Ultrasound-targeted Microbubble Destruction Group Research Articles

Ultrasound-targeted microbubble destruction rapidly improves left ventricular function in rats with ischemic cardiac dysfunction

BackgroundPrevious studies have demonstrated the efficacy of ultrasound-targeted microbubble destruction (UTMD) in the treatment of ischemic heart failure (HF). The purpose of this study was to explore the mechanism by which UTMD improves ischemic HF. MethodsAn ischemic heart failure model was established using Sprague-Dawley rats. Rats were randomly divided into 7 groups: sham group, HF group, HF + MB group, HF + ultrasound (US) group, HF + UTMD group, HF + UTMD+LY294002 group, and HF + LY294002 group. Serum BNP level and echocardiographic parameters were measured to evaluate cardiac function. PI3K/Akt/eNOS signaling pathway protein levels were detected by immunohistochemistry (IHC) and western blotting. The concentrations of nitrous oxide (NO) and ATP were detected by ELISA, and hematoxylin and eosin (HE) staining was used to evaluate myocardial tissue. ResultsUTMD rapidly improved ejection fraction (EF) (HF: 37.16 ± 1.21% vs. HF + UTMD: 46.31 ± 3.00%, P < 0.01) and fractional shortening (FS) (HF: 18.53 ± 0.58% vs. HF + UTMD: 24.05 ± 1.84%, P < 0.01) in rats with ischemic HF. UTMD activated the PI3K/AKT/eNOS signaling pathway (HF vs. HF + UTMD, P < 0.01) and promoted the release of NO and ATP (HF vs. HF + UTMD, both, P < 0.05). Inhibition of the PI3K/AKT/eNOS signaling pathway by LY294002 worsened EF (HF: 37.16 ± 1.21% vs. HF + LY294002: 32.73 ± 3.05%, P < 0.05), and the release of NO and ATP by UTMD (HF + UTMD vs. HF + UTMD+LY294002, P < 0.05). ConclusionsUTMD can rapidly improve cardiac function in ischemic HF by activating the PI3K/Akt/eNOS signaling pathway and promoting the release of NO and ATP.

Amplification of Plasma MicroRNAs for Non-invasive Early Detection of Acute Rejection after Heart Transplantation With Ultrasound-Targeted Microbubble Destruction.

Acute rejection (AR) screening has always been the focus of patient management in the first several years after heart transplantation (HT). As potential biomarkers for the non-invasive diagnosis of AR, microRNAs (miRNAs) are limited by their low abundance and complex origin. Ultrasound-targeted microbubble destruction (UTMD) technique could temporarily alter vascular permeability through cavitation. We hypothesized that increasing the permeability of myocardial vessels might enhance the abundance of circulating AR-related miRNAs, thus enabling the non-invasive monitoring of AR. The Evans blue assay was applied to determine efficient UTMD parameters. Blood biochemistry and echocardiographic indicators were used to ensure the safety of the UTMD. AR of the HT model was constructed using Brown-Norway and Lewis rats. Grafted hearts were sonicated with UTMD on postoperative day (POD) 3. The polymerase chain reaction was used to identify upregulated miRNA biomarkers in graft tissues and their relative amounts in the blood. Amounts of six kinds of plasma miRNA, including miR-142-3p, miR-181a-5p, miR-326-3p, miR-182, miR-155-5p and miR-223-3p, were 10.89 ± 1.36, 13.54 ± 2.15, 9.84 ± 0.70, 8.55 ± 2.00, 12.50 ± 3.96 and 11.02 ± 3.47 times higher in the UTMD group than those in the control group on POD 3. Plasma miRNA abundance in the allograft group without UTMD did not differ from that in the isograft group on POD 3. After FK506 treatment, no miRNAs increased in the plasma after UTMD. UTMD can promote the transfer of AR-related miRNAs from grafted heart tissue to the blood, allowing non-invasive early detection of AR.

Ultra-small micelles together with UTMD enhanced the therapeutic effect of docetaxel on Glioblastoma

Owing to the difficult-to-penetrate blood-brain barrier (BBB), glioblastoma (GBM) doesn't respond well to the current chemical therapeutics. In this study, ultra-small micelles (NMs) self-assembled by RRR-a-tocopheryl succinate-grafted-ε-polylysine conjugate (VES-g-ε-PLL) as the delivery vehicle of chemical therapeutics in conjunction with ultrasound-targeted microbubble destruction (UTMD) to surmount BBB and treat GBM. Docetaxel (DTX) as a hydrophobic model drug was incorporated into NMs. DTX-loaded micelles (DTX-NMs) with 3.08% of drug loading exhibited a hydrodynamic diameter (33.2 nm) and positive Zeta potential (16.9 mV), having a remarkable tumor-permeating capacity. Furthermore, DTX-NMs presented good stability in physiologic condition. The sustained- release profile of DTX-NMs was also displayed by dynamic dialysis. Treatment of DTX-NMs together with UTMD led to more pronounced apoptosis of C6 tumor cells than DTX-NMs alone. Moreover, compared with the DTX solution or DTX-NMs alone, the combination of DTX-NMs with UTMD had a stronger inhibitory effect on tumor growth for GBM-bearing rats. The median survival period of GBM-bearing rats was extended to 75 days in the DTX-NMs+UTMD group from under 25 days in the control group. The invasive growth of glioblastoma was largely inhibited by the combination of DTX-NMs with UTMD, which was demonstrated by staining of Ki67, caspase-3, and CD31, together with TUNEL assay. In conclusion, the combination of ultra-small micelles (NMs) with UTMD may be a promising strategy to overcome the limitations of the first-line chemotherapeutics against GBM.

Delivery of astragalus polysaccharide by ultrasound microbubbles attenuate doxorubicin-induced cardiomyopathy in rodent animals

ABSTRACT The aim of this study was to investigate the cardioprotective effects and probable mechanism of ultrasound-targeted microbubble destruction (UTMD) combined with astragalus polysaccharide (APS) on diabetic cardiomyopathy (DCM) model rats. The DCM rats with diabetes and cardiomyopathy were induced via chronic treatment of doxorubicin and then randomly divided into the (1) DCM model group; (2) APS microbubble group; (3) UTMDgroup; and (4) APS microbubbles combined with UTMD group. After 4-week intervention, the fasting blood glucose levels, body weight, %HbA1c level and glucose tolerance of DCM rats received combination therapy were significantly improved as compared with those of UTMD or saline-treated ones. Moreover, the heart/body weight ratio, and myocardial contractility were all improved after receiving combination therapy groups compared with others. In addition, significantly upregulated activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) as well as significantly downregulated malondialdehyde (MDA) levels were all observed in the ones received combined treatment compared to others. Furthermore, the lipid accumulation and the expression levels of inflammatory factors were all significantly down-regulated in those ones received combination therapy compared with others (all P < 0.05). Further pathological analysis demonstrated that combination therapy effectively ameliorated fibrosis and myocardial morphological changes of DCM rats via activating the upregulation of AMPK and PPAR-γ signaling pathway, and inhibiting NF-κB activity in myocardial tissues of DCM rats. In conclusion, APS microbubbles combined with UTMD effectively protect the myocardial injury of DCM rats via activating AMPK signaling pathway to alleviate inflammation response, fibrosis and oxidative stress in myocardial tissues.

Ultrasound-Targeted Microbubble Destruction Enhances Inhibitory Effect of Apatinib on Angiogenesis in Triple Negative Breast Carcinoma Xenografts.

Ultrasound-targeted microbubble destruction (UTMD) has been proven as an effective technique to assist drugs to cross the vascular wall and cell membrane. This study was aimed at evaluating the synergistic antiangiogenic and growth-inhibiting effects of apatinib (APA) and UTMD on the triple negative breast cancer (TNBC). The TNBC xenograft model was established in nude mice (n = 40) which were then randomly divided into the APA plus UTMD (APA-U) group, UTMD group, APA group, and model control (M) group (n = 10 per group). Corresponding treatment was done once daily for 14 consecutive days. The general condition and body weight of tumor-bearing nude mice were monitored. Routine blood test and detection of liver and kidney function were done after treatments. The tumor size and microcirculation were examined by two-dimensional ultrasonography (2DUS) and contrast-enhanced ultrasonography (CEUS), respectively. Then, the tumor tissues were harvested for the detection of vascular endothelial growth factor (VEGF) by immunohistochemistry and for CD31-PAS double staining to assess microvessel density (MVD) and heterogeneous vascular positivity rate. After treatments, the tumor growth and angiogenesis were significantly inhibited in the APA group and the APA-U group, and these effects were more obvious in the APA-U group. The tumor volume, CEUS parameters, VEGF expression, and MVD in the APA-U group were significantly lower than those in the APA group (P < 0.05), while there were no marked differences in the heterogeneous vascular positivity rate, body weight, and blood parameters between the two groups (P > 0.05). In the UTMD group, the tumor growth and angiogenesis were not significantly inhibited, and all the parameters were similar to those in the M group (P > 0.05). During the experiment, all mice survived and generally had good condition. In conclusion, APA combined with UTMD may exert synergistic antiangiogenic and growth-inhibiting effects on the TNBC and not increase the heterogeneous vasculature and the severity of APA-related systemic side effects.

Therapeutic Angiogenesis for Ovarian Transplantation through Ultrasound-Targeted Microbubble Destruction

Timely angiogenesis and effective microcirculation perfusion are essential for the survival and functional recovery of transplanted ovaries. Ultrasound-targeted microbubble destruction (UTMD) can lead to angiogenesis and increase flow perfusion by causing transient inflammation. The purpose of this study was to evaluate the effects of UTMD on transplanted ovarian revascularization and survival. In vitro, for the criteria of cell viability and tube formation capability, the optimal exposure parameters were determined to be a microbubble concentration of 1 × 108/mL, mechanical index of 1 and exposure time of 30 s. After ovarian transplantation, 40 female Sprague Dawley rats were divided into four groups: transplantation alone, ultrasound alone, microbubbles alone and ultrasound and microbubbles (UTMD). At 7 d after transplantation, ovarian perfusion was assessed using qualitative and quantitative methods. The effect of angiogenesis was assessed by contrast-enhanced ultrasound, laser Doppler perfusion imaging and histologic analysis. The results, in which ovarian perfusion was highest in the UTMD group, suggest that UTMD can effectively improve ovarian perfusion. Compared with the other three groups, the number of follicles, microvascular density and rate of Ki-67-positive cells increased significantly in the UTMD group, while apoptosis decreased significantly (p < 0.05). The study indicates that UTMD promoted ovarian re-vascularization after ovarian transplantation and maintained follicular reserve.

Ultrasound-targeted microbubble destruction-mediated Ang1 gene transfection improves left ventricular structural and sympathetic nerve remodeling in canines with myocardial infarction.

BackgroundThe present study aimed to determine whether ultrasound-targeted microbubble destruction (UTMD)-mediated angiopoietin 1 (Ang1) gene transfection can improve angiogenesis and potentially reverse left ventricular (LV) structural and sympathetic nerve remodeling in canines with myocardial infarction (MI).MethodsThirty dogs were randomly divided into groups (n=10/group) as follows: the MI group (MI dogs without UTMD treatment), the UTMD group (MI dogs with UTMD-mediated negative control plasmid treatment), and the UTMD-Ang1 group (MI dogs with UTMD-mediated Ang1 plasmid treatment). LV dimensions, systolic function, and synchrony were used to reflect the structural remodeling. The density of tyrosine hydroxylase (TH)- and growth-associated protein 43 (GAP43)-positive nerve fibers were calculated to assess the sympathetic nerve remodeling.ResultsOne month after treatment, the UTMD-Ang1 group showed lower LV end-diastolic dimension (LVEDD: 31.2±2.3 mm) and higher LV ejection fraction (LVEF: 44.6%±4.3%) than the MI group (LVEDD: 34.5±2.2 mm, t=2.282, P=0.014; LVEF: 37.3%±3.1%, t=3.718, P=0.003) and the UTMD group (LVEDD: 34.1±2.8 mm, t=2.264, P=0.040; LVEF: 39.3%±4.5%, t=2.408, P=0.030). LV synchrony was higher in the UTMD-Ang1 group compared with the MI group by 2-dimensional speckle-tracking echocardiography. Angiogenic density was higher in the UTMD group than the MI group but was highest in the UTMD-Ang1 group according to immunohistochemistry of CD31 and α-smooth muscle actin staining. The density of TH- and GAP43-positive nerve fibers were decreased in the UTMD-Ang1 group (TH: 1,928.2±376.6 μm2/mm2; GAP43: 2,090.8±329.2 μm2/mm2) compared with the MI group (TH: 2916.5±558.4 μm2/mm2, t=4.069, P=0.001; GAP43: 3,275.4±548.6 μm2/mm2, t=5.153, P=0.000) and the UTMD group (TH: 2,552.7±408.1 μm2/mm2, t=3.181, P=0.007; GAP43: 2,630.5±419.3 μm2/mm2, t=2.863, P=0.013). The relative Ang1 and sarcoplasmic reticulum Ca2+-ATPase 2a protein levels were significantly higher in the UTMD-Ang1 group than the UTMD and MI groups by Western blot, while the phospholamban levels exhibited the opposite trend. Plasma norepinephrine and N-terminal pro-B-type-natriuretic peptide were significantly reduced in the UTMD-Ang1 group from day 1 to 1 month after MI.ConclusionsUTMD-mediated Ang1 transfection can promote angiogenesis, reverse LV structural and sympathetic nerve remodeling, and improve LV synchrony after MI.

Targeted galectin-7 inhibition with ultrasound microbubble targeted gene therapy as a sole therapy to prevent acute rejection following heart transplantation in a Rodent model

BackgroundDespite significant advances in transplantation, acute cellular rejection (AR) remains a major obstacle that is most prevalent in the first months post heart transplantation (HT). Current treatments require high doses of immunosuppressive drugs followed by maintenance therapies that have systemic side effects including early infection. In this study, we attempted to prevent AR with a myocardial-targeted galectin-7-siRNA delivery method using cationic microbubbles (CMBs) combined with ultrasound targeted microbubble destruction (UTMD) to create local immunosuppression in a rat abdominal heterotopic heart transplantation acute rejection model. Methods and resultsGalectin-7-siRNA (siGal-7) bound to CMBs were synthesized and effective ultrasound-targeted delivery of siGal-7 into target cells confirmed in vitro. Based on these observations, three transplant rat models were tested：①isograft (ISO); ② Allograft (ALLO) +UTMD; and ③ALLO + PBS. UTMD treatments were administered at 1, 3, 5, 7 days after HT. Galectin 7 expression was reduced by 50% compared to ALLO + PBS (p < 0.005), and this was associated with significant reductions in both galectin 7 and Interleukin-2 protein levels (p < 0.001). The ALLO + UTMD group had Grade II or less inflammatory infiltration and myocyte damage in 11/12 rats using International Society For Heart and Lung Transplantation grading, compared to 0/12 rats with this grading in the ALLO + PBS group at 10 days post HT (p < 0.001). ConclusionsUltrasound-targeted galectin-7-siRNA knockdown with UTMD can prevent acute cellular rejection in the early period after allograft heart transplantation without the need for systemic immunosuppression. Key wordsMicrobubble, Acute Rejection, Heart Transplantation, Galectin-7, RNA.

Effects of modified acidic fibroblast growth factor mediated by nanoliposomes combined with ultrasound-targeted microbubble destruction on left ventricular systolic function in diabetic rats

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. Key words: Ultrasound-targeted microbubble destruction; Diabetic cardiomyopathy; Ventricular function, left; Nanoliposomes; Modified acidic fibroblast growth factor

Assessment of the Preventive Effect Against Diabetic Cardiomyopathy of FGF1-Loaded Nanoliposomes Combined With Microbubble Cavitation by Ultrasound

Acidic fibroblast growth factor (FGF1) has great potential in preventing diabetic cardiomyopathy. This study aimed to evaluate the preventive effect of FGF1-loaded nanoliposomes (FGF1-nlip) combined with ultrasound-targeted microbubble destruction (UTMD) on diabetic cardiomyopathy (DCM) using ultrasound examination. Nanoliposomes encapsulating FGF1 were prepared by reverse phase evaporation. DM model rats were established by intraperitoneal injection of streptozotocin (STZ), and different forms of FGF1 (FGF1 solution, FGF1-nlip, and FGF1-nlip+UTMD) were used for a 12-week intervention. According to the transthoracic echocardiography and velocity vector imaging (VVI) indexes, the LVEF, LVFS, and VVI indexes (Vs, Sr, SRr) in the FGF1-nlip+UTMD group were significantly higher than those in the DM model group and other FGF1 intervention groups. From the real-time myocardial contrast echocardiography (RT-MCE) indexes, the FGF1-nlip+UTMD group A and A×β showed significant differences from the DM model group and other FGF1 intervention groups. Cardiac catheter hemodynamic testing, CD31 immunohistochemical staining, and electron microscopy also confirmed the same conclusion. These results confirmed that the abnormalities, including myocardial dysfunction and perfusion impairment, could be suppressed to different extents by the twice weekly FGF1 treatments for 12 consecutive weeks (free FGF1, FGF1-nlip, and FGF1-nlip+UTMD), with the strongest improvements observed in the FGF1-nlip+UTMD group. In conclusion, the VVI and RT-MCE techniques can detect left ventricular systolic function and perfusion changes in DM rats, providing a more effective experimental basis for the early detection and treatment evaluation of DCM, which is of great significance for the prevention of DCM.

Combined treatment with ultrasound-targeted microbubble destruction technique and NM-aFGF-loaded PEG-nanoliposomes protects against diabetic cardiomyopathy-induced oxidative stress by activating the AKT/GSK-3β1/Nrf-2 pathway

The present study sought to investigate the effect of non-mitogenic acidic fibroblast growth factor (NM-aFGF) loaded PEGylated nanoliposomes (NM-aFGF-PEG-lips) combined with the ultrasound-targeted microbubble destruction (UTMD) technique on modulating diabetic cardiomyopathy (DCM)and the mechanism involved. Animal studies showed that the diabetes mellitus (DM) group exhibited typical myocardial structural and functional changes of DCM. The indexes from the transthoracic echocardiography showed that the left ventricular function in the NM-aFGF-PEG-lips + UTMD group was significantly improved compared with the DM group. Histopathological observation further confirmed that the cardiomyocyte structural abnormalities and mitochondria ultrastructural changes were also significantly improved in the NM-aFGF-PEG-lips + UTMD group compared with DM group. The cardiac volume fraction (CVF) and apoptosis index in the NM-aFGF-PEG-lips + UTMD group decreased to 10.31 ± 0.76% and 2.16 ± 0.34, respectively, compared with those in the DM group (CVF = 21.4 ± 2.32, apoptosis index = 11.51 ± 1.24%). Moreover, we also found significantly increased superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) activity as well as clearly decreased lipid hydroperoxide levels and malondialdehyde (MDA) activity in the NM-aFGF-PEG-lips + UTMD group compared with those in the DM group (p < .05). Western blot analysis further revealed the highest level of NM-aFGF, p-AKT, p-GSK-3β1, Nrf-2, SOD2 and NQO1 in the NM-aFGF-PEG-lips + UTMD group. This study confirmed using PEGylated nanoliposomes combined with the UTMD technique can effectively deliver NM-aFGF to the cardiac tissue of diabetic rats. The NM-aFGF can then inhibit myocardial oxidative stress damage due to DM by activating the AKT/GSK/Nrf-2 signaling pathway, which ultimately improved the myocardial structural and functional lesions in diabetic rats.

Focused ultrasound-mediated microbubble destruction for glioblastoma treatment

Glioblastoma is the most common primary brain tumor with a poor prognosis despite advances in various treatment modalities, such as radiation therapy (RT). This study compared the tumor growth inhibition effects of focused ultrasound-targeted microbubble destruction (UTMD) therapy with the RT using an orthotopic mouse glioma model. Mice were implanted with GL261 glioblastoma cells and divided into three groups: control group (no treatment); RT group (2 Gy/day, 5 days/week, 3 consecutive weeks); and UTMD group (FUS sonication in the presence of systemically injected microbubbles at the peak negative pressure of 1.5 MPa, frequency of 1.44 MHz, and 2 treatments/week for 3 consecutive weeks). Contrast-enhanced magnetic resonance imaging (MRI) was performed once every four days for measuring the tumor volume. Both UTMD and RT caused significant growth inhibition compared to the control group; however, there was no significant difference between the UTMD and RT groups. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining showed that the number of apoptotic tumor cells in both RT and UTMD groups were significantly higher than the control group without the difference between these two groups. This study suggests that UTMD suppressed glioblastoma tumor growth and this effect was comparable with that achieved by RT.

A Combination of UTMD-Mediated HIF-1α shRNA Transfection and TAE in the Treatment of Hepatic Cancer.

To explore the antitumor effect of hypoxia-inducible factor-1α short hairpin RNA (HIF-1α shRNA) delivered by ultrasound targeted microbubble destruction (UTMD) and transcatheter arterial embolization (TAE) on rats with hepatic cancer. After the models of transplantation hepatoma were established, Wistar rats were randomly divided into 4 groups: Control group, UTMD group, TAE group, and UTMD+TAE group. Contrast-enhanced ultrasound (CEUS) was used to monitor tumor size on day 14 after four different treatments. Western blotting and immunohistochemistry were applied to measure the protein level of HIF-1α and VEGF in the hepatic cancer tissue. In comparison with UTMD+TAE group (21.25±10.68 days), the mean survival time was noticeably shorter in the Control group and TAE group (13.02±4.30 days and 15.03±7.32 days) (p<0.05, respectively). There was no statistical difference between UTMD+TAE group and UTMD group of the mean survival time (p>0.05). In addition, our results proved that the tumor sizes in UTMD+TAE group were obviously smaller than those in other groups (p<0.05, respectively). By CEUS, we clearly found that the tumor size was the smallest on day 14 in the UTMD+TAE group. The western blotting and immunohistochemistry results proved that the protein levels of HIF-1α and VEGF in UTMD+TAE group were obviously lower than those in TAE group and Control group on days 7 and 14 (p<0.05, respectively). However, there was no statistical difference between UTMD+TAE group and UTMD group (p>0.05). In this study we tried to explore the antitumor effect through a combination of UTMD-mediated HIF-1α shRNA transfection and TAE on rats with hepatic cancer. Our results showed that UTMD-mediated HIF-1α shRNA transfection and TAE can obviously silence HIF-1α and VEGF expression, thereby successfully inhibiting the growth of the tumor.

The promoting effect of ultrasound targeted microbubble destruction on establishment of type I diabetic nephropathy in rats

Objective To explore the feasibility of the promoting effect of ultrasound targeted microbubble destruction(UTMD) on establishment of type Ⅰ diabetic nephropathy in rats. Methods Forty male SD rats were randomly and equally divided into four groups(n=10): control group(group A), streptozocin group(group B), streptozocin and ultrasound microbubble group(group C) and streptozocin and UTMD group(group D). The fasting blood-glucose (FBG) were tested, the 24 h′s urine were collected and the 24 h′s urine microalbumin(mAlb) were measured and then urinary albumin excretion rate(UAER) were calculated twice a week at fixed time in all groups. When greater than 3-fold increase in UAER compared with controls at the same age and gender, the diabetic nephropathy model in rats was considered to be established successfully, then the change of time and weighed were recorded and rats were killed and collected the blood of left atrial appendage to measure, blood urea nitrogen (BUN), serum creatinine (SCr) and Alanine aminotransferase(ALT). The left kidneys were weighted and then observed glomerular pathological changes under light microscope and detected the expression of CD34 in kidney of rats in each group by immunohistochemical method. Results ①The time of establishing diabetic nephropathy model in group D was obviously shorter than that in group B and C(all P 0.05). ②The pathological changes of diabetic nephropathy appeared in rats of group B, C and D; the expression of CD34 in B, C and D groups were raised. Conclusions UTMD can obviously shorten the molding time of type I diabetic nephropathy rats, which has feasibility. Key words: Microbubbles, target; Diabetes mellitus, type 1; Diabetic nephropathies; Rat

Myocardium-Targeted Transplantation of Phd2 Shrna-Modified Mesenchymal Stem Cells by Ultrasound-Targeted Cationic Microbubble Destruction Improves Cardiac Function in Myocardial Infarction of Rats

Mesenchymal stem cell therapy has been proposed as a promising therapy for acute myocardial infarction (AMI). However, some studies showed little effect of MSCs on AMI. One of the limitations are low homing efficiency of MSCs at the site of injury. We hypothesis that Ultrasound targeted microbubble destruction (UTMD) technique combined with PHD2 shRNA-modified MSCs enhances the homing efficiency of transplanted stem cells and improves cardiac function in myocardial infarction of rats. ① Cationic lipid microbubble was prepared by filming-rehydration and machine vibration. Its concentration, average diameter and zeta potential were measured. The binding capacity with plasmid and contrast-enhanced ultrasound effect was also assessed. ② The plasmid that silence PHD2 gene and carry GFP gene was constructed. Firstly, the cultured MSCs were divided into three groups, including Plasmid, Plasmid + Ultrasound and Plasmid + UTMD to prove that UTMD enhances gene transfection and find optimal transfection parameters by UTMD. Then, to demonstrate that shPHD2-EGFP silence PHD2 gene and induce HIF-1α and angiogenic factor gene, the experimental regimens included Control, EGFP and shPHD2-EGFP; to prove that PHD2 silencing enhances MSCs survival, the MSCs were divided into four groups, including Control, BMSC, BMSC-EGFP and BMSC-shPHD2-EGFP. ③ Rat myocardial infarction models were conducted. Firstly, 30 rats were divided into three groups, including MI, MI-MSCEGFP and MI-MSCshPHD2-EGFP. The MSCs transplantation effect was tested. Then, other 120 rats were divided into four groups, including Control, MI-PBS, MI-BMSCsEGFP and MI-BMSCsshPHD2-EGFP. The echocardiography, histological character and myocardial apoptosis were evaluated. ① The concentration of CMB was (4.12±0.29) × 109/ml. Its average diameter and zeta potential was (1.67±0.32) μm and (29.18±1.36) mV respectively. The saturated DNA loading capacity for 1×108CMB was (3.56±0.31) μg. And enhancement effect in rat heart was good. ② Compared with Plasmid and Plasmid + Ultrasound groups, the gene transfection efficiency was highest. US intensity (1.0W/cm2 ) and exposure time（30s）were relative optimal transfection parameters. The shPHD2-EGFP down-regulated PHD2 and up-regulated HIF-1α after it was delivered into MSCs by UTMD. Meanwhile, the shPHD2-EGFP decreased MSC apoptosis. ③ The number of MSCs for transplantation in MI-Plasmid + UTMD group was higher than MI-Plasmid and MI-Plasmid + ultrasound groups. Four weeks after transfection, compared with the other groups, myocardial apoptosis and scar size at the infarct area reduced in the MI-BMSCsshPHD2-EGFP group (P<0.05). Capillary densities in MI-BMSCsshPHD2-EGFP group were increased and left ventricular function had improved (P<0.05) over other groups. UTMD successfully promoted transfection efficiency of plasmid DNA in vitro and increased marrow stem cell homing in vivo, thus improving the efficacy of therapeutic angiogenesis and myocardial function.

Myocardium-Targeted Transplantation of Phd2 Shrna-Modified Mesenchymal Stem Cells by Ultrasound-Targeted Cationic Microbubble Destruction Improves Cardiac Function in Myocardial Infarction of Rats

Mesenchymal stem cell therapy has been proposed as a promising therapy for acute myocardial infarction (AMI). However, some studies showed little effect of MSCs on AMI. One of the limitations are low homing efficiency of MSCs at the site of injury. We hypothesis that Ultrasound targeted microbubble destruction (UTMD) technique combined with PHD2 shRNA-modified MSCs enhances the homing efficiency of transplanted stem cells and improves cardiac function in myocardial infarction of rats. ①Cationic lipid microbubble was prepared by filming-rehydration and machine vibration. Its concentration, average diameter and zeta potential were measured. The binding capacity with plasmid and contrast-enhanced ultrasound effect was also assessed. ② The plasmid that silence PHD2 gene and carry GFP gene was constructed. Firstly, the cultured MSCs were divided into three groups, including Plasmid, Plasmid + Ultrasound and Plasmid + UTMD to prove that UTMD enhances gene transfection and find optimal transfection parameters by UTMD. Then, to demonstrate that shPHD2-EGFP silence PHD2 gene and induce HIF-1α and angiogenic factor gene, the experimental regimens included Control, EGFP and shPHD2-EGFP; to prove that PHD2 silencing enhances MSCs survival, the MSCs were divided into four groups, including Control, BMSC, BMSC-EGFP and BMSC-shPHD2-EGFP. ③ Rat myocardial infarction models were conducted. Firstly, 30 rats were divided into three groups, including MI, MI-MSCEGFP and MI-MSCshPHD2-EGFP. The MSCs transplantation effect was tested. Then, other 120 rats were divided into four groups, including Control, MI-PBS, MI-BMSCsEGFP and MI-BMSCsshPHD2-EGFP. The echocardiography, histological character and myocardial apoptosis were evaluated. ① The concentration of CMB was (4.12±0.29) × 109/ml. Its average diameter and zeta potential was (1.67±0.32) μm and (29.18±1.36) mV respectively. The saturated DNA loading capacity for 1×108CMB was (3.56±0.31) μg. And enhancement effect in rat heart was good. ② Compared with Plasmid and Plasmid + Ultrasound groups, the gene transfection efficiency was highest. US intensity (1.0W/cm2 ) and exposure time（30s）were relative optimal transfection parameters. The shPHD2-EGFP down-regulated PHD2 and up-regulated HIF-1α after it was delivered into MSCs by UTMD. Meanwhile, the shPHD2-EGFP decreased MSC apoptosis. ③ The number of MSCs for transplantation in MI-Plasmid + UTMD group was higher than MI-Plasmid and MI-Plasmid + ultrasound groups. Four weeks after transfection, compared with the other groups, myocardial apoptosis and scar size at the infarct area reduced in the MI-BMSCsshPHD2-EGFP group (P<0.05). Capillary densities in MI-BMSCsshPHD2-EGFP group were increased and left ventricular function had improved (P<0.05) over other groups. UTMD successfully promoted transfection efficiency of plasmid DNA in vitro and increased marrow stem cell homing in vivo, thus improving the efficacy of therapeutic angiogenesis and myocardial function.

Using basic fibroblast growth factor nanoliposome combined with ultrasound-introduced technology to early intervene the diabetic cardiomyopathy.

Basic fibroblast growth factor (bFGF)-loaded liposome (bFGF-lip) combined with ultrasound-targeted microbubble destruction (UTMD) technique was investigated to prevent diabetic cardiomyopathy (DCM). Cardiac function and myocardial ultrastructure were assessed. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining, immunohistochemistry staining, and Western blot assay were used to investigate the signal pathway underlying the expression of bFGF in DCM treatment. From Mason staining and TUNEL staining, bFGF-lip + UTMD group showed significant differences from the diabetes group and other groups treated with bFGF or bFGF-lip. The diabetes group showed similar results (myocardial capillary density, collagen volume fraction, and cardiac myocyte apoptosis index) to other bFGF treatment groups. Indexes from transthoracic echocardiography and hemodynamic evaluation also proved the same conclusion. These results confirmed that the abnormalities including diastolic dysfunctions, myocardial fibrosis, and metabolic disturbances could be suppressed by the different extents of twice-weekly bFGF treatments for 12 consecutive weeks (free bFGF or bFGF-lip +/− UTMD), with the strongest improvements observed in the bFGF-lip + UTMD group. The group combining bFGF-lip with UTMD demonstrated the highest level of bFGF expression among all the groups. The bFGF activated the PI3K/AKT signal pathway, causing the reduction of myocardial cell apoptosis and increase of microvascular density. This strategy using bFGF-lip and UTMD is a potential strategy in early intervention of DCM in diabetes.

Prevent diabetic cardiomyopathy in diabetic rats by combined therapy of aFGF-loaded nanoparticles and ultrasound-targeted microbubble destruction technique

Acidic fibroblast growth factor (aFGF) has shown the great potential to prevent the structural and functional injuries caused by diabetic cardiomyopathy (DCM). The present study sought to investigate the preclinical performance and mechanism of the combination therapy of aFGF-nanoparticles (aFGF-NP) and ultrasound-targeted microbubble destruction (UTMD) technique for DCM prevention. From Mason staining and TUNEL staining, aFGF-NP+UTMD group showed significant differences from the diabetes group and other groups treated with aFGF or aFGF-NP. The cardiac collagen volume fraction (CVF) and cardiac myocyte apoptosis index in aFGF-NP+UTMD group reduced to 4.15% and 2.31% respectively, compared with those in the diabetes group (20.5% and 11.3% respectively). Myocardial microvascular density (MCD) in aFGF-NP+UTMD group was up to 35n/hpf, much higher than that in the diabetes group (14n/hpf). The diabetes group showed similar results (MCD, CVF and cardiac myocyte apoptosis index) to other aFGF treatment groups (free aFGF±UTMD or aFGF-NP). Indexes from transthoracic echocardiography and hemodynamic evaluation also proved the same conclusion. These results confirmed that the abnormalities including diastolic dysfunctions, myocardial fibrosis and metabolic could be suppressed by the different extents of twice weekly aFGF treatments for 12 consecutive weeks (free aFGF or aFGF-NP±UTMD), with the strongest improvements observed in the aFGF-NP+UTMD group. Western blot and immunohistochemical analyses of heart tissue samples further revealed the high efficiency of heart-targeted delivery and effective cardioprotection with this combination approach. Overall, this study has generated supportive data that are critical for the translation of a promising DCM prevention strategy.

Effects of diagnostic ultrasound-targeted microbubble destruction on the homing ability of bone marrow stromal cells to the kidney parenchyma.

Bone marrow stromal cells (BMSC) transplantation proves successful in treating kidney disease and injury in many studies. However, their reparative capacity is limited by the poor homing ability in vivo, which is decided mainly by the local expression of chemoattractants. Our study explored the mechanical effects of ultrasound targeted microbubble destruction (UTMD) on BMSCs homing ability in treated kidney tissues. Rats were injected with red fluorescent protein (RFP)-labelled BMSCs and sonicated with microbubble-mediated ultrasound. Then, we tested kidney micro-environment changes induced and their influence on stem cell homing ability. The results showed that the mechanical effects of UTMD would increase local and transient levels of chemoattractants (i.e. cytokines, integrins and growth factors) in targeted kidney tissues. Transmission electron microscopy showed that vascular endothelial cell was discontinuous in the UTMD group post-treatment, becoming smooth 72h later. Confocal laser scanning microscopy and RT-PCR showed up to eight times more stem cells in the peritubular regions of experimental kidneys on days 1 and 3 post-treatment compared with the contralateral kidney. These results confirmed that renal micro-environment changes caused by appropriate UTMD may promote BMSC homing ability toward treated kidney tissues without renal toxicity and cell damage. • This experiment showed a feasible strategy in promoting stem cell homing ability. • The treatment uses diagnostic ultrasound during enhancement with IV microbubbles. • A suitable micro-environment was important for targeted stem cell homing and retention. • The method is effective for stem cell homing to kidney diseases. • More work is required with larger animals before potential human trials.

Ultrasound-targeted microbubble destruction-mediated downregulation of CD133 inhibits epithelial-mesenchymal transition, stemness and migratory ability of liver cancer stem cells.

Hepatocellular carcinoma (HCC) is an aggressive disease with a poor outcome due to the high incidence of metastasis. Cancer stem cells (CSCs) have been identified to be responsible for tumor progression and may be generated by epithelial-mesenchymal transition (EMT) characteristics. CD133 is a specific surface marker for liver cancer stem cells (LCSCs), which is also considered as an important functional factor for tumorigenesis and overall survival in HCC. Ultrasound-targeted microbubble destruction (UTMD) has recently been used as a novel, safe and effective gene transfection technology. The aim of the present study was to elucidate the regulatory mechanism of CD133 and EMT in LCSCs and whether the UTMD-based shRNA delivery system facilitated gene delivery in LCSCs. In the present study, CD133+ cells were isolated from the SMMC-7721 HCC cell line and then transfected with shCD133 mediated by UTMD and liposomes, respectively. Compared to the liposomes group, the UTMD group resulted in significantly improved transfection efficiency. The downregulation of CD133 reversed the EMT program, attenuated self-renewal, proliferation and migration of CD133+ LCSCs and suppressed the growth of CSC tumor xenografts. Additionally, the downregulation of CD133 led to downregulation of the nuclear factor-κB (NF-κB) pathway. The present study demonstrated that CD133 plays a critical role in the regulation of the EMT process, tumor-initiating properties and migratory ability of LCSCs. The UTMD technique targeted for CD133 downregulation may be examined as a potential therapeutic strategy for HCC.