Positively charged microbubbles to target nucleic acid delivery with ultrasound

Congrès sous l’égide de la Société Française de Génie Biologique et Médical (SFGBM).National audienceNucleic acid delivery in vivo via physical means or non-viral vectors still need improvements in particular to reach deep tissues. Sonoporation is highly advantageous for this purpose as ultrasound can be focalised to a particular tissue leading to targeted gene delivery, without alteration of the environing tissues. Few examples of transfection using cationic microbubbles (MBs) and DNA have been reported. We ought to improve the existing systems by developing positively charged MBs able to oscillate in order to follow their fate in vivo and gain in understanding of the pharmacokinetic of the MBs, then target DNA delivery with ultrasound. After optimisation, we obtained MBs in the 1-3 !m range (98% below 10 !m) able to adsorb nucleic acid on their surface. In vitro parameters have been optimised to obtain in vitro transfection both with DNA and siRNA. The systemic injection in mice led to the observation of these MBs in the liver in less than 10 seconds. This investigation allows to address the key parameters to tentatively obtain reproducible gene transfection in vivo

Ultrasound-assisted microbubbles gene transfer in tendons for gene therapy

International audienceOur 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 mu g of plasmid in the presence of 5 x 10(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

Characterization of Positively Charged Lipid Shell Microbubbles with Tunable Resistive Pulse Sensing (TRPS) Method: A Technical Note

International audienceMicrobubbles are polydisperse microparticles. Their size distribution cannot be accurately measured from the current methods used, such as optical microscopy, electrical sensing or light scattering. Indeed, these techniques present some limitations when applied to microbubbles, which prompted us to investigate the use of an alternative technique: tunable resistive pulse sensing (TRPS). This technique is based on the principle of the Coulter counter with the advantage of being more flexible compared to other methods using this principle, since the flow rate, the potential difference and the pore size can be modulated. The main limitation of TRPS is that more than one size of nanopore membrane is required to obtain the full size distribution of polydisperse microparticles. To evaluate this technique, the concentration and the size distribution of positively charged microbubbles were studied using TRPS and compared to data obtained using optical microscopy. We describe herein the parameters required for the accurate measurement of microbubble concentration and size distribution by TRPS and present a statistical comparison of the data obtained by TRPS and optical microscopy

Arginine reduces bacterial invasion in rats with head injury

International audienceOBJECTIVES:The benefit of arginine in intensive care unit patients with severe sepsis is still controversial. An excessive supply of arginine could lead to an overproduction of nitric oxide and could be responsible for septic shock and multiorgan failure. However, this claim is not supported by any experimental or clinical data. We set out to determine whether an enteral supply of arginine would modulate bacterial invasion in rats with head injury.METHODS:Male Sprague-Dawley rats with head injury were randomized into two groups. Group 1 included rats with head injury fed a standard enteral nutrition (Sondalis HP, n = 10) and group 2 included rats with head injury fed the standard enteral nutrition plus arginine (4 g/kg/d, n = 11). Two days after head injury, the rats received a single enteral bolus of luminescent Escherichia coli Xen 14. Bacterial proliferation was evaluated in vivo at time + 2 hrs and time + 6 hrs after E. coli challenge. Four days after head injury, blood was sampled for arginine and fibrinogen assay. Muscles, intestine, spleen, and thymus were removed and weighed.RESULTS:There was no mortality in either group. The luminescence signal was similar in the two groups at time +2 hrs (group 1: 414 [5-823] vs. group 2: 496 [0.1-993] (median value[min-max]; not significant) and was significantly lower at time +6 hrs in group 2 (group 1: 71 [0-142] vs. group 2: 8.5 [0-17]; p = .026). Arginine treatment did not improve any nutritional parameters.CONCLUSIONS:Arginine was not responsible for mortality in rats with head injury with infectious complications and reduced the intensity of bacterial invasion

Optimisation of intradermal DNA electrotransfer for immunisation

The development of DNA vaccines requires appropriate delivery technologies. Electrotransfer is one of the most efficient methods of non-viral gene transfer. In the present study, intradermal DNA electrotransfer was first optimised. Strong effects of the injection method and the dose of DNA on luciferase expression were demonstrated. Pre-treatments were evaluated to enhance DNA diffusion in the skin but neither hyaluronidase injection nor iontophoresis improved efficiency of intradermal DNA electrotransfer. Then, DNA immunisation with a weakly immunogenic model antigen, luciferase, was investigated. After intradermal injection of the plasmid encoding luciferase, electrotransfer (HV 700 V/cm 100 micros, LV 200 V/cm 400 ms) was required to induce immune response. The response was Th1-shifted compared to immunisation with the luciferase recombinant protein. Finally, DNA electrotransfer in the skin, the muscle or the ear pinna was compared. Muscle DNA electrotransfer resulted in the highest luciferase expression and the best IgG response. Nevertheless electrotransfer into the skin, the muscle and the ear pinna all resulted in IFN-gamma secretion by luciferase-stimulated splenocytes suggesting that an efficient Th1 response was induced in all case

Thiamine Deficiency in Cultured Neuroblastoma Cells: Effect on Mitochondrial Function and Peripheral Benzodiazepine Receptors

When neuroblastoma cells were transferred to a medium of low (6 nM) thiamine concentration, a 16-fold decrease in total intracellular thiamine content occurred within 8 days. Respiration and ATP levels were only slightly affected, but addition of a thiamine transport inhibitor (amprolium) decreased ATP content and increased lactate production. Oxygen consumption became low and insensitive to oligomycin and uncouplers. At least 25% of mitochondria were swollen and electron translucent. Cell mortality increased to 75% within 5 days. [3H]PK 11195, a specific ligand of peripheral benzodiazepine receptors (located in the outer mitochondrial membrane) binds to the cells with high affinity (KD = 1.4 +/- 0.2 nM). Thiamine deficiency leads to an increase in both Bmax and KD. Changes in binding parameters for peripheral benzodiazepine receptors may be related to structural or permeability changes in mitochondrial outer membranes. In addition to the high-affinity (nanomolar range) binding site for peripheral benzodiazepine ligands, there is a low-affinity (micromolar range) saturable binding for PK 11195. At micromolar concentrations, peripheral benzodiazepines inhibit thiamine uptake by the cells. Altogether, our results suggest that impairment of oxidative metabolism, followed by mitochondrial swelling and disorganization of cristae, is the main cause of cell mortality in severely thiamine-deficient neuroblastoma cells

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

International audienceDespite 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 50 μg 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 2 days 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 2 days. The 12-fold difference (p < 0.05) between MicroMarker™ and our positively charged microbubbles was maintained over a period of 6 months. 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

Magnetic and Photoresponsive Theranosomes: Translating Cell-Released Vesicles into Smart Nanovectors for Cancer Therapy

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