Volume 65, Issue 2 p. 714-731
RESEARCH ARTICLE
Open Access

Mechanical and antimicrobial properties of green and photoactive AgTiO2/poly(3hydroxybutyrate) (PHB) electrospun membranes

Safa Ladhari

Safa Ladhari

Department of Chemistry, Biochemistry, and Physics, Université du Québec à Trois-Rivières (UQTR), Trois-Rivières, Québec, Canada

Laboratory of Advanced Materials for Energy and Environment, Université du Québec à Trois-Rivières (UQTR), Trois-Rivières, Québec, Canada

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Nhu-Nang Vu

Nhu-Nang Vu

Department of Chemistry, Biochemistry, and Physics, Université du Québec à Trois-Rivières (UQTR), Trois-Rivières, Québec, Canada

Laboratory of Advanced Materials for Energy and Environment, Université du Québec à Trois-Rivières (UQTR), Trois-Rivières, Québec, Canada

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Alireza Saidi

Alireza Saidi

Laboratory of Advanced Materials for Energy and Environment, Université du Québec à Trois-Rivières (UQTR), Trois-Rivières, Québec, Canada

Institut de Recherche Robert-Sauvé en Santé et Sécurité du Travail (IRSST), Montréal, Québec, Canada

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Amine Aymen Assadi

Amine Aymen Assadi

College of Engineering, Imam Mohammad Ibn Saud Islamic University, IMSIU, Riyadh, Saudi Arabia

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Phuong Nguyen-Tri

Corresponding Author

Phuong Nguyen-Tri

Department of Chemistry, Biochemistry, and Physics, Université du Québec à Trois-Rivières (UQTR), Trois-Rivières, Québec, Canada

Laboratory of Advanced Materials for Energy and Environment, Université du Québec à Trois-Rivières (UQTR), Trois-Rivières, Québec, Canada

Correspondence

Phuong Nguyen-Tri, Department of Chemistry, Biochemistry, and Physics, Université du Québec à Trois-Rivières (UQTR), 3351 Boulevard des Forges, Trois-Rivières, QC G8Z 4M3, Canada.

Email: [email protected]

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First published: 16 December 2024

Abstract

The rise of antibiotic-resistant microbes and concerns over non-biodegradable waste highlight the need for innovative materials with integrated functionalities that address these critical issues. In this study, we synthesized poly(3hydroxybutyrate) (PHB) electrospun nanofibers blended with gelatin (Ge) and loaded with photoactive AgTiO2 nanoparticles with improved mechanical and biological properties. Biological tests revealed excellent antibacterial activity of the prepared membrane, with efficiency exceeding 99% against Escherichia coli and 95% against Staphylococcus epidermidis after 90 and 60 min of exposure to low-power commercial LED lights, respectively. Filtration studies using a dead-end stainless-steel cell reveal that both bacteria are eliminated (>99% after one filtration cycle). Results of the viability test showed that blending PHB with Ge improves the membrane's anti-biofouling properties. The membranes were also characterized using SEM and EDX mapping techniques for morphological and elemental analysis, DSC and TGA to evaluate thermal properties and crystallinity, and FTIR to confirm chemical structure. Moreover, the electrospun membranes exhibited enhanced mechanical properties with the addition of Ge. PHB/Ge/3 wt% AgTiO2 sample showed 2.2 times better tensile strength and 1.89 times improved Young's modulus compared to PHB membranes. Finally, the breakdown of PHB/Ge/AgTiO2 membranes occurred progressively over only 8 weeks, showing the membranes' green and sustainable nature.

Highlights

  • Antibacterial efficacy of 99% against E. coli and 95% against S. epidermidis.
  • Microfiltration efficiency >99% achieved in one filtration cycle.
  • Mechanical strength is enhanced by 2.2 times with the addition of gelatin.
  • Effective anti-biofouling is confirmed by CLSM and SEM tests.
  • Membranes degraded within 8 weeks in natural soil.

Abbreviations

  • CLSM
  • confocal laser scanning microscopy
  • DSC
  • differential scanning calorimetry
  • E. coli
  • Escherichia coli
  • Ge
  • gelatin
  • PHA
  • polyhydroxyalkanoates
  • PHB
  • poly(3hydroxybutyrate)
  • ROS
  • reactive oxygen species
  • S. epidermidis
  • Staphylococcus epidermidis
  • SEM
  • scanning electron microscope
  • TGA
  • thermogravimetric analysis
  • 1 INTRODUCTION

    Clinical or other healthcare equipment, a hospital-acquired infection, often known as nosocomial, is an infection contracted when obtaining medical treatment.1 Bacterial infections are collected in surgical operating rooms, clinics, hospitals, and diagnostic labs. Microbes in large quantities can be harmful to humans and cause death.2, 3 Combining chemical entities to achieve antibacterial effects is an innovative method for minimizing the spread of infectious pathogens. Microbes adhered to manufactured surfaces tend to survive and multiply in a humid environment. Meanwhile, microbial cells form a biofilm, which grows in number on the surface. The biofilm consists of polysaccharides and encapsulated cells, nourishing microbial cells and allowing them to endure harsh environments.4 Controlling microbial development on artificial surfaces is a crucial focus of health and material science. Sterilizing the surrounding environment can prevent surface infection. The most often used disinfectants include hydrogen peroxide, hypochlorite, and reactive oxygen species (ROS). Other often-used chemicals include alcohols, quaternary ammonium compounds, silver, and triclosan.5 Regular use of disinfectants, particularly triclosan, can lead to significant environmental issues, as the aseptic state is temporary.6 Another option is to use antimicrobial surfaces to avoid bacteria biofilm formation. Corresponding surfaces reject or kill microorganisms, preventing adhesion.7 Using antibacterial materials to prevent bacterial contamination in some medical areas is one of the most promising methods for managing pathogenic bacteria.8 As antibiotics lose efficacy, resulting in treatment failure, infectious diseases become more difficult to treat.9, 10 Various prominent antimicrobial compounds, such as carbon nanotubes, metallic oxide nanoparticles, metal–organic frameworks (MOF), and anti-biofilm gel, have evolved as substitutes for antibiotics to stop bacterial resistance.11, 12

    The current drive to discover novel antimicrobial products with unique properties, such as the capacity to combat bacteria resistant to multiple drugs, makes it imperative to enhance the productive techniques for creating nanostructured materials with antibacterial qualities for human health and environmental fields. Specifically, the characteristics of electrospun nanofibers, like their diameter, alignment, high porosity, high surface area to volume, linked pores, controlling the release of active compounds, and increasing the dispersion of nano-additives into biopolymer matrices, can be engineered to kill bacteria.13-15 poly(3hydroxybutyrate) (PHB), the simplest member of the poly (3-hydroxy alkanoates) family, is one of the most studied microbial polyesters due to its thermoplastic behavior and mechanical properties that make it suitable for drug-loading applications. Furthermore, it has better renewability, biodegradability, and biocompatibility than other aliphatic polyesters [such as polylactic acid (PLA) and polycaprolactone (PCL)].12, 16 However, the surface hydrophobicity of materials based on PHB is one of its drawbacks. Regarding biomedical applications, PHB's hydrophobic properties influence its regulated biodegradation and effective interactions with biological media and cells.17, 18 Furthermore, due to its high crystallinity, PHB is stiff and brittle,19 resulting in poor mechanical characteristics and a limited extension at the break, limiting its applicability. Another barrier to the successful use of these polymers is their production costs, determined mainly by the costs of the beginning feedstocks. Since the discovery of PHB, researchers have tackled these challenges, and numerous ways to improve the polymer's processability and reduce brittleness have been developed. Blending with other bio-based polymers provides a diverse way to modify polymer characteristics while maintaining biodegradability,20 specifically gelatin (Ge). Ge is a biodegradable, biocompatible polymer that is hydrophilic and non-antigenic. It also demonstrates plasticity, and due to the qualities mentioned, it is frequently used in several biomedical applications.21 In addition to being non-toxic and biodegradable, Ge, derived from collagen, is commonly chosen as a biofunctionalization agent because of its bioaffinity to bio-cells and its lack of antigenicity.22 The literature states that Ge reduces the intermolecular interactions that hold PHB structures together.23 Hence, electrospinning of blend PHB/Ge will mitigate potential issues by overcoming the limitations of individual polymers, resulting in a novel biomaterial with high biocompatibility and better mechanical and physical/chemical qualities. Nonetheless, although this blended material presents improved properties, it still lacks antibacterial activity. As a result, enhancing antibacterial characteristics is crucial to make PHB/Ge appropriate for antimicrobial and biodegradable applications.

    In recent years, nanotechnology has presented numerous chances to control compounds at the nanoscale, transforming them into potential antibacterial agents. Nanomaterials can disrupt bacterial membranes, form holes and pits on cell walls, generate ROS, bind to sulfhydryl groups of metabolic enzymes, inhibit respiratory activity, and integrate with DNA.24 This prevents microorganisms from developing resistance against them and would be more effective than antibiotics at inhibiting bacterial resistance. As a result, nanomaterials are particularly effective at killing multidrug-resistant bacteria strains. In particular, photocatalysts, such as titanium dioxide (TiO2)-based nanomaterials, have been developed and altered to improve their photocatalytic antibacterial performance in antibacterial research.25 UV light can activate TiO2 photocatalytic activity, which then goes through electron transfer processes and produces photogenerated electron holes. Consequently, many ROS are produced, including hydroxyl (HO·) and superoxide radical (O2), which then interact with biological molecules such as proteins, macromolecules, DNA, and phospholipids in the cellular membrane to cause bacterial cell death. It has been discovered that the antibacterial activity of TiO2 can be enhanced through assembly with functional nanomaterials, which also helps to separate charges and maximizes their use in visible light for a variety of practical applications. Silver nanoparticles (Ag NPs) are a class of functional nanomaterials well-known for their antibacterial properties. They have been applied extensively in the biomedical area to eradicate germs.26, 27 Ag exhibits antibacterial action without light activation, in contrast to TiO2. Due to these factors, the photoactive impact of hybrid Ag-TiO2 NPs, when added to polymeric materials, may produce effective and consistent antibacterial activity. In our recent study,28 Ag-TiO2 hybrid NPs decorating an electrospun PHB microfiber membranes were found to improve their antibacterial characteristics, especially when exposed to low-power commercial LED light, where the best-performing sample demonstrated over 99% antibacterial efficacy against Escherichia coli and Staphylococcus epidermidis. In another study, Rodríguez-Tobías used PHB to create nanofibers by the mixing and electrospinning-electrospray methods while using the same concentration of ZnO NPs.29 The former had an antibacterial efficiency value of 3.20 ± 0.15, while the latter had only 1.20–1.40. Although nanofibers generated utilizing the electrospinning-electrospray approach did not show the expected enhanced antibacterial characteristics, the antibacterial effectiveness against E. coli and S. aureus depended on the elaboration technique. The design of ZnO-embedded PHB fibers was more suitable for bactericidal effects.

    PHB/Ge composite scaffolds have been investigated in hybrid PHB-based materials for regenerative tissue and controlled drug release in medical applications.30, 31 They show decreased hydrophobicity, reduced burst effect, and more regulated drug release, but studies of their antibacterial properties have not yet been investigated. In addition, attempts to create hybrid antibacterial materials using PHB, Ge, and AgTiO2 NPs have been still limited. Thus, this work focuses on the fabrication of hybrid PHB membranes doped with both Ge and AgTiO2 NPs and a thorough examination of their structure, morphology, thermal and biological properties, and biodegradability as potential materials for various biomedical applications.

    Herein, PHB is produced and blended with Ge, loaded with hybrid Ag-TiO2 NPs, and optimized for antibacterial activity against E. coli and S. epidermidis. Scanning electron microscope (SEM), FTIR, Differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA) were used to characterize the electrospun nanofibers. The contact angle is determined using the drop method to determine the wettability of the nanofibers. Furthermore, the antibacterial activity, microfiltration, and cell viability of the functionalized nanofibers' efficiency are assessed. These produced membranes also exhibited excellent antifouling capabilities and high biodegradability. Our studies primarily seek to create an efficient, simple antibacterial material that employs hybrid Ag-TiO2 NPs to suppress bacterial growth and biofilm formation, demonstrating the feasibility of low-cost biodegradable PHB/Ge membranes and a comprehensive study of their structure, morphology, thermal, mechanical, and biological properties. With such high antibacterial properties, enhanced mechanical strength, effective antibiofilm formation, and biodegradability, these membranes could be considered in biomedical applications, water filtration, and environmental protection.

    2 MATERIALS AND METHODS

    2.1 Materials

    The polymer poly(3hydroxybutyrate) (PHB) was acquired in the form of pellets from Goodfellow (Huntingdon, England). It has a weight-average molecular mass (Mw) of 550 Kg/mol and a polydispersity index (PI) of 1.2. Ge was purchased in granular form from Fisher Scientific (New Jersey, USA). Dichloromethane with a purity of at least 99.8% and chloroform with a purity of at least 99.8% were provided by Alfa Aesar (Mississauga, United States) and Thermo Scientific (United States), respectively. These solvents were used to dissolve PHB. Acetic acid was used to dissolve Ge. The biological assays utilized E. coli (ATCC 11229) and S. epidermidis (ATCC 12228) obtained from Cedarlane in Southern Ontario, Canada. In addition, the bacteria cell fixation process involved using Glutaraldehyde 5% obtained from Sigma Aldrich and sodium cacodylate buffer (0.2 M, pH 7.4) purchased from Fisher Scientific. The Live/Dead Bacterial Viability Kits (L7012) were acquired from Thermofisher Scientific. AgTiO2 NPs synthesis is summarized in our previous published research.28

    2.2 Preparation of PHB/Ge/AgTiO2 complex electrospun microfibers

    PHB pellets (1.4 g; 7% w/v) were dissolved in a co-solvent of chloroform/dichloromethane (1:1) and heated to 60°C using a reflux condenser for 24 h. Meanwhile, Ge powder (0.2 g; 2% w/v) was dissolved in acetic acid by heating at 70°C for 30 min. The two polymer solutions were left to cool down at room temperature. 7 mL of PHB solution was gradually mixed with 2 mL of Ge solution while stirring gently. Then, AgTiO2 NPs were added to the mixture. The final solution was maintained at a consistent temperature at room temperature and agitation for 15 min, as sudden changes can cause phase separation. The mixture was injected into a syringe using a metallic needle to create fiber membranes. The electrospinning procedure was performed using a custom-made laboratory instrument with a high-voltage direct current (DC) source capable of generating voltages ranging from 0 to 25 kV. The electrospinning process was carried out with the following parameters: applied voltage 13 kV, average temperature 23°C, average relative humidity 30%, distance between needle tip and collector 12 cm, needle diameter 0.9 mm, and feed rate 0.5 mL/min. To ensure the evaporation of the total solvents, PHB membranes were put in a vacuum oven for 24 h at room temperature and 10 h at 40°C. Obtained PHB/Ge membranes were named PGx, where x is the wt% of AgTiO2 NPs added to the blended polymer solution. Control bio-based samples, PHB with 0%AgTiO2 NPs and 4%AgTiO2 NPs, P0 and P4, respectively, are examined to determine the role of Ge in the antibacterial effect and bacterial adhesion.

    2.3 Characterizations

    The SEM (JEOL-JSM 5500) was used to analyze the surface morphologies of the electrospun fibers both before and after the addition of AgTiO2 NPs. Briefly, a small portion of a microfiber sample was vacuum coated with gold after being attached to a brass stub with double adhesive tape. The specimens were examined with a magnification of 2500 times. The elemental composition of AgTiO2 NPs was determined using an X-ray spectrometer (SEM–EDX) line mapping analysis. The average diameter of each sample was determined by measuring the diameters of 100 randomly selected nanofibers using ImageJ Software. This software was also used to measure the electrospun membranes' porosities. SEM images of the membranes were first acquired at high magnification, ensuring clear resolution of individual pores, and then they were imported into ImageJ. At least 10 regions of each image were analyzed to obtain statistically reliable results, and the measurements were repeated across three SEM images from different membrane areas. The average pore size was then calculated and analyzed for consistency. The membrane surface was evaluated using a high-resolution 3D laser confocal microscope (VK-X1000 3D laser confocal microscope). The nanofiber samples were analyzed using FTIR to identify their functional groups within a 500–4000 cm−1 spectral range using an FTS 45 instrument equipped with a Universal ATR sample adapter with a diamond crystal. The wettability of the nanofibers utilizing the Surface Energy (Theta Flex optical tensiometer manufactured by Attension) was gauged by measuring the contact angle of water using the drop method. In this procedure, deionized water was placed on the surfaces of the membranes. The measurements were conducted three times for each sample, and the mean contact angle was calculated. A Perkin Elmer TGA (Perkin Elmer Instruments, USA) was employed with a gas flow rate of 20 mL/min and a 10°C/min heating rate in a nitrogen atmosphere. DSC studies were conducted on a (TA Waters D.S.C. 25, dynamic) in a nitrogen atmosphere. An axial tensile testing equipment was used to measure the mechanical properties of the PG membranes. Before measurements, samples were preconditioned for 24 h at 50% relative humidity. The specimen was divided into 30 × 15 mm pieces and affixed between two clamps. Tensile strength refers to the highest force a test specimen creates before rupture. The measurement was conducted on an Instron 4201 universal testing instrument with a 500 N load cell and a 10 mm/min crosshead speed. The length of the gauge was continuously measured until the sample failed at room temperature. The measurements were conducted five times for each sample. As reported, the electrospun membranes were subjected to biodegradation by a soil burial test.28 The samples were extracted from the soil every 2 weeks, meticulously washed with deionized water, and subsequently subjected to a 24-h drying process in an oven set at 50°C to determine their weight decrease.

    2.4 Antibacterial performance

    2.4.1 Bacterial reduction assays

    Gram-positive S. epidermidis (ATCC 12228) and Gram-negative E. coli (ATCC 11229) were the two test organisms used for assessing the antibacterial activity of the membranes, applying a straightforward technique previously described.28 First, all the (2 × 2 cm) samples were sterilized with UV light for 30 min, then they were put in a 6-well plate. Next, 100 μL bacterial solution (1.5 × 105 CFU/mL) and 1 mL of physiological saline were added to all wells. The 6-well plate was then exposed to white light irradiation provided by the LED-L16 Photoreactor (4.8 mW/cm2 intensity, Luzchem Inc). After appropriate irradiation times, 2 mL of physiological saline was added to each well, and 100 μL of the combined solution was dispersed on agar plates. Finally, the well plates were incubated at 37°C for 24 h. The average of three replicates for each sample is given for the results. The following formula was used to calculate the survival ratio (SR):
    SR = A A 0 × 100 % (1)

    The average number of microorganisms on the control untreated and antibacterial membranes is denoted by A and A0, respectively. The findings are derived from the average of the three replicates.

    2.4.2 Microfiltration analysis

    The microfiltration performance was evaluated using a dead-end stainless-steel filtration unit (HP4750 from Sterlitech, USA) with a total surface area of 14.6 cm2, as reported.32 Before conducting studies, electrospun membranes were sterilized using UV light for 10 min, while the filter unit was autoclaved at 120°C for 15 min. Before filtering, the specimens were moistened and compressed at a pressure of 69 kPa for 10 min. This process ensured a consistent water flow, and all the pores were fully open. Next, 10 mL of liquid bacterial cultures containing (104 cells/mL) was passed through the specimens at room temperature at a pressure of 69 kPa. The bacterial concentration decrease following each filtration was assessed using the pour plate technique and quantified using the Log reduction value (LRV, Equation 2) and the microfiltration efficiency (Equation 3). Every microfiltration test was repeated three times.
    LRV = Log A Log B (2)
    A 1-log reduction signifies the deactivation of 90% of the bacteria. Consequently, the quantity of bacteria is decreased by a factor of 10. Similarly, a 2-log drop represents a 99% decrease, indicating a reduction of germs by a factor of 100, and so forth.
    Microfiltration efficiency = A B A × 100 % (3)

    A and B represent the feed and filtrate bacteria concentrations, measured in CFU/mL.

    2.5 Anti-biofouling tests

    To assess bacterial adherence and biofilm formation on the specimen, 1 mL volume of bacterial culture containing 106 cells/mL was applied to the surface of the samples in a 6-well polystyrene culture plate, and then they were incubated for 18 h at 37°C without agitation. Subsequently, the bacteria culture was extracted, and the samples were washed three times with distilled water to eliminate nonadherent cells. The samples were further examined using SEM to capture images, while Confocal Laser Scanning Microscopy (CLSM) was used to monitor both living and dead bacteria. For SEM analysis, the bacterial cells were initially fixed in the samples at room temperature for 1 h using a 5% (v/v) glutaraldehyde in a 0.2 M sodium cacodylate buffer. After being washed twice with sodium cacodylate buffer, the samples were gradually dehydrated with 25%, 50%, 70%, 90%, and 100% ethanol. Finally, the samples were scanned with a SEM microscope at a voltage of 10 kV.

    The assay to determine the viability of bacteria cells was conducted using the LIVE/DEAD L7012 Kit. The staining solutions were made following the manufacturer's directions. In summary, two dyes, SYTO 9 and Propidium iodide dyes, were utilized for staining. The components (A and B) were thoroughly combined in a microfuge tube at a 1:1 ratio. Subsequently, 6 μL of the pre-mixed staining solution was introduced into 2 mL sterile 0.85% sodium chloride solution (NaCl). Subsequently, the microfiber sample was submerged in 200 μL of the staining solution for 30 min at 37°C in dark conditions. The staining was eliminated, and the sample was washed once with a 0.85% NaCl solution and seen using CLSM. The filters employed are FITC, which has an excitation range of 480–500 nm, and Propidium iodide, which has an excitation range of 550–650 nm.

    3 RESULTS AND DISCUSSION

    3.1 Morphological screening of the electrospun membranes

    SEM studied the influence of adding Ge and AgTiO2 NPs on the electrospun membrane's morphology, structure, and properties. The electrospinning conditions (applied field, inner needle diameter, and flow rate) were kept constant for the different experiments. The surface morphologies of the electrospun samples (Figure 1) reveal the production of homogenous, uniform, and bead-free fibers. P0 shows a well-distributed surface with a primarily open pore network, while blended PHB/Ge membranes show a straightened pore arrangement. The size-distribution histograms demonstrate that the diameter distribution of the fibers is in the micron range, with P0 and P4 fibers, and in the nano range with the blend PHB/Ge fibers. The average fiber size of pure PHB is about 1.3 μm, possibly because PHB has a higher molecular weight (Figure 1B). The combined PHB/Ge fibers are thinner than the pure PHB fibers; their average diameter is around 786.5 nm ± 221 nm (Figure 1D,F,G,K). The decreased diameter of electrospun fibers compared to the neat PHB sample could be attributed to the addition of Ge, which reduced the viscosity of the spinning solution.33 The Ge was added at a low concentration, so the solution was less viscous, mainly when dissolved in acetic acid.34, 35 Electrospinning often produces finer fibers because lower solution viscosity influences the jet's stretching and thinning as it moves from the needle to the collector. Moreover, histogram analyses revealed a reduction in the diameters of PHB/Ge fibers upon the introduction of AgTiO2 NPs. The fibers' diameters dropped from 969 nm of PG0 to 512 nm of PG4 as the concentration of AgTiO2 NPs increased. The decrease in nanofiber diameter could be attributed to an elevated electric field resulting from a higher charge density and the conductivity of the electrospinning fluid during the operation. The higher the charge density, the smoother and thinner the resulting fibers due to increased jet-whipping instability.36 The charge density of the solution increased according to the concentration of AgTiO2 NPs in the mixed polymer solution compared to the neat PHB solution. Consequently, thinner fibers were generated. It is hypothesized that this phenomenon can be explained by the increased concentration of repulsive charges resulting from residual ions and charges produced on the NPs when a voltage bias is applied. This promotes the formation of consistent and thinner fiber structures, which have a greater ratio of surface area to volume.37 An additional cause of diameter reduction in PHB/Ge nanofibers is enhanced electrical conductivity when adding NPs.38 However, PG5 showed an increase in fiber diameter. Our investigation demonstrated comparable findings with previous research,23 in which the incorporation of 5% w/v AgTiO2 NPs led to increased fiber diameters, indicating successful integration of AgTiO2 NPs with the PG nanofibers. Furthermore, the accumulation of AgTiO2 NPs during the electrospinning procedure can also result in the extensive dispersion of nanofibers of diverse dimensions.39 Meanwhile, the agglomeration of AgTiO2 NPs was detected within the PG5 fibers. The distribution of AgTiO2 NPs within the PG fibers was examined using energy dispersive spectroscopy (EDS; Figure 1G). The distribution of AgTiO2 components in agglomeration clusters indicates that AgTiO2 was effectively loaded into the PG fibers through electrospinning. However, due to their low concentration in the PG matrix, AgTiO2 NPs did not significantly appear on the surfaces of the PG3, PG4, and P4 fibers. Moreover, considering the SEM sensitivity depth of up to 3 nm, the AgTiO2 NPs were located within the polymer fibers at a more significant depth than the SEM study's sensitivity.

    Details are in the caption following the image
    SEM images and histograms of the fiber diameter distribution of (A) P0, (B) PG0, (C) PG3, (D) PG4, (E) P4, and (F) PG5 fibers. Each histogram was plotted with 100 fibers measured. (G) Energy Dispersive Spectroscopy (EDS) mapping of PG5.

    3.2 Composition and physical–chemical analysis

    The water contact angle (WCA) measurements (Figure 2A) were used to examine the wettability of the electrospun nanofibers. The PG nanofibers exhibited a decreased hydrophobicity, showing a high level of wettability attributed to the presence of Ge and AgTiO2 NPs. The enhanced water-attracting properties of the nanofibers were primarily caused by the addition of Ge and AgTiO2 NPs, which contain -COOH and -OH groups by forming more hydrogen bonds. This resulted in the PG nanofibers having a strong affinity for water compared to pure PHB, which is naturally water-repellent with a water contact angle of 143.7 ± 6°. The water contact angle of P4 was measured to be 127.2 ± 1° (Figure SI2), slightly lower than the pure PHB. This angle is attributed to the presence of AgTiO2 NPs. Hence, Ge can adjust the hydrophobicity of PHB to achieve the unique needs of different applications. Studies have shown that hydrophilic surfaces are less prone to biofouling than hydrophobic surfaces.40 As the hydrophobicity of a material's surface rises, hydrophobic organic molecules, like bacteria, are attracted to it, causing surface contamination. Hydrophilic materials typically have better antifouling properties.41, 42 Surface-associated proteins, such as carboxylates and phosphate groups of teichoic acids of the peptidoglycan layer in Gram-positives and fimbriae in Gram-negatives bacteria, have been shown to contribute to hydrophobicity in bacterial cells significantly.43 This suggests that PG/AgTiO2 NPs composite films have the potential to be effective anti-biofouling materials for antibacterial application.

    Details are in the caption following the image
    (A) WCA of P0, PG, and P4 membranes; (B) FTIR spectra of (A) P0, (B) PG0, (C) PG3, (D) PG4, (E) PG5, and (F) gelatin; (C) TGA curves; (D) Derivative thermogravimetry (DTG) curves, (E) DSC curves; and (F) Stress–strain curves of P0 and PG membranes.

    FTIR analyses were conducted on the electrospun membranes to determine the structural modifications due to PHB blending with Ge. PG3, PG4, and PG5 had similar IR spectra. Figure 2B displays the blended fibers' FTIR spectra of P0, PG, and Ge. No discernible solvent peaks were observed, indicating that the solvent had evaporated entirely throughout the electrospinning process. Peaks that are related to Ge and PHB were found to overlap. Three areas in the spectra 3600–2300 cm−1 (Amide A) (N-H stretch), 1656–1644 cm−1 (Amide I) (C=O stretch), and 1560–1335 cm−1 (Amide II) (C-N stretch) can be used to establish the presence of Ge in the sample.44 Amides A, I, and II bands were slightly visible in the PG electrospun spectra, and their intensity lightly increased as the Ge was added compared to P0. The spectrum demonstrates the FTIR's sensitivity and capacity to detect Ge peaks even when blended at low concentrations. The presence of an amide group verifies the existence of Ge in nanofibers formed during polymer blending and electrospinning. Amide groups in Ge can establish hydrogen bonds with water molecules. Thus, Ge enhanced the hydrophilicity of the resulting PG membrane, as described earlier. The Ge bands were attenuated due to shrinkage within the fiber blends and higher PHB-nanofiber exposure. As previously stated,31 PHB-nanofibers supported Ge-nanofibers. Comparable results were observed in an FTIR investigation for fibers made by combining PHB and Ge polymers. In addition, the FTIR spectra exhibited a prominent peak at 1720 cm−1, indicating the stretching vibration of the ester (C=O) bond. This peak is exclusive to PHB and is associated with the conformation of the polymer backbone.45

    Studying the crystallization and melting behavior of PG membranes is crucial due to its impact on the crystalline structure and the macroscopic properties of the materials. The thermal characteristics of the electrospun membranes were assessed using a TGA and DSC combined testing analysis (Figure 2C–E). The thermal parameters derived from these analyses are collected in Table 1. The thermal stabilities of PGO, PG3, PG4, and PG5 were diminished compared to the PO fibers, possibly due to the addition of Ge. The Ge enhanced the surface wettability of the PG nanofibers, resulting in a larger area for water adsorption and a decrease in its thermal stability.46 The thermal stability of PG nanofibers was also affected by the incorporation of AgTiO2 NPs. The initial temperature at which thermal degradation occurs decreases as the amount of AgTiO2 NPs increases, as indicated by the Tonset value (Table 1). As previously reported, TiO2 nanofillers improve heat transmission properties from the heat source to the inside of the polymer, increasing the thermal deterioration of PHB.47

    TABLE 1. Thermal properties of P0 and PG electrospun materials.
    Sample Decomposition temperaturesa Residuea (%) Cold crystallization temperature, Tccb (°C) Heat of cold crystallization, ΔHccb (J g−1) Melting temperature, Tmb (°C) Heat of melting, ΔHmb (J g−1) Degree of crystallinity, Xcc (%)
    Tonset Tmax Tendset
    P0 262.3 290.3 409.9 0.22 52.8 3.2 173.5 68.3 44.65
    PG0 261.1 286.4 399.8 0.23 49.9 4.3 169.7 69.1 31.07
    PG3 257.6 287.3 413.3 0.24 50 4.4 167.4 69 30.97
    PG4 251.4 278.8 368.1 0.26 51 4.6 163.7 65.7 29.27
    PG5 243.8 277.1 407 0.47 50.2 4 165.5 54.8 24.37
    • a Were determined from TGA analysis.
    • b Tg, Tcc, ΔHcc, Tm, and ΔHm were determined from the second heating curve using DSC analysis. The peak maximum was designated as Tm. The values of ΔHm and ΔHcc were obtained by analyzing the endothermic melting and exothermic cold crystallization peaks, respectively.
    • c Was calculated using Equation (4) (SI).

    The DSC curves were produced for P0 and PG nanofibers. The pure PHB membrane exhibits a distinct endothermic peak at 173.5°C, corresponding to the polymer's melting point.48 Including Ge in PG reduces the melting temperature, which decreases to 169–165°C compared to the P0 sample. According to reports, PHB and Ge are thermodynamically immiscible polymers. Consequently, phase separation occurs during electrospinning, developing a core-shell structure composed of PHB (core) and Ge (shell).49 Because Ge has a polyelectrolyte (polyampholyte) character, it tends to move toward the outer layer of the PHB/Ge solution jet due to electrostatic repulsion, forming a shell layer.50 Thus, the variations in the melting temperature could be linked to the core-shell composition of the fibers, which in turn impacts the passage of heat from the Ge layer to the PHB core. The crystallinity of pure PHB membrane aligns with the information provided in the literature.23 The combination of PHB and Ge, as well as PHB, Ge, and AgTiO2 NPs, reduced crystallinity. This tendency can be attributed to the development of a thin gelatinous layer (shell), which hinders the crystallization of PHB (confinement effect). Adding AgTiO2 NPs to the polymer matrix also induces a reduction in the crystallinity of PHB. AgTiO₂ NPs act as nucleating agents and disrupt the crystalline lattice of PHB.

    The tensile properties of P0 and PG membranes were tested to measure their durability and elasticity. A tensile stress–strain curve for electrospun samples was generated, and Young's modulus, the tensile strength, and the strain break were estimated from the curve. Figure 2F depicts the elastic deformation of electrospun nanofibers. As the deformation progressed, the sample hit the yield stage or elastic limit, and the material was deformed beyond recovery. Tensile strength is the maximum stress value (y-axis), whereas strain break is the maximum strain point (x-axis), as determined by the curve. The functionalized nanofibers' Young's modulus was calculated using the stress–strain curve's starting slope or gradient. The findings are summarized in Table 2. Including Ge with PHB nanofibers improved the samples' tensile strength and modulus while lowering their elongation percentage. Similar findings were discovered when adding Ge to PCL microfibers.51 It could be because, when stretched, the Ge nanofibers' stress on PHB nanofibers is somewhat distributed, improving the composite scaffolds' tolerance and minimizing their deformation. Ge creates a stiffer polymer matrix that may enhance mechanical performance.

    TABLE 2. Mechanical properties of P0 and PG electrospun materials.
    Young's modulus (MPa) Tensile strength (MPa) Strain break (%)
    P0 62.12 ± 2.31 1.47 ± 0.3 15.48 ± 1.16
    PG0 89.86 ± 8.62 1.23 ± 0.26 15.87 ± 0.8
    PG3 179.01 ± 13.30 4.70 ± 0.27 13.43 ± 1.03
    PG4 126.68 ± 13.58 3.74 ± 0.32 11.23 ± 2.26
    PG5 117.07 ± 0.72 2.85 ± 0.77 11.14 ± 1.82

    Additionally, the reinforcing impact of AgTiO₂ NPs may contribute to the improved mechanical properties observed. Tensile strength increased with AgTiO2 NPs inclusion into the PHB-Ge matrix, suggesting that AgTiO₂ NPs may play a role in efficient stress transfer across the polymer matrix. These nanoparticles may exert a strengthening impact due to their actions as fillers, which allow them to distribute stress more uniformly throughout the matrix, enhancing mechanical performance. The highest tensile stress was obtained in the PG3 samples and then PG4, reaching 4.7 and 3.74 MPa, respectively. Previous research on reinforcing electrospun fibers shows that low concentrations of nanoparticles enhance mechanical characteristics. Meanwhile, agglomerations can also cause fibers to become less mechanically strong by forming structures resembling defects,52 as observed with PG4 and PG5. Increased AgTiO₂ NPs content causes fiber agglomeration and exposure of nanoparticles, resulting in decreased tensile strength and brittleness.53 Samadian et al.54 stated that adding nanohydroxyapatite to cellulose/Ge nanofibrous films reduced their tensile strength from 3.01 MPa to 2.68 MPa at weight ratios ranging from 12.5% to 50%.

    3.3 Antibacterial response

    3.3.1 Quantitative antimicrobial assay

    The plate-counting assay was used to assess the antibacterial efficacy of the nanofibers against E. coli and S. epidermidis, with PG0 nanofibers serving as control samples. P0 and P4 were examined to study the effect of Ge on the antibacterial activity. The studies were conducted in light conditions as our previous work confirmed that AgTiO2 NPs displayed enhanced antibacterial activity under low-power commercial LED light.28 Antibacterial efficiency, represented as a percentage, demonstrates the absence of viable colonies on the electrospun mats after 90 min for E. coli and 60 min for S. epidermidis. The results are shown in Figure 3.

    Details are in the caption following the image
    (A) Antibacterial activity of P0, PG, and P4 membranes against E. coli after 90 min of light illumination and against S. epidermidis after 60 min of light illumination, (B) Microfiltration efficiency of P0, PG, and P4 membranes against E. coli and S. epidermidis, and (C) Photographs of agar plates after microfiltration of E. coli and S. epidermidis with the P0, PG, and P4 membranes.

    The P0 and PG0 nanofibers lacked antibacterial efficacy against both bacteria strains. After incubation with PG nanofibers, a limited amount of E. coli colonies were identified on the agar plate, although no S. epidermidis colonies grew. Furthermore, the number of bacteria in the colonies decreases from PG0 to PG5, and bacterial development is significantly suppressed with PG4 and PG5. This suggests that AgTiO2 NPs induce the antibacterial activity of electrospun membranes. Furthermore, employing AgTiO2 NPs on a nanometric scale results in larger surface areas, which may expedite the destruction and wrinkling of bacterial cell walls.55 Moreover, when exposed to visible light, AgTiO2 NPs can create ROS through oxidation–reduction processes, including hydroxyl radicals (OH) and superoxide radicals (O2). ROS are highly oxidative chemicals that can tear bacterial cell membranes and destroy biomolecules, resulting in cell death.56 AgTiO2 NPs content was the key factor impacting the antibacterial activity of membranes. In addition, as expected, blending PHB with Ge decreases the Tg and the crystallinity of the polymeric membrane, resulting in increased AgTiO2 NPs release. The gradual release of AgTiO2 NPs from PG electrospun nanofibers increases their ability to kill bacteria by slowly releasing antimicrobial agents over time. The PHB and Ge mixture controls the NP's release rate and ensures a long-lasting antibacterial effect, which is more effective than burst release. As a biocompatible material, Ge makes the nanofibers more compatible with biological systems. This enhances the antibacterial activity by promoting the interaction between AgTiO2 NPs and the bacterial cells.

    Nonetheless, PG nanofibers were more successful in killing the Gram-positive bacteria S. epidermidis than the Gram-negative bacterium E. coli. As previously reported, the difference in antibacterial impact between Gram-positive and Gram-negative bacteria is primarily due to changes in their cell wall architectures.57 Gram-positive bacteria's outer cell wall comprises peptidoglycan and acidic polysaccharides (teichoic acids), which include many holes that may allow the adhesion or penetration of antibacterial agents into the cell. Furthermore, Gram-negative bacteria have an outer membrane composed of proteins, lipids, and lipopolysaccharides, which may act as a barrier to the entry of bioactive compounds into the cell.58 The antibacterial activity of the PG fibrous membrane showed strong antibacterial effects due to the sustained antibacterial action of AgTiO2 NPs against Gram-positive and Gram-negative bacteria.

    3.3.2 Microfiltration action

    The nanofibrous membranes' filtration ability was challenged using a representative waterborne bacterium, E. coli and S. epidermidis. Table 3 displays the bio-based specimens' efficiency in eliminating both bacteria. After one filtration cycle, PG3, PG4, and PG5 retained >95% of the cells related to a 1-log decrease value (Figure 3B,C). The microfiltration process depends on a size exclusion mechanism, so the pore diameter of the membranes is correlated with the microfiltration efficiency. The quantitative assessments of pore average size were obtained using the image analysis software program ImageJ. The samples with the lowest pore size diameter provided the best retention rate. Given their average pore diameter of 126 nm, which was the lowest among the other specimens examined, except P4, it was unsurprising that the PG4 and PG5 nanofibers showed the highest retention rates.

    TABLE 3. Microfiltration efficiency and pore average diameter of electrospun membranes.
    LRV Microfiltration efficiency (%) Pore average diameter (nm)
    E. coli S. epidermidis E. coli S. epidermidis
    P0 0.57 ± 0.07 0.59 ± 0.09 60.13 ± 4.04 65.33 ± 5.06 528 ± 1.61
    PG0 0.39 ± 0.06 0.60 ± 0.21 43.98 ± 5.40 75.00 ± 10.26 223 ± 1.05
    PG3 1.03 ± 0.87 1.76 ± 0.12 90.58 ± 7.79 98.26 ± 1.59 129 ± 0.74
    PG4 2.72 ± 0.00 1.98 ± 0.00 99.71 ± 0.50 100.00 ± 0.00 126 ± 0.52
    P4 0.85 ± 0.10 0.90 ± 0.10 78.70 ± 4.94 87.50 ± 2.76 120 ± 0.89
    PG5 1.54 ± 0.04 1.76 ± 0.00 97.10 ± 0.25 100.00 ± 0.00 126 ± 1.33

    These results also fit rather well with the diameters stated in the literature of bacterial cells for E. coli; the average bacterial cell size is 1–2 μm in length and 0.5 μm in width, and a diameter of 0.5–1.5 μm for S. epidermidis.32, 59 Nevertheless, the size of the pores of the membranes is not the only determinant of the LRV and microfiltration efficiency recorded in Table 3. The ImageJ software test for estimating the maximal pore diameter of the bio-based specimens makes assumptions that the pores are cylindrical tubes. However, this is not the case with electrospun nonwoven materials in which the pores are interconnected as a bundle of tortuous tubes. This generates resistance to liquid movement, impeding bacterial invasion via the porous structure. Moreover, as previously stated, AgTiO2 NPs are responsible for cell inactivation. Because the method employed to quantify the filtered cells only counts the number of living cells, those deactivated were excluded from the filtration data. This explains why PG4 has a better microfiltration efficiency than P4, although P4 has a smaller pore size. This is consistent with the antibacterial results reported in the quantitative antimicrobial assay. Finally, our results show that our PG membranes, especially PG4 and PG5, had higher (≥99.9%) microfiltration efficiencies for both Gram-positive and Gram-negative bacteria.

    3.3.3 Bacterial adhesion and anti-biofouling properties

    Researchers have employed two ways for surface modification to reduce the biofouling potential of polymer membranes.60 The first approach uses an anti-adhesion technique to reduce membrane biofouling.61 Hydrophilic or polyelectrolyte materials can be added to the membrane surface to minimize bacterial adhesion to polymer membranes. For instance, hydrophilic compounds reduce the hydrophobic interactions between extracellular polymeric substances of the biofoulants (bacteria) and membranes.62 The second strategy involves an antibacterial technique that regulates membrane biofouling by applying biocides.61

    Following 18 h of incubation with E. coli and S. epidermidis, the surfaces of the nanofibers were examined using SEM and CLSM (Figure 4) to obtain insight into the bacterial attachment on the electrospun membranes. The green fluorescence on the nanofibers' surfaces indicated a living bacterium cell, while the red fluorescence indicated a dead cell.

    Details are in the caption following the image
    SEM and CLSM images of (A) P0, (B) PG0, (C) PG3, (D) PG4, (E) P4, and (F) PG5 membranes after being in contact with (1) E. coli and (2) S. epidermidis cells.

    SEM images detected large clusters of living E. coli and S. epidermidis cells on the unloaded P0 and PG0 surfaces, showing significant growth of the extracellular matrix and a high level of biofilm formation. Corresponding CLSM images showed that the bacteria were stained green, indicating the formation of healthy living bacterium cells on the P0 and PG0. In contrast, visual inspection revealed that most of the bacteria in AgTiO2 NPs loaded nanofibers were dead. This indicates that the bacterial cells have been injured and become red due to penetration into their cell membranes. This was especially evident in PG4 and PG5, where the membranes displayed a distributed appearance, a notable decrease in the number of cells, and a significant reduction in biofilm. The bacterial cells turned red when the AgTiO2 NPs content rose, indicating that they had been destroyed, showing the fouling-resistant property of the membranes. This suggests that AgTiO2 NPs were responsible for killing bacteria, as previously stated.

    Our findings show that blending PHB with Ge may improve the membrane's anti-biofouling properties in addition to the antibacterial effect of AgTiO2 NPs. By increasing the hydrophilicity of polymer membranes, hydrophilic PG electrospun nanofibers can potentially decrease membrane biofouling.63 The WCA confirmed the decrease in hydrophobicity when Ge was added. This explains the enhanced anti-biofouling property of PG4 against P4. Furthermore, by contact killing, the PG nanofibers loaded with AgTiO2 NPs may lessen membrane biofouling caused by the antibacterial activity of AgTiO2 NPs.

    3.4 Biodegradability

    The surge in demand for plastic materials is driving up the exploitation of fossil fuels, which poses a severe environmental hazard with implications for human health, ecosystem toxicity, and global warming. This situation has led to the requirement for new materials that can solve and provide sustainable solutions orthogonal to these immediate challenges.

    To evaluate the biodegradation capacity of the nanofiber membranes, PG samples were buried in natural soil, and their morphological and weight variations were monitored over time (Figure 5 and Figure SI3). It has been confirmed that PHB degrades naturally in soil,28 and it occurs in three steps, as previously reported64: from a high-molecular molecule to monomers and oligomers, the self-enzymatic breakdown of biomass generated during the first stage, and finally, from biomass to CO2 and H2O. The initial stage of degradation of the material is depicted in Figure 5, which shows changes in its structure. This phase is primarily characterized by the microbial activation and mechanical action of soil adhesion, as well as the onset of oxidative and hydrolytic action mediated by soil constituents and enzymes.65 The fibers' microstructure underwent a substantial alteration after 4 weeks of exposure to natural soil (Figure SI3). The breakdown of PG membranes occurred progressively over 8 weeks, with over 50% decomposition already evident by week 5. The pace at which the membrane broke down in soil was unaffected by the presence of Ge. The primary cause of weight loss exceeding 50% after 5 weeks is the microbial breakdown of PHB in the soil.66

    Details are in the caption following the image
    Confocal profiler 3D image and laser images of P0, PG0, PG3, PG4, P4, and PG5 membranes before and after 8 weeks of burial test.

    The hydrophobicity of the nanofiber membrane makes it difficult for soil bacteria to survive and cling to the surface, which explains why P0 degrades slowly at first. However, it is speculated that microbial aggregation infiltrates from the membrane surface pores, causing direct contact with the inner PG nanofibers and accelerating the internal degradation of the polymer substrate, significantly increasing the degradation rate. Furthermore, investigations have shown that PG degraded quickly (Figure SI3), aided by its enhanced hydrophilicity, which promotes microbial aggregation. Microbial enzymes can break down PHB and Ge molecules into smaller monomers or low-molecular-weight chemicals. Microorganisms can use these breakdown products to participate in organic cycling in soil, resulting in fast degradation and the formation of carbon dioxide and water.67 The quick breakdown of PG could possibly be ascribed to the decrease in the crystallinity, as shown by the thermal analysis data.

    Furthermore, it was shown that soil particles were included in the material's pores and structure (Figure 5). This increase may be explained by adding soil particles to the material's surface, which could cause the membrane to retain more moisture or microbiological components than usual—a characteristic of the initial stages of soil degradation. It should be mentioned that there was a noticeable amount of sample embrittlement and fragmentation after 8 weeks of burial test. The existence and the size of membrane pores have a significant influence on biodegradation. Pores provide better microbial access, resulting in more pronounced enzymatic activity. As degradation advances, biofilms grow within the pores, which might clog smaller ones. Continuous microbial activity degrades the membrane structure, leading to fractures, pore expansion, and fragmentation (Figure 5).

    Moreover, as reported previously, including AgTiO2 NPs did not inhibit the membranes' biodegradation.28 Unlike previous investigations, introducing antimicrobial agents can slow or stop biodegradation. The soil burial experiment results show that PG membranes are biodegradable in soil, which perfectly aligns with the study's initial goal of preventing secondary contamination from antibacterial membranes. This study investigates a more sustainable, ecologically friendly environment with little environmental impact. Bio-based and biodegradable bioplastics PHB blended with Ge can provide qualities similar to standard plastics while generating extra benefits due to their low carbon footprint.

    4 CONCLUSION

    Blended fibers, PHB, and Ge loaded with AgTiO2 NPs were electrospun with chloroform, dichloromethane, and acetic acid as co-solvents. The resulting fibers had a diameter ranging between 500 and 1000 nm. SEM, FTIR, wettability measurements, TGA and DSC studies, and tensile strength measurements confirmed the fabrication of the antibacterial nanofibers. It was discovered that Ge decreased PHB's crystallinity, which affected wettability and enhanced the antibacterial effects due to the sustained antibacterial action of AgTiO2 NPs against E. coli and S. epidermidis. Moreover, it was surprising that adding Ge enhanced the fibers' tensile characteristics. It was also found that adding Ge reduced the fibers' pore size, which improved the microfiltration efficiency. In addition, the biodegradation control was systematically elucidated with 99% soil biodegradation rates in 8 weeks.

    In summary, based on the findings of the characterizations, PG4 showed the highest level of performance, including thermal, physical stability, and mechanical performance. It also exhibited an outstanding antibacterial and microfiltration efficiency of ≥99.99% against E. coli and S. epidermidis when exposed to LED light and good resistance to biofouling. The results showed that AgTiO2 NPs are responsible for these antibacterial characteristics. Additional research may yield a deeper comprehension of the processes and effectiveness of bactericidal interaction. This could lead to the creation of novel antibacterial biomaterials that effectively inhibit the growth of biofilms, resulting in the creation of innovative approaches to the problems affecting human health and the environment today.

    ACKNOWLEDGMENTS

    Thank to students and technicians from the laboratory of advanced materials for energy and environment for their kind help during the realization of this project.

      FUNDING INFORMATION

      We would like to thank: Natural Sciences and Engineering Research Council of Canada (NSERC), l'Institut de recherche Robert-Sauvé en santé et en sécurité du travail (IRSST), Canada, and le Centre de Recherche sur les Systèmes Polymères et Composites à Haute Performance (CREPEC) to financial support of this work.

      CONFLICT OF INTEREST STATEMENT

      Authors will be asked to provide a conflict of interest statement during the submission process. For details on what to include in this section, see the “Conflict of Interest” section in the Editorial Policies and Ethical Considerations section above. Submitting authors should ensure they liaise with all coauthors to confirm agreement with the final statement.

      DATA AVAILABILITY STATEMENT

      Data will be made available on request.