The Study Of Building Of Bufalin-loaded Pluronic Polyethylenimine Nanoparticles And Its Inhibitory Effect On Colorectal Cancer

[Background and objective]Background:Colorectal cancer is the third most common cancer and the third leading cause of cancer death in men and women in the United States. The survival and prognosis of colorectal cancer patients is dependent on the stage of the tumor at the time of detection. Unfortunately, about50%of patients display regional or distant metastases at the time of diagnosis. Therefore, the treatment of colon cancer has become a focus of research worldwide. The formation of metastasis is an enormously complex process, whereby tumor cells disseminate from the primary tumor to colonize distant organs. This multistep process involves initial malignant cell invasion of the microenvironment, filtration into the bloodstream, survival during migration, and extravasation into distant organs. Subsequent steps, including proliferation, induction of angiogenesis, and evasion of apoptotic death, must be overcome by the malignant cell for successful metastasis. Although tumor invasion and metastasis is fatal, the molecular mechanism is not yet fully clear. Therefore, we are now looking for new treatments. Bufalin is a soluble digoxin-like immunoreactive component of Chansu (a traditional Chinese medicine), and is an extract of dried toad venom from the skin and parotid venom glands of Bufo gargarizans or B. melanostictus, and can produce a significant antitumor effect in a variety of tumors, including hepatocellular carcinoma, colorectal cancer, leukemia, and gastric cancer. A large number of studies have shown that antitumor effects of bufalin include inhibition of cell proliferation and induction of apoptosis of tumor cells. Because of its toxicity, insolubility in water, fast metabolism, short half-life, and other shortcomings, its application in cancer therapy is limited. However, studies have shown that nanocomposites can combine bufalin to overcome these shortcomings.Nanoparticulate drug delivery systems are making a significant contribution to the improvement of drug delivery in cancer. Targeting nanoparticulate drug delivery systems, especially biodegradable nanoparticles, provides opportunities to meet these existing challenges, with the advantages of improved pharmacokinetics, favored tumor accumulation, and reduced side effects. Hydrophilic pluronic polymers have a novel type of amphiphilic, nonionic surfactant micelle structure that is insoluble in the aqueous solution of the compound, which provides good solubility in the hydrophobic microenvi-ronment, thereby increasing the solubility of water-soluble sub-stances. The potential use of pluronic polymers as biomedical drug carriers, for controlled drug release and gene therapy, has been given much attention. polyethylenimine (PEI) is a class of cationic polymer with a large group structure, which increases the stability of pluronic polymeric composites.Objective:To prepare bufalin loaded Pluronic-PEI nanoparticles with the characteristic of targeting and sustained-release effect, and establish the quality evaluation standard; Determination of the nanoparticles in vitro release and target distribution, to explore its inhibitory effect on colorectal cancer in nude mice, in order to reveal the pharmacological mechanism of bufalin loaded Pluronic-PEI nanoparticles.[Methods]1. preparation method of encapsulation for bufalin Pluronic-PEI nanoparticles: select the low molecular weight PEI (Mw=2000) as the crosslinker, improve the Pluronic thermodynamic instability, preparing the bufalin Pluronic-PEI nanoparticles by ultrasonic emulsification method.2. Study on physicochemical properties of encapsulation of bufalin Pluronic-PEI nanoparticles:observe the particle size distribution and nano microcapsule characterizationby transmission electron microscope; Determine the nanoparticles encapsulation efficiency and drug loading, create the curve of vitro release, test the toxicity of blanknano microcapsule cell.3. Targeting Distribution Research of encapsulated bufalin Pluronic-PEI nanoparticles:using RhodamineB as a fluorescence probe, packaged in the nano microcapsule, observes the distribution situation of nano microcapsule intake in cells under confocal laser scanning microscope.4. Inhibitory effect of bufalin loaded Pluronic-PEI nanoparticles in nude mice with colorectal cancer:establish a HCT116colon cancer tumor model in nude mice, were randomly divided into control group, blank nanoparticles group, bufalin group, encapsulation of bufalin nano microcapsule group, oxaliplatin group, and intravenous injection. After treatment,compare the body weight changes; targeting fluorescence imaging and systemic metastasis of nude mice.[Results]1. The preparation and physicochemical properties of encapsulation of bufalin Pluronic-PEI nanoparticles:overcomes pluronic thermodynamic instability by chemical crosslinking with crosslink-modifier, the particle size of prepared nanoparticles is uniform, the shapes are round, the minimum diameter is30nm and80nm maximum diameter, the average is65±0.214nm, the Zeta potential is5.87±0.42mV. The highest loading is at the time When bufalin dosage is0.4mg.2. The vitro release of encapsulated bufalin Pluronic-PEI nanoparticles is obviously lower than that of free bufalin. In4hours, the release rate of free bufalin has exceeded60%already. But for nanoparticles, in the first5hours, it shows the burst facies, the cumulative release amount is up to40%～50%, after5hours, nanoparticles in vitro release obviously slowed down, at32hours, the cumulative release amount of bufalin reached70%～80%.3. Research of Targeting drug distribution:in HCT116cells, red fluorescent drug loaded nanoparticles group Rhodamine B was stronger than the free Rhodamine B, and is mainly distributed in the cytoplasm of a cell, only a small amount of distribution in the nucleus, and the red fluorescence could not be observed almost at free Rhodamine B nucleus.4. Inhibitory effect of bufalin loaded Pluronic-PEI nanoparticles on nude mice bearing colon cancer:comparison between of bufalin Pluronic-PEI nanoparticles and dose of bufalin after treatment, for nanoparticles, the inhibitory effect for metastasis growth and movement of nude mice are more obvious, and the survival of higher quality.[Conclusion]1. This study established the methods of preparation and quality evaluation of encapsulation of bufalin Pluronic-PEI nanoparticles using the standard method of ultrasonic emulsification.2. Compared with free bufalin, encapsulation of bufalin Pluronic-PEI nanoparticles showed obvious sustained-release characteristic, with the extension of time to gradually release the drug in32hours, the cumulative release up to70-80%.3. Cellular uptake efficiency of Rb-bufalin-Pluronic-PEI nanoparticles significantly increased, the nanoparticles can more effectively target the tumor, has good effect to the tumor target.4. Cell proliferation rate of blank nanoparticles is over80%,its Shows almost no cytotoxicity.5. Encapsulation of bufalin Pluronic-PEI nanoparticles has significantly inhibited metastasis on cancer growth and movement, has better therapeutic effect on colorectal cancer in nude mice, and can significantly improve the quality of life in nude mice.