【摘要】：第一部分攜氧載ICG納米粒制備及特性檢測目的:制備攜氧液態(tài)氟碳載吲哚菁綠PLGA納米粒并進行其特性檢測方法:采用雙乳化-溶劑揮發(fā)法(w/o/w)制備攜氧液態(tài)氟碳載吲哚菁綠PLGA納米粒(OPI-NPs)、載攜氧液態(tài)氟碳PLGA納米粒(OP-NPs)和載吲哚菁綠PLGA納米粒(I-NPs),光鏡下觀察形態(tài)、Malvern粒徑測定儀測定OPI-NPs的粒徑大小分布,UV-Vis分光光度計測定ICG的包封率及載藥量,光聲成像儀觀察其光聲顯影能力,并與OP-NPs+free ICG及I-NPs的光聲顯影能力進行比較。結果:OPI-NPs為球形,平均粒徑大小(275.24±7.28)nm,大小分布范圍在80 nm—1000 nm之間,ICG的包封率約55%。OP-NPs+free ICG、I-NPs及OPI-NPs的光聲顯影強度分別為0.336±0.022,0.744±0.029及1.112±0.072。OPI-NPs及I-NPs的光聲顯影強度明顯強于OP-NPs+free ICG,而OPI-NPs的光聲顯影強度明顯強于I-NPs,差異具有統(tǒng)計學意義(P0.05)。結論:成功制備OPI-NPs納米粒,粒徑相對比較均勻,且具有良好的光聲顯影能力。第二部分納米粒激發(fā)的適宜激光、超聲參數(shù)及納米粒濃度的篩選目的:篩選出可以激發(fā)納米粒相變及其破裂的適宜激光及超聲參數(shù)及用于光聲動力治療的納米粒ICG濃度。方法:1、激光參數(shù)篩選:將制備好的納米粒稀釋20倍,按照激光強度及輻照時間分組如下:0.5W/cm2,30s、0.5W/cm2,60s、1.0W/cm2,30s,1.0 W/cm2,60s、1.5 W/cm2,30s、1.5 W/cm2,60s、2.0 W/cm2,30s和2.0 W/cm2,60s。分別給予上述不同處理條件后,光鏡下觀察微球形態(tài)變化。2、超聲參數(shù)篩選:將制備好的納米粒稀釋20倍,激光(2.0W/cm2,30s)輻照后,根據(jù)超聲強度及輻照時間分組如下:0.44W/cm2,30s、0.44 W/cm2,60s、0.66 W/cm2,30s、0.66 W/cm2,60s、0.88 W/cm2,30s、0.88 W/cm2,60s、1.10 W/cm2,30s和1.10 W/cm2,60s。分別給予相應處理,處理后即刻光鏡觀察微球破碎情況。3、納米粒ICG濃度篩選:取對數(shù)生長期的SKOV3細胞,根據(jù)納米粒中ICG的濃度分組如下:對照組、0.5μg/ml組、1.0μg/ml組、2.0μg/ml組、4.0μg/ml組和8.0μg/ml組,共6組。分別將納米粒與細胞共培養(yǎng)24h后,CCK-8檢測細胞存活率。4、適宜激光、超聲作用下對微球相變及擊碎情況觀察:,將制備好的納米粒稀釋20倍,分別取等量的納米粒懸浮液放入凝膠孔中,分別在激光(2.0W/cm2,30s)輻照前后、激光(2.0W/cm2,30s)加超聲(0.88W/cm2)輻照30秒及60秒后,診斷超聲觀察凝膠管回聲變化、光鏡觀察微球形態(tài)變化、粒徑測定儀測定微球粒徑大小分布。5、適宜激光、超聲作用下對細胞增殖的影響:取對數(shù)生長期的SKOV3細胞,分組如下:對照組、激光組、超聲組及激光聯(lián)合超聲組。分別給予相應處理。應用CCK-8檢測細胞存活率,觀察各組對細胞增殖抑制情況。結果:1、當激光輻照能量達到1.5W/cm2時納米粒發(fā)生明顯相變,伴隨著輻照時間的延長相變數(shù)目進一步增加,當激光能量達到2.0W/cm2時納米粒相變數(shù)目明顯增多,但隨著時間的延長,相變數(shù)目無明顯變化。2、當超聲輻照能量為0.44W/cm2和0.66W/cm2時相變的納米粒未見明顯變化,伴隨著輻照時間的延長仍未見明顯的變化;當超聲輻照能量達到0.88W/cm2時相變的納米粒數(shù)目明顯減少,伴隨著輻照能量增加到1.10W/cm2時相變的納米粒數(shù)目減少不明顯,但隨著輻照時間的延長至60s時,相變的納米粒數(shù)目明顯減少。3、被包裹的ICG濃度0.5μg/ml至4μg/ml之間對細胞無明顯抑制作用,被包裹的ICG濃度達8μg/ml時細胞增殖明顯受到抑制。4、激光激發(fā)前后的超聲顯影明顯增強,光鏡下觀察納米粒粒徑增大,納米粒平均粒徑分別為275.24nm和1533.65nm;激光輻照后再進行超聲輻照30s和60s較激光激發(fā)后的超聲顯影明顯減弱,納米粒平均粒徑分別為1060.56nm和771.38 nm。5、激光(2.0W/cm2,30s)及超聲(0.88W/cm2,60s)對細胞無明顯抑制作用。結論:激光(2.0W/cm2,30s)可有效激發(fā)納米粒發(fā)生相變,超聲(0.88W/cm2,60s)可有效使相變的納米粒破碎,且此能量下的超聲及激光無論單獨或聯(lián)合作用,對SKOV3細胞生長均無影響。納米粒ICG濃度≤4μg/ml對SKOV3細胞生長無抑制作用,故選擇激光(2.0W/cm2,30s)、超聲(0.88W/cm2,60s)及納米粒ICG濃度4μg/ml作為本實驗適宜參數(shù)用于后續(xù)研究中。第三部分攜氧載ICG納米粒協(xié)同光聲動力對卵巢癌SKOV3細胞增殖抑制及其機制的實驗研究目的:觀察光動力和聲動力聯(lián)合攜氧納米粒對卵巢癌SKOV3細胞的增殖抑制及其機制。方法:1、不同處理方式對細胞生長抑制情況檢測:將對數(shù)生長期的SKOV3細胞隨機分6組:ICG+L+U組、I-NPs+L+U組、PI-NPs+L+U組、OPI-NPs+L+U組、OPI-NPs+L組和OPI-NPs+U組。分別進行相應處理,24小時后應用CCK-8檢測各組細胞存活率;2、不同的納米粒ICG濃度對細胞生長抑制情況檢測:將不同ICG濃度(0.25μg/ml、0.5μg/ml、1.0μg/ml、2.0μg/ml和4.0μg/ml)的納米粒加入到對數(shù)生長期的SKOV3細胞共培養(yǎng)24小時后,分別用激光(2.0W/cm2,30s)及超聲(0.88W/cm2,60s)處理,處理后24小時,用CCK-8檢測細胞存活率;3、不同方式處理后無細胞體系溶液內活性氧檢測:按照ICG終濃度為4μg/ml的各樣本加入96孔培養(yǎng)板中,然后各孔中加入濃度為50m M SOSG 0.02ml,按照不同處理方式分組如下:對照組、PBS+L+U組、ICG+L+U組、I-NPs+L+U組、PI-NPs+L+U組、OPI-NPs+L+U組、OPI-NPs+L組和OPI-NPs+U組;處理后20分鐘,用酶標儀測各組溶液的熒光強度;4、納米粒ICG濃度對無細胞體系溶液內活性氧產生情況的影響:將不同ICG濃度(0.25μg/ml、0.5μg/ml、1.0μg/ml、2.0μg/ml和4.0μg/ml)的納米粒分別加入到96孔板鐘,然后再加入濃度為50m M SOSG 0.02ml,用激光(2.0W/cm2,30s)聯(lián)合超聲(0.88W/cm2,60s)處理,處理后20分鐘,用酶標儀測溶液的熒光強度;5、不同方式處理后細胞內活性氧檢測:將對數(shù)生長期的SKOV3細胞分8組:對照組、PBS+L+U組、ICG+L+U組、I-NPs+L+U組、PI-NPs+L+U組、OPI-NPs+L+U組、OPI-NPs+L組和OPI-NPs+U組,分別加入ICG或納米粒使溶液中的ICG濃度為4μg/ml,與SKOV3細胞共培養(yǎng)24h后,分別加入2μl DCFH-DA(10m M)共培養(yǎng)20分鐘,然后用PBS沖洗3次,加入無血清培養(yǎng)液2ml,再予以激光和/或超聲進行處理,處理后20分鐘分別用流式儀檢測活性氧熒光強度及熒光顯微鏡下觀察細胞內活性氧指示劑顯影情況。結果:1、PI-NPs+L+U組,OPI-NPs+L+U組和OPI-NPs+L組對細胞的增殖抑制率分別為(30.2±1.291)%,(77.24±0.4634)%和(59.06±2.003)%,明顯高于其他各處理組(P0.05);OPI-NPs+L+U組和OPI-NPs+L組對細胞的增殖抑制率明顯高于PI-NPs+L+U組,OPI-NPs+L+U組對細胞的增殖抑制率明顯高于OPI-NPs+L組,差異均有統(tǒng)計學意義(P0.05)。2、伴隨著納米粒ICG濃度的增大細胞增殖抑制率也隨著增加,4μg/ml組達到(72.04±5.273)%,明顯高于其他各濃度組,差異均有統(tǒng)計學意義(P0.05)。3、PI-NPs+L+U組,OPI-NPs+L+U組和OPI-NPs+L組無細胞體系溶液中的活性氧指示劑的熒光強度分別為185.3±19.46、440.1±11.47和355.6±31.15明顯高于其他各處理組(P0.05);OPI-NPs+L+U組和OPI-NPs+L組的活性氧含量明顯高于PI-NPs+L+U組,OPI-NPs+L+U組的活性氧含量明顯高于OPI-NPs+L組,差異均有統(tǒng)計學意義(P0.05)。4、伴隨著納米粒濃度增加,細胞外活性氧的產生進一步增加,4μg/ml組達到440.1±11.47,明顯高于其他各濃度組,差異均有統(tǒng)計學意義(P0.05)。5、熒光顯微鏡見PI-NPs+L+U組,OPI-NPs+L+U組和OPI-NPs+L組細胞內均出現(xiàn)明顯綠色熒光,,OPI-NPs+L+U組和OPI-NPs+L組的熒光較PI-NPs+L+U組明顯;流式儀定量檢測細胞內活性氧指示劑熒光強度可見:PI-NPs+L+U組,OPI-NPs+L+U組和OPI-NPs+L組細胞內活性氧指示劑的熒光強度分別為1935±53.14,2939±300.2和2935±295.3,明顯高于其他各處理組,OPI-NPs+L+U組和OPI-NPs+L組細胞內活性氧的熒光強度明顯高于PI-NPs+L+U組,差異均有統(tǒng)計學意義(P0.05),而OPI-NPs+L+U組細胞內活性氧的熒光強度與OPI-NPs+L組無明顯差異(P0.05)。結論:激光聯(lián)合超聲激發(fā)攜氧納米粒能夠明顯抑制卵巢癌SKOV3細胞的增殖,其機制可能與細胞內外活性氧的產生有關。
[Abstract]:The preparation and characterization of oxygen loaded ICG nanoparticles in the first part: preparation of oxygen carrying liquid fluorocarbon indocyanine green PLGA nanoparticles and its properties detection methods: the preparation of oxygen carrying liquid fluorocarbon indocyanine green PLGA nanoparticles (OPI-NPs), oxygen carrying liquid fluorocarbon PLGA nanoparticles (OP-NPs) and indoles carrying oxygen carrying liquid fluorocarbon indocyanine green (w/o/w) Cyanine green PLGA nanoparticles (I-NPs) were observed under light microscope. The size distribution of OPI-NPs was measured by Malvern particle size analyzer. The encapsulation efficiency and drug loading of ICG were measured by UV-Vis spectrophotometer. The photoacoustic imaging ability of ICG was observed by the photoacoustic imaging instrument. The results were compared with that of OP-NPs+free ICG and I-NPs. The results showed that OPI-NPs was spherical and average. The size of the particle size (275.24 + 7.28) nm, the size distribution range from 80 nm to 1000 nm, the encapsulation efficiency of ICG is about 55%.OP-NPs+free ICG, the photoacoustic intensity of I-NPs and OPI-NPs is 0.336 + 0.022,0.744 + 0.029 and 1.112 + 0.072.OPI-NPs and I-NPs is obviously stronger than that of OP-NPs+free. Better than I-NPs, the difference is statistically significant (P0.05). Conclusion: the successful preparation of OPI-NPs nanoparticles with relatively uniform particle size and good photoacoustic development ability. The suitable laser, ultrasonic parameters and nanoparticle concentration of the second part nanoparticles are screened. The suitable laser can be selected to select the suitable laser to stimulate the phase transition and rupture of nanoparticles. And ultrasonic parameters and the concentration of nanoparticles ICG for photoacoustic power therapy. Method: 1, laser parameters screening: the prepared nanoparticles are diluted 20 times, according to laser intensity and irradiation time as follows: 0.5W/cm2,30s, 0.5W/cm2,60s, 1.0W/cm2,30s, 1 W/cm2,60s, 1.5 W/ cm2,30s, 1.5 W/cm2,60s, 2 W/cm2,30s and 2 W/cm2,60s. respectively given. After the above treatment conditions, the morphology of the microspheres was observed under the light microscope.2, the ultrasonic parameters were screened: the prepared nanoparticles were diluted 20 times. After the laser (2.0W/cm2,30s) irradiation, the ultrasonic intensity and the irradiation time were grouped as follows: 0.44W/cm2,30s, 0.44 W/cm2,60s, 0.66 W/ cm2,30s, 0.66 W/cm2,60s, 0.88 W/cm2,30s, 0.88 W/cm2,60s, 1.10 W/cm2,30s. And 1.10 W/cm2,60s. were treated respectively. After treatment, the microsphere fragmentation was observed by immediate light microscopy (.3) and nanoparticles ICG concentration was screened: the logarithmic growth period SKOV3 cells were selected according to the concentration of ICG in the nanoparticles as follows: the control group, the 0.5 mu g/ml group, the 1 mu g/ml group, the 2 mu g/ml group, the 4 micron g/ml group and 8 micron g/ml group, and 6 groups respectively. The nanoparticles and the group were respectively. After the cell co culture of 24h, CCK-8 was used to detect the cell survival rate.4, suitable for laser. Under ultrasonic action, the microsphere phase transformation and fragmentation were observed: the prepared nanoparticles were diluted 20 times, and the same amount of nanoparticle suspension was put into the gel pores respectively. Before and after the laser (2.0W/cm2,30s) irradiation, the laser (2.0W/cm2,30s) and ultrasonic (0.88W/cm2) irradiated 30 respectively. After second and 60 seconds, the echo changes of gel tube were observed by diagnostic ultrasound. The morphology of microspheres was observed by light microscope. The size distribution of microspheres was measured by particle size measuring instrument.5. The effect of laser and ultrasound on cell proliferation: SKOV3 cells of logarithmic growth period were taken as follows: control group, laser group, ultrasound group and laser combined ultrasound group. The cell survival rate was detected by CCK-8, and the inhibition of cell proliferation was observed. The results were as follows: 1, when the laser irradiation energy reached 1.5W/cm2, the nanoparticles had obvious phase transition, and the number of phase transition was further increased with the prolongation of irradiation time. The number of nanoparticles phase transition was significantly increased when the laser energy reached 2.0W/ cm2, but with the time, the number of phase transition was significantly increased. There is no obvious change in the number of phase changes of.2. When the energy of ultrasound irradiation is 0.44W/cm2 and 0.66W/cm2, the phase transition nanoparticles do not change obviously. There is no obvious change with the prolongation of the irradiation time. When the energy reaches 0.88W/cm2, the number of nanoparticles decreases obviously, with the increase of radiation energy to 1.10W/cm2. The decrease of the number of phase transition nanoparticles was not obvious, but with the prolongation of the irradiation time to 60s, the number of phase transition nanoparticles decreased obviously by.3, and the concentration of the encapsulated ICG between 0.5 and 4 Mu was not obviously inhibited. The proliferation of the encapsulated ICG was 8 mu g/ml and the cell proliferation was obviously inhibited by.4 and the ultrasonic development before and after laser excitation. The size of nanoparticles was increased by light microscopy. The average particle size of nanoparticles was 275.24nm and 1533.65nm, respectively. The ultrasonic development of 30s and 60s after laser irradiation was obviously weakened after laser irradiation. The average particle size of nanoparticles was 1060.56nm and 771.38 nm.5 respectively, and the laser (2.0W/cm2,30s) and ultrasound (0.88W/cm2,60s) were used to the cells. Conclusion: laser (2.0W/cm2,30s) can effectively stimulate the phase transition of nanoparticles. Ultrasound (0.88W/cm2,60s) can effectively break the phase of phase transition, and the ultrasound and laser under this energy have no effect on the growth of SKOV3 cells either alone or in combination. The ICG concentration of nanoparticles is less than 4 mu g/ml to the growth of SKOV3 cells. Use, select laser (2.0W/cm2,30s), ultrasound (0.88W/cm2,60s) and nanoparticles ICG concentration of 4 mu g/ml as the appropriate parameters of this experiment in the follow-up study. Third experimental study on the inhibition of the proliferation of ovarian cancer SKOV3 cells with oxygen loaded ICG nanoparticles and the mechanism of the inhibition of proliferation of ovarian cancer cells: the observation of photodynamic and acoustic power combined oxygen carrying nanoparticles Inhibition and mechanism of the proliferation of SKOV3 cells in ovarian cancer cells. Methods: 1, detection of cell growth inhibition by different treatments: SKOV3 cells in logarithmic growth period were randomly divided into 6 groups: ICG+L+U group, I-NPs+L+U group, PI-NPs+L+U group, OPI-NPs+L+U group, OPI-NPs+L group and OPI-NPs+U group. 24 hours after 24 hours, CCK-8 detection was used. Group cell survival rate; 2, different nanoparticles ICG concentration for cell growth inhibition detection: the nanoparticles with different ICG concentrations (0.25 g/ml, 0.5 mu g/ml, 1 mu g/ml, 2 micron g/ml and 4 mu g/ml) were added to the logarithmic growth period SKOV3 cells for 24 hours, and respectively treated with stimulated light (2.0W/cm2,30s) and ultrasound (0.88W/cm2,60s), and 24 smaller after treatment. At the time, the cell survival rate was detected by CCK-8. 3, the reactive oxygen species in the cell solution was detected in different ways after different treatments: the samples were added to the 96 hole culture plate according to the final concentration of 4 mu g/ml, and the concentration was 50m M SOSG 0.02ml in each hole. The control group, PBS+L+U, ICG+L+U, I-NPs+L+U, PI-NPs+L were grouped according to the different treatments. Group +U, group OPI-NPs+L+U, group OPI-NPs+L and group OPI-NPs+U; 20 minutes after treatment, the fluorescence intensity of each solution was measured by an enzyme scale. 4, the effect of ICG concentration on the production of reactive oxygen in the cell solution: the nanoparticles with different ICG concentrations (0.25 mu g/ml, 0.5 mu g/ml, 1 micron ml, 2 mu g/ml and 4 micron g/ml) were added to the 96 hole clock, respectively. Then the concentration of 50m M SOSG 0.02ml was added with the laser (2.0W/cm2,30s) combined ultrasound (0.88W/cm2,60s) treatment, after 20 minutes, the fluorescence intensity of the solution was measured by the enzyme scale. 5, the intracellular reactive oxygen species were detected in different ways: the logarithmic growth period SKOV3 cells were divided into 8 groups: control group, PBS+L+U group, ICG+L+U group, I-NPs+L+U group, PI-NPs+L+U. Group OPI-NPs+L+U, group OPI-NPs+L+U, group OPI-NPs+L and OPI-NPs+U were added to ICG or nanoparticles to make ICG concentration in the solution respectively. After co culture 24h with SKOV3 cells, 2 micron DCFH-DA (10m M) were added for 20 minutes respectively. Then 3 times were washed with serum free culture solution and then treated with laser and / or ultrasound for 20 minutes after treatment. The fluorescence intensity of reactive oxygen species was detected by flow meter and the development of active oxygen indicator was observed under the fluorescence microscope. Results: 1, PI-NPs+L+U group, OPI-NPs+L+U group and OPI-NPs+L group were respectively (30.2 + 1.291)%, (77.24 + 0.4634)% and (59.06 + 2.003)%, respectively, higher than the other treatment groups (P0.05); OPI-NPs+L+U The inhibitory rate of cell proliferation in the group and OPI-NPs+L group was significantly higher than that in the PI-NPs+L+U group. The proliferation inhibition rate of the OPI-NPs+L+U group was significantly higher than that in the OPI-NPs+L group. The difference was statistically significant (P0.05).2, with the increasing of the ICG concentration of the nanoparticles, the proliferation inhibition rate was also increased, and the 4 mu g/ml group reached (72.04 + 5.273)%, obviously higher than the others. The difference of the concentration group was statistically significant (P0.05).3, group PI-NPs+L+U, group OPI-NPs+L+U and OPI-NPs+L group, the fluorescence intensity of active oxygen indicator in no cell solution was 185.3 + 19.46440.1 + 11.47 and 355.6 + 31.15 respectively higher than that of other treatment groups (P0.05), and the active oxygen content in OPI-NPs+L+U and OPI-NPs+L groups was significantly higher than that of the group OPI-NPs+L+U and OPI-NPs+L. In group PI-NPs+L+U, the content of active oxygen in group OPI-NPs+L+U was significantly higher than that in group OPI-NPs+L, and the difference was statistically significant (P0.05).4. With the increase of the concentration of nanoparticles, the production of extracellular reactive oxygen species increased further, the group of 4 micron g/ml reached 440.1 + 11.47, obviously higher than the other concentration groups, the difference was statistically significant (P0.05).5, and the fluorescence microscope was PI -NPs+L+U group, OPI-NPs+L+U group and OPI-NPs+L group showed obvious green fluorescence, and the fluorescence intensity of OPI-NPs+L+U group and OPI-NPs+L group was more obvious than that of PI-NPs+L+U group. The fluorescence intensity of intracellular reactive oxygen indicator quantitative detection by flow meter was visible: the fluorescence intensity of active oxygen indicator in PI-NPs+L+U group, OPI-NPs+L+U group and OPI-NPs+L group was respectively. 1935 + 53.142939 + 300.2 and 2935 + 295.3, significantly higher than the other treatment groups, OPI-NPs+L+U group and OPI-NPs+L group intracellular reactive oxygen fluorescence intensity was significantly higher than the PI-NPs+L+U group, the difference was statistically significant (P0.05), but the fluorescence intensity of active oxygen in the OPI-NPs+L+U group was not significantly different from that of the OPI-NPs+L group (P0.05). Conclusion: laser coupling Oxygen-carrying nanoparticles stimulated by ultrasound can inhibit the proliferation of ovarian cancer SKOV3 cells, and the mechanism may be related to the production of reactive oxygen species inside and outside the cells.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.31

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1 楊凱,陳紹維,陳睿,溫玉明;隱形順鉑聚乳酸納米微粒對口腔鱗癌原發(fā)灶的靶向性研究[J];華西口腔醫(yī)學雜志;2005年05期

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