Microbubbles for Nucleic Acid Delivery in Liver Using Mild Sonoporation.

Ultrasound-mediated gene delivery is an interesting approach, which could help in increasing gene transfer in deep tissues. Moreover, it allows for performing experiments guided by the image to determine which elements are required. Microbubbles complexed with a eukaryotic expression cassette are excellent agents as they are responsive to ultrasounds and, upon oscillation, can destabilize membranes to enhance gene transfer. Here, we describe the preparation of positively charged microbubbles, plasmid free of antibiotic resistance marker, their combination and the conditions of ultrasound-mediated liver transfection post-systemic administration in mice. This association allowed us to obtain a superior liver gene expression at least over 8 months after a single injection.

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.

Evaluation of a new concept of immune-enhancing diet in a model of head-injured rat with infectious complications: A proof of concept study.

Immune-enhancing diet (IED) utilization in critically ill septic patients is still debated. A new concept of IED has been proposed combining extra glutamine sequentially with either antioxidants or other amino acids, in order to match patient requirements according to their response to injury. We evaluated whether this new IED elicits a more favorable response to stress when compared with two existing IEDs both enriched in arginine but with different levels of anti-oxidants, in a validated rat model combining head injury (HI) and infectious complications. Forty-eight HI rats were randomized into four groups (n = 11-13 per group) to receive, for 4 days, standard enteral nutrition (S), one of the two existing IEDs (IED1, IED2), or the new IED (IED3; providing glutamine and antioxidants for two days and glutamine and specific amino acids for two days). Two days after HI, the rats received an enteral bolus of luminescent Escherichia coli Xen14 to induce infection, and bacterial dissemination was evaluated. Body weight (BW) was recorded daily. Four days after HI, animals were euthanized; blood was sampled; organs were weighed; cumulated nitrogen balance (CNB) and nitrogen efficiency were determined. IED3 was more efficient than IED1 and IED2 in improving BW recovery from D3 (D3 vs. D1, p < 0.05) after HI. It significantly improved CNB and net protein utilization (IED3 vs. S, IED1, IED2, p < 0.05). An IED with sequential administration of anti-oxidants and glutamine may be better suited to meeting nutritional requirements in severe catabolic states.

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

Microbubbles 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.

Magnetic and photoresponsive theranosomes: translating cell-released vesicles into smart nanovectors for cancer therapy.

Cell-released vesicles are natural carriers that circulate in body fluids and transport biological agents to distal cells. As nature uses vesicles in cell communication to promote tumor progression, we propose to harness their unique properties and exploit these biogenic carriers as Trojan horses to deliver therapeutic payloads to cancer cells. In a theranostic approach, cell-released vesicles were engineered by a top-down procedure from precursor cells, previously loaded with a photosensitizer and magnetic nanoparticles. The double exogenous cargo provided vesicles with magnetic and optical responsiveness allowing therapeutic and imaging functions. This new class of cell-derived smart nanovectors was named "theranosomes". Theranosomes enabled efficient photodynamic tumor therapy in a murine cancer model in vivo. Moreover the distribution of this biogenic vector could be monitored by dual-mode imaging, combining fluorescence and MRI. This study reports the first success in translating a cell communication mediator into a smart theranostic nanovector.

Mucosal imprinting of vaccine-induced CD8⁺ T cells is crucial to inhibit the growth of mucosal tumors.

Although many human cancers are located in mucosal sites, most cancer vaccines are tested against subcutaneous tumors in preclinical models. We therefore wondered whether mucosa-specific homing instructions to the immune system might influence mucosal tumor outgrowth. We showed that the growth of orthotopic head and neck or lung cancers was inhibited when a cancer vaccine was delivered by the intranasal mucosal route but not the intramuscular route. This antitumor effect was dependent on CD8⁺ T cells. Indeed, only intranasal vaccination elicited mucosal-specific CD8⁺ T cells expressing the mucosal integrin CD49a. Blockade of CD49a decreased intratumoral CD8⁺ T cell infiltration and the efficacy of cancer vaccine on mucosal tumor. We then showed that after intranasal vaccination, dendritic cells from lung parenchyma, but not those from spleen, induced the expression of CD49a on cocultured specific CD8⁺ T cells. Tumor-infiltrating lymphocytes from human mucosal lung cancer also expressed CD49a, which supports the relevance and possible extrapolation of these results in humans. We thus identified a link between the route of vaccination and the induction of a mucosal homing program on induced CD8⁺ T cells that controlled their trafficking. Immunization route directly affected the efficacy of the cancer vaccine to control mucosal tumors.

In vivo bioluminescent imaging of a new model of infectious complications in head-injury rats.

Infectious complications are responsible for 10-25% of mortality in head-injured patients. In the present work we developed a model of infectious complications in head-injury rats using Escherichia coli (E. coli) with a stable copy of the lux operon, and monitored the infection in vivo by optical imaging. Rats were randomized into three groups: AL (healthy rats), HI (head-injury rats), and HI-EC (HI rats+single enteral bolus of E. coli, 1.3×10(9)/rat given 2 days after HI). Infection was evaluated with a camera at 2 and 6 h after E. coli challenge. Blood and organs were sampled to assess biological parameters. HI was associated with body weight loss, muscle atrophy, and plasma amino acid disturbances, in particular glutamine depletion (AL 919±37 versus HI 647±25 and HI-EC 717±20 µmol/L; p<0.05). In the HI-EC rats, the luminescence signal was observed at T+2 (mean [range]: 34,778 cpm [1617-2,918,810]), and was significantly decreased at T+6 (0 cpm [0-847,922]; p<0.05). Bacterial challenge was associated with a specific body weight loss and a decrease in gastrocnemius protein content, in alanine (AL 512±41 versus HI-EC 395±29 µmol/L; p<0.05), and in sulfur plasma amino acids. In conclusion, we propose a controlled model of HI with infectious complications characterized by specific metabolic alterations. Combined with the in vivo monitoring of the infection by bioluminescence, this model offers a valuable tool to evaluate specific strategies for HI patients.

A comprehensive study in triblock copolymer membrane interaction.

Poloxamers are triblock copolymers made of poly(ethylene glycol)-(poly(propylene glycol))-poly(ethylene glycol). They have been shown to enhance gene transfer in the muscle, and co-administration of polymers and DNA appeared to be crucial to obtain this effect. It is questionable then if some interaction occurs between polymers and DNA. Polymer interaction with membranes represents a second crucial point due to the central hydrophobic part of the triblock copolymers. Besides, the question of the polymer spanning or adsorbing to the surface has not been solved by now. We addressed these issues by means of sensitive techniques that allowed working in diluted conditions and gaining in comprehension of gene transfection. By means of simultaneous time-correlated single-photon counting and fluorescence correlation spectroscopy, we have shown that the diffusion time of a single DNA molecule and PicoGreen lifetime was not altered in the presence of the triblock copolymer L64. Polypropylene (glycol) interactions with dodecylphosphocholine micelles were shown to occur at a deep level by (1)H NMR using doxyl probes located at the head or the lipid extremity of the micelles. The polypropylene (glycol) also interacted with lipid bilayers in a manner dependent on the cholesterol content, as shown by differential scanning calorimetry using liposomes. This interaction destabilised the membrane and allowed the release of small molecules. Finally, molecular dynamic simulation of the copolymer L64 in the presence of dodecylphosphocholine showed that the hydrophobic core of the polymer formed an extremely tight cluster, whose dimensions excluded the possibility of polymer spanning across the lipidic micelles. The simulation positively correlated with the destabilising effect observed on the liposomal membrane models.

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.

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.

Plasmid DNA electrotransfer for intracellular and secreted proteins expression: new methodological developments and applications.

In vivo electrotransfer is a physical method of gene delivery in various tissues and organs, relying on the injection of a plasmid DNA followed by electric pulse delivery. The importance of the association between cell permeabilization and DNA electrophoresis for electrotransfer efficiency has been highlighted. In vivo electrotransfer is of special interest since it is the most efficient non-viral strategy of gene delivery and also because of its low cost, easiness of realization and safety. The potentiality of this technique can be further improved by optimizing plasmid biodistribution in the targeted organ, plasmid structure, and the design of the encoded protein. In particular, we found that plasmids of smaller size were electrotransferred more efficiently than large plasmids. It is also of importance to study and understand kinetic expression of the transgene, which can be very variable, depending on many factors including cellular localization of the protein, physiological activity and regulation. The most widely targeted tissue is skeletal muscle, because this strategy is not only promising for the treatment of muscle disorders, but also for the systemic secretion of therapeutic proteins. Vaccination and oncology gene therapy are also major fields of application of electrotransfer, whereas application to other organs such as liver, brain and cornea are expanding. Many published studies have shown that plasmid electrotransfer can lead to long-lasting therapeutic effects in various pathologies such as cancer, blood disorders, rheumatoid arthritis or muscle ischemia. DNA electrotransfer is also a powerful laboratory tool to study gene function in a given tissue.

Applications of plasmid electrotransfer.

The use of electric pulses to transfect cells has recently been extended to show the utility of this procedure in vivo. Electrotransfer has been performed in vivo on several tissue types including skin, blood vessels, liver, tumor, muscle, cornea, brain and spleen. The most widely targeted tissue has been skeletal muscle. In addition to its potential use in gene therapy, in vivo DNA electrotransfer is also, because of its simplicity, a powerful laboratory tool to study in vivo gene expression and function in a given tissue. Many published studies have now shown that plasmid electrotransfer can lead to a long-lasting therapeutic effect in various pathologies, such as cancer, blood disease, or muscle ischemia. The future potential for this gene therapy approach will include delivery for both local action or distal effect by secretion of the transgenic proteins in the circulation.