PLGA/Fe₃O₄ Implants Carrying Cisplatin For Synergistic Magnetic Hyperthermal Ablation Of Rabbit VX2 Tumor

PARTⅠ PREPARATION AND CHARACTERIZATION OFPLGA/FE3O4 IMPLANTS CARRYING CISPLATINObjective To prepare the Fe₃O₄-loaded PLGA implants carrying cisplantin（DDP/PLGA-Fe₃O₄）,and investigate their characterization,AMF-induced magnetic heating ability in vitro and biosafety.Methods 3 formulations were prepared containing Fe₃O₄ with a mass fraction of 10%,20%,and 30%,respectively.The ultimate mass fraction of DDP in the gel was 1%.The phase-transformation process of DDP/PLGA-Fe₃O₄ was recorded with ultrasound in an agar gel phantom.The DDP/PLGA-Fe₃O₄（50 μl）with a different Fe₃O₄ mass fraction were placed at the center of the AMF.The temperature was continuously monitored by a far-infrared thermometer and the optimum Fe₃O₄ mass fraction of DDP/PLGA-Fe₃O₄ was selected.The morphology,microstructure of DDP/PLGA-Fe₃O₄ were investigated with scanning electron microscopy（SEM）.Elemental analysis was performed via an energy dispersive X-ray spectrometer（EDS）.Ddifferent concentrations of immersed solution of PLGA-30%Fe₃O₄（without DDP）,which were prepared using different concentrations of PLGA-30%Fe₃O₄ in RPMI-1640 medium were incubated with the human ovarian cancer cell line SKOV3 for 24 h.CCK-8 assay was used to detect the viability of the cells.Results After contacting with water,liquid-solid phase transformation occurred immediately and the ultrasound signal intensity of the material appeared hyperechoic.DDP/PLGA-Fe₃O₄ had great magnetic thermal conversion efficiency depending on the Fe₃O₄ content and extended time of exposure to AMF.DDP/PLGA-Fe₃O₄ with Fe₃O₄ mass fraction of 30% can generate heat up to the temperature to lead tumor tissue coagulation necrosis in a short time.The morphology and structure of the material was loose and porous under SEM.EDS exhibited that Fe element was well-distributed within the carbonaceous and oxygen,platinum frameworks uniformly.The results of a CCK8 assay showed that there was no obvious cytotoxicity to SKVO3 cells in various concentrations of immersed solution groups compared to the control group（P > 0.05）.Conclusion DDP/PLGA-Fe₃O₄ had a good heating ability when exposure to AMF.It is a new biologically safe magnetic-hyperthermia materials.PART Ⅱ IN VITRO AMF-INDUCED DRUG RELEASEFROM DDP/PLGA-FE3O4Objective To investigate the drug release profiles of DDP/PLGA-30% Fe₃O₄ when exposure to AMF.Methods Prepared liquid DDP/PLGA-30%Fe₃O₄（50 μl）was immersed in 1 ml of phosphate-buffered saline（PBS,PH 7.4）in a dialysis bag and placed in a centrifuge tube（45 ml）with an additional 29 ml of PBS.At the time points of 2 and 8 h,the centrifuge tubes were exposed to the AMF for 10 s and 30 s,respectively.One ml of the buffer medium was sampled from each tube at 0.5,1,2,4,8,12,24,48,72,and 96 h and 1 ml of fresh PBS was compensated in order to maintain a constant volume of 30 ml.The amount of released DDP was quantified by the high-performance liquid chromatography（HPLC）system.The group without AMF treatment was assessed under the same condition as the control.Results In comparison,a higher drug release was found with exposure to AMF than without exposure to AMF.In addition,the magnetic responsive drug release profile is strongly related to the temperature,the drug release increased with prolonged time exposure to AMF.Conclusion The release of DDP from DDP/PLGA-Fe₃O₄ can be boosted and controlled by the AMF.PART Ⅲ PLGA/FE3O4 IMPLANTS CARRYINGCISPLATIN FOR THERAPY OF RABBIT VX2 TUMORObjective To investigate the efficacy of magnetic-induced hyperthermia ablation in excised bovine liver and select the optimum mass of DDP/PLGA-30%Fe₃O₄ and ablation time.To explore the efficacy of DDP/PLGA-30% Fe₃O₄ for treatment of VX2 metastatic tumor in rabbits.Methods Liquid DDP/PLGA-30%Fe₃O₄ with different amounts（50 μl and 100 μl,respectively）were injected into the center of the excised bovine liver blocks via syringe under the guidance of ultrasound.The DDP/PLGA（100 μl）were employed for control.Then,the blocks were exposed to the same AMF for 1 min,3 min,and 5 min,respectively.The temperature of the liver blocks was recorded every 10 s.The ablation tissue volumes of the bovine livers were estimated.Liquid DDP/PLGA-30%Fe₃O₄（100 μl）was injected into excised bovine liver cuboids,then exposed to the same AMF for 3 min.The peak surface temperature of the liver at different distances（0,1,2,and 3 cm away from the injection point）was measured at the time point of 180 s by a far-infrared thermometer.The 48 rabbits with intramural VX2 tumors in their thighs were randomly divided into 4 groups of 12: blank control group（PLGA/NMP）,pure chemotherapy group（DDP/PLGA）,pure magnetic hyperthermia ablation group（PLGA-30%Fe₃O₄ + AMF）,and magnetic hyperthermia ablation with chemotherapy group（DDP/PLGA-30%Fe₃O₄ + AMF）.The rabbits received an intratumoral injection with the same dose of PLGA/NMP,DDP/PLGA,PLGA-30% Fe₃O₄,and PLGA/DDP-30%Fe₃O₄,respectively.The four groups were exposed to AMF for 3 min after administration.The temperature of tumors were monitored by a far infrared thermometer and recorded every 10 s.At 1 day after treatment,tumor pathology examination by hematoxylin and eosin（HE）staining and a transmission electron microscope（TEM）were performed.The expression of heat shock proteins 70（HSP70）was detected by immunohistochemistry.At 4 days after treatment,for the incomplete ablated tumor tissue in each group,expression of proliferation cell nuclear antigen（PCNA）for tumor cell proliferation assessing was detected by immunohistochemistry.Apoptosis of tumor cell was detected by Td T-mediated d UTP nick end labeling（TUNEL）.The serum was collected using the ear artery of the rabbits at days 1,4 and 7 to detect soluble interleukin-2 receptor（s IL-2R）by enzyme-linked immunosorbent assay（ELISA）.The size of the tumor was measured by US images.The concentration of platinum in the tumor tissue（center and edge）was detected by inductively coupled plasma atomic emission spectroscopy（ICP-AES）at days 1,4,and 7.All of the remaining rabbits were fed for further observation of tumor growth trends.Results The necrotic volumes of ablated bovine livers increased with the mass of the DDP/PLGA-Fe₃O₄ and the duration time under AMF.The temperature decreased significantly with the distance to the location of the implants,Which provided a basis for the in vivo experiment.HE staining showed the edge of the coagulative necrosis in the partially ablated tumor was obvious,which indicated that the residual tumor models were built successfully.TEM showed irreversible destruction of cells in the necrotic tissue with lysis or apoptotic body occurrence.The tumor volumes in the DDP/PLGA-30%Fe₃O₄ + AMF group presented a decreasing trend while the tumor volumes in the 3 other groups continued to increase at varying degrees after treatment.The tumor cell apoptotic index（AI）of PLGA/DDP-30% Fe₃O₄ + AMF group was highest among all groups（P<0.05）,while the tumor proliferation index（PI）was lowest among all groups（P<0.05）.Platinum in the center of the tumor as well as in the edge of the tumor kept a higher concentration during the treatment and decreased over time.The s IL-2R level of the serum in the DDP/PLGA-30%Fe₃O₄ + AMF group and the PLGA-30%Fe₃O₄ + AMF group decreased with time and were eventually lower than those in the groups without exposure to AMF.The expression intensity and expression rate of HSP70 in 2 hyperthermia groups were much higher than that in the control group and in pure chemotherapy group.Conclusion Magnetic hyperthermia ablation combined with chemotherapy could effectively improve the therapeutic efficiency of tumor,which may provied a novel and minimally invasive strategy for the treatment of tumors.