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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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 72 h 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. CONCLUSIONS: 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. KEY POINTS: • 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.

Molecular imaging of vulnerable plaques in rabbits using contrast-enhanced ultrasound targeting to vascular endothelial growth factor receptor-2.

PURPOSE: Increased neovascularization has been identified as a feature of atherosclerotic plaque vulnerability and can be traced by microbubble ultrasound contrast agents (UCA). We investigated the relationship between retention of a vascular endothelial growth factor receptor 2 (VEGFR-2) targeted UCA and VEGFR-2 expression in a vulnerable plaque model in rabbits. METHODS: Microbubbles targeting to VEGFR-2 were prepared by conjugation of biotinylated microbubbles with biotinylated VEGFR-2 antibody via streptavidin. Vulnerability was created by delivering recombinant p53 adenovirus to atherosclerotic plaques obtained in abdominal aorta by a high cholesterol diet and balloon endothelial injury. Twelve week later, the average video intensity of pre- and postcontrast ultrasound images was measured. VEGFR-2 expression and vascular density were quantified by immunohistochemical staining. RESULTS: Retention of targeted UCA in plaques was higher than that of nontargeted UCA (144 ± 18 dB versus 107 ± 9 dB; Z= -3.984, p = 0.000). VEGFR-2 expression was correlated with video intensity of targeted (r(2) = 0.78, p = 0.001), but not of nontargeted, UCA (r(2) = 0.17, p ≥ 0.05). CONCLUSIONS: The magnitude of the sonographic signal from retained VEGFR-2 targeted UCA correlates with VEGFR-2 expression. These results validate the use of targeted UCA for sonographic imaging of vulnerable abdominal artery plaques in rabbits.

Enhanced Homing of CXCR-4 Modified Bone Marrow-Derived Mesenchymal Stem Cells to Acute Kidney Injury Tissues by Micro-Bubble-Mediated Ultrasound Exposure.

Although the curative effects of bone marrow stromal cells (BMSCs) for acute kidney injury (AKI) have been recognized, their in vivo reparative capability is limited by the low levels of targeted homing and retention of intravenous injected cells. Stromal cell-derived factor-1 (SDF-1) plays an important role in stem cell homing and retention through interaction with its specific functional receptor, CXCR4, which is presumably related to the poor homing in AKI therapy. However, most of the functional CXCR4 chemokine receptors are lost upon in vitro culturing. Ultrasound-targeted micro-bubble destruction (UTMD) has become one of the most promising strategies for the targeted delivery of drugs and genes. To improve BMSC homing to AKI kidneys, we isolated and cultured rat BMSCs to third passage and enhanced CXCR-4 transfection efficiency in vitro by applying UTMD and polyethylenimine. Transwell migration assay showed that the migration ability of CXCR4-modified BMSCs was nine-fold higher than controls. Then, mercuric chloride-induced AKI rats were injected with transfected BMSCs through their tail veins. We showed that enhanced homing and retention of BMSCs were observed in the CXCR-4 modified group compared with other groups at 1, 2 and 3 d post-treatment. Collectively, our data indicated that UTMD was an effective method to increase BMSCs' engraftment to AKI kidney tissues by increasing CXCR-4 expression.

Transfection of CXCR-4 using microbubble-mediated ultrasound irradiation and liposomes improves the migratory ability of bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) have proven useful for the treatment of various human diseases and injuries. However, their reparative capacity is limited by their poor migration and homing ability, which are primarily dependent on the SDF-1/CXCR4 axis. Most subcultured BMSCs lack CXCR4 receptor expression on the cell surface and exhibit impaired migratory capacity. To increase responsiveness to SDF-1 and promote cell migration and survival of cultured BMSCs, we used a combination of ultrasound-targeted microbubble destruction (UTMD) and liposomes to increase CXCR4 expression in vitro. We isolated and cultured rat BMSCs to their third passage and transduced them with recombinant plasmid pDsRed-CXCR4 using microbubble-mediated ultrasound irradiation and liposomes. Compared to some viral vectors, the method we employed here resulted in significantly better transfection efficiency, CXCR4 expression, and technical reproducibility. The benefits of this approach are likely due to the combination of "sonoporation" caused by shockwaves and microjet flow resulting from UTMD-generated cavitation. Following transfection, we performed a transwell migration assay and found that the migration ability of CXCR4-modified BMSCs was 9-fold higher than controls. The methods we describe here provide an effective, safe, non-viral means to achieve high levels of CXCR4 expression. This is associated with enhanced migration of subcultured BMSCs and may be useful for clinical application as well.

Enhanced Homing Ability and Retention of Bone Marrow Stromal Cells to Diabetic Nephropathy by Microbubble-Mediated Diagnostic Ultrasound Irradiation.

Bone marrow stromal cell (BMSC) transplantation can successfully treat diabetic nephropathy (DN), but the lack of a specific homing place for intravenously injected cells limits the effective implementation of stem cell therapies. The migration and survival of transplanted BMSCs are determined by inflammatory reactions in the local kidney micro-environment. We tested the hypothesis that microbubble-mediated diagnostic ultrasound irradiation could provide a suitable micro-environment for BMSC delivery and retention in DN therapy. In this study, red fluorescent protein-labeled BMSCs were administered combined with microbubbles to streptozotocin-induced DN rats 4 wk after diabetes onset. We observed enhanced BMSC homing and retention in microbubble-mediated diagnostic ultrasound-irradiated kidneys compared with the contralateral kidneys on days 1 and 3 post-treatment. The results from immunohistochemical analysis, Western blot and enzyme-linked immunosorbent assay indicated that the local and transient expression of various chemo-attractants (i.e., cytokines, integrins and trophic factors) found to promote BMSC homing was much higher than observed in non-treated kidneys. The local capillary endothelium rupture observed by transmission electron microscopy may account for local micro-environment changes. Histopathologic analysis revealed no signs of kidney damage. These results confirmed that renal micro-environment changes caused by appropriate microbubble-mediated diagnostic ultrasound irradiation may promote BMSC homing ability to the diabetic kidney without renal toxicity and cell damage. This non-invasive and effective technique may be a promising method for BMSC transplantation therapy.

Ultrasound-Targeted Microbubble Destruction Improves the Migration and Homing of Mesenchymal Stem Cells after Myocardial Infarction by Upregulating SDF-1/CXCR4: A Pilot Study.

Mesenchymal stem cell (MSC) therapy shows considerable promise for the treatment of myocardial infarction (MI). However, the inefficient migration and homing of MSCs after systemic infusion have limited their therapeutic applications. Ultrasound-targeted microbubble destruction (UTMD) has proven to be promising to improve the homing of MSCs to the ischemic myocardium, but the concrete mechanism remains unclear. We hypothesize that UTMD promotes MSC homing by upregulating SDF-1/CXCR4, and this study was aimed at exploring this potential mechanism. We analyzed SDF-1/CXCR4 expression after UTMD treatment in vitro and in vivo and counted the number of homing MSCs in MI areas. The in vitro results demonstrated that UTMD not only led to elevated secretion of SDF-1 but also resulted in an increased proportion of MSCs that expressed surface CXCR4. The in vivo findings show an increase in the number of homing MSCs and higher expression of SDF-1/CXCR4 in the UTMD combined with MSCs infusion group compared to other groups. In conclusion, UTMD can increase SDF-1 expression in the ischemic myocardium and upregulate the expression of surface CXCR4 on MSCs, which provides a molecular mechanism for the homing of MSCs assisted by UTMD via SDF-1/CXCR4 axis.

Microbubbles coupled to methotrexate-loaded liposomes for ultrasound-mediated delivery of methotrexate across the blood-brain barrier.

Methotrexate (MTX) is the single most effective agent for the treatment of primary central nervous system lymphoma. Currently, the delivery of MTX to the brain is achieved by high systemic doses, which cause severe long-term neurotoxicity, or intrathecal administration, which is highly invasive and may lead to infections or hemorrhagic complications. Acoustically active microbubbles have been developed as drug carriers for the noninvasive and brain-targeted delivery of therapeutics. However, their application is limited by their low drug-loading capacity. To overcome this limitation, we prepared microbubbles coupled to MTX-loaded liposomes using ZHIFUXIAN, a novel type of microbubbles with a superior safety profile and long circulation time. MTX-liposome-coupled microbubbles had a high drug-loading capacity of 8.91%± 0.86%, and their size (2.64 ± 0.93 µm in diameter) was suitable for intravenous injection. When used with ultrasound, they showed more potent in vitro cytotoxicity against Walker-256 cancer cells than MTX alone or MTX-loaded liposomes. When Sprague-Dawley rats were exposed to sonication, administration of these MTX-liposome-coupled microbubbles via the tail vein led to targeted disruption of the blood-brain barrier without noticeable tissue or capillary damage. High-performance liquid chromatography analysis of the brain MTX concentration showed that MTX delivery to the brain followed the order of MTX-liposome-coupled microbubbles + ultrasound (25.3 ± 2.4 µg/g) > unmodified ZHIFUXIAN + MTX + ultrasound (18.6 ± 2.2 µg/g) > MTX alone (6.97 ± 0.75 µg/g) > MTX-liposome-coupled microbubbles (2.92 ± 0.39 µg/g). Therefore, treatment with MTX-liposome-coupled microbubbles and ultrasound resulted in a significantly higher brain MTX concentration than all other treatments (P<0.01). These results suggest that MTX-liposome-coupled microbubbles may hold great promise as new and effective therapies for primary central nervous system lymphoma and other central nervous system malignancies.

Ultrasound-targeted stromal cell-derived factor-1-loaded microbubble destruction promotes mesenchymal stem cell homing to kidneys in diabetic nephropathy rats.

Mesenchymal stem cell (MSC) therapy has been considered a promising strategy to cure diabetic nephropathy (DN). However, insufficient MSCs can settle in injured kidneys, which constitute one of the major barriers to the effective implementation of MSC therapy. Stromal cell-derived factor-1 (SDF-1) plays a vital role in MSC migration and involves activation, mobilization, homing, and retention, which are presumably related to the poor homing in DN therapy. Ultrasound-targeted microbubble destruction has become one of the most promising strategies for the targeted delivery of drugs and genes. To improve MSC homing to DN kidneys, we present a strategy to increase SDF-1 via ultrasound-targeted microbubble destruction. In this study, we developed SDF-1-loaded microbubbles (MB(SDF-1)) via covalent conjugation. The characterization and bioactivity of MB(SDF-1) were assessed in vitro. Target release in the targeted kidneys was triggered with diagnostic ultrasound in combination with MB(SDF-1). The related bioeffects were also elucidated. Early DN was induced in rats with streptozotocin. Green fluorescent protein-labeled MSCs were transplanted intravenously following the target release of SDF-1 in the kidneys of normal and DN rats. The homing efficacy was assessed by detecting the implanted exogenous MSCs at 24 hours. The in vitro results showed an impressive SDF-1 loading efficacy of 79% and a loading content of 15.8 µg/mL. MB(SDF-1) remained bioactive as a chemoattractant. In the in vivo study, SDF-1 was successfully released in the targeted kidneys. The homing efficacy of MSCs to DN kidneys after the target release of SDF-1 was remarkably ameliorated at 24 hours compared with control treatments in normal rats and DN rats. In conclusion, ultrasound-targeted MB(SDF-1) destruction could promote the homing of MSCs to early DN kidneys and provide a novel potential therapeutic approach for DN kidney repair.

Investigation into the impact of diagnostic ultrasound with microbubbles on the capillary permeability of rat hepatomas.

Ultrasound-targeted microbubble destruction (UTMD) takes advantage of transiently increased capillary permeability to enhance the release of tumor-specific drugs from blood vessels into sonicated tumor tissues. However, the application of focused ultrasound is limited because of the lack of an appropriate image-monitoring system. In this study, hepatoma-bearing Sprague-Dawley rats were insonicated with low-frequency diagnostic ultrasound and injected with Evans Blue (EB) dye and microbubbles through their tail veins to test changes in capillary permeability. We studied how the mechanical index, sonication duration and the injected microbubble (MB) concentration affect the hepatoma vascular permeability by quantitatively evaluating the EB delivery efficiency. Confocal laser scanning microscopy was used to observe the deposition of red fluorescence-dyed EB in tumor tissues. In addition, P-selectin, a type of biochemical marker that reflects vascular endothelial cell activation, was identified using an immunoblotting analysis. The experimental results reveal that EB delivery efficiency in tumor tissues was greater in groups with the diagnostic ultrasound-mediated UTMD (8.40 ± 0.71 %ID/g) than in groups without UTMD (1.73 ± 0.19 %ID/g) and EB delivery efficiency could be affected by MI, sonication duration and MB dose. The immunoblotting analysis indicates that diagnostic ultrasound-induced UTMD results in the vascular endothelial cell activation to increase capillary permeability, justifying the high quantity of EB deposited in tumor tissues.

Kidney-targeted transplantation of mesenchymal stem cells by ultrasound-targeted microbubble destruction promotes kidney repair in diabetic nephropathy rats.

We test the hypothesis that ultrasound-targeted microbubble destruction (UTMD) technique increases the renoprotective effect of kidney-targeted transplantation of bone-marrow-derived mesenchymal stem cells (BM-MSCs) in diabetic nephropathy (DN) rats. Diabetes was induced by streptozotocin injection (60 mg/Kg, intraperitoneally) in Sprague-Dawley rats. MSCs were administered alone or in combination with UTMD to DN rats at 4 weeks after diabetes onset. Random blood glucose concentrations were measured at 1, 2, 4, and 8 weeks, and plasma insulin levels, urinary albumin excretion rate (UAER) values, the structures of pancreas and kidney, the expressions of TGF- ß 1, synaptopodin, and IL-10 were assessed at 8 weeks after MSCs transplantation. MSCs transplantation decreased blood glucose concentrations and attenuated pancreatic islets/ ß cells damage. The permeability of renal interstitial capillaries and VCAM-1 expression increased after UTMD, which enhanced homing and retention of MSCs to kidneys. MSCs transplantation together with UTMD prevented renal damage and decreased UAER values by inhibiting TGF- ß 1 expression and upregulating synaptopodin and IL-10 expression. We conclude that MSCs transplantation reverts hyperglycemia; UTMD technique noninvasively increases the homing of MSCs to kidneys and promotes renal repair in DN rats. This noninvasive cell delivery method may be feasible and efficient as a novel approach for personal MSCs therapy to diabetic nephropathy.

Myocardium-targeted transplantation of mesenchymal stem cells by diagnostic ultrasound-mediated microbubble destruction improves cardiac function in myocardial infarction of New Zealand rabbits.

BACKGROUND: Therapeutic ultrasound-mediated microbubble destruction has been applied in the targeted delivery of genes, drugs and stem cells. We intended to study whether diagnostic US irradiating lipid-coated microbubble destruction combined with bone-marrow derived MSC infusion could enable the targeted delivery of MSCs into the myocardium and improve cardiac function of the myocardial infarction of New Zealand rabbits. METHODS: Diagnostic ultrasound was applied to the anterior chest for 10 min after intravenous injection of lipid-coated microbubble followed by infusion of BM-MSCs. Echocardiography, histological examination, and western blotting were performed 4 weeks after cell transplantation. RESULTS: The cardiac function (assessed by fractional shortening and ejection fraction) was markedly improved by US+Microbubble+MSC treatment. The number of capillaries stained by HE in US+Microbubble+MSC group (47+/-23) was much greater than that of the MSCs infusion group (26+/-7), US+Microbubble group(22+/-5) and PBS infusion group (19+/-10), P<0.01. US+Microbubble stimulation induced the expression of adhesion molecule (VCAM-1) in capillaries and enhanced the myocardial permeability of microvessels. US+Microbubble-mediated supply of MSCs increased the level of VEGF in ischemic myocardium. Area of cardiac fibrosis in the US+Microbubble+MSC group was significantly decreased by 25.6%,40.1% and 46.8% when compared with MSC infusion group, US+Microbubble group and PBS infusion group, respectively. CONCLUSIONS: This non-invasive cell delivery system may be useful as a novel and efficient approach for angiogenic cell therapy to the infarcted myocardium.