| PART Ⅰ PREPARATIONN AND CHARACTERIZATION OF DOXORUBICIN AND INDOCYANINE GREEN CO- LOADED MULTIFUNCTIONAL LIPOSOMESObjectTo prepare doxorubicin and indocyanine green co-loaded multifunctional liposomes(FA-DOX-ICG-PFP@Lip)and characterize their basic properties,biosafety,targeting ability,photothermal properties,phasechanging ability,and drug release behaviour.Method1.The liposomes encapsulating DOX and ICG were synthetized through film hydration and two-step emulsion method.The morphology of liposomes was observed under by optical microscope and transmission electron microscope.The size distribution and surface charge were measured with a Malvern laser particle size analyzer.The encapsulation efficiency(EE)and loading capacity(LC)of DOX and ICG were measured by UV spectrophotometer.The expression of folic acid on the surface of the liposomes were determined by confocal laser scanning microscopy(CLSM).The particle size change of FA-DOX-ICG-PFP@Lip dissolved in phosphate buffer solution(PBS)was detected by Malvern laser particle size analyzer.2.The cells were co-incubated with different concentrations of liposomes without DOX,and the cell survival rate was determined by cell counting kit-8(CCK-8).The healthy mice were randomly divided into blank control group and FA-ICG-PFP@Lip group.The mice were sacrificed on days 1,7 and 15 after administration,and blood samples were collected for routine blood tests and biochemical examinations.3.To explore the targeting characteristics of the liposomes,FA-DOXICG-PFP@Lip and DOX-ICG-PFP@Lip incubated with Y79 folate receptor-overexpressing cells and Y79 cells with antagonized folate receptors,then observed under CLSM and flow cytometer(FCM).The Y79tumor-bearing mice model was established for the distribution experiment in vivo.The fluorescence distribution in tumor-bearing mice was monitored by fluorescence imaging system at different time points after injecting Di Rlabeled FA-DOX-ICG-PFP@Lip and DOX-ICG-PFP@Lip,and relevant images were collected and the fluorescence signal intensity at the tumor site was measured.The mice were sacrificed and the tumor and main organs were isolated for fluorescence imaging after 24 h,and the fluorescence signal intensity of the tumor and different organs was compared.4.Different concentrations of FA-DOX-ICG-PFP@Lip(0.625,1.25,2.5,5 mg/m L)were irradiated by 808 nm laser and the temperature changes were monitored by thermal imager.Meanwhile,the process of optical droplet vaporization(ODV)of liposomes was observed under an optical microscope.FA-DOX-ICG-PFP@Lip and DOX-ICG-PFP@Lip were intravenously injected into Y79 tumor-bearing mice.The tumor sites were irradiated by808 nm laser,and the temperature changes were monitored by thermal imager and corresponding images were collected.5.FA-DOX-ICG-PFP@Lip(2.5 mg/m L)were irradiated by 808 nm laser.Samples at different time points before and after laser irradiation were collected and the cumulative release amount of DOX in the samples was detected by UV spectrophotometer.Results1.FA-DOX-ICG-PFP@Lip liposomes were successfully prepared by film hydration and two-step emulsion method,and their solution was gray green.The results of transmission electron microscopy(TEM)showed that the liposome was spherical,uniform in size and well dispersed.The particle size of FA-DOX-ICG-PFP@Lip liposome was(309.5±16.7)nm,and the zeta potential was about(-23.5±8.6)m V.The EE of DOX was(62.04±5.10)%and LC was(5.17±0.43)%,the EE of ICG was(91.85±2.98)% and LC was(7.65±0.25)%.The high expression of folic acid on liposomes was detected by immunofluorescence method under CLSM.Moreover,the mean particle size increased slightly when dissolved in PBS,indicating that the liposome had good stability.2.The cell survival rate after incubation with FA-ICG-PFP@Lip was above 90%.No abnormality was found in normal condition and related blood tests in healthy mice after liposomes injection.3.The CLSM results showed that a large number of FA-DOX-ICGPFP@Lip liposomes were phagocytosed by Y79 cells,while the number of liposomes that were not modified with FA was significantly reduced.Moreover,when FRs on the surface of Y79 were antagonized by FA,the amount of FA-DOX-ICG-PFP@Lip around the cell was also reduced.The same result was obtained by flow cytometry.The fluorescence imaging of tumor-bearing mice showed that the accumulation of liposomes in tumor site reached the maximum 3 h after injection of FA-DOX-ICG-PFP@Lip.4.The heating effect of FA-DOX-ICG-PFP@Lip after 808 nm laser irradiation was obvious,and was positively correlated with liposome concentration.The photothermal stability of liposomes was significantly higher than that of ICG solution.It was found that liposomes could rapidly transform into microbubbles after irradiation by 808 nm laser.Liposomes also showed good photothermal conversion efficiency in the test of photothermal conversion ability in vivo.5.The UV spectrophotometer results showed that the release rate of DOX increased significantly after 808 nm laser irradiation.ConclusionThe multifunctional liposome FA-DOX-ICG-PFP@Lip containing DOX and ICG was successfully prepared by thin film hydration and double emulsion method.At the same time,the FA-DOX-ICG-PFP@Lip with uniform size,good stability,high drug loading.At the same time,the prepared liposomes have good biosafety,targeting ability,photothermal conversion ability.Moreover showed good controlled release performance in vitro,which lays a foundation for the imaging and treatment of retinoblastoma.PART Ⅱ STUDY OF THE DUAL-IMAGING CAPABILITY OF FA-DOX-ICG-PFP@LIPObjectTo investigate the photoacoustic and ultrasonic imaging capabilities of FA-DOX-ICG-PFP@Lip liposomes and provide a basis for the diagnosis of RB.MethodSix groups of samples [FA-DOX-ICG-PFP@Lip(0.625,1.25,2.5,5mg/m L),DOX-ICG-PFP@Lip(5 mg/m L)and saline group,n=5] were tested and analyzed by PA imaging system and ultrasonography in vitro.In addition,Y79 tumor-bearing mice were randomly divided into 2 groups(FADOX-ICG-PFP@Lip and DOX-ICG-PFP@Lip,n=5).The liposomes were injected intravenously,and the photoacoustic and ultrasonic images of tumor sites in tumor-bearing mice were collected at different time points,the photoacoustic and ultrasonic signal intensity of each group was quantitatively analyzed.ResultsIn vitro photoacoustic imaging experiments,photoacoustic signal was enhanced with the increase of FA-DOX-ICG-PFP@Lip concentration.There was no statistical difference in photoacoustic signal intensity between FADOX-ICG-PFP@Lip and DOX-ICG-PFP@Lip at the same concentration,and no photoacoustic signal was detected in the saline group.In vivo photoacoustic imaging experiments,1 hour after injection of FA-DOX-ICGPFP@Lip and DOX-ICG-PFP@Lip,obvious photoacoustic signals began to appear at the tumor site,and then gradually increased to the peak at 3 h,and then gradually decreased.In vitro ultrasound imaging experiments,the echo signal of each liposome gradually increased during the irradiation of 0-5 min by 808 nm laser,and reached the maximum at 4min,and the intensity of echo signal was positively correlated with the concentration of liposome.There was no statistical difference in ultrasonic signal intensity between FA-DOX-ICGPFP@Lip and DOX-ICG-PFP@Lip at the same concentration,and no echo signal was detected in the saline group.In vivo ultrasound imaging experiments,after injection of FA-DOX-ICG-PFP@Lip and DOX-ICGPFP@Lip liposomes,the tumor site was irradiated with 808 nm laser.The echo signal enhancement of tumor site in FA-DOX-ICG-PFP@Lip group was more obvious than that in DOX-ICG-PFP@Lip group.ConclusionFA-DOX-ICG-PFP@Lip liposomes have excellent photoacoustic and ultrasonic imaging capabilities,providing conditions for targeted imaging of retinoblastoma.PART Ⅲ EFFICACY OF NEAR-INFRARED LASER COMBINED WITH FA-DOX-ICG-PFP@LIP CHEMO/PHOTOTHERMAL THERAPY FOR RETINOBLASTOMAObjectTo evaluate the chemo/photothermal therapeutic efficacy induced by FA-DOX-ICG-PFP@Lip liposomes to retinoblastoma.MethodsThe survival rate of Y79 cells treated with 7 groups of samples(control,laser only,DOX only,FA-DOX-ICG-PFP@Lip,FA-ICG-PFP@Lip+laser,DOX-ICG-PFP@Lip+laser and FA-DOX-ICG-PFP@Lip+laser)were measured by CCK-8.Y79 tumor-bearing mice were randomly divided into 7 groups and given different treatments,the weight and tumor volume of tumor-bearing mice were observed for 14 days.After 14 days,the mice were sacrificed,tumor tissues and main organs were collected for hematoxylin-eosin(HE)staining,and the apoptosis and proliferation of cancer cells in each group were detected by TUNEL and PCNA techniques.ResultsIn vitro experiments,the survival rates of Y79 cells treated by DOXICG-PFP@Lip + laser and FA-DOX-ICG-PFP@Lip + laser were the lowest,while laser irradiation only had no killing effect on Y79 cells.The results of treatment in vivo showed that the tumor volume of the FA-DOX-ICGPFP@Lip + laser group gradually decreased,indicated the best inhibitory effect on tumor growth.The results of TUNEL and PCNA also showed that the apoptosis index of tumor cells was the highest and the proliferation index was the lowest in FA-DOX-ICG-PFP@Lip + laser group.ConclusionThe combination of near-infrared laser and FA-DOX-ICG-PFP@Lip liposome has achieved dual-mode image-guided chemical/photothermal treatment for retinoblastoma,which effectively inhibiting tumor growth and recurrence. |
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