Size-tunable lipid vectors for controlled local delivery of siRNA from gene activated matrix.

Tissue engineering aims to restore or replace different types of biological tissues through the association of cells, biologic factors and biomaterials. Currently, stem cells arise as a major cell source for many therapeutic indications, and their association with 3D scaffolds allow increasing regenerative medicine efficiency. In this context, the use of RNA interference to enhance or control stem cell differentiation into the desired phenotype appears as a promising strategy. However, achieving high transfection efficiency of cells in a 3D structure requires the use of a vector allowing for the spatiotemporally controlled release of the genetic material from these scaffolds. In this study, we report a new siRNA nanovector, called solvent exchange lipoplexe formulation (SELF), which has a tunable size, is stable over time in cell culture conditions and possess a high efficiency to transfect primary human mesenchymal stromal cells (hMSC). We associated SELFs with porous 3D collagen microspheres and demonstrated that the loading capacity and release kinetics were different depending on the size of the associated SELF. Interestingly, these different release profiles resulted in differences in the transfection kinetics of hMSCs. This original and unique type of gene activated matrix, with adaptable release kinetics, could be of interest for long-term and/or sequential transfection profiles of stem cells in 3D culture. STATEMENT OF SIGNIFICANCE: This work combines the use of human mesenchymal stromal cell (hMSC) and gene therapy for tissue engineering. Here, a gene-activated matrix was elaborated with collagen microspheres supporting hMSCs and acting as a reservoir for transfection vectors. This injectable GAM allows for the local and sustained delivery of nucleic acids, hence long-lasting transfection of the supported cells. With the original synthesis protocol presented herein, the size of the nanocarriers can be easily adapted, resulting in different siRNA release profiles from the microspheres. Most interestingly, different siRNA release profiles gave rise to different cell transfection profiles as assessed by the downregulation of a target gene. This highlights the versatility of the system and its suitability for various pathophysiological needs in regenerative medicine.

Formulation and Evaluation of SNEDDS Loaded with Original Lipophenol for the Oral Route to Prevent Dry AMD and Stragardt's Disease.

Dry age-related macular degeneration (Dry AMD) and Stargardt's disease (STGD1) are common eye diseases, characterized by oxidative and carbonyl stress (COS)-inducing photoreceptor degeneration and vision loss. Previous studies have demonstrated the protective effect of photoreceptors after the intravenous administration of a new lipophenol drug, phloroglucinol-isopropyl-DHA (IP-DHA). In this study, we developed an oral formulation of IP-DHA (BCS Class IV) relying on a self-nanoemulsifying drug delivery system (SNEDDS). SNEDDS, composed of Phosal® 53 MCT, Labrasol®, and Transcutol HP® at a ratio of 25/60/15 (w/w/w), led to a homogeneous nanoemulsion (NE) with a mean size of 53.5 ± 4.5 nm. The loading of IP-DHA in SNEDDS (SNEDDS-IP-DHA) was successful, with a percentage of IP-DHA of 99.7% in nanoemulsions. The in vivo study of the therapeutic potency of SNEDDS-IP-DHA after oral administration on mice demonstrated photoreceptor protection after the induction of retinal degeneration with acute light stress (73-80%) or chronic light stress (52-69%). Thus, SNEDDS formulation proved to increase the solubility of IP-DHA, improving its stability in intestinal media and allowing its passage through the intestinal barrier after oral force-fed administration, while maintaining its biological activity. Therefore, SNEDDS-IP-DHA is a promising future preventive treatment for dry AMD and STGD1.

Antioxidative properties and ability of phenolic compounds of Myrtus communis leaves to counteract in vitro LDL and phospholipid aqueous dispersion oxidation.

UNLABELLED: Antioxidant activities of Myrtus communis leaf phenolic compounds (McPCs) were investigated on 2,2'-9-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS(+) •) and oxygen radical absorbance capacity (ORAC) tests or on oxidation of biological models, human low-density lipoprotein (LDL) and phospholipid aqueous dispersion (L-α-phosphatidylcholine stabilized by bile salts). Two extraction techniques, microwave-assisted (MAE) and conventional (CE), were used to isolate McPCs, producing similar results of phenolic compound content. ABTS(+) • assay showed clearly that myrtle extracts exhibited a stronger scavenging effect than butylated hydroxyanisole and α-tocopherol, with a slight advantage for myrtle CE extract. In ORAC assay, the both McPC extracts were similarly less effective than the pure compounds as caffeic acid and myricitrin (myricetin 3-O-rhamnoside) but stronger than butylated hydroxytoluene. Moreover, myrtle CE and MAE extracts, and myricitrin were able to inhibit similarly the production of conjugated dienes and to prolong the lag phase (Tlag) during Cu(2+)-induced LDL oxidation with a dose-response effect. The cryo-electron microscopy observations on studied phospholipid dispersion stabilized by bile salts (BS) revealed the presence of bilayer vesicles and micelles. In 2,2'-azobis (2-amidinopropane) hydrochloride-induced phospholipid/BS oxidation, myrtle CE and MAE extracts gave similar effects to α-tocopherol and caffeic acid but myricitrin showed a higher protective effect than myrtle extracts. We showed also that no synergic or additive effect between α-tocopherol and myrtle extracts or caffeic acid in α-tocopherol-enriched phospholipid/BS dispersion, but myricitrin showed an additive effect and thus promoted the total antioxidant activity. These data showed that myrtle extract could be used as potential natural antioxidants, food stabilizers, or natural health products. PRACTICAL APPLICATION: We show that microwave-assisted extraction could be an alternative method for plant phenolic compound recovery allowing important gain in time extraction.We report inhibition of low-density lipoprotein oxidation in vitro initiated by Cu(2+) ions. We report that myrtle extract may be a source of natural antioxidants to counteract phospholipid peroxidation as well as α-tocopherol.

MgATP regulates allostery and fiber formation in IMPDHs.

Inosine-5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in nucleotide biosynthesis studied as an important therapeutic target and its complex functioning in vivo is still puzzling and debated. Here, we highlight the structural basis for the regulation of IMPDHs by MgATP. Our results demonstrate the essential role of the CBS tandem, conserved among almost all IMPDHs. We found that Pseudomonas aeruginosa IMPDH is an octameric enzyme allosterically regulated by MgATP and showed that this octameric organization is widely conserved in the crystal structures of other IMPDHs. We also demonstrated that human IMPDH1 adopts two types of complementary octamers that can pile up into isolated fibers in the presence of MgATP. The aggregation of such fibers in the autosomal dominant mutant, D226N, could explain the onset of the retinopathy adRP10. Thus, the regulatory CBS modules in IMPDHs are functional and they can either modulate catalysis or macromolecular assembly.

On the kinetics of adsorption and two-dimensional self-assembly of annexin A5 on supported lipid bilayers.

Annexin A5 is a protein that binds to membranes containing negatively charged phospholipids in a calcium-dependent manner. We previously found that annexin A5 self-assembles into two-dimensional (2D) crystals on supported lipid bilayers (SLBs) formed on mica while a monolayer of disordered trimers is formed on SLBs on silica. Here, we investigated in detail and correlated the adsorption kinetics of annexin A5 on SLBs, supported on silica and on mica, with the protein's 2D self-assembly behavior. For this study, quartz crystal microbalance with dissipation monitoring and ellipsometry were combined with atomic force microscopy. We find, in agreement with previous studies, that the adsorption behavior is strongly dependent on the concentration of dioleoylphosphatidylserine (DOPS) in the SLB and the calcium concentration in solution. The adsorption kinetics of annexin A5 are similar on silica-SLBs and on mica-SLBs, when taking into account the difference in accessible DOPS between silica-SLBs and mica-SLBs. In contrast, 2D crystals of annexin A5 form readily on mica-SLBs, even at low protein coverage (< or =10%), whereas they are not found on silica-SLBs, except in a narrow range close to maximal coverage. These results enable us to construct the phase diagram for the membrane binding and the states of 2D organization of annexin A5. The protein binds to the membrane in two different fractions, one reversible and the other irreversible, at a given calcium concentration. The adsorption is determined by the interaction of protein monomers with the membrane. We propose that the local membrane environment, as defined by the presence of DOPS, DOPC, and calcium ions, controls the adsorption and reversibility of protein binding.

The basic framework of VE-cadherin junctions revealed by cryo-EM.

Artificial adherens junctions were reconstituted in vitro by assembly of cadherin fragments at the surfaces of liposomes. The architecture of the adherens junctions was revealed by cryo-electron microscopy (cryo-EM). The formation of these artificial adherens junctions was shown to result from the two-dimensional (2D) self-assembly of cadherin fragments at membrane surfaces. The molecular architecture of the junctions was resolved by combining information from several cryo-EM views. This study concludes to the 2D ordered nature of the cadherin assembly and shows that the minimal information required to build up an adherens junction is contained within the extracellular moiety of cadherin molecules.

Structural characterization by (13)C-NMR spectroscopy of products synthesized in vitro by polysaccharide synthases using (13)C-enriched glycosyl donors: application to a UDP-glucose:(1-->3)-beta-D-glucan synthase from blackberry (Rubus fruticosus).

A simple and sensitive method for the characterization of products synthesized in vitro by polysaccharide synthases is described. It relies on the use of (13)C-enriched nucleotide sugars as substrates and on the analysis of the newly synthesized polysaccharides by (13)C-nuclear magnetic resonance (NMR) spectroscopy. The method was validated with a (1-->3)-beta-d-glucan synthase from blackberry, but it may be applied to the study of any glycosyltransferase. The chemical synthesis of UDP-d-[U-(13)C]glucose was achieved in a classical procedure with an overall yield of 50%. A uniformly labeled (1-->3)-beta-d-glucan was synthesized from this substrate, using detergent extracts of blackberry cell membranes as a source of synthase. One hundred micrograms of product was sufficient for liquid and solid-state (13)C-NMR spectroscopy analyses. The method is at least 100 times more sensitive than in the case of nonenriched polysaccharides. It allows the unequivocal identification and direct structural characterization of the products synthesized in vitro, as opposed to conventional methods that rely on the use of radioactive substrates and enzymatic hydrolysis of the polysaccharides with specific glycoside hydrolases. The method proves that the glycan analyzed was synthesized de novo because the final product is enriched in (13)C. Information on the 3D organization of the polymer may also be obtained by solid-state NMR spectroscopy.