| 1. The research backgroundTreatment of malignant tumor is a difficult problem in the field of life science and medicine.The high incidence of breast cancer in women, is an important health problem in the world, especially in low and middle income countries. Paclitaxel is extracted and separated from the bark of short leaves taxus chinensis as antitumor drugs. Paclitaxel is an effective anticancer drugs for clinical treatment of many solid tumors.Taxol(?) solubility is extremely low in water, thus it always dissolved polyoxyethylene castor oil and anhydrous ethanol (50:50, v/v) in the clinical.However, polyoxyethylene castor oil release histamine duing metabolism in vivo, then a severe allergic reaction will happen. Paclitaxel lack of selectivity of target tissue, there are also such as bone marrow suppression, thrombocytopenia, leukopenia, anemia and other systemic side effects. Negative effects of polyoxyethylene castoroil, non-selective systemic toxic and less cancer cells consume, greatly limits the application of Paclitaxel.Therefore, carring Taxol(?) with carrier bags to improve efficacy, reduce the toxic and improve bioavailability is imperative.Liquid fluorocarbon (perfluooctylbromide) nanoparticles are composed of perfluooctylbromide (perfluooctylbromide PFOB) parcelled with lipid monolayer. According to report, liquid fluorine carbon nanoparticles (NPs) have excellent security to substitute blood in clinical. When liquid fluorine carbon nanoparticles (NPs) are studied as a new generation of ultrasound contrast agents, it were found have advantages as a drug carrier. Targeted nanoparticles drug delivery system, is a rising technology in recent years, and its potential as a drug delivery system is more and more taken seriously in recent years. Breast cancer, ovarian cancer cells express high cell medium glycoprotein, and cerebral fat sulfate (sulfatide) is combined with cell matrix glycoprotein on the outside or inside cells, especially with TN-C.In this study, we connect sulfatide and NPs as targeted drug delivery system (SNPs) of PTX on treatment of breast cancer. At present most of target NPs are clone antibody as target material, because the clone antibody were immunogenic,poor stability and complex operation etc. It greatly restricted the further development of targeted imaging and drug delivery. Sulfatide as target material, have strong stability, and advantages of easy operation. The most important is that it not only can be used as a target material, and in the SNPs can also act as one of the ingredients of outer membrane lipid. After targeted NPs contact with cells, it can exchange phospholipids with the phospholipid bilayer of cell membrane, thus it can delivery wrapped drugs in carrier to the cells quickly and effectively. Therefore, SNPs as a targeted NPs, can pass more drugs through faster way into the target cells more efficient. This study use SNPs as PTX targeting nano delivery carrier, have two advantages:one is the SNPs have targeted effection of breast cancer, the second is after targeted NPs contact with cells, it can exchange phospholipids with the phospholipid bilayer of cell membrane, thus it can delivery wrapped drugs in carrier to the cells quickly and effectively.Therefore, this study used liquid fluorine carbon nanoparticles of PTX which containing sulfatide (PTX-SNPs) as the PTX targeting delivery systems to characterize, cell toxicity, as well as pharmacodynamics research in vivo.2. The research methodshi the first part of this paper, PTX-SNPs was successful made by using reverse evaporation method.Use drug loadings (DLC), the coating rate (DLE) and particle size as the main indexes, and through the single Factor analysis that lipid concentration (Factor A), the quality ratio of EPC and sulfatide as (Factor B), the content of PFOB (Factor C) and the amount of PTX (Factor D) as the main influence factors, and through the four factors three levels orthogonal experiment that the best prescription of PTX was successfully achieved PTX-SNPs, and carries on the characterization and research.The second part of this paper inspected cell inhibitory effection in breast cancer cells of mice (EMT6), at the same time by using flow cytometry to detect cell apoptosis. And made blank liquid fluorine carbon nanoparticles (NPs), blank liquid fluorine carbon nanoparticles contain cerebroside sulfate (SNPs), paclitaxel liquid fluorine carbon nanoparticles (PTX-NPs), containing cerebroside sulfate liquid fluorine carbon nanoparticles of paclitaxel (PTX-SNPs) and Taxol(?) (Taxol(?)). Using MTT and flow cytometry to examine their cell inhibitory effect. At the same time used Coumarin 6 as a fluorescence probe to do cells engulf fluorescence experiments, and verify the prescription of cell damage is due to relatively high intracellular drug concentration after the prescription action.In the third part of this paper, the rat caudal vein respectively injected Taxol(?)(?), PTX-NPs, PTX-SNPs with dosage for PTX 10 mg/kg. To establish the HPLC analysis method for determining the PTX plasma concentration in rat, and draw blood drug concentration curve. In the end it also studied the pharmacokinetic behavior of Taxol(?), PTX-NPs, PTX-SNPs in rats.For determining the drug distribution of Taxol(?), PTX-NPs, PTX-SNPs in various organizations of tumor-burdened mice, firstly, need to build mould. Inoculate tumor in mice with the exponential growth (EMT6) breast cancer cells. First of all, digest and collect the cells trypsin, and washing in culture medium. Then used the hemocytometer to count cells, centrifugal, remove the supernatant. With PBS to suspension, centrifugal, remove the supernatant. According to the above methods, wash three times with PBS, avoid the mice immune rejection caused by residual FBS. Then took the concentration of 1.5×107 with precipitate saline EMT6 breast cancer cells of mice for 0.2 mL to inject in the right side of 5 weeks BALB/c mice’s back. When the mice tumor diameter reached around 5-8mm two weeks later, and mice tumor even volume grow to about 200 mm, building successful. With EMT6 tumor mice were injected in caudal vein of Taxol(?), PTX-NPs, PTX-SNPs, given the dose of 10mg/kg.Use HPLC to determine the drug concentration in organization, observe and examine the Taxol(?), PTX-NPs and PTX-SNPs tissue distribution in mice. Frozen section in Coumarin 6 as a model drug, and inject the Coumarin 6, Coumarin 6-SNPs and Coumarin 6-NPs in mice tail vein, respectively kill mice in the 1h and 8h, take out the tumor tissue and dipped in paraformaldehyde, pay attention to avoid light preservation, detect slice under the fluorescence microscope.Breast cancer in mice model was established in the fourth part of this paper, At first it need to build mould.Building method consult to the third part of this article, after the mould building, respectively inject saline solution (control), blank SNPs, Taxol(?), PTX-NPs and PTX-SNPs in the tail vein, and give each group the same PTX dose of 10 mg/kg. Once every four days injection, and injection for 20 days. Mice must be weighed, mice tumor volume be measured and record before each injection.At 20th day, kill mice, and weighed the tumor, then soaked in paraformaldehyde, paraffin embedding, at last slice for immunohistochemical experiment. By drawing the mice tumor tissue volume growth curve, calculating the change of each index and tumor biopsies of TUNEL apoptosis detection, vascular density (MVD) and Bad immunohistochemical experiments, and then evaluate of Taxol(?), PTX-NPs, PTX-SNPs treatment for breast cancer in mice.2. The experimental results1. The PTX-SNPs preparation and evaluationThe preparation of PTX-SNPs was through rotary evaporation method. Use DLC, DLE and particle size as the main indicators, through the single Factor analysis and calculation, determine the lipid concentration (Factor A), the quality ratio of EPC and sulfatide (Factor B), the content of PFOB (Factor C) and the amount of PTX (Factor D) four factors as the main influence factors, and through the four factors 3 levels orthogonal experiment design, and eventually got the best prescription for lipid concentration (Factor A) was 1.2 mg/mL, EPC and sulfatide quality ratio (Factor B) is 40:1, volume of PFOB (Factor C) was 15%, the dosage of the PTX (Factor A) was 1.3 mg/mL.By measured of high performance liquid (HPLC), to optimize the prescription after the PTX-SNPs, the PTX coating rate was 96.20%±0.14%, and the drug was 1.24 mg/mL. The particle size of PTX-SNPs was 237.5nm, Zeta potential was 14.7±0.7, dispersion coefficient was 0.28±0.045, and the particle size distribution measured by melvin determination was uniformity. PTX-SNPs was with blue opalescence appearance, under the electron microscope observation PTX-SNPs appearance spherical or class of spherical, and particle size was basically identical with melvin test results. By HPLC determining the release of Taxol(?), PTX-NPs and PTX-SNPs. In the dialysis 14h, PTX release rate had been close to 100% in Taxol(?); PTX release rate were 52.78%±1.29% and 49.78%±1.28% respectively in PTX-SNPs and PTX-NPs. Dialysis 60h, PTX release rate were 87.44%±1.78% and 80.80%±1.74% respectively in PTX-SNPs and PTX-NPs. The release of PTX character was the initial burst release, then was continuous, slow release in PTX-SNPs and PTX-NPs.2. The PTX-SNPs cytotoxic effectIn order to investigate PTX-SNPs cytotoxic effect of breast cancer cell (EMT6) in mice, the comparison of several kinds of preparation cytotoxic effect on breast cancer cells (EMT6) in mice was evaluated by MTT method. After PTX-SNPs effect compared the activity of breast cancer cells (EMT6) was lower than the free PTX-NPs in mice. In PTX solution with PTX 10μ g/mL, PTX-SNPs and PTX-NPs effection, after 48h of breast cancer in mice cells (EMT6) early apoptosis rate were 3.9%±0.3%,4.6%±0.6%, and 13.6%±0.5%. When the concentration of PTX up to 30μg/mL, early apoptosis rate increased to 13.1%±0.2%,13.3%±0.2%, and 44.9%±1.7% respectively.We speculated from the results that PTX-SNPs and breast cancer cells in mice EMT6 cell co-culture, the drug concentrations in the cells were higher than PTX-NPs combine with free PTX cellular drug concentration. Took coumarin 6 as model drug, joint target block experiment, and the results showed that with the Coumarin 6-SNPs work together for 2h, the fluorescence intensity in cells were 1.9 times than Coumarin 6-NPs and 2.0 times than Coumarin group. At 12h with Coumarin 6-SNPs combination, the trend of the fluorescence intensity in cells uniformity to 2 h.3. The PTX-SNPs concentrations in the plasma and in the organization of ratsTaxol(?) concentration in plasma of rats presents the fast elimination phase drug within 2h, in the dosing 8h plasma concentrations of already could not detect PTX. PTX-SNPs and PTX-NPs concentration present a rapid elimination phase in plasma of rats within 2h, then with comparatively slow elimination between 2-8h, between 8h and 24h after a with very slow speed to eliminate, the blood drug concentration had maintained at lower levels.PTX-SNPs and PTX-NPs distribution in tissue was different from the Taxol(?), as nano preparation after injection of PTX-SNPs and PTX-NPs, PTX distribution of the liver and spleen in mice were better than injections of Taxol(?) group. After PTX-SNPs dosing, PTX concentrations of tumor tissue in mice were significantly greater than Taxol group drug concentration after the treatment.4. The PTX-SNPs therapeutic effection of breast cancer in mice (EMT6)After treatment by PTX-SNPs, PTX-NPs and Taxol(?), TIR respectively were 70.8%,45.6% and 34.0%; And DT respectively were (11.8±11.8) days, (6.3±0.4) and (5.8±0.4) days. This showed PTX-SNPs treatment effection in mice breast cancer (EMT6) was better than that of PTX-NPs and Taxol. Tumor growth curve, and the tumor weight of 20 days are coincide with the above results. Experimental results of TUNEL tumor tissue apoptosis showed that the PTX-SNPs group apoptosis rate was 46.83%±4.9%, and obviously higher than that of PTX-NPs (30.91%±3.52%) and Taxol(?) (24.27%±5.32%) and other groups. At the same time immunohistochemical experiments such as MVD and Bad experiment also proves that the PTX-SNPs can promote the secretion of tumor Bad and reduce tumor growth of new blood vessels, thus effectively promote breast cancer cells apoptosis.4. Research conclusionThis research successfully got the preparation of PTX liquid fluorine carbon nanoparticles containing sulfatide targeting drug delivery system. Blue opalescence appearance, the shape was spherical or class spherical under tem. The encapsulation rate was 96.20%±0.14%, and the drug was 1.24mg/mL. PTX-SNPs particle size was 237.5nm, Zeta potential was 14.7±0.7, dispersion coefficient was 0.28±0.045, melvin determination of size distribution was uniform. In tumor status, endothelial gap widened, and allowed diameter less than 700nm particles to pass through. PTX-SNPs particles size at around 200nm, and made condition for SNPs to reach the tumor cells passing PTX through the capillary. PTX-SNPs appearance with blue light. In vitro condition imitating dialysis at 14h in vivo, PTX release rate of Taxol(?) had been close to 100%; PTX release rate of PTX-SNPs and PTX-NPs were 52.78%±1.29% and 49.78%±1.28% respectively. Dialysis at 60h, PTX release rate of PTX-SNPs and PTX-NPs were 87.44%±1.78% and 80.80%±1.74% respectively. PTX released characteristic of PTX-SNPs and PTX-NPs was burst release in initial, then was continuous and slow release. In vitro simulation environment, release had delayed effection than Taxol, that was because phospholipid monolayer played valve and control of the membrane on drug release.By measuring and comparing the PTX-SNPs, PTX-NPs and Taxol(?) three preparations’influence on breast cancer cell (EMT6) activity in mice, and the results showed that PTX-SNPs had stronger cytotoxic effect than PTX-NPs and Taxol(?) of breast cancer cells (EMT6) in mice. Then through early apoptosis rate determination showed at 48h, PTX-SNPs had better effect than PTX-NPs and Taxol(?) on promoting apoptosis of breast cancer cell (EMT6) in mice. Took Coumarin 6 as a model drug, by cell swallow carrier bag to gobble up drugs in the experiment, confirmed that the Coumarin 6-SNPs compared Coumarin 6-NPs and Coumarin 6, can pass more Coumarin 6 to EMT6 breast cancer cells in mice. Because high drug concentration in target cells, so medicine is also much greater lethality.By measuring the rat blood drug concentration curve and tissue distribution in mice, PTX-SNPs had changed the pharmacokinetic behavior of PTX. PTX-SNPs half-life in rat plasma is longer than Taxol(?), the distribution of the liver, spleen and EMT6 tumors in mice got more Taxol(?). Target parts had high concentrations of drugs, therapeutic effect was strong. Therefore, we also conducted PTX-SNPs treatment of preliminary pharmacodynamics study on breast cancer EMT6 in mice. By measuring the tumor volume, drawing volume-time growth curve, calculating tumor weight, DT and TIR, to verify the PTX-SNPs curative effection of breast cancer EMT6 in mice is much better than the PTX-NPs and Taxol(?). TUNEL and immunohistochemistry experiment results show that the PTX-SNPs can promote breast cancer tissue cell apoptosis and tumor necrosis factor Bad secretion, at the same time reduce the growth of tumor MVD. All of these helped to inhibit the growth of tumor tissue and promote tumor cell death. The treatment of breast cancer in mice, PTX-SNPs treatment effection is much better than PTX-NPs and Taxol(?), and due to sulfatide targeting function on EMT6 breast cancer cells in mice or glycoprotein extracellular matrix. |
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