Lyp - 1 Modified Magnetic Nanoparticles Mediated Tumor Magnetic Targeted Heat

ObjectiveThe aims of the present study are to investigate the preparation of LyP-1modified magnetic nanoparticles (LyP-1-MNPs) which could home to the cancer overexpressing protein P32, to study the biocompatibility and targeting properties of LyP-1-MNPs in vitro, and to test the targeting therapy of LyP-1-MNPs in vivo.Methods1. Preparation and characterization ofLyP-1modified MNPsFirstly, Polyethylene glycol(PEG)-coated magnetic nanoparticals (PEG-MNPs) were synthesized by "single-step method", which is chemical co-precipitation method substantially, but it is more simple and cost less time. PEG-MNPs, modified by special agent, were conjugated with cell-penetrating peptide LyP-1. The size, zeta potential, and coating characterizations of LyP-1-MNPs were analyzed by transmission electron microscope (TEM), zetaplus zeta potential measurement, and fourier transform infrared spectroscopy (FTIR).2. In vitro studyThe expressions of P32of cell lines were detected by RT-PCR and western-blot. Cytotoxicity of functional magnetic nanoparticles was evaluated by CCK-8assay, and the aggregation state of LyP-1-MNPs in cells was studied by Prussian blue staining and transmission electron microscopy (TEM). Subcellular localization of LyP-1-MNPs in different cell lines was observed by Laser Scanning Confocal Microscope(LCSM).The iron content of cellular uptake was measured by Inductively coupled plasma mass spectrometry (ICP-MS).3. In vivo studyThe tumor-bearing mice model was built by transplanting human breast cancer cell line MCF-7cells into the subcutaneous tissue of the right hind limb of BALB/c nude mouse. And then all animals were divided into three groups randomly:negative control group, hyperthermia group, and targeting therapy group. The experimental groups were injected with magnetic fluid, after magenetic hyperthermia treatment, the body weight of animals and the tumor volume were measured, then the body weight curves and the tumor volume curves were plotted. When30days had passed after therapy, the mice were euthanized with an intraperitoneal injection of pentobarbital sodium(120mg/kg), the important organs, such as heart, liver, spleen, lungs, kidneys and tumor,were obtained and prepared for histological analysis, then the samples were cut into about5μ m thick section and the sections were stained with either hematoxylin and eosin (H&E) or Prussian blue. Biocompatibility of MNPs was analysed by H&E staining, and biodistrbution of MNPs was observred by Prussian blue staining.Results1. Magnetic nanoparticles had good stability, well magnetism response, and great dispersibility. When exposed to alternating magnetic field, MNPs had good warming performance. The morphology of particals modified with LyP-1was approximately spherical and the mean size of the magnetic nanoparticles was about15nm.After being coated by Polyethylene glycol (PEG), the surface of MNPs displayed the negative charge properties, and when LyP-1was conjugated with the PEG-MNPs, the zeta potential was changed into positive charge from negative charge.2. We found that human breast cancer MCF-7is a P32-high-expressing cell line and relatively,colorectal cancer cell line CT-26is a P32-low-expressing cell line using RT-PCR and western-blot assay.3. The result of CCK-8assay showed that PEG-MNPs modified with LyP-1had no significant cytotoxicity at a concentration of less than60μg/mL, cytotoxicity grade had a narrow range between0grade and1grade. The cellular uptake of targeting nanoparticals was higher than that of Polyethylene glycol (PEG)-coated magnetic nanoparticals (PEG-MNPs) which has no targeting properties, and the cellular uptake of LyP-1-MNPs in overexpressing P32cell MCF-7was much higher than that in low-expressing P32cell CT-26. The targeting nanoparticals mainly localized in the cytoplasm, and some were found in the nucleus, we speculated that the distribution of LyP-1-MNPs maybe effected by different mechanisms of uptake routines, because of the size of LyP-1-MNPs, the most MNPs aggregated and were uptaked through cells swallowed way into the cytoplasm, while some that had good dispersibility and small size could be transported across the cell wall and nucleus wall through the receptor-mediated swallowed way. 4. The result of in vivo experiment demonstrated that the tumor volume of the experimental groups was suppressed effectively,and the curative ratio of hyperthermia group was30%(n=10), and the curative ratio of targeting therapy group was60%(n=10),which was the twice of that of hyperthermia group. Histological analysis indicated that the biocompatibility of magnetic nanoparticals was very well, because there was no any inflammation and immunoreaction. Prussian blue staining of sections of samples illustrated that the biodistribution of MNPs in body mainly accumulated in the tumors and also showed in the spleen and lungs.ConclusionsMagnetic nanoparticles had good stability, well magnetism response, great dispersibility, good warming performance, nice biocompatibility. After modified with LyP-1, the LyP-1modified magnetic nanoparticles could home to the overexpressing P32cell lines MCF-7, and the cellular uptake of magnetic nanoparticals was much higher than the non-targeting nanoparticals significantly. We also found that targeting magnetic nanoparticals had a greater inhibitive effect on tumor growth than hyperthermia group or control group.