Eaz nanobiocomposite patches along with native ESM samples were cut into square patches (dimensions: 1 cm x 1 cm) and sterilized under UV irradiation for 10 mins on each side prior to the assay. The standard antibiotic solution containing 1x streptomycin - penicillin was first soaked in sterilized (as mentioned above) 1 cm x 1 cm square shaped Whatman Filter Paper (42) for 15 mins prior to the assay. This was done in order to maintain similar experimental 9 ACS Paragon Plus Environment


conditions as compared to ESM, Ez and Eaz samples. The sterilized specimens of ESM, Ez, 1Eaz, 2-Eaz, 3-Eaz, 4-Eaz nanobiocomposites and antibiotic loaded Whatman Filter Paper were plated in quadruplicate against E. coli or P. aeruginosa and S. aureus or B. subtilis bacterial cultures. The above plates along with samples were incubated at 37°C for 24 h to study the zone of inhibition for each sample and the standard antibiotic solution. Plate Count Method. Further the inhibition kinetics of natural tissue and as fabricated nanobiocomposites were ratified under dynamic conditions using plate count technique by evaluating the surviving colony forming units (CFU)/ ml. In order to perform the same, gram positive bacteria, S. aureus and gram negative bacteria, E. coli were chosen as model organisms. All samples were portioned equally (2 cm x 2 cm) and sterilized using the above narrated procedure. Post Sterilization, all the samples were suspended in 2-day old bacterial cultures comprising of ~ 2 x 106 cfu/ ml of E. coli and S. aureus cultures in MH broth at 37º C. After regular interval of 1, 3, 5, 10 and 24 h, 200 µl of bacterial culture was aliquoted and serially diluted for the range 10-1 to 10-6. The dilutions were plated on MH based agar plates and incubated for 24 hrs at 37° C. Post incubation, the bacterial colonies were counted using a click-counter and plotted against time to study the inhibition kinetics of the nanobiocomposites and control samples (ESM). Moreover, the % reduction in bacterial growth or killing efficiency (Ke) of the nanobiocomposites as compared to natural membrane post 1 h of incubation was also calculated using the formula mentioned below: 𝐾𝑒 = ((𝐸𝑖 − 𝑆𝑖) ÷ 𝐸𝑖) × 100………………… (1) Where Ei represents the number of surviving bacterial colonies present in the control sample and Si corresponds to that of the nanobiocomposites.


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In-vitro cytocompatibility of nanobiocomposite The in-vitro cytocompatibility of bare ESM and nanobiocomposites was evaluated against NIH 3T3 cell line using MTT and LDH assay. Firstly, 5 x 103 NIH 3T3 cells were seeded per well in a 96 well plate and allowed to grow for 2 days at 37º C, 85% relative humidity and 5% CO 2. Cells were supplemented with complete DMEM media (Gibco, Invitrogen) containing 10% FBS and 1% antibiotic (penicillin-streptomycin). All the test samples including bare ESM were cut into disc shapes and sterilized under UV irradiation for 20 mins on each side just prior to the assay. After the cells were allowed to grow for 2 days, the sterilized test samples were introduced in triplicates for each time point. In order to determine cell viability and cytotoxicity of test samples, the cells along with composites were incubated for 24 h, 48 h and 72 h at 37º C, 85% relative humidity and 5% CO2. In order to determine cell viability, MTT assay was conducted wherein along with the above test samples wells containing only cells were considered as control whereas another well containing only DMSO and devoid of cells or samples were marked as blank. At every interval, 100 µl of MTT solution (5 mg/ml) was added and incubated for 4 h at 37º C. After 4 h, 200 µl DMSO was added to each well in order to dissolve the formazan crystal. The absorbance intensity for each sample and controls were measured at 570 nm using a plate reader (Biorad). The metabolic activity or % cell viability of NIH 3T3 cells were determined by the following equation: % Cell Viability = OD595[Sample] – OD595[Blank]/ OD595[Control] – OD595[Blank] X 100 In order to study the cytotoxicity of above samples, LDH assay was conducted. In this study along with the above mentioned samples, two other controls containing only cells and media were designed and introduced at every interval. At each time point, one of the controls were treated with Triton X-100 in order to lyse the cells so that maximum LDH leakage in the media could be 11 ACS Paragon Plus Environment


studied and this control was treated as “positive control”. The other control containing only cells and media was kept untreated and considered as “negative control”. Further, LDH assay was conducted on all the wells containing test samples and controls at each time point according to manufacturer’s protocol to determine toxicity of test samples. At every time interval, the amount of LDH released in cell culture media was demonstrated as a percentage (%) of the negative control. It was also equally important to assess the interaction of cells with nanoparticle loaded matrices so that the same could be explored for further biomedical applications as well. Initially, the nanobiocomposites were sterilized following the above protocol. Post sterilization, NIH 3T3 cells were seeded at a density of 2 x 104 cells/ sample and incubated at 37 ºC, 5% CO2 and relative humidity of 85% for 24 h, 48 h and 72 h. At every time interval the cell loaded nanobiocomposites were fixed using 4% paraformaldehyde in PBS. For microscopic studies, the fixed cell loaded samples were dehydrated using a series of ethanol from 50% - 100% and gold sputtered prior to imaging under FESEM (FESEM, Carl Zeiss-Supra 35VP). Further, the fibroblast cell loaded nanobiocomposites were stained using Rhodamine–phalloidin (R415, ThermoFisher Scientific) and DAPI (ThermoFisher Scientific) following manufacturer’s protocol. Image acquisition was done using Axio Observer Z1 microscope (Carl Zeiss, Germany). Statistical Analysis All data sets were analyzed using GraphPad Prism software (version 5.02, La Jolla, CA, USA), following one-way ANOVA t test. The level of significance between data sets were considered significant at p < 0.05. All experiments were conducted in triplicates, and data were represented as mean ± standard deviation (SD) for n = 3 except when mentioned separately.


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Results & Discussion The usage of nanoparticle embedded matrices have evolved as potential alternatives in the study of antibacterial platforms. Of late, biopolymers have been deployed to fabricate fibrous or non-fibrous matrices for anchoring nanoparticles to fabricate nanobiocomposites with bactericidal activity. In the present study, microfibrous eggshell membrane has been exploited as a three dimensional platform for hierarchical decoration of ZnO nanoflowers and Ag nanoparticles to form a nanobiocomposite. The innate antibacterial activity of ESM coupled with easy availability and feasible extraction methodology makes ESM a potential alternative biopolymer matrix for preparing such nanobiocomposites. Further, ZnO nanoflowers were implanted on the microfibers of ESM followed by loading of varying concentration of Ag nanoparticles. The hierarchical arrangement of nanoflake like structures to form ZnO nanoflowers which facilitated efficient loading of Ag nanoparticles. An optimum concentration of nanoparticles was desired as elevated levels may elicit toxic response thus release kinetics of prepared nanobiocomposites were studied in order to determine the same. Subsequently, the synergistic bactericidal properties of nanobiocomposites were demonstrated using Gram-negative, E. coli or P. aeruginosa and Grampositive, S. aureus or B. subtilis as model organisms. The nanobiocomposites were able to illustrate excellent antibacterial activity when exposed to bacterial cultures and incorporation of nanoparticles elevated the bactericidal properties by ~ 97% as compared to the natural tissue. Moreover, toxicity profile of the nanobiocomposites were also studied on NIH 3T3 fibroblast cells. It was witnessed that concentration of nanoparticles loading on the nanobiocomposite was appropriate enough for eliciting bactericidal effects on smaller sized prokaryotic bacterial cells while keeping a low toxic profile when exposed to bigger eukaryotic 3T3 fibroblast cells.



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Microstructural Evaluation In accordance to the prime focus of this study which aspires to develop ZnO implanted ESM bionanocomposite, it is imperative to assess the synthesized product under electron microscope. Representative FESEM images of the synthesized ESM/ ZnO (Ez), Ag loaded ESM/ ZnO (Eaz), bare ESM and Ag – NPs are collated in (Figure 1a-i). The images clearly depict anchored ZnO nanoflowers spatially located on the ESM strands. It might be inferred from

(a)

(b)

(c)

(d)

(e)

(f)

(g)

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Figure 1. Microstructural Evaluation of Ez, Eaz, ESM, and Ag-NP were carried out. The FESEM micrographs of (a-c) Ez and (d-f) Eaz samples clearly depict successful implantation of ZnO Nanoflowers and further decoration of Ag-NPs on as fabricated samples (Red arrows signify ZnO Nanoflower whereas yellow arrows points towards the Ag-NP decorated on top of nanoflakes). Further microstructure of bare ESM was also revealed in (g). (h) Micrograph demonstrating the result of using non-ionic surfactant in formation of Ez complexes and thus leading to non-specific deposition of ZnO Nanoflowers in between as well as on the fibers. The morphology of Ag-NP was further studied using TEM analysis as reported in (i).


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microscopic analysis that the nucleation had transpired exclusively on the polymer fibers as all the flowers are evidently located on the network and not on interstitial spaces. Closer assessment of an individual nanoflower (Figure 1c) reveals its diameter to be approximately 2 µm and comprising of multiple nanoflakes fused together. The nucleation promoting role of CTAB in the development of ZnO flakes/sheets was investigated in our previous work.35 An additional role for CTAB can be ascribed to the formation of CTA+- [Zn(OH)4]2- ionic pairs. Under system’s reduced surface tension, the CTA+ ions carry the zincate ions to the ESM backbone and implant the primary growth units on the fibers. Following which, imparted sonochemical energy is utilized by zincate ions to dehydrate and nucleate as ZnO nanocrystals at room temperature. Under sustained sonication, the crystals would have developed nanorods due to the characteristic anisotropic growth of ZnO crystals along c-axis. However, the formation of nanoflowers comprising an assembly of nanoflakes might be explained from the fact that the system contains excess OH- ions due to the potassium hydroxide used for synthesis. Moreover, the dehydration of zincate ions is associated with simultaneous generation of additional hydroxyl ions.36 These OH- ions are expected to dominate the zincate ions in a competitive binding process, and cap the [001] plane comprising of Zn2+ ions, thus neutralizing the surface charge.37 This impedes the normal growth condition, where negatively charged zincate ions alternately stacks on the [001] plane to produce nanorods. Once the evolution of the plane with fastest kinetics is impeded, the other planes with relatively faster growth rates develop to produce nanoflakes. The assembly of nanoflakes to form nanoflowers might be perceived as the systems’ effort to optimize the surface energy. Further presence of Ag-NP was also confirmed from point EDX analysis and magnified FESEM micrographs as represented in Figure S1. The hierarchical decoration of ZnO nanoflowers with Ag-NP is attributed to the attractive van der Waals forces between particles.38



Further detailed explanation suggesting the interaction between ZnO and Ag-NPs was provided in the XPS section. To confirm the role of the cationic surfactant in implanting the flowers, a control experiment was conducted with non-ionic Poly Ethylene Glycol (PEG) replacing CTAB, with all other parameters remaining constant. The FESEM image (Figure 1h) for the nanoflowers produced with this process differed from those depicting CTAB mediated synthesis products. It is observed that ZnO nanoflowers produced with PEG are not anchored to the ESM backbone, while they are completely rooted to the fibers when CTAB is used. Therefore, the cationic surfactant is inferred to have a discerning role in anchoring the particles. The disparity among the ESM anchored nanoflowers synthesized with CTAB or PEG originates from the isoelectric point of the ESM network. The extremely basic environment created due to the KOH employed for synthesis renders the system’s pH to cater around 10. The high value of pH implies a negatively charged surface for the ESM network39-40 which attracts the ionic pair of CTA+-[Zn(OH)4]2- and anchors the moiety on the surface. An extremely relevant study in this field has been reported by Li et.al. which suggests CTAB as a facilitator to anchor zincate ions on negatively charged surface of silk templates.41 The size distribution of bare ESM microfibers, ZnO nanoflowers and Ag-NPs were carried out using Image J software (NIH, USA). The plots for ZnO nanoflowers and Ag-NPs were found to portray narrow size distribution wherein highest frequency of occurrence for ZnO nanoflowers was in the range of 1.5 - 1.8 µm while for Ag-NPs, it was in the range of 15 – 25 nm [Figure 2a & 2b]. The fiber diameter was witnessed to spread across a wide range starting from 0.5 to 3.0 µm, however, highest distribution of fiber diameter was observed in the size range of 1 – 2 µm [Figure 2c]. Further, it was important to determine surface area of as prepared nanobiocomposites since


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(a)

(b)

(d)

(g)

(h)

(c)

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Figure 2. Size Distribution curve for fibers of bare ESM along with nanoparticles of ZnO Nanoflower or Ag-NP (a-c). (d) Further plot suggesting surface area analysis performed using Micro-CT and its corresponding images (e-j).

composites with higher surface area can be expected to exhibit better exposure to bacterial cells thus evidently increasing the bactericidal efficacy. In the present study, the native ESM was decorated with ZnO nanoflowers followed by Ag-NP as mentioned before. Notably, the nanobiocomposites were sequentially decorated with elements whose density varies over a wide range and thus conventional surface area measurements (unit mass-1 based approach) was avoided to minimize ambiguity. Herein the surface area was measured per unit volume by Micro-CT analysis for different samples with varying compositions. Each unit volume was represented by one voxel having a volume of 5 µm x 5 µm x 5 µm. It was observed that native ESM possessed an average surface area (SA) of 736 ± 59 µm2 per voxel whereas with loading of ZnO nanoflowers, 17 ACS Paragon Plus Environment


SA increased to 1104.12 ± 88 µm2 for Ez samples. The SA further increased with decoration of Ag-NPs and substantially reached a maximum of 2538.63 ± 203 µm2 for 3-Eaz samples. Figure 2d also depicted that SA for 4-Eaz samples decreased to 1835.18 ± 146.81 µm2 which could have attributed to the fact that nanoparticle agglomeration leads to decrease in overall surface area of composites which was also supported by Figure S2.42 The corresponding Micro-CT images for all the samples are represented in Figure 2 (e-j). Surface area of all the samples were reported in table S1 as mentioned in the supplementary information. Physico-chemical Properties of Nanobiocomposites A more detailed molecular footprint of prepared nanobiocomposites was provided by ATRFTIR and XPS analysis. Crystallographic interpretation of above prepared samples was also performed and reported in supplementary information. FTIR spectra (Figure S3) suggest that peaks pertinent to vibrations originating from metallic bonds often appear at lower wavenumbers of IR spectra. Such a characteristic peak is indeed observed for the Zn-O bond at within 400-550 cm-1 for Ez and Eaz samples corresponding to the bending vibrations.43 A small peak is simultaneously visible at 720 cm-1 which might be ascribed to the C-S stretching mode vibrations originating from the cysteine moieties present within the egg shell membrane.44 Additional bands appearing at 3400 cm-1 may be ascribed to the aromatic amide N-H stretching vibration from the membrane while the sharper peak at 1650 cm-1 are attributed to the C=O stretching vibrations.22, 45 Other visible bands appearing from the chemical moieties present within the membrane can be located at 1065 cm-1 (polysaccharides) and 2850 cm-1 (C-H bonds from the lipids). The FTIR spectra thus represents a strong interaction between the zinc oxide and the host membrane through the visible peaks. However, footprints pertaining to the presence of Ag nanoparticles were unclear from the spectral plot.


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(a)

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Figure 3. XPS spectra for Zn 2p, O 1s, and Ag 3d elemental composition of Ez (a-b), and Eaz, (c-e).

In an attempt to further affirm chemical constituency of the nanobiocomposites and decode the chemical states of individual constituents, XPS analysis was carried out. Figure 3 demonstrates the high resolution XPS spectra of Ez and Eaz samples. While an anticipated peak of Zn 2p 3/2 appears at 1022 eV which is ascribed to the Zn2+ state in the zinc oxide nanoflowers, a doublet peak arises at 1044 eV from the Zn2p1/2 state.46 The peak at 530 eV is related to the O1s state of the oxygen and originates due to contributions from O2- ionic moieties in the ZnO lattice as well as oxygen vacancies and from the chemisorbed oxygen adatoms. Another doublet peaks at binding energies of 367.26 eV and 373.26 eV are visible for the Eaz sample corresponding to Ag 3d5/2 and Ag 3d3/2 states respectively. A spin-orbital splitting of 6 eV implies that the silver nanoparticles are in their metallic state Ag (0) and not oxidized.47 Additionally, a slight shift for the Ag 3d5/2 and Ag 3d3/2 peaks towards lower binding energy levels as compared to bulk Ag (368.2 eV and 374.2 eV for Ag 3d5/2 and Ag 3d3/2 respectively) implies transfer or interaction of electrons from Ag Np 19 ACS Paragon Plus Environment


to ZnO nanoflakes present at the surface of microfibers.48 It was also indicative of the fact that anchoring of Ag-NP on the surface of ZnO nanoflowers leads to adjustment of their respective fermi level to similar or same value. Further tunneling of free electrons from the new fermi level to the conduction band of ZnO nanoflowers may also shift the binding energy of Ag 3d5/2 to a lower level as compared to bulk Ag which was apparent from our XPS data.49 Lastly, it was also observed that binding energy of ZnO nanoflakes (Zn 2p) in Eaz composite shifted slightly towards the higher level (1021.11 and 1044.20 eV) as compared to Ez samples. The above results were in good agreement with previously reported literature.50-51 Moreover, the interaction between Ag Np and ZnO nanoflakes at the interface was also confirmed from the above observation. Crystallographic studies of as prepared nanobiocomposites were performed using X-ray diffraction studies as illustrated in supplementary information and Figure S4. The presence of [101] plane of ZnO crystals was clearly visible from the peak at 36.3º of the XRD plot. Meanwhile, presence of Ag nanoparticles (Ag-NP) was not evident from the above plot. However, the presence of Ag-NP was quite evident from EDX analysis, FESEM micrographs and XPS analysis. Further studies like zeta potential, ICP-MS and Thermal analysis were also performed to provide clear evidences of Ag loading. The evaluation of zeta potential for composites intended to be used for bactericidal activity is of paramount importance as bacterial cells possess a net negative charge so depending on the overall surface charge the composite will either attract or repel the microbial cells. Figure 4a depicts that natural ESM possess a net negative charge of -6.45 ± -1.45 mV whereas Ez samples possessed a zeta potential of 2.34 ± 0.5 mV. Further, it was experienced that with introduction of Ag-NPs it further increased to 19.9 ± 1.393 mV for 1-Eaz and 45.7 ± 3.199 mV for 2-Eaz samples. The zeta potential reached a maximum of 50.8 ± 3.556 mV for 3-Eaz samples while for 4-Eaz


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samples, it dropped to 31.1 ± 2.17 mV. A unimodal charge distribution was observed for all the samples which indirectly justifies excellent loading of ZnO or Ag-NP nanofibers on the microfibrous ESM (Figure S5). The negative charge on ESM surface also justifies the utility of CTAB in implanting zincate ions on the microfibers of ESM. A surge of ZnO nanoflowers and Ag-NPs shifted the negative potential of ESM towards the positive end. Poor loading of Ag-NPs for 4-Eaz samples owing to the agglomeration of Ag-NPs lead to a drop in zeta potential for 4-Eaz samples. The shift of zeta potential towards the positive side also provides a direct evidence of

(a)

(b)

(c)

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Figure 4. Zeta Potential and Thermal analysis of all samples were performed and represented in 3a &3b. Mechanical properties of as fabricated nanobiocomposites and bare ESM. Above plots depicts the effect of nanoparticle decoration on the tensile strength (a) and Elastic Modulus (b) of the natural tissue.



nanoparticle loading which may further attract the negatively charged bacterial cells and facilitate in bactericidal activity of the nanobiocomposite. The thermal properties of bare ESM and nanobiocomposites (Eaz samples) were evaluated using thermogravimetric analysis (TGA). Thermal analysis of all carried out until 600 ºC wherein the rate of increase in temperature was maintained at 10 ºC/ min. Figure 4b clearly depicts that significant weight loss took place between the temperature range 210 ºC – 450 ºC. It was apparent that notable thermal degradation started around ~ 210 ºC for bare ESM whereas ZnO or Ag-ZnO NPs loaded composites started to degrade around ~ 220 ºC, which was in agreement with reported literature.52 Notably, the weight residue after heat treatment increased gradually after consecutive decoration of ESM with ZnO nanoflowers or Ag-NP mainly due to increase in non-volatile metal ions. After heat treatment at 600 ºC, the residue was about ~ 15% for ESM and ~20% for Ez samples whereas for 3-Eaz or 4-Eaz samples it was recorded to be 24%. Additionally, the remaining residual weight for 1-Eaz and 2-Eaz was ~21% and ~24% respectively. The residue percentage increased gradually with increase in decoration with higher amount of Ag-NP. Further, it was also ascertained that Ag-NP loading was highest for 3-Eaz and 4-Eaz samples suggesting that loading concentration of Ag-NP on the prepared nanobiocomposite reached a point of saturation. Polymeric nanobiocomposites designed for biomedical applications demands reasonable strength along with comparable elastic modulus to become self-supporting and also sustain handling damages. It was often witnessed that inorganic nanofillers are incorporated into the matrix of biopolymers in order to fabricate a resultant composite with improved mechanical properties. The presence of ZnO nanoparticles were reported to drastically improve mechanical properties of biopolymers.7 Herein, mechanical properties of as fabricated nanobiocomposites


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were evaluated at room temperature under wet condition to understand the effect of nanoparticle decoration on the same. The measured tensile strength and elastic modulus of composites were plotted as a function of nanoparticle loading as shown in Figure 4c-d. It was witnessed that bare ESM possessed a tensile strength of 5.82 ± 0.291 MPa which gradually increased with increasing concentration of nanoparticles to a maximum of 9.7 ± 0.485 MPa for 3-Eaz thus posing an approximate ~ 67% increment in the tensile strength of nanobiocomposites. It was also important to monitor the elastic modulus of nanobiocomposites at the same time so that increasing loading of nanoparticles doesn’t alter elasticity of the matrices. Figure 4d demonstrated that loading of nanoparticles progressively increased the elastic modulus to 234.6 ± 9.54 MPa for 3-Eaz samples as compared to 149.7 ± 6.24 MPa for the natural tissue thus recording an increase of ~ 57%. It was apparent from above observation that mechanical characteristics of the polymer composite were influenced by nanoparticle loading. Besides distribution of nanofiller along the matrix, interactions between filler and matrix also plays a crucial role in determining the mechanical properties. It could be related that the strong improvement in elastic modulus along with tensile strength could be a cumulative effect of homogenous distribution of nanoparticles and excellent interactions between the nanoparticles with bare ESM fibers at the interface. Moreover, synergistic relation between the above two phases can also be related to the fact that a nucleation mediated process was adopted to synthesize ZnO nanoflakes directly on the microfibrous ESM. Further, the interaction between Ag-ZnO48 and Ag – NH – R53 groups are well established facts thus justifying the above results. However, it was apparent that increased loading for 4-Eaz caused a drop in tensile strength as well as elastic modulus. Owing to higher loading, the nanocomposites of ZnOAg might have formed clusters thus leading to reduced interfacial contact between the matrix and nanoparticle hence limiting the stress transfer. The reported improvement in mechanical properties



post decoration of nanoparticles were in good agreement with previous literatures.54 More importantly it was ratified from the study that mechanical properties of biodegradable polymers could be reinforced using inorganic nanofillers. Release Kinetics of the Nanoparticles The demonstration of Ag and ZnO nanoparticle migration into surrounding medium is an important phenomenon when considering the possible application of nanobiocomposites in bactericidal activity. The release kinetics of nanoparticles in a particular environment relies entirely on pH and moisture content of the surrounding. The nanobiocomposites were incubated in LB solution at varying pH of 5.5, 7 and 8.5 maintained at 37º C. The release kinetics of nanoparticles from matrices were evaluated at regular interval as reported in Figure 5. ICP-MS plot revealed that 3-Eaz nanocomposite was able to release 2.1 ppm and 0.13 ppm of Ag and ZnO nanoparticles respectively, post 3 h of incubation at pH 5.5. Subsequently with increasing pH the release rate of Ag and ZnO dropped to 1.3 ppm and 0.8 ppm respectively at pH 7 and further at alkaline pH (pH – 8.5), the rate dropped to 0.75 ppm and 0.05 ppm respectively after 3 h. However, it was apparent from the plots that a burst release of nanoparticles was observed in first few hours of contact following which a steady pattern was followed. Further it was manifested that with decreasing pH, a surge of nanoparticle release was witnessed in the MH medium which was in agreement with previous studies.55 The release of AgNP were facilitated by dissolved oxygen and the same was explained by the following equation56: 4𝐴𝑔(𝑠) + 𝑂2 (𝑎𝑞) + 4𝐻 + = 4𝐴𝑔+ (𝑎𝑞) + 2𝐻2 𝑂 ………………… (2) The above reaction clearly suggests that the release of Ag+ is directly guided by oxidation of Ag from the surface of ZnO and ESM. Lower pH value of the medium potentially explains the


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1 2 3 4 5 6 7 8 (a) (c) (b) 9 10 11 12 13 14 15 16 17 18 19 20 (d) 21 22 23 24 25 26 27 28 29 30 31 32 33 Figure 5. Plot for ICP-MS analysis. The rate of silver release from Eaz nanobiocomposites were observed at (a) pH 5.5 (b) pH 7.5 (c) pH 34 8.5 at varying time interval. Further, the rate of ZnO release from Ez nanobiocomposite (d) was also studied at varying pH and as a function 35 of time 36 37 38 enhanced release kinetics of Ag nanoparticles from the nanobiocomposites. The release kinetics 39 40 for ZnO nanoparticles under varying pH conditions was represented by the following equations57: 41 42 43 𝑍𝑛𝑂 (𝑠) + 2𝐻 + (𝑎𝑞) = 𝑍𝑛2+ (𝑎𝑞) + 𝐻2 𝑂(𝐼)--------------------- (3) (acidic condition) 44 45 46 𝑍𝑛𝑂 (𝑠) + 𝐻2 𝑂(𝑙) = 𝑍𝑛2+ (𝑎𝑞) + 2𝑂𝐻 − (𝑎𝑞) --------------------- (4) (neutral condition) 47 48 49 𝑍𝑛(𝑂𝐻)2 (𝑠) = 𝑍𝑛2+ (𝑎𝑞) + 2𝑂𝐻 − (𝑎𝑞) --------------------- (5) (basic conditions) 50 51 52 It was reported that Zn2+ ions possessed best release kinetics under acidic conditions while 53 54 lowest solubility was witnessed under alkaline conditions. It was noted that post 72 h of incubation, 55 56 57 58 25 59 ACS Paragon Plus Environment 60


3-Eaz nanocomposite released 3.2 ppm and 0.22 ppm concentration of Ag and ZnO nanoparticles respectively at pH 5.5. The three dimensional hierarchical structure of ZnO nanoflakes, guided an extensive release of Ag nanoparticles when exposed to appropriate conditions. Moreover, the microfibrous architecture of ESM also delivered a cylindrical platform and huge surface area for facilitating extensive loading of ZnO and Ag nanoparticles. In brief, structural assembly of nanobiocomposites provided an excellent framework for extensive release of nanoparticles in surrounding environment. The matrices demonstrated varying nanoparticle release kinetics in acidic or alkaline aqueous environment thus endorsing the opportunity for different biomedical applications. Furthermore, it is to be noted that ZnO nanoparticles were impregnated onto the matrices supporting the higher release kinetics of Ag-NPs. However, when the natural matrix was completely digested, the ratio of Zn2+ ion to Ag+ ions was 1.24 suggesting that ZnO was the base or bulk material and also justifying the decoration of Ag-NPs (Figure S6). Evaluation of Antibacterial Activity

In order to evaluate antibacterial efficacy of nanobiocomposites and the natural tissue, Kirby−Bauer’s diffusion assay and plate count method were deployed. Clinically important and potential human pathogens like Gram negative, E. coli or P. aeruginosa and gram positive, S. aureus or B. subtilis bacterial cultures were studied as test organism to determine the bactericidal activity. Hierarchical decoration of ESM with ZnO and Ag nanoparticles rendered it with excellent biocidal activity against all the microbes. It was observed that when exposed to E. coli bacterial cultures, Ez samples presented a clear zone of inhibition (ZOI) of 1.87 ± 0.12 cm2 and with inclusion of Ag nanoparticles in 3-Eaz samples, the ZOI increased to 2.59 ± 0.15 cm2 [Figure 6a]. Further it was also witnessed that 1-Eaz, 2-Eaz and 4-Eaz possessed a ZOI of 2.07 ± 0.12 cm2, 2.46 ± 0.15 cm2 and 2.44 ± 0.14 cm2 respectively as compared to 1.15 ± 0.07 cm2 for ESM. 26 ACS Paragon Plus Environment

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Moreover, when incubated with P. aeruginosa, the ZOI was recorded as 1.09 ± 0.07 cm2, 1.71 ± 0.1 cm2, 1.98 ± 0.12 cm2, 2.43 ± 0.15 cm2, 2.85 ± 0.17 cm2, 2.3 ± 0.14 cm2 for ESM, Ez, 1-Eaz, 2-Eaz, 3-Eaz and 4-Eaz samples respectively [Figure 6b]. In comparison, for S. aureus bacterial culture, Ez samples exhibited a clear ZOI of 1.51 ± 0.07 cm2 and loading of Ag nanoparticle further increased it to 2.49 ± 0.12 cm2 for 3-Eaz samples (Figure 6a). It also revealed that 1-Eaz, 2-Eaz and 4-Eaz portrayed a ZOI of 1.98 ± 0.09 cm2, 2.25 ± 0.11 cm2 and 2.39 ± 0.12 cm2 respectively, in contrast to a lesser ZOI of 1.04 ± 0.05 cm2 for ESM. Additionally, for B. subtilis the ZOI was recorded as 1.06 ± 0.05 cm2, 1.56 ± 0.07 cm2, 1.87 ± 0.09 cm2, 2.28 ± 0.12 cm2, 2.43 ± 0.13 cm2, 2.25 ± 0.12 cm2 for ESM, Ez, 1-Eaz, 2-Eaz, 3-Eaz and 4-Eaz samples respectively [Figure 6b]. Finally, 1x streptomycin - penicillin standard antibiotic soaked filter papers reported a maximum ZOI of 5.1 ± 0.4 cm2, 5.3 ± 0.45 cm2, 4.33 ± 0.3 cm2 and 4.45 ± 0.37 cm2 for E. coli, P. aeruginosa, S. aureus and B. subtilis respectively [Figure 6c]. The digital images for above culture plates were (c)

(b)

(a)

(e)

(f)

(d)

(g)

Figure 6. Evaluation of Antibacterial Efficacy and bactericidal kinetics. (a) & (b) depicts the Zone of Inhibition (ZOI) of as fabricated nanobiocomposites and bare ESM against gram negative, E. coli or P. aeruginosa and gram positive, S. aureus or B. subtilis bacterial strain and compared it against standard antibiotic (Pen-Strep) as revealed in 5c. (d) depicts digital photograph of sample (3-Eaz), demonstrating the highly flexible nature of developed material. The bactericidal kinetics of prepared nanobiocomposites and bare ESM against E. coli (e) and S. aureus (f) were determined by plate count method and plotted as a function of time. (g) demonstrates the effect of nanoparticles decoration on increasing killing efficiency (Ke) of nanobiocomposites as compared to bare ESM. Error bars depict standard deviation (n ≥ 4) of independent samples per group; single and double asterisks indicate P < 0.001 and P < 0.03 respectively.



represented in Figure 7. The above observation demonstrated a decreasing trend for 4-Eaz samples as compared to 3-Eaz samples for all the bacterial cultures and this aspect could be related to lowered loading efficiency and release kinetics of Ag-NP at high concentration which was also apparent from ICP-MS studies. The assay clearly affirms the effect of nanoparticle decoration on increased antibacterial efficacy of nanobiocomposites in contrast to bare ESM.

In order to evaluate inhibition kinetics of the nanobiocomposites under dynamic condition as a function of time, submerge culture or plate count method was adopted. It was also important to observe the effect of various loading percentage of Ag on the biocidal activity. It was apparent that the results of plate count method were concurrent with above Kirby−Bauer’s diffusion assay. In E. coli bacterial culture, 3-Eaz samples significantly reduced the bacterial growth to 3 x 103 CFU/ml and 1 x 101 CFU/ml from an initial concentration of 2 x 106 CFU/ml post 1 h and 3 h of incubation [Figure 6e]. The results suggested a 5 log reduction in bacterial colony size post 3 h of incubation while no colonies were witnessed after 5 hrs of incubation. Similar response was also witnessed for 2-Eaz samples wherein the colony size was reduced to 4 x 103 C FU/ ml and 2 x 101 CFU/ml post 1 h and 3 h of incubation. Alike disc diffusion method, 4-Eaz samples encountered lower bactericidal activity as compared to 3-Eaz and 2-Eaz, wherein the colony size was calculated to be 1 x 104 CFU/ml and 4 x 102 CFU/ml after 1 h and 3 h of incubation. In case of 4-Eaz and 1Eaz samples, bacterial colonies were witnessed to survive until 5 h of incubation. A 5 log reduction in colony size was observed for Ez sample after 10 h of incubation and no colonies were witnessed after 24 h of incubation. Further, similar inhibition kinetics were observed when the nanobiocomposites were exposed to S. aureus bacterial suspension for varying time intervals [Figure 6f]. 3-Eaz and 2-Eaz samples recorded a colony count of 5 x 103 CFU/ ml and 7 x 103 CFU/ ml post 1 h of exposure respectively. Figure 6e also stated that there was a 5 log reduction 28 ACS Paragon Plus Environment

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Figure 7. Digital images of antibacterial plates for bare ESM and nanobiocomposites when exposed to gram negative, E. coli or P. aeruginosa and gram positive, S. aureus or B. subtilis bacterial strain and compared against standard antibiotic (Pen-Strep). Scale bar: 1 cm

in colony size for both 3-Eaz and 2-Eaz specimens after 3 h of incubation while 5 h onwards, no colonies were witnessed. Owing to the phenomena of nanoparticle agglomeration for 4-Eaz samples, only 3 log (1 x 103 CFU/ ml) reduction in bacterial colonies were observed after 3 h of incubation indicating reduced bactericidal activity as compared to 2-Eaz or 3-Eaz. For Ez samples, a 4 log reduction in colony size was registered after 10 h of incubation while no colonies were seen after 24 h. However, for both E. coli and S. aureus bacterial cultures, few colonies were observed in case of bare ESM post 24 h of incubation. Thus the above results substantiate that 329 ACS Paragon Plus Environment


Eaz samples displayed better bactericidal effect compared to other samples. Further, it was witnessed that the inclusion of Ag loaded nanobiocomposites reduced bacterial colony size by ~ 98% and 97% for E. coli and S. aureus bacterial suspension, respectively post 1 h of incubation as compared to bare ESM (Figure 6g). Additionally, the synergistic effect of Ag – ZnO was also evident as Eaz nanobiocomposites possessed ~ 39% higher inhibition rate as compared to Ez specimens. The above results clearly ratify the effect of nanoparticle decoration on the enhanced bactericidal activity of nanobiocomposites as compared to ESM samples.

Further, FESEM was deployed to understand the mechanism of action underlying the antibacterial activity of nanocomposite matrices. After exposure to bacterial colonies, the cell loaded matrices were fixed and morphology of attached cells were studied. Figure 8a & 8d illustrates that bacterial cells with elongated and deformed morphology were seen to migrate along the edges of ESM samples. E. coli cells possessed an elongated and deformed morphology whereas S. aureus cells demonstrated a flattened and wrinkled morphology. As anticipated, most of the bacterial cells witnessed on Ez and Eaz membranes possessed a distorted and collapsed morphology with nanoparticles surrounding the edges of their cell membrane (Figure 8). Figure 8c and 8f clearly suggest that nanoparticles attach on cell membrane aided in distortion of the membrane thus describing an important aspect responsible for antibacterial activity of the nanobiocomposites. The micrographs illustrated in figure 8 were in agreement with previous studies describing inactivation of E. coli and S. aureus cells upon contact with Ag decorated nano or microfibers.58

There were various mechanisms proposed for explaining the antibacterial activity of ZnO and Ag nanoparticles.59-60 ZnO nanoparticles alone disrupted the cell membrane of bacterial cells


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Figure 8. FESEM analysis of bactericidal activity of nanobiocomposites and bare ESM. (a & d) reveals deformation in cell morphology of E. coli and S. aureus bacterial strains owing to the bactericidal activity of bare ESM due to presence of naturally occurring GAGs. (b & e) shows the efficacy of ZnO nanoparticles of Ez samples in killing bacterial cells of E. coli and S. aureus by rupturing the cell membrane. (c & f) further demonstrates the proficiency of hierarchical nanostructure of Eaz composites in destroying the microbes with presence of dead cells been observed on the microfibers. Bare ESM and composite samples from plate count experiment were withdrawn, fixed and imaged under FESEM to witness the process. The microfiber indeed provides a matrix for attracting microbial cells thus facilitating the bactericidal activity of the nanobiocomposites.

and released Zn2+ ions which led to H2O2 generation from the nanoparticle surface.7,

61

The

generation of reaction oxygen species (ROS) in the form of H2O2, can be coupled to the moisture absorption phenomena of ZnO which in turn facilitates H2O2 generation. H2O2 can penetrate cell membrane much easily than .O2- and can initiate a series of cellular pathways that can lead to oxidative stress induce apoptosis of bacteria cells.62 Moreover, it is also believed that the introduction of H2O2 may overwhelm bacterial defense system and initiate programmed cell death (PCD) genetic module of microbial cells which may led to an outburst of oxidative stress and free radicals which is supposed to impart lethal effects on the bacterial cells.63 Further, the three dimensional hierarchical architecture of the ZnO nanoflakes impregnated ESM substrate not only 31 ACS Paragon Plus Environment


facilitates better loading of Ag NPs but also pushes more amount of Ag+ ions into bacterial cytoplasm thus elevating the bactericidal response. These Ag NPs tend to release Ag+ ions which react with thiol group of bacterial enzymes and tend to inactivate them.64 The Ag+ ions are also reported to possibly affect the respiratory system of the bacterial cells. The toxic character of Ag NPs is associated with surface oxidation and capability to generate Ag+ ions following the reaction in equation (1).65 Additionally, Ag NPs are also believed to react with sulphur and phosphorus forming complexes with DNA backbone of bacterial cells thus destroying the oligonucleotides leading to cell death.66-67 Moreover, Ag nanoparticles tend to dephosphorylate phosphotyrosine residue of bacterial peptides thus interrupting signal transduction pathway leading to bacterial growth inhibition in gram negative bacteria.68 Moreover, the ability of Ag-NP to adhere on bacterial cell and generate free radicals led to alteration and disruption of bacterial cell wall.69-70

Moreover, the three dimensional hierarchical structure adopted for Eaz and Ez, facilitated spontaneous leaching of Ag nanoparticles from Eaz film within the first 4 hrs which can be seen from the ICP – MS graph (Figure 4). The Eaz membranes are able to release 0.1 ppm of Ag+ and 0.23 ppm of Zn2+ which is enough for showing good antibacterial activity against both E. coli (gram negative bacteria) and S. aureus (gram positive bacteria).

Cytotoxicity of Nanobiocomposites

The excellent antibacterial property of Ag and ZnO nanoparticles are often accompanied or related to its cytotoxic effects towards mammalian cell lines. Herein, NIH 3T3 fibroblast cells were deployed to examine cell viability and cytotoxicity of prepared nanobiocomposites by MTT or LDH assay. Figure 9a-c demonstrated the results for MTT assay post incubation of nanobiocomposites in 3T3 fibroblast cells after 24 h, 48 h and 72 h. It was observed that exposure 32 ACS Paragon Plus Environment

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Figure 9. Evaluation of Cell Viability and Cytotoxicity. (a-c) demonstrates results of MTT assay after incubation of NIH 3T3 cells with prepared nanobiocomposites or bare ESM. (d-f) illustrates LDH activity of 3T3 cells post exposure to Ez, 1-Eaz, 2-Eaz, 3-Eaz, 4-Eaz and bare ESM samples for 24 h, 48 h and 72 h. (g-i) indicates the effect on cell adhesion and morphology of NIH 3T3 cells when incubated with bare ESM and nanoparticle decorated ESM composites (Ez and 3 – Eaz) for 3 days. Error bars depict standard deviation (n ≥ 3) of independent samples per group; double and triple asterisks indicates P < 0.03 and P < 0.05 respectively. (Scale Bar: 10 µm)



of nanobiocomposites for the first 24 h and 48 h had no significant impact on the mitochondrial activity in cells. After 72 h of incubation, nanobiocomposites comprising of higher Ag loading particularly, 3-Eaz and 4-Eaz, exhibited slightly lower cell viability of 76% and 74% respectively as compared to its counterparts. However, the decrease in cellular activity for higher loading of Ag nanoparticles was statistically insignificant. Further, cytotoxicity study of the samples was performed by analysing LDH activity from supernatants post exposure of 3T3 fibroblast cells for varying time interval. LDH assay was performed to investigate impact of nanoparticles on the integrity of cell membrane (Figure 9d-f). Subsequently, no elevation in LDH levels were observed in nanobiocomposite treated samples post 24 h of incubation. Even after 2 days of incubation, the LDH level witnessed for nanocomposite treated groups were in admissible range of 107% – 116% of the negative control.71 Further, it was reported that post 72 h of exposure, LDH levels for 3-Eaz and 4- Eaz samples moderately went up to 124% and 127% of the negative control, which was just above the permissible limit. However, elevated LDH activity of 3-Eaz and 4-Eaz samples were statistically insignificant as compared to the bare ESM. The results and plots narrated above for LDH activity substantiate that the above observation was in congruent with cell viability studies.

Cell viability and cytotoxicity studies conclude that the effect of nanoparticles on cellular integrity was observed with increase in time interval. It was apparent from the above results that sustained leaching of nanoparticles over time and longer exposure period moderately elevated the cytotoxicity levels. Moreover, since the culture medium was not replaced until the third day, the leached Ag and ZnO-NP accumulated within the environment thus leading to an obvious cytotoxic response on day 3. It was previously hypothesized from ICP-MS study that Ag nanoparticles tend to agglomerate to form bigger particles with increasing loading concentration. Previously Kim et.


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al. reported that cytotoxicity of Ag nanoparticles elevates with increasing particle size thus justifying the increased cytotoxicity of 4-Eaz samples as compared to its counterparts. In general, Ag and ZnO nanoparticles are categorized as cytocompatible at low concentrations as was obvious from the above study.72-73 High doses of > 50 ppm (µg/ ml) or > 20 ppm (µg/ ml) for Ag and ZnO nanoparticles respectively are considered cytotoxic against NIH 3T3 cell line.74-75 It was evident from ICP-MS studies that at pH – 5.5, a maximum release of 3.2 ppm and 0.22 ppm was recorded for Ag and ZnO NP respectively from the nanobiocomposites which were much lower than the toxicity range. The above explanations justify the biocompatible and less toxic nature of the as prepared samples. Further, experimental evidences also demonstrates that presence of appropriate dosage of Ag NP in biomaterial coatings may enhance osteoblast proliferation76-77 and also facilitate fibroblast attachment along with increased endothelial cell response.78 .

In order to understand the interaction of 3T3 cells with the fibers of nanobiocomposites,

FESEM analysis or rhodamine – DAPI staining was carried out. It was apparent from the FESEM images (Figure 9g-i) that 3T3 cells were able to adhere on the fibrous surface of Ag – ZnO or ZnO loaded nanobiocomposites post 3 days of incubation. Also, presence of nanoparticles didn’t cause any deformation in cellular morphology. Instead, formation of flower shaped ZnO nanoflakes led to reduction in inter-fiber space thus promoting anchorage of the lamellipodial extensions of fibroblast cells unlike the bare ESM. According to our previous study, ESM samples failed to provide a good platform for adhesion of fibroblast cells owing to large inter-fiber space and rough surface topography.39 Similar instances were also observed in this study thus presence of ZnO nanoflakes in low concentration might facilitate the attachment of fibroblast cells. Figure 10 illustrates fluorescence micrographs with rhodamine phalloidin - DAPI staining were in agreement 35 ACS Paragon Plus Environment


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Figure 10. Rhodamine – DAPI images of NIH 3T3 cells loaded nanobiocomposites and bare ESM post incubation for 3 days at 37ºC under ambient conditions. The study demonstrates cell attachment, proliferation and cell – cell interaction in on the surface of nanobiocomposites. (Scale Bar: 50 µm)

with FESEM results. It was observed that after 3 days of incubation, cells possessing intact morphology and glowing blue cell nuclei were trying to adhere on the surface of samples. It was clear from the micrographs that cells were able to maintain their cyto-skeleton until 2-Eaz samples whereas some distortions in membrane periphery was witnessed for 3-Eaz and 4-Eaz matrices. Higher loading and accumulation of nanoparticles in culture medium might have marginally affected membrane integrity. Above explained adhesion properties of cells on the prepared nanobiocomposites ratify the fact that if engineered accordingly, the composites can also be exploited further as a biomaterial in tissue regeneration studies. It was also observed previously that lower doses of Ag do not exhibit cytotoxic effects on eukaryotic cells. Usually eukaryotic cells are larger and possess higher structural or functional redundancy in contrast to prokaryotic cells. Thus lower dosages of Ag or ZnO NPs can be used to


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eradicate bacterial colonies while maintaining integrity of fibroblast cells.73 In this study, similar strategy was deployed wherein the three dimensional fibrous platform of ESM was exploited to develop biocompatible nanoparticle loaded composites for bactericidal activity. Further it can be concluded that 2-Eaz and 3-Eaz samples not only possess highest bactericidal activity against E. coli, P. aeruginosa, S. aureus or B. subtilis strains but also acted as a cytocompatible material. Conclusions The study narrates a unique route for in-situ synthesis of ZnO nanoflakes on microfibrous ESM and its hierarchical decoration with Ag – Nps for antibacterial applications. FESEM micrographs of as fabricated polymeric nanobiocomposite demonstrated presence of well-spaced ZnO nanoflakes impregnated microfibrous ESM with presence of Ag – NPs, covering the whole surface of matrix. It was evident that sonochemical assisted synthesis of the nanobiocomposite didn’t disrupt the three dimensional architecture of ESM. Physico-chemical characterization of the composite revealed excellent interaction not only between the nanoparticles but also at the interface where nanoparticles adhere onto the matrix. XPS studies clearly suggest presence of Ag – NPs in their metallic state and also confirms the interaction between Ag – ZnO NPs. Moreover, presence of nanoparticles also led to increased mechanical strength of the nanobiocomposites. The prepared nanobiocomposite demonstrated excellent antibacterial properties when exposed to both static as well as dynamic culture conditions thus validating the fact that Ag and ZnO nanoparticles could remain functional when decorated onto a polymeric matrix. It was apparent from above study that nanoparticle decoration enhanced bactericidal efficacy of the natural tissue by many folds. For suspension cultures, near complete elimination of bacterial colonies were obtained within 3 h post incubation. Additionally, it can also be stated that the natural tissue acted as an excellent carrier for nanoparticle loading which was further substantiated 37 ACS Paragon Plus Environment


by ICP-MS studies. The nanobiocomposites not only showed excellent release rate in the first few hours of contact but also possessed a sustained release profile with time. It was further ratified that the nanobiocomposites were biocompatible and encouraged cell adhesion with incubation time. The nanoparticle release rate along with bactericidal and cytocompatibility profile of nanobiocomposites conclude to the fact that 3-Eaz was best suited for varying biomedical applications like tissue engineering, antibacterial patches, insect repellant patches and others. The hierarchical nature of developed nanobiocomposite can also be explored as a platform for optical biosensor or also as a piezo-electric based nanogenerator application. Acknowledgement The authors of present work want to acknowledge fellowship support received from Council of Scientific and Industrial Research (CSIR) for Preetam Guha Ray (File No. 31/015(0134)/2017-EMR-I). The authors are also thankful to Indian Institute of Technology, Kharagpur for providing financial support and research facilities. The authors would also like to acknowledge Mr. Subhodeep Jana from Micro-CT lab at Central Research Facility, IIT Kharagpur for facilitating in Micro-CT related experiments. Supporting Information Available: The particulars of experimental methodology implemented for microstructural evaluation, biochemical analysis, and physico-mechanical characterization of as fabricated nanobiocomposites were detailed. Figures (Figures S1 - S6) and explanations related to magnified FESEM Micrographs, EDX, ATR-FTIR, XRD, charge distribution of zeta potential, ICP-MS of acid digested nanobiocomposite were also included. Table S1 containing average surface area of all the samples was also included.


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Sonication assisted facile approach to hierarchically decorate Ag-NP on ZnO nanoflowers impregnated eggshell membrane for bactericidal activity and evaluation of its in vitro cytocompatibility



Sonication assisted facile approach to hierarchically decorate Ag-NP on ZnO nanoflowers impregnated eggshell membrane for bactericidal activity and evaluation of its in vitro cytocompatibility 540x265mm (150 x 150 DPI)

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Sonication Assisted Hierarchical Decoration of Ag-NP on Zinc

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