Scale club represents 100?nm. by OX26 and CTX adjustment. Our outcomes also demonstrated that OX26 endowed the PLs using the transportation ability over the BBB. Using the BMVECs/C6 cells co-culture model, the viability of C6 cells was reduced to 46.0% after OX26/CTX-PL/pC27 transfection. The OX26/CTX-PL/pC27 complexes exhibited enhanced therapeutic effects on C6 cells. Moreover, the dual-targeting therapeutic effects were further conformed with diminished tumor volumes (18.81??6.15?mm3) and extended median survival time (46?days) in C6 glioma-bearing rats. Immunohistochemical analysis revealed the therapeutic effects derived from enhanced hTERTC27 expression in the tumor site. Conclusions The PEGylated liposomes altered with OX26 and CTX are able to significantly promote cell transfection, increase the transport of plasmid DNA across the BBB and afterwards target the brain glioma cells and BBB model was established and confirmed by the permeation experiment and transendothelial electrical resistance (TEER) values ( 250 cm2). No obvious reduction of TEER values was observed during the experiment, indicating that transport of pC27 did not disrupt the BBB barrier. Physique?4A showed the transport ability of different lipoplexes across BBB model at the same concentration of pDNA. The transport ratios were 3.23% for PL/pC27, 6.50% for OX26-PL/pC27, 6.42% for OX26/CTX-PL/pC27, 3.08% for PL/pC27 preconditioned with OX26, 3.00% for OX26-PL/pC27 preconditioned with OX26, 3.13% for OX26/CTX-PL/pC27 preconditioned with OX26 at Sulfaphenazole 4?h, respectively. When the model was pre-incubated with free OX26 MGC102953 to saturate the TfR around the BBB, the transport ratios of OX26-PL/pC27 and OX26/CTX-PL/pC27 decreased significantly, which displayed no difference with that of PL/pC27. There was no obvious alternation for PL/pC27 preconditioned with and without OX26. The results revealed OX26 modification around the PL/pDNA complexes played a critical role in the transport assay while CTX did not exert effect on the transport across BBB, and the increased transport ability of the PL altered with OX26 may be mediated by TfR. Our results were supported with the research that this TF-conjugated liposomes were able to increase the delivery of drug across the BBB mediated by TfR [40]. Open in a separate window Physique 4 Transport ratios across the BBB and dual-targeting effects (Physique?5A?~?F). Sulfaphenazole The average tumor volumes on day 18 were 53.61??3.71?mm3 for PBS group, 47.1??3.02?mm3 for OX26/CTX-PL/pEGFP group, 44.87??3.12?mm3 for PL/C27 group, 33.49??2.83?mm3 for OX26-PL/pC27 group and 18.81??6.15?mm3 for OX26/CTX-PL/pC27 group, respectively. As shown in Physique?5G, the OX26/CTX-PL/pC27 therapy significantly diminished the tumor size when compared with the controls (p? ?0.01), PL/pC27 (p? ?0.01) and OX26-PL/pC27 therapy (p? ?0.05). On the contrary, OX26/CTX-PL/pEGFP did not exhibit inhibitory effects around the tumor, compared with the PBS control (p? ?0.05). Treatment effects of different PL complexes were also reflected by the Kaplan-Meier survival curves (Physique?5H). The median survival time of rats treated with OX26/CTX-PL/pC27complexes (46?days) was significantly longer than that of rats treated with PBS (13?days, p?=?0.000), OX26/CTX-PL/pEGFP (14?days, p?=?0.000), PL/pC27 (21?days, p?=?0.002) and OX26-PL/pC27 (29?days, p?=?0.038), respectively. These results indicated that OX26-PL/pC27 was able to improve the treatment efficacy while the dual-targeting OX26/CTX-PL/pC27 led to the most significant tumor inhibition and the most significant improvement in the median survival time of tumor-bearing rats. These findings offered the Sulfaphenazole strong evidence for the dual-targeting therapeutic effects. Open in a separate window Physique 5 Dual-targeting effects of Sulfaphenazole OX26/CTX-PL/pC27 complex and survival monitoring and and therapeutic efficacy in brain glioma-bearing rats. The ligand OX26 plays a critical role in transporting the lipoplexes across the BBB, and CTX functions as a major role in targeting brain glioma cells. Furthermore, OX26 also contributes to the glioma-targeting effect of the lipoplexes. The results would encourage further developments for non-invasive targeting therapy of brain gliomas by intravenous injection. Materials and methods Materials Soybean phosphatidylcholine (S100-PC) was purchased from Lipoid GmbH (Ludwigshafen, Germany). DC-chol, 2-Iminothiolane hydrochloride (2-IT, Trauts reagent), CTX and sulforhodamine B (SRB) were provided by Sulfaphenazole Sigma-Aldrich (Saint Louis, MO, USA). Mal-PEG2000-DSPE was obtained from Avanti Polar Lipids (Alabaster, AL, USA). Anti-Transferrin Receptor antibody (OX-26) and anti-Telomerase reverse transcriptase antibody were purchased from Abcam (Cambridge, UK). Sephadex G-50 and Sepharose CL-4B were provided by Science and Technology Development Co., Ltd. (Beijing, China). Centriprep-10 concentrators were purchased from Millipore Amicon (Bedford, MA, USA). NanoOrange? Protein Quantitation Kit was purchased from Invitrogen (California, USA). YOYO-1 and LysoTracker Red were purchased from Molecular Probes (Eugene, OR, USA). pIRES2-EGFP (pEGFP) and pIRES2-EGFP/hTERTC27 (pC27) were purified using a E.Z.N.A.TM.