Peripheral Neuron Survival and Outgrowth on Graphene

Graphene displays properties that make it appealing for neuroregenerative medicine, yet its interaction with peripheral neurons has been scarcely investigated. Here, we culture on graphene two established models for peripheral neurons: PC12 cells and DRG primary neurons. We perform a nano-resolved analysis of polymeric coatings on graphene and combine optical microscopy and viability assays to assess the material cytocompatibility and influence on differentiation. We find that differentiated PC12 cells display a remarkably increased neurite length on graphene (up to 27%) with respect to controls. Notably, DRG primary neurons survive both on bare and coated graphene. They present dense axonal networks on coated graphene, while they form cell islets characterized by dense axonal bundles on uncoated graphene. These findings indicate that graphene holds potential for nerve tissue regeneration and might pave the road to novel concepts of active nerve conduits.

. (a) Characteristic AFM topography of an intercalated graphene sample, showing atomically flat terraces separated by steps (scale bar: 400 nm). (b) Raman spectrum of an intercalated graphene sample, obtained using a 532 nm laser and a 50x objective lens. The insert shows the single Lorentzian fitting of the 2D peak, with a narrow FWHM of 28 cm-1. (c) 2D peak position (left) and FWHM (right) distribution in a large area (scale bar: 2 µm).

AFM Topography of SiC after three different incubation times with polymeric coatings
AFM analysis of SiC substrates after different polymeric coating show similar topographies with a homogeneous carpet of spots of few nanometers (Fig. S2(e)). The substrates were incubated with the following coating solutions at 37 °C for 1, 4 and 12 h: (a) PLL, 100 µg/ml Poly-L-lysine in water; (b) COLL, 200 µg/ml Collagene Type I in deionized (DI) water; (c) PDL, 30 µg/ml Poly-D-lysine in PBS; (d) PDL/laminin, 30 µg/ml PDL and 5 µg/ml laminin in PBS. Figure S2. AFM topography images of SiC samples after three different times of incubation (1, 4 and 12 h) with a coating solution of: (a) PLL, (b) collagen, (c) PDL, (d) PDL/laminin (scale bar: 500 nm). The insets show phase images of the same areas, which are not sensitive to slow changes in height and improve identification of nanometric structures. (e) All the samples are coated with a homogeneous carpet of spots of few nanometers, as showed in the AFM line profile of a SiC sample after 4 h incubation with PDL/laminin.

Topography and hydrophilicity of gold and glass
Gold (Au) and glass show a relatively high surface roughness already before the coating, with a rootmean-square (rms) roughness comparable to the features of the polymeric layer. However, the variation in the hydrophilicity confirmed the presence of the coating, as shown by the contact angle measurements reported in figure S3(c). Non-coated gold was more hydrophobic than non-coated glass. The coatings had opposite effects on the substrates, increasing hydrophilicity for gold and increasing hydrophobicity for glass. Contact angles were measured using a CAM 101 contact angle meter, from KSV Instruments Ltd. (Finland) and estimated by measuring the angles between the baseline of the droplet and the tangent at the droplet boundary. Figure S3. AFM topography and roughness profiles of gold (a, Au) and nitric-acid-treated glass (b, Glass) before protein coating and after 4h incubation with Poly-L-lysine (4h PLL) and Collagen Type I (4h COLL) (scale bar: 200 nm). Both the surfaces revealed an initial roughness comparable to the one after any coating, preventing the recognition of nanometric details of the coatings. (c) Contact angle measurements of Au and Glass before protein coating and after 4h incubation with Poly-Llysine (PLL) and Collagen Type I (Collagen). All measurements were made using DI water as a probe liquid. Values are the mean ± standard deviation for 3 samples.

AFM Topography of graphene after different coating solutions
We tested the effect of different PDL coating solutions on graphene substrates. We obtained similar network-like structures for all the conditions: (a) PBS and water solution of PDL/laminin (30 µg/ml PDL and 5 µg/ml laminin), (b) PBS solution of PDL alone (30 µg/ml) and (c) water solution of PDL at higher concentration (100 µg/ml).

Topography and hydrophilicity of SiC and graphene
AFM analyses of graphene and SiC substrates evidence a similar surface topography with a comparable rms roughness (Fig. S5(a) and (b)). However, the two substrates present distinct differences in hydrophilicity, with SiC significantly more hydrophilic than graphene, as shown by contact angle measurements (Fig. S5(c)). Figure S5. AFM topography of SiC (a) and bare graphene, G (b) samples, with characteristic line profiles across the terraces. Scale bar: 2µm. (c) Contact angle measurements of silicon carbide (SiC) and graphene (G) before protein coating and after 4h incubation with Poly-L-lysine (PLL) and Collagen Type I (Collagen). All measurements were made using DI water as a probe liquid. Values are the mean ± standard deviation for 3 samples. Non-coated graphene was more hydrophobic than non-coated SiC.

Dorsal root ganglion cell body area
Cell bodies show an increased area with culture time. To estimate the body area, cell bodies were approximated to an oval shape and relative areas were evaluated using ImageJ. Figure S6. Increase of the cell body area with time in dorsal root ganglion (DRG) cells. For cell soma analyses more than 100 cells per sample were analysed.

DRG neurons on coated and uncoated graphene
DRG neurons show a uniform distribution on coated graphene and interconnected cell islets on uncoated graphene, already after 24h from seeding ( Fig. S7(a)). With time, neurons covered homogeneously the coated samples, while cells islets with significant axonal fasciculation were observed on uncoated graphene (Fig S7(c) and (d)). Cell body area, calculated for the isolated cells, was comparable with the one on coated graphene as shown in Fig S7(