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The Fundamental Research Of Gold Nanocrystal Powder-Loaded,Folater-Targeted, Phase-Change Polymer Contrast Agent

Posted on:2015-03-24Degree:DoctorType:Dissertation
Country:ChinaCandidate:M P LiFull Text:PDF
GTID:1224330467987916Subject:Medical imaging and nuclear medicine
Abstract/Summary:PDF Full Text Request
PART I:Preparation and Identification of Foliate-targeted PolymersObjective Preparing folate-targetedPLGA-PEG-FA and establishing method to monitor the crucial preparation steps, in the meantime, identifying its chemical structure and targeting specificity in vitro.Methods Preparation of PLGA-PEG-FA by five-step:①Synthesis PLGA-NHS:Activating carboxyl group by coupling PLGA and N-hydroxysuccinimide (NHS);②Synthesis mono-amino blocked NH2-PEG-BOC:Reacting NH2-PEG-NH2with BOC, NH2-PEG-NH2+BOC'NH2-PEG-NH-BOC;③Synthesis PLGA-PEG-NH-BOC:Coupling of NH2-PEG-NH-BOC and PLGA, NH2-PEG-NH-BOC+PLGA-NHS'PLGA-PEG-NH-BOC;④Synthesis PLGA-PEG-NH2:Replacing BOC with TFA and de-protecting amino group, PLGA-PEG-NH-BOC+TFA'PLGA-PEG-NH2; ⑤Synthesis PLGA-PEG-FA:Coupling PLGA-PEG-NH2and FA, PLGA-PEG-NH2+FA'PLGA-PEG-FA;Monitoring the key steps of preparation by thin-layer chromatography and nuclear magnetic resonance spectroscopy and identifying the structure of PLGA-PEG-FA by1H-NMR and targeting specificity in vitro with flow cytometry.Result①One new peak is identified at the chemical shift of2.6ppm, which is the same with methylene peak of NHS except proton characteristic peak of-CH3、-CH2and-CH-in1H-NMR of PLGA-NHS, meaning the carboxyl group of PLGA is replaced by NHS successfully;②TLC results:the color of lane A (positive control) and lane D (PLGA-PEG-NH2) is obvious while there is no color of PLGA, PLGA-NHS, NHS and FA, indicating PEG has connected with PLGA through chemical bond;③Identifying the structure of PLGA-PEG-FA with’H-NMR: proton characteristic peak of-CH3,-CH2and-CH-could be found in PLGA; the new peak at chemical shift of3.4ppm is the same with-CH2-of PEG and at6.5n6.8and7.1ppm is aromatic proton of folic acid; the peak at chemical shift of5.7and4.1ppm belongs to the amide proton and aromatic amines proton of folic acid, suggesting the chemical shift is folic acid;④The positive cells of SKOV3increase significantly and saturate when adding more and more PLGA-PEG-FA measured by flow cytometer and also the trend shows an S curve, which is compliance the binding rule of receptors and ligands. The positive cells decrease when adding different concentrations of FA and SKOV3and disappear when adding enough amount of FA, indicating FA inhibits the binding of PLGA-PEG-FA and FR of SKOV3competitively.Conclusion①PLGA-PEG-FA is prepared successfully and the purity is98%while the structure is right and could bind to FR specifically;②The preparation method of PLGA-PGE-FA is established and the processing could be monitored and critical parameters are obtained;③NMR and TLC are used as the processing control of NHS activation and PEG connection;④Laying foundation for preparing folate-targeted golden radio contrast polymer. PART Ⅱ Study of preparation and properties of Gold nanocrystal powder-loaded, Folater-targeted,phase-change polymer contrast agentObjectives Using the Folater-targeted polymer (PLGA-PEG-FA) successfully prepared in the first part to coat perfluoro pentane (PFP) and gold nanoparticles (GNP), so as to prepare Gold nanocrystal powder-loaded, Folater-targeted, phase-change polymer ultrasound and photoacoustic contrast agent (GNP/PFP/PLGA-PEG-FA). Establish and optimize preparation methods, and detect physical properties and in vitro targeting of polymer ultrasound and photoacoustic contrast agents.Methods①Prepare phase change contrast agent:use double emulsion to wrap in PFP and perfluoro hexane (PFH), preparing modified nanoparticles contrast agent. The precision constant temperature controller heat them with a microscope real-time observing phase transformation, and compare the differences between the two phase transition temperature; and use the diagnostic ultrasound real-time observing the phase transformation; low intensity ultrasound priming package liquid fluorocarbon contrast agent targeted nano phase change. Through the above three methods to determine whether to use PFP or PFH as the phase contrast agent kernel;②Prepare folate targeted transformation of gold loaded polymer contrast agent:Using the double emulsion to prepare folate targeted transformation of gold loaded polymer contrast agent. Precision constant temperature controller gradually warming with real time observation of phase change optical microscope, finding differences between no gold and gold loaded polymer contrast agent transformation temperature of high intensity focused ultrasound induced difference of hair transformation and the laser irradiation induced phase transition;③Optimize the preparation conditions of GNP/PFP/PLGA-PEG-FA polymer contrast agent system:use the destruction of microsphere method to determine coating weight, according to which to determine the best quality ratio of PHP and PLGA-PEG-FA; use laser irradiation with observation of the amount of air bubbles under the light microscope, phase GNP and PLGA-PEG-FA best mass ratio determines;④With the blood cell counting plate, pH, scanning electron microscopy, transmission electron microscopy, Malvera laser, the NP/PFP/PLGA-PEG-FA polymer ultrasound and photoacoustic contrast agent general physicochemical diameter instrument were studied. Its in-vitro targeting specific is studied by flow cytometry.Result⑤Two kinds of phase change nanoparticles contrast agent PFP/PLGA-PEG-FA and PFH/PLGA-PEG-FA in the conventional two-dimensional ultrasound are densely punctate hyperechoic, uniform distributed. Particle size distribution, showed a single peak, the average particle size were:the diameter of (280.9±33.5) nm, the latter is (749±116) nm. Phase transition temperature of the heating plate and the water is divided into43and42℃, the latter were71℃and68℃; LIFU can promote their transformation, but the latter transformation needs lower energy;②Compare phase-contrast agent after gold (GNP/PFP/PLGA-PEG-FA) with non-gold (PFP/PLGA-PEG-FA), the phase transition temperature has no obvious change; high intensity focused ultrasound (HIFU) can be the two phase excitation, excitation intensity had no significant difference; after laser irradiation, the former can change, the latter does not. Temperature, HIFU and laser irradiation can not only stimulate the contrast of gold (GNP/H2O/PLGA-PEG-FA) phase change;③The best quality ratio of PFP and PLGA is11:1, the rate of package is highest; GNP and PLGA mass ratio is1:12.5, after laser irradiation, the phase transition is strongest;④The general physicochemical properties:the appearance of brownish black suspension concentration;3.657±1013/ml; pH=7.12; the scanning electron microscope is spherical, regular shape, the size is uniformed, good dispersion degree; transmission electron microscopy showed spherical, irregular patchy and visible particulate matter is not evenly distributed in the nanoparticles; particle diameter of (268.1±90.87) nm, Zeta potential (-67.6±12.7) mV;⑤Flow cytometry show with the GNP/PFP/PLGA-PEG-FA increasing, the number of FR positive cells in SKOV3and HL-60cells expressed increased significantly. And reach the saturation (close to100%), with an "S" curve, in line with the regulation of receptor and ligand. No expression of FR in Wish cells was not detected positive cells. By adding different concentrations of FA and SKOV3were incubated together, with the increase of FA concentration, the number of positive cells decreased rapidly, when the FA reaches a certain amount, have not detected positive cells. FA competitively inhibited the binding of GNP/PFP/PLGA-PEG-FA polymer contrast agent and SKOV3FR.Conclusion①After PLGA-PEG-FA packages, phase transition of PFP is closer to physiological conditions than PFH, and choose PFP as the phase change material can be taken into account both in the treatment and meeting the demand of clinical diagnosis and detection;②Loading GNP does not affect the temperature and induced phase transition conditions, but increases the properties of light induced phase transformation, making the contrast agent more multi modal imaging potential; ③The general physicochemical properties showed that the contrast agent is of regular shape, uniform size nano microspheres;④The contrast agent and two strains with high expression of different subtype FR cells, FA could competitively inhibit the binding, and no expression of FR cells do not bind, indicated that the contrast agent and FR specific binding;⑤Temperature, HIUF, LIUF and light could stimulate GNP/PFP/PLGA-PEG-FA to transform. The contrast agent with ultrasound contrast agent and photoacoustic contrast agent characteristics;⑥The above indicates the success of preparing folate targeted transformation of gold loaded polymer ultrasound and photoacoustic contrast agent (GNP/PFP/PLGA-PEG-FA). PART Ⅲ Gold nanocrystal powder-loaded, Folater-targeted, phase-change polymer contrast agent for increasing ultrasound imagingObjective To explorer the gold nanocrystal powder-loaded, Folater-targeted, phase-change polymer contrast agent (GNP/PFP/PLGA-PEG-FA) for improving ultrasound imaging after be phase-changed by ultrasound and laser in vitro and in vivo.Methods①We investigated the increasing of ultrasound imaging by GNP/PFP/PLGA-PEG-FA after be phase-changed by ultrasound and laser in vitro. Four groups have been studied:GNP/PFP/PLGA-PEG-FA group and GNP/H2O/PLGA-PEG-FA group as a control, PFP/PLGA-PEG-FA group and H2O/PLGA-PEG-FA as a control. Each one was treated by low-intensity focused ultrasound (LIFU) and laser. The fundamental and harmonic ultrasound imaging before and after LIFU and laser treated were observed by ultrasonic Instrument;②The liver of pig was choused as isolated tissue. Two different methods were studied:local injection method and perfusion method. GNP/PFP/PLGA-PEG-FA and PFP/PLGA-PEG-FA were injected into the shallow surface of the liver, respectively. For perfusion method,5ml of the two above agents were perfused into hepatics through the branches of portal vein, and sonovue was used as a control. Each agent was treated by LIFU and laser. The fundamental and harmonic ultrasound imaging before and after LIFU and laser treated were observed by ultrasonic Instrument;③SKOV3tumor-bearing nudes were choused as objects of the experiment. GNP/PFP/PLGA-PEG-FA group and PFP/PLGA-PEG-FA group as experimental groups, and sonovue was used as a control. Intraperitoneal injection was used. For sonovue group, ultrasound contrast imaging was observed immediately after sonovue injection and before sonovue injection as a control. Two experimental groups were injected intraperitoneally immediately after,20min,2h and4h using LIFU and laser treated. The fundamental and harmonic ultrasound imaging were observed.Results①In vitro, after treated with LIFU, both PFP-contained agents: GNP/PFP/PLGA-PEG-FA and PFP/PLGA-PEG-FA were observed a lot of punctate hyperechoic in the ultrasound harmonic mode. While the two free of PFP agents:GNP/H2O/PLGA-PEG-FA and H2O/PLGA-PEG-FA were observed no significant changed in fundamental and harmonic ultrasound imaging. After laser treated, only gold nanocrystal powder-loaded agent: GNP/PFP/PLGA-PEG-FA was observed punctate hyperechoic in harmonic ultrasound imaging, and no significant changed was observed in free gold nanocrystal powder-loaded agent;②For isolated tissue experiment, the result of local injection is fully consistent with the result of gels in vitro. After sonovue perfusion, agents were rapidly into the liver parenchyma through portal vein branches. The hyperechoic distributed in the liver parenchyma was observed in ultrasound contrast imaging. After5-10min, The hyperechoic distributed beneath the liver capsule was observed. Before treated with LIFU, no significant changed were observed in fundamental and harmonic ultrasound imaging in both homemade contrast agents, while after LIFU treated, the hyperechoic distributed in the interesting region were measured in fundamental and harmonic ultrasound imaging, especially in harmonic ultrasound imaging. After laser treated, only GNP/PFP/PLGA-PEG-FA observed hyperechoic in interesting region in fundamental and hannonic ultrasound imaging, especially in harmonic ultrasound imaging;③In vivo experiment, after sonovue injected intraperitoneally, under ultrasound contrast imaging mode, the tumor began to image at30s and performed uneven high enhancement, and after5min, the contrast agents almost all dissipated. After GNP/PFP/PLGA-PEG-FA injected intraperitoneally immediately after,20min,2h and4h, before using LIFU and laser treated, no significant changed were observed in both fundamental and harmonic ultrasound imaging. After GNP/PFP/PLGA-PEG-FA injected intraperitoneally immediately after using LIFU and laser treated, the significant punctate hyperechoic in the treated region was observed in fundamental and harmonic ultrasound imaging. While there were no punctate hyperechoic in untreated region. At20min, LIFU and laser treated again, in the treated region strong punctate hyperechoic was still observed. At2h, LIFU and laser treated once more, in the treated region punctate hyperechoic was still observed, and the intensity of hyperechoic was decreased. After4h, LIFU and laser treated again, in the treated region, no hyperechoic was observed. For PFP/PLGA-PEG-FA group, after LIFU treated, the results were the same with GNP/PFP/PLGA-PEG-FA group. However, no hyperechoic was observed under laser.Conclusion①GNP/PFP/PLGA-PEG-FA can be phase-changed by LIFU and laser in vitro;②GNP/PFP/PLGA-PEG-FA can be phase-changed by LIFU and laser in isolated tissue, and can improve ultrasound imaging;③The retention time of GNP/PFP/PLGA-PEG-FA is obviously extended in vivo. GNP/PFP/PLGA-PEG-FA can be phase-changed by LIFU and laser in treated region;④The imaging of GNP/PFP/PLGA-PEG-FA is passive imaging which different from sonovue. Only external excitation can cause imaging in the corresponding region;⑤These results indicate that GNP/PFP/PLGA-PEG-FA may have immeasurable application in clinical.
Keywords/Search Tags:Targeting polymer materials, Polylactic acid-glycolic acid, Polyethylene glycol, folic acidFolater-targeted, phase-changem, Gold nanocry stalpowder, ultrasound contrast agent, photoacoustic contrast agentSound-induced phase, light-induced phase
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