*3.3. Biomedical Application of Nanocellulose*/*Graphene Composites*

The biomedical applications of nanocellulose/nanographene composites include photothermal ablation of pathogenic bacteria, combined chemo-photothermal therapy against cancer, drug delivery, biosensorics, isolation and separation of various biomolecules, wound dressing and particularly tissue engineering (Table 1).

For photothermal ablation of pathogenic bacteria, a composite paper containing nanocellulose with Au linked to GO using quaternized carboxymethyl chitosan was developed. When excited by near-infrared laser irradiation, the paper generated a rise in temperature of more than 80 ◦C, sufficient for photothermal ablation, both on Gram-positive bacteria (*Bacillus subtilis* and *Staphylococcus aureus*) and on Gram-negative bacteria (*Escherichia coli* and *Pseudomonas aeruginosa*). Additionally, the composite paper showed a remarkable enhancement in tensile strength, bursting index and tear index in comparison with the properties of pure nanocellulose paper [93].

For chemophotothermal synergistic therapy of colon cancer cells, dual stimuli-responsive polyelectrolyte nanoparticles were developed by layer-by-layer (LbL) assembly of aminated nanodextran and carboxylated nanocellulose on the surface of chemically modified GO. Tests on the HCT116 human colon cancer cell line revealed that these nanoparticles allowed for the intracellular delivery of curcumin, which was released in response either to acidic environments or to near-infrared excitation [94]. In this context, nanocellulose/graphene composites are good candidates as carriers for controlled drug delivery, particularly of anticancer drugs. Systems releasing doxorubicin, a model drug with broad-spectrum anticancer properties, were developed. These systems included nanocomposite carboxymethyl cellulose/GO hydrogel beads [95], nanocomposite films made of graphene quantum dots incorporated into a carboxymethyl cellulose hydrogel [72] or macroporous polyacrylamide hydrogels. These hydrogels were prepared using an oil-in-water Pickering emulsion, containing GO and hydroxyethyl cellulose with a quaternary ammonium group [96].

Sensing and biosensing is another important application of nanocellulose/graphene composites. These sensors can be divided into electrochemical, piezoelectric, optical and acoustic wave-based sensors. Electrochemical sensors were constructed for detecting cholesterol [98], glucose and pathogenic bacteria [110], avian leucosis virus [111] and organic liquids [112]. The biosensor for detecting cholesterol was based on chemically-modified nanocellulose, grafted with silylated GO and enriched with ZnO nanoparticles in order to enhance its electrical conductivity [98]. The biosensor for detecting glucose and pathogenic bacteria was based on nanocellulose paper coated with GO, reduced by vitamin C and functionalized with platinum nanoparticles with a cauliflower-like morphology in order to enhance the electrical conductivity of the composite. The platinum surface was functionalized either with glucose oxidase (via chitosan encapsulation) or with an RNA aptamer [110]. The biosensor for the avian leucosis virus was an immunosensor, based on graphene-perylene-3, 4, 9, 10-tetracarboxylic acid nanocomposites as carriers for primary antibodies, on composites of nanocellulose and Au nanoparticles as carriers for secondary antibodies, and on the alkaline phosphatase catalytic reaction [111]. The sensor for organic liquids, mainly organic solvents, was based on cellulose nanocrystal-graphene nanohybrids, selectively located in the interstitial space between the natural rubber latex microspheres [112].

Piezoelectric nanocellulose/graphene-based sensors have usually been designed for strain sensing, i.e., as wearable electronics for monitoring the motion of various parts of the human body, e.g., fingers [63,99,113]. For these purposes, the flexibility and stretchability of nanocellulose was further enhanced by adding other polymers, such as elastomers, represented e.g., by polydimethylsiloxane (PDMS) [113], or hydrogels, represented e.g., by poly(vinyl alcohol) (PVA), crosslinked (together with cellulose nanofibers and graphene) with borax [63].

Optical sensors can be based on surface-enhanced Raman spectroscopy (SERS) or fluorescence. Cellulose SERS strips decorated with plasmonic nanoparticles, termed graphene-isolated-Au-nanocrystals (GIANs), were developed for constructing portable sensors for detecting complex biological samples, e.g., for detecting free bilirubin in the blood of newborns [100]. A fluorescence sensor, based on sulfur and nitrogen-co-doped graphene quantum dots, immersed into nanocellulosic hydrogels, was developed for detecting laccase. This enzyme is widely used in industrial and technological applications, such as bleaching of fabrics, tooth whitening, decoloration of hair, water purification and in oxidizing dyes in beer, must and wines [71].

An example of an acoustic wave-based sensor is an ammonia sensor, based on a quartz crystal microbalance (QCM) with a sensing coating. This coating is composed of negatively-charged electrospun cellulose acetate nanofibers, positively-charged polyethylenimine and negatively-charged GO, and it was created by the electrostatic LbL self-assembly technique [101].

For protein isolation, a metal affinity carboxymethyl cellulose-functionalized magnetic graphene was prepared by successive modifications of GO nanosheets with magnetic nanoparticles, carboxymethyl cellulose and iminodiacetic acid, and then chelated with copper ions. This composite exhibited high adsorption selectivity toward histidine-rich proteins, which was utilized for isolating hemoglobin from human whole blood, and also for isolating a polyhistidine-tagged recombinant

protein from *Escherichia coli lysate*, namely *Staphylococcus aureus* enterotoxin B [77]. For macromolecular separation, cellulose acetate nanocomposite ultrafiltration membranes were fabricated using 2D layered nanosheets, e.g., GO and exfoliated molybdenum disulfide (MoS2), and were successfully tested using macromolecular bovine serum albumin [79].

Nanocellulose/graphene composites are also important components of tissue engineering scaffolds, improving their mechanical properties and their bioactivity. In the studies by Pal et al. (2017), mentioned above, a PLA/CNC/rGO nanocomposite film showed antibacterial activity against Gram-positive *Staphylococcus aureus* and against Gram-negative *Escherichia coli*. At the same time, this film exhibited negligible cytotoxicity against a mouse NIH-3T3 fibroblast cell line, as revealed by an MTT assay of the activity of mitochondrial oxidoreductase enzymes [87]. Nanocomposites of CNCs and rGO, incorporated into PLA matrix through the melt-mixing method, were noncytotoxic and cytocompatible with epithelial human embryonic kidney 293 (HEK293) cells [68]. PLA incorporated with rGO and TEMPO-oxidized CNCs, grafted with poly(ethylene glycol) (PEG), displayed radical scavenging activity and negligible toxicity and cytocompatibility to mouse embryonic C3H10T1/2 cells [69]. A composite film consisting of hydrophilic bacterial cellulose nanofibers and hydrophobic rGO, prepared from GO using a bacterial reduction method, supported the adhesion, viability and proliferation of human bone marrow mesenchymal stem cells in a similar way to standard cell culture polystyrene, and better than pure rGO films [37]. Incorporating GO into electrospun cellulose acetate nanofibrous scaffolds enhanced the adhesion and growth of human umbilical cord mesenchymal stem cells. It also enhanced osteogenic differentiation of these cells, manifested by the activity of alkaline phosphatase, and biomineralization of the scaffolds in a simulated body fluid [59]. Nanofibrous composites of bacterial nanocellulose, a conductive poly(3,4-ethylene dioxythiophene) (PEDOT) polymer and GO, mimicking the native extracellular matrix and allowing electrical stimulation of neural PC12 cells, induced specific orientation and differentiation of these cells [105]. Polyvinyl alcohol/carboxymethyl cellulose (PVA/CMC) scaffolds loaded with rGO nanoparticles, prepared by lyophilization, enhanced the proliferation of EA.hy926 endothelial cells in vitro and angiogenesis in vivo using a chick chorioallantoic membrane model [107]. Polyacrylamide-sodium carboxymethylcellulose hybrid hydrogels reinforced with GO and/or CNCs also have potential for tissue engineering applications due to their tunable mechanical properties [135]. Genetically modified hydrophobin, a fungal cysteine-rich protein, was used to connect nanofibrillated cellulose of wood origin and graphene flakes in order to construct biomimetic mechanically-resistant materials similar to nacre and combining high toughness, strength and stiffness [136].

Nanocellulose/graphene composites also have great potential for the fabrication of antibacterial textiles and for advanced wound dressing. Antibacterial textiles were prepared by electrospinning a mixture containing cellulose acetate, TiO2 and GO sheets. These textiles showed high antibacterial activity with an inhibition rate higher than 95% against *Bacillus subtilis* and *Bacillus cereus*[137]. Bacterial cellulose is considered as one of the most suitable materials for advanced wound dressing, due to its appropriate mechanical properties, such as strength, Young's modulus, elasticity and conformability, and also due to its great capacity to retain moisture in the wound (for a review, see [2]). These favorable properties can be further enhanced by adding graphene-based materials and by crosslinking with synthetic polymers, such as poly (ethylene glycol), poly (vinyl alcohol), poly (acrylic acid) and poly (acrylamide). In a study by Chen et al. (2019), a bacterial nanocellulose-grafted poly (acrylic acid)/GO composite hydrogel was prepared as a potential wound dressing. The inclusion of GO improved the attachment and proliferation of human dermal fibroblasts in cultures on the composites [108]. Similarly, hydroxypropyl cellulose matrix incorporated with GO and silver-coated ZnO nanoparticles showed improved tensile strength, and also anti-ultraviolet, antibacterial and immunostimulatory effects, which promoted wound healing in an in vivo mouse model [109].
