Ultrasound-mediated Microbubble Destruction Research Articles

Caveolin Delivered by Ultrasound-Mediated Microbubble Destruction Prevents Endothelial Cell Proliferation.

The nitric oxide synthase (eNOS) is an important regulator of vascular homeostasis. eNOS is modulated by intracellular mechanisms that include protein-protein interaction with Caveolin-1 (Cav). Cav binds to and impairs eNOS activation reducing vascular permeability and angiogenesis. Blocking of eNOS by Cav has been proposed as therapeutic antiangiogenic approach. However, the efficient and controlled delivery of the peptide requires to be solved. The effect of antennapedia (AP)-Cav loaded into microbubbles (MBs) and delivered by ultrasound-mediated microbubble destruction (UMMD) into brain endothelial cells (bEnd.3 cells) was evaluated on NO production using DAF2-DA, cell migration assessed by the wound healing assay, cell proliferation with BrdU, and ex-vivo angiogenesis in rat aortic rings. An enhanced inhibitory effect of AP-Cav was observed on cells treated with UMMD. MBs and ultrasound disruption delivery of AP-Cav increased acetylcholine-induced NO release, wound healing, cell proliferation, and angiogenesis inhibition on bEnd.3 cells, compared to free AP-Cav administration. We demonstrated that the delivery of Cav via AP-Cav-loaded MBs and UMMD may be an administration method for Cav that would increase its therapeutic potential by enhancing efficacy and cellular specificity.

High‐Performance Delivery of a CRISPR Interference System via Lipid‐Polymer Hybrid Nanoparticles Combined with Ultrasound‐Mediated Microbubble Destruction for Tumor‐Specific Gene Repression (Adv. Healthcare Mater. 10/2023)

CRISPR Interference Systems The main objective of this study in article 2203082 by Yujin Zong, Mingxi Wan, and co-workers, is to develop novel pH-responsive lipid-polymer hybrid nanoparticles (PLPNs) to achieve optimal CRISPRi system delivery via ultrasound-mediated microbubble destruction (UMMD) technology, thereby inducing efficient target gene repression.

Ultrasound-Triggered Microbubbles: Novel Targeted Core-Shell for the Treatment of Myocardial Infarction Disease.

Myocardial infarction (MI) is known as a main cardiovascular disease that leads to extensive cell death by destroying vasculature in the affected cardiac muscle. The development of ultrasound-mediated microbubble destruction has inspired extensive interest in myocardial infarction therapeutics, targeted delivery of drugs, and biomedical imaging. In this work, we describe a novel therapeutic ultrasound system for the targeted delivery of biocompatible microstructures containing basic fibroblast growth factor (bFGF) to the MI region. The microspheres were fabricated using poly(lactic-co-glycolic acid)-heparin-polyethylene glycol- cyclic arginine-glycine-aspartate-platelet (PLGA-HP-PEG-cRGD-platelet). The micrometer-sized core-shell particles consisting of a perfluorohexane (PFH)-core and a PLGA-HP-PEG-cRGD-platelet-shell were prepared using microfluidics. These particles responded adequately to ultrasound irradiation by triggering the vaporization and phase transition of PFH from liquid to gas in order to achieve microbubbles. Ultrasound imaging, encapsulation efficiency cytotoxicity, and cellular uptake of bFGF-MSs were evaluated using human umbilical vein endothelial cells (HUVECs) in vitro. In vivo imaging demonstrated effective accumulation of platelet- microspheres injected into the ischemic myocardium region. The results revealed the potential use of bFGF-loaded microbubbles as a noninvasive and effective carrier for MI therapy.

High-Performance Delivery of a CRISPR Interference System via Lipid-Polymer Hybrid Nanoparticles Combined with Ultrasound-Mediated Microbubble Destruction for Tumor-Specific Gene Repression.

The dCas9-based CRISPR interference (CRISPRi) system efficiently silences genes without causing detectable off-target activity, thus showing great potential for the treatment of cancer at the transcriptional level. However, due to the large size of the commonly used CRISPRi system, effective delivery of the system has been a challenge that hinders its application in the clinic. Herein, a combination of pH-responsive lipid-polymer hybrid nanoparticles (PLPNs) and ultrasound-mediated microbubble destruction (UMMD) is used for the delivery of the CRISPRi system. The core-shell structure of PLPNs can effectively be loaded with the CRISPRi plasmid, and increases the time spent in the circulating in vivo, and "actively target" cancer cells. Moreover, the combination of PLPNs with UMMD achieves a higher cellular uptake of the CRISPRi plasmid in vitro and retention in vivo. Furthermore, when PLPNs loaded with a CRISPRi plasmid that targets microRNA-10b (miR-10b) are used in combination with UMMD, it results in the effective repression of miR-10b in breast cancer, simultaneous disturbance of multiple cell migration and invasion-related signaling pathways, and a significant inhibition of lung metastasis. Thus, the established system presents a versatile, highly efficient, and safe strategy for delivery of the CRISPRi system both in vitro and in vivo.

Dual-Modality Molecular Imaging of Tumor via Quantum Dots-Liposome-Microbubble Complexes.

Molecular imaging has demonstrated promise for evaluating the expression levels of biomarkers for the early prediction of tumor progression and metastasis. However, most of the commonly used molecular imaging modalities are relatively single and have difficulties imaging complex biological processes. Here, we fabricated αvβ3-integrin-targeted quantum-dots-loaded liposome-microbubble (iRGD-QDLM) complexes that combined ultrasound imaging with optical imaging. The resulting iRGD-QDLM has excellent binding capability to 4T1 breast cancer cells. Ultrasound molecular imaging of 4T1 tumors demonstrated that significantly enhanced ultrasound molecular signals could be observed in comparison with non-targeted QDLM. Importantly, our study also suggested that iRGD-QDL on the surface of microbubbles could be delivered into a tumor by ultrasound-mediated microbubble destruction and adhered to αvβ3 integrin on breast cancer cells, achieving transvascular fluorescent imaging. Our study provides a novel approach to dual-modality molecular imaging of αvβ3 integrin in the tumor tissue.

Targeting nanoparticle-conjugated microbubbles combined with ultrasound-mediated microbubble destruction for enhanced tumor therapy

The stress of the abnormal stromal matrix of solid tumors is a major limiting factor that prevents drug penetration. Controlled, accurate, and efficient delivery of theranostic agents into tumor cells is crucial. Combining ultrasound with nanocarrierbased drug delivery systems have become a promising approach for targeted drug delivery in preclinical cancer therapy. In this study, to ensure effective tumor barrier penetration, access to the tumor microenvironment, and local drug release, we designed targeted nanoparticle (NP)-conjugated microbubbles (MBs); ultrasound could then help deliver acoustic energy to release the NPs from the MBs. The ultrasound-targeted MB destruction (UTMD) system of negatively charged NPs was conjugated with positively charged MBs using an ionic gelation method. We demonstrated the transfer of targeted NPs and their entry into gastric cancer cells through ligand-specific recognition, followed by enhanced cell growth inhibition owing to drug delivery-induced apoptosis. Moreover, the UTMD system combining therapeutic and ultrasound image properties can effectively target gastric cancer, thus significantly enhancing antitumor activity, as evident by tumor localization in an orthotopic mouse model of gastric cancer. The combination of ultrasound and NP-based drug delivery systems has become a promising approach for targeted drug delivery in preclinical cancer therapy.

Release of vascular agonists from liposome-microbubble conjugate by ultrasound-mediated microbubble destruction: effect on vascular function.

The endothelium is a single cell layer of the vessel wall and a key regulator of blood flow in vascular beds. Local and systemic pathologies have been associated with alterations in endothelial function. However, targeting the endothelium with vasoconstrictor or vasodilator drugs is often accompanied by systemic effects. Here, we evaluated a liposome-microbubble delivery system as a vascular hydrophilic agonist carrier. Phenylephrine (Phe) or acetylcholine (Ach)-loaded liposomes were conjugated to microbubbles. The drug release was triggered by ultrasound (US), and the vascular response was assessed in rat aortic rings using an isolated organ chamber. Aortic rings incubated with Phe-liposome-microbubble conjugate, exposed to US showed a marked contractile response (0.79±0.04g) compared to empty liposomes conjugated to microbubbles, aortic rings exposed only to US, and Phe-liposome-microbubble conjugate without US exposure that elicited a minimal or no response. Expressed as %, contractile responses were 85.24±4.31% and 12.62±3.23% for Phe-Chol-liposome-microbubble conjugate and empty Chol-liposome-microbubble conjugate exposed to US, respectively. Addition of 1×10-5M Ach to pre-contracted aortic rings decreased the contraction response from 1 to 0.21g. The addition of Ach-liposome conjugate and exposure to US decreased the contraction response to 0.32g. Additionally, the ED50 values for Phe and Ach released by US from liposome-microbubble conjugates were 3.6×10-8M±2.8×10-9M for Phe and 2.0×10-8M±1.8×10-9M. In conclusion, we evaluated a hybrid delivery system that consisted of loaded liposomes conjugated to microbubbles to deliver and release vascular agonists using UMMD.

Ultrasound-Mediated Microbubble Destruction Inhibits Skin Melanoma Growth by Affecting YAP1 Translation Using Ribosome Imprinting Sequencing.

Cutaneous melanoma (CMM) is a skin tumor with a high degree of malignancy. BRAF resistance imposes great difficulty to the treatment of CMM, and partially contributes to the poor prognosis of CMM. YAP is involved in the growth and drug resistance of a variety of tumors, and mechanical signals may affect the activation of YAP1. As a novel ultrasound treatment technology, ultrasound-mediated microbubble destruction (UMMD) has been reported to have a killing effect on isolated CMM cells. In this study, the tumor tissue samples were collected from 64 CMM patients. We found that YAP1 mRNA expression was irrelevant to the clinicopathological characteristics and prognostic survival of the CMM patients. The drug-resistant cell line was constructed and subcutaneously implanted into nude mice, which were further separately treated with UMMD, ultrasound (US), and microbubbles (MB). The result showed that UMMD significantly inhibited the growth of tumor tissues. Ribosome imprinting sequencing (Ribo-seq) is a genetic technology for studying protein translation at genetic level. Ribo-seq, RNA-seq, and RT-qPCR were applied to detect YAP1 expression in CMM mouse tumor tissues. Ribo-seq data revealed that UMMD greatly up-regulated the expression of YAP1, interestingly, the up-regulated YAP1 was found to be negatively correlated with the weight of tumor tissues, while no significant change in YAP1 expression was detected by RNA-seq or RT-qPCR assay. These results indicated that UMMD could inhibit the tumor growth of drug-resistant CMM by affecting the translation efficiency of YAP1, providing a strong basis for the clinical treatment of UMMD in CMM.

Localized Delivery of Caveolin-1 Peptide Assisted by Ultrasound-Mediated Microbubble Destruction Potentiates the Inhibition of Nitric Oxide-Dependent Vasodilation Response

In the endothelium, nitric oxide synthase (eNOS) is the enzyme that generates nitric oxide, a key molecule involved in a variety of biological functions and cancer-related events. Therefore, selective inhibition of eNOS represents an attractive therapeutic approach for NO-related diseases and anticancer therapy. Ultrasound-mediated microbubble destruction (UMMD) conjugated with cell-permeable peptides has been investigated as a drug delivery system for effective delivery of anticancer molecules. We investigated the feasibility of loading antennapedia-caveolin-1 peptide (AP-Cav), a specific eNOS inhibitor, onto microbubbles to be delivered by UMMD in rat aortic endothelium. AP-Cav-loaded microbubbles (AP-Cav-MBs) and US parameters were characterized. Aortas were treated with UMMD for 30 s with 1.3 × 108 MBs/mL AP-Cav (8 μM)-MBs at 100-Hz pulse repetition frequency, 0.5-MPa acoustic pressure, 0.5 mechanical index and 10% duty cycle. NO-dependent vascular responses were assessed using an isolated organ system, 21 h post-treatment. Maximal relaxation response was inhibited 61.8% ± 1.6% in aortas treated with UMMD-AP-Cav-MBs, while in aortas treated with previously disrupted AP-Cav-MBs and then US, the inhibition was 31.6% ± 1.6%. The vascular contractile response was not affected. The impact of UMMD was evaluated in aortas treated with free AP-Cav; 30 μM of free AP-Cav was necessary to reach an inhibition response similar to that obtained with UMMD-AP-Cav-MBs. In conclusion, UMMD enhances the delivery and potentiates the effect of AP-Cav in the endothelial layer of rat aorta segments.

Acoustic Radiation or Cavitation Forces From Therapeutic Ultrasound Generate Prostaglandins and Increase Mesenchymal Stromal Cell Homing to Murine Muscle.

Non-ablative ultrasound (US)-based techniques to improve targeted tropism of systemically infused cell therapies, particularly mesenchymal stromal cell (MSC), have gained attention in recent years. Mechanotransduction following targeted US sonications have been shown to modulate tissue microenvironments by upregulating cytokines, chemokines, and trophic factors in addition to vascular cell adhesion molecules (CAM) that are necessary to promote tropism of MSC. While numerous US treatment parameters have demonstrated increased MSC homing, it remains unclear how the different mechanical US forces [i.e., acoustic radiation forces (ARF) or cavitation forces] influence tissue microenvironments. This study sonicated murine muscle tissue with pulsed focused ultrasound (pFUS) at 0.5 or 1.15 MHz each over a range of US intensities. Following sonication, tissue was assayed for the prostaglandins (PG) PGH2 and PGE2 as indicators of microenvironmental changes that would support MSC tropism. PGH2 and PGE2 levels were correlated to physical pFUS parameters and acoustic emissions measured by hydrophone. While ARF (pFUS with absence of cavitation signatures) was sufficient to increase PGH2 and PGE2, non-linear curve fitting revealed a frequency-independent relationship between prostaglandin production and mechanical index (MI), which accounts for increased cavitation probabilities of lower frequencies. The prostaglandin data suggested molecular changes in muscle would be particularly sensitive to cavitation. Therefore, low-intensity pulsed ultrasound (LIPUS) at 1 MHz was administered with low ARF (MI = 0.2) in combination with intravenous (IV) infusions of microbubble (MB) contrast agents. This combination upregulated prostaglandins and CAM without ultrasound-mediated microbubble destruction and ultimately promoted tropism of IV-infused MSC. This study revealed that accentuating non-destructive MB cavitation by US using parameters similar to diagnostic US contrast imaging increased MSC homing. Such approaches are particularly attractive to overcome clinical translation barriers of many still-experimental US parameters used in previous stem cell tropism studies.

Functional Activity and Endothelial-Lining Integrity of Ex Vivo Arteries Exposed to Ultrasound-Mediated Microbubble Destruction

Ultrasound-mediated microbubble destruction (UMMD) is a promising strategy to improve local drug delivery in specific tissues. However, acoustic cavitation can lead to harmful bioeffects in endothelial cells. We investigated the side effects of UMMD treatment on vascular function (contraction and relaxation) and endothelium integrity of ex vivo Wistar rat arteries. We used an isolated organ system to evaluate vascular responses and confocal microscopy to quantify the integrity and viability of endothelial cells. The arteries were exposed for 1–3 min to ultrasound at a 100 Hz pulse-repetition frequency, 0.5 MPa acoustic pressure, 50% duty cycle and 1%–5% v/v microbubbles. The vascular contractile response was not affected. The acetylcholine-dependent maximal relaxation response was reduced from 78% (control) to 60% after 3 min of ultrasound exposure. In arteries treated simultaneously with 1 min of ultrasound exposure and 1%, 2%, 3% or 5% microbubble concentration, vascular relaxation was reduced by 19%, 58%, 80% or 93%, respectively, compared with the control arteries. Fluorescent labeling revealed that apoptotic death, detachment of endothelial cells and reduced nitric oxide synthase phosphorylation are involved in relaxation impairment. We demonstrated that UMMD can be a safe technology if the correct ultrasound and microbubble parameters are applied. Furthermore, we found that tissue-function evaluation combined with cellular analysis can be useful to study ultrasound–microbubble–tissue interactions in the optimization of targeted endothelial drug delivery.

Ultrasound-Targeted Microbubble Destruction Enhances the Antitumor Efficacy of Doxorubicin in a Mouse Hepatocellular Carcinoma Model

The aim of the study described here was to investigate whether ultrasound-mediated microbubble destruction (UTMD) of targeted microbubbles conjugated with an anti-vascular endothelial growth factor receptor 2 (anti-VEGFR2) antibody can enhance the therapeutic effect of doxorubicin (DOX) on a mouse hepatocellular carcinoma (HCC) model bearing HEP-G2-RFP tumors. The growth of liver tumors in mice was inhibited by using Visistar VEGFR2 plus ultrasound irradiation and by DOX alone. DOX plus UTMD had an inhibitory effect on tumor growth beginning on the seventh day of treatment, while Visistar VEGFR2 alone and DOX alone had inhibitory effects beginning on the 11th day. DOX + UTMD significantly decreased tumor volume and tumor weight compared with DOX alone (p < 0.05) and Visistar VEGFR2 alone (p < 0.05). Compared with DOX alone and Visistar VEGFR2 alone, DOX + UTMD had the highest inhibitory effect on tumor angiogenesis and the highest apoptosis index. UTMD-targeted microbubbles can significantly enhance the antitumor effect of DOX on a mouse HCC model, inhibit angiogenesis and induce apoptosis in tumor cells.

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.

Ultrasound-mediated microbubble destruction: a new method in cancer immunotherapy.

Immunotherapy provides a new treatment option for cancer. However, it may be therapeutically insufficient if only using the self-immune system alone to attack the tumor without any aiding methods. To overcome this drawback and improve the efficiency of therapy, new treatment methods are emerging. In recent years, ultrasound-mediated microbubble destruction (UMMD) has shown great potential in cancer immunotherapy. Using the combination of ultrasound and targeted microbubbles, molecules such as antigens or genes encoding antigens can be efficiently and specifically delivered into the tumor tissue. This review focuses on the recent progress in the application of UMMD in cancer immunotherapy.

Real-time Optical Imaging of Microbubble Destruction with an Acoustic Lens Attached Ultrasonic Diagnostic Probe in Microfluidic Capillary Models.

In this work, an ultrasonic diagnostic system with an attachable acoustic lens was demonstrated for real-time optical imaging of ultrasound-mediated microbubble destruction in a microfluidic capillary model using an inverted microscope. Microbubble destruction under ultrasonic pressure was monitored via an EM-CCD camera with the frame rate of 70 fps. The acoustic field distribution of the transducer with the attachable acoustic lens was simulated via a finite element method (FEM) and measured by a hydrophone. The result of acoustic field distribution shows unfocused beam profiles with 50% decreased pressure of original focal area. With the unfocused beam, inertial cavitation of the microbubbles as a function of transducer input voltages of 30-60 Vpp was studied. In addition, the acoustic cavitation parameters such as frequency of 2 MHz, pulse length of 16 μs, and pulse repetition frequency (PRF) of 1 kHz were investigated under static and dynamic flow conditions in the microfluidic model. In our system, above 45 Vpp, the microbubbles were destroyed more than 50% within 20 seconds so that the threshold for the inertial cavitation was determined to be 45 Vpp in the channel without flow. In the microfluidic capillary model with fluidic flow, it is investigated that shape of microbubble mass continuously changed with acoustic pressure with 60 Vpp.

Ultrasound-mediated microbubbles destruction for treatment of rabbit VX2 orthotopic hepatic tumors

The aim of this study was to determine whether ultrasound (US) mediated microbubbles (MBs) destruction (UMMD) could inhibit VX2 orthotopic hepatic tumor growth in rabbit models. Twenty-four VX2 orthotopic hepatic tumor rabbit models were randomly divided into four groups (n = 6 each): saline group, SonoVue alone group, US alone group, US + SonoVue group. Tumor volume (TV), peak intensity (PI) of contrast enhanced ultrasound (CEUS) in US + SonoVue group were significantly lower than that in other groups after treatment (P < 0.05, for all). Immunohistochemical analysis of tumor tissue showed that microvascular density (MVD) in US + SonoVue group was significantly lower than that in other groups (P < 0.05, for all). Extensive interstitial hemorrhage, intravascular thrombosis, destroyed nucleus membrane, mitochondria vacuolation and chromatin condensation were observed in US + SonoVue group, whereas these changes were rarely appeared in other groups. Our study indicated that UMMD could inhibit the growth of VX2 hepatic tumors in rabbits by irreversible destroying tumor microvessel and tumor cells.

CoQ10-loaded liposomes combined with UTMD prevented early nephropathy of diabetic rats.

Nephropathy is one of the most severe complications of diabetic patients. The therapeutic strategies for diabetic patients should not only focus on the control of blood glucose but also pay attention to the occurrence of diabetic nephropathy (DN). Coenzyme Q10 (CoQ10) has great therapeutic potential for DN. However, the clinical application of CoQ10 has been limited because of its low water-solubility and non-specific distribution. Liposomes were supposed to be an effective way for delivering CoQ10 to kidney. CoQ10 was effectively encapsulated into the liposome (CoQ10-LIP) with a high entrapment efficiency of 86.15 %. The CoQ10-LIP exhibited a small hydrodynamic diameter (180 ± 2.1 nm) and negative zeta potential (−18.20 mV). Moreover, CoQ10-LIP was combined with ultrasound-mediated microbubble destruction (UTMD) to enhance specific distribution of CoQ10 in kidney. In early stage of diabetic mellitus (DM), rats were administrated with CoQ10-LIP followed by UTMD (CoQ10-LIP+UTMD) to prevent occurrence of DN. Results revealed that CoQ10-LIP+UTMD effectively prevented the renal morphology and function of diabetics rats from damage. The protective mechanism of CoQ10-LIP was highly associated with protecting podocyte, promoting vascular repair and inhibiting cell apoptosis. Conclusively, CoQ10-LIP in combination with UTMD might be a potential strategy to prevent occurrence of DN.

Bioeffect of different parameters on four tumor cell lines by ultrasound-mediated microbubble destruction

Objective To explore the bioeffect of different parameters on 4 cell lines by ultrasound-mediated microbubble destruction. Methods The orthogonal experimental design was used to investigate the effect of three factors on the bioeffects of four cell lines under three levels. Three factors included microbubble concentration, sound intensity, irradiation time. Human breast tumor (MCF-7) cells, ovarian tumor (A2780) cells, liver tumor (Bel7402) cells and thyroid tumor (ARO) cells were exposed to ultrasound in the presence of SonoVue. The cell survival rate was determined by MTT methods and the cell luminosity factor was detected by flow cytometry. Results The optimum parameters for Bel7402 and ARO cell were the same(A2B3C2), and they were different from those from MCF-7(A3B1C1) and A2780(A1B3C3) cell. The cell survival rates for 4 cell lines were above 75%, and the cell luminosity factors were different among 4 cell lines. Conclusions The optimum parameters by ultrasound-mediated microbubble destruction for different cell lines are different, and under the optimum parameters the bioeffects of different cell lines are different. Key words: Ultrasonic therapy; Microbubbles; Bioeffect; Cell membrane permeability

The therapeutic effect of low frequency ultrasound mediated microbubbles destruction on rabbit VX2 orthotopic hepatic tumors

Objective To determine whether low frequency ultrasound mediated microbubbles destruction (UMMD) could inhibit VX2 orthotopic hepatic tumor growth in rabbit models. Methods Twenty-four New Zealand white rabbits were implanted with VX2 tumor in left hepatic lobe to establish a homograft rabbit model of liver neoplasms in situ, which were randomly divided into four groups(6 rabbits in each group): group A (intravenous saline only), group B (intravenous microbubbles only), group C (intravenous saline+ low frequency focused ultrasound exposure), and group D (intravenous microbubbles+ low frequency focused ultrasound exposure). After 3 days consecutive treatment, tumor volume(TV), and peak intensity (PI) were monitored by conventional ultrasound and contrast enhanced ultrasound(CEUS) on 0, 1, 7, 14 and 21 days after treatment. The rabbits were euthanized at the end of the experiment. Tumor tissues were evaluated by HE stain. Results The parameters of TV and PI of each tumor had no significant difference among four groups before treatment(all P>0.05). TV had no significant difference among four groups on 1 day after treatment(all P>0.05); PI in group C and group D were significantly lower than those in group A and group B (all P 0.05). The pathological changes of necrosis tissue, hemorrhagic damage of microvessel and thrombosis were observed in the tumors of group D only, whereas these changes occurred rarely in other groups. Conclusions UMMD can inhibit the growth of VX2 hepatic tumors in rabbits, and be used as a promising novel therapeutic strategy to liver neoplasms. Key words: Contrast-enhanced ultrasound; Microbubbles; Liver neoplasms, experimental; Ultrasonic therapy

Research of enhanced green fluorescent protein gene transfer with ultrasound-mediated microbubble destruction in bone defects

To investigate the effect of ultrasonic irradiation time on enhanced green fluorescent protein (EGFP) gene transfection efficiency and local tissue in bone defects using ultrasound-mediated microbubble destruction. Thirty 3-month-old New Zealand rabbits (2.5-3.0 kg in weight) were randomly divided into 5 groups ( n=6) and bone defect models were made on the right ulna. At 10 days after modeling, suspension of microbubbles and EGFP plasmids were locally injected (0.3 mL/kg) and then ultrasound was performed on defect at a frequency of 1 MHz, a intensity of 0.5 W/cm 2, and a duty ratio of 20% for 1, 2, 3, 4, and 5 minutes respectively (in 1, 2, 3, 4, and 5 minutes groups respectively). The survival condition was observed. Rabbits were sacrificed for gross observation at 7 days after transfer. The gene expression was observed by fluorescence staining. HE staining and transmission electron microscopy were used to observe the local tissue damage. The animals all survived. New soft tissue formed in bone defects area at 1 week after transfer, the surrounding muscle tissue was partly filled in it. Green fluorescence expression was observed in all rabbits. The expression was the strongest in 2 minutes group, and was the weakest in 1 minute group. The absorbance ( A) value showed significant differences when compared 1 minute and 2 minutes groups with other groups ( P<0.05), but no significant difference was found between 3, 4, and 5 minutes groups ( P>0.05). Tissue damage was observed in all groups and it was aggravated with the increase of irradiation time. EGFP transfection efficiency in bone defect by ultrasound-mediated microbubble destruction is related to irradiation time. EGFP gene can be efficiently transfected without obvious toxicity at 1 MHz, 0.5W/cm 2, and duty ratio of 20% for 2 minutes in bone defects of rabbits.