| Background Artemisinin(ART) is an active antimalarial drug which is isolated from the Chinese medicinal herb Artemisia annua L (Qinghao). It is an endoperoxide containing sesquiterpene lactone structure, which is considered essential for antimalarial activity. Dihydroartemisinin(DHA) is one of important derivatives of artemisinin and its derivatives and it is an active metabolite of them in vivo. DHA can be synthesized from artemisinin in fewer steps and deoxidated by sodium borohydride. Recent studies have suggested that DHA has excellent antitumor effects, in spite of its preeminent antimalarial effects. DHA is shown to have anticancer effects in a wide variety of cancer models in vitro and in vivo.The main mechanism of action is cell damage mediated by ferric ion. At the same time, DHA has therapeutical effect in various xenograft nude mice models. DHA has poor water and oil solubility, and special endoper-oxide bridge in the molecular structure, so it’s likely disintegrated by the effect of light, heat, oxygen and so on. Many dosage forms of DHA are in clinical use, such as tablet, suppository and suspension, but DHA in those forms has low bioavailability for oral administration due to slow drug dissolution and decomposition in the stomach and intestine. Therefore, the development of the new formulation of DHA which enables quick availability to the body is in great need. It also can bring certain significance for anticancer use of DHA in the near future.Amphiphilic block copolymer mPEG-PLLA could absorb and excrete in vivo after being hydrolyzed and enzymatic hydrolyzed. In recent years, mPEG-PLLA has been widely used as carriers in hydrophobic drug delivery, due to its biocompatibility, biodegradability and non-toxic properties. Meanwhile, block copolymer micelles are generally formed by the self-assembly of either amphiphilic or oppositely charged copolymers in aqueous medium. The hydrophilic and hydrophobic blocks form the corona and the core of the micelles in nanoscale, respectively. The hydrophobic drugs enter into the core of the micelles, on the one hand, the solubility of drug and the stability of micellar system are improved; on the other hand, the presence of a nonionic water-soluble shell as well as the scale (10-200nm) of polymeric micelles are expected to restrict their uptake by the reticuloendothelial system(RES), liver, spleen and other tissues. As a result, the circulation time in blood and half-life of drug was prolonged. In a word, mPEG-PLLA has great advantage in drug delivery system, and has good application prospect as a carrier for hydrophobic drug delivery.Lyophilization is a kind of technology used the principle of sublimation. The samples are freezed soon under low temperature, then the frozen water sublimes into water vapor directly under appropriate vacuum environmental conditions. The thermal instability micellar system is made into lyophilized product by freeze drying technique in order to prolong the long-time stability of the drug nanocarrier which contributes to the storage, transport and employ of this preparation.Objective To increase the dissolution rate in ivtro, prolong the release time and enhance bioavailability of DHA, hydrophobic drug DHA encapsuled by mPEG-PLLA which has good biocompatibility and biodegradability was synthesized and then made into freeze-powder injection. DHA/mPEG-PLLA micelles would have small partical size, stable properties and good shapes. In addition, the aim of the work was to investigate anticancer effect for specific cancer cells in vitro and therapeutic effect for nude mice with specific tumour in vivo in order to provide a basic study for anticancer research and a probable dosage form for clinical use of DHA.Methods and contents The main contents of this work were on DHA-loaded mPEG5000-PLLA3200block copolymer micelles(DHA micelles for short), including it’s preparation and properties in vitro and in vivo. This study includes five parts, including:process optimization, characterization, exploration of properties in vitro, anticancer effect in vitro and antitumor effect in vivo of DHA micelles.Chapter1. Process optimization of DHA micellesHPLC was employed in measuring the amount of DHA in DHA micelles. The separation was performed on a Diamonsil C18column(4.6x150mm,5μm), the mobile phase consisted of acetonitrile and ultrapure water(60:40), the flow rate was lml/min, the detective wavelength was210nm and the column temperature was (25±2)℃. Single-factor index method investigated the effect of producing methods, type of organic solvents, ratio of solid DHA to solid mPEG5000-PLLA3200and ratio of double distilled water phase to organic solvent phase on the partical size and entrapment efficiency (EE) of DHA micelles. Employing modified solvent evaporation method to prepare; choosing three main factors as influencing factor, those was type of organic solvents, ratio of DHA to mPEG5ooo-PLLA32oo and ratio of water phase to organic phase; taking partical size, drug loading(DL) and EE as evaluation indices; an L9(34) orthogonal experimental design was used to systemically optimize the formulations. After adding some kinds of cryoprotectants, liquid DHA micelles was made into lyophilized products.Results:The calibration curve of DHA was linear(R=0.9999, n=6) in the range of10to400μg/ml, and the method was reproducible. The mean recovery of DHA micelles was105.32%, and RSD was3.20%(n=3). Therefore, the above method could measure the amount of DHA in DHA micelles reliably. The partical size and EE of DHA micelles were affected by producing methods, type of organic solvents, ratio of DHA to mPEG5ooo-PLLA32oo and ratio of water phase to organic phase. Taking all factors into account, the optimized DHA micelles were obtained at organic solvent of dichloromethane, mass ratio of DHA to mPEG500-PLLA32oo of1:8and volume ratio of water phase to organic phase of2:15, respectively. According to the optimized procedure, DHA micelles with (119.6±7.04)nm as mean partical size,(13.57±2.57)%as mean DL,(94.23±2.61)%as mean EE were obtained. In lyophilization process, five percent of trehalose was added in liquid micelles, and products with good shape and redissolution, high EE and small mean diameters were prepared.Chapter2. Characterization of DHA micelles The structure of diblock copolymer was confirmed by1H NMR. And segment length of the PLLA was calculated from1H NMR spectrum according to the fixed number-average molecular weight(Mn) of mPEG. Particle size analyzer (Malvern-3000Hs) was used to measure the size and size distribution of the micelles. Transmission electron microscope(TEM) was employed to detect the morphology of blank and drug-loaded micelles by negative staining with phosphotungstic acid. To make sure formation of DHA micelles, comparisons between infrared(IR) spectra of blank polymersomes, DHA-loaded micelles and the mixture of DHA and blank polymersomes were made.Results:The aggregates of the copolymer (mPEG-PLLA) used in this study, had a hydrophilic PEG block with Mn of5000and a hydrophobic PLLA block with Mn of3200. And it well meeted the experimental requirements. The mean sizes of the optical DHA micelles were (110-130)nm. TEM clearly showed the morphology of micelles, confirming that the mPEG5ooo-PLLA32oo copolymers form spherical micelles coexisting with short cylindrical micelles in aqueous media. The hydrophobic core could not be dyed by phosphotungstic acid and showed to be bright whereas the hydrophilic shell to be grey under TEM. IR spectra of blank polymersomes and DHA-loaded micelles were similar. The infrared spectrometry could distinguish the micelles from the physical mixture with many characteristic peaks of DHA. The IR spectra could show the formation of drug-loaded micelles, and drug was encapsulated in the inside of block copolymer micelles.Chapter3. Exploration of properties of DHA micelles in vitroThe critical micelle concentration (CMC) of the copolymers was determined by fluorescence spectroscopy using pyrene as fluorescence probe. A known amount of pyrene in acetone was added to various concentrations of copolymer micelles.The sample solutions were heated at37℃for5h, and then left to cool overnight at room temperature in dark. Pyrene fluorescence intensity at excitation wavelength of333nm and338nm were measured at emission wavelength of390nm. The intensity ratio (338nm/333nm) from pyrene emission spectra vs. logarithm of the copolymers concentration was plotted. The intersection point in the curve gave CMC value. For drug release study, dialysis method was used. DHA micelles and DHA suspension were transferred into dialysis bags and then placed in a shaker and shaken horizontally. Comparations between release curves in different release media were made. The media were phosphate buffer in different pH (pH6.5, pH7.0and pH7.5), containing sodium dodecyl sulfate(SDS) solution (0.3%, w/v). Accumulated release amounts were fitted to analyze the release mechanism of DHA micelles. Long-time stability of DHA micelles was observed by room temperature sample observing test to investigate DHA micelles’stability.Results:The CMC value of blank micelles was2.25×10-3mg/ml(that was2.25mg/l or2.32×10-7mol/l), which meaned that polymeric micelles prepared in this study had good resistance to dilute conditions. The micelles would have good stability under dilute coditions(e.g. intravenous injection). At the limited time, DHA released quickly and completely in phosphate buffer with pH=6.5. In the same release media, the release rate of DHA in DHA micelle was faster than in DHA suspension. The release mechanism of drug released from DHA micelles was first order model in phosphate buffer with pH=6.5and7.0, while the release mechanism was Higuchi model in pH=7.5. The release behaviour of DHA from mPEG-PLLA caused by not only diffusion mechanism, but also frame erosion mechanism. As sample store time was prolonged, partical size increased gradually and EE decreased gradually, but the shape and redissolution of DHA micelles had changed little.Chapter4. Effects of DHA micelles on different cells in vitroMTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide] assay was used to evaluate the cytotoxicity of DHA micelles. Human hepatoma cell line HepG2, human oral carcinoma cell line KB, human ovarian cell line A2780and human hepatocyte cell line L02were used in this study. Experimental data was analysed by SPSS13.0statistical software. The morphology of A2780cell and L02cell after dealing with durg was observed by converted fluorescence microscope.Results:The effects of DHA suspension and DHA micelles on the cells HepG2, KB, A2780and L02viability were significantly dose-dependent, the survival ratios declined with increasing of concentration of each drug. The inhibition reached a maximum after the cell HepG2incubated with them for48h, and the IC50values were (3.00±1.85) and (2.45±2.16)μg/ml, respectively. IC50values were (6.98±0.32) and (5.32±0.28)μg/ml after the cell KB incubated with them for72h; and the IC50values were (29.30±0.41) and (13.17±0.37)μg/ml after the cell A2780incubated with them for72h. As for HepG2, KB and A2780cells, IC50values of DHA micelles group were lower than DHA suspension group. After72h, A2780cell dealed with drug was observed cell shrinkage by converted fluorescence microscope, but the morphology of L02cell was basically no change.Chapter5. Antitumor effect of DHA micelles in vivoTwenty nude BALB/c mice were modeling in this experiment. Tumors were established by subcutaneous injection of107HepG2cells into the flanks of each mouse. When tumors reached around110mm3, the hosting mice were randomly assigned to four groups, and then DHA suspension and DHA micelles were administered daily by tail vein injection. Tumors’sizes were measured once per two days. The mice were closely monitored for14days before euthanized, and the tumors removed. The removed tumors were weighed to compare the difference of tumor mass between different groups. Experimental data was analyzed by SPSS13.0statistical software.Results:Before the treatment, volumes of tumors didn’t differ significantly among the groups(F=0.052,P=0.983). After14days of treatment, those presented significant differences among the four groups(F=10.357,P=0.003). The volumes of high dose(DHA micelles) group were less than that of control group and low dose(DHA micelles) group. Along with the extending of time, volumes of tumors in vivo of all nude mice groups gradually increased. The isolated tumor mass had significant differences between high and low dose group (DHA micelles)(P=0.028) and between high dose group and control group (P=0.003). The inhibition rates of high dose group, DHA group and low dose group were71.70%,40.25%and26.42%, respectively. |
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