Construction Of Magnetic Targeted Drug Delivery System For Phytosterol And Preliminary Study On Integrated Diagnosis And Treatmen

Euphorbiasteroid is one of the main constituents isolated from Euphorbia lathyris which was reported to have significant growth inhibitory effects on cancer and reversal activity against P-glycoprotein(P-gp)mediated MDR in resistant cells.However it is restricted to use in clinical because of its poor water solubility and low bioavailability.Based on biodegradable carrier poly(Lactide-co-glycolic acid),a multi-functional targeted delivery system for insoluble drugs has the advantages of prolonging drug half-life,reducing toxic and side effects,and simultaneously targeting tumors.How to construct a specific targeted drug delivery system with high drug loading and high entrapment efficiency according to the physicochemical properties of drug is still a common problem in the current PLGA targeted drug delivery system research.Therefore,based on the theoretical basis of folate receptor-ligand-mediated targeted therapy and the integration of theranostics,we explored Euphorbiasteroid targeted delivery system that are biodegradable,high clinical safety,and having stable drug loading and encapsulation efficiency with a view to improving the efficacy of its tumor-targeted therapy from the standpoint of pharmaceutical science.Objective:Development of Euphorbiasteroid magnetic and targeted drug delivery system based on magnetic multi-functional nano materials;Pharmacokinetic in rats and tissue distribution studies in mice on Euphorbiasteroid of different PLGA-loaded drug delivery system by UPLC-MS/MS after inject administration to compare the differences;CCK-8 method to evaluate the biocompatibility and safety of blank drug delivery system,targeting of folic and and magnetic role of Fe3O4 was accessed in vivo on KB tumor bearing mice in order to preliminary discuss the potentiality of dual Euphorbiasteroid(EFL1)/superparamagnetic iron oxide(SPIO)-loaded FA targeted PLGA-based nanoparticles for a theranostic purpose.Method:1.In this study,monodisperse Fe3O4 was synthesized by using thermal decomposition of oil phase,and PEG-3,4-dihydroxy benzyl amine(DIB-PEG-NH2)basing on Schiff base reaction was synthesis using DCC/NHS as a condensation agent fistly.DIB-PEG-NH2 was exchanged onto the nanocrystal surface.The water-soluble and biocompatibility Fe3O4-DIB-PEG-NH2 was obtained.Characteristics of the nanosystems were investigated by Fourier transform infrared spectroscopy(FT-IR),vibrating sample magnetometer(VSM),X-ray diffraction(XRD),TEM and SEM to prove the successful synthesis of OA-Fe3O4 and Fe3O4-DIB-PEG-NH2.2.With the anti-cancer drug Euphorbiasteroid(EFL1)as the model drug,folic acid as a targeting factor on magnetic FA-PEG-PLGA-Fe3O4 based Euphorbiasteroid loading drug delivery system was prepared using premix membrane emulsification method.The physico-chemical properties of nanoparticles were characterized by different techniques such as transmission electron microscopy(TEM),dynamic light scattering(DLS)method,X-ray diffraction(XRD),Differential Scanning Calorimeter(DSC)and inductively coupled plasma mass spectroscopy(ICP-MS).The HPLC method for calculate the encapsulation efficiency(EE)and drug loading(DL)was established.The in vitro release behaviors of PLGA-EFL1 and PLGA-EFL1-Fe3O4 were respectively investigated in pH 6.8 and pH 7.4.3.An UPLC-MS/MS method was developed for the determination of EFL1 in the rat plasma and mice tissue biological samples.The pharmacokinetics of RY,PE,PEF and PEFM with 10 mg/kg EFL1 was evaluated in healthy rat;the tissue distribution of RY,PE,PEF and PEFM with 14 mg/kg EFL1 was evaluated in healthy mice to compare and evaluate the differences after developing the magnetic drug delivery system.4.The cytotoxicity of Fe3O4-DIB-PEG-NH2 and FA-PEG-PLGA-Fe3O4 with different concentrations of Fe which were incubated with A549(FA-),Hela(FA+)and KB(FA++)cancer cells was determined by a CCK-8 cell proliferation assay to evaluate their biocompatibility.The spin-spin relaxation time(T2)of Fe3O4-DIB-PEG-NH2 and FA-PEG-PLGA-Fe3O4 were detected using a 3.0 T MRI scanner to,according to the r2 to judge the magnetism.Enhanced in vivo tumor actively and magnetic target ability of FA-PEG-PLGA-Fe3O4 and Fe3O4-DIB-PEG-NH2(with magnetic field),compared to Fe3O4-DIB-PEG-NH2(without magnetic field)in BALB/c-nu KB tumor model was verified by MRI imaging study.Results:1.Magnetic Nanoparticles of Fe3O4 presented a spherical morphology and monodispersed with relatively uniform sizes ranging from 8 to 18 nm which could be obtained from SEM image,DLS figures.The saturated magnetization of Fe3O4 was 39.93 emu/g and showed superparamagnetism.After modification,Fe3O4-DIB-PEG-NH2 showed water-souble and be fairly stable in the aqueous phase with a little increasing in particle size(changing 12nm to 13.55nm).While its saturated magnetization were decreased 2.72 emu/g lower than that of free Fe3O4 due to the modification DIB-PEG-NH2 in the surface of Fe3O4.Although the magnetism has decreased,nanoparticles can still be adsorbed quickly and firmly by the magnet,which still have superparamagnetism.2.With the criteria of EE and DL,factors,such as the ratio of PLGA/EFL1,the concentration of PLGA,the concentration of PVA,and the ratios of O/W2 were studied to optimize the formation and the technology.Optimum conditions was as follows:using W1/O/W2 emulsification method,membrane pore was size 0.5μm(3-stage series connection),ratio of oil and water phase 2 was 1:4;water phase 1 and oil ratio was 1:5,PLGA and EFL1 ratio was 1:15,oil phase concentration was 20 mg/mL;PVA concentration was 20 mg/mL,kind of curing liquid was 0.9%saline.Membrane pressure was 0.7 Mpa,curing time was 4h.Before entering the emulsifier,an ultrasound(power 150W,work3s)was used after filling W2 into mixture solutions.Centrifuge l0min at the speed of 10000r/min and washing with pure water for 3 times.Process validation shows no difference between batches and had good repeatability on EE and DL of PLAG-EFL1.The physicochemical properties of PLGA drug delivery system was evaluated using DSC,XRD,DLS.The zeta potential Absolute value of all the PLGA preparations were among 15 to 22,indicating that drug in a PLGA delivery system was stable.Results showed that EFL1 release from PLAG-EFL1 and PLAG-EFL1Fe3O4 drug delivery system in pH 6.8 release conditions was significantly quicker than pH 7.4 system but both showed sustained-release which is consistent with Ritger-Peppas Fickian model.3.The UPLC-MS/MS method which was established had a good linearity ranging from 10 ng/mL to 1000 ng/mL.All the precision,stability and recovery meet the requirements.The method is suitable for the determination of EFL1 in rat plasma and mice tissue homogenates.Compared to EFL1 solution,the initial concentration of PLAG-EFL1(PE),PLGA-EFL1-FC3O4(PEF)and PLGA-EFL1-Fe3O4(PEF with magnetic field)all was lower than EFL1 solution.Vz were improved from 0.03L/kg to 0.078 L/kg,0.109 L/kg and 0.067 L/kg,respectively,which is 2.6,3.63 and 2.23 times to EFL1 solution.MRT values were 1.43,1.46 and 1.20 times to EFL1 solution respectively,and T1/2z was 2.54,2.34 and 1.72 times than EFL1 solution.These results suggested that PE,PEF and PEF with magnetic field have sustained release trend,plasma climinate rate was cut down.Analysis on the tissue distribution data,we could conclude that PE,PEF and PEF with magnetic field groups distribution features is:Lung>Liver>Kidney.The three groups were targeted to lung and liver obviously on the previous time,and the magnetic group distributed more EFL1 to Lung than PEF without magnetic field.4.Cell viability(%)was measured after 72 h incubation of blank FA-PEG-PLGA-Fe3O4 and Fe3O4-DIB-PEG-NH2 in A549,Hela and KB cells.In the tested concentration range(0-304.8 μM),both FA-PEG-PLGA-Fe3O4 and Fe3O4-DIB-PEG-NH2 did not show any severe cytotoxicity which indicate that their biocompatibility and biodegradability in vitro.The r2 values were 165.94 and 179.12 mM-1s-1,respectively,which showed good relaxation performance.To demonstrate the mice tumor targeting ability of FA-PEG-PLGA-Fe3O4 and Fe3O4-DIB-PEG-NH2 and also to validate the T2 MR imaging performance,we investigated the KB and Hela cells with folate receptor(FR)over-expression tumor mice model.In contrast with blank tumor model,the T2-weighted MR images of the Fe3O4-DIB-PEG-NH2 targeted(with magnetic field)group showed an obviously marked signal drop at 10 h time points,77.13%and 59.83%,respectively;FA mediated FA-PEG-PLGA-Fe3O4 group showed an obviously marked signal drop from 2 h(with 34.64%and 47.10%signal drop)to 24 h(with 70.93%and 70.94%significant signal drop),even at 48 h time points(52.26%signal drop)on Hela tumor mice;FA mediated actively and magnetically target group induced 48 h long-term 67.91%signal drop while Fe3O4-DIB-PEG-NH2 without magnetic field showed no significant signal drop and on 24h,the image started brightening.Conclusion:In conclusion,a dual Euphorbiasteroid/Euphorbia Factor 1(EFL1)/superparamagnetic iron oxide(SPIO)-loaded folate-PEG-PLGA-based nanoparticle drug delivery system for a theranostic purpose was designed and prepared using premix membrane emulsification method.We successfully co-encapsulated SPIO and EFL1 in PLGA-based nanoparticles.The particle size,polydispersity,and zeta potential of this DDS were measured by dynamic light scattering(DLS)and laser Doppler methods,the EE and DL were stability which were successfully detected by HPLC.Sustained and pH-dependent drug release from PLAG-EFL1(PE),PLGA-EFL1-Fe3O4(PEF)NPs was observed.Through pharmacokinetic on rat and tissue distribution studies in mice,we demonstrated that these nanoparticles drug delivery system could slow down the emilation EFL1,prolong the T1/2,and be targeted to lung and liver tissue under magnetic field.Relaxometry studies and phantom MRI demonstrated their efficacy as T2 contrast agent.FA-PEG-PLGA-Fe3O4 and Fe3O4-DIB-PEG-NH2 nanoparticles after injection via the tail vein could be used as potential tumor-targeting MRI contrast agents,thanks to the high uptake of nanoparticles by cells as well as their magnetic characteristics.Blank FA-PEG-PLGA-Fe3O4 NPs and Fe3O4-DIB-PEG-NH2 NPs did not show any severe cytotoxicity in A549,Hela and KB cells after 72 h incubation using CCK-8 method.Together these results,these findings indicate the usefulness of FA-PEG-PLGA-Fe3O4 NPs as tumor-targetable nano-carriers for cancer therapy.These multifunctional nanoparticles may be considered as future nanomedicine for Folate mediated drug delivery,magnetic targeting,and MRI imaging.In the future,our studies could provide experimental basis and theoretical support for the construction of active and magnetical targeted drug delivery system for other esters and steroidal steroids of Euphorbiaceae.