Preparation And Study Of CD30 Targeting Doxorubicin PEG-PLGA Nano-delivery Systerm

The research aims at exploring release effect and target drug delivery system loaded with doxorubicin(DOX)which can specifically target at CD30 aptamers,providing a novel idea of pharmaceutic preparation for treating malignant lymphoma with high CD30 expression.The segmented compound polyethylene glycol(PEG)-poly(lactic-glycolic acid)(PLGA)consists of PEG and PLGA,which is synthesized as the hydrophilic end and hydrophobic block,respectively.The Apt-DOX-NPs with specific binding of CD30 ligands is synthesized using this method.An anti-tumor drug–DOX is encapsulated inside the PEG-PLGA nanoparticles,while the CD30 aptamer,which can target at CD30 specifically,is bound to the surface of the PEG-PLGA nanoparticles.The DOX-NPs and Blank-NPs are prepared using emulsion solvent evaporation method,while CD30 aptamer is bonded through EDC/NHS.In this thesis,the researches focus on optimizing the synthesis method of PEG-PLGA nanoparticles,exploring and analyzing the physicochemical properties of drug-loaded nanoparticles.Furthermore,the in vitro release behavior of the drug-loaded nanoparticles and mathematical model are studied.Finally,the biocompatibility and the therapeutic effect in anti-tumor of the targeted drug-loaded nanoparticles are assessed.The details are as followed:(1)The purchased PEG-PLGA material is characterized and analyzed using ~1H-NMR to determine whether the material meets the study requirements.Besides,the static contact angle of the material is measured to judge the hydrophilic character of the material.The blank nanoparticles are prepared using single emulsion method,dialysis method,and emulsion solvent evaporation method,respectively.Through analyzing data of size,zeta potential and polydispersity index,and observing micromorphology using SEM to evaluate different methods,it is found that the particle size and zeta potential of the blank nanoparticles prepared using the emulsification solvent volatilization method are more suitable.Therefore,this method is using for preparing nanoparticles in subsequent experiments.(2)Four factors have a great impact on the preparation of DOX-NPs using the solvent evaporation method are selected to carry on L9(4~3)orthogonal test,and the results are analyzed by SPSS software.The results show that the ultrasonic time has the greatest influence on the encapsulation efficiency and particle size while preparing the DOX-NPs.Therefore,through exploring and analyzing,the optimum conditions for obtaining the maximum encapsulation efficiency are as followed:PEG-PLGA concentration is 10 mg/mL,internal aqueous phase volume is 200?L,ultrasonic power is 220 W,and ultrasonic time is 5 min.(3)The-COOH on PEG-PLGA-NPs is activated by EDC/NHS,and then reacted with the-NH2 on CD30 Aptamer,in order to make CD30 aptamer conjugate with DOX-NPs.It was proved that the CD30 aptamer and DOX-NPs were successfully conjugated through FT-IR,PAGE,and X-ray electron spectroscopy analyzing.The connection efficiency of the CD30aptamer was 71.51±2.09%.(4)The physicochemical properties Apt-DOX-NPs were systematically analyzed.The results showed that the particle size of Apt-DOX-NPs was 187.87±2.03 nm and zeta potential was 30.77±0.153 mV.Besides,the results of SEM and TEM suggested the drug-loaded nanoparticles were spherical and uniform,and the TGA result showed that the physicochemical properties of the nanoparticles have not been changed when the aptamer and DOX-NPs were conjugated.The drug loading capacity and encapsulation efficiency of DOX-NPs was1.63±0.10%and 86.84±1.07%,while Apt-DOX-NPs was 1.58±3.60%and 85.83±0.98%,respectively.(5)The result of in vitro drug release presented two stages.There was obvious drug burst within 24h,but the drug was released slowly in 24-240h.It was found that the cumulative release rate of DOX-NPs in pH=5.5 release medium was higher than that in pH=7.4.Also,through fitting the model of the release curve,the release characteristics of DOX-NPs in vitro were most consistent with the Weibull equation.(6)The Karpass 299 cell line(K299)was selected to assess the biocompatibility because K299 is the high expression receptor to CD30.The study of competition with anti-CD30antibody revealed that anti-CD30 antibody and CD30 Aptamer targeted different epitopes on the surface of CD30 receptor.Apt-DOX-NPs are superior to DOX-NPs in cytotoxicity,cellular uptake,and apoptosis,which attributed to role of the aptamer-mediated targeting.