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Objective:To investigate the effect of vancomycin (Vm)-loaded microbubbles (MBs) combined with ultrasound targeted microbubble destruction (UTMD) technique on the morphological structure, thickness and bacterial viability of methicillin-resistant Staphylococcus aureus (MRSA) biofilms.Methods:Vm-MBs were prepared by thin film hydration. Sterile coverslips in a diameter of 13 mm were placed in 24-well plates to construct in vitro biofilm models using MRSA as the test strain, and the biofilm morphology was observed by naked eye and light microscopy after crystal violet staining. LIVE/DEAD, SYTO59 and DIL were used to stain biofilms and MBs, respectively. After staining, the biofilm morphology and position of the biofilm in relation to MBs were observed using laser confocal scanning microscopy. The biofilms were divided into control group, Vm group, Vm-MBs group, UTMD group and Vm-MBs+UTMD group according to the random number table method, with 9 samples in each group. After biofilms of each group were treated accordingly for 24 hours, the morphological and structural changes of biofilms in each group were observed using laser confocal scanning microscopy and scanning electron microscopy following LIVE/DEAD staining; the difference in biofilm density in each group was measured with the aid of an enzyme marker following crystal violet staining; the difference in biofilm thickness and bacterial viability in each group were observed by laser confocal scanning microscopy. Results:The prepared Vm-MBs met the experimental requirements. The constructed biofilm model observed by naked eye, light microscopy and laser confocal scanning microscopy showed that the biofilm structure was dense with a relatively uniform thickness of (13.8±0.2)nm, a small amount of dead bacteria inside the membrane and the percentage of live bacteria of (94.9±0.3)%. Laser confocal scanning microscopy showed that MBs could penetrate into deeper layers of biofilms. After the respective treatment was given to each group for 24 hours, Laser confocal scanning microscopy and scanning electron microscopy following LIVE/DEAD staining showed that the biofilm morphological structure was most significantly disrupted in Vm-MBs+UTMD group compared to control, Vm, Vm-MBs and UTMD groups. In Vm-MBs+UTMD group, a large number of dead bacteria was observed, with only a few scattered planktonic bacteria and irregular changes in cell membrane morphology. Crystal violet staining showed that the biofilm density was significantly lower in Vm-MBs+UTMD group compared to control group ( P<0.05), while the differences between Vm, Vm-MBs and UTMD groups were not statistically significant (all P>0.05). Laser confocal microscopy showed that the biofilm thickness was thinner in Vm-MBs, UTMD and Vm-MBs+UTMD groups compared to control group (all P<0.05), with no significant difference between Vm group and control group ( P>0.05) and that the biofilm thickness was thinner in Vm-MBs+UTMD group compared to Vm, Vm-MBs and UTMD groups (all P<0.01), with no significant differences between the other groups (all P>0.05). Bacterial activity in Vm, Vm-MBs, UTMD and Vm-MBs+UTMD groups was significantly lower than that in control group (all P<0.01), with lower in Vm-MBs+UTMD group compared to Vm, Vm-MBs and UTMD groups (all P<0.01), but without significant difference between the other groups (all P>0.05). Conclusion:Vm-MBs combined with UTMD technology can effectively destroy the biofilm morphological structure to reduce biofilm thickness. Meanwhile, Vm-MBs combined with UTMD technology can release antibiotics and significantly decrease bacterial viability to improve antibiotic bactericidal efficacy.
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Objective:To investigate the effects of ultrasound microbubble-mediated RasGAP SH3-binding protein 1 (G3BP1) transfection on the proliferation and migration of human liver cancer HepG2 cells.Methods:HepG2 cells were treated with ultrasound targeted microbubble destruction (UTMD) technology-mediated si-G3BP1. The expression of G3BP1 in HepG2 cell lines was detected by Western blot, and the silencing efficiency was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot. HepG2 cell proliferation and migration were analyzed by flow cytometry, methyl thiazolyl tetrazolium (MTT) , EdU staining, colony formation experiment, wound healing experiment, Transwell experiment and Western blot.Results:After silencing G3BP1 in HepG2 cells, its mRNA and protein levels were significantly reduced (1.01±0.03 vs 0.27±0.03, 1.02±0.01 vs 0.33±0.04) ; UTMD-mediated si-G3BP1 could significantly reduce the proliferation rate (31.49±3.09 vs 12.51±1.02) , proliferation activity (1.20±0.13 vs 0.46±0.31) , EdU-positive cell rate (99.23±1.01 vs 36.75±4.03) , colony formation rate (96.45±1.21 vs 32.67±2.62) , scratch healing rate (97.58±1.04 vs 42.33±2.56) , migration rate (94.28±2.33 vs 39.36±2.51) and Ki67, Cyclin D1, Vimentin protein levels, increase E-cadherin protein levels.Conclusion:UTMD-mediated si-G3BP1 can inhibit the proliferation and migration of human liver cancer HepG2 cells.
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Objective@#To investigate the effects of modified acidic fibroblast growth factor (MaFGF) mediated by nanoliposomes combined with ultrasound-targeted microbubble destruction (UTMD) on left ventricular systolic function in early diabetes mellitus(DM) rats.@*Methods@#The nanoliposomes containing MaFGF(MaFGF-nlip) were prepared by reverse phase evaporation method. Among 60 male Sprague Dawley (SD) rats, 50 rats were randomly selected and were induced to be DM models by streptozotocin(STZ) through intraperitoneal injecting, the other 10 rats as control group. Then DM rats were randomly divided into 4 groups: DM model group, MaFGF solution group, MaFGF-nlip group and MaFGF-nlip+ UTMD group. After the successful induction of DM model, the intervention was performed twice a week.After 12 weeks of intervention, all rats underwent conventional echocardiography and velocity vector imaging (VVI). Left ventricular ejection fraction (LVEF) and left ventricular fraction shortening(LVFS) were measured by conventional echocardiography. The mean peak systolic radial velocity (Vs), radial strain (Sr) and radial strain rate (SRr) of six walls at the papillary muscle level were measured in left ventricular short-axis view by VVI. At last, myocardial tissue of all rats were stained with Sirius red to evaluate myocardial interstitial fibrosis. The level of myocardial apoptosis was evaluated by TUNEL staining, and the changes of myocardial ultrastructure were observed by transmission electron microscopy.@*Results@#The prepared MaFGF-nlip were more rounded, evenly dispersed, and of good stability and high encapsulation efficiency. Twelve weeks later after intervention, LVEF, LVFS, Vs, Sr and SRr in the DM model group were significantly lower than those in the control group (all P<0.05). LVEF, LVFS, Vs, Sr and SRr in the MaFGF-nlip+ UTMD group were significantly higher than those of the DM model group and other intervention groups (all P<0.05). The results of Sirius red staining and Tunel staining showed that CVF and AI in the DM model group were significantly higher than those in the control group (all P<0.05). For MaFGF-nlip+ UTMD group, CVF and AI were significantly decreased compared with the DM model group and other intervention groups(all P<0.05). According to the results of transmission electron microscopy, compared with the DM model group, the improvement of myocardial ultrastructure was the most obvious in the MaFGF-nlip+ UTMD group.@*Conclusions@#MaFGF delivered by using nanoliposomes combined with UTMD can improve the left ventricular systolic function in diabetic rats by inhibiting the myocardium cardiac fibrosis and reducing the cardiomyocyte apoptosis.
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Ultrasound contrast agent microbubbles combined with low frequency ultrasound named as low-frequency ultrasound-targeted microbubble destruction technology has become an effective and non-invasive anti-tumor therapy for deep tumors.It can enhance the efficacies of chemotherapy,gene therapy,immunotherapy,and anti-angiogenic therapy by improving cell membrane permeability and destroying tumor neovasculature.It can be applied to sonodynamic therapy and realize multimodal synergistic therapy on the basis of nanoparticles,which increases the anti-tumor efficiency and offers a promising target therapy for tumors.
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Humans , Contrast Media , Genetic Therapy , Microbubbles , Neoplasms , UltrasonographyABSTRACT
Objective To explore the application value of IL-8 monoclonal antibody microbubble combined with ultrsound targeted microbubble destruction ( UTMD) on alleviating myocardial ischemia reperfusion/injury ( MIRI) in rabbits .Methods Forty-two rabbits were randomly divided into closed chest group ( n =7) ,open chest control group ( n = 7) and ischemia-reperfusion ( I/R) group ( n = 28) .I/R group were randomly divided into 30 min reperfusion group( n =7) ,60 min reperfusion group( n =7) ,120 min reperfusion group ( n = 7 ) and 180 min reperfusion group ( n = 7 ) .All rabbits were examined by electrocardiogram , echocardiography and HE staining after MIRI . Targeted myocardial contrast echocardiography ( MCE) was performed and ELISA was used to detect IL-8 content in rabbit myocardium before and after UTMD . Results Electrocardiogram and wall motion returned to normal at 60 min after reperfusion .Targeted MCE showed that with the prolongation of reperfusion after I/R ,the video intensity of myocardium in reperfusion area increased gradually , reaching its peak at 120 min and 180 min after reperfusion .After UTMD ,the video intensity decreased ,and the change rate of video intensity in 30 min reperfusion group was higher than those in other reperfusion groups(all P<0 .05) .The content of IL-8 and its neutralization rate in the ELISA results were consistent with the video intensity and rate of change of targeted MCE .HE staining and scanning electron microscopy showed that myocardial injury was found in I/R group .With the prolongation of reperfusion time ,the degree of myocardial injury was gradually aggravated ,and the injury was alleviated after irradiation .Conclusions IL-8 monoclonal antibody combined with UTMD has the advantages of non-invasive and highly effective in alleviating MIRI .It provides a new way to treat MIRI .
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Objective To investigate the therapeutic effect and mechanism of non-mitogenic acid fibroblast growth factor 1( NMFGF1) on diabetic cardiomyopathy ( DCM) by using PEG-modified nano-liposomes combined with ultrasound-targeted microbubble destruction technique ( UTMD ) . Methods The NMFGF1 loaded PEG-modified nano-liposomes were prepared by a water-in-water emulsion method and their quality inspections were also investigated. Type 1 diabetes animal model was induced by intraperitoneal injection of streptozotocin ( 70 mg/kg) in male SD rats. The diabetic rats were raised twelve weeks after the diabetes model was established and DCM rats were selected by ultrasonic heart function examination. After two weeks of intervention, all rats were kept for another two weeks and then underwent transthoracic echocardiography examination. The rats were sacrificed and myocardial tissue was obtained to quantify myocardial collagen fraction ( CVF ) and cardiac myocyte apoptotic index by Sirius red staining and TUNEL staining. Results NMFGF1-loaded PEG-nano-liposomes showed a good morphology and 90.3％± 1.4％ NMFGF1 encapsulation efficiency. Compared with DCM group, NMFGF1group, and NMFGF1-PEG-nano-liposomes group, NMFGF1-loaded PEG-nano-liposome plus UTMD group showed increased left ventricular end diastolic diameter (LVIDd) [(7.36±0.42) vs (5.75±0.24), (6.64±0.27), (6.72±0.24)mm, all P<0.05]and leftventricularfractionshortening(LVFS) [(50±3) vs (33±2), (44±5), (43±3)mm, all P<0.05], and decreased left ventricular posterior wall thickness (LVPW) [(1.65±0.07) vs (1.89±0.08), (1.73±0.11), (1.73 ±0.07) mm, all P<0.05], with decreased CVF and apoptotic index(all P<0.05). Conclusion PEG-nano-liposomes combining with UTMD technique has a greater translational potential in the delivery of NMFGF1 for the treatment of DCM by attenuating oxidative stress-induced injury and may provide a promising strategy for treating diabetes cardiomyopathy.
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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 ( 3878 .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 .
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Objective To silence the expression of Itch gene of T-lymphocytes by ultrasound-targeted microbubble destruction (UTMD) and to investigate the cytotoxicity of transfected T lymphocytes against LA795 lung neoplasms cells in vitro. Methods T lymphocyte were isolated by magnetic bead,and an targeted shRNA to silence Itch gene of T lymphocytes was established.48 hours after transfection by UTMD,the transfection efficiency was detected and analyzed by fluorescence microscopy and flow cytometry;the expression of Itch protein was measured with Western blot;72 hours after transfection,the secretion of IL-2 and IFN-γ in the cell supernatant was measured by enzyme-linked immunosorbent assay (ELISA). The cytotoxicity activity changes against LA795 lung neoplasms cells was compared between transfected T lymphocytes and negative control or simplex T lymphocytes in vitro. Results The transfection rate to silence Itch gene of T lymphocytes by UTMD was 52.3%±3.8%. At 48 h after transfection,the Itch gene expression can be effectively suppressed by UTMD. Seventy-two hours after transfection,the secretion level of cytokines including IL-2 and IFN-γ,was significantly increased in the group of targeted shRNA to silence Itch gene of T lymphocytes by UTMD(P<0.05) and transfected T lymphocyte also showed more efficient killing ability against LA795 lung neoplasms cells than negative control or blank group at E : T of 10 : 1,20 : 1 and 40 : 1 (P<0.05). Conclusions Silencing the expression of Itch by UTMD can significantly promote immune activity of T lymphocyte,enhance the cytotoxicity activity of T lymphocyte against LA795 lung neoplasms cells in vitro.
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Objective To investigate the advanced preventive effect of acid fibroblast growth factor (aFGF) on diabetic cardiomyopathy(DCM) by using heparin-modified nano-liposomes combined with ultrasoundtargeted microbubble destruction technique (UTMD).Methods aFGF-loaded nano-liposomes (aFGF-lips) were prepared by lyophilization technique.Type Ⅰ diabetes model was induced by intraperitoneal injection of streptozotocin (STZ,70 mg/kg) in male SD rats.Before and twelve weeks after intervention,all rats underwent the transthoracic echocardiography.The segmental mean peak systolic radial velocity (Vs),systolic circumferential strain (Sc),and systolic circumferential strain rate (SRc) were measured.The expression of aFGF in DCM rats was detected by western blot.The rats were sacrificed and myocardial tissue were stained with masson staining and Tunel staining to quantify myocardial collagen fraction(CVF) and cardiac myocyte apoptosis index(AI).Results aFGF-lips showed good morphology and aFGF encapsulation efficiency (89.4±1.2)％ with high stability.From the animal experiments,the echocardiographic indexes including Vs,Sc and SRc had significantly improvements over DM group (P＜0.05) and all other treatment group (P＜0.05).The Masson's trichrome staining demonstrated that CVF was significantly higher in DM group than in the control group and was significantly lower in the aFGF-loaded nano-liposome+UTMD group than other groups(all P＜0.05).The TUNEL results showed that AI was significantly higher in DM group than in the control group and was significantly lower in aFGF-loaded nano-liposome +UTMD group than other groups (all P＜0.05).Conclusion aFGF nano-liposome combining with UTMD technique can improve the functions and pathologies of the hearts in type 1 diabetes mellitus model,which might provide a novel technique for aFGF in DCM prevention.
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In recent years, research about microRNA has been increasing.How to safely and effectively introduce microRNA into target cells and target tissues is the key to successful treatment of diseases using microRNA.Many studies have reported that ultrasound-targeted microbubble destruction technology (UTMD) mediated gene transfection has the advantages of safety, targeting and high transfection efficiency.The research of UTMD mediated microRNA transfection has also increased year by year.The mechanism of UTMD mediated microRNA transfection and the application in the treatment of diseases such as cancer and cardiovascular system are reviewed in this paper.
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Objective To improve the canines myocardial infarction curative effect by using ultrasound targeted microbubble destruction (UTMD) combining with nuclear localization signal(NLS) peptide to increase hAng-1 gene transfection efficiency.Methods Forty-six canines were randomly divided into 4 groups (n=10 in each group) after the models of myocardial infarction were prepared.Group A were untreated control;Group B were transfected with hAng-1;Group C were transfected with UTMD+hAng-1;Group D were transfected with UTMD+NLS nuclear localization signal+hAng-1.The therapeutic agents were intravenously injected at one week after myocardial infarction in each group,and the ultrasound were irradiated at the precardium in group C and D.①Echocardiography was used before and at one week after myocardial infarction and 28 days after gene transfection.Two-dimensional echocardiography was used to detect cardiac function,the left ventricular ejection fraction,the left ventricular wall motion and the myocardial contrast echocardiography were used to detect myocardial perfusion of all canines in the four groups.②On twenty-eight days after gene transfection,mRNA and Western Blot were used to detect the expression of hAng-1.Immunohistochemistry was used to detect capillary density of peri-infarct area and microvessel density (MVD).Masson′s trichromatic staining and gross specimen were used to evaluate the degree and the area of myocardial fibrosis.The gene transfection efficiency and the curative effect of all the four groups were evaluated and compared.Results ①Before myocardial infarction,in four groups canine ventricular wall motion and cardiac function were normal,and myocardial filling defect was not showed by myocardial contrast echocardiography.At one week after myocardial infarction,the left ventricular anterior and interval walls motion and the left ventricular ejection fraction in the four groups were significantly decreased.Myocardial contrast echocardiography showed anterior and interval walls myocardial filling defect.There was no significant difference among the four groups(P>0.05);On 28 days after gene transfection the left ventricular ejection fraction in the four group were increased in an order of group A,B,C,D,there was significant difference when comparing group C and D with other groups separately(P<0.05).Myocardial contrast echocardiography showed much contrast filling in the infarction and surrounding area in group D,a little contrast filling in group C and filling defect in group A and group B,there was significant difference when comparing group C and D with other groups separately(P<0.05).②RT-PCR and Western Blot showed the hAng-1 mRNA and protein expression in group D were higher than those in the other group.There was significant difference when comparing group C and group D with other groups separately(P<0.05).③Immunofluorescence showed the capillary densities were(4.7±1.6)/mm2,(11.2±2.8)/mm2,(70.0±6.4)/mm2 and (85.3±7.0)/mm2 in group A,group B,group C and group D.The differences were statistically significant compared group C and group D with other groups (P<0.05).④Masson′s trichromatic staining and cardiac gross specimen showed that the degree and area of myocardial fibrosis were gradually reduced in an order of group A,B,C,D.Conclusions UTMD and NLS peptide could effectively transfect hAng-1 gene and it provided a novel strategy of gene treatment for ischemic heart disease.
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Objective@#The therapeutic effect of acid fibroblast growth factor 1(FGF1) on rats with diabetic cardiomyopathy (DCM) was evaluated by using nano-liposomes combined with ultrasound-targeted microbubble destruction technique (UTMD).@*Methods@#The FGF1-loaded nano-liposomes were prepared by water-in-water emulsion method combined with lyophilization technique.TypeⅠdiabetes model was induced by intraperitoneal injection of streptozotocin (STZ, 70 mg/kg) in 60 male SD rats.Sixteen weeks later, diabetic rats were randomly divided into: placebo group (saline treatment), FGF1 group, FGF1-loaded nano-liposomes group, and FGF1-loaded nano-liposomes plus UTMD group (n=15 each). After two weeks of intervention followed by 2 weeks intervention stop, all rats underwent cardiac catheterization, and the left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP) and the maximal increase/decrease rate of left ventricular pressure (LV±dp/dtmax) were measured.Then, the rats were sacrificed and myocardial tissue were obtained for Masson trichrome staining, TUNEL apoptotic staining and CD31 immunohistochemistry staining to quantify myocardial collagen fraction (CVF), cardiac myocyte apoptotic index and myocardial microvascular density (MVD).@*Results@#(1)Scanning electron microscope results revealed good morphology and FGF1 encapsulation efficiency (84.3±2.8)% with high stability and dispensability of FGF1 loaded nano-liposomes.(2)The hemodynamic evaluation showed that LVESP, LV + dp/dtmax and LV -dp/dtmax were all significantly higher, while LVEDP was significantly lower in the FGF1-loaded nano-liposome+ UTMD group than in DCM group, FGF1 solution group, and FGF1 nano-liposome group(all P<0.05). (3)The Masson trichrome staining demonstrated that CVF was significantly higher in all DCM groups than in control group and was significantly lower in the FGF1-loaded nano-liposome+ UTMD group than in DCM group, FGF1 solution group, and FGF1 nano-liposome group (all P<0.05). (4)The CD31 immunohistochemical staining results showed that MVD was significantly lower in all DCM groups than in control group and was significantly higher in the FGF1-loaded nano-liposome+ UTMD group than in DCM group, FGF1 solution group, and FGF1 nano-liposome group (all P<0.05). (5)The TUNEL results showed that apoptotic index was significantly higher in all DCM groups than in control group and was significantly lower in the FGF1-loaded nano-liposome + UTMD group than in DCM group, FGF1 solution group, and FGF1 nano-liposome group (all P<0.05).@*Conclusion@#FGF1 nano-liposomes combining with UTMD technique can significantly improve cardiac functions and attenuate myocardial CVF and apoptosis and enhance myocardial MVD in DCM rats.
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Objective To evaluate gene transfection in liver , lung and kidney by ultrasound , microbubble and recombinant adenovirus mediated exogenous stromal cell derived factor-1α ( SDF-1α) gene transfer to the heart in rats with acute myocardial infarction( AMI) . Methods Forty AMI SD rats were randomly divided into control and experimental groups:myocardial infarction + ultrasound irradiation group (M+ U/control group , n = 10) ,and 3 experimental subgroups on the basis of pAd-EGFP/SDF-1α ( The biotin recombinant adenovirus expressing enhanced green fluorescent protein and SDF-1α) . Genes transfection length of time:1 day ,2 days and 3 days of transfection ( M +S1+U ,M+S2+U and M+S3+U group) . The expression of EGFP in liver ,lung and kidney were detected by laser scanning confocal microscopy at 7 days after administration . Results There was a little expression of EGFP in the liver ,lung and kidney in the drug delivery group and no expression in the control group .The differences in the expression of EGFP between all the gene delivery groups and the control group were statistically significant ( P <0 .05) . With the increase of the number of medication days , the target gene transfection increased slightly ,but there was no significant difference among the different drug delivery groups . Conclusions When the ultrasound ,microbubble and recombinant adenovirus mediated exogenous SDF-1αgene transfer to the heart in AMI rats ,liver ,lung and kidney tissues will also be transfected . However ,with the increasing of the days of administration , the transfection of target gene in non-target tissue produces only a slight accumulation . The transfection area of target gene in non-target tissue is not linear correlated with the days of administration .