Effective intraperitoneal gene transfection system using nanobubbles and ultrasound irradiation.
Ontology highlight
ABSTRACT:
SUBMITTER: Nishimura K
PROVIDER: S-EPMC8241157 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:In this study, stable nano-sized bubbles (nanobubbles [NBs]) were produced using the mechanical agitation method in the presence of perfluorocarbon gases. NBs made with perfluoropropane had a smaller size (around 400 nm) compared to that of those made with perfluorobutane or nitrogen gas. The lipid concentration in NBs affected both their initial size and post-formulation stability. NBs formed with a final lipid concentration of 0.5 mg/ml tended to be more stable, having a uniform size distribution for 24 h at room temperature and 50 h at 4 °C. In vitro gene expression revealed that NBs/pDNA in combination with ultrasound (US) irradiation had significantly higher transfection efficacy in colon C26 cells. Moreover, for in vivo gene transfection in mice left limb muscles, there was notable local transfection activity by NBs/pDNA when combined with US irradiation. In addition, the aged NBs kept at room temperature or 4 °C were still functional at enhancing gene transfection in mice. We succeeded in preparing stable NBs for efficient in vivo gene transfection, using the mechanical agitation method.
| S-EPMC8241156 | biostudies-literature
Project description:This paper aims to evaluate the efficacy of intravitreal ultrasound (US) irradiation for green fluorescent protein (GFP) plasmid transfer into the rabbit retina using a miniature US transducer. Intravitreal US irradiation was performed by a slight modification of the transconjunctival sutureless vitrectomy system utilizing a small probe. After vitrectomy, the US probe was inserted through a scleral incision. A mixture of GFP plasmid (50??L) and bubble liposomes (BLs; 50??L) was injected into the vitreous cavity, and US was generated to the retina using a SonoPore 4000. The control group was not exposed to US. After 72?h, the gene-transfer efficiency was quantified by counting the number of GFP-positive cells. The retinas that received plasmid, BL, and US showed a significant increase in the number (average ± SEM) of GFP-positive cells (32 ± 4.9; n = 7; P < 0.01?). No GFP-positive cells were observed in the control eyes (n = 7). Intravitreal retinal US irradiation can transfer the GFP plasmid into the retina without causing any apparent damage. This procedure could be used to transfer genes and drugs directly to the retina and therefore has potential therapeutic value.
| S-EPMC3307015 | biostudies-literature
Project description:A prevailing problem in neuroscience is the fast and targeted delivery of DNA into selected neurons. The development of an appropriate methodology would enable the transfection of multiple genes into the same cell or different genes into different neighboring cells as well as rapid cell selective functionalization of neurons. Here, we show that optimized femtosecond optical transfection fulfills these requirements. We also demonstrate successful optical transfection of channelrhodopsin-2 in single selected neurons. We extend the functionality of this technique for wider uptake by neuroscientists by using fast three-dimensional laser beam steering enabling an image-guided "point-and-transfect" user-friendly transfection of selected cells. A sub-second transfection timescale per cell makes this method more rapid by at least two orders of magnitude when compared to alternative single-cell transfection techniques. This novel technology provides the ability to carry out large-scale cell selective genetic studies on neuronal ensembles and perform rapid genetic programming of neural circuits.
| S-EPMC3836031 | biostudies-literature
Project description:We previously developed anionic ternary bubble lipopolyplexes, an ultrasound-responsive carrier, expecting safe and efficient gene transfection. However, bubble lipopolyplexes have a low capacity for echo gas (C3F8) encapsulation (EGE) in nonionic solution such as 5% glucose. On the other hand, we were able to prepare bubble lipopolyplexes by inserting phosphate-buffered saline before C3F8 encapsulation. Surface charge regulation (SCR) by electrolytes stabilizes liposome/plasmid DNA (pDNA) complexes by accelerated membrane fusion. Considering these facts, we hypothesized that SCR by electrolytes such as NaCl would promote C3F8 encapsulation in bubble lipopolyplexes mediated by accelerated membrane fusion. We defined this hypothesis as SCR-based EGE (SCR-EGE). Bubble lipopolyplexes prepared by the SCR-EGE method (SCR-EGE bubble lipopolyplexes) are expected to facilitate the gene transfection because of the high amount of C3F8. Therefore, we applied these methods for gene delivery to the brain and evaluated the characteristics of transgene expression in the brain.First, we measured the encapsulation efficiency of C3F8 in SCR-EGE bubble lipopolyplexes. Next, we applied these bubble lipopolyplexes to the mouse brain; then, we evaluated the transfection efficiency. Furthermore, three-dimensional transgene distribution was observed using multicolor deep imaging.SCR-EGE bubble lipopolyplexes had a higher C3F8 content than conventional bubble lipopolyplexes. In terms of safety, SCR-EGE bubble lipopolyplexes possessed an anionic potential and showed no aggregation with erythrocytes. After applying SCR-EGE bubble lipopolyplexes to the brain, high transgene expression was observed by combining with ultrasound irradiation. As a result, transgene expression mediated by SCR-EGE bubble lipopolyplexes was observed mainly on blood vessels and partially outside of blood vessels.The SCR-EGE method may promote C3F8 encapsulation in bubble lipopolyplexes, and SCR-EGE bubble lipopolyplexes may be potent carriers for efficient and safe gene transfection in the brain, especially to the blood vessels.
| S-EPMC5907898 | biostudies-other
Project description:Ultrasound application in the presence of microbubbles has shown great potential for non-viral gene transfection via transient disruption of cell membrane (sonoporation). However, improvement of its efficiency has largely relied on empirical approaches without consistent and translatable results. The goal of this study is to develop a rational strategy based on new results obtained using novel experimental techniques and analysis to improve sonoporation gene transfection. In this study, we conducted experiments using targeted microbubbles that were attached to cell membrane to facilitate sonoporation. We quantified the dynamic activities of microbubbles exposed to pulsed ultrasound and the resulting sonoporation outcome, and identified distinct regimes of characteristic microbubble behaviors: stable cavitation, coalescence and translation, and inertial cavitation. We found that inertial cavitation generated the highest rate of membrane poration. By establishing direct correlation of ultrasound-induced bubble activities with intracellular uptake and pore size, we designed a ramped pulse exposure scheme for optimizing microbubble excitation to improve sonoporation gene transfection. We implemented a novel sonoporation gene transfection system using an aqueous two phase system (ATPS) for efficient use of reagents and high throughput operation. Using plasmids coding for the green fluorescence protein (GFP), we achieved a sonoporation transfection efficiency in rate aortic smooth muscle cells (RASMCs) of 6.9%±2.2% (n=9), comparable with lipofection (7.5%±0.8%, n=9). Our results reveal characteristic microbubble behaviors responsible for sonoporation and demonstrated a rational strategy to improve sonoporation gene transfection.
| S-EPMC3758692 | biostudies-literature
Project description:Research in the field of high-intensity focused ultrasound (HIFU) for intracranial gene therapy has greatly progressed over the years. However, limitations of conventional HIFU still remain. That is, genes are required to cross the blood-brain barrier (BBB) in order to reach the neurological disordered lesion. In this study, we introduce a novel direct intracranial gene delivery method, bypassing the BBB using human serum albumin-based nanobubbles (NBs) injected through a less invasive intrathecal route via lumbar puncture, followed by intracranial irradiation with low-frequency ultrasound (LoFreqUS). Focusing on both plasmid DNA (pDNA) and messenger RNA (mRNA), our approach utilizes LoFreqUS for deeper tissue acoustic penetration and enhancing gene transfer efficiency. This drug delivery method could be dubbed as the "Spinal Back-Door Approach", an alternative to the "front door" BBB opening method. Experiments showed that NBs effectively responded to LoFreqUS, significantly improving gene transfer in vitro using U-87 MG cell lines. In vivo experiments in mice demonstrated significantly increased gene expression with pDNA; however, we were unable to obtain conclusive results using mRNA. This novel technique, combining albumin-based NBs and LoFreqUS offers a promising, efficient, targeted, and non-invasive solution for central nervous system gene therapy, potentially transforming the treatment landscape for neurological disorders.
| S-EPMC10856598 | biostudies-literature
Project description:Because of their size (1-10 μm), microbubble-based drug delivery agents suffer from confinement to the vasculature, limiting tumor penetration and potentially reducing the drug efficacy. Nanobubbles (NBs) have emerged as promising candidates for ultrasound-triggered drug delivery because of their small size, allowing drug delivery complexes to take advantage of the enhanced permeability and retention effect. In this study, we describe a simple method for production of nested-nanobubbles (Nested-NBs) by encapsulation of NBs (∼100 nm) within drug-loaded liposomes. This method combines the efficient and well-established drug-loading capabilities of liposomes while utilizing NBs as an acoustic trigger for drug release. Encapsulation was characterized using transmission electron microscopy with an encapsulation efficiency of 22 ± 2%. Nested-NBs demonstrated echogenicity using diagnostic B-mode imaging, and acoustic emissions were monitored during high-intensity focused ultrasound (HIFU) in addition to monitoring of model drug release. Results showed that although the encapsulated NBs were destroyed by pulsed HIFU [peak negative pressure (PNP) 1.54-4.83 MPa], signified by loss of echogenicity and detection of inertial cavitation, no model drug release was observed. Changing modality to continuous wave (CW) HIFU produced release across a range of PNPs (2.01-3.90 MPa), likely because of a synergistic effect of mechanical and increased thermal stimuli. Because of this, we predict that our NBs contain a mixed population of both gaseous and liquid core particles, which upon CW HIFU undergo rapid phase conversion, triggering liposomal drug release. This hypothesis was investigated using previously described models to predict the existence of droplets and their phase change potential and the ability of this phase change to induce liposomal drug release.
| S-EPMC7333229 | biostudies-literature
Project description:Ultrasound (US) imaging is a widely used imaging technique. The use of US contrast agents such as microbubbles, which consist of phospholipids and are filled with perfluorocarbon gases, has become an indispensable component of clinical US imaging, while molecular US imaging has recently attracted significant attention in combination with efficient diagnostics. The avidin-biotin interaction method is frequently used to tether antibodies to microbubbles, leading to the development of a molecular targeting US imaging agent. However, avidin still has limitations such as immunogenicity. We previously reported that lipid-based nanobubbles (NBs) containing perfluorocarbon gas are suitable for US imaging and gene delivery. In this paper, we report on the development of a novel antibody modification method for NBs using Fc-region-binding polypeptides derived from protein A/G. First, we prepared anti-CD146 antibody-modified NBs using this polypeptide, resulting in high levels of attachment to human umbilical vein endothelial cells expressing CD146. To examine their targeting ability and US imaging capability, the NBs were administered to tumor-bearing mice. The contrast imaging of antibody-modified NBs was shown to be prolonged compared with that of non-labeled NBs. Thus, this antibody modification method using an Fc-binding polypeptide may be a feasible tool for developing a next-generation antibody-modified US imaging agent.
| S-EPMC6631014 | biostudies-literature
Project description:Herein we showcase the potential of ultrasound-responsive nanobubbles in enhancing macromolecular permeation through layers of the retina, ultimately leading to significant and direct intracellular delivery; this being effectively demonstrated across three relevant and distinct retinal cell lines. Stably engineered nanobubbles of a highly homogenous and echogenic nature were fully characterised using dynamic light scattering, B-scan ultrasound and transmission electron microscopy (TEM). The nanobubbles appeared as spherical liposome-like structures under TEM, accompanied by an opaque luminal core and darkened corona around their periphery, with both features indicative of efficient gas entrapment and adsorption, respectively. A nanobubble +/- ultrasound sweeping study was conducted next, which determined the maximum tolerated dose for each cell line. Detection of underlying cellular stress was verified using the biomarker heat shock protein 70, measured before and after treatment with optimised ultrasound. Next, with safety to nanobubbles and optimised ultrasound demonstrated, each human or mouse-derived cell population was incubated with biotinylated rabbit-IgG in the presence and absence of ultrasound +/- nanobubbles. Intracellular delivery of antibody in each cell type was then quantified using Cy3-streptavidin. Nanobubbles and optimised ultrasound were found to be negligibly toxic across all cell lines tested. Macromolecular internalisation was achieved to significant, yet varying degrees in all three cell lines. The results of this study pave the way towards better understanding mechanisms underlying cellular responsiveness to ultrasound-triggered drug delivery in future ex vivo and in vivo models of the posterior eye.
| S-EPMC5444814 | biostudies-literature
Project description:PurposeThe purpose of this study was to investigate whether ultrasound-targeted cationic microbubble destruction could effectively deliver endostatin-green fluorescent protein (ES-GFP) plasmids to human retinal vascular endothelial cells (HRECs).MethodsCationic microbubbles (CMBs) were prepared and then compared with neutral microbubbles (NMBs) and liposomes. First, the two types of microbubbles were characterized in terms of size and zeta potential. The cell viability of the HRECs was measured using the 3-(4,5-dimthylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) assay. The transcription and expression of endostatin, VEGF, Bcl-2, and Bcl-xl were measured via quantitative real-time PCR (qPCR) and western blotting, respectively.ResultsCMBs differed significantly from NMBs in terms of the zeta potential, but no differences in size were detected. Following ultrasound-targeted microbubble destruction (UTMD)-mediated gene therapy, the transcription and expression of endostatin were highest in the CMB group (p<0.05), while the transcription and expression of VEGF, Bcl-2, and Bcl-xl were lowest compared with the other groups. Moreover, the inhibition of HREC growth was enhanced following treatment with CMBs compared with NMBs or liposomes in vitro (p<0.01).ConclusionsThis study demonstrated that ultrasound-mediated cationic microbubbles could enhance the transfection efficiency of ES-GFP, which had obvious impacts on the inhibition of the growth process of HRECs in vitro. These results suggest that the combination of UTMD and ES-GFP compounds might be a useful tool for gene therapy targeting retinal neovascularization.
| S-EPMC4548789 | biostudies-literature