Introduction
In view of the growing interest in using graphene-based materials (GBMs) for biomedical applications, it is paramount to evaluate and improve their biocompatibility.[1] Covalent and non-covalent surface modification with polymers is a strategy to overcome possible toxicity of GBMs. Covalent functionalization often implies using toxic solvents, while procedures for non-covalent surface modification are simple and easily up-scalable.[2] This work aims to study the effect of graphene nanoplatelets (GNP) non-covalent surface modification with biocompatible polymers on biocompatibility.
Materials and Methods
Polymers (poly(vinyl alcohol) - PVA, hydroxyethyl cellulose, poly(ethylene glycol), poly(vinyl pirrolidone), chondroitin, glucosamine, hyaluronic acid) and GNP-C (750, XG Sciences) were dispersed in water (1:1 ratio) by sonication, centrifuged and supernatant discarded, removing excess polymer. Physico-chemical characterization was performed by Scanning electron microscopy (SEM), Dynamic Light Scattering (DLS), X-ray photoelectron spectroscopy (XPS), Raman Spectroscopy and Thermogravimetric analysis (TGA). Hemolysis was evaluated for different concentrations of materials, as well as biocompatibility towards human fibroblasts (HFF-1) - cell metabolic activity (resazurin), viability (LIVE/DEAD), membrane damage (TEM), and ROS production (DCFH-DA).
Results and discussion
Materials were characterized, but results for GNP-C modified with PVA are highlighted, since they unveiled the best biocompatibility. SEM images reveal PVA forms a film at GNP-C surface. DLS shows surface adsorption of PVA increases GNP-C average particle size from 0.5-2 to 25 µm, due to encapsulation and induction of sheet interactions, leading to agglomeration. XPS, Raman and TGA confirm the presence of 21% PVA at GNP-C surface. Hemolysis induced by all GNP-polymer is below 1.7%, in concentrations up to 500 µg/mL (3h). Resazurin assay show 50 µg/mL GNP-C causes toxicity towards HFF-1 at 24h, which decreases at 48 and 72h. GNP-C-PVA is non-toxic up to 100 µg/mL until 72h. LIVE/DEAD assay show that at 72h, 20 and 50 µg/mL GNP-C-PVA induce significantly lower cell death than GNP-C. For 50 µg/mL, ROS production is increased by 4.4 fold when GNP-C contacts cells, while only by 3.3 fold for GNP-C-PVA. TEM images show GNP-C was almost completely exfoliated interacting with plasma membrane, being internalized without membrane damages, and in some cases being found interacting with mitochondria. GNP-C-PVA was more agglomerated, presenting larger volume than GNP-C, being found more often outside plasma membrane.
Conclusions
Polymer adsorption decreases hemolysis of GNP-C. Small sized GNP-C enter cells inducing ROS production, and therefore, toxicity. PVA encapsulation of GNP-C increases particle size, decreasing internalization and avoiding ROS production. Thus, GNP-C-PVA has potential for use as coating or filler of medical devices, with the purpose of improving mechanical/thermal properties, while reducing acute toxicity.
Foundation for Science and Technology - PhD grant SFRH/BD/86974/2012
References:
[1] Pinto AM, Gonçalves IC, Magalhães FD. Graphene-based materials biocompatibility: A review. Colloids and Surfaces B: Biointerfaces 2013, 111, 188–202.
[2] AM Jason A, RD William. Noncovalent Functionalization of Graphene by Molecular and Polymeric Adsorbates. Journal of Physical Chemistry Letters, 2013, 4, 2649–2657.