Insight concerning the mechanism of therapeutic ultrasound facilitating gene delivery: increasing cell membrane permeability or interfering with intracellular pathways?

Nonviral gene delivery methods encounter major barriers in plasmid DNA (pDNA) trafficking toward the nucleus. The present study aims to understand the role and contribution of therapeutic ultrasound (TUS), if any, in pDNA trafficking in primary cells such as fibroblasts and cell lines (e.g., baby hamster kidney [BHK]) during the transfection process. Using compounds that alter the endocytic pathways and the cytoskeletal network, we show that after TUS application, pDNA trafficking in the cytoplasm is not mediated by endocytosis or by the cytoskeletal network. Transfection studies and confocal analyses showed that the actin fibers impeded TUS-mediated transfection in BHK cells, but not in fibroblasts. Flow cytometric analyses indicated that pDNA uptake by cells occurs primarily when the pDNA is added before and not after TUS application. Taken together, these results suggest that TUS by itself operates as a mechanical force driving the pDNA through the cell membrane, traversing the cytoplasmic network and into the nucleus.

Effect of peptides bearing nuclear localization signals on therapeutic ultrasound mediated gene delivery.

BACKGROUND: One of the major limitations of nonviral gene delivery methods is nuclear transport of plasmid DNA (pDNA). Peptides bearing nuclear localization signal (NLS) were shown to mediate nuclear import of macromolecules. We have explored the use of cell-permeable peptides (CPP) bearing NLS sequences to enhance transfection mediated by a nonviral approach: therapeutic ultrasound (TUS). METHODS: Two CPP-NLS peptides which differ in the location of the NLS relative to the CPP were used: S4 13-PV and PV-S4 13. The peptides were attached to pDNA using electrostatic interactions. Gel-electrophoresis and fluorescent assays were performed to evaluate pDNA-peptide interactions and condensation effects. Confocal microscopy was used to evaluate pDNA-peptide interaction inside cells. Transfection studies were conducted with the luciferase gene, using pDNA-peptides alone, or with the application of TUS. RESULTS: Attachment of both peptides to pDNA condensed the pDNA, with higher affinity for the S4(13)-PV peptide. This interaction protected pDNA from endonucleases, but was also reversible. Both peptides mediated pDNA delivery to cell cytoplasm, but less significantly to the nucleus. Thus, both peptides produced transfection in cells, when added after incubation with DNA, with higher transfection-level for PV-S4 13. Application of TUS increased transfection mediated by these peptides, but was not higher compared to transfection using TUS and pDNA alone. CONCLUSIONS: This study suggests that CPP-NLS peptides may be used for condensing pDNA and bringing it into the cell cytoplasm, but their ability to mediate nuclear import of pDNA is insignificant.

Therapeutic ultrasound facilitates antiangiogenic gene delivery and inhibits prostate tumor growth.

Gene therapy clinical trials are limited due to several hurdles concerning the type of vector used, particularly, the viral vectors, and transfection efficacy when non-viral vectors are used. Therapeutic ultrasound is a promising non-viral technology that can be used in the clinical setting. Here, for the first time, we show the efficacy of therapeutic ultrasound to deliver genes encoding for hemopexin-like domain fragment (PEX), an inhibitor of angiogenesis, to prostate tumors in vivo. Moreover, the addition of an ultrasound contrast agent (Optison) to the transfection process was evaluated. Prostate cancer cells and endothelial cells (EC) were transfected in vitro with cDNA-PEX using therapeutic ultrasound alone (TUS + pPEX) or with Optison (TUS + pPEX + Optison). The biological activity of the expressed PEX was assessed using proliferation, migration, and apoptosis assays done on EC and prostate cancer cells. TUS + pPEX + Optison led to the inhibition of EC and prostate cancer cell proliferation (<65%), migration (<50%), and an increase in apoptosis. In vivo, C57/black mice were inoculated s.c. with prostate cancer cells. The tumors were treated with TUS + pPEX and TUS + pPEX + Optison either once or repeatedly. Tumor growth was evaluated, after which histology and immunohistochemistry analyses were done. A single treatment of TUS + pPEX led to a 35% inhibition in tumor growth. Using TUS + PEX + Optison led to an inhibition of 50%. Repeated treatments of TUS + pPEX + Optison were found to significantly (P < 0.001) inhibit prostate tumor growth by 80%, along with the angiogenic indices, with no toxicity to the surrounding tissues. These results depict the efficacy of therapeutic ultrasound as a non-viral technology to efficiently deliver genes to tumors in general, and to deliver angiogenic inhibitors to prostate cancer in particular.

The effects of albumin-coated microbubbles in DNA delivery mediated by therapeutic ultrasound.

The application of therapeutic ultrasound (TUS) in combination with contrast agents (USCA) to mediate gene delivery relies on the understanding of the bioeffects involved. The objective of this study was to evaluate the various bioeffects generated by albumin-coated microbubbles: Optison, an USCA, when applied with TUS operated for 10-30 min, on cells and on DNA transfection. This study reveals that Optison microbubbles were still acoustically active after long-term TUS application of 30 min. Optison enhances TUS-gene transfection by increasing the number of plasmids in the cells and also by distributing the plasmids to more cells, without significant decrease in cell viability. Optison also interacts with the DNA to further enhance transfection in a mechanism not necessarily involving cavitation. However, Optison affects mainly the cell cytoplasmatic membrane, without interfering with DNA intracellular trafficking. Using high-resolution scanning electron microscopy (HRSEM), the bioeffects on cell membrane induced by TUS-Optison were observed, demonstrating that Optison lead to a rougher surface, characterized by depressions that are reversible within 24-h post TUS. These effects are different from those observed when only TUS was applied. The findings from this study suggest that albumin-coated microbubbles enhances transfection when using TUS for 10-30 min, and that microbubbles play a major role in elevating cell transfection level and efficiency.

Therapeutic ultrasound optimization for gene delivery: a key factor achieving nuclear DNA localization.

When applying therapeutic-ultrasound (TUS) for gene-delivery, there is a great need to understand the contribution of different parameters to the transfection process. The aim of this study is to optimize a wide range of parameters associated with the TUS system concurrent with parameters associated with the transfection, achieving high transfection level and efficiency (total number of cells), while localizing the DNA in the nucleus. Exposure of different cell-types (BHK, LNCaP, BCE) to TUS resulted in high gene expression (1200 fold) and efficiency (28%) with minimal loss in cell viability (<20%). The optimal transfection level and efficiency was achieved using TUS at 2 W/cm2 (0.159 MPa), 30% duty cycle (DC) for 30 min (1080 J/cm2), by placing the transducer above the cells. Long-term TUS application was found to overcome the rate-limiting step of this technology-driving DNA to the cell nucleus. The effect of cell density and DNA concentrations were studied. Increasing DNA concentration contributes to the increase in total gene expression, but not necessarily to transfection efficiency. Our findings support the feasibility of TUS to deliver genes to cells and contribute to the understanding of wide range of parameters that affect the capability of TUS to efficiently deliver genes.

Continuous delivery of endogenous inhibitors from poly(lactic-co-glycolic acid) polymeric microspheres inhibits glioma tumor growth.

PURPOSE: There is an urgent need for modalities that can localize and prolong the administration of the antitumor agents, particularly antiangiogenic, to achieve long-term tumor inhibition. However, one of the major obstacles is designing a device in which the biological activity of sensitive endogenous inhibitors is retained. We have designed a biodegradable polymeric device, which provides a unique and practical means of localizing and continuously delivering hemopexin (PEX) or platelet factor 4 fragment (PF-4/CTF) at the tumor site while maintaining their biological activity. The potential and efficacy of this system is shown in vitro and in vivo in a human glioma mouse model. EXPERIMENTAL DESIGN: Polymeric microspheres made of poly(lactic-co-glycolic acid) (PLGA) were loaded with very low amounts of PEX and PF-4/CTF. The release profiles of these factors from PLGA and their biological activity were confirmed in vitro using proliferation assays done on endothelial and tumor cells. Tumor inhibition using this system was studied in nude mice bearing a human s.c. glioma. RESULTS: PEX and PF-4/CTF released in vitro from PLGA microspheres were biologically active and significantly inhibited the proliferation of human umbilical vein endothelial cells, bovine capillary endothelial cells, and U87-MG cells. A single local s.c. injection of PLGA microspheres loaded with low amounts of PEX or PF-4/CTF resulted in an 88% and 95% reduction in glioma tumor volume 30 days post-treatment. Immunohistochemical analysis of the treated tumors showed a marked decrease in tumor vessel density compared with untreated tumors. CONCLUSION: Our findings show that polymeric microspheres are a very promising approach to locally and efficiently deliver endogenous inhibitors to the tumor site leading to a significant inhibition of the tumor.