| The purpose of this experiment was focused on the preparation of methoxy polyethylene glycol-poly (lactic acid)-MMC micelles. First we prepared methoxy poly (ethylene glycol)-poly (lactic acid) block copolymer (mPEG-PLA) and analyed the structure and its characteristics with infrared spectroscopy (IR) and H1NMR. Adopting acetone dissolved–volatilization methods, the mPEG-PLA-MMC micelle was prepared, mPEG-PLA micelle was filtered through 0.45um micropore membrane and the filtrate was collected. The collected filtrate was freezingly dried to botain the solid of mPEG-PLA-MMC nanomicelles. The nanomicelle sizes and shape were characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The encapsulation efficiency and drug loading of mPEG-PLA-MMC nanomicelles were examined by UV spectrophotometry. Effect of micellar nano-particles about burst release, sustained release were also measured by UV spectrophotometry. The critical micelle concentration (CMC) of micellar mPEG-PLA nanoparticles was measured by fluorescence spectrophotometery. With PBS as buffer solution, MMC release from micellar mPEG-PLA nanoparticles was measured by UV spectrophotometry. MTT test was used to study the influence of mPEG-PLA block polymer on the MMC pharmacological action in vitro. The main experimental cells were human stomach adenocarcinoma BGC-823 cells and human liver cancer HepG2 cells. The mouse sarcoma S180 and ascites hepatoma H22 models were used to study the pharmacological action of the mPEG-PLA-MMC nanomicelles in vivo, and the results were compared with the single use of MMC to study the effects of micellar mPEG-PLA-MMC nanoparticles to target tumors and to enhance the pharmacological action of MMC.1. The methoxy poly(ethylene glycol) - PLA block polymers (mPEG-PLA) Preparation and CharacterizationTaking the methoxy polye (thylene glycol) and D, L- lactide as raw materials, the mPEG-PLA block polymer was synthesized by direct ring-opening polymerization. The mPEG and D, L-lactide were dried inside the vacuum drying oven, at 70℃, 0.1mPa pressure for 2h. The toluene was purified by distillation before used. The mPEG and D, L- lactide were invested according to certain mass ratio into the airtight three-necked bottle, taking the stannous octoate as the catalyst, and reflowed for 8h at 140℃under nitrogen protection. The primary product of methoxy poly (ethylene glycol)-ploy (lactic acid) block polymer (mPEG-PLA) could be obtained at the end of the reaction. The mPEG-PLA block polymer was white or mike-like cream after purification. The mPEG-PLA block copolymer was characterized by IR and H1NMR. After analysis we could make sure that the samples were methoxy poly (ethylene glycol)–poly (lactic acid) block copolymer (mPEG-PLA).2. Critical micelle concentration (CMC) DeterminationThe CMC value of mPEG-PLA block polymers was measured by fluorescence substance pyrene which was as a probe. With the increase in the concentration of mPEG-PLA copolymer concentration, pyrene excitation light power increased and excitation wavelength red-shifted. When the copolymer concentration got above CMC value, some of pyrene entered in the micelles, the I338/1333 ratio changed significantly, so we could determine the CMC values by this obvious change point of the copolymer. Prepared mPEG-PLA solution that contained a certain amount of pyrene, the value of CMC 1.9mg / L was obtained by calculation with the determined fluorescence values.3. Preparation and Characterization of mPEG-PLA-MMC micelles3.1 Preparation of mPEG-PLA-MMC micellesAccording to different quantity ratio, mPEG-PLA block copolymer and MMC were dissolved into a certain volume of acetone solution and kept stirring till all the solids were dissolved. In the ventilation chamber and dark conditions the solution was added to a beakea with certain amount of triplply distilled water at speed of 1 drop/s, a magnetic stirrer being used for the complete mix of the dropped solution witn the water. Acetone may volatilize completely by ventilation. Certain period of time later, the solution was removed and transferred into the previously treated dialysis bags, the MMC could be educed from the bags while the micelles could be preserved in them in the constant temperature water bath oscillator. When the dialysis ended, the micelles in the dialysis bag were transferred to 0.45um sterile pore membrane for sterilization. Filtrate was collected by autoclaving glassware and pre-freezed under -20℃. The pre-freezed filtrate was moved into freeze-drying machine when the pre-frozen finished, and solid preparation of micelles were obtained when freeze-dried completed. The micellar mPEG-PLA-MMC nanoparticles were preserved in the sealed dark surroundings under normal temperature.3.2 Micelle sizes and morphology characterizationMicellar particle sizes and shape were mainly characterized by transmission electron microscopy (TEM) and atomic force microscope (AFM).3.2.1 Transmission electron microscopy (TEM)First of all, a certain concentration of micellar solution was prepared and then dropped to 200 mesh carbon-coated copper screen, fixed 2min and then the liquid was sucked with filter paper. Waited 5min, the micelles were stained with 3% (m/v, pH=5.0) of phosphotungstic acid negatively for 1min, dried with filter paper and observed under TEM for the particle size and shape. The results showed that micelles are spherical particles and have core-shell structure with bright in the middle because of the large density of the PLA hydrophobic core and the less desity of the peripheral layer of the hydrophilic mPEG. Blank micelle particle sizes are about 50nm, while the drug-loaded micelle particle sizes are between 80~100nm with a more uniform distribution.3.2.2 Atomic force microscopy (AFM)A certain concentration of micellar solution was prepared and scattered as far as possible by ultrasonic to reduce reunion. After the ultrasound,the stripping solution was added to the freshly splited mica surface, fixed 5min and then the solution was sucked as rigourously as possible with filter water. At last, the samples were dried completely in the air. The dried samples were observed under atomic force microscope (AFM). The results showed that the particle sizes of micelles are consistent with that of the TEM.3.2.3 Infrared spectroscopy (IR)Three different samples including mPEG-PLA-MMC micelles, mPEG-PLA blank micelles and mPEG-PLA blank micelles physically mixed with MMC were analysed by using KBr tablet. By comparing the infrared spectra, we can make sure that the MMC really existed in the compound.3.2.4 H1NMR spectroscopyWeighed appropriate samples and dissolved them into CH2DCCl3, then transferred the solutions to the NMR tube, the samples can be analyzed under the conditions of 400MHz. By comparing the H1NMR images of mPEG-PLA-MMC micelles, mPEG-PLA blank micelles and physical mixture of mPEG-PLA blank micelles and MMC, we could make sure that the MMC really existed in the mPEG-PLA micelles. So we could say that we had prepared mPEG-PLA-MMC micellar nanoparticles.4. mPEG-PLA-MMC nanomicelle drug loading and encapsulation efficiency and release propertyMMC has absorption at 365nm and mPEG-PLA polymer do not in here so that there was no material interference with the determination of MMC. Therefore the MMC content of mPEG-PLA-MMC micelle can be determined by UV.Collecting the MMC solution outside of the dialysis bag in the process of mPEG-PLA-MMC micelle preparation and the MMC absorbance in the solution was determined. Brought MMC absorbance into the MMC standard curve A = 0.0042 +0.0607 * C (R = 1.0000, P<0.0001) we could get the free MMC quantity in the solution. Subtracted free MMC quantity from MMC total quantity, we could obtain MMC quantity in the mPEG-PLA-MMC nanomicelles. Drug loading and entrapment efficiency of the micelles could be accounted with the following formula. When the quantity ratio of mPEG-PLA block copolymer to the MMC is 10:3, the micelle drug loading and encapsulation efficiency are better. Therefore all of the following experiments were done with the quantity ratio of 10:3.Weighed a certain amount of the drug micelles and dissolved it in certain volume of PBS, transferred the solution to the dialysis bag that was previously treated. The dialysis bag was sealed and then put into the beaker that had a large amount of PBS. The beaker was placed in the constant temperature water bath oscillator at setting temperature and speed and a fixed amount of fresh PBS around the bag in the beaker was taken out, which was supplemented with the same amount of fresh PBS at the scheduled time point. The MMC in the taken-out PBS was determined by ultraviolet method. The PBS replacement and MMC determination were constantly carried out until the MMC could not be detected. We can obtain micellar mPEG-PLA-MMC nanoparticle burst release and sustained-release properties by making figure about accumulated content of MMC to that time point. The results showed that the mPEG-PLA-MMC nanomicelles burst release is not obvious, but the effect of sustained-release is obvious with a release period of 18±1d (n=3).5. The effects of micellar mPEG-PLA-MMC in vitroThe objective of the experiment was to observe whether mPEG-PLA could affect the anti-cancer effect of MMC or not in vitro. Inhibition rate was measured by MTT test to prove whether mPEG-PLA have influences on the tumor cell-killing effect of MMC. BGC-823 cell and HpeG2 cell were used in the MTT test. In vitro results showed that mPEG-PLA block copolymer cann't enhance the activity of MMC, in contrast, the lower dose of mPEG-PLA-MMC micelles's anti-tumor activity is lower than the same amount of MMC alone, there was no significant difference between high dose (200μg/ml and above) mPEG-PLA-MMC micelles and single MMC. The results of this experiment were reasonable because mPEG-PLA-MMC nanomicelles have sustained release ability, in the low doses only less of MMC can be freed from the mPEG-PLA-MMC nanomicelles, so the inhibitory effect is lower; But in the high dose of mPEG-PLA-MMC nanomicelles that MMC can be released more, when the MMC achieved a certain anount there was no significant difference between mPEG-PLA-MMC micelles and MMC used alone.6. The effects of micellar mPEG-PLA-MMC in vivo The objective of the experiment was to observe whether mPEG-PLA could affect the anti-cancer effect of MMC or not in vivo. The whole experiment included two experiments, one of which used the model of S180 mice sarcoma to observe the tumor control rate and the other used the H22 ascitic tumor to observe the life elongation rate. BABL/C mice were used in the toxicity studies, a series of doses of drugs were used in the experiment and tissue injury can be observed.6.1 The effects on mice sarcoma S180Model of mice sarcoma S180 can estimate the effect of anti-cancer drug by calculating the tumor control rate. The results indicated that mPEG-PLA could reinforce the anti-cancer effect of MMC remarkably in vivo. When the mPEG-PLA-MMC micelle doses were 2, 6, 18 mg/kg the tumor inhibition were 31.23%, 51.78%, 66.80%, while that of the MMC of 1mg/kg was 34.78%. The MMC content of mPEG-PLA-MMC nanomicelle is about 1/6, therefore 6mg/kg of mPEG-PLA-MMC nanomicelle has identical MMC content with single use of MMC 1mg/kg. The test consequences demonstrated that 2mg/kg mPEG-PLA-MMC micelle has the accordant tumor inhibition with 1mg/kg of singly-used MMC. In the middle and high doses the tumor inhibition of mPEG-PLA-MMC nanomicelles were significantly increased in compared with 1mg/kg of MMC, there were statistical significance (P <0.001).6.2 The effects on mice ascitic tumor H22Model of mice ascetic tumor H22 can estimate the effect of anti-cancer drug by calculating the life elongation rate. The results indicated that mPEG-PLA-MMC nanomicelle could lengthen the living time of the tumor-bearing mice in vivo. The life-extension rates of mPEG-PLA-MMC micelles were 76.6%, 171.0%, 206.5% at dose of 2, 6, 18 mg/kg, while the life-extension rate of MMC1mg/kg was 123.4%. The results demonstrated that life-extension rate of mPEG-PLA-MMC micelles with dose of 2mg/kg is lower than that of MMC 1mg/kg with statistical difference (P <0.01). When doses of the mPEG-PLA-MMC micelles were 6, 18 mg/kg, survival time is significantly increased and exist statistical significance (P <0.01)6.3 Local tissue injury studyThe results showed that mPEG-PLA-MMC micelles can significantly reduce the damage role of MMC on the tissue. Compared with the single MMC group, mPEG-PLA-MMC micelle group have the following feature: tissue damage occurs later, the incidence is lower and damage area is smaller. In the mPEG-PLA-MMC micelle group the minimum dose for tissue damage is 0.08mg while single MMC experimental group is 0.02mg. The mPEG-PLA-MMC nanomicelle groups tissue damage areas are smaller than the same dose of MMC groups with statistically significant differences (P <0.05 or P <0.01).
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