| BackgroundBreast cancer, one of the most common malignant tumors for women, has seen its elevation every year. According to statistics, there are about1.3million people suffering breast cancer and500000dying of that each year in the world. Obviously, breast cancer has become one of the biggest threats for women’s health.Treatments for breast cancer at present mostly include surgery, radiotherapy, chemotherapy, biological therapy etc.. Among them, the chemotherapy plays a significant role and anthracycline has been one of the most accepted and effective drugs. However, administration of adriamycine has been limited for its toxic and side effects, especially its cumulative dose cardiotoxicity. To solve this problem, nanotechnology has been widely studied.Compared with traditional material, nanomaterials,1-100nm in diameter, have different properties such as surface effect, volume effect and quantum size effect, which made it widely used in engineering, chemistry, biology, medicine and so on.In the field of medicine, nanomaterials could improve curative effect and the bioavailability of some chemotherapy drugs and reduce their toxicity by encapsulating them. In addition, they could carry some special factors, such as cytokines, antibodies, directly to target tumor cells. Nano drug carriers are mainly divided into four categories:1. nanoparticles can encapsulate drugs to enhance their physical and chemical stability which protect them against degradating.2. nanoparticles not only encapsulate drugs but also tumor targets, which can guide the drug to the tumor lesions selectively and reduce toxicity in vivo.3. nanoparticles encapsulate drugs and iron oxide to guide these nanoparticles to the expected tissue in the additional magnetic field, showing the function of targeting and imaging based on their superparamagnetism.4.Multi-functional nanoparticles, encapsulating drugs, targets, ferric oxide and so on, could exert the effects of smart drug release, targeting, imaging and real-time monitoring.At present, nanomaterials are mainly applied as drug carriers in various cancers and have been widely developed. Ideal nanomaterial carriers should meet the following properties:stability, no toxicity, no irritate, no teratogenicity, small particle and narrow size distribution; protecting drugs and increasing its stability; ability to respond to external environment, such as PH, temperature, enzyme and redox, etc.; carrying a variety of drugs;imaging,etc. Nowadays, some nanomaterials have been widely studied, including liposomes, iron oxide, quantum dots and polymers, etc. Polymer is divided into inorganic molecules and organic molecules. The former contains inorganic nanometer hydroxyapatite and mesoporous silicon, while the latter includes small molecules and macromolecule polymer materials, which are divided into natural and artifical macromolecular such as poly(lactic acid/glycolic acid) copolymer, amphiphilic copolymer, etc. Macromolecular polymer material has good biological compatibility and biodegradable properties, and is a very promising nanomaterial. Liposome, with single or multiple double phospholipid membrane vesicles, is the double molecular capsule similar to the biological membrane and its main ingredient is phospholipids. One end of the phospholipids is the phosphate group which makes it hydrophilic, while the hydrocarbon chain at the other end makes it hydrophobic. Also liposome has low immunogenicity and good biological compatibility and could prolong the biological half-life and duration of drug action with drug controlled release. Besides, the liposome can increase the curative effect of drugs and reduce adverse reactions with the help of high capillary permeability of tumor tissue and low permeability of normal tissues. Otherwise, as one of the most promising nanoparticles, iron oxide has been widely studied. Its superparamagnetism help magnetic nanoparticles make of directional migration possible, so that to attain the goal of drug targeting, tracking and monitoring in vivo. Unfortunately, pure iron oxide nanoparticles are not very stable in the water. However, some hydrophobic materials such as oleic acid, polyethylene, Planck, PLGA can increase their biocompatibility obviously by encapsulating them. Researchers are finding ways to increase the biocompatibility of the magnetic nanoparticles as well as to explore methods of intelligent drug release. Previously, studies have reported that some nanomaterials could degrade and achieve the intelligent drug release under the influence of some certain environmental stimulations such as redox, temperature, PH etc.. Most of these studies, however, stay in the level of the chemical reactions, while a few achieve the cell level and even fewer reach the animal level.To achieve the aim that adriamycin could be guided to tumor tissue specially, we use the new adriamycin nanocarrier DOX/rPAA@SPION to study the curative effect, toxicity, biological distribution and MRI imaging in animal level. Since DOX/rPAA@SPION can focus on tumor tissues with the help of external magnetic effect, we’re able to achieve targeted drug delivery and nuclear magnetic resonance (NMR) imaging tracer monitoring purposes. Moreover, rPAA is sensitive to acids and reducing agents which are abundant in the tumor environment, so that the nanoparticles reaching the tumor site can release adriamycin intelligently and effectively and kill tumor cells and improve curative effect. Besides, the unique structure of nanoparticles can also help reduce the chemotherapy drug’s adverse reaction and prolong its half-life.Objective1. Establish the MDA-MB231nude mouse model to explore the anti-tumor effect of DOX/rPAA@SPION magnetic nanoparticles in vivo.2. To understand the toxcity of the DOX/rPAA@SPION magnetic nanoparticles in vivio by observing the state of nude mice, weighting the mice and performing HE stain to the heart, liver and kidney of the mice.3. To detect DOX/rPAA@SPION distribution in vivo by Prussian blue staining to the heart, liver and kidney of the mice.4. To image, monitor and visualize DOX/rPAA@SPION magnetic nanoparticles by performing MRI on nude mice before intravenous administration,12h and24h after giving DOX/rPAA@SPION injection respectively.Methods1. MDA-MB231breast cancer cell lines:resuscitate and culture cells with L15culture medium containing10%Fetal bovine serum, digest cells in logarithmic growth phase, count, and lastly dissolve cells in150ul serum-free L15medium.2. The nude mice aged5w were purchased from southern medical university laboratory animal center. Being raised in SPF condition, activities and mental state of them were closely observed during the adaptive phase.3. Establish MDA-MB231breast cancer nude mice model:serum-free cell suspension solution was injected subcutaneously to right flanks of the nude mice.4. DOX/rPAA@SPION magnetic nanoparticles solution and magnetic validation: dissolve DOX/rPAA@SPION nanoparticles into sterile PBS solution by using the ultrasonic instrument with ultrasonic probe under the PBS liquid surface for at least 30min. After the nanoparticles were fully dissolved, observe its paramagnetism by putting the nanoparticle suspension in magnetic field.5. The curative effect of DOX/rPAA@SPION:When subcutaneous tumors of the mice were～50mm3, mice were divided into three groups (n=7) randomly. Group one (control group) was treated with0.9%normal saline, group two (adriamycin group) was treated with doxorubicin alone, and group three (DOX/rPAA@SPION group) was treated with DOX/rPAA@SPION and immediately place a magnet on the tumor for1.5hour after injection every time. Group two and three were injecting with doxorubicin at a dose of7mg/kg once weekly for2consecutive weeks. All mice were injected though the tail vein. Measure tumors’ length (a) and width (b) every two days until24days after treatment, tumor volume (v) was calculated as v=a×b2/2, weigh tumor tissues after nude mice were killed.6. The toxicity and biological distribution of DOX/rPAA@SPION:observe the activities and mental state of mice and weigh them every two days during the study. On24th day after treatment, all mice were put to death. Some tissues (tumor tissue, heart, liver, kidney) of the mice were stripped and routinely stained with Prussian blue and HE.7. The MRI of DOX/rPAA@SPION:The mice were anaesthetized with pentobarbital sodium (1%) at a dose of70mg/kg. MRI experiments were carried out with a1.5tesla (T) MRI machine (TR/TE=3500/120ms, FOV=170,68,17mm, matrix=260×157, NEX=4, thickness/interval=2/0.6mm). The coronal and transversal cross sections images were acquired before and after administration of DOX/rPAA@SPION solution at a dose of70mg (Fe)/kg body weight through the tail vein on12h and24h respectively.Results1. The DOX/rPAA@SPION nanoparticles were fully dissolved by the ultrasonic instrument and revealed good paramagnetism at a magnetic field.2. On the4th day after the treatment, the tumor size of control group, adriamycin group, and DOX/rPAA@SPION group mice were137.47±20.62mm3,122.79±21.19mm3, and51.70±7.92mm3respectively. The tumor volume of DOX/rPAA@SPION group was significantly smaller than that of the control group (p=0.000) and the adriamycin group (p=0.000), while no significant difference (p=0.138) was found between the adriamycin group and the control group. On the8th day after the treatment, tumor size of control group, adriamycin group and DOX/rPAA@SPION group mice were259.88±7.70mm3,195.48±9.77mm3and72.08±6.06mm3respectively. The tumor size of the adriamycin group and the DOX/rPAA@SPION group were obviously smaller compared with the control group. And the tumor size of the DOX/rPAA@SPION group was smaller than that of the adriamycin group with statistical significance (p＜0.05). On the24th day after the treatment, tumor size of control group, adriamycin group and DOX/rPAA@SPION group mice were1378.29±104.85mm3,838.02±189.45mm3and247.31±60.14mm3respectively. The tumor size of the DOX/rPAA@SPION group was obviously smaller compared with the control group and the adriamycin group (p＜0.05)。And the inhibition ratio of adrimycin and DOX/rPAA@SPION was41.67%and76.96%respectively.3. The mice weight of the control group, adriamycin group, and DOX/rPAA@SPION group were19.36±0.35(g),19.39±0.37(g), and19.37±0.35(g) respectively on the first day of the treatment. And there is no statistical significance (p＞0.05) within these three groups. Compared with the control group, the mice weight of the adriamycine and DOX/rPAA@SPION group had no significant changes (p＞0.05) in the study. Besides, the mental status and activity of all the mice were normal. Different degrees of tumor necrosis and apoptosis can be seen in the tumor tissues of all the three groups, while the other tissues (heart, liver, kidney) did not show obvious cell apoptosis, necrosis, fibrosis and other pathologic changes in the three groups.4. The results of Prussian blue showed that the tissues (tumor, heart, liver, kidney) of the adriamycine group and the DOX/rPAA@SPION group had no obvious iron deposition on the24th day after the treatment when compared with the control group.5. The MRI results:the signal of the tumor area, lungs and the liver were normal before the administration of DOX/rPAA@SPION. The signals of the tumor areas became lower, as well as the signals of the the lungs and the liver at the12th hour after the administration of DOX/rPAA@SPION. And24hours after the administration the signals of the tumor areas, the lungs and the liver became lower than the signals of those areas before the administration and became slightly higher than those12hours after the administration.Conclusion1. DOX/rPAA@SPION nanoparticles on MDA-MB231breast cancer mice models significantly inhibited tumor growth, demonstrating the obvious curative effect of this nanoparticle.2. The mice with DOX/rPAA@SPION nanoparticles were in good mental state and no obvious pathologic changes were observed in their organs in the study, suggesting this nanoparticle had low toxicity and prominent safety.3. There were not any iron deposition in tumor tissues and organs on24th day after treatment, which demonstrated this nanoparticle metabolized quickly and had good biocompatibility.4. MRI can be used to visualize and real-time monitor DOX/rPAA@SPION nanoparticles in tumor lesions. MRI imaging on24th hour after injection showed remaining drugs in tumor area, indicating the DOX/rPAA@SPION has long half-life. Above all, DOX/rPAA@SPION magnetic nanoparticle is a mult-fuctional particle that has good curative effect, low toxcity and MRI constract effect in vivo and maybe have wide clinical application prospect. |
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