Carbon nanotubes prolong the regulatory action of nerve growth factor on the endocannabinoid signaling

Introduction: Carbon nanotubes (CNTs) have shown enormous potential in neuroscience. Nerve growth factor (NGF)-CNTs complex promotes the neuronal growth, however, the underlying mechanism(s) have remained elusive. Based on the interplay between NGF and the endocannabinoid system, involvement of the neuroprotective endocannabinoid, 2arachidonoyl glycerol (2-AG), was investigated in the mechanism of action of NGF. Materials and Methods: Multi-walled CNTs (MWCNTs)-NGF complex was prepared using amino-functionalized COOH-MWCNTs. MWCNTs were characterized by Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). In vitro cytotoxicity was evaluated by MTT assay. Following three times daily intracerebroventricular injections of NGF solution (2, 5, and 10 μg), and 5, 10, and 20 μg of acidor amine-modified MWCNTs, or MWCNTs-NGF complex for either one or 7 days, 2-AG contents were quantified in the frontal cortex and hippocampus of rats by isotope-dilution liquid chromatography/mass spectrometry. Results: FTIR confirmed the amino-functionalization of COOH-MWCNTs and NGF immobilization on the aminated MWCNTs. Aminated MWCNTs and MWCNTs-NGF complex showed less cytotoxicity than COOH-MWCNTs (P<0.05, P<0.01, and P<0.01). Chronic, but not acute, administration of MWCNTs-NGF complex and NGF solution at the highest dose tested led to the elevation of 2-AG at 1 h from the last injection (P<0.01 and P<0.001). 2-AG enhancement induced by MWCNTs-NGF complex lasted for up to 5 and 12 h post-injection (P<0.01 and P<0.001). 2-AG contents remained at the baseline level in the sham and groups receiving vehicle, acidor amine-modified MWCNTs (P>0.05). Conclusion: Functionalized MWCNTs-NGF complex induces a long-lasting increase of brain 2-AG content indicating the efficiency of this nanostructure to provide a sustained concentration of NGF. Implication of 2-AG in the mechanism of action of NGF might be of great therapeutic significance in the neurological disorders. D ow nl oa de d fr om w w w .p hy ph a. ir at 2 0: 07 IR S T o n T hu rs da y O ct ob er 1 3t h 20 16 168 | Physiol Pharmacol 19 (2015) 167-176 Hassanzadeh et al. engineering (Cottrell, 2006; Lindsay, 1994). Despite the therapeutic significance of growth factors, their short half-lives, slow tissue penetration, or vulnerability to a variety of environmental factors (Lindsay, 1994; Pfister et al., 2007) usually limit their effectiveness. During the last decades, outstanding breakthroughs in the emerging field of nanotechnology have resulted to the development of novel theranostic strategies. In order to provide a better efficacy profile and patient compliance, nanoparticulate delivery systems were designed for the delivery of growth factors, however, several disadvantages prevented the effective delivery (Zhang and Uludağ, 2009) leading to the development of more efficient technologies including the advanced nanovectors for the delivery of compounds with short half-life or poor solubility. In this context, carbon nanotubes (CNTs) with immense potential in various scientific fields including the nanomedicine, have attracted a considerable interest for the protection or targeted delivery of a wide variety of compounds (Foldvari et al., 2008). These nanosystems with high thermoelectrical conductivities, superior mechanical properties, improved biocompatibility and solubility, are becoming increasingly attractive for the application in theranostic settings (Cellot et al., 2009; Kam and Dai, 2005). Besides the application for high-resolution and non-invasive imaging, biosensing, tissue engineering and regenerative medicine (Mohammadi et al., 2009; Fabbro et al., 2012), CNTs may be used as the nanoreserviors for the controlled release of drugs or growth factors (Son et al., 2006; Bhirde et al., 2009) that might be of great therapeutic significance. Moreover, CNTs promote neurite outgrowths and modulate the synaptic plasticity (Cellot et al., 2011). Meanwhile, functionalization of CNTs is necessary to improve their solubility, bioactivity, and biocompatibility (Ya-Ping et al., 2002). In a rat model of ischemic brain injury, functionalized CNTs showed greater protective capacity and less adverse effects. These nanomaterials reduced the neuronal apoptotic markers and postischemic inflammation and improved the behavioural functions (Al-Jamal et al., 2011). In the spinal cord injury (SCI), post-treatment with polyethylene glycolfunctionalized single-walled CNTs has been shown to reduce the lesion volume, promote the axonal regeneration, and improve the hind-limb locomotor recovery (Roman et al., 2011) indicating their effectiveness against SCI. The therapeutic potential of functionalized CNTs against stroke and glioblastoma have also been reported (Lee et al., 2011; Zhao et al., 2011). The ability of neurotrophin-coated MWCNTs to promote the neurite outgrowth has been previously reported (Matsumoto et al., 2007). The prototypical neurotrophin, nerve growth factor (NGF), which plays a pivotal role in the survival and maintenance of neurons in the peripheral and central nervous systems, is trophic for the cholinergic neurons which are critically involved in the cognitive processes. Furthermore, NGF has shown therapeutic potential in the neurological disorders such as SCI and Alzheimer’s disease (Huang et al., 2006; Lad et al., 2003). In this respect, MWCNTs-NGF complex has been designed to promote the neurite outgrowth (Chen et al., 2014; Meng et al., 2013), however, the underlying mechanism(s) have remained elusive. In recent years, the interaction between NGF and the endocannabinoid system has been well-documented (Calatozzolo et al., 2007; Hassanzadeh and Rahimpour, 2011; Hassanzadeh and Hassanzadeh, 2011; Hassanzadeh and Hassanzadeh, 2012; Hassanzadeh and Hassanzadeh, 2013). For instance, endocannabinoids have been shown to activate transient receptor potential vanilloid subfamily type 1 (TRPV1) channels (Zhang et al., 2005) and NGF modulates the functions of TRPV1 channel and endocannabinoid signaling (Keimpema et al., 2014). Contribution of endocannabinoid signaling to the formation of neuronal networks and neuroprotective processes has been the focus of intense research. This ubiquitous signaling system is implicated in the survival signaling pathways and plays a pivotal role against the neuronal insult and excitotoxic damage. The endogenous or exogenous cannabinoids have shown neuroprotective effects in a variety of in vivo and in vitro models of neuronal injury (Hassanzadeh, 2014; van der Stelt and Di Marzo, 2005). The therapeutic potential of the endocannabinoid system in neurological diseases has also been well documented (van der Stelt and Di Marzo, 2003; Centonze et al., 2007). The endocannabinoid, 2-arachidonylglycerol (2-AG), due to its involvement in the neuroprotective processes, has attracted a considerable interest. Indeed, the endocannabinoid-induced axonal growth and guidance D ow nl oa de d fr om w w w .p hy ph a. ir at 2 0: 07 IR S T o n T hu rs da y O ct ob er 1 3t h 20 16 Carbon nanotubes, nerve growth factor, and endocannabinoid signaling Physiol Pharmacol 19 (2015) 167-176 | 169 majorly depend on the integrity of 2-AG signaling networks (Panikashvili et al., 2001; Keimpema et al., 2010). Based on this background, we aimed to investigate; i) the suitability of functionalized MWCNTsNGF complex for providing longer-lasting effect of NGF, ii) involvement of 2-AG in the central mechanism of action of NGF. Materials and methods Preparation of MWCNTs-NGF complex Amino functionalization of CNTs improve their dispersibility and reduce the toxicity (Lee et al., 2011; Chen et al., 2014). Meanwhile, we used COOHMWCNTs instead of the direct aminization of MWCNTs as the carboxylation of CNTs before aminization has been shown to enhance the reactivity of CNTs and improve further aminization (Hamdi et al., 2015). Amine-modified MWCNTs-NGF complexes were prepared as previously described in detail (Freitas et al., 2014; Hamdi et al., 2015; Matsumoto et al., 2010) with some modifications. In brief, 500 mg of COOHfunctionalized MWCNTs (Plasmachem GmbH, Berlin, Germany) and 50 ml of 98% SOCl2 (Sigma Aldrich, Germany) were sonicated using ultrasonic system (Tecna 6, Tecno-Gaz, Italy) at 70% amplitude for 40 min and stirred using a magnetic stirrer (IKA, Germany) at 25 ° C for 48 h. The suspension was filtered with 0.45 μm pore-sized microporous membrane (Sartorius, Germany), washed with tetrahydrofuran for 5 times to remove the excess SOCl2, and vacuumed for 25 min at 25 ° C. The residue was reacted with 50 ml of ethylenediamine (EDA) (Sigma Aldrich, Germany) and stirred for 10 h. Afterwards, the suspension was filtered, washed with tetrahydrofuran for 5 times, vacuumed for 25 min, dialyzed in the deionized distilled water using a dialysis bag (MW cut-off 14 KD) for 72 h, and vacuumed to obtain the aminated MWCNTs. In order to prepare amine-modified MWCNTs-NGF complex, rat nerve growth factor (Sigma Aldrich, Germany) in phosphatebuffered saline (PBS) (30 μg/ml) was added to the mixture of aminated MWCNTs and PBS (0.25% w/v), stirred for 24 h at 25 ° C, and centrifuged using the sigma-3k30 centrifuge (Sigma, Germany) at 10,000 rpm for 15 min. Following the supernatant removal, the sample was washed with PBS, re-centrifuged (10,000 rpm for 15 min), and dispersed in 10 ml of PBS. Characterization of the MWCNTs Fourier transform infrared (FTIR) spectroscopy, a powerful tool for the comprehensive characterization of the chemical structures of MWCNTs, was performed using the FTIR spectrophotometer (Shimadzu, Japan). The surface morphology and dispersion of MWCNTs were evaluated by the scanning electron microscopy (SEM, KYKY-EM3200, KYKY Technology Development Ltd., Beijing, China). Cytotoxicity of MWCNTs Cell viability was evaluated by MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (Sigma Aldrich, Germany) colorimetric assay (Mosmann, 1983). Briefly, rat pheochromocytoma PC12 cells (Pasteur Institute, Tehran, Iran) in the phase of exponential growth were seeded in 96-well plates (Nunc, Denmark) at a density of 10 4 cells/well and incubated in a 5% CO2 incubator for 24 h at 37 ° C. The viability of cells exposed to the serial dilutions of MWCNTs in PBS (10, 20, 30, 50, 100, and 200 μg/ml) was evaluated at 1 st , 3 rd , and 7 th day of incubation in 5% CO2 incubator at 37 ° C. In this respect, MTT was dissolved in PBS to provide a stock solution (5 mg/ml) and 20 μl of it was added to each well and incubated for 4 h in a 5% CO2 incubator at 37 ° C. Afterwards, the culture medium was carefully aspirated and 100 μl of dimethyl sulfoxide was added to each well to dissolve the formazan crystals. In order to completely dissolve the crystals, the plate was subjected to low-speed oscillation for 10 min and the absorbance (Abs) was measured at 570 nm using a microplate reader (Anthos 2020, Anthos Labtec Instruments, Austria). Other groups including a blank experimental group (MWCNTs without PC12 cells), a control group (non-exposed cells), and a blank control group (culture medium) were also considered for the calculation of cell viability as Formula 1. The results were reported as the mean±SE of six independent experiments (n=6). Formula 1. Cell viability (%) = Abs of treated group Abs of blank experimental group Abs of control group Abs of blank control group ×100 D ow nl oa de d fr om w w w .p hy ph a. ir at 2 0: 07 IR S T o n T hu rs da y O ct ob er 1 3t h 20 16 170 | Physiol Pharmacol 19 (2015) 167-176 Hassanzadeh et al.