Hybrid AlginateâProtein-Coated Graphene Oxide Microcapsules

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Hybrid Alginate-protein-coated Graphene Oxide Microcapsules Enhance the Functionality of Erythropoietin Secreting C2C12 Myoblasts Laura Saenz del Burgo, Jesús Ciriza, Argia Acarregui, Haritz Gurruchaga, Francisco Javier Blanco, Gorka Orive, Rosa Maria Hernandez, and José Luis Pedraz Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b01078 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on February 5, 2017

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Full-length manuscript

Hybrid Alginate-protein-coated Graphene Oxide Microcapsules Enhance the Functionality of Erythropoietin Secreting C2C12 Myoblasts AUTHORS: Laura Saenz del Burgo†a, Jesús Ciriza†a, Argia Acarreguia, Haritz Gurruchagaa, Francisco Javier Blancob, Gorka Orivea, Rosa María Hernándeza, Jose Luis Pedraz*a.

AFFILIATIONS: a

NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque

Country (UPV/EHU), Paseo de la Universidad 7, 01006, Vitoria-Gasteiz, Spain. Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Vitoria-Gasteiz, Spain b

INIBIC-Hospital Universitario La Coruña, 15006, La Coruña Spain. Biomedical Research

Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), La Coruña, Spain. †

These authors contributed equally

*Corresponding author. E-mail: [email protected] (Jose Luis Pedraz)

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ABSTRACT The beneficial effect of combining alginate hydrogel with graphene oxide (GO) on microencapsulated C2C12-myoblasts viability has recently been described. However, the commercially available GO lacks homogeneity in size, being this parameter of high relevance for the cell fate on two-dimension studies. In three-dimension applications the capacity by this material of binding different kind of proteins can result in the reduction of de novo released protein that can effectively reach the vicinity of the microcapsules. Undoubtedly, this could be an important hurdle on its clinical use when combined with alginate-PLL microcapsules. Here, we demonstrate that the homogenization of GO nanoparticles is not a mandatory preparation step in order to get the best of this material on cell microencapsulation. In fact, when the superficial area of these particles is increased, higher amounts of the therapeutic protein erythropoietin (EPO) is adsorbed on their surface. On the other hand, we have been able to improve even more the favorable effects of this graphene derivative on microencapsulated cell viability by forming a protein bio-corona. These proteins block the potential binding sites of EPO and, therefore, enhance the amount of therapeutic drug that is released. Finally, we prove that these hybrid alginate-protein-coated GO-microcapsules are functional in vivo. KEYWORDS: graphene oxide, cell microencapsulation, alginate, fetal bovine serum, albumin



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1 INTRODUCTION Cell microencapsulation represents one of the strategies committed to the development of artificial three dimensional (3D) scaffolds in order to give support for the growth and maintenance of living cells. This technology consists in the immobilization of cells within a hydrogel-based matrix, usually surrounded by a polymeric membrane. This membrane will control the passage of nutrients and oxygen to the core of the capsule while permitting the release of the therapeutic products produced de novo by the enclosed cells. At the same time, this system protects the cells from the external environment avoiding the entrance of high molecular weight immune system components such as immunoglobulins and immune cells.1 Therefore, this scaffold provides an adequate environment for the maintenance of different kind of therapeutically active cells and can be used for both drug and cell delivery in the treatment of several disorders and the regeneration of tissues.2-10 Alginate has been considered as the biomaterial of choice for this technology and it is possible to find many studies in the literature analyzing the viability of different kind of cells enclosed in alginate microbeads. However, differences between the chemical structure, viscosity, concentration, cell density or encapsulation method used, might have a great impact on microcapsule porosity, and consequently, permeability, its mechanical strength and also, cell viability levels. All this makes it difficult to compare results among different laboratories. Thus, it is necessary to provide internal controls in order to analyze any improvement that changes on the traditional plain alginate microcapsules might have on cell viability. Unfortunately, alginate is an inert polymer that is unable to provide cell–matrix interactions as, inherently, it lacks cell-adhesivity.11 Consequently, the tailoring of this material in order to create a more bio-mimetic support for the enclosed cells is one of the main areas of interest in the field. In this sense, the modification of this scaffold with integrin-mediated cell adhesion sequences, such as arginine-glycine-aspartic acid (RGD, derived from fibronectin), has improved the viability of the cells and, therefore, the functionality of the implant in vivo.12,

13

Moreover, the RGD moieties 3

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showed to enhance the mechanical properties and resistance of the capsule, another characteristic that need to be optimized in order to enhance the biosecurity of the grafts.13 Importantly, chemical modification gives a huge potential for tailoring different types of alginates for specific clinical applications. However, these modifications entail a significant knowledge of chemistry. As a result, it can be propose to combine alginate with other compounds in order to form alginate composites that provide some of the interesting adhesion properties that RGD has shown in this cell encapsulation technology. In the search of other materials beneficial for the improvement of the cell microencapsulation technology, graphene and its derivatives appear as good candidates, because of their exceptional mechanical properties. In fact, they could, potentially, reinforce the physical characteristics of different materials such as alginate hydrogels. Mostly, these materials have been applied in electronics, optics and energy because of their excellent chemical and physical properties.14-16 However, due to their tunable characteristics they are expected to have a big impact also in biomedicine, including approaches related to drug delivery, gene delivery and contrast imaging.17-20 Nevertheless, the published biological effects of these novel carbon materials have shown, sometimes, to be conflicting.21,

22

While some of these studies did not detect particular cellular

alterations, some others have observed that these materials could induce cellular damage.23-26 One of the most studied graphene derivatives is graphene oxide (GO). GO is usually synthesized by oxidation and exfoliation of natural graphite powder with various oxidants in acidic media. The typical method of synthesizing GO was developed by Hummers and co-workers.27 This material presents many oxygen containing functional groups providing a hydrophilic surface and, therefore, the possibility of forming stable dispersions in water.28,

29

Besides, this characteristic enables

different biochemical reactions to occur on its basal plane as well as on its edges.30 On the other hand, fascinating properties of graphene regarding cell adhesion and proliferation on 2D cultures of different types of cells have also been reported.31-37 Moreover, this material has shown to decrease



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the swelling effect by sodium citrate on alginate-poly-L-lysine-alginate (APA) microcapsules, indicating a higher resistance against osmotic changes, and also an enhancement on their apparent rigidity due to an interaction between alginate and GO particles.18, 38, 39 Based on our experience, the valuable effect of GO on microencapsulated C2C12 myoblasts viability depends on the concentration to which the cells are exposed.18 However, some other parameters such as the dimension of the nanoparticles or their surface chemistry could also have a great impact on their biological effects.40 In fact, in 2D cultures, GO particles interact with the mouse mesenchymal progenitor C2C12 cell membrane in a size-dependent manner, being the cellular uptake mechanisms phagocytosis for large particles and clathrin-mediated endocytosis for smaller size sheets.41 Moreover, the induction of oxidative stress on A549 cells has been reported as doseand size-dependent even when GO shows good biocompatibility.42 Unfortunately, GO sheets show a wide size distribution after the synthesis and several approaches are being developed to narrow this heterogeneity in size.43 Thus, our first goal was to homogenize the particle size of GO nanoparticles to clarify the size effect on alginate microencapsulated erythropoietin (EPO) producing C2C12 myoblasts (C2C12-EPO) viability and therapeutic drug production and release. Previously, we described the ability of GO particles to adsorb the therapeutic protein EPO, probably by electrostatic and hydrogen bonding through their oxidized groups and the extensive surface defects of GO sheets that could serve as biding sites. The adsorption reduced the amount of synthesized protein released to the vicinity of microcapsules when co-encapsulated with C2C12EPO.18 Undoubtedly, when the use of GO nanoparticles in combination with cell microencapsulation would be translated into clinical relevant situations, this effect would be a significant problem to be overcome. Therefore, our second goal was to investigate if the preincubation of GO with different protein solutions could be enough to form a protein bio-corona that would avoid the adsorption of EPO and, consequently, allow the release of this therapeutic drug from the alginate scaffolds.


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Our group has previously shown how alginate-PLL microcapsules with a diameter of 350 µm containing C2C12–EPO myoblasts are able to increase the hematocrit of rodents after subcutaneous implantation.6, 44 However, for taking this microencapsulation technology into the clinic, in some cases, smaller capsules with similar potency that can be implanted into more delicate tissues or organs, where the volume of the implant is the limiting factor, could broaden the possibilities of this technology.45 Thus, our final goal was to prove if smaller diameter microcapsules containing GO nanoparticles and C2C12-EPO myoblasts could enhance the hematocrit levels on mice, which will prove the functionality of the graft and therefore, the beneficial use of this new material on cell microencapsulation technologies.



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2 MATERIAL AND METHODS 2.1 Biomaterials GO slurry 3% (w/w) was obtained from Graphenea (Spain). The product was suspended either in water, fetal bovine serum (FBS) or 10 mg/ml of bovine serum albumin (BSA) and sonicated for 1 hour in order to obtain a higher percentage of monolayer flakes. Ultra pure low-viscosity and high guluronic (LVG) acid alginate was purchased from FMC Biopolymer (Norway). Poly-L-Lysine hydrobromide (PLL, 15-30 kDa) and BSA were purchased from Sigma-Aldrich (St Louis, MO). FBS was purchased from Life Technologies (Carslbad, CA). 2.2 Microfluidization procedures GO at concentrations of 100 and 250 µg/ml and suspended either in water, FBS or 10 mg/ml BSA, was crushed using a LV1 Low Volume Microfluidizer (Microfluidics). GO suspensions went through a microfluidized treatment either once, twice or three times using 1000 or 1500 psi. Treated suspensions were immediately used for EPO adsorption studies, particle size measurement and/or microencapsulation procedures. For aggregation studies, suspensions were stored up to 48 hours at 4oC. 2.3 Particle size and zeta potential measurements The mean particle size and the polydispersity index (PDI) of GO suspensions either in water, FBS or 10 mg/ml BSA were analyzed by Dynamic Light Scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments), to ascertain the impact of microfluidization on GO particles breakdown, as well as aggregation after 48 hours. The particle size reported as the hydrodynamic diameter was obtained by cumulative analysis. Zeta potential was obtained by Laser Doppler Velocimetry (LDV) using the Zetasizer Nano ZS. The Smoluchowski approximation was used to support the calculation of the zeta potential from the electrophoretic mobility. Only data that met the quality criteria


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according to the software program DTS 5.0 were included in the study. All measurements were carried out in triplicate. 2.4 Transmission electron microscopy (TEM) Samples were diluted in mili-Q H2O (1:50 for non- microfluidized samples and 1:10 for microfluidized samples) and mixed properly in a vortex. Then, 5 µl of each sample were deposited on Carbon coated Copper grids and dried overnight. Samples were visualized in a JEOL JEM 1400 Plus TEM at 120kV and images were acquired with a Hamamatsu Flash sCMOS digital camera 2.5 EPO adsorption assay For EPO adsorption studies, similar volumes of a solution of 100 mIU/ml of recombinant EPO and 100 µg/ml of GO processed or not through microfluidization and suspended either in water, FBS or 10 mg/ml BSA were incubated in rotation for 24 hours. Next, samples were spun for 30 minutes and supernatants were collected to quantify the non-adsorpted EPO with the Quantikine IVD EPO ELISA kit (R&D Systems). All samples and standards were measured at least in duplicate. A minimum of three independent experiments were analyzed for each condition. The percentage of adsorbed EPO was estimated as follows: Adsorbed EPO (%) = [(Initial quantity of EPO–Final quantity of EPO)/Initial quantity of EPO]x100 2.6 Cell culture conditions Murine C2C12 myoblasts genetically engineered to secrete EPO (C2C12-EPO) were grown in Tflasks with Dulbecco´s modified Eagles´s medium (Gibco) supplemented with 10% FBS, 2 mM Lglutamine (Gibco) and 1% antibiotic/antimycotic solution (Gibco) at 37ºC in humidified 5% CO2/95% air atmosphere. Cells were passaged every 2-3 days. 2.7 Cell microencapsulation procedure



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Sterile 1.87% (w/v) alginate solution were prepared by dissolving LVG alginate in a 1% (w/v) mannitol solution and filtered through a 0.20 µm syringe filter (Millipore, MA, USA). GO was suspended in sterile water, FBS or 10 mg/ml BSA at a concentration of 250 µg/ml and sonicated for 1 hour. GO suspensions were mixed with the 1.87% alginate solution obtaining a final concentration of 1.5% alginate and 50 µg/ml GO. Murine C2C12-EPO cells were harvested using 0.25% trypsin-EDTA (Life Technologies, Carslbad, CA) and resuspended into the aforementioned sterile 1.5% alginate solutions containing 50 µg/ml GO at a density of 4x106 cells/ml. As controls, C2C12-EPO cells were resuspended into a 1.5% alginate solution without GO. These cell suspensions were extruded through a disposable nebulizer using a 5 ml sterile syringe in a pneumatic atomization generator (Bioencapsulation portable platform Cellena®, Ingeniatrics Technologies, Spain). The resulting alginate microbeads were maintained in agitation for 10 minutes in a 55 mM CaCl2 solution for complete ionic gelation. Afterwards, microcapsules were electrostatically coated with 0.05% (w/v) PLL for 5 minutes and coated with 0.1% (w/v) LVG alginate for another 5 minutes. All microcapsules were prepared at room temperature, under aseptic conditions and were cultured in C2C12 medium. The diameters (160 ± 20 µm) and overall morphology of microcapsules were characterized using an inverted optical microscopy (Nikon TSM). 2.8 Early apoptosis quantification and cell viability studies Early apoptosis of C2C12–EPO myoblasts microencapsulated with alginate and 50 µg/ml of GO suspended in either water, FBS or 10 mg/ml BSA was quantified with the Annexin-V-FITC apoptosis

Detection

Kit

(Sigma-Aldrich)

at

days

1,

10

and

20

post-encapsulation.

Microencapsulated C2C12-EPO myoblasts in alginate matrices without GO were studied as controls. Briefly, 250 µl of microcapsules (106 cells) were incubated with alginate lyase (Sigma-Aldrich) for 30 minutes at 37oC to release the C2C12-EPO myoblasts. Next, cells were rinsed twice with DPBS, resuspended in 10 mM HEPES/NaOH containing 0.14 M NaCl and 2.5 mM CaCl2 (binding buffer, 9 ACS Paragon Plus Environment

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pH 7.5) and stained with annexin V-FITC and propidium iodide for exactly 10 minutes at room temperature protected from light. Fluorescence was determined immediately with a BD FACS CaliburTM flow cytometer (BD Biosciences). Unstained samples or samples stained only with annexin V-FITC or propidium iodide were used to establish the appropriate acquisition parameters for the analyzed samples. For cell viability studies by flow cytometry, the same samples and controls were quantified with the LIVE/DEAD® Viability/Cytotoxicity Kit (Invitrogen™) at days 1, 10 and 20 postmicroencapsulation. Cells were released from microcapsules by incubation with alginate lyase for 30 minutes at 37oC, and stained with 100 nM calcein AM and 8 µM ethidium homodimer-1 solution for 20 minutes at room temperature, protected from light. Fluorescence was determined immediately with a BD FACS Calibur flow cytometerTM. Unstained samples or samples stained only with 100 nM calcein AM or 8 µM ethidium homodimer-1 were ran as controls to establish the appropriate acquisition parameters for the analyzed samples. For cell viability studies by fluorescence microcopy, samples were also stained with the LIVE/DEAD® Viability/Cytotoxicity Kit. First, microcapsules were washed four times in a test tube with DPBS and allowed to sediment. Then, they were mixed with the optimal studied concentration of dies (0.5 µM calcein AM and 0.5 µM ethidium homodimer-1 in DPBS) and placed on 96-well plates. After incubation at room temperature for 40 minutes in the dark, samples were observed under a Nikon TMS microscope with the excitation/emission settings for calcein AM (495/515 nm) and ethidium homodimer (495/635 nm). At least three independent experiments were analyzed for each condition. 2.9 Metabolic activity, membrane integrity and EPO secretion studies The metabolic activity, the membrane integrity and the EPO secretion of encapsulated C2C12–EPO myoblasts in alginate and 50 µg/ml of GO suspended in water, FBS or 10 mg/ml BSA was



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determined at day 1, 10 and 20 post-encapsulation and compared with cells encapsulated in alginate without GO as controls. For metabolic activity 5 µl of microcapsules per well were placed in 100 µl of medium on 96-well plates. Afterwards, 10 µl of Cell Counting Kit-8 (CCK-8) solution (Sigma Aldrich) were added to each well. Plates were incubated inside a humidified chamber for 4 hours at 37ºC and read on an Infinite M200 (TECAN Trading AG, Switzerland) microplate reader at 450 nm with reference wavelength set at 650 nm. At least five wells were placed for each condition. The membrane integrity was determined with the in vitro toxicology Lactic Dehydrogenase (LDH) based Assay Kit, (Sigma-Aldrich). 500 µl of medium was incubated with 50 µl of microcapsules for 24 hours and 50 µl of supernatants were collected to determine the amount of released LDH. At the same time, 50 µl of microcapsules were incubated for 24 hours with 500 µl of medium and lysed to determine the total LDH activity. All collected media were subjected to enzymatic analysis based on the reduction of NAD by LDH and its further reaction with tetrazolium dye by following manufacture´s recommendations. The resulting colored compound absorbance was read out on the Infinite M200 microplate reader at a wavelength of 490 nm, with absorbance reading at 690 nm as background. For EPO secretion determination, 12-well plates were plated with 100 µl of microcapsules in 1 ml of media and supernatants were collected after 24 hours. The amount of released EPO was assayed with the Quantikine IVD EPO ELISA kit (R&D Systems). All samples and standards were measured at least in duplicate. Also, three independent experiments were analyzed for each experiment. 2.10 In vivo assay Syngeneic six weeks old C3H mice were purchased from Janvier Labs (France) and housed with sterile feed and autoclaved water. Four different groups of microcapsules were produced and 11 ACS Paragon Plus Environment

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implanted into animals: alginate-PLL microcapsules without cells (empty microcapsules), alginatePLL microcapsules containing C2C12 myoblasts no secreting EPO (C2C12), alginate-PLL microcapsules containing 50 µg/ml of GO suspended in FBS and C2C12 myoblasts secreting EPO (GO-FBS C2C12-EPO), and alginate-PLL microcapsules containing 50 µg/ml of GO suspended in 10 mg/ml BSA and C2C12 myoblasts secreting EPO (GO-BSA C2C12-EPO). Nine animals per group were implanted subcutaneously, using an 18-gauge catheter, with 200 µl of microcapsules prepared the day before implantation and suspended in phosphate-buffered saline solution (PBS) in a final volume of 1 ml. During the procedure all animals were maintained under anesthesia by isofluorane inhalation. Blood samples were collected weekly from the submandibular area with heparinized capillary tubes (Deltalab). Then, capillaries were spun down at 760xg for 15 minutes. Hematocrits were determined using a standard microhematocrit method and expressed as mean + standard deviation. Animals were sacrificed at 3 and 6 days, and 2 months after implantation to retrieve microcapsules. All the experimental procedures were performed in compliance with protocols approved by the institutional animal care and use committee of the University of Basque Country UPV/EHU (Permit Number: CEEA/360/2014/Ciriza Astrain). 2.11 Histological and macroscopical analysis: evaluation of the immune reaction At 6 days and 2 months after implantation, three animals from each group were sacrificed, and the implants were retrieved and fixed in a 4% paraformaldehyde solution for histological analyses. The overall evaluation of the immune reaction toward transplanted microcapsules was performed in a blind way by two different pathologists and from each treatment group. Serial horizontal cryostat sections (14 µm) were processed for hematoxylin and eosin (H&E) staining. Photographic images were taken using a Nikon D-60. 2.12 Statistical analysis Statistical analysis was performed using SPSS software, version 21.00.1, or GraphPad Prism 5.01 (GraphPad Inc., San Diego, CA). Data are expressed as mean ± standard deviation. Values with p< 12 ACS Paragon Plus Environment


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0.05 were considered significant for comparison of groups using ANOVA and Tukey's Post Hoc Test.


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3 RESULTS AND DISCUSSION 3.1 Homogenization and size reduction of GO particles Commercially available GO particles, when diluted and slightly sonicated, showed an average surface size of 1535.33 ± 435.31 nm as previously described.18 However, the dispersion on this dimension was high, which could affect the way these flakes interact with the co-encapsulated cells. We wanted to determine the effect that different GO sizes might have on encapsulated myoblasts (3D scaffold). In fact, there are contradictory results in the bibliography regarding this effect on 2D cell cultures. Some authors have claimed that micro-sized GO led to higher cytotoxicity than nanosized GO due to a faster sedimentation rate that reduces nutrient availability for cell growth in 2D cultures.46 Also, these micro-sized GO particles were able to induce a stronger inflammatory response on macrophages.23 However, there are many procedures for evaluating the GO cytotoxicity in vitro and also, different ways of preparing the GO particles.42, 47 Although, inside our alginate-PLL microcapsules, GO and cells will have less direct contact as a consequence of being suspended in a gelified matrix, we attempted to reduce and homogenize the particle size of the commercial concentrated GO through microfluidization procedures. GO flakes were crushed by passing once, twice or three times through a LV1 Low Volume Microfluidizer at pressures of 1000 or 1500 psi. As Figure 1A shows, the non-microfluidized GO suspension showed a high heterogeneity in size, which was confirmed by Transmission Electron Microscopy (TEM) images (Supplementary Figure 1). However, after going through the microfluidizer, GO particle size was reduced up to 5 times ending up with size orders around 300 + 7 nm, independently of the pressure applied or the number of times passed through the microfluidizer system (see Supplementary Figure 1). Moreover, the homogeneity of the particles increased significantly. On the other hand, the polydispersity index (PDI) was also reduced at least



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1.5 times by all the pressure conditions and number of passes tested, although no significant differences were detected when compared with non-crushed GO at day 0 (Figure 1B). As specified by the manufacturer, at a concentration of 4 mg/ml, GO flakes tend to agglomerate requiring dilution followed by slight sonication in order to obtain a higher percentage of monolayer flakes. Therefore, we stored microfluidized GO suspensions for 48 hours and studied their agglomeration properties compared with non-crushed GO particles. As expected, non-crushed GO flakes agglomerated and almost doubled in size as observed with the Zetasizer Nano ZS (Figure 1A, day 2). However, homogenized GO suspensions kept the same particle size with no evidence of significant particle agglomeration (Figure 1A, day 2). Size homogeneity and PDI were maintained in similar values after 48 hours of storage at all the microfluidized conditions tested and statistically significant differences (p

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