Targeted Pegylated Plga Coated Prussian Blue Nanocomposite For PA/MR Multimodal Imaging And Synergistic Chemo-thermal Tumor Therapy

PART I PREPARATION AND CHARACTERIZATION OF PRUSSIAN BLUE LOADED NANOCOMPOSITEObjective1.To prepare a PEGylated poly(lactic-co-glycolic acid)(PLGA)targeting with folic acid(FA)coated Prussian blue nanoparticles(PB NPs)and paclitaxel(PTX),to construct multifunctional PLGA-PB-PTX-PEG-FA nanocomposite and to test their physical properties.2.To research the effects of cell targeting in vitro of PLGA-PB-PTX-PEG-FA by the MDA-MB-231 breast cancer cells,and to measure the viability of the cells treated by PLGA-PB-PTX-PEG-FA.Methods1.Paclitaxel(PTX)-loaded Folic acid(FA)targeted PEGylated PLGA nanoparticles encapsulating Prussian blue(PB)(PLGA-PB-PTX-PEG-FA)nanocomposite was fabricated by a modified double emulsion(water/oil/water)evaporation process.The size,morphology,UV–vis–NIR absorbance spectra,Fourier transfer infrared(FTIR)spectrum and confocal laser scanning microscopy(CLSM)were tested to evaluate the structural characterization of PLGA-PB-PTX-PEG-FA.To study the photothermal effect and synergistic therapeutic efficacy,PLGA-PB-PTX-PEG-FA nanocomposite suspensions of various concentrations nanoparticle dispersions were exposed to a laser with the wavelength of 808 nm and the output power of 0.647W/cm2 for 10 min in vitro.Drug Loading and releasing of PLGA-PB-PTX-PEG-FA nanocomposite were assessed by high performance liquid chromatography(HPLC).2.Cell targeting in vitro was verified by confocal laser scanning microscopy(CLSM)to assess the targeting effect of PLGA-PB-PTX-PEG-FA nanocomposite.CCK8 method was used to measure the viability of the cells and synergistic therapeutic efficacy of combining PLGA-PB-PTX-PEG-FA with laser.Results1.The highly dispersed PLGA-PB-PTX-PEG-FA nanocomposite with spherical morphology with smooth surface was Di Rectly observed by TEM and SEM and has good uniformity with an average hydrodynamic diameter of 236.6±55.04 nm.Both PB and PLGA-PB-PTX-PEG-FA nanocomposite displayed a broad absorption band from 500 nm to 900 nm with a strong absorption peak at ~702 nm.While the IR spectrum of the PLGA-PB-PTX-PEG-FA nanocomposite showed both the corresponded peaks of PLGA-FA-PEG(1756.23 cm-1)and PB NPs(2086 cm-1).The CLSM image of dye conjugation of PB NPs and Di I-conjugated PLGA-PTX-PEG-FA showed green and red,separately,while the merged image was orange.The excellent photothermal performance and remarkable photostability of PLGA-PB-PTX-PEG-FA were vertified.Obvious concentration-dependent temperature increases were found for PLGA-PB-PTX-PEG-FA nanocomposite under laser irradiation.The photothermal heating effect was also dependent on the power of the incident laser and irradiation duration.Drug loading efficiency and capacity tested by HPLC were 77.82% and 7.22%,respectively.Laser irradiation can accelerate the PTX release rate.2.Under CLSM observation,numerous red dots representing PLGA-PB-PTX-PEG-FA nanocomposite could be found gathered around the cell membrane stained with Di O(green fluorescence),while there were limited dots in the cytoplasm of MDA-MB-231 cells,and fewer nanoparticles around the membrane and within cancer cells in the non-targeted and free folate antagonism groups.The CCK8 results revealed that the cell viability of the PLGA-PB-PTX-PEG-FA combined with NIR group was lowest,verifying the effect of synergistic chemothermal therapy in vitro.ConclusionWe developed a versatile folate-targeted nanocomposite with a core-shell structure by coating PB NPs with a PLGA shell and then modifying with PEG.The prepared PLGA-PB-PTX-PEG-FA nanocomposite showed good targeting effect in vitro,excellent photothermal transformation capacity,higher drug loading capacity,excellent drug controlled release effect and possessed synergistic therapeutic efficacy combined photothermal and chemical therapy effects in vitro.PART II PRUSSIAN BLUE LOADED NANOCOMPOSITE FOR PA/MR MULTIMODAL IMAGING AND IN VIVO TARGETINGObjectiveTo investigate the potential of PLGA-PB-PTX-PEG-FA enhancing photoacoustic(PA)and magnetic resonance imaging(MRI)as contrast agent in vitro;to research in vivo tumor targeting of PLGA-PB-PTX-PEG-FA nanocomposite and to observe the PA/MR imaging effect of PLGA-PB-PTX-PEG-FA on MDA-MB-231 xenograft tumor-bearing nude mice in vivo,and investigate the enhanced capacity and the principle.Methods1.Ten MDA-MB-231 xenograft tumor-bearing nude mice were randomly divided into two groups: targeted(PLGA-PB-PTX-PEG-FA)and non-targeted(PLGA-PB-PTX-PEG)for vital fluorescence imaging(VFI).After anesthesia,about 200 ?L(20 mg/m L)folate-targeted or non-targeted nanocomposite stained with Di R were injected through the tail vein.The mice were observed with a VFI system(Xenogen IVIS Spectrum,Perkin Elmer,USA)before and 1,2,4,6,and 24 h after treatment.Tissues including the heart,liver,spleen,lung,kidney,brain,and tumor nodes were extracted completely after observation for additional ex vivo fluorescence observation.The quantitative fluorescence intensities of tissues at the similar size of region of interest(ROI)were measured on tumors before and after injection or isolated organs at different time points and were recorded.2.Different concentrations of PLGA-PB-PTX-PEG-FA nanocomposite were evaluated with small animal PAI equipment or 3.0T MR Scanne.PLGA-PTX-PEG-FA nanocomposite at the same concentration and deionized water were tested for comparison.PA signals and the MRI signal intensity(SI)within the ROI were further measured.Fifteen MDA-MB-231 xenograft tumor-bearing nude mice were randomly divided into three equal groups and injected with 200 ?L PLGA-PB-PTX-PEG-FA nanocomposite intravenously,PLGA-PB-PTX-PEG at a dose of 20 mg/m L,or normal saline(NS group).PA signals(absorption of 700 nm)of the tumor before and 1,2,4,6,and 24 h after tail intravenous injection were recorded.The ratio of the tumor PA was calculated as PApose/PApre.MRI was performed with the same divided group on a 3.0T MR scanner with a small animal coil.T1-weighted images were acquired before and at 1,2,4,6,and 24 h after intravenous injection.Signal intensity of the tumor(SItumor)and thigh muscle(SImuscle)at similar sized ROIs were measured before and after injection at different time points.The relative signal intensity(SIr)and percentage of signal intensity enhancement(PSIE)were calculated.Results1.No animals showed fluorescence in the body before injection.Strong reddish yellow fluorescence regions were seen in the tumors,spine,brain,and spleen of nude mice in the folate-targeted group 1 h after intravenous injection.Over time,VFI showed that the fluorescence intensities of the tumor,spine,and brain gradually reduced,while the intensity of the liver enhanced.At 24 h,there was still minimal fluorescence in the tumor,spleen,and liver.In contrast,no obvious red fluorescence was observed in the tumor regions of the control nude mice after administration of the non-targeted agent.Fluorescence only appeared in the liver area of the non-targeted group and disappeared at 24 h.Ex vivo fluorescence imaging showed that tumors in the targeted group had obvious fluorescence,while the non-targeted group had none,and the liver,spleen and lung tissues of the two groups had stronger fluorescence intensity.The quantitative fluorescent intensity of the spleen and tumors in the targeted group were obviously higher than in the non-targeted group(P < 0.05).2.The results of PA imaging and MRI indicated the great potential of PLGA-PB-PTX-PEG-FA as a PA/MR imaging contrast agent.The corresponding PA value and PSIE were positively correlated with PLGA-PB-PTX-PEG-FA nanocomposite concentration.The PA value and PSIE of PLGA-PB-PTX-PEG-FA nanocomposite were far higher than that of PLGA-PTX-PEG-FA(P < 0.05).In vivo,there was obvious variation of PA signals and MRI images contrast enhancement in the tumor region 1 h after injection into the targeted group.Although the PA signal weakened over time,there was still a clear PA signal and remarkable enhancement of T1-weighted MRI contrast in tumor sites compared to the non-targeted and NS groups at 24 h after injection.The ratio of PA signal and PSIE of the targeted group was significantly higher than that of the non-targeted and NS groups at each time point(P < 0.05).ConclusionThe prepared PLGA-PB-PTX-PEG-FA nanocomposite showed good targeting effect in vivo and enhanced both PA and MR imaging in vitro and in vivo as a multimodal biological imaging contrast agent.PART III SYNERGISTIC THERAPEUTIC EFFICACY OF PRUSSIAN BLUE LOADED NANOCOMPOSITE ON BREAST CANCERObjectivesTo evaluate the combined photothermal and chemical therapy effect of PLGA-PB-PTX-PEG-FA nanocomposite on MDA-MB-231 xenograft tumor-bearing nude mice.MethodsThirty-five MDA-MB-231 xenograft tumor-bearing nude mice were divided into seven groups randomly: control group ? PTX group ?PLGA-PB-PEG-FA group ? PLGA-PB-PTX-PEG-FA group ?PLGA-PB-PEG-FA+NIR group?PLGA-PB-PTX-PEG-FA+NIR group and NIR group.Each group were injected relatively reagent by tail intravenous injection.The tumor of MDA-MB-231 tumor-bearing mice intravenously injected with the PLGA-PB-PTX-PEG-FA nanocomposite or the PLGA-PB-PEG-FA were locally irradiated with 808 nm laser(0.647 W/cm2,10 min).The NIR group was only irradiated with laser as the same parameters.During treatment,thermal images of the tumor were recorded with an infrared thermal camera every 10 s.The tumor size of each mouse was measured before and after treatment and the relative tumor volumes(RTV)were calculated.After 21 days of treatment,tumors were excised and weighed and calculated by tumor inhibition rate(TIR).Cell apoptosis and cell proliferation were detected by Td T-mediated d UTP nick end labeling(TUNEL)and proliferating cell nuclear antigen(PCNA)methods,respectively.The proliferative index(PI)and apoptotic index(AI)expressed were estimated.The heart,liver,spleen,lungs,and kidney of nude mice in the PLGA-PB-PTX-PEG-FA and control groups and tumor of every group were collected after treatments for 21 days and subjected to hematoxylin and eosin(H&E)staining.ResultsMice bearing MDA-MB-231 tumors were intravenously injected with PLGA-PB-PTX-PEG-FA nanocomposite and subjected to 808 nm laser exposure.The surface temperature of tumors rapidly increased from 34.9 ± 0.8 to 52±3.1°C in 10 min.After laser irradiation,tumors in the PLGA-PB-PTX-PEG-FA combined with NIR group formed black scabs 3 days post irradiation.The tumors were nearly eliminated 12 d after irradiation and did not continue to grow until 21 d after irradiation.In marked contrast,the PLGA-PB-PTX-PEG-FA combined with NIR group showed significantly inhibited tumor growth compared with other groups.Notably,the relative tumor volume decreased most after treatment with PLGA-PB-PTX-PEG-FA and laser irradiation.The tumors in other groups continued to grow.Significant differences were observed between any two groups(P<0.05),besides PLGA-PB-PEG-FA group,NIR group and control group.PLGA-PB-PTX-PEG-FA+NIR group compared with other groups with stronger antitumor properties(98.86%)than the other groups of which TIR declined.The results of PCNA and TUNEL showed that the lowest PI and highest AI were found in the PLGA-PB-PTX-PEG-FA combined with NIR group.Significant PI and AI differences were observed between any two groups(*P<0.05),besides PLGA-PB-PEG-FA group,NIR group and control group.H&E staining demonstrated that the largest necrosis area of tumors was in the PLGA-PB-PTX-PEG-FA combined with NIR group.In addition,H&E staining of the heart,liver,spleen,lungs,and kidney revealed no observable changes between the PLGA-PB-PTX-PEG-FA combined with NIR group and control group.ConclusionOur results demonstrated that active targeting of the PLGA-PB-PTX-PEG-FA nanocomposite to tumor possessed synergistic therapeutic efficacy combined photothermal and chemical therapy effects,which can further enhance breast cancer treatment.And the prepared PLGA-PB-PTX-PEG-FA nanocomposite was a potential targeting nanometer material for PA/MR multimodal imaging and imaging-guided synergistic therapeutic efficacy combining photothermal therapy and chemotherapy.