The Preparation And Preliminary Application Of Aptamer Modified PLGA Nanoparticles For Targeted Gene Delivery

Background and ObjectiveProstate cancer is the second most common malignancy and the sixth leadingcause of cancer morbidity among males in the worldwide. About half of the prostatecancer has progressed to advanced disease, or translated to hormone refractoryprostate cancer. Traditional methods such as surgery, radiotherapy, hormone therapyand chemotherapy do nothing well for advanced prostate cancer, new safe andefficient methods need to be explored. Gene therapy is an effective andsequence-specific treatment, which may be benefit for advanced prostate cancer.Antigene therapy appears to be a promising strategy, which has the potential toefficiently inhibit transcription and cell proliferation by conforming DNA triplex atthe target location using triplex forming oligonucleotide (TFO). It is necessary to findan optimum gene delivery system. Polymer nanoparticles (NP) have been widelyexplored as gene carriers due to desired characteristics. However, nanoparticle genevectors lack active targeting. Aptamers which binding to target with high affinity andspecificity become the hot pot in tumor targeted therapy. In this study, we aimed toestablish TFO-encapsulated nanoparticle-aptamer bioconjugates (TFO-NP-Apt), using biocompatible and biodegradable polymer PLGA nanoparticles as the gene deliverysystem, and A10aptamer targeting the prostate specific membrane antigen. Weevaluate the nanoparticle characteristics and pharmacological properties ofTFO-NP-Apt, and explore the anti-proliferation effects for prostate cancer cells,which providing a new drug for prostate cancer’s therapy.Materials and Methods1、The PLGA-COOH and NH2-PEG-COOH were synthesized toPLGA-PEG-COOH by acid amide condensation reaction, the same as NH2-A10Aptconjugated to PLGA-PEG-COOH.1H-nuclear magnetic resonance (1H-NMR) wasused to identify the two condensation reactions. The resulting TFO-encapsulatednanoparticle-aptamer bioconjugates (TFO-NP-Apt) which targeted prostate cancerLNCaP cells for antigene therapy were prepared using the double emulsion solventevaporation method. Dynamic light scattering was used to evaluate the mean particlesize and polydispersity index, and potential analyze light scattering was used tomeasure the surface zeta potential of nanoparticle. The surface morphology and sizedistribution were determined by scanning electron microscopy. The TFOencapsulation efficiency and loading efficiency of nanoparticles were calculated bymeasuring TFO content indirectly with nucleic acid/protein analyzer. In vitro drugrelease study was made to verify the controlled and delayed release of nanoparticles.2、Fluorescein labeled TFO-NP-Apt, TFO-NP and TFO were prepared for cellularuptake experiment. We observed the LNCaP cells carrying fluorescence usingfluorescent microscope after incubation with drugs30min,1h and2h. Flow cytometrywas used to assess the cell uptake efficiencies quantitatively. In cell counting kit-8(CCK-8) test, group TFO-NP-Apt, TFO-NP, TFO and NP, and group24h,48h and72h were designed to explore the time-effect and dose-effect in inhibiting LNCaPcells’ proliferations. Prostate cancer PC3cells were used as the control cells in cellexperiments. Results1、In1H-NMR graphs, the characteristic peaks of PEG and aptamer were observedat3.3-3.9ppm and1.2-1.7ppm, respectively. Nanoparticle size measured by dynamiclaser light scattering was225.2±8.1nm (Mean±SD, n=3), polydispersity index was0.096±0.02, and zeta potential was-35.5±3.3mV. Scanning electron microscopygraphs show that the nanoparticles look round, having smooth surfaces and uniformsize distributions. The TFO encapsulation efficiency and drug loading efficiency were25.4%±3.1%(n=3) and1.34±0.16μg/mg, respectively. In vitro drug release study withTFO-NP-Apt, a sustained slow release was observed after an initial burst.2、In cellular uptake experiment, cells in TFO-NP-Apt group had significantlyhigher uptake efficiencies when compared with TFO-NP or TFO group at each period(30min,1h,2h), and the uptake of TFO-NP-Apt in LNCaP (PSMA+, AR+) was betterthan that in PC3(PSMA-, AR-). Flow cytometry indicated quantitatively that theuptake efficiency of TFO-NP-Apt in LNCaP increased with the time prolonging. InCCK-8test, TFO-NP-Apt was significantly more cytotoxic than TFO-NP and TFO(p<0.001). The anti-proliferation effects became better with time and dosageincreasing (p=0.004, p<0.001). NP showed no toxicity. Besides, the effects in LNCaPwere better than in PC3at each period (p<0.05).ConclusionsWe established the nanoparticle gene delivery system TFO-NP-Apt for targetedantigene anti-proliferating prostate cancer cells successfully. The nanoparticles havedesired characteristics and high TFO encapsulation efficiency and loading percentage.Besides, the nanoparticle had a sustained slow release after an initial burst.The anti-proliferation effects were verified in cell experiments. TFO-NP-Aptenhanced the cell uptake efficiency and improved the anti-proliferation effect forLNCaP. The anti-proliferation demonstrated favorable time-effect and dose-effectrelations.