Ultrasound-targeted Microbubble Destruction Research Articles

Inducing Tumor Cell Apoptosis: Mediated Survivin miRNA by Ultrasound and Cationic Lipid Contrast Agent.

Non-viral delivery systems is a promising method for gene or drug delivery. Polyethyleneimine (PEI) is a double-edged sword. It internalizes itself into the cell through endocytosis and promotes gene transfer efficiency. However, the strong positive charge also makes PEI highly toxic to cells. Ultrasound-targeted microbubble destruction (UTMD) is a promising non-viral method for gene and drug delivery, but its efficiency still needs to be improved. The aim of this study was to explore a system that combines ultrasound with non-viral gene delivery for the treatment of cervical cancer HeLa cells. In this study, we synthesized a kind of cationic ultrasound contrast agent(CUCA) that the physical and chemical properties, gene carrying capacity and cytotoxicity were verified. On the basis of previous studies, we further optimized the following transfusion parameters including ultrasound parameters, microbubble concentration, plasmid concentration, cell density and other parameters. The experiment was designed to compare the following six groups: (1) Plasmid group (P group), plasmid 15 μg; (2) PEI + plasmid group (PEI + P group),1 μl of PEI containing 10 nmol nitrogen and 1 μg of DNA containing 3 nmol phosphate for a PEI/DNA ratio equal to a nitrogen/phosphate ratio of 7 for transfection; (3) Ultrasound + plasmid group (US + P), plasmid 15 μg; (4) Ultrasound + cationic liposomal ultrasound contrast agent + plasmid group (UTMD + P group), plasmid 15 μg and cationic liposomal ultrasound contrast agent 5%; (5) Ultrasound + cationic liposomal ultrasound contrast agent + PEI + plasmid group (UTMD + PEI + P group), PEI/DNA ratio equal to a nitrogen/phosphate ratio of 7 for transfection and cationic liposomal ultrasound contrast agent 5%; and (6) Blank group, no treatment), The influence on Hela cells was observed under microscope, the efficiency of apoptosis was measured by flow cytometry, and cell viability was tested in CCK 8. The optimized transfection parameters can improve the transfection efficiency of ultrasound combined with C-UCA to a certain extent, but its transfection efficiency is still lower than that of branched polyethyleneimine (bPEI) 25 kDa. By investigating the effect of HeLa cells apoptosis induced by UTMD in combination with PEI mediated survivin miRNA, we found that both PEI alone and ultrasound in combination with CUCA were able to transfect cells with survivin miRNA to effectively induce HeLa cell apoptosis. However, the synergistic effect between the two methods was not significant. In contrast, the combined use of ultrasound, C-UCA and PEI may significantly reduce the transfection efficiency of UTMD and PEI, and the specific mechanism remains to be further studied.

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.

Synergistic Anti-tumor Mechanisms of Low-frequency Ultrasound-targeted Microbubble Destruction:Mechanisms and Application

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.

Ultrasound targeted microbubble destruction assisted exosomal delivery of miR-21 protects the heart from chemotherapy associated cardiotoxicity

Chemotherapy related cardiotoxicity is now becoming one of the biggest hurdles for the prognosis of cancer patients. Therapeutically delivering protective small RNAs holds promise for the cardiotoxicity prevention and therapy. However, heart is intrinsically refractory to the nanoparticle-mediated drug delivery. In this study, we found that the exosome-mediated miRNA delivery into the heart could be significantly augmented with the aid of ultrasound targeted microbubble destruction (UTMD). Moreover, we found that UTMD assisted exosomal miR-21 delivery into the heart significantly decreased the cell death, and restored the cardiac function in a doxorubicin induced cardiotoxicity mouse model. Our study here not only provides a promising strategy to protect the heart from the chemotherapy related cardiotoxicity, but also sheds light on gene therapy of other heart diseases.

Role of GRK4 in the regulation of the renal ETB receptor in hypertension.

The endothelin receptor type B (ETBR) regulates water and electrolyte balance and blood pressure, in part, by inhibiting renal sodium transport. Our preliminary study found that the ETBR-mediated diuresis and natriuresis are impaired in hypertension with unknown mechanism. Persistently increased activity of G protein-coupled receptor kinase 4 (GRK4), caused by increased expression or genetic variants (eg, GRKγ142V), impairs the ability of the kidney to excrete a sodium load, in part, by impairing renal dopamine D1 receptor function through persistent phosphorylation. Our present study found that although renal ETBR expression was not different between Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs), renal ETBR phosphorylation was higher in SHRs. The role of hyper-phosphorylation in impaired ETBR-function was supported by results in human (h) GRK4γ transgenic mice. Stimulation of ETBR by BQ3020-induced natriuresis in human (h) GRK4γ wild-type (WT) mice. However, in hGRK4γ 142V transgenic mice, the renal ETBR was hyperphosphorylated and ETBR-mediated natriuresis and diuresis were not evident. There were co-localization and co-immunoprecipitation of ETBR and GRK4 in renal proximal tubule (RPT) cells from both WKY and SHRs but was greater in the latter than the former group. SiRNA-mediated downregulation of GRK4 expression, recovered the impaired inhibitory effect of ETBR on Na+ -K+ -ATPase activity in RPT cells from SHR. In vivo downregulation of renal GRK4 expression, via ultrasound-targeted microbubble destruction, decreased ETBR phosphorylation and restored ETBR-mediated natriuresis and diuresis in SHRs. This study provides a mechanism by which GRK4, via regulation of renal ETBR function, participates in the pathogenesis of hypertension.

Acute Cardiac Rejection: Antagomir‐155 Attenuates Acute Cardiac Rejection Using Ultrasound Targeted Microbubbles Destruction (Adv. Healthcare Mater. 14/2020)

In article number 2000189 by Li Zhang, Mingxing Xie, and co-workers, cationic microbubbles composed of DOTAP, DSPC, and DSPE-PEG2000 are synthesized to carry antagomir-155 and deliver it to allograft heart in a murine model by ultrasound targeted microbubbles destruction (UTMD). Sonoporation and cavitation make antagomir-155 penetrate vascular endothelium into intracellular region of allograft heart. This therapy downregulates microRNA-155 level in graft heart and attenuates degree of acute cardiac rejection.

Transfection of TGF-β shRNA by Using Ultrasound-targeted Microbubble Destruction to Inhibit the Early Adhesion Repair of Rats Wounded Achilles Tendon In vitro and In vivo.

Tendon injury is a major orthopedic disorder. Ultrasound-targeted microbubble destruction (UTMD) provides a promising method for gene transfection, which can be used for the treatment of injured tendons. The purpose of this study was to investigate the optimal transforming growth factor beta (TGF-β) short hairpin RNA (shRNA) sequence and transfection conditions using UTMD in vitro and to identify its ability for inhibiting the early adhesion repair of rats wounded achilles tendons in vivo. The optimal sequence was selected analyzing under a fluorescence microscope and quantitative real-time reverse transcription polymerase chain reaction in vitro. In vivo, 40 rats with wounded Achilles tendons were divided into five groups: (1) control group, (2) plasmid group (3) plasmid + ultrasound group, (4) plasmid + microbubble group, (5) plasmid + microbubble + ultrasound group, and were euthanized at 14 days post treatment. TGF-β expression was evaluated using adhesion scores and pathological examinations. The optimal condition for UTMD delivery in vitro was 1W/cm2 of output intensity and a 30% duty cycle with 60 s irradiation time (P < 0.05). The transfection efficiency of the plasmid in group 5 was higher than that in other groups (P < 0.05). Moreover, the lowest adhesion index score and the least expression of TGF-β were shown in group 5 (P < 0.05). When compared with the other groups, group 5 had a milder inflammatory reaction. The results suggested that UTMD delivery of TGF-β shRNA offers a promising treatment approach for a tendon injury in vivo.

Antagomir-155 Attenuates Acute Cardiac Rejection Using Ultrasound Targeted Microbubbles Destruction.

Antagomir-155 is an artificial inhibitor of miRNA-155, which is expected to be a promising therapeutic target to attenuate acute cardiac rejection (ACR). However, its vulnerability of being degraded by endogenous nuclease and potential off-target effect make the authors seek for a more suitable way to delivery it. In attribution of efficiency and safety, ultrasound targeted microbubbles destruction (UTMD) turns out to be an appropriate method to deliver gene to target tissues. Here, cationic microbubbles to deliver antagomir-155 downregulating miRNA-155 in murine allograft hearts triggered by UTMD are synthesized. The viability of this therapy is verified by fluorescent microscopy. The biodistribution of antagomir-155 is analyzed by optical imaging system. The results show antagomir-155 delivered by UTMD which significantly decreases the levels of miR-155. Also, this therapy downregulates the expression of cytokines and inflammation infiltration. And allograft survival time is significantly prolonged. Therefore, antagomir-loaded microbubbles trigged by UTMD may provide a novel platform for ACR target treatment.

Phosphorus dendrimer-based copper(II) complexes enable ultrasound-enhanced tumor theranostics

Abstract Design of simple-component theranostic agents with enhanced tumor accumulation represents a novel strategy in the area of precision nanomedicine for effective tumor theranostics. Herein, we report an innovative theranostic strategy based on ultrasound-enhanced tumor accumulation of a theranostic agent of phosphorus dendrimer-copper(II) complexes (1G3-Cu). The employed 1G3-Cu complexes represent a drug-free nanotheranostic agent with a simple composition possessing an r1 relaxivity of 0.7024 mM−1 s−1 for T1-weighted magnetic resonance (MR) imaging and an IC50 of 1.24 μM to inhibit the growth of a pancreatic cancer cell line. We show that cancer cells can be inhibited by the 1G3-Cu complexes through the activation of the apoptotic process with the upregulation of Bax, P53 and PTEN, downregulation of Bcl-2, and decrease of intracellular ATP. With the assistance of ultrasound targeted microbubble destruction (UTMD) technology that can increase cell membrane permeability temporarily via sonoporation effect, the 1G3-Cu complexes enable enhanced tumor MR imaging and chemotherapy due to their improved tumor accumulation. With the proven minimal toxicity to normal tissues at imaging and therapeutic doses, the prepared 1G3-Cu complexes may be potentially employed for UTMD-enhanced theranostics of different types of cancer.

A Cleverly Designed Novel Lipid Nanosystem: Targeted Retention, Controlled Visual Drug Release, and Cascade Amplification Therapy for Mammary Carcinoma in vitro.

ObjectiveTo construct an ideal theranostic nanoplatform (LIP3); to clarify its physicochemical properties; to confirm its characteristics of dual-modality imaging, active-targeting, and cascade amplification therapy for mammary carcinoma; and to perform a preliminary exploration of the cytotoxicity mechanism.DesignA self-prepared liposome nanosystem, LIP3, can actively target 4T1 cells because the surface is linked with C-RGD. Haematoporphyrin monomethyl ether (HMME), an excellent sonosensitizer entrapped in the lipid bilayer, can function in photoacoustic imaging. Low-intensity focused ultrasound (LIFU) of ultrasound-targeted microbubble destruction (UTMD) promotes localized drug delivery into tumours because PFH, a phase-change substance, is loaded in the LIP3 core, achieving visualization of targeted drug release, and sonodynamic therapy (SDT) can kill tumour cells. SDT provides a favourable environment for AQ4N, resulting in amplification of LIP3 treatment. Therefore, LIP3 shows targeted aggregation and targeted release, integrating dual-mode imaging and precise treatment.ResultsThe self-prepared lipid nanosystem, LIP3, meets the above expectations and has ideal physicochemical properties, with a regular sphere with uniform distribution. Contrast-enhanced ultrasound (CEUS), photoacoustic imaging, and bimodal imaging were effective in vitro. In 4T1 cell experiments, the cell capacity was as high as 42.9%, and the cytotoxicity to 4T1 was more than 5 times that of LIP1 (containing AQ4N only) and more than 2 times that of LIP2 (containing only HMME), achieving comparable results as cascade therapy for mammary cancer.ConclusionLIP3, a theranostic nanoplatform, was successfully constructed and conformed to the physicochemical characterization of ideal nanoparticles, with active-targeting, dual-modality imaging, visualized drug release, and precise treatment under the action of LIFU. SDT provides a favourable environment for AQ4N, resulting in amplification of LIP3 treatment. Therefore, LIP3 shows targeted aggregation and targeted release, integrating dual-mode imaging, and precise cascade treatment. This unique theranostic NPS with multiple capabilities is expected to be a favourable anti-cancer method in the future.

UTMD inhibit EMT of breast cancer through the ROS/miR-200c/ZEB1 axis

As a potential drug/gene delivery system, the ultrasound-targeted microbubble destruction (UTMD) system can be used as a vehicle as well as increasing the permeability of biological barriers to enhance the effect of tumor treatment. However, the effect of UTMD in the tumor EMT process is unknown. In this study, we aimed to investigate the potential and mechanism of UTMD induced oxidative stress in inhibiting EMT of breast cancer. Human breast MDA231 cells were treated with microbubble (MB), ultrasound (US) and UTMD, respectively. The generation of oxidative stress, the levels of miR-200c, ZEB1 and vimentin, and the numbers of migratory cells were evaluated quantitatively and qualitatively by the measurement of intracellular reactive oxygen species (ROS), qRT-PCR, western blot assay, and transwell assay. Then, to evaluate the role of UTMD-induced oxidative stress and miR-200c in the epithelial-mesenchymal transition (EMT) inhibition, the ROS scavenger N-acetyl-L-cysteine (NAC) and miR-200c inhibitor were used before UTMD treatment. We found that UTMD induced oxidative stress, upregulated the expression of miR-200c, downregulated the expression of ZEB1 and vimentin and suppressed the MDA231 cell migration. The addition of NAC and miR-200c inhibitor had an opposite impact on the expression of miR-200c and ZEB1, thus hindered the effects of UTMD on MDA231 cells EMT. In conclusion, UTMD can inhibit the EMT characteristics of MDA231 cells. The mechanism may be related to the regulation of the miR-200c/ZEB1 axis through the generation of ROS induced by UTMD, which may provide a new strategy to prevent the tumor cells EMT under UTMD treatment.

Ultrasound Assisted Exosomal Delivery of Tissue Responsive mRNA for Enhanced Efficacy and Minimized Off-Target Effects

Exosome-mediated nucleic acids delivery has been emerging as a promising strategy for gene therapy. However, the intrinsic off-target effects due to non-specific uptake of exosomes by other tissues remain the big hurdle for clinical application. In this study, we aimed to enhance the efficacy and minimize the off-target effects by simultaneously encapsulating engineered mRNA translationally activated by tissue-specific microRNA (miRNA) and increasing targeted delivery efficiency via ultrasound-targeted microbubble destruction (UTMD). Briefly, the upstream of interest transcript was engineered to harbor an internal ribosome entry site (IRES) modified with two miRNA recognition sites. In vitro reporter experiments revealed that the engineered mRNA could be encapsulated into exosomes and can be translationally activated by corresponding miRNAs in the recipient cells. By a proof-of-principle in vivo experiment, we encapsulated miR-148a (an adipose relatively specific miRNA)-responsive PGC1α mRNA into exosomes and delivered the exosomes into the adipose tissue with the aid of UTMD. Efficient PGC1α translation was activated in the adipose tissue, together with obvious browning induction. Moreover, there was much lower off-target translation of PGC1 α in lungs and other tissues. Taken together, our study establishes a novel adipose-specific exosome delivery strategy to enhance efficacy and minimize off-target effects simultaneously.

Ultrasound-targeted microbubble destruction optimized HGF-overexpressing bone marrow stem cells to repair fibrotic liver in rats

Background/aimsBone marrow mesenchymal stem cells (BMSCs) have shown their therapeutic potential in cytotherapy for liver fibrosis. However, the insufficient homing of BMSCs and undefined proliferation of BMSCs represent a significant challenge and largely limit the effective implementation. The aims of the present study were to determine whether stable expression of hepatic growth factor (HGF) in BMSCs coupled with ultrasound-targeted microbubble destruction (UTMD) technique could effectively and definitely alleviating carbon tetrachloride (CCl4)-induced liver fibrosis in rats.Materials and methodsA rat model of liver fibrosis was acquired by injection of carbon tetrachloride (CCl4). The experimental rats were randomly assigned to the four groups: normal, CCl4, BMSCs-HGF/US, and BMSCs-HGF/UTMD groups. The BMSCs, transfected by recombinant adeno-associated virus vector encoding human genome sequence of HGF (BMSCs-HGF), were transplanted in rat via the tail vein. The homing efficiency of BMSCs was observed by immunofluorescence staining. The liver function and its morphological changes were analyzed by biochemical tests and liver histology. The expression of liver fibrosis markers including α-smooth muscle actin (α-SMA), collagen I, and vimentin were examined by immunohistochemistry and quantitative real-time polymerase chain reaction.ResultsThe homing efficiency of BMSCs in the fibrotic liver was significantly greater with the application of UTMD. The biochemical markers of liver function and histopathological results showed significantly better improvement in BMSCs-HGF/UTMD group than the other groups, and the serum levels of biochemical markers returned to normal ranges in 12 weeks in this group. Furthermore, the expression levels of liver fibrosis markers (α-SMA, collagen I, and Vimentin) were all significantly lower in BMSCs-HGF/UTMD group in comparison with other groups.ConclusionsOur findings have demonstrated that stable expression of HGF in BMSCs and application of the UTMD technique facilitate the homing of BMSCs, and more importantly, which could further improve their alleviation of liver fibrosis. Therefore, these findings have an important clinical implication that AAV-BMSCs-HGF and UTMD hold promise as a novel therapeutic approach for liver fibrosis.

Ultrasound Responsive Magnetic Mesoporous Silica Nanoparticle-Loaded Microbubbles for Efficient Gene Delivery

Purpose: Gene therapy is an important therapeutic strategy for cancer. Nanoparticles are used for noninvasive gene delivery, which has great potential in tumor therapy. However, it is a challenge to construct a targeted gene delivery vector with high gene delivery efficiency, good biocompatibility, and multiple functions. Method: Herein, we designed magnetic mesoporous silica nanoparticle loading microbubbles (M-MSN@MBs) for ultrasound-mediated imaging and gene transfection. The plasmid DNA (pDNA) was encapsulated into the pores of M-MSNs. Also, the pDNA-carrying M-MSNs were loaded in the lipid microbubbles. Results: The gene vector presented good biocompatibility, DNA binding stability, ultrasound imaging performance, and magnetic responsiveness. The polyethyleneimine (PEI)-modified M-MSNs effectively protected the loaded pDNA from enzyme degradation. The cytotoxicity of M-MSNs was significantly reduced via encapsulating in lipid microbubbles. Upon the magnetic field, M-MSN@MBs were attracted to the tumor area. Then, ultrasound-targeted microbubble destruction (UTMD) not only released loaded M-MSNs but also facilitated M-MSNs delivery to tumor tissue by opening blood-tumor barrier and increasing the cytomembrane permeability, and ultimately improved the pDNA delivery efficiency. Conclusion: Our findings suggested that the developed ultrasound-responsive gene delivery system was a promising platform for gene therapy, which could noninvasively enhance tumor gene transfection.

Ultrasound-targeted microbubble destruction-mediated miR-767 inhibition suppresses tumor progression of non-small cell lung cancer.

MicroRNAs (miRNAs/miRs) have important roles in tumor progression in various human cancers. Ultrasound-targeted microbubble destruction (UTMD)-mediated gene transfection has been considered a useful tool for improving cancer treatment. The present study aimed to investigate the role of miR-767 in non-small cell lung cancer (NSCLC) and further analyze the effects of UTMD-mediated miR-767 inhibition on tumor progression. The expression of miR-767 was measured by reverse transcription-quantitative PCR. UTMD-mediated miR-767 inhibition was achieved by the co-transfection of microbubbles and miR-767 inhibitor in NSCLC cells. Cell proliferation was assessed by a CCK-8 assay and cell migration and invasion were examined by a Transwell assay. The expression of miR-767 was increased in NSCLC serum, tissues and cells compared with controls. The reduction of miR-767 in NSCLC cells led to the inhibition of cell proliferation, migration and invasion. UTMD increased the transfection efficiency of the miR-767 inhibitor in NSCLC cells, and UTMD-mediated miR-767 inhibition resulted in a more significant suppressive effect on tumor cell proliferation, migration and invasion. Taken together, the results indicated that miR-767 expression is upregulated in both NSCLC clinical samples and cells. The downregulation of miR-767 can inhibit tumor cell proliferation, migration and invasion, and these effects are further promoted by UTMD-mediated miR-767 inhibition, which indicated the potential of a UTMD-mediated miR-767 inhibition as a novel therapeutic strategy for NSCLC treatment.

Ultrasound-responsive neurotrophic factor-loaded microbubble- liposome complex: Preclinical investigation for Parkinson's disease treatment

Ultrasound-targeted microbubble destruction (UTMD) in conjunction with neurotrophic factors (NFs) gene delivery has the potential to facilitate the penetration of therapeutic genes into the brain for neuroprotective therapy against neurodegenerative diseases. We previously presented a gene delivery system that conjugates gene-carrying liposomes with microbubbles (MBs) to open the blood-brain barrier (BBB) for the delivery of genes into the brain. Since both glia cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) can protect dopaminergic neurons from neurotoxicity demonstrated in Parkinson's disease (PD) animal models, the present study seeks (1) to develop a novel gene-nanocarrier MB complex carrying BDNF or GDNF gene and (2) to protect dopaminergic neurons in a mouse model of PD via the proposed UTMD system. In the experimental design, PD animals received treatment that delivered GDNF, BDNF, or combined GDNF/BDNF in conjunction with UTMD treatment, and pathological changes in dopamine neurons were histologically examined. Rotarod assay was employed to evaluate the motor behavior. Our results demonstrate that either BDNF or GDNF gene delivery via the UTMD system provides a neuroprotective effect with evidence of improvements of behavioral deficits, decreased calcium influx, GFAP and caspase 3 expression, and rescued dopaminergic neuronal loss. Simultaneously performing GDNF/BDNF gene delivery did not show additional benefits beyond individually delivering BDNF or GDNF genes, possibly due to a hampering effect of simultaneous GDNF/BDNF competing expressions, thus dampening the overall therapeutic effect. In conclusion, these results suggest that UTMD in conjunction with delivery of GDNF or BDNF gene can synergistically serve as an effective gene therapy strategy for neurodegenerative diseases.

A novel UTMD system facilitating nucleic acid delivery into MDA-MB-231 cells.

Gene therapy is emerging as a promising method for the treatment of various diseases. The safe and efficient delivery of therapeutic nucleic acids is a gene therapy prerequisite. Ultrasound, particularly in combination with microbubbles composed of biocompatible materials such as lipid, PLGA and chitosan, is a novel non-viral tool for gene transportation. Under ultrasound irradiation, microbubbles explode and generate pores in the cell membrane. Hence, genes can enter cells more easily. In order to transfect nucleic acids into MDA-MB-231 cells in a low-cost and non-viral manner for further breast cancer gene therapy studies, we explored ultrasound targeted microbubble destruction (UTMD) technology and evaluated the efficiency and safety of the delivery of plasmid encoding enhanced green fluorescent protein (pEGFP) and a microRNA-34a (miR-34a) mimic by UTMD. Sonovitro ultrasonic apparatus was employed to generate ultrasonic field, which was developed by our group. Ultrasonic parameters, including acoustic intensity (AI), exposure time (ET) and duty cycle (DC), were optimized at 0.6 W/cm2 AI, 20 s ET and 20% DC, the cell viability was not obviously impaired. Under these conditions, the UTMD-mediated transfection efficiency of pEGFP was greater than 40%. In addition to plasmid DNA, an miR-34a mimic was also successfully introduced into the cytoplasm by UTMD and found to inhibit proliferation, induce apoptosis of MDA-MB-231 cells and regulate downstream molecules. The present study indicates that further in vivo UTMD-mediated gene therapy studies are warranted.

Applications of Micro/Nanotechnology in Ultrasound-based Drug Delivery and Therapy for Tumor.

Ultrasound has been broadly used in biomedicine for both tumor diagnosis as well as therapy. The applications of recent developments in micro/nanotechnology promote the development of ultrasound-based biomedicine, especially in the field of ultrasound-based drug delivery and tumor therapy. Ultrasound can activate nano-sized drug delivery systems by different mechanisms for ultrasound- triggered on-demand drug release targeted only at the tumor sites. Ultrasound Targeted Microbubble Destruction (UTMD) technology can not only increase the permeability of vasculature and cell membrane via sonoporation effect but also achieve in situ conversion of microbubbles into nanoparticles to promote cellular uptake and therapeutic efficacy. Furthermore, High Intensity Focused Ultrasound (HIFU), or Sonodynamic Therapy (SDT), is considered to be one of the most promising and representative non-invasive treatment for cancer. However, their application in the treatment process is still limited due to their critical treatment efficiency issues. Fortunately, recently developed micro/nanotechnology offer an opportunity to solve these problems, thus improving the therapeutic effect of cancer. This review summarizes and discusses the recent developments in the design of micro- and nano- materials for ultrasound-based biomedicine applications.

Ultrasound-mediated delivery of miRNA-122 and anti-miRNA-21 therapeutically immunomodulates murine hepatocellular carcinoma in vivo

Hepatocellular carcinoma (HCC) is the most common cause of cancer-related mortality, and patients with HCC show poor response to currently available treatments, which demands new therapies. We recently developed a synthetic microRNA-based molecularly targeted therapy for improving HCC response to chemotherapy by eliminating drug resistance. We used ultrasound-targeted microbubble destruction (UTMD) to locally deliver microRNA-loaded nanoparticles to HCC. Since the immune microenvironment plays a crucial role in HCC disease development and response to treatment, and UTMD and microRNAs have the potential to interfere with the immune system, in this study we analyzed the immunomodulatory effects of UTMD and miRNAs in HCC. We used an immunocompetent syngeneic HCC mouse model for the study. We conducted cytokine profiling in tumor, lymph nodes, and serum of animals within the first 24 h of treatment to analyze changes in the level of pro- and antitumoral cytokines. The results showed: (1) Hepa1–6 syngeneic tumors expressed HCC-related cytokines, (2) UTMD-microRNA combination therapy triggered transient cytokine storms, and (3) delivery of microRNA-122 and anti-microRNA-21 affected the immune microenvironment by decreasing the level of GM-CSF in tumors while modulating protumoral IL-1α, IL-1β, IL-5, IL-6 and IL-17 and antitumoral IL-2 and IL-12 in tumor-proximal lymph nodes, and increasing IL-2 in the serum of tumor-bearing mice. Local delivery of targeted therapy by UTMD significantly reduced the concentration of IL-12 and IL-17 in lymph nodes of treated and contralateral tumors suggesting a systemic response. ConclusionUTMD-mediated delivery of microRNA-122 and anti-microRNA-21 modulated the immune microenvironment of Hepa1–6 tumors at the level of cytokine expressions. Exploiting antitumoral immune effects could enhance the therapeutic efficacy of the proposed combination therapy for HCC.

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