New development and application of ultrasound targeted microbubble destruction in gene therapy and drug delivery.

Ultrasound is a common used technique for clinical imaging. In recent years, with the advances in preparation technology of microbubbles and the innovations in ultrasound imaging, ultrasound is no longer confined to detection of tissue perfusion, but extends to specific ultrasound molecular imaging and target therapy gradually. With the development of research, ultrasound molecular imaging and target therapy have made great progresses. Targeted microbubbles for molecular imaging are achieved by binding target molecules, specific antibody or ligand to the surface of microbubbles to obtain specific imaging by attaching to target tissues. Meanwhile, it can also achieve targeting gene therapy or drug delivery by ultrasound targeted microbubble destruction (UTMD) mediating genes or drugs to specific target sites. UTMD has a number of advantages, such as target-specific, highly effective, non-invasivity, relatively low-cost and no radiation, and has broad application prospects, which is regarded as one hot spot in medical studies. We reviewed the new development and application of UTMD in gene therapy and drug delivery in this paper. With further development of technology and research, the gene or drug delivery system and related methods will be widely used in application and researches.

Optimization and apoptosis induction by RNAi with UTMD technology in vitro.

Apoptosis induction by short hairpin RNA (shRNA) expression vectors may be an efficient and promising strategy for cancer gene therapy. Ultrasound-targeted microbubble destruction (UTMD) is an appealing technique; however, there few data are available to demonstrate the feasibility and to optimize the methodology for this technology. The aim of this study was to optimize this technique and to elucidate the effects on gene inhibition and apoptosis induction in vitro. Human cervical cancer cell lines were obtained and cultured.shRNA vectors were constructed, and the UTMD technique was examined to determine whether or not it was suitable for shRNA transfection into cells. Cells were then examined using flow cytometry. The results revealed that the optimal irradiation parameters obtained higher transfection efficiency and did not affect the integrity of plasmid DNA. We concluded that survivin downregulation with shRNA expression vectors, mediated by the optimal UTMD parameters, markedly induced cell apoptosis and caused cell cycle arrest, laying a foundation for further investigation of this cancer therapy.

Augmentation of transgenic expression by ultrasound­mediated liposome microbubble destruction.

Non-invasive, efficient and tissue-specific transgenic technologies could be valuable in gene therapy. Although non-viral carriers may be safer and cheaper, they have a much lower transfection efficiency than viral gene carriers. The present study was designed to test the transgenic expression and safety of red fluorescent protein (RFP) in HeLa cells in vitro and in transplanted tumors of nude mice in vivo under ultrasound-mediated liposome microbubble destruction (UMLMD) conditions. Plasmids containing RFP were gently mixed with liposome microbubbles (LMs). The mixture was added to HeLa cells or injected into BALB/c mice by the tail vein under various ultrasound exposure and LM parameters, and then the transfection efficiencies were examined. The results in vivo and in vitro demonstrated that, following a comparison of the plasmid group, the ultrasound + plasmid group and the LM + plasmid group, UMLMD significantly increased the transgenic expression (P<0.01) without causing any apparent detrimental effect. From the study, we concluded that UMLMD could be a non-invasive, effective and promising non-viral technique for gene therapy and transgenic research.

Ultrasound- and liposome microbubble-mediated targeted gene transfer to cardiomyocytes in vivo accompanied by polyethylenimine.

OBJECTIVES: Gene transfer to cardiomyocytes in vivo has received much research attention in the last decade but remains a substantial hurdle. Gene transfer using ultrasound-targeted microbubble destruction is a promising tool for gene therapy. Little data have shown the feasibility and optimization of this method for primary myocardial disease. In this study, we sought to determine the feasibility and efficiency of in vivo gene transfer to the myocardium mediated by ultrasound-targeted microbubble destruction accompanied by polyethylenimine. METHODS: Three plasmids (luciferase reporter, red fluorescent protein reporter, and enhanced green fluorescent protein reporter) were used in this study. The ultrasound parameters were also optimized. A solution containing phosphate-buffered saline, a plasmid, plasmid complex, or polyethylenimine/plasmid, and liposome microbubbles was injected via a tail vein with (study) or without (control) transthoracic ultrasound irradiation. The efficiency of reporter gene transfer was determined by detection of luciferase activity or microscopy, and histologic investigations of the tissue specimens were performed. RESULTS: Ultrasound-targeted microbubble destruction significantly increased luciferase activity in vivo compared to plasmids and microbubbles alone (P < .001). More importantly, the increase in transgene expression was significantly related to ultrasound-targeted microbubble destruction in the presence of polyethylenimine (P < .001). In addition, fluorescein expression was present in all sections that received ultrasound-targeted microbubble destruction. The fluorescent reporter genes and luciferase plasmid all had similar results. Regardless of ultrasound exposure, expression in other organs was close to a background level except for the liver and lung. Hematoxylin-eosin staining showed no notable myocardial injury or death in control and treated mice. CONCLUSIONS: An atraumatic targeted gene delivery technique based on ultrasound-targeted microbubble destruction and polyethylenimine has been developed to transfect cardiomyocytes in vivo. If a suitable target gene is added, the novel technique could be highly effective in many kinds of heart disease.

Enhancing microRNA transfection to inhibit survivin gene expression and induce apoptosis: could it be mediated by a novel combination of sonoporation and polyethylenimine?

Apoptosis is a physiologically essential mechanism of cell and plays an important role in reducing the development and progression of tumors. The appealing strategy for cancer therapy is to target the lesions that induce apoptosis in cancer cells. Survivin, the smallest member of the mammalian inhibitors of the apoptosis protein family, is upregulated in various malignancies to protect cells from apoptosis. Survivin knockdown could induce cancer cell apoptosis and inhibit tumor-angiogenesis. Survivin expression would be silenced by microRNA (miRNA)-mediated RNA interference. However, noninvasive and tissue-specific gene delivery techniques remain absent recently and the utilizations of miRNA expression vectors have been limited by inefficient delivery technique, especially in vivo. On the other hand, safe and promising technologies of gene transfection would be valuable in clinical gene therapy. Successful treatment of gene transfer method would lead to a new and readily available approach in the anticancer research. Sonoporation is an alternative technique of gene delivery that uses ultrasound targeted microbubble destruction to create pores in the cell membrane. Based on our previous studies, in this article, we postulated that the transfection of miRNA could be mediated by the combination of sonoporation and polyethylenimine (PEI) which was one of the most effective poly-cationic gene vectors and enhance the endocytosis of plasmids DNA and hypothesized that the gene silencing and apoptosis induction with miRNA targeting human Survivin would be improved by this novel technique. In our opinion, this novel combination of sonoporation and PEI could enhance targeted gene delivery effectively and might be a feasible, novel candidate for gene therapy.

Targeted gene delivery in tumor xenografts by the combination of ultrasound-targeted microbubble destruction and polyethylenimine to inhibit survivin gene expression and induce apoptosis.

BACKGROUND: Noninvasive and tissue-specific technologies of gene transfection would be valuable in clinical gene therapy. This present study was designed to determine whether it could enhance gene transfection in vivo by the combination of ultrasound-targeted microbubble destruction (UTMD) with polyethylenimine (PEI) in tumor xenografts, and illuminate the effects of gene silencing and apoptosis induction with short hairpin RNA (shRNA) interference therapy targeting human survivin by this novel technique. METHODS: Two different expression vectors (pCMV-LUC and pSIREN) were incubated with PEI to prepare cationic complexes (PEI/DNA) and confirmed by the gel retardation assay. Human cervical carcinoma (Hela) tumors were planted subcutaneously in both flanks of nude mice. Tumor-bearing mice were administered by tail vein with PBS, plasmid, plasmid and SonoVue microbubble, PEI/DNA and SonoVue microbubble. One tumor was exposed to ultrasound irradiation, while the other served as control. The feasibility of targeted delivery and tissue specificity facilitated by UTMD and PEI were investigated. Moreover, immunohistochemistry analyses about gene silencing and apoptosis induction were detected. RESULTS: Electrophoresis experiment revealed that PEI could condense DNA efficiently. The application of UTMD significantly increases the tissue transfection. Both expression vectors showed that gene expressions were present in all sections of tumors that received ultrasound exposure but not in control tumors. More importantly, the increases in transgene expression were related to UTMD with the presence of PEI significantly. Silencing of the survivin gene could induce apoptosis effectively by downregulating survivin and bcl-2 expression, also cause up-regulation of bax and caspase-3 expression. CONCLUSIONS: This noninvasive, novel combination of UTMD with PEI could enhance targeted gene delivery and gene expression in tumor xenografts at intravenous administration effectively without causing any apparently adverse effect, and might be a promising candidate for gene therapy. Silencing of survivin gene expression with shRNA could be facilitated by this non-viral technique, and lead to significant cell apoptosis.