Anthony Delalande

Sonoporation: Mechanistic insights and ongoing challenges for gene transfer

Microbubbles first developed as ultrasound contrast agents have been used to assist ultrasound for cellular drug and gene delivery. Their oscillation behavior during ultrasound exposure leads to transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. In this review, we summarize current state of the art concerning microbubble-cell interactions and cellular effects leading to sonoporation and its application for gene delivery. Optimization of sonoporation protocol and composition of microbubbles for gene delivery are discussed.

Accelerated Achilles tendon healing by PDGF gene delivery with mesoporous silica nanoparticles

We report the ability of amino- and carboxyl-modified MCM-41 mesoporous silica nanoparticles (MSN) to deliver gene in vivo in rat Achilles tendons, despite their inefficiency to transfect primary tenocytes in culture. We show that luciferase activity lasted for at least 2 weeks in tendons injected with these MSN and a plasmid DNA (pDNA) encoding the luciferase reporter gene. By contrast, in tendons injected with naked plasmid, the luciferase expression decreased as a function of time and became hardly detectable after 2 weeks. Interestingly, there were neither signs of inflammation nor necrosis in tendon, kidney, heart and liver of rat weekly injected with pDNA/MSN formulation during 1.5 months. Our main data concern the acceleration of Achilles tendons healing by PDGF-B gene transfer using MSN. Biomechanical properties and histological analyses clearly indicate that tendons treated with MSN and PDGF gene healed significantly faster than untreated tendons and those treated with pPDGF alone.

Sonoporation at a low mechanical index

AbstractThe purpose of this study was to investigate the physical mechanisms of sonoporation, in order to understand and improve ultrasound-assisted drug and gene delivery. Sonoporation is the transient permeabilisation and resealing of a cell membrane with the help of ultrasound and/or an ultrasound contrast agent, allowing for the trans-membrane delivery and cellular uptake of macromolecules between 10 kDa and 3 MDa. The authors studied the behaviour of ultrasound contrast agent microbubbles near cancer cells at low acoustic amplitudes. After administering an ultrasound contrast agent, HeLa cells were subjected to 6·6 MHz ultrasound with a mechanical index of 0·2 and observed with a high-speed camera. Microbubbles were seen to enter cells and rapidly dissolve. The quick dissolution after entering suggests that the microbubbles lose (part of) their shell while entering. The authors have demonstrated that lipid-shelled microbubbles can be forced to enter cells at a low mechanical index. Hence, if a therap...

Ultrasound and microbubble-assisted gene delivery in Achilles tendons: Long lasting gene expression and restoration of fibromodulin KO phenotype

The aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 μg of plasmid encoding luciferase gene in the presence of 5×10⁵ BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods.

Ultrasound-assisted microbubbles gene transfer in tendons for gene therapy

Our study aimed at evaluating the use of ultrasound-assisted microbubbles gene transfer in mice Achilles tendons. Using a plasmid encoding luciferase gene, it was found that an efficient and stable gene expression for more than two weeks was obtained when tendons were injected with 10 microg of plasmid in the presence of 5x10(5) BR14 microbubbles with the following acoustic parameters: 1 MHz, 200 kPa, 40% duty cycle and 10 min of exposure time. The rate of gene expression was 100-fold higher than that obtained with naked plasmid injected alone without ultrasound or with ultrasound in absence of microbubbles. The long term expression of transgene makes ultrasound-assisted microbubble a suitable method for gene therapy in tendons.

EFFICIENT GENE DELIVERY BY SONOPORATION IS ASSOCIATED WITH MICROBUBBLE ENTRY INTO CELLS AND THE CLATHRIN-DEPENDENT ENDOCYTOSIS PATHWAY

Microbubble oscillation at specific ultrasound settings leads to permeabilization of surrounding cells. This phenomenon, referred to as sonoporation, allows for the in vitro and in vivo delivery of extracellular molecules, including plasmid DNA. To date, the biological and physical mechanisms underlying this phenomenon are not fully understood. The aim of this study was to investigate the interactions between microbubbles and cells, as well as the intracellular routing of plasmid DNA and microbubbles, during and after sonoporation. High-speed imaging and fluorescence confocal microscopy of HeLa cells stably expressing enhanced green fluorescent protein fused with markers of cellular compartments were used for this investigation. Soft-shelled microbubbles were observed to enter cells during sonoporation using experimental parameters that led to optimal gene transfer. They interacted with the plasma membrane in a specific area stained with fluorescent cholera subunit B, a marker of lipid rafts. This process was not observed with hard-shelled microbubbles, which were not efficient in gene delivery under our conditions. The plasmid DNA was delivered to late endosomes after 3 h post-sonoporation, and a few were found in the nucleus after 6 h. Gene transfer efficacy was greatly inhibited when cells were treated with chlorpromazine, an inhibitor of the clathrin-dependent endocytosis pathway. In contrast, no significant alteration was observed when cells were treated with filipin III or genistein, both inhibitors of the caveolin-dependent pathway. This study emphasizes that microbubble-cell interactions do not occur randomly during sonoporation; microbubble penetration inside cells affects the efficacy of gene transfer at specific ultrasound settings; and plasmid DNA uptake is an active mechanism that involves the clathrin-dependent pathway.

The Orai-1 and STIM-1 Complex Controls Human Dendritic Cell Maturation

Ca2+ signaling plays an important role in the function of dendritic cells (DC), the professional antigen presenting cells. Here, we described the role of Calcium released activated (CRAC) channels in the maturation and cytokine secretion of human DC. Recent works identified STIM1 and Orai1 in human T lymphocytes as essential for CRAC channel activation. We investigated Ca2+ signaling in human DC maturation by imaging intracellular calcium signaling and pharmalogical inhibitors. The DC response to inflammatory mediators or PAMPs (Pathogen-associated molecular patterns) is due to a depletion of intracellular Ca2+ stores that results in a store-operated Ca2+ entry (SOCE). This Ca2+ influx was inhibited by 2-APB and exhibited a Ca2+permeability similar to the CRAC (Calcium-Released Activated Calcium), found in T lymphocytes. Depending on the PAMPs used, SOCE profiles and amplitudes appeared different, suggesting the involvement of different CRAC channels. Using siRNAi, we identified the STIM1 and Orai1 protein complex as one of the main pathways for Ca2+ entry for LPS- and TNF-α-induced maturation in DC. Cytokine secretions also seemed to be SOCE-dependent with profile differences depending on the maturating agents since IL-12 and IL10 secretions appeared highly sensitive to 2-APB whereas IFN-γ was less affected. Altogether, these results clearly demonstrate that human DC maturation and cytokine secretions depend on SOCE signaling involving STIM1 and Orai1 proteins.

Enhanced Achilles tendon healing by fibromodulin gene transfer

UNLABELLED Tendon injury is a major musculoskeletal disorder with a high public health impact. We propose a non-viral based strategy of gene therapy for the treatment of tendon injuries using histidylated vectors. Gene delivery of fibromodulin, a proteoglycan involved in collagen assembly was found to promote rat Achilles tendon repair in vivo and in vitro. In vivo liposome-based transfection of fibromodulin led to a better healing after surgical injury, biomechanical properties were better restored compared to untransfected control. These measures were confirmed by histological observations and scoring. To get better understandings of the mechanisms underlying fibromodulin transfection, an in vitro tendon healing model was developed. In vitro, polymer-based transfection of fibromodulin led to the best wound enclosure speed and a pronounced migration of tenocytes primary cultures was observed. These results suggest that fibromodulin non-viral gene therapy could be proposed as a new therapeutic strategy to accelerate tendon healing. FROM THE CLINICAL EDITOR Tendon injury is relatively common and healing remains unsatisfactory. In this study, the effects of liposomal-based delivery of fibromodulin gene were investigated in a rat Achilles tendon injury model. The positive results observed would provide a new therapeutic strategy in clinical setting in the future.

Improved gene transfer with histidine-functionalized mesoporous silica nanoparticles

Mesoporous silica nanoparticles (MSN) were functionalized with aminopropyltriethoxysilane (MSN-NH2) then L-histidine (MSN-His) for pDNA delivery in cells and in vivo. The complexation of pDNA with MSN-NH2 and MSN-His was first studied with gel shift assay. pDNA complexed with MSN-His was better protected from DNase degradation than with MSN-NH2. An improvement of the transfection efficiency in cells was observed with MSN-His/pDNA compared to MSN-NH2/pDNA, which could be explained by a better internalization of MSN-His. The improvement of the transfection efficiency with MSN-His was also observed for gene transfer in Achilles tendons in vivo.

Cationic microbubbles and antibiotic-free miniplasmid for sustained ultrasound–mediated transgene expression in liver

Despite the increasing number of clinical trials in gene therapy, no ideal methods still allow non-viral gene transfer in deep tissues such as the liver. We were interested in ultrasound (US)-mediated gene delivery to provide long term liver expression. For this purpose, new positively charged microbubbles were designed and complexed with pFAR4, a highly efficient small length miniplasmid DNA devoid of antibiotic resistance sequence. Sonoporation parameters, such as insonation time, acoustic pressure and duration of plasmid injection were controlled under ultrasound imaging guidance. The optimization of these various parameters was performed by bioluminescence optical imaging of luciferase reporter gene expression in the liver. Mice were injected with 50μg pFAR4-LUC either alone, or complexed with positively charged microbubbles, or co-injected with neutral MicroMarker™ microbubbles, followed by low ultrasound energy application to the liver. Injection of the pFAR4 encoding luciferase alone led to a transient transgene expression that lasted only for two days. The significant luciferase signal obtained with neutral microbubbles decreased over 2days and reached a plateau with a level around 1 log above the signal obtained with pFAR4 alone. With the newly designed positively charged microbubbles, we obtained a much stronger bioluminescence signal which increased over 2days. The 12-fold difference (p<0.05) between MicroMarker™ and our positively charged microbubbles was maintained over a period of 6months. Noteworthy, the positively charged microbubbles led to an improvement of 180-fold (p<0.001) as regard to free pDNA using unfocused ultrasound performed at clinically tolerated ultrasound amplitude. Transient liver damage was observed when using the cationic microbubble-pFAR4 complexes and the optimized sonoporation parameters. Immunohistochemistry analyses were performed to determine the nature of cells transfected. The pFAR4 miniplasmid complexed with cationic microbubbles allowed to transfect mostly hepatocytes compared to its co-injection with MicroMarker™ which transfected more preferentially endothelial cells.