Lipids for Nucleic Acid Delivery: Cationic or Neutral Lipoplexes, Synthesis, and Particle Formation

International audienc

Lipids for Nucleic Acid Delivery: Cationic or Neutral Lipoplexes, Synthesis, and Particle Formation

International audienc

Prdm13 forms a feedback loop with Ptf1a and is required for glycinergic amacrine cell genesis in the Xenopus retina

Background: Amacrine interneurons that modulate synaptic plasticity between bipolar and ganglion cells constitute the most diverse cell type in the retina. Most are inhibitory neurons using either GABA or glycine as neurotransmitters. Although several transcription factors involved in amacrine cell fate determination have been identified, mechanisms underlying amacrine cell subtype specification remain to be further understood. The Prdm13 histone methyltransferase encoding gene is a target of the transcription factor Ptf1a, an essential regulator of inhibitory neuron cell fate in the retina. Here, we have deepened our knowledge on its interaction with Ptf1a and investigated its role in amacrine cell subtype determination in the developing Xenopus retina. Methods: We performed prdm13 gain and loss of function in Xenopus and assessed the impact on retinal cell fate determination using RT-qPCR, in situ hybridization and immunohistochemistry. Results: We found that prdm13 in the amphibian Xenopus is expressed in late retinal progenitors and in about half of the mature amacrine cells, predominantly in glycinergic ones. Clonal analysis in the retina reveals that prdm13 overexpression favours amacrine cell fate determination, with a bias towards glycinergic cells. Conversely, knockdown of prdm13 specifically inhibits glycinergic amacrine cell genesis. We also showed that, as in the neural tube, prdm13 is subjected to a negative autoregulation in the retina. Our data suggest that this is likely due to its ability to repress the expression of its inducer, ptf1a. Conclusions: Our results demonstrate that Prdm13, downstream of Ptf1a, acts as an important regulator of glycinergic amacrine subtype specification in the Xenopus retina. We also reveal that Prdm13 regulates ptf1a expression through a negative feedback loop.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Functionalization of single- and multi-walled carbon nanotubes with cationic amphiphiles for plasmid DNA complexation and transfection

International audienceThe possibility of delivering DNA effi ciently to cells represents a crucial issue for the treatment of both genetic and acquired diseases. However, even although the effi ciency of non-viral transfection systems has improved in the last decade, none have yet proven to be suffi ciently effective in vivo. We report herein our results on the functionalization of single-walled carbon nanotubes (SWNT) and multi-walled carbon nanotubes (MWNT) by two cationic amphiphiles (lipid RPR120535 and pyrenyl polyamine), their use for the complexation of plasmid DNA, and their efficiency in transfecting cells in vitro. The experiments have shown that the efficiency of transfection is higher when using SWNT instead of MWNT, and that transfection effi ciency is similar or slightly higher when using nanoplexes (SWNT/lipid RPR120535/DNA) instead of lipoplexes (lipid RPR120535/DNA) and several orders of magnitude higher than that of naked DNA. This study therefore shows both that the transfection is better when using SWNTs and that it is dependent on the nature of the amphiphilic molecules adsorbed on the nanotubes

Iminothiol/thiourea tautomeric equilibrium in thiourea lipids impacts DNA compaction by inducing a cationic nucleation for complex assembly

International audienceOur research on lipidic vectors for transfection led us to develop thiourea lipids able to interact with DNA. Hence, we developed a series of lipopolythioureas based on the strong hydrogen bond donor ability of thiourea. More recently we have reported a branched hydroxylated bis-thiourea derivative with interesting transfecting properties. The last step of the syntheses involved a strong acidic condition, leading to an unstable product upon storage. Therefore we designed a new synthesis in mild acidic conditions. Though they exhibit the same mass, the lipids obtained in the two different conditions differ by their interaction with DNA. We therefore explored the physicochemical properties of these two lipids by different means that we describe in this article. In order to insure easier and reliable (13)C-NMR studies of the thiourea group we have designed the synthesis of the corresponding (13)C-labeled thiourea lipids. We have thus shown that when the lipid was submitted to mildly acidic medium; only the thiourea group was observed; while a thiourea/charged and/or uncharged iminothiol tautomeric equilibrium formed when the last step of the synthesis was submitted to low pH. NMR experiments showed that this tautomeric equilibrium could not form in polar solvents. However, UV experiments on the liposomal form of the lipopolythiourea showed the presence of the tautomers. Lipid/DNA interaction consequently differed according to the acidic treatment applied. Eventually, these results revealed that on this particular thiourea lipid, electrostatic interactions due to cationic thioureas are likely to be responsible for DNA compaction and that this tautomeric form of the thiourea could be stabilised by hydrogen bonds in a supramolecular assembly. Nevertheless, this does not reflect a general thiourea lipid/DNA interaction as other thiourea lipids that are able to compact DNA do not undergo an acidic treatment during the final stage of their synthesis

Lipothioureas as Lipids for Gene Transfection: A Review

International audienceNon-viral gene therapy requires innovative strategies to achieve higher transfection efficacy. A few years ago, our group proposed bioinspired lipids whose interaction with DNA was not based on ionic interactions, but on hydrogen bonds. We thus developed lipids bearing a thiourea head which allowed an interaction with DNA phosphates through hydrogen bonds. After a proof of concept with a lipid bearing three thiourea functions, a molecular and cellular screening was performed by varying all parts of the lipids: the hydrophobic anchor, the spacer, the linker, and the thiourea head. Two lipothiourea-based structures were identified as highly efficient in vitro transfecting agents. The lipothioureas were shown to reduce non specific interactions with cell membranes and deliver their DNA content intracellularly more efficiently, as compared to cationic lipoplexes. These lipids could deliver siRNA efficiently and allowed specific cell targeting in vitro. In vivo, thiourea lipoplexes presented a longer retention time in the blood and less accumulation in the lungs after an intravenous injection in mice. They also induced luciferase gene expression in muscle and tumor after local administration in mice. Therefore, these novel lipoplexes represent an excellent alternative to cationic lipoplexes as transfecting agents. In this review we will focus on the structure activity studies that permitted the identification of the two most efficient thiourea lipids

Design, Synthesis, and Evaluation of Enhanced DNA Binding New Lipopolythioureas

International audienceNonviral gene delivery is limited to a large extent by the cationic nature of most of the chemical vector. We have shown that lipopolythioureas interact with DNA. However, lipopolythioureas were not very efficient at transfecting cells, probably due to reduced interaction between the noncationic synthetic lipid and the cell membrane. Here, we report that liposomes made from a new thiourea lipid, DPPC, and a lipid bearing an RGD ligand allowed very efficient entry of the lipopolythioureas into integrin R v 3 expressing cells. In addition, we show that a stable interaction between DNA and lipopolythiourea could be obtain with two thiourea groups. Moreover, the addition of a hydrophilic terminus improves the formulation of these new DNA binding agents

Europium labeled lactosylated albumin as a model workflow for the development of biotherapeutics

International audienceLactosylated albumin is currently used as a radiopharmaceutical agent to image the liver asialoglycoprotein receptors and quantify hepatic liver function in various diseases. A lactosylated protein (LACTAL) conjugate showed excellent liver uptake compared to non-lactosylated protein and a high signal to noise ratio, based on the biodistribution in mice using 99mTc-scintigraphy. However, in the laboratory, it is useful to have a method that can be used in daily practice to quantify cellular targeting or biodistribution. We propose a methodology from synthesis validation to pre-clinical demonstration and introduce a new practical detector (LACTAL.Eu) of the LACTAL molecule in biological media. We confirmed the purity and colloidal stability of the sample through physical analytical techniques, then showed the absence of in vitro toxicity of the agent and demonstrated in vitro targeting. Taking advantage of the fluorescence decay of the lanthanide, we performed measurements directly on the cell media without any further treatment. Finally, biodistribution in mice was confirmed by ex vivo measurements