The mixture was stirred for 6 h at room temperature following the second time addition of EDC (1 mg/ml) after being sonicated with PEI (2.5 mg/ml) for 5 min. efficacy against HCC both and in combination with the chemotherapeutic drug sorafenib. We found that NGO-PEG-PEI significantly enhanced the cellular uptake of C6-ceramide. By investigating the mechanism of cellular delivery, we determined that the internalization of NGO-PEG-PEI/Cer progressed primarily via a clathrin-mediated mechanism. The combination of NGO-PEG-PEI/Cer and sorafenib exhibited synergy between these two drugs. Further work revealed that NGO-PEG-PEI/Cer may play a role in subverting multidrug resistance (MDR) in HCC cells by inactivating HOE 32020 MDR and Akt signaling. NGO-PEG-PEI/Cer also significantly inhibited tumor growth and improved survival times and suppresses xenograft tumor growth (Tagaram et al., 2011), exerting an inherent tumor-killing effect. However, ceramide is highly hydrophobic, which largely limits its application is closely related to its surface functionalization (Hu et al., 2011). Zhang et SRA1 al. developed DOX-loaded NGO-PEG (Polyethylene Glycol) as a strategy for chemo-photothermal synergistic therapy in one system, which significantly enhanced the therapeutic efficacy of cancer treatment and (Zhang W. et al., 2011). NGO has great potential for use as delivery vehicles designed to enhance cancer treatment, So our collaborator developed PEG and PEI (Polyethylenimine) co-conjugated ultra-small nano-GO (NGO-PEG-PEI) as a novel gene delivery carrier, and found that it showed excellent stability against salts HOE 32020 and serum (Feng et al., 2013). In the present study, we used these nanoparticles for loading C6-ceramide, and we found that this formulation allows C6-ceramide to travel through the bloodstream and target tumor cells via enhanced cellular permeability and retention, facilitating its potential clinical use as a novel therapeutic strategy. Additionally, through and studies we also investigated the antitumor efficacy and molecular mechanisms of NGO-PEG-PEI/Cer combined with other chemotherapy drugs in HCC. Materials and Methods Synthesis and Characterization of NGO-PEG-PEI/Cer NGO-PEG-PEI was kindly provided by Dr. Kai Yang at the School of Radiation Medicine and Protection (SRMP) of Soochow University (Suzhou, China). Briefly, GO was obtained by oxidation of graphite following the modified Hummers method. Preparation of NGO-PEG-PEI was performed according to previous description (Feng et al., 2013). A mixture of GO solution (0.5 mg/ml) with 6-armed amine-terminated PEG (0.5 mg/ml) was under sonication for 5 min. Then EDC (0.5 mg/ml) was added, after another 5 min sonication, the mixture was stirred gently for 10 min at room temperature. The mixture was stirred for 6 h at room temperature following the second time addition of EDC (1 mg/ml) after being sonicated with PEI (2.5 mg/ml) for 5 min. After that, the mixture was washed with deionized water by 100 nm Milli-Q membrane filter (Millipore, Bedford, MA, United States) 3 times, and we obtained NGO-PEG-PEI re-suspended in water. NBD C6-ceramide (6-((N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)amino)hexanoyl)Sphingosine) (N1154, Thermo Fisher Scientific, MA, United States) solution with gradient concentration was prepared and its absorbance at 536 nm was measured. The standard curve was drawn according to different concentrations. Then C6-ceramide was mixed with a certain concentration of NGO-PEG-PEI solution in equal volume and oscillated overnight. After centrifuging for 30 min at 8000 rpm, the absorbance of supernatant was determined, and the concentration of free drug in supernatant was obtained according to the standard curve. Then NGO-PEG-PEI/Cer was prepared according to the maximum loading of C6-ceramide carried by NGO-PEG-PEI. After loadinging the C6-Ceramide with NGO-PEG-PEI, PBS was added to make the final volume of 1.0 ml. The average size and zeta potential of the NGO-PEG-PEI/Cer complex were then measured with dynamic laser scattering (DLS) and a Zetasizer 3000HS particle analyzer (Malvern Instrument Inc., Worcestershire, United Kingdom), respectively. The sizes and zeta potential values were presented as the average values of three measurements. Cell Culture and Maintenance The human HCC cell lines HepG2, HuH7, and HOE 32020 PLC/PRF/5 were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). HuH7-SR is sorafenib-resistant HuH7 cell line, which was retained in our lab. All the cell lines were authenticated by short-tandem repeat profiling and cultured in Dulbeccos Modified Eagles Medium (GIBCO, Carlsbad, CA, United States) supplemented with 10% heat inactivated fetal bovine serum (GIBCO). Cells were incubated in a 5% CO2 humidified incubator at 37C. Cell Uptake of NGO-PEG-PEI/Cer.
The mixture was stirred for 6 h at room temperature following the second time addition of EDC (1 mg/ml) after being sonicated with PEI (2