RGDyC/PEG Co-Modified PAMAM Dendrimers Loaded With Arsenic Trioxide As Targeting Drug Delivery System For Glioma Therapy

OBJECTIVE G5 PAMAM dendrimer was used as the carrier material,and it was modified by non-toxic,non-immunogenic polyethylene glycol(PEG)and tumor targeting functional group RGDyC.Fluorescein isothiocyanate(FITC)was labelled and the arsenic trioxide(ATO)was encapsulated into the carrier,in order to construct the targeting drug delivery system(TDDS)for glioma therapy(RGDyC-mPEG-PAMAM/ATO).The modified PAMAM dendrimer was characterized and evaluated through in vitro cytology study and the related TDDS was also studied both in vitro and in vivo.The purpose of this study was encapsulating ATO in the modified PAMAM dendrimers to solve the problem that the poor BBB penetration,narrow treatment window and deficient body distribution specificity of ATO and so on,which provide a novel angle for the study of glioma treatment.METHODS RGDyC-mPEG-PAMAM was synthesized by mPEG3000,MAL-PEG3500-NHS,G5 PAMAM and RGDyC.Nuclear magnetic resonance(1H-NMR)and fourier transform infrared(FT-IR)spectra were performed to confirm the structure of RGDyC-mPEG-PAMAM and also determined its grafting rate.The particle size and Zeta potential of the nanoparticles were determined by Nano-particle size-Zeta potential analyzer and the morphology was observed by transmission electron microscopy(TEM).After FITC labeling,the structure and FITC grafting rate was identified by 1H-NMR.The toxicity in vitro,cellular uptake and intracellular localization of the modified dendrimers were tested by hemolysis assay,cytotoxicity assay,cellular uptake test and laser scanning confocal microscope images respectively.After encapsulating ATO,inductively coupled plasma emission spectrum(ICP)was used to determined the concentration of As and the drug loading and encapsulation efficiency were calculated.The in vitro release characteristics of ATO solution and RGDyC-mPEG-PAMAM/ATO were studied by dialysis bag method.MTT assay was used to investigate the cytotoxicity of ATO and the anti-tumor effect of ATO formulation in vitro.In vitro BBB and C6 cell co-culture models were established to investigate the inhibitory effect of ATO and its formulation after transporting across BBB.The pharmacokinetic behavior of ATO solution,PEG-PAMAM/ATO and RGDyC-mPEG-PAMAM/ATO was evaluated on Wistar rats.RESULTS 1H-NMR and FT-IR demonstrated the successful synthesis of RGDyC-mPEG-PAMAM.After modification,the particle size increased and the Zeta potential decreased,but still remained electropositive.Most of the particles of RGDyC-PAMAM,PEG-PAMAM and RGDyC-mPEG-PAMAM were kept at about 20 nm,and the Zeta potentials were(12.50±0.70)mV,(9.27±0.40)mV and(5.36±0.22)mV respectively.The modified carriers showed spherical appearances and had the good overall dispersibility.The hemolytic toxicity study and cytotoxicity study showed that G5 PAMAM dendrimers has huge toxicity especially at high concentrations but it could be markedly decrease due to PEG modification.However,with the increase of PEG modification,the ability of cell uptake decreased.While the modification of RGDyC could increase the tumor cell uptake to a certain extent.After encapsulating ATO into the modified PAMAM dendrimers,the concentration of As was measured by ICP.The drug loading of RGDyC-mPEG-PAMAM/ATO and PEG-PAMAM/ATO were 2.82%and 2.67%,and the entrapment efficiencies were 34.82%and 32.97%,respectively.In vitro drug release experiments showed that ATO was released rapidly at both pH 7.4 and pH 5.5,and RGDyC-mPEG-PAMAM/ATO illustrated obviously sustained and pH-dependent drug release,at pH 7.4 and pH 5.5,the cumulative release within 48 h were 68.1%and 79.5%,respectively.In vitro cytotoxicity and BBB and C6 cell co-culture model experiments showed that ATO had the best inhibitory effect directly to C6 cells in vitro.However,by constructing the in vitro BBB model,it was found that compared with ATO,the formulation had a better anti-tumor ability after penetrating BBB.The modification of RGDyC could further significantly improve the targeting ability to C6 cells and increase the inhibition rate.The concentration of ATO in the plasma of Wistar rats was determined by ICP-MS.The results demonstrated that T1/2 β,AUC0→t,MRT of the formulation increased and CL was significantly decreased.This indicated that the retention time of free ATO was short and the elimination was extremely fast but loading ATO into modified PAMAM dendrimers could significantly slow its plasma clearance and showed the pharmacokinetic characteristics of long circulation.CONCLUSION RGDyC-mPEG-PAMAM was successfully synthesized.PEG modification could indeed decrease the cytotoxicity in vitro of PAMAM dendrimers and RGDyC modification could actually enhance the affinity between the dendrimers and the targeting cells,increase the C6 cell uptake and finally boost the anti-tumor ability in vitro.Compared with free ATO solution,this RGDyC-mPEG-PAMAM/ATO illustrated obviously sustained and pH-dependent drug release and has a better anti-tumor ability in vitro after penetrating BBB.The pharmacokinetic behavior of ATO was markedly improved by PEG-PAMAM/ATO and RGDyC-mPEG-PAMAM/ATO with longer retention time,lower clearance and bigger AUC values.