Pretargeting To Ovarian Cancer For Ultrasonic Molecular Imaging And Chemotherapy

BackgroundThe morbidity of ovarian cancer ranks third in the female reproductive system tumor,but the fatality is ranking first.Although the death of the patients with ovarian cancer is associated with a variety of factors,but the early diagnosis,early and correct treatment is no denying the key to reduce the mortality.Therefore,how to realize the early diagnosis of ovarian cancer,and choose suitable treatment for different patients with proper scheme,has long been a diligent direction of the researchers.At present,the various tumor targeting PLGA nanoparticles,which loaded drugs,gene et al,become the hotspot in the ultrasonic molecular imaging and therapy.Loading chemotherapeutic drug paclitaxel in the phase variant PLGA nanoparticles and targeting to ovarian cancer,not only can realize ultrasonic molecular imaging,for the early diagnosis of ovarian cancer,but also can prevent from the drug delivery limitation caused by hydrophobic character,and can increase drug effectiveness and reduce the side effects.Direct targeting method as one of the most common used traditional ways of tumor targeting,which has simplicity of operating,is susceptible to the micro-environment in vivo.In addition,as an example of antibody,the randomness of covalent binding between the ligand and effector might cause the partially competitive inhibition of connection between functional groups and body receptor,and leads to a reduced targeting ability.Therefore,how to improve the efficiency of targeting tumor becomes a crucial problem which scholars are trying to solve.Pretargeting technology is originated in radionuclide imaging research and found that this method can effectively improve the tumor target/non-target ratio.But it is seldom to be adopted in ultrasonic molecular imaging research.This study intends to prepare paclitaxel loaded and phase-shifting PLGA nanoparticles,and through two-step biotin-streptavidin pretargeting technology,for targeting to ovarian cancer cells in vitro,aims to explore whether the pretargeting technology can be with the feasibility in tumor targeting of ultrasonic molecular imaging and treatment,and providing a new approach to the accurate diagnosis and treatment of ovarian cancer.Objective1.To prepare the streptavidinated and paclitaxel loaded phase-shifting PLGA nanoparticles(PTX-PLGA-SA/PFPs),detect the loading efficient of paclitaxel and observe the drug release characteristics in vitro,observe the enhanced ultrasonography after phase transformation of nanoparticles caused by Low Power Focused Ultrasound(LIFU).2.To explore the ability of PTX-PLGA-SA/PFPs as an ultrasonic molecular contrast agents uniting the two-step pretargeting technology to target to the ovarian cancer cells and affect the cell viability in vitro,in order to further study of the pretargeting technology used in vivo,and to provide the basis of achieving the accurate ultrasonic molecular imaging and targeting treatment for tumor.Methods1.The PTX-loaded and phase-shifting PLGA nanoparticles(PTX-PLGA/PFPs)were prepared by the single emulsion(O/W)solvent evaporation method.And PTX-PLGA-SA/PFPs were manufactured by streptavidinating PTX-PLGA/PFPs using carbodiimide method.The encapsulation efficiency of paclitaxel capsuled in PTX-PLGA-SA/PFPs was determined by high performance liquid chromatography(HPLC),and the drug release characterize was studied by dialysis method in constant temperature shaker.The phase shifting of PTX-PLGA-SA/PFPs was induced by heating and Low-intensity focused ultrasound(LIFU),and observed by microscope and ultrasonography respectively,and the average acoustic intensity after phase transformation induced by LIFU was analyzed by DFY software.2.The anti-CEA antibody(Ab)were covalently linked via carbodiimide method on the PTX-PLGA/PFPs to prepare the Ab-carried drug-loaded nanoparticles(PTX-PLGA-Ab/PFPs)as the contrast group of direct targeting.Both PTX-PLGA-SA/PFPs and PTX-PLGA-Ab/PFPs were observed by confocal laser scanning microscopy to evaluate the status of SA and Ab binding to PTX-PLGA/PFPs.And the ligation efficiency was determined by flow cytometry(FCM).3.By confocal laser microscope and flow cytometry,the targeting efficiency for ovarian cancer SKOV3 cells in vitro were comparably evaluated in each group as below,PTX-PLGA-SA/PFPs uniting two-step pretargeting technology group(drug-loaded pretargeting group),PTX-PLGA-Ab/PFPs group(drug-loaded and directly targeting group),PTX-PLGA/PFPs group(drug-loaded and non-targeting group)and antibody blocking group.The cell viabilities in drug-loaded pretargeting group,drug-loaded and directly targeted group,drug-loaded and non-targeting group,simple nanoparticles group(PLGA/PFPs)and pure PTX group were detected through the CCK 8 drug-loading reagent method to evaluate the ability of PTX-PLGA-SA/PFPs uniting the two-step pretargeting methods for targeting and killing cancer cellsResultsThe nanoparticle sizes of PTX-PLGA-SA/PFP were 383.0±75.59 nm.The PTX-PLGA-SA/PFPs were observed as with the homogeneous distribution under optical microscope,and the spherical appearance under electron microscope.The encapsulation efficiency and drug loading efficiency of PTX were(71.56±6.51)% and(6.57±0.61)%,respectively.In the study of drug release in vitro,the sudden release characteristics was observed with the ratio of approximately 70% in short time of 2-3 days.With the LIFU power of 7.5 W in 3 min,the PTX-PLGA-SA/PFPs can obviously enhance the ultrasonography(*P<0.05).And when the temperature was up to 45?,the nanoparticles start to shift their phase into micro-bubble? When temperature was reaching to 60?,most nanoparticles changed phase to micro-bubble,and continually fractured with the temperature rising.2.The PE-SA had been successful conjugated on the PTX-PLGA/PFPs,proved by laser confocal microscope with appearing red fluorescence on the nanoparticles.And the Ab had been successfully combined with the nanoparticles in the same way with yellow fluorescence,mixed by Green fluorescent from FITC labeled second antibody and red fluorescent from DiI staining nanoparticles.Flow cytometry showed that the combination efficiency between the SA and PTX-PLGA/PFPs,the Ab and PTX-PLGA/PFPs were(97.16+1.20)% and(92.74 + 5.75)%,respectively.3.The experimental result of targeting to the ovarian cancer SKOV3 cells showed that more nanoparticles adhered to ovarian cancer SKOV3 cells in the drug-loaded pretargeting group than the drug-loaded and directly targeting group and the rest groups,and the flow cytometry showed significantly higher mean cell fluorescence in the drug-loaded pretargeting group than in others(*P<0.05).The viability of cells in the drug-loaded pretargeting group was lower than the drug-loaded and directly targeting group and other groups(*P<0.05)significantly,and only higher than the pure PTX group(*P<0.05).Conclusions1.In this study,the streptavidinated and paclitaxel loaded phase-shifting PLGA nanoparticles(PTX-PLGA-SA/PFPs)were successfully manufactured.The size of nanoparticles distributed homogeneously,with a high encapsulation efficiency of paclitaxel and conjugating efficiency to streptavidin.With a burst release in vitro,the PTX released from the nanoparticles at a high rate.The phase transition can be induced by the LIFU and heating,and after phase transformation the nanoparticles can obviously increase the contrast enhanced ultrasonic imaging.2.By comparing with directly targeting way,the two-step biotin-streptavidin of pretargeting technology can significantly enhance the targeting ability of the paclitaxel loaded phase-shifting PLGA nanoparticles to ovarian cancer cells in vitro,and obtain a well efficacy of targeted therapy.It provides a possible way of targeting to ovarian cancer for ultrasonic molecular imaging and precise chemotherapy.