Superparamagnetic Iron Oxide-loaded Polymer Contrast Agents For Multi-modality Imaging And High Intensity Focused Ultrasound Cancer Ablation

PART PREPARATION AND CHARACTERIZATION OFIRON OXIDE-LOADED POLYMER CONTRAST AGENTSObjective To prepare the superparamagnetic Fe₃O₄-loaded polymermicrocapsules as a novel multi-functional contrast agents （Fe₃O₄/PLGA）,and investigate their properties of physics, optics, magnetism and biosafety.Methods Fe₃O₄/PLGA microcapsules were fabricated by a typicaldouble emulsion evaporation method. The surface morphology andstructure, the mean size and zeta potential distribution, the concentration ofFe₃O₄loading in PLGA microcapsules, the absorption spectrum and themagnetic properties of Fe₃O₄/PLGA were assessed using several analyticalmethods. The DiI labeled Fe₃O₄/PLGA （DiI-Fe₃O₄/PLGA） microcapsules,which were prepared using the same process as Fe₃O₄/PLGA wereincubated with MDA cells for different hours （4h,12h,24h）, then thephagotrophic effect of these microcapsules in MDA cells was observed by light microscope, MTT assay was used to detect the viability of the cells.The Fe₃O₄/PLGA microcapsules were injected into normal rabbits by veinto investigate the effect of these microcapsules on rabbit liver, kidney andheart at different time （1d,3d, and7d）.Results Fe₃O₄/PLGA microcapsules became brown emulsion whendissolved in double distilled water. The microcapsules exhibited a perfectspherical morphology with not very smooth surface under light microscope（LM） and scanning electron microscope （SEM）. A lot of Fe₃O₄particlesdistributed in the shell structure were observed under transmission electronmicroscope （TEM）. The diameter of Fe₃O₄/PLGA was （885.6±133.2） nm,and zeta potential was （-2.32±3.84） mV measured by a Laser Particle SizeAnalyzer System. The concentration of Fe₃O₄loading in PLGAmicrocapsules was46.58μg/ml detected by an atomic absorptionspectrophotometer （AAS）. The Fe₃O₄/PLGA absorption spectrum wasaround590nm and650nm measured by a UV-Vis spectrophotometry. TheM-H hysteresis loop behavior showed the Fe₃O₄/PLGA typicalsuperparamagnetic properties. A time-dependent Fe₃O₄/PLGA uptake byMDA cells was observed under the light microscope. The amount ofFe₃O₄/PLGA uptake increased with the increasing incubation time. MTTresults showed the cells proliferation activity was not affected afterFe₃O₄/PLGA engulfed by MDA cells. And the Fe₃O₄/PLGA had nosignificant effect on rabbit liver, kidney and heart after injection into the rabbits.Conclusion Fe₃O₄/PLGA microcapsules have been successfullysynthesized; these microcapsules have good spherical morphology, uniformsize, high encapsulation efficiency and good biosafety. They have excellentoptical absorption and magnetic properties, which make them a potentialcandidate for photoacoustic （PA） and magnetic resonance （MR） imaging. PART Ⅱ IRON OXIDE-LOADED POLYMERCONTRAST AGENTS FOR ENHANCED US, MR ANDPA IMAGINGObjective To observe the effect of Fe₃O₄/PLGA for enhanced US, MRand PA imaging in vitro, to investigate the feasibility of thesemicrocapsules as a multi-modality imaging agents; to observe the influenceof these agents on rabbits liver tumor MR imaging in vivo, to investigatethe enhanced capacity and the principle of Fe₃O₄/PLGA in MR imaging.Methods The prepared Fe₃O₄/PLGA microcapsules with differentconcentrations were applied to perform US and MR imaging in vitro. Awavelength of532nm pulsed laser excitation was used to carry outphotoacoustic imaging. A single Fe₃O₄/PLGA microcapsule andFe₃O₄/PLGA microcapsules that were phagocytized by MDA cells wereselected to detect the PA signal.21days after implantation of VX2tumorsin liver,12rabbits were randomly divided into three equal groups with theinjection of saline, PLGA, and Fe₃O₄/PLGA1ml/kg via ear veinrespectively. MR imaging was performed before and after injection of theabove three agents, the signal intensity （SI） of liver parenchyma and livertumor were measured, then, the signal ratio of liver tumor to liverparenchyma was calculated before and after enhancement. After MR scanning, Prussian blue and HE staining were done.Results In vitro, the Fe₃O₄/PLGA showed hyperechogenicity in USimaging, the echo intensity （EI） decreased with the concentration andmechanical index decreasing. The Fe₃O₄/PLGA exhibited MRI negativelyenhanced effect in T2*-weighted images, the signal intensity （SI） decreasedwith the increasing Fe concentration in PLGA. On the other hand,Fe₃O₄/PLGA could produce strong PA signal even were phagocytized byMDA cells under532nm laser. The PA signal increased with increasinglaser energy. In vivo MR imaging, after injection of Fe₃O₄/PLGA, the SI ofliver parenchyma reduced markedly, however, no significant changes of SIwere found in liver tumor, the signal ratio of liver tumor to liverparenchyma increased significantly compared with the other two groups （P<0.05）. Prussian blue stain showed accumulation of the blue particleswithin liver parenchyma in Fe₃O₄/PLGA group, this phenomenon was notoccurred in liver tumor tissue and groups with injection of saline andPLGA.Conclusion The Fe₃O₄/PLGA microcapsules are successfully acted asmulti-modality biological imaging contrast agents for US, MR and PAimaging in vitro. And The Fe₃O₄/PLGA microcapsules can effectivelyenhanced the signal intensity contrast between the liver tumor and normaltissue in rabbits, which have a potential application value in the detection of liver carcinoma. PART Ⅲ IRON OXIDE-LOADED POLYMERCONTRAST AGENTS FOR HIGH INTENSITY FOCUSEDULTRASOUND CANCER ABLATIONObjective To investigate the efficacy of Fe₃O₄/PLGA combined withHIFU in bovine liver tissue ablation, and to investigate the influence ofFe₃O₄/PLGA combined with HIFU in rabbits bearing breast tumor.Methods The degassed bovine liver in vitro was directly injected into200μl Fe₃O₄/PLGA in PBS with different Fe concentrations just beforeHIFU ablation, the PBS （200μl） and pure PLGA （200μl） were employedfor control. After HIFU ablation, the gray-scale value change and thevolume of coagulated tissue irradiated by HIFU was calculated.14daysafter implantation of VX2tumor in breast,32rabbits were randomlydivided into four equal groups. In group Ⅱ, Ⅲ, and Ⅳ, the rabbits receiveda percutaneous injection of0.5ml saline, PLGA, and Fe₃O₄/PLGA solutioninto breast tumor at3,6,9,12dots, about3mm around the tumorrespectively, and massaged for1min before receiving HIFU ablation, whileHIFU group received HIFU ablation only. HIFU ablation parameters werethe same:“ablated-dot” mode, acoustic power was150W, exposureduration was5s. After HIFU ablation, the gray-scale value change and thevolume of coagulative necrosis was calculated, HE staining, TEM andimmunohistochemical examination of PCNA were performed to detect the structure changes of the targeted tissue caused by HIFU ablation.Results The gray-scale value change and the volume of coagulatedtissue in bovine liver tissue irradiated by HIFU after local injection ofFe₃O₄/PLGA was larger than that of PBS and pure PLGA （P<0.05）. Inrabbits with breast cancer, the gray-scale value change and the volume ofcoagulative necrosis caused by HIFU ablation was substantially larger andthe positive index （PI） of PCNA was lower in the necrotic region after theinjection of Fe₃O₄/PLGA microcapsules compared to the group withoutinjection agents, the group of saline and the group of pure PLGAmicrospheres under the same irritation （P<0.05）. Pathological examinationdemonstrated the targeted tissue was destroyed severely, Fe₃O₄nanoparticles were seen depositing in residual tumor cells under TEM.Conclusion The Fe₃O₄/PLGA microspheres can effectively enhanced thebiomedical effect of HIFU ablation both on bovine liver tissue and onrabbits bearing VX2breast tumor, The Fe₃O₄/PLGA was a potentialsynergistic agent for HIFU cancer surgery.