Antibacterial Graphene-Based Hydroxyapatite/Chitosan Coating with Gentamicin for Potential Applications in Bone Tissue Engineering

Electrophoretic deposition process (EPD) was successfully used for obtaining graphene (Gr) reinforced composite coating based on hydroxyapatite (HAP), chitosan (CS) and antibiotic gentamicin (Gent), from aqueous suspension. The deposition process was performed as a single step process at a constant voltage (5 V, deposition time 12 min) on pure titanium foils. The influence of graphene was examined through detailed physico-chemical and biological characterization. Fourier transform infrared spectroscopy, field emission scanning electron microscopy, thermogravimetric analysis, Xray diffraction, Raman and X-ray photoelectron analyses confirmed the formation of composite HAP/CS/Gr and HAP/CS/Gr/Gent coatings on Ti. Obtained coatings had porous, uniform, fracturefree surfaces, suggesting strong interfacial interaction between HAP, CS and Gr. Large specific area of graphene enabled strong bonding with chitosan, acting as nanofiller throughout the polymer matrix. Gentamicin addition strongly improved the antibacterial activity of HAP/CS/Gr/Gent coating that was confirmed by antibacterial activity kinetics in suspension and agar diffusion testing, while results indicated more pronounced antibacterial effect against Staphylococcus aureus (bactericidal, viable cells number reduction >3 logarithmic units) compared to Escherichia coli (bacteriostatic, <3 logarithmic units). MTT assay indicated low cytotoxicity (75 % cell viability) against MRC-5 and L929 (70 % cell viability) tested cell lines, indicating good biocompatibility of HAP/CS/Gr/Gent coating. Therefore, electrodeposited HAP/CS/Gr/Gent coating on Ti can be considered as a prospective material for bone tissue engineering as a hard tissue implant.


Introduction
Titanium and its alloys are preferred biomaterials for hip, knee and joint replacements. Pure Ti is considered a reliable implant material that is inert in the body environment with a long-lasting feature. 1,2 Although it possesses a lot of qualities, pure Ti cannot sufficiently induce osseointegration process causing ineffective bone ingrowths. However, the biocompatibility and bioactivity of Ti can be significantly improved by modifying its surface by various processes. For developing biocompatible composite films on the Ti surface, electrophoretic deposition process (EPD) is considered a favorable technique that allows formation of diverse composite materials. 3,4 An interesting choice for bone tissue regeneration coating material is hydroxyapatite (HAP), due to its biocompatibility and similarity with bone tissue. 5 HAP is a bioactive ceramic material, characterized by high biocompatibility and ability to form a direct chemical bond with bone tissue. 6 However, due to its brittleness and poor adhesion properties it is necessary to combine HAP with an adequate polymer to serve as a binder. The prospective solution represents the combination of biopolymer matrix and bioceramics, e.g. hydroxyapatite-based composite coatings with natural or synthetic polymers. 7 The most commonly used natural polymers are collagen, 8 gelatin, 9 alginate 10 and chitosan. 9,11,12 Synthetic polymers that are most often used in bone tissue engineering are poly(lactideco-glycolide) (PLGA), 13 polycaprolactone (PCL), 14 poly(vinyl alcohol) (PVA), 11 poly(glycolic acid) (PGA), 15 poly(lactic acid) (PLA). 16,17 Chitosan (CS) is intensively investigated in the field of tissue engineering due to its unique properties, such as biodegradability, biocompatibility, adhesiveness and intrinsic antibacterial properties. 18,19 It is very important to point out that chitosan can mimic the structure of glycosaminoglycans in the bone extracellular matrix, contributing to the sponginess of the developed bone. 20,21 Hydrophilic surface of CS improves cell adhesion, differentiation and proliferation, minimizing, at the same time, the body response upon implantation. 22 In combination with HAP, chitosan contributes to improved mechanical, adhesion and antibacterial coating properties, which is of particular importance for further application. 23 Also, the possibility of applying bone tissue composite coatings as a carrier of anti-infection drugs at the implantation site, as well as growth factors that stimulate the osteoblasts activity is the subject of investigation. 24 Considering HAP brittleness, as well as low fracture toughness and poor tensile strength, graphene (Gr) inclusion in composite structures could improve mechanical, thermal and electrical properties of the composites, while good biocompatibility makes it a promising biomaterial. 25,26 When used as reinforcement filler in composites with HAP and polymers, Gr could induce increased bone formation ability, increased specific area, smaller grain size and could elevate the negative surface charge attracting more calcium and forming bone-like layer, 27 as well as good cytocompatibility. 28 Nowadays, there is a growing need for development of antibacterial drug-eluting coatings which will prevent biofilm occurrence and treat implant-associated infections. 29 This type of coating should ensure the long-term drug action through controlled release of the drug at the implantation site overcoming the problems associated with bone infection occurrence. Locally administrated antibiotic has less susceptibility to promote antibiotic resistance achieving at the same time long-term antibiotic release with lower applied antibiotic doses. 30 Antibiotic-loaded composites obtained by EPD technique have gained much attention in recent studies when vancomycin, 31 tobramycin, 32 ciprofloxacin, 4 ampicillin, 33 tetracycline, 34 levofloxacin, 35 ibuprofen 34 and gentamicin 18 have been successfully included in composites aimed for controlled drug delivery. Depending on the nature of the infection or the inflammatory process, different types of antibiotics can be selected for treatment.
One of the antibiotics that are often used to treat post-surgery osteomyelitis is gentamicin.
Gentamicin, a water-soluble aminoglycoside antibiotic, is known to have very potent antibacterial activity for the treatment of wide range of infections, caused by both Gram-negative and Grampositive bacteria. 18,36,37 The principal objective of the research was to obtain improved Gr-reinforced antibacterial composite HAP/CS coatings, with and without gentamicin, on Ti substrate using electrophoretic deposition technique from aqueous suspension since antibiotic-loaded coatings are intended for medical use. To our knowledge, based on previously published literature, this is the first time that the single step deposition from four-component HAP/CS/Gr/Gent aqueous suspension, with no additional treatment, was performed.

Materials
The following materials were all purchased from Sigma-Aldrich and used to assemble composite coatings on Ti surface: powders of HAP (particles < 200 nm), chitosan (medium molecular weight Merck, Germany) were used for gentamicin derivatization procedure. The mobile phase consisted of sodium octanesulfonate and glacial acetic acid (Merck, Germany).

Electrophoretic deposition
Aqueous suspensions containing 1 wt % HAP nanopowder, 0.05 wt % chitosan, 0.01 wt % graphene and 0.1 wt % gentamicin sulfate were used for EPD process in order to obtain HAP/CS/Gr and HAP/CS/Gr/Gent composite coatings, according to our previously published procedure. 18 Suspensions pH value was adjusted to 4.4. Cataphoretic deposition was performed on pure Ti plate, serving as a working electrode (cathode), at constant voltage of 5 V for deposition time of 12 min. Two parallel platinum plates were used as counter electrodes (anodes). After deposition was performed, coatings were dried at room temperature for 24 h, while the thicknesses were 3.0±1.5 μm and 3.2±0.9 μm for HAP/CS/Gr and for HAP/CS/Gr/Gent coating, respectively.

Characterization
The instrument LEO SUPRA 55 (Carl Zeiss AG, Germany) operating at 10 kV voltage acceleration was used for field-emission scanning electron microscopy (FE-SEM) equipped with In-Lens detector and combined SE-BSE mode was used. Fourier transform infrared spectroscopic studies (FTIR) were carried out on a machine Nicolet IS-50 (Thermo Fisher Scientific, USA) in ATR mode in the range of 400−4000 cm −1 (4 cm −1 spectral resolution). X-ray photoelectron spectroscopy (XPS) spectra were recorded using a K Alpha System photoelectron spectroscope (Thermo Electron, USA), with monochromatic Al Kα X-ray (1486.6 eV) excitation source. The adventitious carbon peak maximum in the C 1s spectra was set as 284.8 eV. OriginPro 9 software was used for peak fitting, with previous Shirley-type background correction. Raman analysis was carried out by a Renishaw Invia (Renishaw plc, UK) Raman spectrophotometer (514-nm argon laser) with 10 % intensity of the total power and in the spectral range from 3500 to 100 cm −1 . Thermogravimetric analysis (TGA) was performed by TGA Q5000 IR/SDT Q600 instrument (TA Instruments, USA), operating in 30-1000 °C range at a heating rate of 20 °C/min in inert atmosphere (N2, 50 mL/min). X-ray diffraction analysis (XRD) was performed by powder diffractometer Philips PW 1710 (Philips, Netherland) with Ni-filtered Cu Kα radiation (λ = 1.5418 Å). Diffraction intensity was recorded at room temperature, between 10−70°, 0.05° step. For phase analysis, PowderCell software was used. Gentamicin content was estimated by HPLC chromatography using Dionex UltiMate 3000 HPLC system (Thermo Fisher Scientific, USA) with Chromolith RP-18 column (Merck, Germany). Gentamicin in the solution was derivatized prior to HPLC (ultraviolet (UV) detector) loading, as described in our previously published paper. 18 Main gentamicin components were detected by UV detector at 330 nm.

Results
In general, the main advantages of EPD technique are: (a) the ability to produce coatings on metal surfaces of complex shapes, (b) the cleanliness of the process without the use of potentially toxic chemicals, thus producing biocompatible materials that are safe for medical use, and (c) coating fabrication at room temperature, which is of particular importance for drug and biologically active molecules processing. Since the gentamicin is water-soluble, deposition of HAP/CS/Gr/Gent coating on titanium surface was performed from aqueous solution in a single step, i.e co-precipitation from a four-component aqueous suspension has occurred. This is a remarkable advantage over the data available so far in the literature relating to bioactive coatings deposited in several steps. [39][40][41] Due to the electrolysis of water, hydrogen and oxygen evolution occurs on the cathode and anode, respectively (Eqs. 1 and 3). anode: Chitosan is soluble in aqueous solutions, at lower pH values (slightly acidified environment), due to the protonation of amine groups, e.g. CS-NH3 + . 3

FTIR analysis
The characteristic FTIR spectral bands for HAP/CS/Gr and HAP/CS/Gr/Gent coatings in the region from 400 to 4000 cm -1 are represented in Figs Spectral carbonate bands for HAP/CS/Gr composite coating were observed in the regions from 800 to 900 cm -1 and from 1350 to 1600 cm -1 (Fig. 1a). 46 Carbonate ions can substitute hydroxyl and/or phosphate groups, leading to formation of carbonate substituted HAP. When the replacement of OHgroups by CO3 2groups occurs, the change in HAP structure is known as A-type substituted HAP.
When PO4 3groups are substituted by CO3 2groups, then such substitution is denoted as B-type hydroxyapatite. 46 In FTIR spectra for HAP/CS/Gr composite coating (Fig. 1a), observed carbonate band at 878 cm -1 represents the vibration mode of O-C-O group. 18,46 With the aim to determine the type of carbonate substitution in HAP, deconvolution of carbonate peak in the region 800-900 cm -1 ( Fig. 1c) and 1350-1600 cm -1 (Fig. 1d) was performed. The deconvolution of carbonate band at 878 cm -1 revealed three different peaks (Fig. 1c). A peak at 879 cm -1 can be assigned to the A-type carbonate substitution, peak at 872 cm -1 suggested the presence of B-type substitution, while the peak at 865 cm -1 represented the non-apatitic carbonate. 47 The deconvolution of FTIR peaks in the region from 1350 to 1600 cm -1 (Fig. 1d) confirmed the presence of A-and B-type of substitution in HAP, as well. Bands at 1421 and 1470 cm -1 , as well as a doublet at 1407 and 1441 cm -1 , can be assigned to Btype, 48 while the presence of a band at 1528 cm -1 , 46 suggested the A-type substitution in HAP (Fig.1d).
A band at 1456 cm -1 can be assigned to the both A and B-type of carbonate substituted HAP. 47 Based on the positions of all carbonate bands, it could be concluded that "AB-type" substitution occurred in HAP/CS/Gr. The carbonate substitution in HAP structure is advantageous regarding bioactivity and similarity to the natural bone. 47 Two distinctive bands at 1654 cm -1 (amide I band) and 1546 cm -1 (amide II band) for HAP/CS/Gr coating (Fig. 1e) were assigned to the C=O stretching vibration of -NHCO-group and the N-H bending in -NH2 group of CS, respectively. 18,49 The chitosan presence in HAP/CS/Gr coating was also verified by bands at 2857 cm -1 and 2926 cm -1 (Fig. 1a), originating from C-H stretching in the CS structure. 49,50 At 1388 cm -1 (Fig. 1d ) the band for CH3 symmetrical deformation in chitosan structure can be distinguish. 51 The bands at 473, 563, 600, 952, 1020 and 1085 cm -1 undoubtedly confirmed the hydroxyapatite presence in the HAP/CS/Gr composite coating.
All these bands were ascribed to the different vibration modes of PO4 3group. 18 Band at 1560 cm -1 (Fig. 1e) can be assigned to the skeletal vibration of Gr, confirming the successful incorporation of graphene in HAP/CS/Gr composite coating. 43,52 Inset in Figure 1a highlights the region from 3000 to 3600 cm −1 , where prominent peak at 3570 cm -1 was observed, corresponding to -OH stretching from HAP structure. 53 A wide band at around 3279 cm -1 (for HAP/CS/Gr) can be attributed to the valence vibrations of hydroxyl groups, sensitive to hydrogen bonding. 49 Therefore, it can be assumed that hydrogen bonding between hydroxyl groups of HAP and hydroxyl and amino groups of CS occurred. In Fig. 1b FTIR spectrum for HAP/CS/Gr/Gent coating is represented.
Generally, characteristic bands, corresponding to HAP, chitosan and graphene can be observed for HAP/CS/Gr/Gent coatings, as it was reported for HAP/CS/Gr coating (Fig. 1a). After gentamicin incorporation in HAP/CS/Gr/Gent coating, a new band at 1640 cm -1 appeared (Fig. 1f). This band can be assigned to the N-H bending vibration of primary aromatic amines, 54 Fig. 1b)) was noticed after gentamicin introduction. These bands were attributed to the valence vibrations of hydroxyl groups, sensitive to hydrogen bonding. 49 Therefore it can be assumed that hydrogen bonding between hydroxyl groups of HAP and amino and hydroxyl groups of CS with newly introduced gentamicin hydroxyl and amino groups occurred.

XRD analysis
XRD analyses of HAP/CS/Gr and HAP/CS/Gr/Gent coatings (Figs. 2a and b,  influence the formation of a large number of nucleation sites. At the same time, as gentamicin is a bulky molecule, there is also a possibility that it prevents further crystal growth. Both of these effects lead to finer crystallite structure and smaller size in the case of HAP/CS/Gr/Gent, which contributes to better osseointegration due to bone-like apatite formation. 56,57 Namely, owing to their large surface-tovolume ratio HAP nanoparticles are considered to improve the in vitro bone-like apatite formation, by increasing the specific area of biomaterial. 58,59 Moreover, it was shown that a larger contact area enhanced the adhesive protein adsorption and thus contributed to the interactions between cells and biomaterial surfaces. 4,60 The higher amount of adsorbed proteins was in correlation with a larger specific area, justifying the statement of improved bioactivity when smaller particles are used. 61

XPS analysis
The deconvoluted high resolution XPS spectra of C 1s, O 1s, and N 1s of HAP/CS/Gr and HAP/CS/Gr/Gent coatings are depicted in Fig. 3. C 1s peak was fitted into five different modes interactions (free amines), 66 while peaks at 401.7 eV for both spectra could be assigned to the C-NH3 + interactions (protonated amines). 18,64 Since gentamicin sulfate solution was used, XPS spectrum of HAP/CS/Gr/Gent composite displayed the S 2p peak, clearly confirming the gentamicin incorporation. S 2p peak (data not shown) was fitted with peak component at 170 eV. 18 Elemental composition, calculated from XPS (Table 3)  along with gentamicin decomposition took place. 73 Moreover, DTG analysis pointed to the prominent peak at 294 °C (ascribed as CS degradation) for HAP/CS/Gent coating (Fig 6a) which was shifted to 305 °C for HAP/CS/Gr/Gent coating (Fig 6b). Bearing in mind that HAP in both coatings is carbonate

Gentamicin content
The overall antibiotic content (measured in triplicate) was determined using the HPLC-UV method after the derivatization procedure. The three most dominant gentamicin components are C1, C1a, and C2 with minor differences in their structure and similar antibiotic activity 80 for which the obtained retention times were approximately 3.3, 6.4, and 9.8 min. The average amount of gentamicin per 1 cm 2 of HAP/CS/Gr/Gent coating was 8.2 ± 0.1 μg. Compared to previously published results 18 for HAP/CS/Gent coating of 7.3 ± 0.1 μg per cm 2 , this increase of more than 10 wt % is substantial and clearly the result of graphene presence in the coating, not surprisingly as Gr represents a powerful nano-platform for antibiotic gentamicin loading. 79

Antibacterial activity and cytotoxicity evaluation
Gentamicin, like most aminoglycosides, irreversibly binds to specific subunit proteins (30S) and 16S rRNA in the bacterial cell. This binding causes t-RNA misreading that prevents the synthesis of vital proteins. More precisely, gentamicin binds to four 16S rRNA nucleotides and a protein S12 amino acid, causing interference in the decoding site close to the nucleotide 1400 in 16S rRNA of 30S subunit. This region interacts with the wobble base in the anticodon of tRNA, leading to initiation complex interference, polysomes breakup into nonfunctional monosomes, as well as a misreading of mRNA that causes incorrect amino acids to be inserted into the polypeptide. [81][82][83] The antibacterial efficacy of HAP/CS/Gr/Gent (1 mg/mL gentamicin) and HAP/CS/Gr coatings was investigated using the agar diffusion method and the results are presented in Fig. 7. Pure gentamicin solution in the presence of S. aureus TL and E. coli ATCC 25922 was used as a control (D =10 mm), causing clear, wide inhibition zone (31 mm in diameter, Fig. 7a) in the S. aureus TL presence.
However, E. coli ATCC 25922 responded with wide inhibition zone consisting of brighter (D=24 mm) and darker (D=30 mm) inhibition regions indicating maximum and moderate bacterial sensitivity, respectively (Fig. 7b). Neither of the bacteria was sensitive to HAP/CS/Gr coating as indicated by inhibition zone absence ( Fig. 7a and b, sample 1). Contrary to HAP/CS/Gr, HAP/CS/Gr/Gent coating exhibited wide inhibition zone (36 × 27 mm) (Fig. 7a, sample 2) against S. aureus TL and 2 sensitivity zones against E. coli (Fig. 7b,         Statistical evaluation for samples done in triplicate was performed using one-way ANOVA, with a multiple comparisons posthoc analysis (* p < 0.01 for the respective bacteria strain).  Accepted Article