Modulation of conductivity of alginate hydrogels containing reduced graphene oxide through the addition of proteins

pharmaceutics Article Modulation of Conductivity of Alginate Hydrogels Containing Reduced Graphene Oxide through the Addition of Proteins Ahmed Raslan 1,† , Jesús Ciriza 2,3,4,† , Ana María Ochoa de Retana 5, María Luisa Sanjuán6, Muhammet S. Toprak 7, Patricia Galvez-Martin 8, Laura Saenz-del-Burgo 1,2,9,* and Jose Luis Pedraz 1,2,9,*   Citation: Raslan, A.; Ciriza, J.; Ochoa de Retana, A.M.; Sanjuán, M.L.; Toprak, M.S.; Galvez-Martin, P.; Saenz-del-Burgo, L.; Pedraz, J.L. Modulation of Conductivity of Alginate Hydrogels Containing Reduced Graphene Oxide through the Addition of Proteins. Pharmaceutics 2021,13, 1473. https://doi.org/10.3390/ pharmaceutics13091473 Academic Editor: Maria Antonietta Casadei Received: 10 June 2021 Accepted: 7 September 2021 Published: 15 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1NanoBioCel Group, Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of the Basque Country UPV/EHU, 01006 Vitoria-Gasteiz, Spain; [email protected] 2Biomedical Research Networking Center in Bioengineering, Biomaterials, and Nanomedicine, CIBER-BBN, Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain; [email protected] 3 Tissue Microenvironment (TME) Lab, Aragón Institute of Engineering Research (I3A), University of Zaragoza, C/Mariano Esquillor s/n, 50018 Zaragoza, Spain 4Institute for Health Research Aragón (IIS Aragón), 50009 Zaragoza, Spain 5Department of Organic Chemistry I, Faculty of Pharmacy and Lascaray Research Center, University of the Basque Country (UPV/EHU), Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain; [email protected] 6Instituto de Ciencia de Materiales de Aragón (Universidad de Zaragoza-CSIC), Facultad de Ciencias, 50009 Zaragoza, Spain; [email protected] 7Biomedical and X-ray Physics, Department of Applied Physics, KTH-Royal Institute of Technology, 10691 Stockholm, Sweden; [email protected] 8R&D Animal and Human Health, Bioibérica S.A.U., 08029 Barcelona, Spain; [email protected] 9Bioaraba Health Research Institute, Jose Atxotegi, s/n, 01009 Vitoria-Gasteiz, Spain *Correspondence: [email protected] (L.S.-d.-B.); [email protected] (J.L.P.); Tel.: +34-945014542 (L.S.-d.-B.); +34-945013091 (J.L.P.) † Authors contributed the same. Abstract: Modifying hydrogels in order to enhance their conductivity is an exciting field with applications in cardio and neuro-regenerative medicine. Therefore, we have designed hybrid alginate hydrogels containing uncoated and protein-coated reduced graphene oxide (rGO). We specifically studied the adsorption of three different proteins, BSA, elastin, and collagen, and the outcomes when these protein-coated rGO nanocomposites are embedded within the hydrogels. Our results demonstrate that BSA, elastin, and collagen are adsorbed onto the rGO surface, through a non-spontaneous phenomenon that fits Langmuir and pseudo-second-order adsorption models. Protein-coated rGOs are able to preclude further adsorption of erythropoietin, but not insulin. Collagen showed better adsorption capacity than BSA and elastin due to its hydrophobic nature, although requiring more energy. Moreover, collagen-coated rGO hybrid alginate hydrogels showed an enhancement in conductivity, showing that it could be a promising conductive scaffold for regenerative medicine. Keywords: hydrogel; alginate; reduced graphene oxide; conductivity; collagen 1. Introduction Hydrogels are three-dimensional scaffolds made up of highly hydrophilic polymers. Because they absorb so much water, these hydrogels swell, representing a high degree of flexibility, closer to that of natural tissue [ 1 ]. Hydrogels also represent high porosity, excellent biocompatibility, and controllable degradability [ 2 ], triggering their application in biomedicine including, applications of soft contact lenses in the correction of vision [ 3 ], developing a tissue engineering process [ 2 , 4 , 5 ], diagnostics [ 6 ], and embolizing cells [ 7 ]. Depending on the type of bonding, these hydrogels can be classified as either physical or chemical. Physical bonding, such as hydrogen bonding, and hydrophobicity result in physical gels, which are often reversible and affected by environmental factors [ 1 ]. Pharmaceutics 2021,13, 1473. https://doi.org/10.3390/pharmaceutics13091473 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021,13, 1473 2 of 18 Chemical gels, in contrast, are formed by covalent bonding between polymers. These hydrogels are permanent and stable [8,9]. However, there are many limitations to the applications of nature hydrogels in clinical applications. These include high water content, large pores, weak mechanical strength, and fast drug release [3,10]. In the course of time, natural hydrogels have been gradually replaced by synthetic hydrogels that have a longer half lifetime and high mechanical strength [11]. Incorporating a special chemical group into the hydrogel will improve its functionality and allow the hydrogel to be switched by heat, light, magnetic fields, chemical agents, or pH alterations [ 2 , 12 , 13 ]. Functionalized hydrogels with therapeutic peptides and proteins are also possible. These can be used to treat diseases, such as cancer, immune disorders, mental disorders, hypertension, and certain cardiovascular and metabolic problems. Extracellular matrix (ECM) is a non-cellular component of tissue that provides physical support to cells. Emerging research has shown that ECM provides tissue-specific biochemical and biophysical cues required for tissue morphogenesis [14]. For decades, alginate was considered one of the best biomaterials for assembling and fabricating functional hydrogels, owing to its excellent biocompatibility and high porosity [ 15 , 16 ]. However, several drawbacks, such as its mechanical strength, weakness, the leak of cell adhesion, and its rapid drug release, have limited its clinical application [ 17 , 18 ]. To solve these drawbacks, different materials have been integrated into the alginate matrix, also creating biomimetic support. In this regard, graphene has been applied in various fields based on this excellent characteristic, including electronics [ 19 ], being considered a strong candidate in the field of biomedicine, both for fabricating drug delivery vehicles and gene therapy [ 19 – 22 ]. However, studies with graphene are contradictory [ 22 ]. On one hand, some reports describe graphene as a material that does not cause any alteration in cell function [ 23 , 24 ], with acceptable hemocompatibility, and without induction of immune response, even at high concentrations [ 24 ]. On the other hand, reports show a cytotoxic effect even at a low dosage [ 23 , 25 ]. Graphene oxide (GO), a derivative from graphene, can be produced through Hummer’s method [ 26 – 30 ] and shows unique physical and mechanical properties, including high thermal conductivity [ 26 , 31 , 32 ], colossal surface area [ 33 , 34 ], and a robust mechanical strength [ 35 , 36 ]. The oxidation process of graphene alters the surface of graphene, increasing its affinity to water [ 37 , 38 ] and, therefore, mediating a vast number of biochemical reactions and bio-conjugations along its surface [ 39 ]. GO biocompatibility is affected in two-dimensional cultures by factors such as GO surface processing and the particle size of surface functionality, with impact on adhesion or cell proliferation [ 40 ]. In this regard, our group combined GO with alginate to modify alginate surface properties and its mechanical strength, showing good biocompatibility with myoblasts in alginate microcapsules, within a range of GO concentrations. Concisely, GO concentrations between 25 and 50 µ g/mL enhanced the viability of C 2 C 12 myoblasts [ 16 , 40 – 42 ]. However, the integration of GO within alginate matrices reduced the release of therapeutic factors, since GO could sorb the secreted therapeutic factors on its surface due to its high surface activity. GO sorption was solved by applying a pre-coating layer on the GO surface with fetal bovine serum [16,40–42]. An alternative graphene derivate is reduced GO, with low surface absorbability compared to GO [ 43 ]. Several techniques have been utilized to reduce GO, including mechanical reduction or chemical reduction [ 43 ], adding alterations in rGO surface, such as the chemical structure and hydrophilicity [ 37 , 44 ]. However, there is again conflicting information comparing the biocompatibility of GO and rGO [ 43 , 45 , 46 ]. It has been described that the irradiated light reduction in GO yields an immense reactive oxygen species generation and oxidative stress [ 43 ], while the small particle size of thermally reduced oxide can stimulate cytotoxicity, facilitating its cell membrane penetration [ 43 ]. However, chemically reduced rGO shows lower toxicity than other rGO forms [ 47 ]. Modified scaffolds with rGO have shown strong mechanical strength and ultra-high electrical conductivity [ 48 ], with favorable impacts on cell viability, proliferation, and differentia- Pharmaceutics 2021,13, 1473 3 of 18 tion [ 48 ]. Hydrothermal processing of alginate and graphene oxide in an aqueous solution yields hybrid alginate-rGO hydrogels with high porosity. In the hydrothermal process, graphene nanosheets and alginate form a porous structure as a result of auto-assembly; afterwards, the hybrid hydrogel is produced by ionically linking polymer networks of alginate [ 49 ]. Thus, rGO has become more applicable in tissue engineering, particularly for neuronal regeneration [ 48 , 50 ] or cardiomyocytes regeneration [ 51 ]. Here, we are using reduced graphene-based materials, as it is one of the best redox species that could be studied on an electrode. The redox peaks will give a clear indication of changes to the double layer on the electrode’s surface. When rGO-protein-alginate is incorporated over an electrode the double layer changes, this can affect the double-layer capacitance and the electron transfer resistance. Therefore, by monitoring the charge transfer resistance, we could understand the charge transfer properties of the double layer. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) are interrelated electroanalytical methods to examine the electrochemical double layer on the electrodes. In this work, we aimed to create conductive protein-rGO-alginate hydrogels using different proteins in order to study their adsorption capacity and electrochemical characteristics to identify the best composition. 2. Materials and Methods 2.1. Materials The chemically reduced graphene oxide (rGO) powder was provided by Graphenea (San Sebastian, Spain). Bovine Serum Albumin (BSA) and type 1 Collagen were purchased from Sigma Aldrich (Saint Louis, MO, USA). Elastin was provided by Bioiberica (Barcelona, Spain). High pure, low-viscosity, and ultra guluronic (LVG) acid alginate was purchased from FMC Biopolymer (Drammen, Norway). 2.2. Protein Adsorption rGO powder in a 3/1 mixture of water and DMSO (v/v) to get a suspension of 4 mg/mL rGO, homogenizing by sonication for 60 min in a bath. The resulting rGO dispersion was diluted to 2.5 mg/mL with 18 M Ω cm resistivity deionize water (DI). Then, 90 µ L of 200 µ g/mL BSA, 200 µ g/mL elastin, or 500 µ g/mL collagen were mixed with 10 µ L of 250 µ g/mL rGO suspension for 120 min at 37 ◦ C under agitation at 400 rpm. The samples were spun down at 15,000 rpm for 15 min to collect supernatants. The lack of adsorbed protein was determined with the BCA kit (Thermo Fisher, Massachusetts, MA, USA) in a M 200 TECAN microplate reader (TECAN Trading AG, Männedorf, Switzerland) at 562 nm. At least three samples were quantified to ensure accuracy and repeatability. The % of protein sorption was estimated by Equation (1), and the adsorption capacity qe ( µ g/ µ g) was estimated by Equation (2), where C 0 ( µ g/mL) and Ce ( µ g/mL) are the original protein concentration and the protein concentration at steadiness, respectively, S is the sample volume (mL), and m is the mass of rGO (µg). Adsorbability (%) = (C0−Ce) ×100/C0(1) Adsorption capacity qe = (C0−Ce) ×S/m (2) Freundlich and Langmuir’s adsorption isotherm models were implemented to estimate the adsorption isotherm. The Langmuir model is displayed in Equations (3) and (4), where the concentration of the adsorbed protein at steady-state is Ce ( µ g/mL), qe is the adsorption capacity ( µ g/ug), qmax ( µ g/ µ g) is the maximum quantity of protein sorbed per unit mass of rGO, K L (mL/ µ g) is the Langmuir factor associated with the surface affinity for the protein, C 0 is the initial protein concentration, and R L is the separation factor that specifies the Langmuir isotherm’s fundamental aspects [52]. Ce/qe = Ce/qmax + 1/(qmax·KL) (3) Pharmaceutics 2021,13, 1473 4 of 18 RL=1/(1+KL·C0) (4) Freundlich model is described as follows (Equation (5)), where K F and m are the Freundlich constant and intensity adsorption, respectively: Log qe = log KF+ 1/m·log Ce (5) 2.3. Kinetics of Protein Adsorption 250 µ g/mL rGO was suspended either in 50 µ g/mL BSA, 200 µ g/mL collagen, or 50 µ g/mL elastin by agitation at 37 ◦ C. After spinning down at 15,000 rpm for 10 min, supernatants were collected after the following incubation times: 5, 10, 20, 30, and 80 min. Adsorbed protein was quantified with a BCA kit (Thermo Fisher) in a M 200 TECAN microplate reader (TECAN Trading AG, Männedorf, Switzerland) at 562 nm. At least three samples were quantified to ensure accuracy and repeatability. Intra-particle diffusion model, pseudo-first-order, and pseudo-second-order rate adsorption were applied to the results to determine the most appropriate adsorption kinetic model. At least three samples were quantified for each condition. Several models were evaluated to elucidate the adsorption mechanism and the adsorption rate (Equations (6)–(9)) [ 16 ], where qt ( µ g/ µ g) is the quantity of the adsorbed protein vs. time (t) (min.), Kp is the intra-particle diffusion rate constant ( µ g/ µ g. min 1/2 ), and C ( µ g/ µ g) is a constant for the intra-particle diffusion model, which provides a piece of information about the thickness of the barrier layer [ 52 , 53 ], qe ( µ g/ µ g) is the adsorption capacity at equilibrium, K 1 (min −1 ) is the constant rate of the pseudo-first-order model, and K 2 ( µ g ·µ g −1 min −1 ) is the pseudo-second-order model’s constant rate. qt= Kp.t1/2 + C (6) log (qe −qt) = log (qe) −(K1·t)/2.303 (7) t/qt=t/qe + 1/K2·1/(qe)2(8) 1/qt= 1/(K2+ qe2)·1/t+ 1/qe (9) 2.4. Thermodynamics of Protein Adsorption 250 µ g/mL rGO was suspended either in 50 µ g/mL BSA, 200 µ g/mL collagen, or 50 µ g/mL elastin solution. Mixtures with each protein were incubated and agitated at the following temperatures: 5, 10, 15, 25, 37, and 39 ◦ C. After spinning down at 15,000 rpm for ten minutes, supernatants were collected to quantify the amount of non-adsorbed protein with BCA kit (Thermo Fisher) in a M 200 TECAN microplate reader (TECAN Trading AG, Männedorf, Switzerland) at 562 nm. At least three samples were quantified for each condition. The fundamental thermodynamic factors, such as entropy change ( ∆ S ◦ ), enthalpy change ( ∆ H ◦ ), and Gibbs free energy change ( ∆ G ◦ ), were calculated using Equations (10)–(13) [ 53 – 55 ], where R is the gas constant (8.314 J/mol K), T the absolute temperature (K), K d the equilibrium constant, qe ( µ g/ µ g) the quantity of protein adsorbed per mass unit of rGO at equilibrium, and Ce ( µ g/mL) the equilibrium concentration of each protein: Kd = qe/Ce (10) ∆G◦=−R·T·lnKd (11) lnKd=−∆H/R·T + ∆S/R (12) ∆G◦=∆H−T∆S (13) 2.5. Characterization of the rGO-Protein Binding The binding of each protein to rGO surface was studied by Raman and Fourier transform infrared spectroscopy. First, 250 µ g/mL rGO was suspended either in 50 µ g/mL BSA, 200 µ g/mL collagen, or 50 µ g/mL elastin solution, and agitated at 37 ◦ C for 2 h. The supernatants were collected after spinning down at rpm for 15 min and lyophilized in a Pharmaceutics 2021,13, 1473 5 of 18 Telstar Lyobeta 15 lyophilizer. rGO without protein incubation and proteins without rGO were also studied. At least three samples were studied for each condition. Raman spectrum was obtained by confocal Raman imaging (Alpha 300 M, Company WITec, Ulm, Germany) with a 532 nm laser (5% laser power, a contact time of the 50 s, and four accumulations). FT-IR, using an attenuated total reflectance (ATR) technique, was performed in a FT-IR Bruker IFS 66/S Spectrometer, with 32 scans at a resolution of 4 cm −1 between the wavelength ranges of 4000–400 cm −1 . Air background was applied as a blank. At least three samples were analyzed for each condition. 2.6. EPO and Insulin Adsorption Blocking Study 100 µ g/mL rGO was suspended either in DI water, 50 µ g/mL BSA, 200 µ g/mL collagen, or elastin and incubated for two hours at 37 ◦ C. After being spun down at 15,000 rpm for 15 min, supernatants were removed, and protein-coated rGO was incubated with 200 µ L of 200 mIU/mL recombinant EPO or 150 mIU/mL recombinant insulin for 24 h at 37 ◦ C. Uncoated rGO was used as a reference. Next, samples were spun down by centrifuging for 15 min at 15,000 rpm and supernatants were collected. Non adsorbed EPO and insulin were quantified with Quantikine IVD EPO (R&D Systems) and Insulin Elisa (Mercodia), respectively, following manufacturer recommendation. At least three samples were analyzed for each condition. 2.7. Preparation of Alginate Hydrogels Containing rGO and Protein-Coated rGO At room temperature, 1.87 g of high pure sodium alginate was dissolved in 1% mannitol by magnetic string at 200 rpm for 2 h; then, it was mixed and homogenized with either rGO or protein-coated rGO suspension, obtaining a final concentration of 1.5% alginate and 50 µ g/mL rGO. To prepare hybrid alginate hydrogels, alginate solutions were mixed with 60 µ L of 1.22 M calcium sulfate through a connector (Braun) between two Luer Lock syringes (BS Syringe). The mixed solutions were dispensed between two glass slides with 2 mm spacing, leaving them for 30 min to form hydrogel disks, 14 mm in diameter. 2.8. Conductivity of Alginate Hydrogels Containing Protein-Coated rGO Electrochemical impedance spectra (EIS) were measured using a potentiostat (Princeton Applied research, Oak Ridge, TN, USA), with a screen-printed electrode (Dropsens, Oviedo, Spain) based on carbon, and a silver electrode as reference. Samples were immersed in 0.1 M PBS buffer at room temperature, applying frequency series from 10 −1 to 105 Hz. Cyclic Voltammetry (CV) measurements were performed in 0.1 M PBS buffer between the potential range from − 0.2 to 0.5 V and at different scan speeds (100 mVs −1 ). Specific capacitance was estimated from CV curves by Equation (14) [ 16 , 56 ], where C (F · g −1 ) is the specific capacitance, Q the mean charge throughout the charging and discharging procedure, the potential range V (Volt), and the mass m (g) of the hydrogel disk: C = Q/(2Vm) (14) 2.9. Statistical Analysis Statistical analysis was performed with GraphPad Prism 9.0 (GraphPad Inc., San Diego, CA, USA) and SPSS (version 27.00, IBM, New York, NY, USA) software. Results were presented as mean ± standard deviation. A normality test was performed, considering p< 0.05 as statistically significant values after ANOVA and Tukey’s post hoc test bivariate correlation testing. 3. Results and Discussion 3.1. Adsorption of Proteins on rGO Surface We began studying the adsorption capacity of rGO to several proteins usually located in FBS. Thus, we observed that rGO adsorption capacity (qe) was enhanced at low protein dose values, indicating the presence of available active groups on the rGO Pharmaceutics 2021,13, 1473 6 of 18 surface. However, no significant modifications in qe values at high initial protein doses (Co) were quantified, suggesting that no further protein loading on the rGO surface was allowed. Among the studied proteins, collagen showed the highest adsorption capacity (qe = 0.022 µg/µg), while other hydrophilic proteins, such as BSA and elastin showed a low affinity for rGO surface (qe between 0.0049–0.0067 µg/µg) (Figure 1a). Pharmaceutics 2021, 13, x FOR PEER REVIEW 6 of 18 3. Results and Discussion 3.1. Adsorption of Proteins on rGO Surface We began studying the adsorption capacity of rGO to several proteins usually located in FBS. Thus, we observed that rGO adsorption capacity (qe) was enhanced at low protein dose values, indicating the presence of available active groups on the rGO surface. However, no significant modifications in qe values at high initial protein doses (Co) were quantified, suggesting that no further protein loading on the rGO surface was allowed. Among the studied proteins, collagen showed the highest adsorption capacity (qe = 0.022 µg/µg), while other hydrophilic proteins, such as BSA and elastin showed a low affinity for rGO surface (qe between 0.0049–0.0067 µg/µg) (Figure 1a). Figure 1. (a). Adsorption capacity (qe) of rGO (250 µg/mL) with initial concentrations of 200 µg/mL BSA, 200 µg/mL elastin and 500 µg/mL collagen after two hours of incubation at 37 °C. (b) Langmuir models for BSA, collagen, and BSA adsorption on the rGO surface. In order to understand the adsorption phenomena involved on rGO surface, Langmuir and Freundlich’s models were applied to the qe values recorded at a constant temperature, calculating the required parameters for the aforementioned models (Table 1 and Figure 1b). Protein adsorption phenomena on the rGO surface was better specified by the Langmuir than the Freundlich model, with R2 values between 0.968 and 0.996 for the Langmuir model and convergence between calculated qmax values and experimental qe results. Therefore, we suggest that adsorption phenomena occur on a homogeneous surface of rGO, with a specific number of adsorption sites on rGO surface binding to protein active sites and forming a monolayer [56]. In fact, Langmuir variable values (RL) < 1 would suggest advantageous adsorption onto the rGO surface for the studied proteins, with irreversibile adsorption for collagen and elastin (RL ≈ 0) [57]. Table 1. Parameters required for Langmuir and Freundlich adsorption isotherms. qe (µg/µg) is the Adsorption capacity at equilibrium, KL (mL/µg) is the Langmuir factor, R2 is the coefficient of determination for the Langmuir model, and qmax (µg/µg) is the maximum adsorption capacity of the proteins by rGO. For the Freundlich model, where KF is the Freundlich constant and m is the intensity adsorption, R2 is the coefficient of determination for the Freundlich isotherm. Langmuir Model Freundlich Model qe µg/µg KL ml/µg RL R2 qmax µg/µg 1/m m Kf R2 BSA 0.0070 0.0297 0.1570 0.968 0.0091 0.345 2.896 0.00197 0.95 Collagen 0.0220 0.0313 0.0660 0.987 0.0230 0.386 2.590 0.00228 0.92 Elastin 0.0049 0.1980 0.0271 0.996 0.0049 0.161 6.184 0.00230 0.83 Figure 1. ( a ). Adsorption capacity (qe) of rGO (250 µ g/mL) with initial concentrations of 200 µ g/mL BSA, 200 µ g/mL elastin and 500 µ g/mL collagen after two hours of incubation at 37 ◦ C. ( b ) Langmuir models for BSA, collagen, and BSA adsorption on the rGO surface. In order to understand the adsorption phenomena involved on rGO surface, Langmuir and Freundlich’s models were applied to the qe values recorded at a constant temperature, calculating the required parameters for the aforementioned models (Table 1and Figure 1b). Protein adsorption phenomena on the rGO surface was better specified by the Langmuir than the Freundlich model, with R 2 values between 0.968 and 0.996 for the Langmuir model and convergence between calculated qmax values and experimental qe results. Therefore, we suggest that adsorption phenomena occur on a homogeneous surface of rGO, with a specific number of adsorption sites on rGO surface binding to protein active sites and forming a monolayer [ 56 ]. In fact, Langmuir variable values (R L ) < 1 would suggest advantageous adsorption onto the rGO surface for the studied proteins, with irreversibile adsorption for collagen and elastin (RL≈0) [57]. Table 1. Parameters required for Langmuir and Freundlich adsorption isotherms. qe ( µ g/ µ g) is the Adsorption capacity at equilibrium, K L (mL/ µ g) is the Langmuir factor, R 2 is the coefficient of determination for the Langmuir model, and qmax ( µ g/ µ g) is the maximum adsorption capacity of the proteins by rGO. For the Freundlich model, where K f is the Freundlich constant and m is the intensity adsorption, R2is the coefficient of determination for the Freundlich isotherm. Langmuir Model Freundlich Model qe µg/µg KL ml/µgRLR2qmax µg/µg1/m m KfR2 BSA 0.0070 0.0297 0.1570 0.968 0.0091 0.345 2.896 0.00197 0.95 Collagen 0.0220 0.0313 0.0660 0.987 0.0230 0.386 2.590 0.00228 0.92 Elastin 0.0049 0.1980 0.0271 0.996 0.0049 0.161 6.184 0.00230 0.83 Studying the adsorption capacity (qt) of rGO over time, we observed a quick adsorption process, completed after 20 min (Figure 2a). The intraparticle-diffusion model was implemented to attain a suitable mechanism that fits the protein adsorption on rGO surface (Figure 2b). However, since qtvs. t 1/2 plotting showed linearity without going across zero, we think that intraparticle-diffusion is not the only process controlling protein adsorption on the rGO surface; film diffusion also contributes to the protein adsorption [ 40 ]. Calcu- Pharmaceutics 2021,13, 1473 7 of 18 lated intraparticle-diffusion parameters, such as the diffusion rate constant (KP) and the impediment layer wideness (C) (Table 2), showed collagen with the highest intra-particle diffusion rate Kp (0.0001 µ g ·µ g −1 min −1/2 ) and boundary layer thickness (0.0132), correlating to a strong hydrophobic attraction between collagen and the hydrophobic rGO surface. This result suggests that π – π bonding between collagen and rGO might also be responsible for this bonding [ 57 ]. However, the hydrophilicity of BSA and elastin would result in the decrease in interference forces and therefore the affinity with rGO. Figure 2. Kinetic protein adsorption models on rGO surface. ( a ) Adsorption capacity over time; ( b ) intra-particle diffusion model plot; ( c ) pseudo-second-order model plot for BSA and collagen; ( d ) nonlinear plot of pseudo-second-order for elastin. Table 2. Calculated parameters from intra-particle diffusion pseudo-first-order and pseudo-second-order model. Calculated parameters from intra-particle diffusion are the intra-particle diffusion rate constant (K p ), constant for the intra-particle diffusion model (C) and coefficient of determination (R 2 ). The presented parameters for the pseudo-first-order are the adsorption capacity at equilibrium (qe), rate constant (K 1 ), and the coefficient of determination (R 2 ). For the pseudo-secondorder model, the parameters are the rate constant (K2) and the adsorption capacity at different times (qt). Intra-Particle-Diffusion Model Pseudo First Order Model Pseudo Second Order Model Kp µg·µg−1min−1/2 CR2K1 min−1 qe µg/µgR2qe µg/µg K2 µg·µg−1min−1R2qt µg/µg BSA 0.00002 0.0037 0.67 22.1 0.0074 0.35 0.009 2.34 0.98 0.0091 Collagen 0.0001 0.0132 0.96 16.3 0.0019 0.65 0.0307 27.27 0.99 0.0294 Elastin 0.00005 0.0004 0.91 67.7 0.0379 0.82 0.0067 2.16 0.93 0.0044 Pharmaceutics 2021,13, 1473 8 of 18 Among adsorption kinetic mathematical models, the low R 2 values calculated in the pseudo-first-order model (0.35–0.82), with vast difference between estimated qe and experimental qt (Table 2), discarded this model for describing adsorption phenomena on the rGO surface. However, the pseudo-second-order model showed high R 2 values (0.93–0.99) and convergence between the estimated qe and the experimental qt values (Table 2); therefore, this model can be considered the best kinetic model to define the studied protein adsorption phenomena on the rGO surface [ 58 , 59 ]. Interestingly, pseudo-secondorder constant K 2 reduced while hydrophilicity increased with the lowest K 2 for elastin (2.16 ·µ g −1· in −1 ) and with the highest for collagen (27.27 µ g ·µ g −1· min −1 ). Representing qt values versus time, while a linear pseudo-second-order plot was more suitable for collagen and BSA (Figure 2c), a nonlinear pseudo-second-order plot was fit for elastin adsorption (Figure 2d) [ 60 ]. Collagen showed a ten-fold higher adsorption rate constant (K 2 ) than BSA and elastin, indicating the highest affinity for rGO than elastin or BSA and due to the strong hydrophobic–hydrophobic interactions between rGO surface and collagen [61]. 3.2. Thermodynamics of Protein Adsorption onto rGO Surface The study of adsorption capacity (q T ) with BSA and elastin on rGO surface increasing temperature revealed the exothermic character ( ∆ H ◦ < 0) of the adsorption (Figure 3a, Table 3). However, collagen showed endothermic adsorption when the temperature was increased ( ∆ H ◦ = 2.44 kJ/mol). We consider that the protein adsorption could start with an endothermic hydration step, followed by exothermic adsorption, but in collagen, there would be a hydration step caused by its hydrophobic nature that requires more energy than the other studied proteins [ 62 ]. This hypothesis would explain why an increase in the temperature would enhance the adsorption capacity of collagen, while it would increase the kinetic energy of BSA and elastin causing their elution from rGO surface. Moreover, the low ∆ G ◦ values (Table 3) indicated the physio-sorption nature of the adsorption [ 58 ], a nonspontaneous phenomenon that is a feature of positive ∆ G ◦ values. It could be attributed to the presence of an energy barrier in the migration of the studied proteins towards the rGO surface, with water forming a hydration shell around the proteins that would hinder their adsorption on rGO. Finally, the remarkable reduction in entropy during the adsorption of the studied proteins [ 58 ] suggests that molecular motion at the solid–liquid interface is more organized [63]. Pharmaceutics 2021, 13, x FOR PEER REVIEW 8 of 18 Collagen 0.0001 0.0132 0.96 16.3 0.0019 0.65 0.0307 27.27 0.99 0.0294 Elastin 0.00005 0.0004 0.91 67.7 0.0379 0.82 0.0067 2.16 0.93 0.0044 Among adsorption kinetic mathematical models, the low R2 values calculated in the pseudo-first-order model (0.35–0.82), with vast difference between estimated qe and experimental qt (Table 2), discarded this model for describing adsorption phenomena on the rGO surface. However, the pseudo-second-order model showed high R2 values (0.93–0.99) and convergence between the estimated qe and the experimental qt values (Table 2); therefore, this model can be considered the best kinetic model to define the studied protein adsorption phenomena on the rGO surface [58,59]. Interestingly, pseudo-second-order constant K2 reduced while hydrophilicity increased with the lowest K2 for elastin (2.16·µg−1·in−1) and with the highest for collagen (27.27 µg·µg−1·min−1). Representing qt values versus time, while a linear pseudo-second-order plot was more suitable for collagen and BSA (Figure 2c), a nonlinear pseudo-second-order plot was fit for elastin adsorption (Figure 2d) [60]. Collagen showed a ten-fold higher adsorption rate constant (K2) than BSA and elastin, indicating the highest affinity for rGO than elastin or BSA and due to the strong hydrophobic–hydrophobic interactions between rGO surface and collagen [61]. 3.2. Thermodynamics of Protein Adsorption onto rGO Surface The study of adsorption capacity (qT) with BSA and elastin on rGO surface increasing temperature revealed the exothermic character (∆H° < 0) of the adsorption (Figure 3a, Table 3). However, collagen showed endothermic adsorption when the temperature was increased (∆H° = 2.44 kJ/mol). We consider that the protein adsorption could start with an endothermic hydration step, followed by exothermic adsorption, but in collagen, there would be a hydration step caused by its hydrophobic nature that requires more energy than the other studied proteins [62]. This hypothesis would explain why an increase in the temperature would enhance the adsorption capacity of collagen, while it would increase the kinetic energy of BSA and elastin causing their elution from rGO surface. Moreover, the low ΔG° values (Table 3) indicated the physio-sorption nature of the adsorption [58], a non-spontaneous phenomenon that is a feature of positive ΔG° values. It could be attributed to the presence of an energy barrier in the migration of the studied proteins towards the rGO surface, with water forming a hydration shell around the proteins that would hinder their adsorption on rGO. Finally, the remarkable reduction in entropy during the adsorption of the studied proteins [58] suggests that molecular motion at the solid– liquid interface is more organized [63]. Figure 3. (a) Influence of temperature on the adsorption of proteins. Where mixtures of 250 µg/mL rGO were suspended either in 50 µg/mL BSA, 200 µg/mL collagen, or 50 µg/mL elastin solution and incubated for 2 h at the following temperatures: 5, 10, 15, 25, 37, and 39 °C. (b) Van’t Hoff linear plot of ln Kd against 1/T for proteins adsorption on rGO. ΔH° was estimated from the slope (= −ΔH°/R) and ΔS° from the y-intercept (= +ΔS°/R). Figure 3. ( a ) Influence of temperature on the adsorption of proteins. Where mixtures of 250 µ g/mL rGO were suspended either in 50 µ g/mL BSA, 200 µ g/mL collagen, or 50 µ g/mL elastin solution and incubated for 2 h at the following temperatures: 5, 10, 15, 25, 37, and 39 ◦ C. ( b ) Van’t Hoff linear plot of ln Kd against 1/T for proteins adsorption on rGO. ∆H◦was estimated from the slope (= −∆H◦/R) and ∆S◦from the y-intercept (= +∆S◦/R). Pharmaceutics 2021,13, 1473 9 of 18 Table 3. Thermodynamic parameters for protein adsorption by rGO: enthalpy of adsorption, ∆ H ◦ (kJ/mol); entropy of adsorption, ∆ S ◦ (kJ/mol · K); Gibbs free energy of adsorption, ∆ G ◦ (kJ/mol); coefficient of determination, R2. ∆H◦ kJ/mol ∆S◦ kJ/mol.K ∆G◦ kJ/mol R2 BSA −40.34 −0.207 23.95 0.99 Collagen 2.44 −0.069 23.68 0.95 Elastin −89.96 −0.358 28.40 0.94 3.3. Surface Chemistry of Protein Adsorbed rGO In order to confirm the adsorption of the studied proteins onto the rGO surface, we studied rGO and protein-adsorbed rGO by Raman spectroscopy and FT-IR. In Raman spectra, G and D bands at ~1595 cm −1 and ~342 cm −1 in rGO Raman spectrum indicated the occurrence of defects due to the reduction process of GO [ 52 ] (Figure 4a), also detected in protein adsorbed-rGO samples (Figure 4b–d). After proteins were adsorbed on the rGO surface, the G and D bands shifted to ~1588–1601 cm −1 and ~1342–1351 cm −1 , respectively. The intensity ratio between those bands (ID/IG) suggested an sp 2 electron distribution in all the samples [ 63 ], being higher than those ID/IG ratios previously described in graphene [ 64 ], and slightly increased when proteins were adsorbed on rGO (Table 4). Finally, the 2D band position and their intensity ratio with G band (2D/G) increase would indicate more structural defects, most likely attributed to protein adsorption on rGO [ 65 ], confirming the adsorption of the studied proteins on the rGO surface [66]. Pharmaceutics 2021, 13, x FOR PEER REVIEW 9 of 18 Table 3. Thermodynamic parameters for protein adsorption by rGO: enthalpy of adsorption, ΔH° (kJ/mol); entropy of adsorption, ΔS° (kJ/mol·K); Gibbs free energy of adsorption, ΔG° (kJ/mol); coefficient of determination, R2. ΔH° kJ/mol ΔS° kJ/mol.K ΔG° kJ/mol R2 BSA −40.34 −0.207 23.95 0.99 Collagen 2.44 −0.069 23.68 0.95 Elastin −89.96 −0.358 28.40 0.94 3.3. Surface Chemistry of Protein Adsorbed rGO In order to confirm the adsorption of the studied proteins onto the rGO surface, we studied rGO and protein-adsorbed rGO by Raman spectroscopy and FT-IR. In Raman spectra, G and D bands at ~1595 cm−1 and ~342 cm−1 in rGO Raman spectrum indicated the occurrence of defects due to the reduction process of GO [52] (Figure 4a), also detected in protein adsorbed-rGO samples (Figure 4b–d). After proteins were adsorbed on the rGO surface, the G and D bands shifted to ~1588–1601 cm−1 and ~1342–1351 cm−1, respectively. The intensity ratio between those bands (ID/IG) suggested an sp2 electron distribution in all the samples [63], being higher than those ID/IG ratios previously described in graphene [64], and slightly increased when proteins were adsorbed on rGO (Table 4). Finally, the 2D band position and their intensity ratio with G band (2D/G) increase would indicate more structural defects, most likely attributed to protein adsorption on rGO [65], confirming the adsorption of the studied proteins on the rGO surface [66]. Figure 4. Raman spectra of rGO before and after the adsorption of (a) BSA (rGO + BSA), (b) collagen (rGO + collagen), and (c) elastin (rGO + elastin). Figure 4. Raman spectra of rGO before and after the adsorption of ( a ) BSA (rGO + BSA), ( b ) collagen (rGO + collagen), and (c) elastin (rGO + elastin). Pharmaceutics 2021,13, 1473 16 of 18 10. Raza, F.; Zafar, H.; Zhu, Y.; Ren, Y.; Ullah, A.; Khan, A.U.; He, X.; Han, H.; Aquib, M.; Boakye-Yiadom, K.O.; et al. 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