Cellulose-Derived Nanographene Oxide Surface-Functionalized

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Cellulose Derived Nano-Graphene Oxide Surface Functionalized 3D Scaffolds with Drug Delivery Capability Nejla B Erdal, Jenevieve G. Yao, and Minna Hakkarainen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01421 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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Cellulose Derived Nano-Graphene Oxide Surface Functionalized 3D Scaffolds with Drug Delivery Capability

Nejla B. Erdal, Jenevieve G. Yao, and Minna Hakkarainen*

Department of Fibre and Polymer Technology, KTH Royal Institute of Technology, 100 44 Stockholm, Sweden *Corresponding author. Email: [email protected]

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Abstract Multifunctional 3D scaffolds were targeted by surface grafting cellulose derived nano-graphene oxide (nGO) on the surface of porous poly(ε-caprolactone) (PCL) scaffolds. nGO was derived from cellulose by microwave-assisted carbonization process and covalently grafted onto aminolyzed PCL scaffolds through an aqueous solution process. Fourier transform infrared spectroscopy and thermogravimetric analysis both verified the successful attachment of nGO and scanning electron microscopy depicted a homogeneous dispersion of nGO over the scaffold surface. Mechanical tests were performed and demonstrated a significant increase in compressive strength for the nGO grafted scaffolds. Grafting of nGO was also shown to induce mineralization with the formation of calcium phosphate precipitates on the surface of the scaffolds with the size increasing with higher nGO content. The potential of surface-grafted nGO as a nano-carrier of an antibiotic drug was also explored. The secondary interactions between nGO and ciprofloxacin, a broad-spectrum antibiotic used in the treatment of osteomyelitis, were optimized by controlling the solution pH. Ciprofloxacin was found to be adsorbed most strongly in its cationic form at pH 5, in which π-π electron-donor acceptor interactions predominate and the adsorbed drug content increased with increasing nGO amount. Further, the release kinetics of the drug were investigated during eight days. In conclusion, the proposed simple fabrication process led to a scaffold with multifunctionality in the form of improved mechanical strength, ability to induce mineralization as well as drug loading and delivery capability.

Keywords: Poly(ε-caprolactone) (PCL), aminolysis, graphene oxide, bioactive, scaffold, ciprofloxacin


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INTRODUCTION As an alternative solution to the conventional therapies of transplantation and implantation of assist devices in case of organ or tissue failure, the aim in tissue engineering is to reconstruct and regenerate damaged tissue and organs by mimicking the physiological features and cellular behavior of the functional tissues in vivo. For these purposes, the selection of a scaffold material that is biocompatible, biodegradable, porous, and exhibits the appropriate surface chemistry and mechanical properties required for its purpose, among other things, is crucial for the scaffold to successfully simulate functional living tissue.1 In terms of polymer-based tissue scaffolds, both natural and synthetic polymers have their own advantages and disadvantages. PCL is a Food and Drug Administration (FDA)-approved biomaterial that has a melting point of 60oC and a glass transition temperature of -60oC, leading to easily tailored properties.1,2 In its pristine state, however, PCL possesses insufficient mechanical strength, slow in vivo degradation rate, weak cellular response, and lack of bioactivity, mostly due to its hydrophobic nature.2,3 One way to enhance the biological performance of synthetic polymers is through the modification of the polymeric backbone or surface grafting to introduce functional groups, and thus creating a biomimetic interface that is more compatible to living cells. The modification of polymeric surfaces can be done by either physical or chemical methods. Some examples of physical methods that have been employed are ion irradiation and adsorption of small peptide sequences on polymer surfaces.4,5 The drawbacks with physical methods, however, include the low level of functional groups that could be attached on the surface and the temporary nature of the adsorbed groups, thus risking exchange or removal during in vitro culture or upon implantation.4 On the other hand, chemical methods of surface modification, which include hydrolysis, aminolysis, plasma treatment, and copolymerization, have been shown to result to the successful



immobilization of bioactive compounds and functional molecules on the polymer surface.4,10 Various studies have been done to illustrate the feasibility and effectiveness of aminolysis to modify the surface of PCL to promote cytocompatibility and functionality as a biomaterial.4-6 Some advantages of aminolysis in the surface treatment of biomaterials are: (1) the minimum influence on the bulk properties of the material, (2) the nontoxicity to cells or tissues, and (3) the ability to neutralize the acid generated during scaffold degradation, thereby reducing inflammation around the implanted scaffold.6,7 In addition to this, the introduction of active sites on PCL surface enables immobilization of biocompatible components such as proteins, polysaccharides, cell growth factors, and peptides.6 The potential of GO in polymer-based nanocomposites for tissue engineering applications has been investigated to improve mechanical properties and to enhance bioactivity of the scaffold material. Jalili-Firoozinezha et al. enhanced the cellular proliferation by coating and subsequent reduction of large GO sheets on a PCL mat.8 In another study, Wu et al showed improved mechanical properties, increased hydrophilicity, good cytocompatibility, and osteo-bioactivity, by developing a poly(lactic acid) (PLA) and poly(ε-caprolactone) (PCL) electrospun nanofibrous scaffold indicating the potential of nGO in bone tissue engineering.3 Novel 0D nGO was previously developed in our group by utilizing cellulose and starch as starting materials.9,10 nGO showed high inductivity for hydroxyapatite (HAP) mineralization in PCL composites and in porous starch scaffolds owing to its highly oxidized nature.3,11 The presence of functional oxygen-containing groups and delocalized π-electrons on its basal plane, along with its nano-sized structure that translates to a high surface area, also makes nGOs highly effective adsorbents for the removal of pharmaceutical contaminants from effluents,12,13 or as nano-carrier for the controlled release and delivery of drugs.14,15 Aside from the incorporation of 4 ACS Paragon Plus Environment

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pharmaceuticals that accelerate osteogenesis to enhance the regeneration of bone to the scaffold material, bone infections still represent a major problem in medicine due to the poor accessibility of the bone-infected site by systemically administered antibiotics. Local antibiotic delivery via scaffolds offers the benefit of enabling the antibiotic to reach the site of action at much higher concentrations, while keeping the systemic antibiotic concentrations at a minimum level. This not only improves the healing process, but also prevents the occurrence of side effects caused by high systemic doses of antibiotics, which are only some advantages over orally administered antibiotic therapy.16 The presence of short-term local antibiotics is particularly useful to avoid contamination and to reduce post-surgical septic complications after scaffold replacement.17-19 Ciprofloxacin (CIP) is a broad-spectrum antibiotic that is widely used in osteomyelitis therapy due to its favorable penetration and bactericidal effect on all the probable osteomyelitis pathogens,20 as well as its high ability to penetrate compact bone structures.21 Our previous studies showed that incorporation of biobased nGO with plethora of oxygen functionalities

in

bioresorbable

scaffolds

enhanced

bioactivity

in

the

form

of

biomineralization,3,11,22 while addition of nGO in chitosan hydrogels improved the drug adsorption efficiency of the hydrogels.13 Therefore we hypothesized that grafting cellulose derived nGO on the surface of the scaffolds could even more effectively induce biomineralization and provide adsorption sides for drug loading to introduce a scaffold with drug delivery capability. In addition, this modification could potentially improve the mechanical properties of the scaffolds. The aim was thus to design a 3D porous PCL-based bone tissue scaffolds with multiple functionalities from improved mechanical properties, to enhanced biomineralization, and drug delivery.



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EXPERIMENTAL Materials. Poly(ε-caprolactone) (PCL) (C6H10O2)n (MW 80 000 g/mol), polyethylene glycol (PEG) (H(OCH2CH2)nOH) (MW 1000 g/mol), hexamethylenediamine (1,6-Hexanediamine, NH2(CH2)6NH2, 98%), ninhydrin (2,2-Dihydroxy-1,3-indanedione, C9H6O4, amino acid detection grade) used for the ninhydrin assay of the scaffolds, α-cellulose, nitric acid (HNO3, 70%) Nhydroxysuccinimide

(NHS,

98%)

(1-Hydroxy-2,5-pyrrolidinedione,

C4H5NO3),

sodium

hydroxide (NaOH), and ciprofloxacin (1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1yl)-1,4dihydroquinoline-3-carboxylic acid, C17H18FN3O3, ≥ 98%) were all obtained from Sigma-Aldrich Chemie GmbH. Isopropanol ((CH3)2CHOH) and ethanol (EtOH, 96%) were obtained from VWR Chemicals. Ninhydrin (2,2-Dihydroxy-1,3-indanedione, C9H6O4, ~99%) used for the ninhydrin assay of the films and EDC (N-(3-Dimethylaminopropyl)-N’ethylcarbodiimide hydrochloride, C8H18ClN3, ≥ 98%) were obtained from Fluka Analytical. Chloroform (CHCl3) and sodium chloride (NaCl, laboratory reagent grade) were obtained from Fisher Scientific. All chemicals were used as received.

Methods Preparation of Scaffolds. Conventional solvent-casting/particulate leaching (SCPL) scaffolds were prepared by dissolving 0.7 g of PCL in 5 mL of chloroform under stirring for 1 h at room temperature. NaCl (300-400 microns) was then added to the polymer solution at a ratio of 30:70 – PCL:NaCl (wt/wt), which is equivalent to 1.63 g. Once the NaCl particles had been dispersed in the solution, the solvent was left to evaporate under the fume hood for 24 h. The resulting composites were then removed from the molds and immersed in deionized water for particulate leaching for 2 days under constant stirring. After 48 h, the scaffolds were collected and dried 6 ACS Paragon Plus Environment

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under vacuum. The same procedure was repeated for the scaffolds using the SCPL with centrifugation technique, except that the polymer mixtures with added NaCl were first centrifuged at 2500 rpm for 10 min prior to solvent casting. For the combined SCPL and polymer leaching method, on the other hand, 0.7 g of PEG was dissolved along with PCL (1:1 ratio) in the solvent. NaCl was then added to the polymer solution at a ratio of 30:70 – PCL:NaCl (wt/wt). The particulate and polymer leaching in deionized water took place for 2 days under constant stirring according to previously reported procedure.23 The scaffolds were then dried in vacuum. The resulting scaffolds had diameters of 2 cm, and thicknesses of 0.3-0.4 cm. Aminolysis of PCL. Aminolysis of PCL films (1.5 x 1.5 cm2), which were produced via solvent casting in chloroform solution, and scaffolds (2 cm in diameters and thicknesses of 0.3-0.4 cm) were carried out in a 10 % (w:w) 1,6-hexanediamine/isopropanol solvent system. The films or scaffolds was then immersed in this solution for 30 min at 37 °C under constant stirring. After the reaction, the films/scaffolds were collected and rinsed with deionized water with constant stirring for 24 h at room temperature. The films/scaffolds were then dried in the vacuum oven for 48 h. Synthesis of nano-graphene oxide (nGO) from cellulose. nGOs were produced from cellulosederived carbon nanospheres (CNs) fabricated by microwave-assisted process as described in previous work.9,

22, 24

Briefly, CNs were synthesized by carbonizing α-cellulose in dilute (0.1

g/mL) sulfuric acid (H2SO4) by subjecting it to microwave-assisted hydrothermal process in a flexiWAVE microwave device (Milestone Inc.) for 2 h at 220oC, using a ramp time of 20 min. The obtained CNs were then sonicated in 70 % nitric acid (HNO3) solution (1:100 w/w) using a sonication bath during 30 min and oxidized for 30 min at 90°C under constant stirring. The reaction was stopped by pouring the solution into cold deionized water, and the red-orange solids were collected by evaporating the solvent via rotary evaporation. The product was then freeze7 ACS Paragon Plus Environment


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dried and stored in vacuum at room temperature for at least 2 days for the removal of any residues of water or acid. Grafting of Scaffolds with nGO. Using the solvent-assisted method, nGO was dispersed in deionized water at the concentrations 0.5 mg/ml and 1.0 mg/ml and sonicated for 20 min in a sonication bath. Dry scaffolds were weighed before grafting. Then, the aminolyzed scaffolds (2 cm diameter, 0.3 – 0.4 cm thick) were added to the solution (50 mL in total volume). The grafting reaction was then carried out with constant stirring for 24 h at room temperature. At the end of the reaction, the scaffolds were collected and rinsed with deionized water in a sonication bath for 10 min to remove the non-covalently attached nGO and dried in the vacuum oven at room temperature for 48 h and then weighed again. The scaffolds were named Neat for neat PCL, Aminolyzed for aminolyzed PCL, nGO0.5 for the scaffold grafted using 0.5 mg/mL initial nGO concentration and nGO1 for scaffolds grafted using 1.0 mg/mL initial nGO concentration. Scaffold Porosity. Scaffold porosities were measured by immersing scaffold samples of known dry masses in ethanol for 12 h. The samples were then collected and their saturated weights were recorded. Eq. 1 was then used to derive the scaffold porosity or void volume fraction (Vf), where m2 is the saturated weight, m1 the dry weight, d1 the density of PCL (1.145 g/mL), and d2 the density of ethanol (0.82 g/mL). 𝑉𝑓(%) =

(𝑚2 ― 𝑚1)𝑑1

(𝑚2 ― 𝑚1)𝑑1 + 𝑚1𝑑2 ∙ 100

(1)

Ninhydrin Assay. Ninhydrin assay was performed for the determination of amine density in the aminolyzed samples. Ninhydrin solutions (0.5 M) were prepared by dissolving 4.4535 g of ninhydrin in 50 mL ethanol. The aminolyzed films/scaffolds were placed in vials and immersed in 2 mL of said ninhydrin solution and left to react for approximately 1 min. The vials were then 8 ACS Paragon Plus Environment

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transferred to a water bath and heated at 80oC for 15 min. Afterwards, the samples were dissolved with the addition of 2 mL of chloroform. 2 mL of isopropanol was also added to stabilize the purple colored compound. Calibration curves which had correlation coefficients (R2) ranging from 0.953 – 0.972, were created using known concentrations of 1,6-hexanediamine in ethanol. For the calibration curve, 100 µL of each standard solution was placed in a vial, and the same procedure as mentioned was followed. The amine densities were measured from the absorbances of the mixtures at 570 nm, using a Shimadzu UV-2550 UV-vis instrument. Nitrogen Analysis. Nitrogen analysis was performed on the neat and aminolyzed PCL films to qualitatively compare the amine density after different aminolysis times. The characterization was performed using an Antek MultiTek (PAK). Measurements were taken on samples weighing 1 – 2 mg, and each measurement was repeated four times. Adsorption of Ciprofloxacin. Sorption experiments were performed by immersing nGO1 scaffold samples (that were cut in pieces with the dimensions 0.5 x 0.5 cm2, 8-9 mg) in 5 mL of aqueous ciprofloxacin solutions with concentrations of 0.5 mg/mL. Two solution with different pH were used – pH 5 and pH 6.5. For the pH 5 solution, pH was adjusted by the drop-wise addition of 1 M HCl. The pH 6.5 solution was used as it was, with no adjustments being made. The adsorption of ciprofloxacin was allowed to take place for 24 h under constant stirring at room temperature. The experiment was performed on three samples for each pH value. The supernatant was then collected and the remaining ciprofloxacin concentration was determined via UV-vis detection at the maximum absorbance wavelength (270 nm). The scaffolds were also weighed before and after the adsorption to measure the difference in weight. The UV absorbances were measured using a Shimadzu UV-2550 UV-vis instrument. For the drug-loading of the nGOgrafted scaffolds (aminolyzed, nGO0.5 and nGO1) with ciprofloxacin, aqueous solutions with 9 ACS Paragon Plus Environment


ciprofloxacin concentration of 0.5 mg/mL were prepared. The pH of the solutions was adjusted to pH 5 through the drop-wise addition of 1 M HCl. The scaffolds were then added to the solution (10 mL total volume per scaffold), and sonicated for 20 min. The adsorption process was allowed to take place under constant stirring for 24 h at room temperature. The ciprofloxacin adsorbed scaffolds were named nGO0.5-CIP and nGO1-CIP with the initial nGO grafting concentrations of 0.5 mg/mL and 1.0 mg/mL, respectively. Non-grafted, aminolyzed scaffolds were used as control. The generated calibration curve had a correlation coefficient (R2) of 0.981. Fourier Transform Infrared Spectroscopy (FTIR). FTIR was used to elucidate the structural composition and to determine the functional groups on the surface of PCL films and scaffolds. The FTIR spectra of neat, aminolyzed, nGO-grafted, and ciprofloxacin-loaded PCL films and scaffolds were recorded by a PerkinElmer Spectrum 2000 FTIR spectrometer equipped with attenuated total reflectance (ATR) accessory (golden gate) from Graseby Specac. All spectra were recorded over the wavenumber range of 4000 – 600 cm-1 using 16 scans at a resolution of 4 cm-1. Thermogravimetric Analysis (TGA). TGA was used to estimate the nGO grafting efficiency, as well as to qualitatively compare the amounts of adsorbed ciprofloxacin on the scaffolds with different nGO contents. A Mettler-Toledo TGA/SDTA 851e instrument was used to obtain the thermograms. Samples weighing between 3.5 – 5 mg were heated under nitrogen flow from 20 – 800oC at a flow rate of 10 oC /min. All measurements were performed in alumina cups and in triplicates. Scanning Electron Microscopy (SEM). SEM was used to examine the structural microarchitecture of the scaffolds fabricated through the different preparation techniques, as well as to investigate the morphological changes that have occurred on neat PCL after aminolysis, 10 ACS Paragon Plus Environment

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nGO grafting, and drug loading. The SEM images were taken using an ultra-high resolution field emission (FE)-SEM Hitachi S-4800 (Hitachi, Tokyo, Japan). The samples were sputter coated with a platinum/palladium (Pt/Pd) coater at 2 nm thickness prior to analysis. The sputter coater used was a Cressington 208 HR Sputter Coater (Cressington Scientific Instruments, Ltd.). Energy Dispersive X-ray Spectroscopy (EDS). Elemental analysis of the surfaces was performed on the non-CIP-loaded (e.g., nGO-grafted) and CIP loaded scaffolds to determine the presence of fluorine and nitrogen atoms, as contributed by the drug compound. Elemental analysis was done using a Hitachi S-4800 SEM equipped with an Oxford Instruments X-MaxN 80 EDS. Mechanical Testing. The mechanical properties of the neat, aminolyzed, nGO-grafted, and ciprofloxacin-loaded scaffolds were determined by measuring their elastic modulus and compressive strengths. The cylindrical scaffolds were tested in vertical compression with an Instron 5566 Universal Testing Machine, using a cross-head speed of 0.5 mm/min and a load cell of 500 N. A compression rate of 10 % thickness per min was used, until 80 % deformation of the scaffolds was achieved. For each sample, three parallel tests were carried out after conditioning at 23 oC and 50 % humidity for 24 h. The test scaffolds had a diameter of 19.35 mm and thicknesses between 3 – 4 mm. The compressive modulus was then determined from the slope of the initial linear portion of the stress−strain curve. The compressive strength was calculated by dividing the maximum load applied to the specimen during the test by the cross sectional area. Drug Release Study. To determine the in vitro release behavior of the ciprofloxacin-loaded scaffolds, release experiments were conducted by immersing drug-loaded scaffold samples of known weights (8 – 9 mg; 0.5 x 0.5 cm2) in SBF solution (pH 7.4; composition described in next section) for 12 days. The test vials were placed in an Orbital shaker incubator (Gallenkamp) set to 11 ACS Paragon Plus Environment


50 oscillations per minute and with a temperature maintained at 37 °C. At predetermined time intervals, the release medium was collected and replaced with fresh SBF. The experiment was performed in triplicates. The amount of drug released was quantified via UV-vis detection at the maximum absorbance wavelength (270 nm). The UV absorbances were measured using a Shimadzu UV-2550 UV-vis instrument. The generated calibration curve had a correlation coefficient (R2) of 0.993. Biomineralization Test. Simulated body fluid (SBF), which mimics the ion concentrations in human blood plasma, was prepared according to the conventional SBF (pH 7.4), which is composed of NaCl (7.996 g/L), NaHCO3 (0.350 g/L), KCl (0.224 g/L), K2HPO4 (0.228 g/L), MgCl2 (0.305 g/L), HCl (1 mol/L, 40 mL), CaCl2 (0.278 g/L), Na2SO4 (0.071 g/L), and tris(hydroxymethyl)aminomethane (Tris, 6.057 g/L). The concentrations of the ions in SBF were thus Na+ 142, K+ 5.0, Mg2+ 1.5, Ca2+ 2.5, Cl- 147.8, HCO3- 4.2, HPO42- 1.0, and SO42- 0.5 (mmol/L). Samples of neat, aminolyzed, nGO-grafted, and ciprofloxacin-loaded scaffolds (0.5 x 0.5 cm2, 8 – 9 mg) were then immersed in the SBF solution for 14 days at a controlled temperature of 37 oC. The SBF was replaced each day with fresh solution. After the incubation period, the samples were taken out and rinsed in water. Characterizations were performed via SEM imaging and energy dispersive X-ray spectroscopy (EDS), measured with a Hitachi S-4800 SEM equipped with an Oxford Instruments X-MaxN 80 EDS (Oxford Instruments, Oxfordshire, United Kingdom). Prior to characterization, all samples were sputter coated with Pt/Pd using a Cressington 208 HR Sputter Coater (Cressington Scientific Instruments, Ltd.)

RESULTS AND DISCUSSION


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Porous 3D PCL scaffolds were fabricated and optimized using modified SCPL techniques. Cellulose derived nano-graphene oxide (nGO) was covalently grafted to the surface of the scaffolds by first modifying the surface via aminolysis. nGO was incorporated to facilitate mineralization and serve as an anchor site to immobilize ciprofloxacin through secondary interactions. The adsorption and release of ciprofloxacin by the nGO grafted scaffolds were studied along with the mechanical properties and mineralization of the resulting scaffolds. Scaffold optimization. Three different processes were evaluated to prepare optimized PCL scaffolds. The surface morphologies and microstructures of the prepared scaffolds were investigated via SEM, and the images are illustrated in Figure 1. The SEM image of the scaffold constructed through the traditional SCPL technique reveals non-uniform pore morphology and poor pore interconnectivity. Pore walls appear to have collapsed, thus resulting in low porosity of 60.5%. Indeed, one major issue of SCPL, as displayed here, is that the leaching of salt particles leads to poor interconnection and abnormal pore shape.1 The application of centrifugal force prior to solvent casting may remedy this problem, as seen from the slightly larger and more interconnected pores on the scaffold prepared with this technique. The porosity of the scaffold was also significantly improved to 89.7% (Figure 1). These results are congruous with the observation made by the group of Yang et al., who found that centrifugal treatment enables the particles to be well mixed with different particle sizes being well-distributed in the entire matrix.25 This is in contrast with the standard SCPL method, where larger particles were concentrated on the lower layer of the construct, while the upper layer consisted of the smaller particles. The microstructure of the scaffold prepared by using the combination of SCPL and polymer leaching technique exhibited high porosity (84 – 86%) and a highly interconnected pore network. 13 ACS Paragon Plus Environment


Compared to the scaffolds prepared using the previous two techniques, the pores formed through this technique were clearly larger and well defined, while appearing to be evenly distributed throughout the scaffold construct. This enhancement in the scaffold’s pore morphology is ascribed to the differences in polarity between PCL and PEG, which allows for the PEG to be spontaneously dispersed into spherical droplets in the PCL matrix.23 The combined SCPL and polymer-leaching technique thus enables the fabrication of scaffolds possessing large interconnected channels beneficial to cell growth and propagation. The scaffold using SPCL combined with polymer leaching with PEG and salt (30:70 PCL:NaCl) shown in Figure 1, yielded adequate porosity and pore interconnectivity without compromising its mechanical properties and was further optimized by removing the excess polymer/solvent prior to solvent casting.

Figure 1. Schematic illustration of the different optimization techniques (left) and SEM images of the scaffolds prepared using SCPL technique, SCPL with polymer leaching using 30:70 PCL: NaCl and PEG and SCPL with centrifugal technique and their respective porosities (right).


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Surface Modification of PCL via Aminolysis. In order to covalently attach nGO on the surface of the scaffolds, the surface was first functionalized by aminolysis. Prior to the aminolysis of the 3D scaffolds, aminolysis was first performed on 2D PCL films at different reaction durations – 15 min, 30 min, 1 h, 2 h, and 3 h. This was done to determine the optimal aminolyzing time at which the maximum density of amine groups is achieved. Ninhydrin assay was employed for the analytical determination of the density of amine groups grafted on the surface of PCL films and scaffolds. The basis of the ninhydrin assay is the spectrophotometric measurement of the absorbance of the purple reaction product, Ruhemann’s purple (λmax 500 - 580 nm), which is formed by the reaction between amino groups and ninhydrin hydrate.26,27 The amine groups on the surface of the films and scaffolds were thus quantified through the calibration curve obtained from the absorbances of solutions with different concentrations of 1,6-hexanediamine. The aminolysis reaction is displayed in Figure 2a together with the results of the optimization experiments (Figure 2cb). The amine density sharply increased during the first 30 min of the reaction and no significant changes in the number of amino group was observed with a prolonged reaction time. This finding was further supported by the result of the nitrogen analysis carried out through elemental nitrogen analysis, as illustrated in Figure 2c, which shows that maximum amine density was achieved after 30 min of reaction. Previous studies have also found that a dynamic equilibrium exists in the attachment of amino groups to the PCL surface, where prolonging the reaction time does not affect amine density, as chain segment migration and the exchange of surface groups with bulk segments via rotational and/or translational motion may occur given an extended reaction period.6 For the calibration curve used to evaluate amine group density see the SI, Figures S1−S2.



Using the optimized aminolyzing time of 30 min, the achieved amine density on the 3D scaffolds was 0.388 x10-7 mol/cm3 mg-1, which coincided with the quantified amine density on the PCL films (0.377 x10-7 mol/cm3 mg-1) at the same reaction duration. For a data table of raw values see the SI, Table S1. This indicates that the optimized aminolysis parameters could be extended from 2D films to 3D scaffolds without compromising the diamine diffusion and penetration through the scaffold’s pore system. In fact, the much larger surface area of the scaffold may even have resulted in a slightly higher amine density compared to that of the films. Figure 2c shows the FITR spectrum of the neat and aminolyzed PCL scaffold. The peaks at 1110 cm-1 and 1720 cm-1 were assigned to the ester C-O and C=O stretches, respectively. The peaks at 2868 cm-1 and 2948 cm-1 were attributed to the hydroxyl C-H symmetric and asymmetric stretches, respectively. Finally, the low intensity peak from 3500 – 3420 cm-1 was assigned to the carboxylic acid –OH stretch. Although most of the characteristic amine peaks overlap with the peaks from PCL, aminolysis of the scaffolds was confirmed by the small peak at 830 – 800 cm-1, which is attributed to the alkyl C-H bend of the methyl groups in the diamine, as well as the notable decrease in the ester C-O peak at 1110 cm-1 due to the nucleophilic attack of the amine group.


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Figure 2. (a) Schematic representation of the aminolysis reaction on the PCL scaffold surface, (b) average amine density at different aminolyzing times determined via ninhydrin assay, (c) average amine density at different aminolyzing determined via nitrogen analysis and (d) FTIR spectra of neat and aminolyzed PCL scaffolds.

Grafting of nGO on the Surface of PCL scaffolds. nGO was grafted on the aminolyzed scaffold surfaces using solvent assisted method with water as a solvent enabling the reaction between the epoxy functionalities of nGO and the free amines of aminolyzed PCL scaffold surfaces.25,28, The successful grafting of nGO was visible by the distinct change in color as compared to the neat PCL scaffold, see Figure 3a. As the concentration of nGO was increased the color change was even more prominent indicating attachment of higher amount of nGO. The



successful grafting of nGO on the surface was further confirmed by FTIR through the appearance of the band at 1670 – 1500 cm-1, which stands for the cyclic alkene C=C stretch from the aromatic bond conjugation on the basal plane of nGO, see Figure 3b. All the scaffolds exhibited similar weight loss profiles according to the TGA results, with a monotonic weight loss at approximately 380oC ascribed to the decomposition of PCL chains, see Figure 3c. Neat PCL scaffold, however, exhibited larger weight loss at this temperature compared to the aminolyzed and nGO-grafted scaffolds. This is presumably due to the higher amount of labile oxygen-containing functional groups on PCL, which generate CO, CO2, and steam at the onset of the decomposition temperature. The thermal stability of PCL was thus enhanced by aminolysis, whereby the amidation of the ester groups of PCL converts said labile groups to more stable oxygen groups. After full decomposition of the polymer 100% mass loss was recorded for the neat scaffold, while a residue corresponding to 1% of original weight was retained in the case of the aminolyzed scaffold, attributed to the additional amino groups, as well as the stability contribution of the covalent –CONH- bond. The aminolyzed scaffold thus underwent full decomposition at approximately 500 – 550 oC, during which the thermal loss of the attached diamine to the PCL surface occurs. This is in accordance with a previous study, which found that covalently bound alkylamines to carbon nanotubes exhibited thermal decomposition temperatures within the range of 250 – 500 oC, whereas physisorbed alkylamines had decomposition temperatures of only 150 – 300 oC.29 The nGO-grafted scaffolds, on the other hand, retained a residue of approximately 3% and 6% of their original weight after PCL chain decomposition, for the scaffolds grafted with the initial nGO concentrations of 0.5 and 1 mg/mL, respectively. It is important to note that nGO0.5 and nGO1 retain different masses after the decomposition of the polymer, where the residue increases 18 ACS Paragon Plus Environment

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with increasing nGO content (3 wt% vs. 6 wt%). This is consistent with the mass differences of the scaffolds before and after grafting with nGO, where averages were found to be equivalent to 1.2 wt% (nGO0.5) and 2.6 wt% (nGO1). See SI, Figure S3 for DTG curves and Table S2 for final residue amount, weight loss and temperature at maximum weight loss rate.

Figure 3. (a) Schematic illustration of the grafting of aminolyzed PCL scaffold surface with nGO flakes and images of neat, nGO0.5 and nGO1 scaffolds, (b) FTIR spectra of aminolyzed, nGO0.5 and nGO1 scaffolds and (c) TGA thermograms of neat, aminolyzed, nGO0.5 and nGO1 scaffolds.



Adsorption of Ciprofloxacin. The potential of the surface grafted nGO groups to adsorb drug molecules on their surface through secondary interactions was investigated to evaluate the possible drug loading and delivering capacity of the scaffolds. As the adsorption of ciprofloxacin on nGO was expected to be pH-dependent, sorption experiments were first performed at different pH to investigate the ciprofloxacin adsorption mechanism. The effect of solution pH to the adsorption of the drug is attributed to the pH-dependent speciation of ciprofloxacin, as well as the surface charge characteristics of nGO. nGO was found to have a zeta potential of zero at pH 3.9, and is thus negatively charged over a majority of the pH range.30 Ciprofloxacin, on the other hand, is a zwitterionic compound, and exists in its cationic form at pH < 6.1, whereas it is anionic at pH > 8.7.31 Various adsorption mechanisms therefore predominate at different solution pH levels, with some interactions even operating simultaneously. Below pH 4.0 and above pH 8.7, electrostatic repulsion could take place between the CIP molecules and the nGO surface, as both are negatively charged.32 Between pH 4.0 – 6.1, increased π-π electron-donor acceptor (EDA) interaction is expected, as the CIP molecule and the nGO surface are oppositely charged. In hydrophobic interaction, the more hydrophobic adsorbate tends to be adsorbed on nGO. Apart from the aforementioned EDA and hydrophobic interaction, electrostatic and hydrogen bonding interactions are other mechanisms that may also simultaneously occur.31 Sorption experiments were thus performed to ascertain which adsorption mechanism will predominate, as well as to elucidate the amount of oxygen-containing functional groups remaining on the nGO surface after being grafted onto PCL. The number of oxygen-containing functional groups on nGO ultimately affects the adsorption mechanism that predominates. Hydroxyl groups induce the benzene rings on nGO to act as π-electron donors, while carboxylic acid groups make the benzene rings π-electron acceptors, thus weakening EDA interactions.31 20 ACS Paragon Plus Environment

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It was determined that more ciprofloxacin per mg of sample was adsorbed on the nGO1 scaffolds grafted at pH 5 than pH 6.5, see Table 1. Based on the results, it is suggested that the major driving force for CIP adsorption on the nGO-grafted scaffolds is via π-π EDA interactions, rather than hydrophobic interaction. This is further supported by the measured mass differences on the nGO1 scaffolds before and after CIP adsorption (Table 1). For the calibration curve used to derive the CIP concentration see the SI, Figure S4. The finding that π-π EDA interactions predominate between nGO and ciprofloxacin suggests that the functional groups on the grafted nGO have not been completely reduced, despite their attachment on PCL via covalent bonds with free amine groups. The remaining oxygen containing functional groups on nGO are still enough to facilitate π-π EDA interactions and also to contribute to its hydrophilicity. It is also possible that the grafting pathway employed, which is the solvent-assisted method using water, does not diminish the amount of functional groups on nGO, and in fact even increases the number of hydroxyl groups through its nucleophilic attack on epoxy groups. Hence, this further enhances π-π EDA interactions, as the presence of hydroxyl groups induces the aromatic group in nGO to act as π-electron donors. Choosing pH 5 as the optimal pH the adsorption was further studied using aminolyzed, nGO0.5 and nGO1 scaffolds. It was found that the adsorption of CIP is enhanced by grafting the aminolyzed surface with nGO, as the aminolyzed scaffold exhibited lower adsorption amounts of CIP, see Table 1. The grafting concentration also seems to have an effect by altering the adsorption capacity further as the concentration of nGO increases.



Table 1. Results from the sorption test using nGO1 at two different pH-values and the results from the adsorption of the aminolyzed, nGO0.5 and nGO1 scaffolds at pH 5. pH

Average adsorbed ciprofloxacin per mg (mg/ml)

Average mass difference of scaffolds before and after adsorption (mg)

5

0.022 ± 0.01

0.10 ± 0.03

6.5

0.016 ± 0.003

0.020 ± 0.06

pH 5 Aminolyzed nGO0.5 nGO1

0.036 ± 0.005 0.043 ± 0.03 0.056 ± 0.008

0.013 ± 0.03 0.06 ± 0.08 0.077 ± 0.02

The FTIR spectra of the ciprofloxacin-loaded scaffolds are illustrated in Figure 4a. All scaffolds exhibited the characteristic peaks of PCL. The CIP adsorption is evidenced by the appearance of peaks at 1600 – 1580 cm-1 and 1670 – 1600 cm-1, which are attributed to amine N-H bend and conjugated ketone C=O stretch of ciprofloxacin. The thermograms of the CIP-loaded scaffolds exhibited similar weight loss profiles with the nGO-grafted scaffolds, signifying that the previously grafted nGO is not lost during the process of CIP adsorption, see Figure 4b. For the CIP-loaded scaffold grafted with 1 mg/mL initial nGO concentration, nGO1-CIP, a significant increase in thermal stability and final residual weight was observed, which was presumed to be due to the effect of the strong π-π EDA and hydrogen bonding interactions between CIP and the functional groups of nGO. The same increase in thermal stability was not observed for the CIP-loaded scaffolds grafted with 0.5 mg/mL initial nGO concentration, nGO0.5-CIP, possibly due to the low amount of nGO present in the scaffold, which may have limited the number of functional groups available for the noncovalent attachment of CIP. Compared to nGO1-CIP, the amount of CIP adsorbed on nGO0.5 could be too


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low to exhibit any thermal stability enhancement. See SI, Figure S3 for DTG curves and Table S2 for final residue amount, weight loss and temperature at maximum weight loss rate. Elemental analysis via EDS was performed in order to qualitatively compare the CIP content in the scaffolds. This was done by the determining the amount of fluorine on the scaffolds which is one of the elements that comprise the ciprofloxacin, see Figure 4a for the chemical structure. While no fluorine could be detected on the surface of nGO1 scaffolds, nGO0.5-CIP and nGO1CIP showed 0.45 ± 0.05 and 0.74 ± 0.13 wt% fluorine. The results presented are average values of measurements performed in triplicate. The fluorine detected in the nGO-grafted scaffolds containing CIP increases by 64% for nGO1-CIP, in comparison to nGO0.5-CIP. This result provides further support to the conclusion that ciprofloxacin adsorption occurs through nGO and is thus dependent on the number of functional groups that are available for attachment.

Figure 4. (a) FTIR spectra and (b) TGA thermograms of nGO0.5, nGO1, nGO0.5-CIP and nGO1-CIP scaffolds.

Morphological Analysis via SEM. To characterize the morphology of the scaffolds, SEM images of the neat, aminolyzed, nGO grafted, and ciprofloxacin-loaded scaffolds were examined, as displayed in Figure 5. The neat PCL scaffold mostly exhibited a homogeneous regular 23 ACS Paragon Plus Environment


morphology. Aminolysis of the PCL surface resulted in some defects on the scaffold’s previously smooth surface, which was observable as dark spots. The enlargement of pores and the introduction of nanoscale pores also notably occurred following this surface modification process. These surface defects and formation of small pores on the bulk of the scaffold provide evidence of the penetration and effective diffusion of the diamine through the scaffold. The most evident modification to the scaffold morphology, however, occurred after grafting with nGO. Figure 5 depicts the rough and flake-filled morphology on the scaffold surface after grafting with nGO using an initial nGO concentration of 1 mg/mL (nGO1). This is expected, as the functional groups on nGO could contribute to nGO sheets that are not completely flat and therefore thicker than monolayer.9 In addition, multilayers are expected due to strong interactions between nGO sheets and some agglomerated nGO sheets are also clearly visible, along with voids and surface fractures caused by the stacking of the agglomerates. Besides this, nGO appeared to have been tightly attached onto the scaffold surface as no obvious sheet pullout could be observed, therefore suggesting some interfacial interactions between the nGO sheets and PCL matrix. The morphology of the two nGO grafted scaffolds before and after adsorption of ciprofloxacin is also ipresented in Figure 5. As seen from the image, a smooth layer of the drug has formed on the surface, hence filling up the voids and surface fractures created by nGO agglomerates. Upon closer look on these scaffolds, the adsorbed CIP becomes distinguishable as irregular needle-like crystals. This needle-like appearance of ciprofloxacin is in accordance with morphological observations that were made in previous studies.33-35 One study suggested that ciprofloxacin forms needle-like structures during supersaturation or at higher temperatures, as this structure is actually its metastable form.35 Noticeably, the needle-like ciprofloxacin structures are mainly 24 ACS Paragon Plus Environment

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observed in nGO1-CIP scaffolds containing higher concentration of nGO leading to larger amount of CIP adsorption. It is therefore likely that ciprofloxacin preferentially adsorbs on the areas rich in nGO, and since the scaffold containing more nGO also exhibited some nGO sheet stacking, these areas adsorbed large enough amounts of ciprofloxacin to cause some saturation of the drug leading to formation of needle-like structures.

Figure 5. SEM images of the neat, aminolyzed, nGO-grafted, and CIP-loaded scaffolds and SEM images comparing the nGO-grafted scaffolds with and without CIP.

Mechanical Testing. The mechanical properties of the scaffolds were evaluated through compression testing, and the results are presented in Figure 6. Neat PCL scaffold was found to have a compressive strength of 0.56 MPa, which is significantly lower than that of human cancellous bone (1.5 – 9.3 MPa).36 In terms of its compressive strength, the scaffold developed in this work could be further improved by decreasing its porosity. One study reported the development of PCL scaffolds with compressive strengths of 2.48 MPa and porosities of 75.4%.6 The neat PCL scaffolds developed herein, on the other hand, had porosities up to 85.3%. 25 ACS Paragon Plus Environment


After aminolysis, the scaffold exhibited a decrease in compressive strength. This is explained by the slight degradation undergone by PCL upon exposure to the aminolyzing solution. As discussed previously, this degradation could be visibly observed in the SEM images of the neat and aminolyzed scaffolds, where pore enlargement and the appearance of nano-sized pores had occurred, Figure 5. As further evidence to the degradation of PCL due to the aminolysis, Zhu et al. found that the mass of PCL decreased continually during the aminolysis reaction, and after a reaction time of 30 min using the same diamine concentration as used in this study, the mass loss exhibited by the PCL membrane corresponded to a loss in thickness of 1 nm.6 The group of Gloria et al. also noted the occurrence of chain scission that begins on the amorphous region as a consequence of aminolysis, thus reducing the density of chain entanglements and resulting to a softening effect.5 The nGO-grafted scaffolds, on the other hand, exhibited a significant increase in compressive strength. This was expected, as graphene nanostructures are known for their excellent mechanical properties owing to the honeycomb-like structure of their carbon bonding network.37 nGO has previously been shown to be effective nano-enhancers in strengthening the mechanical properties of polymer composites.3 The significant effect of nGO to the mechanical properties of polymer composites could be due to its ability to anchor polymer chains with its functional groups, thus forming ordered lamellae structures that could stretch, rearrange, and redistribute stress concentrations. A homogeneous dispersion of the nGO particles, however, is crucial in order to achieve efficient interfacial interaction with the polymer matrix, a prerequisite to maximum reinforcing efficiency.38 However, as PCL is hydrophobic, the highly hydrophilic nature of nGO, by virtue of its ample functional groups, could adversely affect its dispersion in the polymer matrix. In the present PCL-nGO system, nGO was grafted on the surface and the non26 ACS Paragon Plus Environment

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dispersibility between nGO and PCL was mediated by the incorporation of a diamine, which enables the formation of covalent bonds between the two components. The improved interaction through covalent attachment supported by possible favorable interfacial interactions between nGO and remaining free amine groups, might allow for a larger interface volume fraction, thereby enabling a reinforcing effect enhanced by the efficient stress transfer between the nGO and the PCL matrix. It is interesting to note that the enhancement in compressive strength and modulus was more pronounced for the scaffold with lower nGO content (nGO0.5), as opposed to that of the scaffold with higher nGO content (nGO1). The nGO concentration of nGO1 may have been too high so that the excess nGO formed bonds with other nGO particles via secondary interactions after the available free amine groups had been consumed. This then resulted in a non-homogeneous dispersion of nGO on the surface, as well as nGO agglomeration. This correlated to the results of a previous study, where the higher concentration of nGO increased its aggregation in the PCL matrix due to the competing intermolecular forces of nGO-nGO and nGO-PCL.11 In the SEM images of the scaffolds, this phenomenon was visible from the stacking of the crumpled nGO sheets and the resultant formation of voids and fractures from said agglomeration. The inhomogeneous surface and the presence of voids and surface fractures clearly adversely affected stress transfer efficiency and reduced the reinforcing effect of nGO. The adsorption of ciprofloxacin on the nGO-grafted scaffolds increased the compressive strength by 29 % when comparing nGO1 with nGO1-CIP. A possible explanation for this could be that the drug has effectively filled in the voids formed by the nGO agglomerates, thus acting as a bridge in favor of efficient load transfer between nGO and the matrix. The adsorption of ciprofloxacin could have improved the coherence between agglomerated nGO sheets and the 27 ACS Paragon Plus Environment


matrix, enabling the direct stress transfer from matrix to nGO. This is in accordance with the observation of the scaffold’s morphology via SEM, where the apparent formation of a drug layer over the scaffold’s surface has engulfed some of the surface fractures observed before the drug loading.

Figure 6. The elastic modulus and compressive strength of the scaffolds.

Release Studies. In vitro drug release studies were conducted to investigate the release behavior of ciprofloxacin adsorbed on the PCL scaffolds. Previous studies have shown that incorporation of nGO in biomaterials can induce mineralization of calcium phosphate on the surface of the scaffolds.3,11 The release tests were therefore performed in SBF as the dissolution medium, in order to control for the effect of the possible formation of a hydroxyapatite surface layer on drug release and to simulate the expected behavior of the scaffolds in vivo. The sampling method employed involved the replacement of the entire volume of the dissolution medium with a fresh solution of SBF after each sample withdrawal. Figure 7 shows the total amount of drug released after certain time periods. For the calibration curve used to derive the CIP concentration for the release study see the SI, Figure S5. 28 ACS Paragon Plus Environment

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Both scaffolds, regardless of nGO grafting concentration, exhibited an initial burst release behavior. After only 2 h, 86% of the drug had been released from nGO0.5-CIP, and 82% had been released from nGO1-CIP. In 24 h, 93 and 91% of the drug had been released from the nGO0.5-CIP and nGO1-CIP, respectively. Total drug release from both scaffolds had occurred after 8 days. While burst release of ciprofloxacin may not be the ideal case for therapeutic purposes of osteomyelitis treatment, the purpose of loading ciprofloxacin in this study was to prevent initial inflammation due to scaffold placement in the body. Gimeno et. al. demonstrated the efficient and preventive action of antibiotic loaded device with antibiotic-release during a week to avoid bacterial contamination.39 In addition, similar release profiles have been suggested before to prevent post-surgical infections in orthopaedic or trauma.40,41 It’s also noteworthy that the sizes of the scaffold samples used for the test were very small (8 – 9 mg; 0.5 x 0.5 cm2 in diameter) thus large exposed surface area of the scaffold samples may have contributed to the fast release of the drug. Samples with larger dimensions could have provided a more accurate release behavior, as they would consist of a larger internal bulk area that would allow for the investigation of the diffusion mechanism of the drug molecules through the scaffold matrix. One possible reason for the difference in the amount of drug released is that the scaffold containing more nGO also had more functional groups, and thus possessed more available sites for the secondary interactions with ciprofloxacin. This effect could also have been amplified by the physical obstruction of nGO particles on the diffusion of the drug through the scaffold. The presence of nGO could also create a longer pathway of diffusion for the ciprofloxacin molecules, thus slowing down its release. A related effect is typically observed in nanocomposite systems. In the study of Scaffaro et al., for instance, the presence of graphene nanoplatelets in ciprofloxacin-



loaded PLA nanocomposites proved to slow down drug release due to the creation of a tortuous pathway; the antimicrobial activity of the composite, however, remained unaffected.33

Figure 7. Release behavior of ciprofloxacin-loaded scaffolds with varying initial nGO concentrations.

Biomineralization Test. The in vitro bioactivity and ability to induce mineralization of the nGO grafted scaffolds was investigated through the incubation of the scaffolds in SBF solution for 14 days. The utilized SBF solution was designed to mimic the ion concentrations of human blood plasma.42 SEM was utilized to analyze the morphologies of the scaffolds after mineralization, and the images are shown in Figure 9. The neat and aminolyzed scaffolds both show the typical morphologies of PCL with some defects and salt particulates that have not been completely leached out. The nGO-grafted and CIP-loaded scaffolds, on the other hand, show the formation of foreign particulates, which are presumed to be aggregated minerals. On the surface of nGO1, it can clearly be seen at higher magnification that these particulates are irregularly shaped, and they form into clusters with a networked structure. The higher degree of mineralization on the scaffolds containing larger amounts of nGO could be explained by the increased number of oxygen containing functional groups available, which have been found to act as anchor sites for mineral deposition.11 In addition, a previous study that utilized similar microwave-derived 30 ACS Paragon Plus Environment

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cellulose-based nGO found that the crystal size of the mineral deposits correlated with the dispersion and aggregation of nGO in the polymer matrix.58 Observations on the morphology and microstructure of the aggregates have been made in other studies, where it has been shown that the formation of macroporous interconnected mineral spheres could potentially be favorable to cell migration and mass transport.43,44 The same clustered formation of minerals is not observed in the CIP-loaded scaffolds, as it appears that the precipitated minerals on these scaffolds were smaller in size and more spread out over the scaffold surface. This may be explained by the fact that the release of ciprofloxacin from the scaffold occurred simultaneously with the growth of minerals. Hence, although the presence of carboxylic groups from ciprofloxacin act as additional Ca2+-binding sites and may enhance mineralization, these nucleation sites are not permanent and would undergo erosion during the incubation period. Based on the SEM images of the mineralized scaffolds, the previous needlelike structure of CIP could no longer be observed, thus providing evidence for the dissolution or release of the drug during incubation in SBF (based on the release test, complete drug release occurred after 8 days). A larger number of nucleation sites were thus formed homogeneously on the scaffold due to the presence of ciprofloxacin, but the growth of minerals on the nucleation sites was restricted due to the release of the drug, resulting in smaller mineralized particles. This is illustrated by the surface of nGO1-CIP, shown in Figure 9, covered with large amount of small circular minerals creating a layer. Elemental analysis by EDS was performed on the scaffolds in order to verify the precipitation of calcium phosphates, which signify active mineralization. A trend existed indicating that the presence of nGO indeed induced mineralization, although the amount of precipitated calcium or phosphorus was not significant. The amount of nGO present on the test scaffolds, however, may 31 ACS Paragon Plus Environment


not have been high enough for pronounced mineralization. EDS also showed that the presence of ciprofloxacin did not diminish the bioactivity of the scaffolds, despite its erosion from the scaffold during the drug release. See the SI, Table S3 for more details.

Figure 8. SEM images of the scaffolds after incubation in SBF for 14 days.

CONCLUSIONS The surface-functionalization of poly(ε-caprolactone)-based scaffolds with nano-graphene oxide (nGO) was successfully demonstrated.

nGO was covalently grafted onto aminolyzed PCL

scaffolds through solvent-assisted method using water as a green solvent. The covalent attachment of nGO was verified by FTIR and TGA, and the amount of nGO grafted onto the 32 ACS Paragon Plus Environment

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scaffolds was quantified by measuring the weights of the scaffolds before and after grafting. Initial nGO concentration of 0.5 mg/mL and 1 mg/mL resulted in the average nGO content of 1.2 wt% and 2.6 wt%, respectively. Functionalization with nGO enhanced the scaffold’s compressive strength and bioactivity. The scaffolds grafted with 0.5 mg/mL initial nGO concentration exhibited a 50% increase in compressive strength, while those grafted using 1.0 mg/mL exhibited a 21% increase. After incubation in SBF the nGO-grafted scaffolds induced formation of mineral precipitates, which was not observed for neat and aminolyzed scaffolds. An antibiotic drug, ciprofloxacin, was adsorbed on the scaffold via secondary interactions with the oxygencontaining functional groups on nGO. Efficient drug loading was achieved at a solution pH 5, where the π-π electron-donor acceptor (EDA) interactions predominated. In addition, the increased nGO concentration on the scaffold enabled an enhanced adsorption of ciprofloxacin. The adsorption of ciprofloxacin further enhanced the compressive strength of the scaffolds, while the scaffolds’ bioactivity was retained. A study on release kinetics revealed a release of ciprofloxacin from the scaffolds during 8 days. This could be useful for the prevention of initial inflammation during the scaffold placement. ACKNOWLEDGEMENTS The Swedish Research Council (VR) is acknowledged for the financial support (Contract Grant No. 2014-4091). SUPPORTING INFORMATION Calibration curves, DTG curves and tabulated data from TGA and DTG curves, raw data for SEM EDS results and ninhydrin assay of scaffolds.



REFERENCES (1) Hokmabad, V.; Davaran, S.; Ramazani, A.; Salehi, R. Design and fabrication of porous biodegradable scaffolds: a strategy for tissue engineering. J. Biomater. Sci. Polym. Ed. 2017, 28, 1797-1825. (2) Mohammadi, S.; Shafiei, S.; Asadi-Eydivand, M.; Ardeshir, M.; Solati-Hashjin, M. Graphene oxide-enriched poly(ε-caprolactone) electrospun nanocomposite scaffold for bone tissue engineering applications. J. Bioact. Compat. Polym. 2017, 32, 325-342. (3) Wu, D.; Samanta, A.; Srivastava, R.; Hakkarainen, M. Nano-Graphene Oxide Functionalized Bioactive Poly(lactic acid) and Poly(e-caprolactone) Nanofibrous Scaffolds. Materials. 2018, 11, 566. (4) Causa, F.; Battista, E.; Della Moglie, R.; Guarnieri, D.; Iannone, M.; Netti, P. Surface Investigation on Biomimetic Materials to Control Cell Adhesion: The Case of RGD Conjugation of PCL. Langmuir 2010, 26, 9875-9884. (5) Gloria, A.; Causa, F.; Russo, T.; Battista, E.; Della Moglie, R.; Zeppetelli, S.; De Santis, R.; Netti, P.A.; Ambrosio, L. Three-Dimensional Poly(ε-caprolactone) Bioactive Scaffolds with Controlled Structural and Surface Properties. Biomacromolecules 2012, 13, 3510-3521. (6) Zhu, Y.; Mao, Z. W.; Shi, H. Y.; Gao, C. Y. In-depth study on aminolysis of poly(εcaprolactone): Back to the fundamentals. Sci. China Chem. 2012, 55, 2419-2427. (7) Zhu, Y.; Gao, C.; Liu, X.; Shen, J. Surface Modification of Polycaprolactone Membrane via Aminolysis and Biomacromolecule Immobilization for Promoting Cytocompatibility of Human Endothelial Cells. Biomacromolecules 2002, 3, 1312-1319.


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Table of Contents graphic (TOC) Cellulose Derived Nano-Graphene Oxide Surface Functionalized 3D Scaffolds with Drug Delivery Capability Nejla B. Erdal, Jenevieve G. Yao, and Minna Hakkarainen


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Cellulose-Derived Nanographene Oxide Surface-Functionalized

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