Ultrasound thrombolysis mediated by transcatheter injection of microbubble in vitro

Objective To fabricate iRGD targeted liposome-microbubble complex containing uPA (iRGD-LMC), and to improve the thrombolytic efficiency and reduce the risk of thrombolysis by iRGD-LMC combining with ultrasound targeted microbubbles destruction (UTMD) to release drug into the thrombus site with the help of microbubble cavitation effect. Methods Biotinylated iRGD-MBs were fabricated by thin-film rehydration method.Biotinylated liposomes containing uPA were fabricated by freeze-thaw method and were conjugated to the biotinylated iRGD-MBs surface through biotin-avidin linkage. The iRGD-LMC was subjected to confocal microscopy to determine the particle morphology. The concentration, average diameter and size distribution were determined by particle sizing instrument. The uPA loading efficiency was measured by BCA Protein Assay Kit. Ultrasound imaging was performed using a Vevo 2100 ultrasound imaging system. The iRGD-LMC was irradiated by different ultrasound time and intensity to release drug. Thrombolytic effect in vitro of iRGD-LMC combined with UTMD was observed on the thrombosis model which was extracted from mouse blood. Results iRGD-LMC was successfully prepared. iRGD-LMC was exhibited a well-defined spherical morphology and homogeneous distribution, like ordinary microbubbles. The concentration of iRGD-LMC was (0.51±0.03)×109/ml and average diameter was (2.62±0.12) μm. Drugs loading efficiency was (3 878.5±97.8) μg uPA per 108 microbubbles. iRGD-LMC could achieve contrast-enhanced ultrasound imaging in vitro. The thrombolytic effect of iRGD-LMC+ US group (87.66±1.69)% was the best in vitro, and had significant difference with others groups (P<0.05), followed by iRGD-LMC group (53.32±4.86)% and uPA group (51.09±9.01)%, Compared with PBS group, US group (23.56±9.46)% had thrombolytic effect. Conclusions iRGD-LMC is successfully prepared, which has the advantages of high drug loading of liposomes and good acoustic properties of microbubbles. iRGD-LMC combined with UTMD achieves a significant thrombolytic effect in vitro. Key words: Ultrasound targeted microbubble destruction; Urinary plasminogen activator; Thrombolytic therapy; iRGD-LMC

Objective To evaluate the efficacy ofmicrobubbles-assisted urokinase thrombolysis under transcranial Doppler ultrasound (TCD) in rabbits with cerebral infarctions.Methods Male New Zealand white rabbits (n=32) were randomly divided into two groups,a urokinase group and a combined urokinase and microbubble group (n=16).The middle cerebral artery (MCA) was occluded by injecting autologous blood clots through the carotid artery.In the combined urokinase and microbubble group,sulfur hexafluoride (SonoVue) microbubbles were injected intravenously immediately after intravenous injection of urokinase.The animals in these two groups were monitored by TCD within 2 h of thrombolysis,and the hemodynamic changes were recorded and the recanalization was noted according to the TCD spectral morphology and blood flow velocity.Results The average blood flow velocity of the middle cerebral artery before performing thrombolysis showed no significant difference between the 2 groups (P＜0.05).One rabbit in the urokinase group had complete recanalization and 4 partial mecanalization with a recanalization rate of 31.3％.Three rabbits in the combined urokinase and microbubble group had complete recanalization and 6 partial recanalization with a recanalization rate of 56.3％.Pathological examination showed no cerebral hemorrhage in either group.The average size of the infarction foci was 13.9％ in the urokinase group and 9.3％ in the combined urokinase and microbubble group with significant difference (P=0.025).Conclusion The addition of microbubbles to urokinase treatment increases vascular recanalization and decreases infarct size,and does not cause side effect of cerebral hemorrhage.During 2 MHz ultrasound monitoring,microbubble can assist urokinase thrombolysis for acute cerebral infarctions. Key words: Tran-cranial Doppler ultrasound; Cerebral infarction; Urokinase; Microbubble; Thrombolysis

Objective To prepare urokinase plasminogen activator (uPA)-loaded anionic lipid microbubbles (uPA-MBs) for thrombolysis with low-frequency ultrasound in vitro.Methods Anionic microbubbles composing of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DPPG),1,2-distearoyl-sn-glycero-3-phosphoethanol amine-N (succinyl PEG2000) (DSPE-PEG2000) and perfluoropropane (C3F8) were prepared by the mechanical vibration method.Then,the resulting anionic microbubbles were incubated with uPA.uPA-MBs were obtained via electrostatic adsorption.Bubble size and distribution were measured by particle size analyzer.FITC-labeled uPA-MBs were obtained and observed under fluorescence microscope.The surface potential of uPA-MBs and plain microbubbles (P-MBs) were detected by Zeta potential analyzer.Sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for confirming the binding of uPA protein and anionic microbubbles.The encapsulation efficiency of uPA-MBs was determined by bicinchoninic acid (BCA) protein assay kit under three different dosages of uPA (10 000,50 000 and 100 000 U).The thrombolysis efficiency of uPA-MBs combined with low-frequency ultrasound was examined in vitro.Two-sample t test,one-way analysis of variance and Bonferroni test were performed to analyze the data.Results UPA-MBs were successfully obtained with the mean particle size of (1.76±0.29) μm.The surface potential of these bubbles was significantly higher than that of P-MBs:(-36.64±0.21) mV vs (-66.33±2.38) mV (t =21.538,P＜0.05).Fluorescence microscope showed a green shell of FITC-labeled uPA-MBs.The encapsulation efficiency of uPAMBs with the added dosage of 10 000 U was (42.01±2.02) ％,which was significantly higher than those of 50000 and 100 000 U ((33.24±1.95)％ and (33.10±1.65)％ respectively,F=22.340,P＜0.05).The thrombolysis efficiency by saline was (4.09±0.80)％,saline + ultrasound (8.50±1.48)％,MBs + ultrasound (14.27± 1.59) ％,uPA-MBs + ultrasound (35.72±6.31) ％ and uPA (16.87±0.46) ％,respectively (F =48.783,t =-8.613,-7.273,-5.942,-6.908,all P＜0.05).Conclusion Anionic microbubbles can successfully load uPA,and achieve significantly better thrombolysis effect when combined with low-frequency ultrasound. Key words: Thrombolytic therapy ; Microbubbles ; Urokinase ; Ultrasonography

Objective To determine the effect of the combined use of urokinase and glycoprotein Ⅱb/Ⅲa-targeted microbubbles prepared by direct conjugation method to dissolve the thromb in vivo and analyse the velocity tracing change of blood flow and explore the possible mechanism. Methods A total of 42 rabbits with platelet-rich thrombi in the femoral artery were randomized into 7 treatment groups ( n = 6 in each group): 1) ultrasound alone (US); 2) ultrasound plus non-targeted microbubbles ( US + M); 3) urokinase alone (UK) ;4) ultrasound, non-targeted microbubble and urokinase (US + M + UK); 5) ultrasound plus platelet-targeted microbubble ( US + R); 6) platelet-targeted microbubble plus urokinase (R + UK); 7)ultrasound, platelet-targeted microbubble and urokinase (US + R + UK). A total of 6 ml of infusion liquor of Urokinase,RGDS and microbubbles (SonoVue) were mixed by 1 ∶ 1 ∶ 1 ratio by the direct conjugation method, infusion via vein within 20 min. Ultrasound was conducted to lyse the clot for 30 min. The recanalization and the velocity tracing change of blood flow in thrombolytic process were evaluated at 120 min post treatment. Results For US, UK, US + M, US + R and US + M + UK groups, recanalization was failed. The R + UK and US + R + UK was recanalizated ( P ＜0.001 ). The blood flow velocity tracing was small and low width in US,UK, US + M, US + R and US + M + UK groups. The wave was high width and disorderly under the thrombolysis therapy in the R + UK and US + R + UK. The thrombolytic effect was demonstrated by the high-width and disorderly resonance changes in the blood flow spectrum during the thrombolytic therapy of US + R + UK. Conclusions The blood flow spectrum of groups had different characteristics in vivo when thrombus was issolved,ultrasonic resonance might be the possible mechanism. Key words: Ultrasonography; Microbubbles; Thrombolytic therapy; Urinary plasminogen activator; RGDS

Intravenous thrombolysis is the only treatment that has the evidence of evidence-based medicine in acute ischemic stroke. However, the narrow time window has limited the treatment opporttmities of many patients. Transcranial ultrasound thrombolysis is a very promising thrombolysis-assisted method, and transcranial ultrasound plus microbubble- assisted thrombolysis is the research hotspot in recent years. At present, more suitable ultrasonic instruments for thrombolysis and a number of ways of ultrasound-assisted thrombolysis are being developed. Key words: stroke; thrombolytic therapy; ultrasonography, Doppler, transcranial

Objective To explore the express of von willebrand factor(vWF) and tissue factor(TF) in ultrasound combined with urokinase and RGDS-targeted microbubbles for recanalization of occluded rabbit femoral artery. Methods A total of 42 rabbits with platelet-rich thrombi in the femoral artery were randomized into 7 treatment groups (n=6 in each group): ①ultrasound alone (US) group; ②ultrasound, non-targeted microbubbles (US+ M) group; ③urokinase alone (UK) group; ④ultrasound, non-targeted microbubble and urokinase (US+ M+ UK) group; ⑤ultrasound, platelet-targeted microbubble (US+ R) group; ⑥platelet-targeted microbubble, urokinase (R+ UK) group; ⑦ultrasound, platelet-targeted microbubble and urokinase (US+ R+ UK) group. A total of 6 ml of infusion liquor of urokinase, RGDS and microbubbles (SonoVue) were mixed by the direct conjugation method, infusion via vein within 20 min. Ultrasound was conducted to lyse the clot for 30 min. The recanalization and the velocity tracing change of blood flow in thrombolytic process and the express of vWF and TF were evaluated at 120 min post treatment. Results For US, UK, US+ M, US+ R and US+ M+ UK groups, recanalization were failed. The R+ UK and US+ R+ UK groups were partly recanalizated or completed recanalized (P<0.001). vWF and TF were positive in US, US+ M, US+ R and R+ UK group. vWF and TF were negative in US+ M+ UK, UK and US+ R+ UK groups. Conclusions The combined effects of low frequency ultrasound and targeted microbubble combined with urokinase can inhibit the expression of vWF and TF, and then promote the thrombolysis, however it can affect the expression of vWF and TF after reperfusion. Key words: Ultrasonography; Targeted microbubbles; Thrombolytic therapy

Objective To study thrombolytic efficiency and influencing factors of low-frequency ultrasound-mediated microbubbles carrying streptokinase.Methods Whole blood clots,contrast agent of ultrasound microbubble and streptokinase solution were prepared.Three groups were set up in this study,including control group (C,2 ml normal saline given),streptokinase group (S,streptokinase alone given) and ultrasound microbubble carrying streptokinase group [UMS,ultrasound (1.2 W/cm2 for 20 min) microbubbles carrying streptokinase].After treatment,the whole blood clots were weighed.Results The ultrasound microbubbles carrying streptokinase were stable.There was significant difference in the clot weight among the three groups (P ＜ 0.01).The thrombolysis rate in UMS group was 50.7％.Conclusion Lowfrequency ultrasound-mediated microbubbles carrying streptokinase may promote the dissolution of deep vein thrombosis effectively. Key words: Deep vein thrombosis; Ultrasound microbubble; Targeted therapy

Sonothrombolysis: The Contribution of Stable and Inertial Cavitation to Clot Lysis

Objective To explore the clinical value of a new tactic with novel dodecafluoropentane (C5F12, DDFP) acoustic vaporizable nano-droplets for the therapy of coronary micro-circulation thrombolysis. Methods Group A was poly butylene succinate buffer solution as blank control; group B was SonoVue microbubbles; group C was DDFP acoustic vaporizable nano-droplets, which were produced with emulsion process by ultrasonic oscillation instrument and rotary evaporator. The size of the nano-droplet was tested by Malvern laser particle size detector and observed with fluorescence microscope. The stability and efficiency of cavitation effect of group C for thrombolysis were tested in an in-vitro artificial vascular system with arterial thrombus model, comparing with group A and B. Weights and pathological sections of the inserted thrombus before and after the thrombolysis in these groups were compared for the thrombolytic efficiency. Results The average size of DDFP nano-droplets were (424.7±30.2)nm. More than 75% of the nano-droplets were ranged from 300-750 nm. The fluorescence microscope showed the DDFP nano-droplets were uniformed round-shaped and stable-structured with favorable dispersity; the nano-droplets had gradually turned into over 3 μm microbubbles under ultrasound irradiation. The thrombus weight loss after thrombolysis in group C [(199.0±35.8)mg, (32.1±4.4)%] was significantly larger than those in group A [(30.2±17.8)mg, (5.0±2.4)%] and B [ (72.6±20.7)mg, (12.7±2.8)%] (all P<0.01). Conclusions DDFP vaporizable nano-droplets with ultrasound irradiation is effective for thrombus elimination in coronary micro-circulation through lasting and stable cavitation effect. Key words: Sonication; Vaporizable nano-droplet; Thromboembolism; Thrombolytic therapy

Objective To explore the adeno-associated virus (AAV) gene transduction and cellular endocytosis mediated by ultrasound combined with microbubbles in two types of cells. Methods HeLa and NIH/3T3 cells were infected by rAAV2-EGFP at a concentration gradient to get the optimal concentrations for enhancement. At these concentrations, HeLa and NIH/3T3 cells were infected by rAAV2-EGFP mediated by ultrasound combined with microbubbles. The gene transduction efficiency were observed and measured by fluorescence microscopy and flow cytometry at 48 h after treatment. The cell viability was tested by CCK-8. The number and distribution of cellular clathrin-coated endocytic pits were observed by confocal fluorescence microscopy and transmission electron microscopy on 45 min after treatment. Results The optimal concentrations for HeLa and NIH/3T3 cells were 2000 v. g./cell and 10000 v. g./cell.Ultrasound combined with microbubbles significantly enhanced the transduction efficiency of rAAV2-EGFP (P 0.05). Confocal fluorescence microscopy and transmission electron microscopy demonstrated that clathrin-coated endocytic pits were more obviously increased in ultrasound combined with microbubbles mediated AAV transduction group than AAV transduction group. Conclusions Ultrasound combined with microbubbles can efficiently enhance the gene transduction of AAV, whose cellular transportation depends on cellular endocytosis, in two types of cells. Stimulating cellular endocytosis might be one of the mechanisms of enhanced cellular transportation of AAV mediated by ultrasound combined with microbubbles. Key words: Sonication; Microbubbles; Adeno-associated virus; Transfection; Cellular endocytosis

Intraclot microbubble combined with urokinase mediated ultrasound thrombolysis: Experiment in vitro

Objective To investigate whether ultrasound microbubble could mediate gene EGFP transfecting retinal ganglial cells (RGCs) in vivo,whether this transfection way is more effective than the orthodox way and whether this way could cause damage of RGCs.Methods Fifty SD rats were randomly divided into 4 groups:the normal control group (n =5),plasmid group (n =15),plasmid + ultrasound group (n =15)and ultrasound microbubble group (n =15).The normal control group was injected 5 μL normal saline into vitreous cavity.The plasmid group was injected 5 μL plasmid.The plasmic + ultrasound group was injected 5 μL plasmid,then we exposed rats eyeballs to 0.5 W/cm2 ultrasonic wave immediately for 60 s( the exposure time accounting for 1/3:the exposure time is 5 s,then pause 10 s,total time 60 s.The ultrasound microbubble group was injected 5 μL suspension of plasmid and microvesicle,then we exposed rats eyeballs as the above way immediately.Seven days later,we made stretched preparation and longitudial frozen section of retina,and RT-PCR of EGFP mRNA of retina.Then fluorescence microscope was used to observe stretched preparation and EGFP expression in RGCs.We counted the number of RGCs to observe the damage situation.RGCs EGFP mRNA was detected through RT-PCR semi-quantitatively.Results The efficiency of ultrasound microbubble mediating gene EGFP transfecting RGCs was significantly higher than the normal control group,plasmid group and plasmic + ultrasound group,and this transfection way didn' t cause damage of RGCs.Conclusion By exposing eyeball to ultrasound of low frequency transduction,ultrasound can mediate EGFP gene transfecting RGCs safely and effectively. Key words: Ultrasonography; Retinal ganglial cells; Gene therapy; Enhanced green fluorescent protein; Gene transfection

Increasing evidence confirms that microbubble (MB)-augmented ultrasound (US) thrombolysis enhances clot lysis with or without tissue plasminogen activator (tPA). Intracranial hemorrhage (ICH) is a major complication militating against tPA use in acute ischemic stroke. We quantified the incidence of ICH associated with tPA thrombolysis and MB + US therapy and compared infarct volumes in a rabbit model of acute ischemic stroke. Rabbits (n = 158) received a 1.0-mm clot, angiographically injected into the internal carotid artery causing infarcts. Rabbits were randomized to 6 test groups including (1) control (n = 50), embolized without therapy, (2) US (n = 18), (3) tPA only (n = 27), (4) tPA + US (n = 22), (5) MB + US (n = 27), and (6) tPA + MB + US (n = 14). US groups received pulsed wave US (1 MHz, 0.8 W/cm) for 1 hour; rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Rabbits with MB received intravenous MB (0.16 mg/kg) given over 30 minutes. Rabbits were killed 24 hours later and infarct volume and incidence, location, and severity of ICH were determined by histology and pathologic examination. Percentage of rabbits having ICH outside the infarct area was significantly decreased (P = 0.004) for MB + US (19%) rabbits compared with tPA + US (73%), US only (56%), tPA (48%), tPA + MB + US (36%), and control (36%) rabbits. Incidence and severity of ICH within the infarct did not differ (P > 0.39). Infarct volume was significantly greater (P = 0.002) for rabbits receiving US (0.97% ± 0.17%) than for MB + US (0.20% ± 0.14%), tPA + US (0.15% ± 0.16%), tPA (0.14% ± 0.14%), and tPA + MB + US (0.10% ± 20%) rabbits; these treatments collectively, excluding US only, differed (P = 0.03) from control (0.45% ± 0.10%). Treatment with MB + US after embolization decreased the incidence of ICH and efficacy was similar to tPA in reducing infarct volume.

To evaluate the impact of the recombined adeno-associated virus encoding soluble tumor necrosis factor related apoptosis inducing ligand gene (rAAV-sTRAIL) on proliferation and apoptosis of human hepatocellular carcinoma (HCC) cells, and to investigate the feasibility and efficiency of transfection of rAAV-sTRAIL into human HCC cells by ultrasound microbubble intensifier. Human HCC cells of the line HepG2 were transfected with rAAV-sTRAIL or rAAV-sTRAIL combined with microbubble echocontrast agent and appropriate dose of ultrasound irradiation. RT-PCR and Western blotting were used to detect the mRNA and protein expression of sTRAIL gene. MTT method was used to detect the proliferation inhibition rate, and the apoptosis rate of the HepG2 cells was evaluated by flow cytometry. The expression levels of sTRAIL mRNA and protein were higher in the rAAV-TRAIL combined with ultrasound microbubble group than in the rAAV-sTRAIL group (both P < 0.05). The proliferation inhibition rate of the rAAV-TRAIL combined with ultrasound microbubble group was significantly higher than that of the rAAV-sTRAIL group (P < 0.05). The apoptotic effect of the rAAV-TRAIL combined with ultrasound microbubble group was greater than that of the rAAV-sTRAIL group (P < 0.05). TRAIL has a potential role to inhibit e proliferation and induce apoptosis of human hepatocellular carcinoma cells. Ultrasound and microbubble echocontrast agent increase the transfection rate of rAAV vector into HCC cells.

Objective To study the effect and possible mechanisms of mesenchymal stem cells （MSCs） transplantion therapy mediated by ultrasound in combination with nitric oxide （NO） microbubbles （MBs）on cardiac function in rats with myocardial infarction （MI）. Methods Twenty-eight rats with MI were randomly divided into the following groups by use of random digits table： phosphate buffered saline group （ injection of PBS into the tail vein） , MSCs group （injection of MSCs into the tail vein） , ultrasound ＋ MBs ＋ MSCs group （ultrasound interven- tion when injection of ordinary MBs into the tail vein followed by injection of MSCs） and ultrasound ＋ NO MBs ＋ MSCs group （ultrasound intervention when injecting NO-MBs into the tail vein followed by injection of MSCs） （n = 7, each group）. After four weeks, the left ventricular systolic function was evaluated with M-mode ultrasound for each group, capillaries density of myocardial ischemic area was counted in each group, and the expression of vascular endothelial growth factor （VEGF） was detected by Western blot and real time PCR. Results The ejection fraction （EF） of NO-MBs group was significantly higher than the other groups （ P 〈 0.05 ）. The number of capillaries in NO- MBs group was also much more than that in the other groups （P 〈 0.05）. The expression of VEGF in infarcted zone was much higher in the NO-MBs group than that in the other groups （ P 〈 0.05 ）. Conclusion Ultrasound and NO MBs-mediated MSCs transplantation therapy could improve the cardiac function of rats after MI, and the possible mechanism was the upregulation of VEGF and angiogenesis. Key words: Myocardial infarction ; Stem cells ; Ultrasound ; Microbubbles agent ; Nitric oxide