Fabrication of magnetite nanoparticle doped reduced graphene oxide grafted polyhydroxyalkanoate nanocomposites for tissue engineering application

Abstract

In tissue engineering, magnetic nanoparticle based polymeric nanocomposites are attractive due to some superior properties that are demonstrated in monitoring the nature of cell proliferation, differentiation and the activation of cell construction in the tissue regeneration phase. Herein, we have developed a non-toxic, antimicrobial, biocompatible and biodegradable magnetic Fe₃O₄/RGO-g-PHBV composite based porous 3D scaffold. The facile and cost-effective green pathways were chosen to reduce the exfoliated graphite oxide using a new microbial strain, Lysinibacillus fusiformis at room temperature. The reduction of exfoliated graphite oxide and the fabrication of iron nanoparticle embedded Fe₃O₄/RGO-g-PHBV nanocomposite were confirmed by X-ray powder diffraction (XRD), X-ray Photoelectron Spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). The analysis of the stretching vibrations by Raman spectroscopy indicated that both the graphite oxide and reduced graphene oxide exhibit frequencies at nearly 1560 cm⁻¹ (G-band) and 1307 cm⁻¹ (D-band). Field Emission Scanning Electron Microscopy (FESEM) and high-resolution transmission electron microscopic studies demonstrated the exfoliated nano sheets of the graphite oxide and the uniform distribution of the deposited ferrite nanoparticles. The inclusion of magnetite nanoparticles and reduced graphene oxide in the network of the PHBV matrix revealed the improvement of the mechanical strength of the nanocomposite, in comparison to the pure PHBV copolymer. The magnetic properties measured by vibrating sample magnetometer (VSM) and magnetic imaging resonance (MRI) confirmed the super-paramagnetic behavior of the nanocomposite, evidenced by the saturation magnetization having low coercive field and dark contrast images in the presence of applied magnetic fields. The confocal and scanning electron microscopy analyses demonstrated the excellent fibroblast cell infiltration, adhesion and proliferation into the micro-porous 3D scaffold, indicating the biocompatibility of the Fe₃O₄/RGO-g-PHBV nanocomposite based supporting biomaterials.

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Introduction

Recently, graphene based nanomaterials have attracted tremendous attention in the field of nanotechnology, due to their unique structures, properties and great potential for applications in areas such as electronics devices, energy storage, catalysts and biomedical devices.^1–8 The inherent structural properties of graphene, particularly π-conjugation, high aspect ratio, bioactive functionality, thermal and mechanical stability and potential biocompatibility render it useful in electronic devices, bio-imaging, drug delivery and tissue engineering applications.^3,5,8 In biomedical fields, graphite oxide and its derivative graphene oxide are considered as promising supporting materials, due to good aqueous dispersibility, bactericidal activity, nontoxicity and biocompatibility. These extraordinary features of graphene are achieved due to the single atomic layer of carbon atoms arranged in a two-dimensional (2D) honeycomb lattice, surrounded by a wide range of functional groups such as epoxy, hydroxyl, and carboxyl groups.^9,10 Unlike graphene oxide, reduced graphene oxide shows excellent electrical, mechanical and thermal properties, which make it superior for usage in biomedical applications.^5,8 The large surface area and functionality of the graphene layers also reinforce the entrapment of different sensing materials (particularly magnetic particles) onto the graphite lattice, which makes graphene an extremely important tool in bio-imaging and biosensors for diagnosis in medical applications.¹¹ However, the large-scale production cost and usage of toxic precursors hinder the use of graphene in a wide range of bio-medical applications.

A variety of chemical and physical strategies have been developed for the large scale production of graphene oxide, including chemical vapor deposition (CVD), epitaxial growth, electric arc discharge and solution based chemical reduction of graphite oxide (GO) to form reduced graphene oxide (RGO).^12–17 However, these strategies are usually time-consuming, involve the usage of toxic reductants and carry a high cost with complicated processes that prevent industrial production. Therefore, an alternative strategy based on the reduction of graphite oxide (GO) by a green pathway has moved into the spotlight, and a facile green and cost-effective process for the large-scale production of reduced graphene based composites with high quality is the prime objective. Herein, microorganisms were used to reduce the graphite oxide to avoid environmental hazards and also to build up an eco-friendly relationship between the production cost and production yield, through the environmental green pathway.^18–20 Recently, Gurunathan et al.²¹ explored the microbial reduction of graphite oxide using biomass of a strain of Escherichia coli. The mechanism of synthesis of stable graphene with a large surface area was reported by Wang et al.,²² in which Shewanella species can transfer metabolically-generated electrons from a cell interior to external electron acceptors, such as epoxy and carbonyl groups in aerobic conditions.

In the present work, we investigate a novel strategy for the cost-effective and benign reduction of graphite oxide (GO) into reduced graphene oxide by the Lysinibacillus fusiformis strain under mild conditions in aqueous solution at room temperature. The synthesized reduced graphene oxide was found to resist the extensive agglomeration of graphene sheets in the graphitic structure, and also restores the π–π conjugation in the electronic structure of graphene.^23,24 In addition, the functionality of the mildly reduced graphene oxide makes an excellent platform for the incorporation of several magnetic particles to facilitate its application in biomedical fields, electronics, optics, electrochemical energy conversion and storage.^25–29 Therefore, the reduced graphene oxide synthesized via a green pathway would be used as a promising tool in biomedical applications.

Biodegradability, hydrophilicity and cell compatibility are the prime conditions for practical applications of biomaterials in the biological field, especially in artificial tissue engineering. Although reduced graphene oxide (RGO) has outstanding mechanical properties, it exhibits low binding capability with neighboring tissues due to the lack of poor osteo-integration ability.³⁰ It has been reported that the highly aqueous, dispersible composite of reduced graphene oxide and polyethylene sorbitan laurate (Tween), formed via non-covalent interactions, is biocompatible with mammalian cell lines and has bactericidal activity against microorganisms.³¹ The pluronic F27-modified, reduced graphene oxide composite exhibited amphiphilic-like molecular assembly and was adsorbed by both hydrophobic and hydrophilic biomolecules through non-covalent bonding interactions.³² Kim et al. demonstrated the intracellular cytosolic delivery of the DOX drug by PEG-conjugated, branched PEI–RGO nanocarriers. NIR irradiation has been employed for triggering endosomal disruption in order to maintain the sustained release of DOX, resulting in higher numbers of damaged cancerous cells.^33,34 Carbon nanotube coated RGO/GO exhibits high biocompatibility, resulting in a higher percentage of gene transfection.³⁵ In tissue maturation, a graphene modified polymer composite was found to be neurite-sprouting and had an outgrowth of stems, which promoted the remodeling of distorted tissues in tissue engineering applications.^36–39 Biodegradable and hydrophobic polymer conjugated reduced graphene oxide composites are therefore most desirable for building up compact bio-devices for tissue engineering applications.

Although researchers have recently explored some reports that deal with the addition of reduced graphene oxide to different biopolymers to improve their mechanical, biocompatible and biodegradable properties, the fabrication of a reduced graphene oxide reinforced PHBV composite has never been reported.

In our present study, we have fabricated a magnetically active Fe₃O₄/RGO-g-PHBV based 3D polymeric scaffold for tissue engineering applications. Fe₃O₄ nanoparticles are used to enhance the biocompatibility of the composite as a trapping agent for therapeutic bio-molecules. In addition, due to the super-paramagnetic behavior of the Fe₃O₄ nanoparticles, they behave as a contrasting agent that can be assigned in MRI/optical multimodal imaging for biological applications.^40–42

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] is a well-known copolymer of polyhydroalkanoates that supports the enormous cell growth on living cell lines due to its semi-crystallinity and bioactivity.⁴³ The biocompatibility of the PHBV polymer is an inherent property, which has been generated due to the presence of the chiral backbone with R [−] configuration. Therefore, integrating the Fe₃O₄ nanoparticles into the RGO-g-PHBV composite to improve the mechanical properties along with biodegradability and biocompatibility in tissue engineering applications is the aim of the present report.

In summary, we initially modified both the RGO and PHBV copolymer using ethylene diamine (EDA) and 1,6-hexadiamine. The aminated RGO and PHBV copolymer were conjugated in the presence of glutaraldehyde [GA], followed by surface modification using magnetically active, Fe₃O₄ nanoparticles. The composite was then turned into a 3D polymeric scaffold to accelerate the fibroblast cell growth and cell proliferation, as shown in Scheme 1.

Scheme 1 Fabrication of the Fe₃O₄ embedded RGO-g-PHBV composite based porous 3D scaffold for tissue engineering application.

Experimental section

Materials

Natural graphite flakes were purchased from Sigma-Aldrich, Germany. Sulfuric acid, potassium permanganate, glutaraldehyde (GA), ethylene diamine (EDA), 1,6 hexadiamine (HAD), sodium nitrite, hydrogen peroxide, and hydrochloric acid were purchased from MERK, India Ltd. The bacterial strain was isolated from waste water. The class of isolated microbial strain is Lysinibacillus species (Lysinibacillus fusiformis) [EMBL accession no. HE648059]. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer was extracted from the Alkaliphilus oremlandii strain⁴⁴ by using waste cooking oil.

Methods

Synthesis of graphite oxide (GO) by a modified Hummers' method. A modified Hummers' method was followed to prepare graphite oxide (GO). Briefly, 1 g of graphite powder and 0.5 g of sodium nitrate (NaNO₃) were dispersed in 25 ml of conc. H₂SO₄ at 0–5 °C in an ice bath. With constant stirring, 3 g KMnO₄ were added to the mixture very slowly. The rate of addition was carefully controlled to keep the reaction temperature lower than 15 °C. The ice bath was then removed and the reaction mixture was heated and kept at 35 °C overnight under stirring until it became a pasty brown color. The reaction mixture was then diluted by adding 400 ml of distilled water under vigorous stirring, followed by addition of 3.0 ml of H₂O₂ (30% v/v) to terminate the reaction; the solution turned yellow. The resulting mixture was centrifuged at 1500 rpm for 30 min and the pellet was washed with 10% hydrochloric acid solution to remove the metal ions, followed by repeated washing with distilled water until the pH of the solution became neutral (pH 7). The purified graphite oxide (GO) was finally dispersed in distilled water and sonicated for 2–4 h to facilitate the exfoliation of graphite oxide.

Isolation of Lysinibacillus fusiformis strain from wastewater for the reduction of graphite oxide. To initiate the microbial reduction, the bacterial strain (Lysinibacillus fusiformis) was developed via the following procedure. The wastewater sample specimen was collected from drainage at the University of Calcutta campus (Kolkata, India). The bacterial strain (Lysinibacillus fusiformis) was then isolated from the wastewater by using a serial dilution method. The specific media used for the isolation of the Lysinibacillus fusiformis strain was composed of 4 g D-glucose, 0.02 g MgSO₄·7H₂O, 0.1 g NH₄NO₃, 0.0256 g K₂HPO₄, 0.08 g K₂HPO₄, 0.002 g FeCl₃·7H₂O, 0.004 g CaCl₂ and 0.01 g NaCl per 100 ml of distilled water at pH 7.0 and in the temperature range of 34–37 °C. In order to prepare the fresh culture of the bacterial strain, 3% inoculums of the isolated bacterial strain were inoculated into the above mineral salts medium. Then, the medium was placed into a shaker incubator at 200 rpm and incubated at 37 °C for 24 h. Finally, the freshly prepared bacterial culture was stored at 4 °C until use.

Microbial reduction of graphite oxide. Microbial reduction of graphite oxide (GO) was carried out by the following method. Freshly prepared bacterial culture [1 ml (10⁵ CFU)] was aseptically added to 100 ml of GO solution (0.5 mg ml⁻¹). The mixture was then shaken at 105 rpm at 37 °C. Finally, the solution was sampled at different time intervals to set an optimum condition for the reduction. On completion of the reduction process, the color changed from brown to black with the simultaneous precipitation of the reduced product. This suggested that the oxygen-containing moieties present in GO might have been removed. The changes in optical density for the growth of the pure bacterial cells in the presence and absence of graphite oxide were recorded using a UV-visible spectrophotometer at 600 nm. Herein, the corrected optical density for the microbial reduction of graphite oxide was calculated according to the method described by Gongming et al.²²

[Corrected OD₆₀₀ = total OD₆₀₀ of the bacterial/GO culture medium − OD₆₀₀ of the pure bacterial culture].

Synthesis of magnetite (Fe₃O₄) nanoparticles. Magnetite nanoparticles were synthesized by using a co-precipitation method.⁴⁰ In brief, 4 g of Mohr's salt [(NH₄)₂Fe(SO₄)₂·6H₂O (0.1 M) and 4.8 g of [(NH₄)Fe(SO₄)₂·12H₂O (0.1 M) were prepared in 100 ml of distilled H₂O. A solution of ammonia was added drop-wise under nitrogen atmosphere at 32 °C for 90 minutes until the pH value of the solution reached 10 and it was then precipitated by the dropwise addition of 0.7 moles of NaOH solution. After precipitation, the supernatant was first decanted and filtered through Whatman qualitative filter paper. Finally, it was washed repeatedly and dried at room temperature.

Preparation of ethylenediamine (EDA) modified reduced graphene oxide. The ethylenediamine (EDA) modified reduced graphene oxide (EDA–RGO) was prepared according to the method reported by Wang et al.⁴⁵ In brief, 65–70 mg of lithium metal were added to the 120 ml of dry ethylenediamine (EDA) solution in a 250 ml four-necked round bottomed flask. The mixture was kept at 37 °C for 80 minutes under nitrogen-purged conditions until the solution turned colorless. Then, 70 mg of as-prepared reduced graphene oxide (RGO) were transferred into the mixed solution and allowed to react for 12 hours at 50 °C with constant stirring at 700 rpm. Finally, the reaction vessel was quenched by bubbling air through it for 60–65 minutes. The product was filtered through Whatman paper, followed by repeated washing with ethanol and distilled water. The black filtrate was dried at 37 °C and stored for further use.

Fabrication of the Fe₃O₄ embedded, PHBV-grafted-RGO porous 3D scaffold. To synthesize the PHBV-grafted-RGO composite, 0.5 g of PHBV copolymer were dissolved in 15 ml of chloroform solution at room temperature and treated with 10 ml (10–12%) of aqueous solution of 1,6-hexanediamine [HDA] solution at 37 °C for 40 minutes with constant mechanical stirring. The copolymer was then precipitated by using methanol solution, followed by repeated washing with distilled water to remove the unreacted amine solution, after which it was vacuum dried at room temperature.

The freshly prepared EDA–RGO was finely dispersed in 30 ml distilled water by using a probe sonicator for 30 minutes. Then, 7 ml [1% (w/v)] of aqueous GA solution were slowly added to the RGO solution, which was then incubated at 25 °C for 4 hours under constant shaking. The solution was centrifuged at 3000 rpm for 10 minutes and washed thrice with de-ionized water to remove the excess GA solution. The GA activated RGO was further dispersed into 25 ml of distilled water and then 15 ml of chloroform solution containing the as-prepared PHBV–HDA were introduced into the system drop-wise at pH 8 under vigorous stirring for 30 minutes at room temperature. The mixed solution was allowed to react for 24 hours at 37 °C and finally, the grey colored product was filtered and washed with de-ionized water and chloroform solvent to remove any unreacted RGO and free PHBV–HMDA copolymer.

The Fe₃O₄ embedded PHBV-grafted-RGO composite was fabricated using a probe sonicator. First, the synthesized PHBV-grafted-RGO composite was dispersed into 30 ml of distilled water for 60 minutes. Then, 100 mg of as-prepared Fe₃O₄ particles were added to the system and sonicated for another 60 minutes until all of the components were finely dispersed. The viscous solution was poured into the Petri dish and vacuum dried at 40 °C and finally, the porous 3D scaffold was stored in a desiccator for different characterizations.

Swelling and in vitro degradation studies of the Fe₃O₄/PHBV-grafted-RGO composite. The water uptake capacity and in vitro degradation of the composite [area = 20 cm² and thickness = 0.3 mm] were conducted in phosphate buffer saline solution at pH 7.4 and temperature of 37 °C for 24 hours, for up to 60 days. The composite films were withdrawn at definite time-intervals and the weight differences were recorded before they were soaked with blotting paper to remove surface water. To find out the percentage of weight loss, the composite was dried in a vacuum oven at room temperature and the percentages of swelling and weight loss were calculated using the following equation:

[%] swelling = [W₂ − W₁]/W₁, [%] weight loss = [D₁ − D₂]/D₁,

where, W₁ and W₂ are the weights of the composite before and after swelling and D₁ and D₂ denote the weights of the composite before and after degradation.

Antimicrobial study

The antimicrobial study of the composite films of pure PHBV copolymer, pure RGO and [Fe₃O₄/RGO-g-PHBV] was performed against several bacterial strains, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis by using a disk-diffusion method. Fresh culture of each bacterial strain was inoculated onto the solid agar containing 13% of nutrient broth [NB]. The disk-like films were then placed on the pre-selected zones of the culture plate and incubated for 24 hours at 37 °C. The inhibition zones were recorded using a zone measurement scale.

Blood hemolytic assay

To determine the degree of lysis of human erythrocyte cells by the polymer composite, a blood hemolytic assay was performed by collecting fresh human blood from 6 healthy women and 6 healthy men with prior informed consent. The human blood was collected with full aseptic pre-cautions by a specialist Doctor [Pathologist] through the national guidelines of the Indian Council of Medical Research [ICMR], and precautions recommended by the ethical committee of Nil Ratan Sirkar Hospital and Medical College, West Bengal, India. 5 ml of fresh blood were centrifuged at 3000 rpm for 10 minutes and the erythrocytes were washed three times with phosphate buffer saline [pH 7.4] solution. The solutions of the composite were prepared in PBS solution with different concentrations [0.5, 0.75, 1.0, 1.25, 1.5 and 2.00 mg ml⁻¹] and the volume of each was made up to 950 μl using PBS solution. Then, 50 μl of RBC were added to each system and incubated for 15 minutes at 37 °C in the dark. After incubation, it was centrifuged for 10 minutes at 3000 rpm. The percentage of cell lysis of RBC membranes was evaluated by the measurement of UV-visible absorbance at 540 nm.

Cell toxicity and cell proliferation studies

The percentage of cell viability of NIH 3T3 fibroblast cells was determined on the Fe₃O₄/RGO-g-PHBV composite in Dulbecco's modified Eagle's medium. The specific growth medium was composed of 10% fetal bovine serum and 1% of an antimycotic antibiotic solution, maintained at 37 °C in a humidified atmosphere with 5% CO₂. 60 μl of specific medium were transferred into each well to assess the cell response for 3 hours. Finally, the cells were treated with 600 μl of DMSO solution and the optical density of the purple solution was recorded at 570 nm by using a UV-visible spectrophotometer.

The nature of cell attachment, and also cell proliferation on the polymeric scaffolds were monitored by using the NIH 3T3 fibroblast cells. The scaffolds prepared from pure PHBV and Fe₃O₄/RGO-g-PHBV polymer composites were cut into small rectangles and placed into the wells. Then, the fibroblast cells [3.1 × 10⁵ cells] were inoculated onto each biomaterial and incubated for 4 days. After incubation, the samples were withdrawn from the medium and washed with phosphate buffer saline solution. Then, they were treated with 2% of glutaraldehyde solution, followed by dehydration in 70% of ethanol for 15 minutes. Finally, the samples were dried under critical-point-dried conditions and stored for spectroscopic analyses. To calculate the average cell spreading area, 5 fields of each experimental set were randomly selected and the average area of 4 cells [total 5 × 4 = 20 cells] in each field was recorded. The experiment was repeated in triplicate for each sample to find out the average cell spreading area.

Characterization

The electronic absorption bands present in GO and RGO were recorded using a UV-visible spectrophotometer (Optizen view, Mecasys) at a resolution of 1 nm. The tensile modulus, strength and percentage of elongation of pure PHBV and Fe₃O₄/RGO-g-PHBV composite films were measured using a Universal Testing Machine (LLOYD, LR10k Plus), at a crosshead speed of 5 mm min⁻¹ at 25 °C under conditions of 65% relative humidity. Fourier transform infrared (FTIR) spectra of both GO and RGO were obtained with an ATR-FTIR (model-Alpha, Bruker, Germany) spectrometer, scanning from 4000 cm⁻¹ to 550 cm⁻¹ for 42 consecutive scans at room temperature. X-ray diffraction studies were carried out using an X-ray diffractometer (Rigaku, RAD-111B) with nickel filtered CuK-α radiation (wave length of 0.154 nm), which was operated at 40 kV and 30 mA over the range of 2θ = 5–50°, with a scanning rate of 20 min⁻¹. The particle sizes of dispersed GO and RGO were analyzed by using a laser particle size analyzer (Malvern, Zetasizer Nano series, nano ZS90). The cross-sectional morphologies of the exfoliated GO and RGO were investigated using a scanning electron microscope (model: Philips XL30, Carl Zeiss, Germany) after coating the samples with gold under vacuum. The microstructures of the samples were analyzed by transmission electron microscope (JEOL JEM 2100 HR with EELS model). The grid for TEM analysis was prepared by placing a drop of the GO, RGO and Fe₃O₄/RGO-g-PHBV composite suspensions on a carbon-coated copper grid and allowing the water to evaporate inside a vacuum dryer. The grid containing GO, RGO and Fe₃O₄/RGO-g-PHBV was scanned by a transmission electron microscope. Thermo-gravimetric analysis (TGA) was carried out on a Q50 TGA (TA instruments, USA) at a heating rate of 10 °C min⁻¹, from 30 to 600 °C under nitrogen flow. Electrochemical studies were carried out in a cell with GO, RGO and Fe₃O₄/RGO-g-PHBV coated electrodes as working electrodes, Pt wire as counter electrode, and a Ag/AgCl electrode as a reference electrode. Cyclic voltammetry and capacitive measurements were collected using an electrochemical workstation (GAMRY Instruments), in 0.5 mol L⁻¹ Na₂SO₄ and 3.0 mol L⁻¹ NaOH aqueous solutions, respectively, as the electrolyte. Raman spectra of the samples were obtained on a Nanofinder 30 Confocal Raman microscope (MODEL 2018 RM (Make Spectra Physics, USA) employing a 532 nm laser beam. The surface morphology of the GO, RGO and Fe₃O₄/RGO-g-PHBV sheets was investigated by an atomic force microscope (VEECO VI INNOVA, Bruker AXS Pte Ltd) in tapping mode at room temperature. The magnetic contrast images of the Fe₃O₄/RGO-g-PHBV composite were recorded by using a 1.5 T clinical Signa HDe scanner [NRSHDe, A156, Spandan Pvt. Ltd.] through agar MRI phantom experiments. The selected parameters for the T₂-weighted images are as follows: magnetic field strength, 1.5 T; repetition time (TR), 2500; echo time (TE), 20–220 ms; FOV, 16 cm²; resolution of 256 × 256 points, respectively. The finely dispersed suspensions of the composite [at varying concentrations of iron] were immobilized in 1.2% (w/v) agarose in a Petri dish. In addition, pure agarose gel was also used in phantom MR imaging studies as the control. The elemental composition was determined by using X-ray photoelectron spectroscopy having monochromatic α-radiation, 1253.6 eV [Omicron ESCA Probe, Taunusstein, Germany). The spectral curve fitting was performed using Advantage software.

Statistical analysis

All experiments were conducted in triplicate and reported values were in the form of triplicate counts ± standard deviations (SD). The Student's t-test was used to compare the statistical significance of test samples against the control [P < 0.05, denoted by *].

Results and discussions

Mechanical properties

The mechanical properties of the pure PHBV copolymer, GO, RGO and Fe₃O₄/RGO-g-PHBV composite were investigated in order to monitor the effect of RGO in the composite. Table 1 shows the values of tensile strength, modulus and the percentage of elongation. Pure PHBV copolymer exhibits comparatively lower values of the tensile strength [4.1 MPa] and elongation percentage [34.6%], but in the case of Fe₃O₄/RGO-g-PHBV, both the tensile strength [8.3 MPa] and elongation percentage [69.3%] were found to be shifted to higher ranges. The results demonstrate that the conjugation of reduced graphene oxide to PHBV copolymer leads to the enhancement of tensile strength and modulus, due to the increase in the stiffness of the composite. Therefore, the vertically aligned PHBV molecules were able to increase the free volume in the composite with the better dispersion of each component into the matrix, resulting in significant improvement of the mechanical properties.

Table 1 Mechanical properties of the pure P(3HB-co-3HV) [PHBV] copolymer and the Fe₃O₄/RGO-g-PHBV composite

Serial	Materials	Tensile strength [MPa]	Modulus [MPa]	Percentage elongation at break
1	Pure P(3HB-co-3HV) [PHBV]	4.1	157.3 ± 0.55	34.6 ± 0.55
2	Fe3O4/RGO-g-PHBV composite	8.3 ± 0.57	167.5 ± 0.77	69.3 ± 0.77

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UV-visible spectroscopic analysis

The microbial reduction of GO by the bacterial strain was monitored using a UV-visible spectrophotometer. From Fig. 1, it was observed that with increasing the incubation period, the optical density values at 600 nm for the growth of pure bacterial cells and bacterial cells in the presence of graphite oxide increased slowly over 7–8 hours. The rate of change of the optical density gradually increased, up to 22–24 hours of incubation, in comparison to the control [pure graphite oxide], followed by the appearance of a steady-state with respect to the incubation period [see Fig. 1B]. Since the growth of bacterial cells in the culture medium was found to be unaffected by the presence of graphite oxide, the corrected OD₆₀₀ values were considered in order to find out the change in optical density at 600 nm during the microbial reduction of graphite oxide.

Fig. 1 UV-visible spectra of graphite oxide and reduced graphene oxide (A). The variation in the optical density (OD₆₀₀) for bacterial cell growth in the presence and absence of graphite oxide, with the incubation period (B).

Graphite oxide (GO) showed a maximum absorption peak at 230 nm and a shoulder peak at nearly 300 nm. The peak at 230 nm was assigned to the π → π* transition of aromatic C–C bonds and another peak at 300 nm was due to the presence of the n → π* transition of C=O bonds. After reduction, a red shift of this characteristic peak was observed at 265 nm for reduced graphene oxide (RGO), suggesting that the conjugated electronic structure was restored after the reaction. The spectrum obtained is in agreement with the previously reported results.⁴⁶

ATR-FTIR spectral analysis

Fig. 2 presents the FTIR spectra of pure GO, RGO, pure P(3HB-co-3HV), RGO–EDA and the RGO-g-PHBV composite. The GO spectra [Fig. 2A] show a strong absorption band at approximately 1722 cm⁻¹ attributed to the carboxylic acid C=O stretching, which was found to disappear in RGO after reduction. The intensities of multiple peaks due to –C–O (epoxy), –C–O (alkoxy) were not significantly changed, but the peak positions were shifted to some extent after reduction.¹⁷ For the GO spectrum, the broad peak at 3112 cm⁻¹ was attributed to the stretching vibrations of the hydroxyl group (–OH). After the reduction of GO, the –OH peak was shifted to higher frequency at 3320 cm⁻¹, which was due to a decrease in intermolecular hydrogen bonding interactions in RGO. Therefore, most of the characteristics peaks were found to be absent in RGO [Fig. 2B], indicating the successful chemical reduction of GO. However, after the modification of RGO with ethylenediamine [EDA], a series of strong absorption peaks was observed at 3367, 2898, 1655, 1570, 1483 and 1214 cm⁻¹, respectively [see Fig. 2C]. The new absorption bands at 1570 cm⁻¹ and 1214 cm⁻¹ were due to the stretching vibrations of adjoined N–H groups and C–N groups. In the case of the RGO-g-PHBV composite [Fig. 2D], the additional stretching frequencies at 1722 cm⁻¹ and 1130 cm⁻¹ were attributed to the presence of the –C=O group and –C–O–C– group, respectively. Therefore, the FTIR spectral analyses indicated the grafting of the PHBV–NH₂ group with the modified reduced graphene oxide. The FTIR spectral reports of 1,2-hexanediamine modified PHBV and EDA modified RGO are in agreement with the reports described by Wang et al.,⁴⁵ Bakare et al.⁴⁷ and Noel et al.⁴⁸

Fig. 2 ATR-FTIR spectra of graphite oxide (A), reduced graphene oxide (B), ethylene diamine modified RGO (C) and the RGO-g-PHBV composite (D).

XRD spectral analysis

XRD diffraction spectra of pure graphite, GO, RGO and the Fe₃O₄/RGO-g-PHBV composite are depicted in Fig. 3. The pristine graphite [Fig. 3D] exhibits a sharp diffraction peak at 2θ = 26.6°, with miller indices (002) corresponding to an interlayer d-spacing of 0.338 nm. After oxidation, the sharp diffraction peak shifted to the lower angle at 2θ = 9.6°. Additionally, the interlayer spacing (d-spacing) of GO (0.92 nm) [Fig. 3C] was increased compared to the d-spacing of pristine graphite. The increase in d-spacing is due to the intercalation of water molecules and the presence of oxygen containing functional groups on both sides of the graphite layer. On the other hand, the XRD patterns of RGO [Fig. 3B] show a broad peak at 2θ = 26.75° with a lower intensity, which indicates the presence of thin graphene layers in the stacks. In the case of RGO, the interlayer spacing (0.34 nm) is slightly larger than that of graphite (0.338 nm), which may be due to the presence of residual oxygen groups in the RGO structure. The pure PHBV copolymer [Fig. 3E] and RGO–EDA [Fig. 3F] exhibit spectral peaks at 12.85° (020), 11.93°, 16.25° (110), 21.6°, 26.13°, attributed to the semi-crystalline nature of the PHBV copolymer and RGO–EDA. For the RGO-g-PHBV/Fe₃O₄ composite [Fig. 3H], the six diffraction peaks at 2θ = 13.16°, 32.23°, 35.9°, 43.3°, 53.5° and 62.9° can be assigned to the (0 2 0), (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0) basal planes of the amorphous PHBV polymer and standard Fe₃O₄ crystal plane [Fig. 3A]. All the new significant diffraction peaks of the RGO-g-PHBV/Fe₃O₄ composite match well with the data from the JCPDS card (19-0629), which indicates that the RGO-g-PHBV/Fe₃O₄ composite consists of disorderly stacked⁴⁹ RGO sheets and well crystallized Fe₃O₄.

Fig. 3 XRD analysis of Fe₃O₄ nanoparticles (A), reduced graphene oxide (B), graphite oxide (C), graphite powder (D), PHBV (E), RGO-EDA (F), RGO-g-PHBV composite (G) and Fe₃O₄ /RGO-g-PHBV composite (H).

Surface morphology analysis

The surface morphology and structure of GO, RGO, RGO-g-PHBV/Fe₃O₄ composite were studied by SEM and TEM analyses. The SEM images of the cross-sections of GO and RGO are presented in Fig. 4. The images reveal that GO [Fig. 4A] and RGO [Fig. 4C] are composed of few layers. The RGO-g-PHBV/Fe₃O₄ composite [Fig. 4D] reveals the uniformly distributed Fe₃O₄ nanospheres on the basal planes of the RGO-g-PHBV sheets. The size of the doped nanosphere was found to be in the range of 20–40 nm. Fig. 5A shows the TEM images for the as prepared GO sheet, from which the sheet seems to be planar and very thin, while the RGO sheet, as shown in Fig. 5B, has wrinkles and a folded geometry. The sheets of layers are found to scroll slightly with the overlays, and collapse to overlay one-another, which are inherent characteristics of graphene oxide.⁵⁰

Fig. 4 SEM analysis of the cross-sectional area of graphite oxide (A), exfoliated graphite oxide (B), reduced graphene oxide (C) and the Fe₃O₄/RGO-g-PHBV composite (D).

Fig. 5 TEM analysis of exfoliated graphite oxide (A), reduced graphene oxide (B) and the Fe₃O₄/RGO-g-PHBV composite (C).

The TEM micrograph [Fig. 5C] of the RGO-g-PHBV/Fe₃O₄ composite exhibits similar features to the SEM morphology and it displays the lattice fringes of Fe₃O₄ with a spacing of 0.48 nm, which agrees well with the d-spacing of the (111) plane of Fe₃O₄. The uniform distribution and coverage of RGO-g-PHBV sheets with magnetic nanoparticles was investigated in the composite.

Thermogravimetric analysis (TGA)

Fig. 6 shows the TGA plots of GO, RGO, PHBV and the Fe₃O₄/RGO-g-PHBV composite, based on the mass loss during heating in the nitrogen atmosphere. For GO [Fig. 2A], the degradation was initiated in two steps. Primarily, the mass loss at nearly 160 °C was due to the breaking of the hydroxyl bond and epoxy group from the ring structure of GO. Secondly, the degradation (65%) in the temperature range of 350–400 °C elucidated the pyrolysis of the carboxyl group, as well as the rupture of the hexagonal ring. However, in the case of RGO [Fig. 6B], the initial degradation was initiated at 100 °C, due to the wash out of weakly bonded water molecules. After that, the major weight loss (20%) was observed above 450 °C, indicating the removal of oxygen containing functional groups. The initial degradation temperature of the Fe₃O₄/RGO-g-PHBV composite was found to be shifted from 100 °C to 180 °C, compared to the pure PHBV copolymer [Fig. 6C] and RGO. The secondary degradation point [260 °C] was also revealed at a comparatively higher range than that of the pure PHBV copolymer [220 °C]. Therefore, the thermal degradation temperature of the Fe₃O₄/RGO-g-PHBV composite [see Fig. 6D] was higher, compared to the PHBV copolymer, which was attributed to the excellent thermal stability of RGO. The improvement may also be attributed to the re-orientation of PHBV molecular chain segments at the interfaces of the PHBV backbone and RGO nanosheets.⁵¹

Fig. 6 TGA analysis of graphite oxide (A), reduced graphene oxide (B), PHBV copolymer (C) and Fe₃O₄/RGO-g-PHBV composite (D).

Energy-dispersive X-ray (EDX) analysis

Energy-dispersive X-ray analysis (EDX) was employed to analyze the elemental composition and weight percentages of the samples, shown in Fig. 7. Each element [C and O atom] in pure GO exhibited high atomic percentage values [C = 56.67% and O = 43.17%], with almost similar values of weight percentages [C = 48.53% and O = 49.25%], due to the presence of different oxygen containing functional groups [epoxy, hydroxyl and carboxylic groups], and the carbon content in the hexagonal ring. The atomic ratio of C and O [C/O] was found to be 1.31, which was lower than that of RGO; this is due to the incorporation of oxygen atoms into the graphitic layers, which destroyed the π-conjugated structure. For RGO, both the atomic and weight percentages were diminished [C_A = 46.82%, C_W = 29.87% O_A = 20.77%, O_W = 17.66%] and the C/O ratio was enhanced to 2.25, indicating the removal of different oxygen containing functional groups [epoxy, hydroxyl and carboxylic group] from the structure.⁵² On the other hand, the Fe₃O₄/RGO-g-PHBV composite presented 4.63% of Fe species (atomic%) along with an enhanced carbon and oxygen content, indicating the reality of the Fe₃O₄ species being coated by RGO-g-PHBV composite networks. In addition, the results of the EDX elemental mapping analysis [Fig. 7E–G] indicated the significant structure and elemental distribution of each component (C, O and Fe). It also revealed that the background of the RGO-g-PHBV composite networks was covered by the uniformly distributed Fe₃O₄ nanoparticles, which were embedded in the RGO-g-PHBV composite. This phenomenon was also supported by SEM and TEM analyses. From the elemental analysis results, we concluded that our reduction process is better than that of the previous report.⁵³

Fig. 7 EDX analysis of graphite oxide (A), reduced graphene oxide (B), Fe₃O₄/RGO-g-PHBV composite (C). EDX elemental mapping analysis of C element (E), O element (F) and Fe element (G) in the Fe₃O₄/RGO-g-PHBV composite image (D).

Cyclic voltammetric analysis

To obtain the electrical conductivity of GO, RGO and the Fe₃O₄/RGO-g-PHBV composite, current–voltage (I–V) curves were measured at room temperature for these samples. The results revealed that GO exhibited the highest resistance (33.04 kΩ) compared to RGO (1.651 kΩ) and the Fe₃O₄/RGO-g-PHBV composite (3.448 kΩ), indicating the improved conductivity in RGO and the Fe₃O₄/RGO-g-PHBV composite. This improvement in electrical conductivity for RGO was attributed to the restoration of π-stacking through the elimination of ‘in situ’ defects and the renewal of the graphite structure in the reduction process.⁵²

Brunauer–Emmett–Teller (BET) analysis

The specific surface area of GO and RGO were determined by Brunauer–Emmett–Teller (BET) analysis. The N₂ adsorption–desorption isotherms of GO and RGO are shown in ESI Fig. S1.† Reduced graphene oxide exhibited a specific area of 56 m² g⁻¹, whereas, the surface area of the three dimensional (3D) graphite oxide was 33 m² g⁻¹. The higher surface area for RGO could be attributed to the reduction of GO using microbes. The microbial reduction process was found to proceed slowly (hours) compared to other reduction processes, which proceed rapidly (minutes, upon heating) when hydrazine or other strong reductants are used on aqueous dispersions of GO. This slow reduction process inhibits the restacking of lamellae in solution⁵⁴ and results in a large surface area compared to that of GO.

Raman spectral analysis

The defects of the expanded lattice and also the dissipated layer of graphene oxide were monitored by Raman spectral analysis. Fig. 8 demonstrates the Raman shift of GO and RGO. The Raman spectra consist of two distinct peaks, the D and G bands. The sharp D-band corresponds to the breathing mode of benzene rings in the k-point phonons of A_1g symmetry, while the G band originates from the in-plane band stretching motions of the sp²-hybridised carbon atom in E_2g phonons.⁵⁵ The oxidized GO exhibited two blue-shifted signals at nearly 1566 cm⁻¹ (G-band) and 1304 cm⁻¹, due to the decrease in the size of the in plane sp² domains. The D/G ratio is the standard for evaluating the defects in the lattice. In the case of GO, the D/G ratio was nearly 1, while after reduction of GO, the ratio was 1.3, indicating large amounts of defects within the crystal lattice. The sharp signal for the G-band was found to broaden in the red-shift region at 1552 cm⁻¹. This is attributed to the restoration of π-conjugation in RGO. The experimental Raman shift is consistent with the previous report.⁵⁶ Furthermore, the in-plane crystallite size [La] was calculated using the following formula:⁵⁷

La [nm] = (2.4 × 10⁻¹⁰)λ_l⁴(I_D/I_G)⁻¹,

where I_D/I_G is the intensity ratio of the D and G band, λ_l is the Raman excitation laser energy, E_l = 2.41 eV [λ_l = 514.5 nm].

Fig. 8 Raman spectral analysis of graphite oxide and reduced graphene oxide.

The observed value of La for GO (12.92 nm) was comparatively higher than that of RGO (11.12). The results suggest that the bacterial reduction of GO was able to recover the aromatic structures by repairing defects.⁵⁸

Atomic force microscopic (AFM) analysis

AFM was used to determine the thickness and surface roughness of GO, RGO and the RGO-g-PHBV composite. The AFM images of the GO, RGO and Fe₃O₄/RGO-g-PHBV composite are shown in Fig. 9. The size and thickness of GO and RGO were observed to be in the range of several nano-meters to 1 micrometer. The thickness of RGO (2.1 nm) was found to be smaller than the parent GO (3.3 nm) and its Fe₃O₄/RGO-g-PHBV composite (4.1 nm). This value is somewhat larger than the average thickness of the single-layer graphene (0.35–1.0 nm) described in a previous report.⁵⁹ The increment in the thickness of RGO and the Fe₃O₄/RGO-g-PHBV composite may be due to the presence of unresolved oxygen containing functional groups and molecular folding in the structures of RGO and the Fe₃O₄/RGO-g-PHBV composite,⁶⁰ resulting in wrapping on the surface of the PHBV copolymer and turning into 3D like structures.

Fig. 9 AFM analysis of graphite oxide (A), reduced graphene oxide (B) and the Fe₃O₄/RGO-g-PHBV composite (C).

Particle size analysis

The light scattering analysis [ESI Fig. S2†] at the scattering angle θ = 90° of the GO and RGO revealed that the particle sizes were in the ranges of 0.385 μm and 0.316 μm, respectively. The zeta potential values at −22 mV also demonstrated the stability of the RGO in aqueous phase. This may be due to the non-spherical shape of the materials and also, the model-derived diameters were not their real sizes. The obtained results regarding the size of GO and RGO were found to be comparatively better than that obtained by Liu et al.⁶¹

Magnetic properties

The magnetic properties of the nanoparticles were analyzed using a vibrating sample magnetometer (VSM) as shown in Fig. 10A. The saturated magnetization (M_s) was calculated from the plot of M_s vs. 1/H (Mat 1/H > 0). The magnetic hysteresis curve exhibited a typical sigmoid (S-like) configuration at zero value of magnetic remanence, which revealed the super-paramagnetic nature of the RGO-g-PHBV/Fe₃O₄ composite. The saturated magnetic moment of the RGO-g-PHBV/Fe₃O₄ composite was found to be 12.43 emu g⁻¹ at 300 K, which indicated the appearance of small particles of Fe₃O₄ in the composite. This value is lower than that of bulk magnetite,⁶² having an M_s value of 480–500 emu g⁻¹. In the biomedical field, the super-paramagnetic behavior has a great advantage in receptor targeting, due to the minimal self-aggregation tendency outside of the targeted region. The zero-field cooled [ZFC] susceptibility test also demonstrated the cusp-like point of intersection at the blocking temperature as shown in Fig. 10B. Field cooling (FC) analysis indicates the variation of magnetic moment with the change in temperature, resulting in the plateau. For ZFC magnetization, a maximum range was revealed and then it decreased to a zero value in the lower ranges of temperature. Since blocking temperature significantly depends on the applied magnetic field, with increasing temperature range, the particles exhibited free alignment with the applied field, due to the super-paramagnetic properties. Further, at lower temperature, the magnetic composite was found to reveal a remanent magnetization with coercivity, related to the temperature dependence of FC magnetization.⁶³

Fig. 10 Determination of the superparamagnetic properties of the Fe₃O₄/RGO-g-PHBV composite (A and B), and MRI analysis (C).

The bio-imaging [Fig. 10C] analysis of the RGO-g-PHBV/Fe₃O₄ composite was also conducted by using the MRI phantom experiment with agar. The T₂-weighed images demonstrated the appearances of darkened circles compared to the control. The results indicate that the dark contrast is related to the concentration of Fe₃O₄ sol in the RGO-g-PHBV/Fe₃O₄ composite. With increasing the Fe₃O₄ nanoparticles concentration [0.1 mM, 0.3 mM, 0.50 mM, 1.0 mM], the circles became more darkened compared to the control. The transverse relaxation rate [R = 1/T₂] exhibited the higher values at 1.5 T. In the case of the RGO-g-PHBV/Fe₃O₄ composite, the specific relaxation values were found to be 149 mM⁻¹ S⁻¹, whereas, for pure Fe₃O₄, it was 114 mM⁻¹ S⁻¹. The experimental results showed better performances compared to reports by Lee et al.,⁶⁴ and are also in agreement with the reports described by Narayanan.⁶⁵

Fluorescence spectral analysis

ESI Fig. S3† shows the fluorescence excitation spectra of GO and RGO for the emission wavelength of 232 nm. For the GO sample, a broad spectral peak was observed in the range of 200 nm to 600 nm, with a sharp excitation peak at 491 nm. The broad red-shifted emission of GO was attributed to the strong interactions between the protonated acid and different polar groups in the geometry.⁶⁶ The quasi-molecular fluorophores present in the isolated –COOH groups are preferentially accelerated by the electronic excitation in the fluorescence spectra of GO layers.⁶⁷ However, the low intensity broad peak in GO revealed the appearance of islands of graphene oxide, which caused the disruption of the network and thus, created a band gap in the electronic structure. The disruption of the network causes quantum confinement of the electron wave, due to the oxidized site.⁶⁸ In the case of RGO, a strong and highly intense emission peak in the visible region at 489 nm was observed [ESI Fig. S3†]; this may be attributed to the existence of quantum confinement in RGO.⁵⁰ The unusual strong intensity at 489 nm may be due to the overlap of the second order emissions associated with the excitation wavelength, irrespective of the fluorescence from the RGO.

X-ray's photoelectron spectroscopic (XPS) analysis

X-ray photoelectron spectroscopic analysis was conducted to investigate the nature of different functional groups present on to the surface of graphite oxide (GO) and reduced graphene oxide (GO). Fig. 11 demonstrates the C1s spectra of graphite oxide and reduced graphene oxide. For graphite oxide [Fig. 11A], the results revealed the significant signal at 284.37 eV and 286.5 eV, attributed to the presence of C=C, C–H and C–C bonds respectively. In addition, the oxygenated functional groups such as C–O group, –COOH group and –C=O group were found to exhibit signals at 288.12 eV, 290.875 eV and 292.5 eV, respectively. After reduction [see Fig. 11B], the peak intensity of the sp³ carbon was found to be diminished compared to deoxygenated groups –C=C, C–H and C–C, which represented the strong signal intensity at 284.7895 eV. The weak intense peak at 287.03 eV was assigned to the presence of partially oxidized –C–O groups. The phenomenon is due to the reduction of graphite oxide by microbial strain and restoration of aromatic C=C conjugation on the graphene surface.⁶⁹

Fig. 11 X-ray photoelectron spectroscopic analysis of graphite oxide (A) and reduced graphene oxide (B).

Swelling and in vitro degradation studies

The water uptake tendency of the Fe₃O₄/RGO-g-PHBV composite scaffold was determined to evaluate the swelling characteristics in phosphate buffer saline (PBS) solution at pH 7.4. Initially at 1–3 hours, the rate of swelling was found to be only 32–58% as shown in Fig. 12A. However, with increasing the incubation time, the rate of water uptake was increased to some extent. The maximum percentage of swelling was recorded to be 156% after a period of 24 hours of swelling. The comparatively lower range of swelling of the composite is attributed to the appearance of a hydrophobic environment in the polymeric-graphene grafted network.⁷⁰

Fig. 12 Swelling study (A) and ‘in vitro’ degradation study (B) of the Fe₃O₄/RGO-g-PHBV composite at pH 7.4 and 37 °C.

‘In vitro’ degradation studies demonstrated the rate of rupturing of the polymer networks in phosphate buffer saline (PBS) solution at 37 °C and pH 7.4, as shown in Fig. 12B. The percentage weight loss of polymer composite was increased with the increase in time. Initially, the weight of the composite was found to be enhanced along with the incubation time in PBS solution, because of their water uptake capacity, resulting in the disruption of polymer networks. After 60 days of incubation, 22.97% of degradation occurred, due to the maximum erosion of the polymer materials. The P(3HB-co-3HV) copolymer is a well-known biodegradable compound, but the rate, as well as percentage of biodegradation was observed to be lower in the composite. This is due to the non-degradable nature of reduced graphene oxide; here, the reduced graphene oxide may be blocking pores in the polymer and hindering the dissipation of different chemical bonds.⁷¹

Study of antimicrobial activity

Fig. 13 represents the antibacterial effect of the PHBV copolymer, reduced graphene oxide and its Fe₃O₄/RGO-g-PHBV composite. Pure PHBV copolymer does not exhibit any antimicrobial effect against both Gram-positive and Gram-negative bacterial strains and it may be consumed as a nutrient during bacterial cell growth.⁷² Reduced graphene oxide and the Fe₃O₄/RGO-g-PHBV composite reveal similar inhibitory activities against both Gram-positive and Gram-negative bacterial strains; however, the greater bactericidal activity was observed against Gram-negative bacterial strains. Both the reduced graphene oxide and its composite demonstrated the maximum zone inhibition of 17.5 ± 0.577 mm against Gram-negative bacteria, whereas, 14.7 ± 0.77 mm of zone inhibition was observed against Gram-positive strains. The predominant antimicrobial activity is attributed to the sharp edge-like geometrical feature of reduced graphene oxide and its composite, which accelerated the membrane stress on the bacterial cells, resulting in the destruction of cytoplasmic organelles, followed by damage of bacterial cells and leaking of RNA.⁷³ The entrapment of bacterial strains within the PHBV grafted reduced graphene oxide sheets may also be another mechanism for the cytotoxicity of the reduced graphene oxide based composite.⁷⁴ The surface embedded Fe₃O₄ nanoparticles are reported to be effective antimicrobial agents,⁷⁵ and therefore, the combined inhibitory effect of both the Fe₃O₄ nanoparticles and the Fe₃O₄/RGO-g-PHBV composite demonstrates significant antimicrobial activity.

Fig. 13 Antimicrobial study of reduced graphene oxide, pure PHBV copolymer and RGO-g-PHBV composite at temperature of 37 °C.

Hemolytic assay

The hemolytic assays of well-known bioactive reduced graphene oxide based composites were evaluated to determine the ‘in vitro’ blood compatibility by using fresh human erythrocytes. The Fe₃O₄/RGO-g-PHBV composite interacted with the membranes of RBC, leading to the excretion of free hemoglobin. Fig. 14 shows that the capacity for lysis of the samples varies with their concentrations. 2.0 mg ml⁻¹ of concentrated samples revealed the maximum percentage [2.75%] of cell hemolysis compared to the positive control. The lower rate of hemolytic activity of the Fe₃O₄/RGO-g-PHBV composite is due to the presence of hydrophobic aromatic environments that have minimum contact area;⁷³ therefore, because the reduced graphene oxide synthesized through the bacterial strain has a low surface area, it is more likely to prevent hemolysis of the erythrocytes than that of graphene oxide.⁷⁶

Fig. 14 Hemolytic study of the RGO-g-PHBV composite in the dark at 37 °C.

Cell viability and cell attachment on polymeric scaffold

The MTT assay was employed to investigate the nature of the bonding interaction between the polymeric composite and NIH 3T3 fibroblast cells. The effective microbial agent, reduced graphene oxide, and its Fe₃O₄/RGO-g-PHBV composite exhibit superior biocompatible properties, and the cytotoxicity of reduced graphene oxide and its composite is solely dependent on the composite concentration (Fig. 15A). At lower concentration (0.5 mg ml⁻¹), the samples revealed significant cell viability [80–85%] for both reduced graphene oxide and its Fe₃O₄/RGO-g-PHBV composite, while on increasing sample concentration to 1.0 mg ml⁻¹, the cell viability was found to decrease by 10–12%. The incorporation of the magnetite nanoparticles and the biocompatible PHBV backbone into the large aromatic surface content of RGO could improve the biocompatibility and antimicrobial activity of reduced graphene oxide and its composite, since PHBV is a well-known biocompatible polymer that accelerates cell proliferation and adhesion, due to better cell–matrix interaction. The presence of the bioactive surface, having both hydrophilic and hydrophobic moieties,^77,78 in the microbially reduced graphene oxide based nanocomposite resulted in a less reactive oxygenated component, enhanced cell attachment and cell proliferation on the Fe₃O₄/RGO-g-PHBV composite based porous scaffold, compared to the chemically reduced graphene oxide that predominantly uses a toxic reducing agent [Fig. 16C and D]. Cell mortality was also found to be a minimum for the microbially reduced graphene oxide and its composite.¹⁶ Recently, the cytotoxic effects of reduced graphene oxide based composites against bacterial strains, and cell-compatibility against mammal cell lines in macrophages or epithelial cells at comparatively lower concentration ranges were investigated, and it was found that some oxidative stress may reduce the cell viability to some extent at higher sample concentrations.^79,80 It was also found that both the cell attachment and proliferation were enhanced significantly with moderately reduced, few-layer RGO in comparison to chemically reduced graphene oxide, which was attributed to the higher density of extracellular matrix (ECM) protein adsorption.⁸¹

Fig. 15 (A) Cell viability assays of the pure PHBV copolymer, reduced graphene oxide and the Fe₃O₄/RGO-g-PHBV composite at 37 °C in a humidified atmosphere of 5% CO₂. (B) Trend of cell spreading area with incubation time on the pure PHBV copolymer and the Fe₃O₄/RGO-g-PHBV composite. The values are shown as means and standard errors (n = 3), *p ≤ 0.05 (significant). * denotes the significant change in comparison to the control.

Fig. 16 SEM morphology of the 3D porous scaffold of the pure PHBV copolymer (A) and the Fe₃O₄/RGO-g-PHBV composite (B). Confocal images of cell proliferation on the pure PHBV copolymer (C), and on the Fe₃O₄/RGO-g-PHBV composite (D). NIH 3T3 fibroblast cell attachment and cell spreading on the pure PHBV copolymer (F) and the Fe₃O₄/RGO-g-PHBV composite (E). Calculation of the average cell spreading area with incubation time [hour] using the Image J program (G).

The micro-sized porous 3D scaffold of the Fe₃O₄/RGO-g-PHBV composite [Fig. 16A and B] uniformly induced the cell spreading and the non-toxicity and biocompatibility of the PHBV copolymer in the conjugated system resulted in minimum cell toxicity. The better cell viability of the Fe₃O₄/RGO-g-PHBV composite compared to pure RGO and GO is due to the aggregation and sedimentation of GO and RGO, preventing the intake of sufficient nutrients for enormous cell growth.

The results [Fig. 16E and F] of cell spreading on the polymeric matrixes revealed the predominant cell migration onto the PHBV copolymer and the Fe₃O₄/RGO-g-PHBV composite based matrixes. The quantification of the average cell area also showed comparatively better cell attachment on the Fe₃O₄/RGO-g-PHBV composite based scaffold, than on the pure PHBV copolymer based scaffold. The better biocompatibility of the Fe₃O₄/RGO-g-PHBV composite may be due the presence of bioactive functional groups and hydrophilic–hydrophobic conjugation in the mildly oxidized, reduced graphene oxide based Fe₃O₄/RGO-g-PHBV composite system. The biocompatibility reinforced the significant cell attachment via the formation of filopodia per mm of composite based film, compared to the pure PHBV copolymer based film.⁸²

The maximum average cell area [Fig. 15B] (501 ± 10 μm²) was observed after 72 hours of incubation for the Fe₃O₄/RGO-g-PHBV composite, indicating maximum spread with a steady morphology on the population scale. The average cell spreading area was calculated using the Image J program [Fig. 16G], consistent with SEM characterization.

Conclusion

In summary, the present work demonstrates the microbial reduction of the exfoliated graphitic lattice, and the synthesis of the magnetically stable and dispersible Fe₃O₄/RGO-g-PHBV composite based 3D scaffold for application in tissue engineering. The expansion of the graphitic layer and restoration of the electronic conjugation was illustrated by spectroscopic analyses. The appearance of lattice defects with the change of I_D/I_G ratio for GO and RGO corresponding to the oxidation of the sp² carbon domain, followed by the removal of functional groups, revealed that the microorganisms were able to reduce the functional groups by enzymatic pathways. The improvement in electrical conductivity in reduced graphene and the Fe₃O₄/RGO-g-PHBV composite is attributed to the restacking of sp² carbon in the electronic structure of graphene. The VSM and MRI analyses of the Fe₂O₃/RGO-g-PHBV composite suggest that the iron nanocrystals were uniformly deposited in the RGO-g-PHBV composite layers, resulting in the appearance of dark contrast images along with the maximum transverse relaxation rate (1/T₂), due to super-paramagnetic character. The cell viability assays also exhibited good fibroblast cell attachment and cell growth in the polymeric scaffold. Therefore, the synthesized electro-conductive, magnetically active, thermally stable and dispersive Fe₂O₃/RGO-g-PHBV composite can potentially be applied as a sensor in imaging areas for tissue engineering applications.

Conflict of interest

No conflict of interest declared.

Acknowledgements

The authors gratefully acknowledge UGC-NFO-20145-2015, CSIR and DST, Government of India for their financial support in this experimental work. Authors acknowledge to Mr Samrat Kundu, Mr Manishankar Ghosh and Nibaron Barui for their technical help during FESEM and MRI analyses.

References

1. J. Wu, W. Pisula and K. Mullen, Chem. Rev., 2007, 107(3), 718–747.
2. M. J. Allen, V. C. Thung and R. B. Kaner, Honeycomb carbon: a review of graphene, Chem. Rev., 2010, 110(1), 132–145.
3. J. H. Jung, D. S. Cheon, F. Liu, K. B. Lee and T. S. Seo, Angew. Chem., Int. Ed., 2010, 49(43), 5708–5711.
4. A. N. H. Castro Neto, F. Guinea, N. M. R. Peres, K. S. Novoselov and A. K. Geim, Rev. Mod. Phys., 2009, 81, 109–162.
5. A. K. Geim and K. S. Novoselov, Nat. Mater., 2007, 6, 183–191.
6. S. Das, S. Adam, E. H. Hwang and E. Rossi, Rev. Mod. Phys., 2011, 83, 407–470.
7. P. Seneor, B. Dlubak, M. B. Martin, A. Anane, H. Jaffres and A. Fert, MRS Bull., 2012, 37, 1245–1254.
8. J. Luo, L. J. Cote, V. C. Tung, A. T. L. Tan, P. E. Goins, J. Wu and J. Huang, J. Am. Chem. Soc., 2010, 132, 17667–17669.
9. K. P. Loh, Q. Bao, G. Eda and M. Chhowalla, Nat. Chem., 2010, 2, 1015–1024.
10. K. Nakada, M. Fujita and G. Dresselhaus, Phys. Rev. B: Condens. Matter Mater. Phys., 1996, 54, 17954–17961.
11. M. Z. Kassaee, E. Motamedi and M. Majdi, Chem. Eng. J., 2011, 172, 540–549.
12. K. S. Kim, Y. Zhao, H. Jang, S. Y. Lee, J. M. Kim and K. S. Kim, Nature, 2009, 457, 706–710.
13. B. Hasia, N. Ferralis, D. G. Senesky, A. P. Pisano, C. Carraro and R. Maboudian, Electrochem. Solid-State Lett., 2011, 14(2), K13–K15.
14. S. Park and R. S. Ruoff, Nat. Nanotechnol., 2009, 4, 217–224.
15. Y. W. Liu, M. X. Guan, L. Feng, S. L. Deng, J. F. Bao, S. Y. Xie, Z. Chen, R. B. Huang and L. S. Zheng, Nanotechnology, 2013, 24, 025604.
16. V. Singh, D. Joung, L. Zhai, S. Das, S. I. Khondaker and S. Seal, Prog. Mater. Sci., 2011, 56, 1178.
17. J. Zhang, H. Yang, G. Shen, P. Cheng, J. Zhang and S. Guo, Chem. Commun., 2010, 46, 1112–1114.
18. Y. Yuan, S. Zhou, B. Zhao, L. Zhuang and Y. Wang, J. Biotechnol., 2012, 03, 118.
19. T. Kuila, S. Bose, P. Khanra, A. K. Misra, N. H. Kim and J. H. Lee, Carbon, 2012, 50, 914–921.
20. X. Huang, X. Qi, F. Boeyab and H. Zhang, Chem. Soc. Rev., 2012, 41, 666–686.
21. S. Gurunathan, J. W. Han, V. Eppakayala and J. H. Kim, Colloids Surf., B, 2013, 102, 772–777.
22. G. Wang, F. Qian, C. W. Saltikov, Y. Jiao and Y. Li, Nano Res., 2011, 4(6), 563–570.
23. X. Hung, S. Li, Y. Huang, S. Wu, X. Zhou, S. Li, C. L. Gan, F. Boey, C. A. Mirkin and H. Zhang, Nat. Commun., 2011, 2, 292.
24. P. A. Dresco, V. S. Zaitsev, R. J. Gambino and B. Chu, Langmuir, 1999, 15, 1945.
25. S. Donglu, M. E. Sadat, A. W. Dunn and D. B. Mast, Nanoscale, 2015, 7, 8209–8232.
26. X. Y. Yang, Y. S. Wang, X. Huang, Y. Ma, Y. Huang, R. Yang and H. Duan, J. Mater. Chem., 2011, 21, 3448–3454.
27. L. M. Zhang, Z. X. Lu, Q. H. Zhao, J. Huang, H. Shen and Z. Zhang, Small, 2011, 7, 460–464.
28. K. Urbas, M. Aleksandrzak, M. Jedrzejczak, R. Rakoczy, X. Chen and E. Mijowska, Nanoscale Res. Lett., 2014, 9, 656.
29. Y. Zhang, H. L. Ma, Q. Zhang, J. Peng, J. Li, M. Zhai and Z. Z. Yu, J. Mater. Chem., 2012, 22, 13064–13069.
30. Y. W. Gu, K. A. Khor and P. Cheang, Biomaterials, 2003, 24, 1603–1661.
31. S. Park, N. Mohanty, J. W. Suk, A. Nagraja, J. An, R. D. Piner, W. Cai, D. R. Dreyer, V. Berry and R. S. Rouff, Adv. Mater., 2010, 22, 1736–1740.
32. H. Hu, J. Yu, Y. Li, J. Zhao and H. Dong, J. Biomed. Mater. Res., Part A, 2012, 100, 141–148.
33. H. Kim, D. Lee, J. Kim, T. Kim and W. J. Kim, ACS Nano, 2013, 7(8), 6735–6746.
34. U. Dembereldorj, M. Kim, S. Kim, E. O. Ganbold, S. Y. Lee and S. W. Joo, J. Mater. Chem., 2012, 22, 23845.
35. S. R. Ryoo, Y. K. Kim, M. H. Kim and D. H. Min, ACS Nano, 2010, 4, 6587–6598.
36. H. L. Fan, L. L. Wang, K. K Zhao, N. Li, Z. Shi, Z. Ge and Z. Jin, Biomacromolecules, 2010, 11, 2345–2351.
37. N. Li, X. M. Zhang, Q. Song, R. Su, Q. Zhang, T. Kong, L. Liu, G. Jin, M. Tang and G. Cheng, Biomaterials, 2011, 32, 9374–9382.
38. S. Y. Park, J. Park, S. H. Sim, M. G. Sung, K. S. Kim, B. H. Hong and S. Hong, Adv. Mater., 2011, 23, H263–H267.
39. T. R. Nayak, H. Andersen, V. S. Makam, C. Khaw, S. Bae, X. Xu, P. L. Ee, J. H. Ahn, B. H. Hong, G. Pastorin and B. Ozyilmaz, ACS Nano, 2011, 5, 4670–4678.
40. Y. Chang, J. Li, B. Wang, H. Luo and L. Zhi, J. Mater. Sci. Technol., 2014, 30(8), 759–764.
41. J. C. Mayer, A. K. Geim, M. I. Katsnelson, K. S. Novoselov, T. J. Booth and S. Rath, Nature, 2007, 446, 60.
42. S. S. J. Aravind, V. Eswaraiah and S. Ramaprabhu, J. Mater. Chem., 2011, 21, 17094–17097.
43. Y. Inoue and N. Yoshie, Prog. Polym. Sci., 1992, 17, 571.
44. N. Pramanik, K. Mukherjee, A. Nandy, S. Mukherjee and P. P. Kundu, J. Appl. Polym. Sci., 2014, 131, 41080.
45. S. Wang, J. Wang, W. Zhang, J. Ji, Y. Li, G. Zhang, F. Zhang and X. Fan, Ind. Eng. Chem. Res., 2014, 53, 13205–13209.
46. Y. Zhang, H. L. Ma, Q. Zhang, J. Peng, J. Li, M. Zhai and Z. Z. Yu, J. Mater. Chem., 2012, 22, 13064–13069.
47. R. A. Bakare, C. Bhan and D. Raghavan, Biomacromolecules, 2014, 15, 423–435.
48. S. Noel, B. Liberelle, L. Robitaille and G. D. Crescenzo, Bioconjugate Chem., 2011, 22(8), 1690–1699.
49. Y. Chang, J. Li, B. Wang, H. Luo and L. Zhi, J. Mater. Sci. Technol., 2014, 30(8), 759–764.
50. J. C. Mayer, A. K. Geim, M. I. Katsnelson, K. S. Novoselov, T. J. Booth and S. Rath, Nature, 2007, 446, 60.
51. X. Wang, H. Z. Bai, A. Liu and G. Sh, J. Mater. Chem., 2010, 20, 9032–9036.
52. S. S. J. Aravind, V. Eswaraiah and S. Ramaprabhu, J. Mater. Chem., 2011, 21, 17094–17097.
53. W. Zhang, X. Zou and J. Zhao, J. Mater. Chem. C, 2015, 3, 2788–2791.
54. S. Park, J. An, J. R. Potts, A. Velamakanni, S. Murali and R. S. Ruoff, Carbon, 2011, 49, 3019–3023.
55. N. Kumar, A. K. Srivastava, H. S. Patel, B. K. Gupta and G. D. Varma, Eur. J. Inorg. Chem., 2015, 2015(11), 1912–1923.
56. N. Mohanty, A. Nagaraja, J. Armesto and V. Berry, Small, 2010, 6(2), 226–231.
57. B. Jayasena and S. Subbiah, Nanoscale Res. Lett., 2011, 6, 1–7.
58. Y. Zhou, Q. Bao, L. A. L. Tang, Y. Zhong and K. P. Loh, Chem. Mater., 2009, 21, 2950–2956.
59. D. Li, M. B. Muller, S. Gilje, R. B. Kaner and G. G. Wallace, Nat. Nanotechnol., 2008, 3, 101–105.
60. M. Ishigami, J. H. Chen, W. G. Cullen, M. S. Fuhrer and E. D. Williams, Nano Lett., 2007, 7, 1643–1648.
61. S. Liu, T. H. Zeng, M. Hofmann, E. Burcombe, J. Wei and R. Jiang, ACS Nano, 2011, 5, 96971–96980.
62. S. Chikazumi, Physics of Ferromagnetism, Clarendon Press, Oxford, 2nd edn, 1997.
63. D. Caruntu, G. Caruntu and C. J. Connor, J. Phys. D: Appl. Phys., 2007, 40, 5801–5809.
64. J. E. Lee, N. Lee, H. Kim, J. Kim, S. H. Choi, J. H. Kim, T. Kim, I. C. Song, S. P. Park, W. K Moon and T. Hyeon, J. Am. Chem. Soc., 2010, 132, 552–557.
65. S. Narayanan, B. N. Sathy, U. Mony, M. Koyakutty, S. V. Nair and D. Menon, ACS Appl. Mater. Interfaces, 2012, 4, 251–260.
66. B. H. Milosavljevic and J. K. Thomas, J. Phys. Chem., 1988, 92, 2997–3001.
67. C. Galande, D. Aditya, A. D. Mohite, V. Anton, A. V. Naumov, W. Gao, L. Ci, A. Ajayan, H. Gao, A. Srivastava, R. B. Weisman and P. M. Ajayan, Sci. Rep., 2011, 1, 85.
68. S. Shukla and S. Saxena, Appl. Phys. Lett., 2011, 98, 073104.
69. P. Wang, Z. G. Liu, X. Chen, F. L. Meng, J. H. Liu and X. J. Huang, J. Mater. Chem. A, 2013, 1, 9189–9195.
70. S. Sayyar, E. Murray, B. C. Thompson, J. Chung, D. L. Officer, S. Gambhir, G. M. Spinks and G. G. Wallace, J. Mater. Chem. B, 2015, 3(3), 481–490.
71. Y. Chen, T. Xi, Y. Zheng, L. Zhou and Y. Wan, J. Biomimetics, Biomater., Tissue Eng., 2011, 10, 55–66.
72. G. R. Saad, M. A. Elsawy and M. Z. Elsabee, Polym.-Plast. Technol. Eng., 2012, 51, 1113–1121.
73. W. Hu, P. Peng, W. Luo, M. Lv, X. Li, D. Li, Q. Huang and C. Fan, ACS Nano, 2010, 7, 4317–4323.
74. O. Akhavan, E. Ghaderi and A. Esfandiar, J. Phys. Chem. B, 2011, 115, 6279–6288.
75. R. A. Ismail, G. M. Sulaiman, S. A. Abdulrahman and T. R. Marzoog, Mater. Sci. Eng., C, 2015, 53, 286–297.
76. K. H. Liao, Y. S. Lin, C. W. Macosko and C. L. Haynes, ACS Appl. Mater. Interfaces, 2011, 3, 2607–2615.
77. Y. Chang, S. T. Yang, J. H. Liu, E. Dong, Y. Wang, A. Cao, Y. Liu and H. Wang, Toxicol. Lett., 2011, 200, 201–210.
78. S. K. Singh, et al., ACS Nano, 2012, 6(3), 2731–2740.
79. A. Sasidharan, et al., Small, 2012, 8, 1251–1263.
80. S. Some, S. M. Ho, P. Dua, E. Hwang, Y. H. Shin, H. J. Yoo, J. S. Kang, D. K. Lee and H. Lee, ACS Nano, 2012, 6(8), 7151–7161.
81. X. Shi, H. Chang, S. Chen, C. Lai, A. Khademhosseini and H. Wu, Adv. Funct. Mater., 2012, 22, 751–759.
82. M. J. Dalby, M. O. Riehle, D. S. Sutherland, H. Agheli and A. S. G. Curtis, Biomaterials, 2004, 25, 5415–5422.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra03233h

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