ASN-GLY-ARG-modified Polydopamine-coated Nanoparticles For Dual-targeting Therapy Of Brain Glioma In Rats

PART ? Preparation and characterization of dual-targeted NPsObjectivesTo synthesize a controlled release and dual-targeting system by using PDA coated MSN as DOX carrier(MSN-DOX-PDA),conjugating NGR to dual-target the endothelial cells and glioma cells.MethodsThe MSN and DOX was stirred in the dark to prepare the DOX-loaded MSN,PDA coating was then modified on the surface of MSN.NGR was functionlized on the PDA coating at last.The encapsulation efficiency(EE)and loading content(LC)of drug were calculated.Particle size,zeta potential and the morphology were defined by dynamic light scattering(DLS)and transmission electron microscope.The drug release profile in vitro was also performed.ResultsThe TEM images showed that the NPs were spherical and had a smooth surface with a size of approximately 160 nm.The zeta potentialwas-22 ± 0.97 Mv.The loading content(LC),encapsulation efficiency(EE)were 19.02%,40.84%,respectively.Only a very small amount of DOX was released in the neutral PBS(p H 7.4).However,the release rate dramatically improved and the cumulative release rate of DOX was approximately 50% within 24 h at p H 4.5.ConclusionWe synthesized a controlled release and dual-targeting NPs.The NPs were almost stable in plasma conditions but allowed sustained drug release in tumorous conditions.PART ? Study on the dual-targeting effects of MSN-DOX-PDA-NGR in vitroObjectivesCell cytotoxicity and uptake assays were investegated in vitro to evaluate the dual-targeting effects of NPs on the glioma cells and glioma neovascular endothelial cells.MethodsC6 cells,AC cells,BCEC cells were cultured,respectively.The models of BCEC cocultured with C6 cells(BCEC-C6),BCEC cocultured with AC cells(BCEC-AC)were established using Millicell Hanging CellCulture Insert.Flow cytometry and western blotting were performed to detect the expression level of CD13 in those cells.Laser scanning confocal microscope and the flow cytometer were used to investigate the cellular uptake of nanoparticles quantitatively and qualitatively,repectively.CCK-8 assay was applied to detect the cytotoxity of different DOX formulations.U251 cells and Gl261 cells were also recruited to evaluate the targeting effects of NPs.The cells incubated with different inhibit agents were detected by the flow cytometer to explore the cellular uptake mechanism.Laser scanning confocal microscopy was used to evaluate the subcellular localization of NPs after lysosome and nuclei were stained.The transport ratio(%)was measured by the HPLC method to assess the ability of the drug carriers across the BBB.For cellular uptake and cell survival,the C6 cells in the coculture model were analysied by flow cytometry and CCK-8 assay,respectively,to evaluate the dual-targeting effects of NPs.ResultsThe flow cytometry and western blotting showed a dramatic elevation of the CD13 level in BCEC-C6.In addition,the level was relatively higher in C6 cells than it was in the primary astrocytes(AC),BCEC,and BCEC-AC.Both C6 and BCEC-C6 cells in the MSN-DOX-PDA-NGR group showed obviously higher fluorescence intensity than those in MSN-DOX-PDA group did.The adding of freeNGR significantly inhibited the uptake of MSN-DOX-PDA-NGR.The NGR-mediated specific binding between MSN-DOX-PDA-NGR and glioma cells was also found in U251 cells but not in GL261.For C6 cells,the cytotoxicity of the different DOX formulations was concentration-and incubation time-dependent.The IC50 values of DOX,MSN-DOX-PDA,MSN-DOX-PDA-NGR,and MSN-DOX-PDA-NGR + free NGR were16.90,138.1,5.09,and 59.71 ?g/m L after a 24 h incubation and 7.608,9.85,1.74,and 10.61 ?g/m L after 48 h,respectively.NGR modification significantly decreased the IC50 values of the undecorated NPs(~27.13-5.66-fold at 24 and 48 h,respectively).However,the addition of free NGR obviously increased the IC50 compared with that of MSN-DOX-PDA-NGR(~11.7-and 6.1-fold at 24 and 48 h,respectively).A similar reversal phenomenon was also displayed in the BCEC-C6 and U251 cells but not in the Gl261 cells.The cell viability was > 80% at a concentration of 1000 ?g/m L of MSN-PDA-NGR and a 96 h treatment period.The uptake of MSN-DOX-PDA-NGR by C6 cells was significantly inhibited by Na N3(energy depletion agent),Cyto-B(macropinocytosis inhibitor),CPZ(clathrin-mediated endocytosis inhibitor),and monensin(lysosome inhibitor),but not by filipin,genistein(caveolae-mediated endocytosis inhibitor)or BFA(Golgi apparatus and intracellular trafficking inhibitor).Furthermore,only genistein did not inhibit the uptake of MSN-DOX-PDA-NGR by the BCEC-C6 cells.Theconfocal laser scanning microscopy showed the MSN-DOX-PDA-NGR was localized in the lysosomes of the C6 and BCEC-C6 cells.The in vitro transport ratios across the BBB increased with incubation time.In BBB model,after the 12-h incubation,the transport ratio was 13.16 and 24.59%for the MSN-DOX-PDA and MSN-DOX-PDA-NGR,respectively.When the excess NGR was pre-incubated to saturate the CD13 binding site of the in vitro BBB,the transport ratio of the MSN-DOX-PDA-NGR was significantly reduced.In the coculture model,the MSN-DOX-PDA-NGR group still exhibited the highest accumulation of DOX and cytotoxicity against the C6 cells.ConclusionThe blank NPs were biocompatible.MSN-DOX-PDA-NGR exhibited a “dual-targeting effect”,which first targeted and transported across the BCEC monolayer and then targeted the C6 cells,to ground for the further in vivo experiments.PART ? Study on the dual-targeting effects of MSN-DOX-PDA-NGR in vivoObjectivesStudy on the bioditribution of different DOX formulations in majororgans,observe the antitumor and antiangiogenesis effects in vivo,and evaluate the applicable safety in vivo.MethodsThe C6 orthotopic glioma model in nude mice was established first,the in vivo and ex vivo distribution of fluorescence in the major organs were visualized using the IVIS animal imaging system after different DOX formulations were injected via the tail vein.In vivo glioma distribution of dual-targeted nanoparticles in glioma-bearing rats was observed using a fluorescence microscope.The concentration of DOX in left(control),right(tumor tissue containing)brain hemispheres and liver tissues were analyzed by HPLC method.The anti-tumor effects in glioma bearing rats were evaluated by H&E staining and TUNEL assay.The anti-angiogenesis was aseesed by immunohistochemical method.The tumour-bearing rats from each group were followed by survival monitoring,and the survival data were analysed using the log-rank test.Healthy adult male Sprague-Dawley rats were treated with MSN-PDA-NGR by tail vein injection.The body mass was recorded and the main organs were H&E stained to examine the toxicity in vivo.ResultsCompared with MSN-DOX-PDA,MSN-DOX-PDA-NGR could accumulate more in the tumor site.The HPLC analysis showed that the DOX concentration in the right brain hemisphere was significantly higherthan that in the left in MSN-DOX-PDA-NGR group.The DOX deposition in liver tissues showed no significant difference among the three groups.H&E staining and TUNEL assay showed obvious apoptotic and necrotic tumor cells in MSN-DOX-PDA-NGR group compared with those in other groups.Immunohistochemistry showed obviously fewer microvessels in the MSN-DOX-PDA-NGR group than that in other groups.Treatment with MSN-DOX-PDA-NGR significantly prolonged the median survival time compared with that for other groups.The increase in body mass of the MSN-PDA-NGR and saline groups showed a comparable tendency over the 4 weeks.Furthermore,H&E staining revealed no obvious pathological lesions or impairment in the major tissues from rats that received MSN-PDA-NGR for 4 weeks.ConclusionMSN-DOX-PDA-NGR has a more powerful antitumor and antiangiogenesis activity since the dual-targeting NPs have powerful BBB penetration capacity and could increase the DOX accumulation at the tumor site.Forthermore,the NPs for drug delivery is compatible.