| PART 1 PREPAATION OF TARGETED MICROBUBBLES AND THE STUDY OF THAT IN DIAGNOSING TRAUMATIC HEPATIC OOZING BLOODObjective To prepare three kinds of ultrasound contrast agents (UCAs):usual microbubbles (MBMal), RGD-targeted lipid microbubbles (MBRGD), controlled RAD lipid microbubble (MBRGD), respectively, and to detect their characters, and then to study the value of MBRGD in the diagnosis of traumatic hepatic oozing blood with the animal experiment.Methods (1) MBs were prepared by mechanical shaking method, and the morphology, dispersion, particle size, potential, counting, connection rate of peptide, the amount of connected peptide were observed and determined; (2) to proceed enhanced imaging in vivo and in vitro with three kinds of MBs,respectively; (3) to observe the target ability of three kinds of MBs in vitro;(4) of 12 New Zealand rabbits were divided into two groups to establish the model of traumatic hepatic oozing blood, which is proved successful when CEUS with MBmal showed that there was no active bleeding but without stopping bleeding within 10 minutes. After that, MBRGD (experimental group) and MBRGD (control group) were injected through veins right now. The perfusion status of MBs were observed in lesions area for 10 minutes. The average grey values (AGVs) of the area of interest were measured at every 1 minute with DFY software. The measurements were compared with that of prior MBMal images respectively, and that of the two groups were campared with each other.Results (1) The observed MBs under the microscope were almost with the same size and good dispersion. The average particle sizes of MBMal, MBRGD and MBRGD were 4.25±0.67 μm,3.71±0.58μm,4.18±0.86μm, respectively. The average potential of that were 0.01± 7.86 mV,37.3±3.44 mV,34.5±5.21 mV, respectively. The concentration of that were 2.52±0.26×108/mL,2.74±0.12×108/mL,2.34±0.46×108/mL, respectively. The combined rate of RGD peptide was 98.62%, and that of RAD polypeptide was 98.94%. The amount of peptide connecting to MBs were 9.76±0.91×104 RGD/MBRGD and 10.13 ±0.34×104 RAD/MBRAD, respectively. (2) Under the condition of 105/mL concentration, the contrast enhanced images were the best of all three kinds of MBs in vitro, and for AGVs, there was no statistic difference among MBs (all P>0.05). In vivo, the enhancement begins at about 10 s after injection, and the duration time was about 10 minutes. There was no statistic difference about the AGVs of three kinds of MBs at the same time (all P values were greater than 0.05). (3) MBRGD obviously gathered around and adhered to the actived and aggregated platelets was obversed under microscope, while these phenomenons of MBMal and MBRGD were not observed. (4)The experimental group showed that MBRGD gathered in the area of trauma, while control group showed no MBRGD gathered. Images of MBRGD were similar with that of MBMal among 1-3 min and there was no statistic difference in AGVs between the two MBs (Pimin, P2min and P3min values were all greater than 0.05). A small quantity of MBRGD gathered in the areas of trauma since 4th min until 6th min, and there was statistical difference in AGVs at these time points (P^mn and P5min values were all less than 0.01, and P6min<0.05). There was no statistic difference in AGVs between the two MBs at the 7th min (P7min>0.05). The AGVs of the 8th min and 9th min of the two MBs decreased than the prior, but there were still statistic difference between MBRGD and MBMai (P8min and P9min values were all less than 0.05). There was no statistic difference at 10th min between the two MBs. There were no statistic difference between MBRGD and MBMal in AGVs at 10 time points, and all P values were all greater than 0.05. Compared with MBRGD and MBRGD , there were statistic difference in AGVs at every time point except 4th and 10th min and both P values were greater than 0.05. There were statistic difference at the rest of the time points in AGVs between the two MBs. Among them, P2min, P3min, P6min and P8min were all less than 0.01, and Pimin, P5min, P7min and P9min were all less than 0.05, with significant statistic difference and statistic difference in AGVs of corresponding time point, respectively.Conclusion The mechanical shaking method of preparing RGD-targeted MBs is easy, with high successful rate, and the MBs had the characters of same size, good dispersion, good targeted ability, and ecxellent contrast imaging in vitro and in vivo. The RGD-targeted MBs could achieve imaging in areas of oozing blood, and could be tried to be used to diagnose traumatic hepatic oozing blood, providing new method for clinical practice.PART 2 PREPARATION OF TARGETED TRANEXAMIC ACID-CARRYING MICROSPHERES AND THE STUDY OF THAT IN TREATING TRAUMATIC HEPATIC OOZING BLOODObjective To prepare four kinds of polymer microspheres:RGD-targeted and tranexamic acid (TXA)-carring microspheres (PLGA-TXA-RGD), TXA-carring microspheres (PLGA-TXA), TXA-carring with non-targeted microspheres (PLGA-TXA-RAD), without drug and RGD-targeted microspheres (PLGA-RGD), and the properties of microspheres were detected. To study whether the PLGA-TXA-RGD is with therapeutic effect and how the value was in traumatic hepatic oozing blood through the animal experiment research.Methods (1) Each PLGA microspheres prepared with double emulsion method, and morphology and dispersion, structure, particle size, potential, peptide connection rate, drug loading efficiency, the encapsulation rate of drug and drug-release rate were observed and determined. (2) 48 male SD rats were randomly divided into 6 groups:normal saline group (N.S), intravenous TXA (I.V TXA) group, PLGA-TXA group, PLGA-TXA-RAD group, PLGA-RGD group, PLGA-TXA-RGD group. After building hepatic traumatic bleeding model,0.5 mL of each reagent was injected through tail vein slowly, and the data, including the time since bleeding until the end of that, blood loss, dry weigh loss, and motality within 3 h were all recorded and compared with each other. (3) The organization of traumatic focal was handled into frozen sections, and distribution of PLGA microspheres along the incision edges and in the blood clot was observed under the fluorescence microscope.Results (1) The microscopic observation of PLGA microspheres were uniform particle size, good dispersion, no reunion phenomenon. Under transmission electron microscope (TEM) the structure of the microspheres is sphericalwith shell structure and,not smooth. The average particle sizes of PLGA-TXA-RGD, PLGA-TXA, PLGA-TXA-RAD, PLGA-RGD were 1811±369.4nm,1747±276.7nm,1831±268.3 nm,1909±305.4nm, respectively. The average potential were -24.7±4.4 mV,-24.3±4.49 mV,-24.6±3.61 mV, -25.5±4.28 mV, respectively. Flow cytometry instrument detecting the connected RAD and RGD peptide rate of drug-loading PLGA microspheres were 92.88% and 93.85%, respectively. The encapsulation efficiency of TXA-carring PLGA microspheres was 18.5± 2.4%, and TXA loading efficiency of that was 3.7±0.5%. Within 3 hours, the drug-release rate of US irradiation was 27.3±1.1%, while that of without US irradiation was 20.0± 1.6%. (2) Comparing with every group, there were significant statistic difference in bleeding time (t), blood lost (W) and dry weight loss (W’), and P values were all less than 0.01. Compared I.V TXA group with N.S group, there were statistic difference on t, W and W, and P values were all less than 0.05. When compared PLGA-TXA-RGD group with I.V TXA group and PLGA-RGD on t, both P values were greater than 0.05 which meant that there were no statistic difference. While compared PLGA-TXA-RGD with PLGA-TXA-RAD, the P value was less than 0.01,which meant that there was significant statistic difference between the two groups. When compared PLGA-TXA-RGD group with PLGA-RGD on W, P value was greater than 0.05, which showed that there was no statistic difference between the two groups. Compared PLGA-TXA-RGD group with I.V TXA group, there was statistic difference and P value was less than 0.05. While compared PLGA-TXA-RGD group with PLGA-TXA group and PLGA-TXA-RAD group, both P values were less than 0.01 and there was significant statistic difference among them. The results of comparison about W’was similar with that of W. For mortality, there was no statistic difference in general and P value was greater than 0.05. And there was neither statistic difference in pairwise comparison and all P values were greater than 0.05. (3) There was no PLGA microspheres gathered along the incisions and only some ones observed within the clots in group PLGA-TXA and group PLGA-TXA-RAD. Besides above observed, in group PLGA-RGD and group PLGA-TXA-RGD, there were visible red fluorescent microspheres gathered aong the incision, with great number, especially there were also some microspheres near the incision forming the blood clots.Conclusion The method of double emulsion preparing TXA-carring and RGD-targeted PLGA microspheres was successful, with the result of uniform size, good dispersion, good encapsulation rate and loading efficiency. Both functions of drug-carring and targeted PLGA microspheres could be as a hemostatic agent injected into vein for the treatment of traumatic hepatic oozing blood. |
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