In modern medicine, biofilm-associated implant-related infections pose a major problem. Bacteria adhere to implant surfaces and evade the immune response and antibiotic therapy by forming biofilms (Kuilla et al., 2010; Zou et al., 2016). The frequent and widespread antibiotic resistance of certain microbes limits the effectiveness of conventional treatments (Mohammed et al., 2020; Er et al., 2022). Bacterial colonization and biofilm formation represent a major challenge in chronic disease management and surgical interventions, contributing significantly to morbidity in conditions such as chronic obstructive pulmonary disease (COPD) and increasing susceptibility to opportunistic infections (Li et al., 2025). Despite extensive research, conventional biomaterials often lack antibacterial properties, increasing the risk of implant failure and revision surgeries (Perreault et al., 2013; Kumar et al., 2019). Although peri-operative or intra-operative bacterial contamination of implants is possible, bacterial colonization via bodily fluids is a more common cause of infection, leading to biofilm formation on medical devices such as catheters and joint prostheses (Wang et al., 2022; Qin et al., 2020). This contributes to high revision surgery rates, affecting pacemakers, heart valves, and stents (Perreault et al., 2013; de Moraes et al., 2015). Implant removal is often necessary, even if only one of multiple implants is affected, which not only increases healthcare expenses and prolongs patient recovery time but also has significant socio-economic consequences (Mohammed et al., 2020; Malisz and Świeczko-Żurek, 2023). To address this, research focuses on developing antimicrobial biomaterials to prevent bacterial adhesion and reduce infection risks (Er et al., 2022; Sundar et al., 2014). An alternative to conventional antibiotics involves searching for different compounds that can provide antibacterial efficacy via different or multiple mechanisms of action.

Graphene-based materials (GBMs) have garnered significant attention due to their unique physicochemical properties, particularly in nanoscience and biomedicine (Kuilla et al., 2010). Their antibacterial and biocompatible properties make them promising for biosensors, tissue engineering, drug delivery, and cancer therapy. Among these, graphene oxide (GO) stands out due to its high surface area and oxygen functional groups, which enhance dispersibility and bioactivity (Bellier et al., 2022; Malisz and Świeczko-Żurek, 2023; Arabinda et al., 2024).

GO exhibits potent antibacterial activities, particularly for Staphylococcus aureus and Escherichia coli, through mechanisms such as membrane disruption, oxidative stress, and bacterial entrapment (Zou et al., 2016; Rojas-Andrade et al., 2017; Kumar et al., 2019). Studies have confirmed GO’s superior antibacterial efficiency compared to other GBMs, with its effectiveness attributed to several factors, such as sheet size, concentration, and dispersion (Liu et al., 2011; 2012; Hu et al., 2010). GO–polyamide and GO–Ag composites further enhance antibacterial activity against multi-drug-resistant strains such as MRSA (Perreault et al., 2013; de Moraes et al., 2015). More recently, GO has also been shown to confer both bactericidal and osteogenic properties when applied to biomedical substrates. For example, a self-assembling GO coating on polyetheretherketone (PEEK) improved antibacterial efficacy while simultaneously promoting osteoblast differentiation and proliferation (Huang et al., 2024). This dual functionality underscores the potential of GO-modified polymer surfaces to prevent bacterial colonization while supporting tissue integration, a combination particularly relevant for long-term implant performance.

Beyond antimicrobial applications, GBMs support osteogenic differentiation, stem cell adhesion, and nerve regeneration (Hoda et al., 2015; Vera-Sanchez et al., 2016; Henriques et al., 2020). Their biocompatibility has been validated in vitro and in vivo, with minimal toxicity in various models (Pinto et al., 2013; Mohan et al., 2018; Williams et al., 2023). GBM-based scaffolds show potential for spinal cord repair and peripheral nerve regeneration, with studies suggesting that their effectiveness may rival that of autologous grafts (Wang et al., 2022; Qian et al., 2021). With their versatility and promising biomedical applications, GBMs continue to be explored as next-generation materials for antibacterial treatments, tissue engineering, and neural repair (Hoda et al., 2015; Vera-Sanchez et al., 2016; Henriques et al., 2020).

Polyhydroxyalkanoates (PHAs) have been used as matrices in biomedical applications for several years. These are promising biopolymers for biomedical composites due to their favorable physicochemical and biological properties (Rai et al., 2008; Możejko-Ciesielska and Kiewisz, 2016). They are produced naturally by bacterial fermentation utilizing renewable resources, offering a sustainable alternative to petroleum-based plastics. They are classified into short-chain-length PHAs (scl-PHAs) and medium-chain-length PHAs (mcl-PHAs), with scl-PHAs being stiff and brittle (melting temperature: 160 °C–180 °C, glass transition ∼0 °C), while mcl-PHAs are more flexible, with lower melting (40 °C–60 °C) and glass transition temperatures (−50 °C to 25 °C) (Możejko-Ciesielska and Kiewisz, 2016; Puppi et al., 2019; Basnett et al., 2018). These biopolymers have demonstrated significant potential in drug delivery, bone and nerve tissue engineering, and wound healing. For example, poly(3-hydroxybutyrate) (P(3HB)) microspheres have been used to encapsulate rifampicin and tetracycline, ensuring prolonged release with minimal side effects (Kassab et al., 1999; Sendil et al., 1999). P(3HB) composites reinforced with hydroxyapatite promoted rapid bone formation and integration (Doyle et al., 1991; Luklinska and Bonfield, 1997), while antibiotic-free antibacterial scaffolds incorporating thioester groups or selenium- and strontium-doped hydroxyapatite have shown promise for bone tissue engineering (Marcello et al., 2021). Moreover, in soft tissue engineering, P(3HB)-based scaffolds supported keratinocyte attachment and proliferation, aiding wound healing (Rai et al., 2008). In nerve tissue engineering, P(3HB) was used for successfully bridging 2 mm nerve gaps in cats and 10 mm gaps in rats, showing results comparable to those of autografts (Misra et al., 2006; Chen, 2009; Rai et al., 2008; Masood, 2016). More recently, mcl-PHA blends, such as poly(3-hydroxyoctanoate-co-3-hydroxydecanoate) (P(3HO-co-3HD)), have been used in nerve conduits, supporting neuronal cell proliferation and promoting neurite growth due to their favorable mechanical properties (Lizarraga-Valderrama et al., 2015). Recent reviews further highlight that blending PHAs with other polymers or incorporating nanofillers can significantly modify their crystallization behavior, morphology, and performance (Eesaee et al., 2022). Such modifications can be tailored to improve stiffness, ductility, or barrier properties, thereby enhancing their suitability for implantable devices and antimicrobial materials.

Despite the increase in biomedical studies with GBMs, there are only a few reports on the correlations between GBM concentration and its effectiveness against bacterial strains without simultaneously negatively affecting eukaryotic cells.

This study investigates the physicochemical and biological aspects of the developed two-dimensional (2D) composites, based on the combination of PHAs with GO, evaluating their in vitro biocompatibility and antibacterial activity with respect to various eukaryotic and prokaryotic cells. The present study explores the feasibility of developing biomedical devices containing effective antibacterial agents that can combat widespread bacterial infections while exhibiting no toxicity toward mammalian cells.

The modern way of living is accompanied by highly mobile populations across countries, which in turn aggravate the global antimicrobial resistance (AMR) problem. Despite technological advances in regenerative medicine, the proliferation and colonization of bacteria on implants remain a major cause of rejection and can result in increased post-surgery mortality. The multidisciplinary era of modern tissue engineering requires exploration of alternative approaches to prevent bacterial colonization and, consequently, biofilm formation on sensitive surfaces such as medical implants. Thus, there is an emerging need to develop implants that inhibit bacterial adhesion while simultaneously allowing eukaryotic cells to proliferate.

In this study, eco-friendly, non-toxic antibacterial materials were developed using a polymer matrix containing two different types of PHAs, scl and mcl PHAs, with antibacterial activity imparted by the addition of the inorganic compound, GO. Only a few studies have investigated the antibacterial properties and, at the same time, the cytotoxicity of PHA/GO composites on mammalian cells. Several mechanisms have been suggested in regard to the antibacterial actions of GO, including oxidative stress by reactive oxygen species (ROS) (Zhang N. et al., 2016; Panda et al., 2018; Wu et al., 2018), “sharp” edges of GO platelets, which cause damage to cell membranes, leading to leakage and death (Nanda et al., 2015; Ervina et al., 2016; Ji et al., 2016; Gao et al., 2017; Liu et al., 2018), and electron transfer interaction from microbial membranes to GO (Li et al., 2014; Ji et al., 2016). At the same time, there is a contradiction in the literature, as in some studies, the antibacterial activity of GO has not been observed (Bianco, 2013).

PHAs, the bio-polyesters with proven biocompatibility and biodegradability, were produced via bacterial fermentation (Mukherjee and Koller, 2023; Koller, 2018). With the aim of developing materials for tissue engineering with a wide range of mechanical properties and intrinsic antimicrobial activity, composites of P(3HB) and P(3HO-co-3HD) were prepared by introducing GO as a particulate filler at loading levels of 0.5, 2, and 5 wt%. The composites were produced by the solvent casting method, with the samples being produced as films with a thickness of approximately 200 µm. The crystallinity of GO used to make the composites drastically degraded, as shown by Raman spectroscopy with a D to G ratio of 1.2. Furthermore, intensities of peaks in GO XRD diffractograms decreased, indicating preservation of only a small fraction of ordered structures. For this ordered structure, the interlayer distance between GO platelets significantly increased.

Both visual inspection and SEM imaging demonstrated aggregation of GO particles within the polymer matrix. It needs to be noted that the solvent casting technique used in sample preparation is not in favor of GO platelet delamination and dispersion in the polymer matrix. Chloroform, one of the best solvents for PHAs, disperses GO only sparingly (Konios et al., 2014). Despite this poor GO dispersibility, its aggregates were uniformly distributed in the composites.

The SEM micrographs revealed the differences in the topography between the P(3HB) and P(3HO-co-3HD) films. The mcl-PHA samples exhibited a smoother surface than the scl-PHA samples, which showed an uneven morphology. The differences of the neat films most probably originated from the different degrees of crystallinity and flexibility of polymer chains between the scl and mcl-PHA. Due to a higher degree of crystallinity and faster crystallization of P(3HB) (Kai et al., 2003; Anbukarasu et al., 2015; Li et al., 2016), it solidifies in patches, resulting in uneven surfaces of the scl-PHA films. The surface morphology of the composites was analyzed following the addition of GO and compared with that of the neat polymers. The addition of different amounts of GO resulted in different effects on the polymer matrix of neat P(3HB) and P(3HO-co-3HD) films. The P(3HO-co-3HD) composites showed the least affected topography, whereas the P(3HB) composites exhibited various-sized pin-holes that were visible even with the lowest GO (0.5 wt%) loading. Analysis of the surface morphology evidenced differences between the P(3HB)-based and P(3HO-co-3HD-co-3HDD)-based samples. Mcl-PHA films obtained by solvent evaporation are usually characterized by a smooth surface, while scl-PHA films usually show a rougher surface with protrusions and pores (Rai et al., 2008; Anbukarasu et al., 2015; Kai et al., 2003; Li et al., 2016).

Surface morphology, along with molecular composition, defines the wettability of the surface, which is very often considered in the evaluation of surface hydrophobicity. The hydrophobicity/hydrophilicity of the samples was determined based on the contact angle: surfaces with a contact angle greater than 90° were considered hydrophobic (water repellent), whereas surfaces with a contact angle less than 90° were considered hydrophilic (Förch et al., 2009; Peschel et al., 2008; Basnett (2014). The water contact angle for the neat P(3HO-co-3HD) sample was higher than for P(3HB). This indicates that mcl-PHA is more hydrophobic, and this feature is ascribed to the long aliphatic chain of P(3HO-co-3HD), which demonstrates its hydrophobic feature. For both polyesters, incorporating hydrophilic GO caused a decrease in water contact angles. This indicates an exposure of the GO platelet at the composite surface. In the case of the P(3HB) composites, the larger decrease in water contact angle was observed for the composite with the 2 wt% filler content. An increase in the water contact angle with a further increase in GO content above 2 wt% is likely to be caused by surface roughening and GO aggregation. A similar trend was observed for P(3HO-co-3HD) composites.

XPS analysis revealed the enrichment of carbonaceous species on the surface of the composites. The deconvolution of the C 1s and O 1s peaks allowed the calculation of the percentage of the average relative atomic concentration for the neat polymers and their composites containing the highest amount of the filler (5 wt% GO). An increase in the presence of carbonyl (C=O) and carboxyl (O-C=OH) groups, attributed to the incorporation of GO in the polymer matrix, was observed only in the P(3HB)/5 wt% composite.

The Raman spectra of the nanocomposites and their respective neat matrices of PHAs were used to investigate the presence of GO, revealing the characteristic peaks of D and G. The presence of the carbonaceous component is more pronounced in the P(3HB)/GO composite than in the P(3HO-co-3HD)/GO composite although the amount of GO was exactly the same in both nanocomposites (0.5 wt%). The P(3HB) composite containing 0.5 wt% of the filler seems to exhibit a stronger resonance Raman effect, indicating that the short aliphatic chains did not disrupt the hydrogen bonds and other non-covalent interactions of the polymer with the oxygen molecules of GO (Bhawal et al., 2016; Keyte et al., 2019). In addition, the close values for the I_D/I_G ratio of the composites compared with the value of GO indicated the well-preserved structure of GO. The P(3HO-co-3HD) composites containing 2 and 5 wt% of GO revealed both the characteristic peaks of D and G bands with a broader shape compared with pristine GO, indicating the incorporation of GO into the polymer host, which forms a much more disordered and defective structure than pure GO (Strankowski et al., 2016). For the 2 wt% composite, the upshift of the G band at 1,603 cm^-1 may result from an enhanced and more efficient dispersion of the GO in the polymer due to the presence of oxygen-containing groups that can form bonds with the ester groups of the polymer and, hence, reduce the GO–GO interactions The intensity ratio (I_D/I_G) of the 2 wt% composite was higher than that of pristine GO, indicating a higher degree of defects in the composite material.

The XRD patterns revealed the solid-state structural properties of the polymers and their respective composites. The XRD peaks of the crystalline P(3HB) are indexed to the orthorhombic crystal planes (020), (110), (021), (111), (121), (040), and (222) of the P(3HB) film. Located at 2θ = 12° and 44° are the two characteristic peaks of GO, which are assigned to (001) and (100) planes with d-spacings of 7.3 Å and 2.1 Å respectively, confirming the successful synthesis of GO (Johra et al., 2014; Ameer and Gul, 2016; Ain et al., 2019). A well-formed network of the GO with polymer matrices of P(3HB) and P(3HO-co-3HD) is indicated by the absence of the characteristic XRD peak of GO located at 2θ = 12°, even for composites containing 5 wt.% GO. Moreover, the shifts of the XRD characteristic peaks of the PHA indicated a change in the crystallinity of the final composites compared with the respective neat matrices. In the case of the P(3HB)/GO composites, a more disordered structure was revealed since the characteristic peaks of the polymer matrix decreased in intensity compared with the respective PHA matrix. With the higher filler content of 5 wt%, a higher peak intensity was observed than the other composites, implying stacking of the graphene oxide layers within the P(3HB) matrix. In the 0.5 and 2 wt% composites of the same biopolymer (P(3HB)), the presence of low-intensity peaks indicates a more amorphous structure and the presence of defects on the final composites (Larsson et al., 2017). Similar findings have been reported in the literature when high molecular weight polyurethanes and ethylene methyl acrylate hybrid nanocomposites were developed with the addition of GO (Bhawal et al., 2016; Strankowski et al., 2016; Larsson et al., 2017).

The DSC analysis revealed the changes in the thermal properties and the crystallinity of the final composites upon the addition of different ratios of GO. The obtained composites showed insignificant changes in their thermal values (T_g and T_m) compared to their respective neat matrices, indicating that compounding with GO has little effect on the thermal properties of the polymer matrices. There are no reported values in the literature for the heat of fusion for 100% crystalline P(3HO-co-3HD). Hence, the degree of crystallinity for P(3HO-co-3HD) could not be evaluated from the DSC results. Nevertheless, the degree of crystallinity is proportional to the enthalpy of fusion; thus, the change in crystallinity from the GO addition to the polymer matrices was considered in terms of the enthalpy of fusion (ΔΗ_f) or the melting enthalpy (ΔΗ_m), determined from the area under the melting endotherm. The higher values of ΔΗ_m indicate higher degrees of crystallinity of the samples. The addition of 2 and 5 wt% into the polymer matrix of P(3HB) enhanced the crystallinity of the composites, indicating that the GO particles acted as nucleation sites. The lower crystallinity observed for the 5 wt% composite, compared with the 2 wt% sample, is attributed to the barrier effect of the GO particles obstructing the growth of the crystals (Larsson et al., 2017). Similarly, the P(3HO-co-3HD) composites with 2 and 5 wt% GO showed an increase in the crystallinity of the polymer matrix. This phenomenon might be attributed to the decrease in free volume in the amorphous phase of the co-polyester due to the presence of GO, suggesting affinity between the filler and the polymer matrix via hydrogen bonds.

At the same time, the addition of GO increased both the crystallinity and rigidity of the respective matrices. For both types of PHAs, the introduction of GO improved the strength of the materials. The hydrogen bonding interactions between the ester groups of the bio-polyesters and the hydroxyl groups of GO enhanced Young’s modulus but reduced the elasticity compared with the neat matrices. The reinforcing effect of the GO on the polymer matrix has also been reported in other studies (Abdala et al., 2013; Sridhar et al., 2013; R. Mahendran et al., 2016; Larsson et al., 2017; Smith et al., 2019; Díez-Pascual, 2021).

More specifically, the P(3HB) composites exhibited greater Young’s modulus values compared with the neat polymer. This indicates that GO acts as a reinforcing filler, promoting more entanglements between the filler and the polymer matrix of the composite and enabling load transfer to the interphase. The composite containing the highest amount of the GO (5 wt%) showed a lower Young’s modulus compared with the 2 wt% composite. Such behavior can be ascribed to the increase in the filler interaction, more likely the restacking of GO, which reduces the reinforcement of the composite structure. Therefore, this agglomeration limits the load transfer from the matrix to GO particles, causing cracks that are easy to propagate. These cracks are reported to be responsible for the reduction in the strength of the developed composites (Ervina et al., 2016). Moreover, at higher GO loading, the bulk structure might have more defects due to GO aggregation in the polymer matrix. These findings are in agreement with the literature. Barrett et al. also mentioned that the addition of GO particles promoted additional entanglements between the polymer chain and the particles, enhancing the stiffness of the polymer matrix compared to the neat PHAs (Barrett et al., 2014; Mahendran et al., 2016; Smith et al., 2019). The elongation at break values in Table 4 show a marked decrease with increasing GO content for both P(3HB) and P(3HO-co-3HD) composites. This reduction in ductility can be attributed to the inherent stiffness of GO nanosheets, which act as rigid fillers that restrict polymer chain mobility. As the GO content increases, strong interfacial interactions between GO’s oxygen-containing functional groups and the polymer chains limit plastic deformation, leading to premature fracture under tensile load. Additionally, at higher loadings, GO aggregation can introduce stress concentration sites that act as crack initiation points, further reducing the material’s capacity to elongate before breaking. These effects explain the inverse relationship between GO content and elongation at break observed in this study. However, the final mechanical properties can further be optimized/tailored according to the required application, improving/weakening the interface between the polymeric matrix and the filler (Misra et al., 2006; Rai et al., 2008). This can be achieved by improving composite processing compared with the solvent-cast method, for example, through melt processing.

The fabrication of the composites aimed at developing 2D structures with antibacterial efficacy against pathogens found in the human body and nosocomial environments. Thus, they could be used as scaffolds for the prevention and reduction of bacterial colonization. The 2D composites were investigated for their antibacterial efficacy using the DCT—ISO 22196/JISZ 2801 (established for measuring antibacterial activity on plastics). Both types of composites containing the different fractions of GO exhibited high antibacterial activity on contact with the bacterial strains. The composites of P(3HB) and P(3HO-co-3HD) showed antibacterial activity against both Gram-positive and Gram-negative bacteria. Antimicrobial activity was concentration-dependent, increasing with the increase in GO content in the case of the P(3HO-co-3HD) composite (Figure 9B). The observed enhancement in antibacterial activity at higher GO loadings can be attributed to several factors. The increase in GO concentration enhances the number of exposed nanosheets on the composite surface, thereby increasing the available active surface area for bacterial contact. GO’s sharp edges can physically disrupt bacterial membranes, while its abundant oxygen-containing functional groups can induce oxidative stress via ROS generation. These mechanisms act synergistically to damage bacterial cells, and the probability of such interactions increases as GO content increases. This dose-dependent behavior is consistent with previous findings on graphene-based materials against both S. aureus and E. coli.

These results are in agreement with those of other studies reporting antibacterial activity of GO against both Gram-positive and Gram-negative species (Akhavan and Ghaderi, 2010; Mejías Carpio et al., 2012; Perreault et al., 2015; Panda et al., 2018; Díez-Pascual, 2021).

The addition of the GO particles into the bio-polyesters resulted in the development of inherently antibacterial composites. A comparison of the antibacterial activity of the P(3HB) and P(3HO-co-3HD) composites indicated that the samples showed greater antibacterial activity against Gram-positive S. aureus (ATCC^® 29213™) than the Gram-negative E. coli (ATCC^® 25922™). Other studies have previously reported that GO is toxic to bacteria and Gram-positive species are more susceptible than Gram-negative species (Akhavan and Ghaderi, 2010; Perreault et al., 2013; Perreault et al., 2015; Díez-Pascual, 2021). Interestingly, the antibacterial activity results showed that P(3HB)/2 wt% GO exhibited the highest antibacterial effectiveness, with an R value approaching 1.0, whereas the P(3HB)/5 wt% GO showed a lower antibacterial effect than both 2 wt% and 0.5 wt% GO composites. This observation suggests that while the addition of GO enhances antibacterial activity, excessive GO loading (5 wt%) may lead to agglomeration, reducing the effective surface area and limiting the contact between GO and bacterial cells. Consequently, 2 wt% GO appears to be the optimal concentration, balancing dispersion and activity to achieve maximum antibacterial performance.

Díez-Pascual (2021) tested the antibacterial activity of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) composites containing a range of GO loading (0.5, 1, 2, and 5 wt%). The fabricated solvent casted films were successfully tested for their antibacterial activity against Gram-positive (S. aureus ATCC^® 12600 and Bacillus subtilis ATCC^® 23857™) and Gram-negative (E. coli, ATCC^® 25922™ and B. cepacia, ATCC^® 25416™). The superior antibacterial activity of the composites toward the Gram-positive species was ascribed to the different bacterial membrane composition. Additionally, Akhavan and Ghaderi (2010) investigated the antibacterial effect of graphene nanosheets against both E. coli and Staphylococcus aureus bacteria and reported that E. coli was more resistant to GO than S. aureus. In another study, Gao et al. (2017) observed that when S. aureus and E. coli bacteria were exposed to the same concentration of GO (40 mg/L), the antibacterial inactivation of the S. aureus strain was completed in a shorter period (12 h) than the E. coli (168 h). The different toxicity is mainly attributed to the different form and structure of the cell membrane of the S. aureus (spherical in shape (840 nm)) and E. coli (rhabdoid in shape (approximately 1,230–3,538 nm)) strains. The different composition of the cell membrane can affect how GO interacts with and disrupts the membrane. This difference in damage was evaluated by comparing the RNA concentration obtained from the supernatant of the bacterial strain samples. It was shown that the RNA released from the S. aureus samples was greater than that of the E. coli samples (Gao et al., 2017). Other suggested chemical and physical mechanisms of the antimicrobial action of GO include electron transfer and oxidative stress resulting from the generation of ROS, membrane stress, and the wrapping/entrapment of bacteria, facilitated by physical contact. Bacterial respiration is essential for the growth and metabolism of bacterial cells based on electron transport to extracellular electron acceptors. When bacterial cells interact with GO, surface proteins exhibiting n-type semiconducting behavior facilitate the formation of a Schottky barrier. This leads to electron transfer from the cell membranes to the graphene materials, which are effective electron acceptors, driven by Fermi level alignment (Prasad et al., 2017; Díez-Pascual, 2021).

The difference in surface electron states of the Gram-positive and Gram-negative bacterial membranes might be a reason for the differences in the antibacterial efficacy of the graphene materials against the two categories of bacterial species.

The ROS species generated from GO as hydroxyl radicals can attack the carbonyl groups of the peptide linkages of the bacterial cell wall and damage the cellular components. More specifically, they are responsible for the oxidation of the cellular components of the bacterial strains, causing peroxidation of lipids, enzyme inhibition, oxidation of proteins, and damage to nucleic acids, which results in microbial death (Prasad et al., 2017; Sharma et al., 2018; Díez-Pascual, 2021; Seifi and Ali, 2021). The physical mechanisms include the wrapping/entrapment of bacterial cells, lipid extraction, and the direct contact of sharp GO edges with bacterial membranes, inducing membrane stress and leading to membrane disruption and subsequent leakage of intracellular material (Akhavan and Ghaderi, 2010; Prasad et al., 2017; Díez-Pascual, 2021; Seifi and Ali, 2021). Hence, the presence of the outer membrane in the Gram-negative E. coli could act as protection against GO damage, while Gram-positive S. aureus lacks an outer membrane, thereby increasing the chances of cell death.

Nevertheless, further studies should be conducted to standardize the media, reagents, and methods, along with ensuring the process of GO origin, size, and other vital physicochemical features prior to antibacterial testing of such materials, so a more comparative correlation would be of most use for the future. Additionally, systematic studies are required for graphene and its derivatives before their incorporation with other constituents.

In vitro direct cell culture experiments were carried out in the presence of the PHA-based composites using L929 murine fibroblasts and NG-10815 neuronal cells. The results showed that the fabricated composites of both polymer types exhibited cytocompatibility toward both cell lines. The quantitative assessment of the samples over period of 24 h revealed that the fabricated composites favor the attachment and the proliferation of L929 murine fibroblasts and NG108-15 neuronal cells.

Chitosan composites loaded with different ratios of GO showed an enhanced bioactivity of the scaffold material when tested with respect to VERO from the African green monkey cell line and murine preosteoblasts MC3T3-E1cells. This behavior appeared to be proportional to the GO concentration (Dinescu et al., 2014). In another study, composites of polyvinyl-N-carbazole with GO exhibited a tolerable cytotoxic effect toward NIH 3T3 fibroblast cells (Mejías Carpio et al., 2012). Akhavan et al. (2014) conducted in vitro viability tests on ginseng-reduced graphene oxide sheets using human neural stem cells (hNSCs). The study showed that the ginseng-reduced graphene oxide showed better proliferation of the hNSCs compared to the rest of the samples. Hybrid nanofibrous scaffolds of PCL-GO were fabricated to guide rat neural stem cell (NSC) differentiation into oligodendrocytes. The authors reported that the selective differentiation of NSCs to mature oligodendrocytes occurred in the absence of inductive factors in the culture medium (Shah et al., 2014). Moreover, proliferation and osteogenic differentiation of adult MSCs isolated from goat bone marrow aspirates were successfully observed after coating culture plates with GO (0.1 mg/mL) (Hoda et al., 2015). Additionally, aligned PLLA nanofibrous scaffolds coated with graphene oxide promoted Schwann cell (SC) growth by regulating cell orientation and induced the differentiation and neurite outgrowth of rat pheochromocytoma 12 (PC12) cells (Zhang K. et al., 2016). When scaffolds of 2D graphene films and nanostructured rGO microfibers were seeded with NSCs, it was shown that the rGO microfibers exhibited superior proliferation and differentiation of the cells compared with the tissue culture plate and 2D graphene films. Over a time period of 5 days, the rGO scaffolds were able to regulate the NSC differentiation into neurons, forming a dense neural network similar to a functional nerve graft (Guo et al., 2017). The ability of pristine graphene to promote cell adhesion and proliferation of monkey renal fibroblast (Cos-7), as well as on primary E18 rat hippocampal neurons, was reported by Rastogi et al. (2017). They found that graphene does not induce any cell stress since no alteration of the mitochondrial membrane potential (MMP), mitochondrial morphology, and autophagy levels was observed after 9 days of incubation.

Despite these promising findings and the favorable attachment and proliferation of cells reported in the literature, some studies have reported that graphene-based materials exert significant toxic effects on PC12 cells in a dose- and time-dependent manner (Kang et al., 2017).

Interestingly, the antibacterial trends were not identical for the two polymer systems. In the case of P(3HB), the 2 wt% GO composites showed the strongest antibacterial activity, while higher loading (5 wt%) seemed to reduce activity, most likely because of GO agglomeration that limited the effective contact with bacterial cells. In contrast, for the P(3HO-co-3HD) composites (Figure 9B), antibacterial activity kept increasing with higher GO content. This difference suggests that the polymer matrix itself plays an important role in how well GO is dispersed. In P(3HO-co-3HD), the GO appears to remain better distributed even at higher concentrations, allowing its antibacterial mechanisms to scale with loading. These results highlight that GO’s antibacterial effect is not only related to concentration, but it also depends strongly on the surrounding polymer environment, which is consistent with other studies reporting GO activity against both Gram-positive and Gram-negative species (Akhavan and Ghaderi, 2010; Mejías Carpio et al., 2012; Perreault et al., 2015; Panda et al., 2018; Díez-Pascual, 2021).

In this research, two types of bio-polyesters served as matrices for creating GO composites. The study investigated both the physicochemical and biological characterization of the resulting scaffolds, aiming to address the global issue of antibacterial resistance in biomedical applications. The introduction of varying GO ratios (0.5, 2, and 5 wt%) had distinct impacts on the physicochemical attributes of the two different types of bio-polyesters (P(3HB) and P(3HO-co-3HD)). Physicochemical analyses confirmed uniform GO incorporation and surface morphological changes, while mechanical testing demonstrated that the addition of 2 wt% GO to P(3HB) increased the Young’s modulus from 776 ± 15 MPa to 1,055 ± 28 MPa, indicating enhanced stiffness. Both types of composites demonstrated antibacterial effectiveness against both Gram-positive and Gram-negative bacterial strains. According to ISO 22196 testing, GO demonstrated a concentration-dependent activity against S. aureus and E. coli, with P(3HB)/2 wt% GO achieving the highest antibacterial efficacy. Cytocompatibility testing using L929 fibroblasts and NG108-15 neuronal cells in direct contact with the specimens showed no adverse effects on cell viability, even in specimens with the highest concentration of GO. The processability of these composites offers a versatile platform for scaffold formulation, allowing adjustment of mechanical and thermal properties by modifying the GO content to meet specific application requirements. Furthermore, the developed drug-free composites exhibited targeted toxicity against bacterial cells without displaying any harm to mammalian cells. Overall, this study underscores the potential of these innovative antibacterial, biodegradable, highly biocompatible, and customizable composites for various medical applications. Future research will focus on fabricating 3D PHA/GO scaffolds and exploring their physicochemical and biological properties for potential use in nerve tissue engineering. In addition, in vivo evaluations, long-term antibacterial studies, and optimization of GO dispersion will be included to maximize efficacy and safety in clinical applications.