Translate this page into:
Biocompatible pure ZnO nanoparticles-3D bacterial cellulose biointerfaces with antibacterial properties
⁎Corresponding author. mocanu_alexandra85@yahoo.com (A. Mocanu) alexandra.mocanu@upb.ro (A. Mocanu)
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
In this paper, we present for the first time the obtaining and characterization of new antibacterial and biocompatible nano-ZnO–bacterial cellulose (BC) material with controlled interfaces for studying in vitro microorganisms (Escherichia Coli (ATCC 8737), B. subtilis Spizizenii Nakamura (ATCC 6633), Candida albicans (ATCC10231)) and mammalian cells (human dermal fibroblast cells) response. The use of BC based material with controlled characteristics in terms of quantity and distribution of ZnO onto BC membrane (with 2D and 3D fibers arrangement) is directly correlated with the surface chemical and topographical properties, the method of preparation, and also with the type of cells implied for the specific application within the bioengineering fields. In our study, the uniform distribution and the control on the quantity of ZnO nanoparticles onto 3D BC were obtained using matrix assisted pulsed laser evaporation (MAPLE) method. The influence on particle distribution onto 3D bio cellulose were investigated based on two types of solvents (water and chloroform) involved in target preparation within MAPLE deposition. The attachment of the nanoparticles to the bacterial cellulose surface and fibrils was demonstrated by SEM and FT-IR studies. The BC-ZnO showed both resistance to bacteria-sticking and non-cytotoxic effect on the human dermal fibroblasts cells at a mass distribution onto surface of 1.68 µg ZnO NPS/mm2. These results represent a good premise in terms of tailoring BC substrates with ZnO particles that could determine or enhance both the biocompatibility and antibacterial properties of BC-composite materials.
Keywords
Bacterial cellulose
Zinc oxide nanoparticles
MAPLE deposition technique
Antibacterial effect
Biocompatibility
1 Introduction
Today, the need for developing new bactericidal materials has been significantly increased due to the concern regarding multidrug-resistant bacterial strains and biofilm associated infections, which can resist to methods commonly used. Bacterial surface contamination, adhesion, persistence and colonization of surfaces by bacteria are increasingly recognized as detrimental to health and society. Despite the numerous existing potent antibiotic drugs and other modern antibacterial means, bacterial infections are still a challenge. Antimicrobial materials used in the clinical setting today are beset by significant shortfalls, including weak antimicrobial activities, risk of microbial resistance, difficulty in monitoring and extending the antimicrobial functions and difficulty in functioning in a dynamic environment. In this context, engineering of materials that would prevent bacterial proliferation onto materials surfaces is an important challenge. Thus, obtaining materials that are both biocompatible and environmentally friendly would substantially contribute to important scientific and clinical advantages in various bio-applications.
Cellulose, which is the most abundant renewable organic material produced in the biosphere, serves as the dominant reinforcing phase in plant structures obtained from wood or annual plants (e.g. cotton, hemp, linen, jute, flax, kenaf, ramie). However, even if wood and annual plants are recognized as the major source of cellulose, there are also a few microorganisms (bacteria, algae, tunicates or fungi) which produce extracellularly certain amounts of cellulose, known as microbial cellulose (MC) (Siró and Plackett, 2010) or bacterial cellulose (BC) which is produced by different bacteria strains such as Acetobactereaceae (Cacicedo et al., 2016; Czaja et al., 2006; Römling, 2002).
The BC is characterized by a 3D multilayer porous structure with a non-woven network of nanofibrous cellulose which exhibits high water content (98–99%), high crystallinity and good high tensile strength (>2 GPa) (Bäckdahl et al., 2006; Petersen and Gatenholm, 2011; Sheykhnazari et al., 2016).
There are various fields of applicability for BC, from research to medicine and industry. For example, in the medical field, BC and its composites proved high biocompatibility in various applications such as: wound dressing materials, artificial skin, vascular grafts, scaffolds for tissue engineering, artificial blood vessels, medical pads and dental implants, just to mention few (Cacicedo et al., 2016; Czaja et al., 2006; Petersen and Gatenholm, 2011).
The major drawback of BC films is that pristine BC does not exhibit antimicrobial activity and in order to enlarge its applications it is necessary to find new methods for synthesis of BC based composites with antimicrobial properties.
In the last decade, many synthetic approaches were used to modify BC with metals and metallic oxides in order to obtain antimicrobial properties. Intensive studies were directed to BC-Ag nanocomposites for wound dressings applications (Maneerung et al., 2008; Wu et al., 2014), while metallic oxides such as TiO2 and Fe3O4 were used to obtain BC-TiO2 and BC-magnetite composites with high photocatalytic activity (Sun et al., 2010) or with magnetic properties which could be used for wastewater purification (Shen et al., 2009).
Due to their antimicrobial activity towards a large spectrum of bacterial microorganisms, zinc oxide nanoparticles (ZnO NPs) has been used frequently in applications related to food industry, agriculture, medical field and photocatalytic applications as well (Shahmohammadi Jebel and Almasi, 2016). Due to its good antibacterial activity (Baruah and Dutta, 2009; Kumar et al., 2011; Sirelkhatim et al., 2015) BC-ZnO composites were obtained by incorporation of ZnO NPs in BC using hydrolysis of zinc acetate in a polyol medium(Bitenc et al., 2009), but also using a newly created method called ‘‘ultrasonic-assisted in situ synthesis’’(Katepetch et al., 2013) or by solution plasma process(Janpetch et al., 2016).
Although ZnO NPs is noxious to certain microorganisms (Shahmohammadi Jebel and Almasi, 2016) its biocompatible properties with human body made it suitable for burn wounds treatment. Thus, functionalizing the BC substrates with ZnO NPs led to antimicrobial effect against both Gram positive and Gram-negative bacteria and faster healing rate (Khalid et al., 2017).
The main issue within the applied methods for NPs incorporation into BC structure is represented by non-uniform distribution or accumulation of NPs on the surface. Furthermore, the quantity of active compound with antimicrobial properties deposited in BC structure is rather difficult to control. In particular, for biological assays, when the initial behavior of cells is influenced by the interfacial characteristics, it is important to provide surfaces with uniform and controllable physical and chemical characteristics.
MAPLE technique has been developed in the recent years as a non-destructive deposition method, for surface functionalization with various proteins, nanoparticles, as well as natural and synthetic polymers while keeping the functionality and control on thickness, roughness and structural characteristics(Aronne et al., 2015; Boanini et al., 2015; Cristescu et al., 2004; Darwish et al., 2015; Dinca et al., 2014; Matei et al., 2011; Pique 2007; Rusen et al., 2016; Sima et al., 2011).
Using MAPLE for transferring small quantity of ZnO in a controlled manner could present interest for medical areas or food packaging applications that request antimicrobial properties of the final material, but also biomedical field, for wound dressing materials. However, there are only few studies related to using MAPLE for obtaining flat surfaces functionalization with ZnO based compound, but none related to coating of 3D configuration samples or for decorating BC. For example, just recently, MAPLE technique was applied to deposition of ZnO nanoparticles functionalized with usnic acid using excimer laser (248 nm) on flat silicon substrates in order to obtain antimicrobial materials with minimum effects on mammalian cells (Stan et al., 2016), as well as nanocomposite coating (ZnO-PEG) using Nd:YAG laser (532 nm) to prevent silicone hydrogel from biofouling (Huang et al., 2016).
In our study, we use MAPLE to decorate biocompatible porous and/or fibrous BC with synthetized ZnO nanoparticles in order to enhance antimicrobial effects of the final materials. Nd:YAG laser working at 266 nm was used to transfer ZnO NPs from chloroform, respectively water matrices onto flat and 3D BC substrates. The functionality of the decorated BC was demonstrated by evaluating its effects on Escherichia coli and human dermal fibroblast cells, aimingfuture applications that require both biocompatibility and antibacterial environments (i.e.: wound protection and healing). Furthermore, the quantity of ZnO NPs deposited onto BC substrates was estimated in all cases in order to evaluate a minimum concentration of the inorganic nanoparticles necessary to inhibit E. coli growth and not interfere with the adherence of human dermal cells. Up to our knowledge it is the first attempt of using this deposition technique for the deposition of pure ZnO NPs onto 3D bio-cellulose membranes in order to obtain nano-ZnO-BC biointerfaces with the respective functionality.
2 Materials and methods
2.1 Materials
Zinc acetate (Sigma-Aldrich), ammonia (25% wt., Aldrich), distilled water and chloroform were used without further purification.
2.2 Methods
2.2.1 Synthesis of bacterial cellulose (BC)
Bacterial cellulose (BC) membranes were produced by Gluconacetobactersp. strain isolated from traditionally fermented apple vinegar in Microbiology Laboratory of Chemical and Biochemical Engineering Department of University Politehnica of Bucharest. The culture was grown in a modified Hestrin-Schramm (MHS) medium containing 2% fructose using a static culture at 28 °C for 7 days. The pellicles obtained were washed with 0.5 N NaOH aqueous solutions at 90 °C for 1 h to eliminate the bacterial cells and then washed repeatedly with deionized water until the pH of water became neutral.
In this experiment BC membranes were used as wet films with 18% wt. humidity (referred as h-BC in the manuscript), respectively, dry films (d-BC).
2.2.2 Synthesis of ZnO nanoparticles
In a 50 mL round flasks 0.07 g of zinc acetate was dissolved in 20 mL of distilled water. Ammonia (25% wt.) was added while keeping a pH = 9. The reaction was maintained under continuous stirring at 60 °C for 2 min. The nanoparticles were recovered by centrifugation as white powder. The ZnO NPs were washed with water and ethanol several times and dried until constant mass.
2.2.3 Obtaining of BC-ZnO films by MAPLE technique
The MAPLE process takes place in a vacuum chamber and it implies the use of an external laser source (Surelite II” pulsed Nd: YAG laser system (Continuum Company) working at a wavelength of 266 nm, with 6 ns pulse duration and 10 Hz repetition rate), a solid cryogenic target and a receiving substrate. In an ideal system for the MAPLE process, the material to be deposited (guest material) is mixed or suspended into a solvent (host matrix), rapidly frozen in a cooper holder using liquid nitrogen, and placed inside the vacuum chamber. In our case, two types of targets, depending on the solvent (water and chloroform were obtained (2% ZnO and 98% solvent). The laser beam was focused on the target (spot size was maintained at 1 mm2 as measured by placing thermally sensitive paper in the plane of the target) under an incidence angle of 45°. The pulse duration was 5–7 ns with a repetition rate of 10 Hz. The laser fluence used was 0.6 J × cm−2. Prior to deposition, the chamber was evacuated to a background pressure of 10−4 mbar with a Pfeiffer-Balzers TPU 170 turbo molecular pump. Films were deposited on silicon and BC substrates located at 40 mm from the target. During deposition, the pressure raised to around 10−4 mbar. The number of pulses used in all the experiments was 36, respectively 90 kpulses. In order to have uniform evaporation the laser beam was translated onto the target surface, while the target was rotated with a motion feed through driven by a motor in order to avoid excessive heating and erosion of a single spot on the target that could lead to damage of the material.
2.2.4 Antimicrobial tests of BC-ZnO films
For antimicrobial activity assay was used Escherichia Coli (ATCC 8737) strain as Gram negative bacteria, B. subtilis Spizizenii Nakamura (ATCC 6633) as Gram positive prokariotic cells, and Candida albicans (ATCC10231) as eukariotic cells. The starter bacterial culture was prepared in liquid LB culture media, growth at 37 °C for 24 h. The cells were filtered and suspend in sterile distilled water in order to obtain an inoculum with concentration of 5.105 CFU/mL. The depletion of the inoculum in amount of 1 µL was made in Petri dishes with nutrient broth. The antimicrobial activity assay for obtained films was the diffusion in solid medium. The films used have square shape with 6 mm side length and were sterilized using short UV radiation. The probes were incubated at 37 °C for 72 h.
2.2.5 Growth of human dermal fibroblast cells on BC-ZnO biointerfaces
2.2.5.1 Sterilization procedure
All tested materials were sterilized for 15 min in antibiotic-antimycotic solution (penicillin 10000Units/ml-streptomycin 10000 μg/ml, 1% in phosphate buffered saline PBS, Gibco) prior to cell culture for preventing bacterial and fungal contamination.
2.2.5.2 Cell culture
Experiments were performed with human FBD (hFBD), human dermal fibroblast (kindly provided by Dr. Lucia Moldovan, INCDSB, Bucharest, Romania). Cells were cultured on material surfaces (0.9 cm2) in a 48-well plate (Costar flat bottom) at a density of 8 × 103 cells/well for 72 h in DMEM, High Glucose, GlutaMAX DMEM medium (Dulbecco’s modified eagle medium) supplemented with 10% Fetal Bovine Serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco) and maintained at 37 °C in a 5% CO2 incubator. After the indicated interval, the materials for testing were removed from culture and placed to a new 48-well plate for further processing.
2.2.5.3 In vitro cytocompatibility evaluation
hFBD viability and proliferation was evaluated through a quantitative cell colorimetric assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega) based on metabolically active cell’s capacity of reducing MTS tetrazolium compound to a colored formazan product. Cells were washed with PBS and incubated with MTS solution for 1 h at 37 °C and then 100 μL of supernatant were place in a 96-well plate clear bottom (Nunc, Thermo Scientific). The quantity of formazan as measured by absorbance at 450 nm using a microplate reader (Mithras LB 940 DLReady) is directly proportional to the number of living cells adhered on the surface of material.
2.2.5.4 Human dermal fibroblast adhesion and morphology
After 72 h of cultivation, hFBD grown on material tested (biocellulose (BC) and BC-ZnO substrates) were fixed for 20 min with 4% paraformaldehyde prepared in PBS, permeabilized for 3 min at room temperature with 0.2% Triton X-100 and then blocked for 1 h with 0.5% bovine serum albumin (BSA) in PBS. After that, samples were incubated with Alexa Fluor 488-conjugated phalloidin (Invitrogen A12379, Life Technologies) dilution 1:100 in 0.5%BSA-PBS for actin filaments detection, washed with PBS and mounted with Prolong Gold Antifade Reagent (Life Technologies). Cells were examined under an Axio Imager.M2 Zeiss Microsope with ApoTome.2 slider module using EC Plan-Neofluar 10x/0.30 M27 objective. Images were captured with AxioCamMRm camera and processed with AxioVisionRel 4.8 program.
2.2.5.5 Sample preparation for scanning electron microscopy
All samples were fixed with 2.5% glutharaldehide in PBS solution for 20 min and then dehydrated for 2 rounds of 15 min incubation with 70%, 90% and 100% ethanol solution. Specimen drying was performed using 50%, 75% hexamethyldisilazane (HDMS electronic grade, Alfa Aesar, Thermo Fisher) solution in ethanol and then 100% pure HDMS solution for 2 rounds of 3 min each. Samples were left to dry in a chemical Asalair 1200 adsorption hood.
3 Characterization
The ZnO NPs and BC films morphology were investigated using a Scanning Electron Microscope (SEM), FEI Co (model Inspect S), 0–30 kV accelerating voltage, working distance 0–30 mm, images have been recorded on different locations of the samples in high vacuum. For microorganism analysis, 10 nm gold was deposited onto the dehydrated samples in order to improve conductivity.
A TecnaiTM G2 F30 S-TWIN high resolution transmission electron microscope (HRTEM) was employed for TEM-SAED/HRTEM investigations, the samples being dispersed in ethanol and strongly ultrasonicated in order to brake the aggregates.
The FT-IR analyses of BC-ZnO biointerfaces were examined on a Jasco FT/IR6200 spectrometer (ABL& E-JASCO Romania) with Intron μ Infrared Microscope with ATR-1000-VZ objective. The spectra were the average of 50 scans recorded at a resolution of 4 cm−1 in a range from 4000 to 500 cm−1 with a TGS detector.
The ZnO crystalline phase was analysed using x-ray powder diffraction (XRD) performed on a Panalytical X'Pert MRD system (λ CuKα = 0.15418 nm).
In order to confirm the presence of ZnO NPs on BC films XPS analysis was performed by EnviroESCA™ (SPECS™ Surface NanoAnalysis GmbH) using monochromatic Aluminum Kα excitation (hν = 1486.7 eV). The samples were mounted on a standard sample plate using double sided Carbon tape. The spectra were measured at 1 mbar of Argon to compensate sample charging. Binding energy scale was corrected using the C 1s peak at 285.0 eV.
In order to estimate the thickness of the ZnO layer deposited on BC substrates, atomic force microscopy (AFM) was performed on Silica plates covered with ZnO in the same conditions and considered as blank substrates for our deposition technique. The AFM measurements were carried out in non-contact mode, using silicon probes (PPP-NCHR, Nanosensors) with 42 N/m force constant, 330 kHz resonance frequency and less than 7 nm radius (nominal values). Topography images were recorded on 40 µm × 40 µm areas.
4 Results and discussion
4.1 Morphological analysis
The MAPLE deposition technique involves the use of different solid targets suspended in various solvents (as described in Section 2.2.3). For MAPLE deposition targets, preformed ZnO NPs obtained by chemical conventional method of reducing a zinc salt precursor in the presence of ammonia were used. The ZnO NPs were separated by centrifugation before being used for MAPLE target preparation and analysed by SEM (Fig. 1a, b). As one can observe, ZnO NPS have dimensions of about few tens of nanometers (Fig. 1b) and aggregate in larger polycrystalline grains.
In order to prove the formation of ZnO NPs with crystalline structure, TEM-SAED/HRTEM investigations were performed on the inorganic powder (Fig. 2). TEM image confirmed the dimensions determined by SEM analysis, namely an average size of 20–30 nm, while HRTEM image highlighted specific lattice spacing parameters for ZnO with wurtzite type structure. Moreover, the SAED pattern indicated a polycrystalline nature of the sample, the diffraction rings being assigned to the interplanar distance of the previously mentioned structure.
In our next step, 2% ZnO NPS dispersed in water, respectively chloroform, were deposited by drop cast method to be used as reference for MAPLE depositions. Both films obtained by this method proved to be inhomogeneous consisting on ZnO NPS agglomerates. No significant influence of solvent was noticed. A typical SEM image of the reference samples is presented in Fig. 3.
Using MAPLE, the deposition of ZnO NPs from aqueous or chloroform matrices on the BC substrate revealed completely different morphologies depending on both solvents (matrices) and the type of substrate used (porous and fibrous BC). By MAPLE, ZnO NPs deposition onto substrates leads to uniform, homogenous distribution of much smaller ZnO NPs agglomerations.
Fig. 4 shows SEM images of ZnO aqueous dispersion deposited on h-BC substrate. ZnO NPs are uniformly distributed onto the substrate surface. Only a small number of larger size agglomerations are present (detail image – Fig. 4b).
Compared with the distribution on the h-BC substrate, the use of d-BC leads also to a ZnO NPS uniform and homogeneous distribution but not only on the substrate surface, but also penetrating between fibers, as shown in Fig. 5(b and c).
Using chloroform as a deposition matrix leads to a substantial change in ZnO NPs distribution on the BC surfaces as shown in the SEM images presented in Fig. 6. Individual ZnO NPs and higher number of aggregates on the surface of the BC substrate are observed compared with the ZnO aqueous suspension case. Furthermore, the ZnO NPs shows higher density on the porous areas of the d-BC or on the irregularities of BC fibers (detail Fig. 6b). The detail image (Fig. 6c) shows the distribution of ZnO NPs and aggregated structures on the surface of d-BC.
It is obvious that the use of ZnO NPs dispersion in chloroform solvent as target for MAPPLE deposition is suitable for a larger ZnO NPs concentration onto BC substrate compared with the use of ZnO aqueous dispersion. These difference between the two dispersions can be attributed to better absorption of the laser source (wavelength 266 nm) energy by chloroform. Furthermore, the nature of the substrate (humid or dry), can lead to competitive processes occurring due to the pressure variation in the deposition chamber during deposition. This could involve the removal of the water from h-BC substrate simultaneously with the fixation of the inorganic particles on the BC film, preventing the adherence of ZnO NPs on the surface. Thus, this suggests that d-BC may be a more suitable material for use as substrate in MAPLE deposition procedure.
4.2 FT-IR analysis
Fig. 7 presents the spectra of pristine BC (a) and the two BC-ZnObiointerfaces obtained by MAPLE technique: on d-BC membrane (b) and h-BC membrane as substrates (c).
Comparing the FTIR spectra, no noticeable differences between them are present. The stretching vibrations of hydroxyl groups are slightly shifted in the BC-ZnO spectra compared with the BC substrate spectrum, fact that indicate an interaction between zinc ions (Zn2+) and hydroxyl groups of BC. The peak at 1638 cm−1 that corresponds to the in-plane –OH vibration of pure BC is slightly shifted to the lower wave numbers in the theBC-ZnO spectra (1625 cm−1 and respectively 1627 cm−1) confirming the surface modification of BC. These observations are in agreement with FT-IR studies previously presented in literature for BC-ZnO composites (Chen et al., 2013; Hu et al., 2011). The FT-IR analysis confirm a slight modification of the substrates in both cases. Due to the fact that MAPLE is a non-destructive deposition technique, this represents an advantage when tailoring properties of different substrates with thin uniform layers of relatively expensive compounds.
4.3 XRD analysis
In the present case, MAPLE technique attained the obtaining of BC-ZnO biointerfaces with uniformly distributed ZnO NPs starting from previously synthesized polydisperse ZnO NPs (Fig. 1). In order to determine the crystalline structure of ZnO deposited via MAPLE, XRD analysis was performed on the BC modified substrates. Due to the very small NPs size and low concentration of ZnO deposited onto substrates, XRD characterization revealed only the cellulose substrate presence (JCPDS no 056-1717). The XRD spectrum presents three major peaks assigned to 100, 010 and 110 crystallographic planes which correspond at 2θ = 14.5°, 16.7° and 22.68° diffraction angles (Fig. S1- Supplementary information), clearly evidencing a typical pattern of cellulose, in good agreement with previous reported data (Wu et al., 2012).
Thus, in order to be able to identify the crystalline structure of ZnO NPs, reference samples fabricated by drop cast method from the solutions used as targets in MAPPLE deposition were used. Fig. 8 shows the hexagonal wurtzite - ZnO crystalline structure characteristic diffraction peaks according to JCPDS no. 036-1451.
4.4 XPS analysis
To provide more information about the surface modification of BC films, XPS analysis was performed (Fig. 9). The samples registered peaks for carbon (C), nitrogen (N) and oxygen (O) in the case of pristine BC, while Zn element was observed for modified BC film.
The deconvolution operation for broad C1s peak was associated with C—H, C—C, or C⚌C binds at the energy of ∼285.0 eV. The signals registered at 286.5 eV, respectively 288 eV were attributed to C—O singly bound (from hydroxyl groups), respectively to C⚌O double bond. The predominant carbon composition (almost 60%) appeared in pristine BC (Fig. 9a) and BC—ZnO samples (Fig. 9b). Scans of N1s was performed and high intensive peaks were observed in both cases: 7 at% for pristine BC (Fig. 9a), and almost 4 at% for the BC modified with ZnO (Fig. 9b). These peaks (registered at 400 eV) are attributed to free amine (H2N—C) and amide (O⚌C—N—C) groups that are coming from some residual bacteria proteins responsible for synthesis process of BC (Taokaew and Phisalaphong, 2015). In Fig. 9b, the XPS spectra revealed that the Zn 2p3/2 spectrum of ZnO was registered at 1021 eV. As it can be observed, the peak of ZnO suffers from overlapping with peaks coming from Zn metal which is in good accordance with literature data (Biesinger et al., 2010). Thus, XPS analysis confirms the presence of ZnO deposited on the surface of BC.
4.5 Bactericide action
Although the bactericidal mechanism of ZnO NPs is still unclear it is known that in solutions, ZnO NPs shows antibacterial activity against both Gram-positive and Gram-negative bacteria. The antibacterial mechanisms for prokaryotic cells were quantified based on different morphologies of ZnO and were attributed to: (i) H2O2 production, (ii) Zn2+ release from dissolution of ZnO NPS, and (iii) the presence of oxygen vacancies onto surface. Thus, the antimicrobial effect of ZnO NPS was proved to be responsible by the production of H2O2 in the absence of light, while maximum Zn2+ release as bactericide mechanism was registered in the case of ZnO micro-disks(Jiang et al., 2016; Sharma et al., 2016). Composites reinforced with ZnO NPs allowed the prokatiotic cells to preferentially adhere on the substrate while H2O2 or Zn2+ were released from ZnO surface which determined a self-entrapment mechanism of the bacteria. As a consequence, this led to the death of Gram negative bacteria at MIC concentrations above 35 μg ZnO NPs/mL of culture medium(Sharma et al., 2016), while for B. subtilis a concentration of 105.17 µg/mL was registered (Arakha et al., 2015), which indicated a high sensibility of these strains in the presence of ZnO NPs. For Candida species, the literature studies about the mechanisms of ZnO NPs action are not conclusive, but was reported larger concentration of ZnO NPs solution to obtain an inhibitory effect on this strain (5.8mg/mL) (Abd and Abbas, 2015).
In our case, the quantity of ZnO NPs deposited onto BC substrates was estimated for all experiments in order to evaluate the optimum concentration of the ZnO NPs needed to inhibit pathogen prokaryotic and eukaryotic cells growth and favor the adherence of fibroblast cells.
The mass distribution of ZnO NPs onto substrates was estimated using the layer thickness determined by AFM. In the case of ZnO aqueous dispersion target deposited on d-BC, the ZnO mass distribution was estimated to 0.28 µg/mm2, while in the case of ZnO chloroform dispersion target, the concentration of antimicrobial particles reached 0.56 µg/mm2 in the case of 90 kpulses MAPLE deposition. Due to the ability of depositing small quantities of bio-functional material in a controlled manner by this technique, the inhibition rate of bacterial growth couldn’t be estimated by classic methods of direct observation of inhibition areas. Therefore, the behavior of BC-ZnO interfaces towards bactericidal inhibition was observed using SEM on surfaces of the functionalized BC cultured with E. coli for 72 h. Fig. 10 shows SEM images of E. coli adherence on: (a, b) BC substrates; (c, d) ZnO aqueous dispersion target deposited on d-BC and (e, f) ZnO chloroform dispersion target deposited on d-BC at different magnifications.
It is obvious that the BC substrates are an enabling environment for bacterial growth, a large number of microorganisms being adhered onto the BC surface, with no change in cell shapes (Fig. 10a, b). However, when BC-ZnO films obtained from aqueous dispersion were used, an inhibitory action on E. coli was observed as evidenced by the small number of bacteria adhering to the surface film (Fig. 10c, d). Moreover, surface morphology of the cells is altered, it looks rough and presenting local membrane invagination, that can be explained by the direct action of Zn2+ ions on the cellular sheath.
Biocellulose films decorated with ZnO NPS (the target prepared from chloroform dispersion) had a pronounced antibacterial action (Fig. 10e, f) which can be correlated with the higher concentration of ZnO NPS deposited on BC substrate. The number of adherent cells on the surface of the film is lower than for BC-ZnO interfaces obtained with ZnO deposited from aqueous dispersion. In Fig. 10f different morphologies of the cells can be observed such as invagination of the membrane’s surface of most cells, disrupted cells and deformities of the cells shape, which indicates a consistent effect of the ZnO NPS presence even at such low concentration (0.56 µg/mm2). The differences between the effects of the two types of coatings on E. coli inhibition can be related to lower quantity of ZnO NPS deposited by MAPLE from water matrix, as compared to chloroform.
Different laser or deposition parameters (laser wavelength, pulse number, target concentration) can be used in order to tailor the quantity of material transferred during deposition. For example, when increasing the number of pulses, the fibrils of d-BC were completely covered with a thick uniform layer of ZnO NPs (Fig. 11a). Although the exposure of the new d-BC with higher quantities of ZnO NPs to E. coli did not led to a clear inhibition area, after SEM examination, E. coli cells were not present on the substrates as in our previous experiment (Fig. 10). Thus, at a 300 nm layer thickness which was correlated with a concentration of 1.68 µg ZnO NPs/mm2, the adherence of Gram negative and Gram positive cells on the substrate’s surface was completely prevented (Fig. 11b, c). The smooth surface of the film corresponding to B subtilis cultivation is due to the exopolysaccharides excreted by the microorganisms in stress conditions induced by the ZnO NPs presence (Fig. 11c) (Arakha et al., 2015). The inhibition of the B. subtilis growth was highlighted by the inhibition zone recorded (IZ = 3 mm Fig. S2 from Supplementary information). Candida cells were not affected by the ZnO NPs concentration; in the solid medium cultivation it was not recorded the inhibition zone, but in the SEM imagine the cells aggregate present a distortion of the usual shape (white arrows) (Fig. 11d).
4.6 In vitro cytocompatibility evaluation using human dermal fibroblast cells
Both BC and ZnO materials are widely used for application from daily care products to sensors or biomedical products. Therefore, a good understanding on the ability of materials prepared in a new approach is required depending on the type of application needed. Good cell proliferation and spreading on native, or modified phosphorylated, and sulfated BC was previously demonstrated (human keratinocytes, human umbilical vein endothelial cells, chondrocytes, etc.) (Chiaoprakobkij et al., 2011; Zang et al., 2015). Although there are various studies implying ZnO particle dissolution inside cells compartments and generation of reactive oxygen species, the responses towards different cells lines are highly variable and are depending on many parameters such as NPs characteristics, NPs synthesis methods, cells type and their exposure mode to NPs, just to mention few (Baptista et al., 2018). In the present study, the experiments on human dermal fibroblasts were performed using samples containing 1.68 µg ZnO NPs/mm2 deposited by MAPLE from chloroform dispersion target, for which the adherence of Gram negative bacteria on BC-ZnO surfaces was prevented. Although the apoptosis induction was reported previously by ZnO NPs in human dermal fibroblasts, our viability tests revealed a different behavior for deposited ZnO NPs. As shown in Fig. 12, the viability tests results, as well as fluorescence and SEM images, indicate a good interaction between the human dermal fibroblasts cells and the BC with and without ZnO.
In 72 h, a slight decrease of cell viability in the BC-ZnO groups when compared to the BC alone or the control was observed. The results were confirmed by the cells morphology analyzed by florescence microscopy after 3 days of culturing (Fig. 12b). Both fluorescence microscopy images and SEM analysis of hFBD after 72 h of cultivation show a higher number of cell spreading onto the control surfaces as compared to BC or BC-ZnO. However, in the case of both type of BC based interfaces, similar spreading was observed, with the actin filaments redistributed into arm-like structures following the anchoring of cells onto the fibrous profile of the BC-ZnO substrates.
Considering these initial results, we could conclude that these BC-ZnO new biointerfaces have a great potential to be used in several biomedical applications that also request antibacterial properties (as new possible types of antibiotics).
5 Conclusions
Pure ZnO NPs have been successfully deposited on humid and dry BC by MAPLE deposition technique achieving a 3D configuration. The inorganic nanoparticles were analyzed by SEM and HRTEM prior to MAPLE deposition onto biocellulose (wet and dry) substrates. This coating technique leads to uniform and homogeneous ZnO NPs distribuiton onto BC films. The BC-ZnO interfaces have been investigated by FT-IR, SEM, and XPS analysis and the BC-ZnO films were tested for antibacterial activity and biocompatiblilty. The BC-ZnO composite materials showed excellent antibacterial activity against B. subtilis and E. coli. as well as good biocompatibility with human dermal fibroblasts cells. The antimicrobial effect of ZnO NPs on the eukaryotic cells, as C. albicans, due to the absence of inhibition zone, was observed only in the SEM images through the modified aggregates. The BC-ZnO showed an excellent prevention of bacteria-sticking at a mass distribution onto surface of 1.68 µg ZnO NPS/mm2, corresponding to a 300 nm ZnO NPs coating. As the antimicrobial test assay demonstrated, even a very thin layer of ZnO has the ability to inhibit completely bacterial growth (Gram positive and Gram negative), without a noticeable effect against eukaryotic cells.
The viability results, as well as the morphology analysis of mammalian cells onto the BC based surfaces, indicated a good interaction between the human dermal fibroblasts cells and the BC with and without ZnO. These results represent a good premise in terms of tailoring BC substrates with ZnO particles that could determine or modulate both the biocompatibility and antibacterial properties of BC-based materials.
Acknowledgement
A.V, V.D. and C.B. acknowledges financial support from Romanian National Authority for Scientific Research (CNCS–UEFISCDI), under the Projects PN-II-RU-TE-2014-1550, PN-II-RU-TE-2014-4-2434, PN-III-P1-1.1-TE-2016-0871 (contract number 66/2018) and Nucleus program-contract. M. S. greatfully acknowledges the Romanian Ministry of Research and Innovation, Project number 15 N/2016 (TEHNOSPEC). AM acknowledges for financial support from University Politehnica of Bucharest through UPB – GEX 2017 Excellence Research Grants Program, project “Active carbon from biomass – BIOPHOTOCARB”, Contract no. 77/25.09.2017, code no. CH 38-17-07.
References
- Effect of zinc oxide nanoparticles on candida albicans of human saliva (in vitro study) Eur. J. Med.. 2015;10:235-244.
- [Google Scholar]
- The effects of interfacial potential on antimicrobial propensity of ZnO nanoparticle. Sci. Rep.. 2015;5:9578.
- [Google Scholar]
- Lipase biofilm deposited by matrix assisted pulsed laser evaporation technique. Appl. Surf. Sci.. 2015;336:196-199.
- [Google Scholar]
- Mechanical properties of bacterial cellulose and interactions with smooth muscle cells. Biomaterials. 2006;27:2141-2149.
- [Google Scholar]
- Nano-strategies to fight multidrug resistant bacteria—“a battle of the titans”. Front Microbiol. 2018:9.
- [Google Scholar]
- Nanotechnology applications in pollution sensing and degradation in agriculture: a review. Environ. Chem. Lett.. 2009;7:191-204.
- [Google Scholar]
- Resolving surface chemical states in XPS analysis of first row transition metals, oxides and hydroxides: Sc Ti, V, Cu and Zn. Appl. Surf. Sci.. 2010;257:887-898.
- [Google Scholar]
- Correlation between morphology and defect luminescence in precipitated zno nanorod powders. Crystal Growth Design. 2009;9:997-1001.
- [Google Scholar]
- Antiresorption implant coatings based on calcium alendronate and octacalcium phosphate deposited by matrix assisted pulsed laser evaporation. Colloids Surf. B: Biointerf.. 2015;136:449-456.
- [Google Scholar]
- Progress in bacterial cellulose matrices for biotechnological applications. Bioresource Technol.. 2016;213:172-180.
- [Google Scholar]
- Polyol mediated synthesis of ZnO nanoparticles templated by bacterial cellulose. Carbohyd. Polym.. 2013;92:1953-1959.
- [Google Scholar]
- Characterization and biocompatibility of bacterial cellulose/alginate composite sponges with human keratinocytes and gingival fibroblasts. Carbohyd. Polym.. 2011;85:548-553.
- [Google Scholar]
- Deposition of biopolymer thin films by matrix assisted pulsed laser evaporation. Appl. Phys. A. 2004;79:1023-1026.
- [Google Scholar]
- Microbial cellulose—the natural power to heal wounds. Biomaterials. 2006;27:145-151.
- [Google Scholar]
- Multi-beam pulsed laser deposition: new method of making nanocomposite coatings. SPIE Opt. Eng. + Appl. SPIE 2015:13.
- [Google Scholar]
- MAPLE-based method to obtain biodegradable hybrid polymeric thin films with embedded antitumoral agents. Biomed. Microdevices. 2014;16:11-21.
- [Google Scholar]
- Solvent-free acetylation of bacterial cellulose under moderate conditions. Carbohyd. Polym.. 2011;83:1575-1581.
- [Google Scholar]
- Nanocomposited coatings produced by laser-assisted process to prevent silicone hydogels from protein fouling and bacterial contamination. Appl. Surf. Sci.. 2016;360:383-388.
- [Google Scholar]
- Fabrication of bacterial cellulose-ZnO composite via solution plasma process for antibacterial applications. Carbohyd. Polym.. 2016;148:335-344.
- [Google Scholar]
- Role of physical and chemical interactions in the antibacterial behavior of ZnO nanoparticles against E. coli. Mater. Sci. Eng. C. 2016;69:1361-1366.
- [Google Scholar]
- Formation of nanocrystalline ZnO particles into bacterial cellulose pellicle by ultrasonic-assisted in situ synthesis. Cellulose. 2013;20:1275-1292.
- [Google Scholar]
- Bacterial cellulose-zinc oxide nanocomposites as a novel dressing system for burn wounds. Carbohyd. Polym.. 2017;164:214-221.
- [Google Scholar]
- Synthesis of well-defined amphiphilic block copolymers by organotellurium-mediated living radical polymerization (TERP) Macromol. Rap. Commun.. 2011;32:1576-1582.
- [Google Scholar]
- Impregnation of silver nanoparticles into bacterial cellulose for antimicrobial wound dressing. Carbohyd. Polym.. 2008;72:43-51.
- [Google Scholar]
- Growth of thin films of low molecular weight proteins by matrix assisted pulsed laser evaporation (MAPLE) Appl. Phys. A. 2011;105:629-633.
- [Google Scholar]
- Bacterial cellulose-based materials and medical devices: current state and perspectives. Appl. Microbiol. Biotechnol.. 2011;91:1277.
- [Google Scholar]
- Deposition of Polymers and Biomaterials Using the Matrix-Assisted Pulsed Laser Evaporation (MAPLE) Process. In: Pulsed Laser Deposition of Thin Films. New Jersey: Wiley; 2007. p. :63-84.
- [CrossRef] [Google Scholar]
- Molecular biology of cellulose production in bacteria. Res. Microbiol.. 2002;153:205-212.
- [Google Scholar]
- In vitro evaluation of poly(ethylene glycol)-block-poly(ɛ-caprolactone) methyl ether copolymer coating effects on cells adhesion and proliferation. Appl. Surf. Sci.. 2016;374:23-30.
- [Google Scholar]
- Morphological, physical, antimicrobial and release properties of ZnO nanoparticles-loaded bacterial cellulose films. Carbohyd. Polym.. 2016;149:8-19.
- [Google Scholar]
- Effect of ZnO morphology on affecting bactericidal property of ultra high molecular weight polyethylene biocomposite. Mater. Sci. Eng. C. 2016;62:843-851.
- [Google Scholar]
- Adsorption of Cu(II) and Pb(II) onto diethylenetriamine-bacterial cellulose. Carbohyd. Polym.. 2009;75:110-114.
- [Google Scholar]
- Bacterial cellulose composites loaded with SiO2 nanoparticles: dynamic-mechanical and thermal properties. Int. J. Biol. Macromol.. 2016;93:672-677.
- [Google Scholar]
- Fibronectin layers by matrix-assisted pulsed laser evaporation from saline buffer-based cryogenic targets. Acta Biomater.. 2011;7:3780-3788.
- [Google Scholar]
- Review on zinc oxide nanoparticles: antibacterial activity and toxicity mechanism. Nano-Micro Lett.. 2015;7:219-242.
- [Google Scholar]
- Microfibrillated cellulose and new nanocomposite materials: a review. Cellulose. 2010;17:459-494.
- [Google Scholar]
- Thin coatings based on ZnO@C18-usnic acid nanoparticles prepared by MAPLE inhibit the development of Salmonella enterica early biofilm growth. Appl. Surf. Sci.. 2016;374:318-325.
- [Google Scholar]
- Bacterial cellulose/TiO2 hybrid nanofibers prepared by the surface hydrolysis method with molecular precision. Nanoscale. 2010;2:287-292.
- [Google Scholar]
- Newby B-mZ Modification of bacterial cellulose with organosilanes to improve attachment and spreading of human fibroblasts. Cellulose. 2015;22:2311-2324.
- [Google Scholar]
- Chemical modifications and characteristic changes in bacterial cellulose treated with different media. J. Polym. Res.. 2012;19:9945.
- [Google Scholar]
- In situ synthesis of silver-nanoparticles/bacterial cellulose composites for slow-released antimicrobial wound dressing. Carbohyd. Polym.. 2014;102:762-771.
- [Google Scholar]
- Investigation on artificial blood vessels prepared from bacterial cellulose. Mater. Sci. Eng. C. 2015;46:111-117.
- [Google Scholar]
Appendix A
Supplementary material
Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2018.12.003.
Appendix A
Supplementary material
The following are the Supplementary data to this article:Supplementary data 1
Supplementary data 1