Stelian S. Maier

Theoretical study on β-cyclodextrin inclusion complexes with propiconazole and protonated propiconazole.

Summary The synthesis of the β-cyclodextrin/propiconazole nitrate inclusion complex and the advantages of the encapsulation of this drug were recently reported, but the experimental data only partially revealed the structure of the supramolecular complex due to the limitations in understanding the intermolecular association mechanism. The present work describes the equilibrium molecular geometries of β-cyclodextrin/propiconazole and β-cyclodextrin/protonated propiconazole, established by the AM1 and PM3 semi-empirical methods. The affinity between different parts of the guest molecule and the cyclodextrin cavity was studied considering that propiconazole possesses three residues able to be included into the host cavity through primary or secondary hydroxyl rims. The results have revealed that the most stable complex is formed when the azole residue of the propiconazole enters the cavity of the cyclodextrin through the narrow hydroxyl’s rim.

Hybrid fullerene conjugates as vectors for DNA cell-delivery

The present study reports fullerene conjugates that act as efficient binders of double stranded DNA (dsDNA) into cytofriendly polyplexes. The conjugates are designed to generate dendrimeric structures, having C60 as the core and bearing linear or branched PEI and polyethyleneglycol (PEG) arms (∼2 kDa). Simple and reproducible synthesis pathways provided C60-PEI and C60-PEG-PEI conjugates. They were able to bind linear and plasmidic dsDNA and they form particulate polyplexes of 50 to 200 nm in diameter. The resulted polyplexes toggle between the anionic and cationic state at nitrogen to phosphorous ratios (N/P) of about 5, as revealed by their zeta potential and became colloidally stable at N/P ratios above 10, as determined by atomic force microscopy (AFM). They are electrophoretically unbreakable starting with N/P ratios of 3 and of 5 when salmon sperm DNA and pEYFP-C1 plasmid, respectively are loaded. Both C60-PEI·pEYFP and C60-PEG-PEI·pEYFP polyplexes are non-cytotoxic against HEK 293T cells in culture and exhibit transfection efficiency better than 25% (N/P ratios above 20) and 6% (N/P ratios above 60) respectively, measured by flow cytometry. For comparison, the commercial SuperFect® from Qiagen (positive control) was able to provide an efficiency of 15-20%, under similar conditions. Moreover, the C60-PEG-PEI conjugate is as performant as the positive control in terms of expression of EYFP reporter gene in cultured cells and exhibited high cytocompatibility, determining cell proliferation up to 200%. Our study proved that C60-PEG-PEI is effective vector for DNA delivery being, in addition, easily synthesizable, practically non-cytotoxic and as efficient the commercially available transfection tools.

Flexible cyclic siloxane core enhances the transfection efficiency of polyethylenimine-based non-viral gene vectors

Transfection of nucleic acid molecules, large enough to interfere with the genetic mechanisms of active cells, can be performed by means of small carriers, able to collectively collaborate in generating cargocomplexes that could be involved in passive mechanisms of cellular uptake. The present work describes the synthesis, characterization, and evaluation of transfection efficacy of a conjugate molecule, which comprises a cyclic siloxane ring (2,4,6,8-tetramethylcyclotetrasiloxane, cD4 H) as the core, and, on average, 3.76 molecules of 2 kDa polyethyleneimine (PEI) as cationic branches, with an average molecular mass of 7.3 kDa. As demonstrated by in silico molecular modeling and dynamic simulation, the conjugate molecule (cD4 H-AGE-PEI) tends to adopt an asymmetric structure, specific for amphipathic molecules (confirmed by a log P value of -1.902 ± 0.06), that favors a rapid interaction with nucleic acids. The conjugate and the polyplexes with the pEYFP plasmid were proved to be non-cytotoxic, and capable of ensuring transfection yields better than 30%, on HEK 293T cell culture, superior to the value obtained using the SuperFect® reagent. We presume that the increased transfection efficacy originates in the ability of the conjugate to locally tightly encompass pDNA molecules by electrostatic interaction mediated by the short PEI branches, and consequently to expose the siloxane hydrophobic moiety, which decreases the interaction energy with the lipid layers.

Biomacromolecular-based ionic-covalent hydrogels for cell encapsulation: The atelocollagen − Oxidized polysaccharides couples

Mixed crosslinked networks of ionic-covalent entanglement type were prepared starting from ternary mixtures of atelocollagen (aK; as fibrillary matrix generator), sodium hyaluronate (NaHyal; a microfibrillation assistant), and oxidized polysaccharides (OxPolys; as both cross-linkers and matrix fillers), and were tested as hydrogels for eukaryotic cell encapsulation. Either oxidized gellan (GellOx) or pullulan (PullOx) were used. An original procedure and optimal hydrogel recipes were developed to encapsulate fibroblasts and adipose-derived stem cells, while preserving their viability and proliferative ability during ex vivo temporarily storage. Physical-chemical, rheological, and biocompatibility properties of the prepared hydrogels were compared against the classic alginate hydrogel used for cell encapsulation. A larger range of material characteristics (from lax to stiff) and better laboratory maneuverability were demonstrated, which permit to design appropriate compositions for particular cell types. All hydrogels undergo fast liquefaction at temperatures between 42 and 50°C, permitting the cell release after a short innocuous thermal shock.

Multivalent polyrotaxane vectors as adaptive cargo complexes for gene therapy

This paper describes the philosophy to design, and a procedure to construct polyrotaxane-type gene carriers, together with the proof of their ability to conjunctively cooperate in order to generate cargo-complexes with dsDNA, able to efficiently transfect cultured cells. The main feature of these entities is their functionality as a cargo-complex that chemomimic the histones, and morphomimic the nucleosome. The polyrotaxane contains a PEG axle end-capped with silatrane cages, allowing the threading of nine cyclodextrin units, functionalized with polyethylenimines (PEI, 2 kDa). The obtained ROT-PEI multivalent architecture is similar to a giant PEI polycation, but devoid of the toxicity of large PEIs. To increase the cargo-complexes’ versatility and to reduce their cytotoxicity, the study has been complemented with two other types of carriers: (i) including a mixture of PEI and short PEG molecules (ROT-PEI-PEG750), and (ii) with PEI branches post-decorated with guanidine or arginine (ROT-PEI-G; ROT-PEI-Arg). The molecular geometry and the overall interactions of the synthesized carriers were investigated in silico. The experimental DNA binding capacity of these carriers in relationship with size, morphology and electrical charge was evaluated. The in vitro tests, showing the cytotoxicity and transfection efficiency of the investigated carriers, provided new information on gene vector design.

Transfection-capable polycationic nanovectors which include PEGylated-cyclodextrin structural units: a new synthesis pathway

Efficient tools are still being searched for to substitute the viral vectors in nucleic acid delivery applications. One of the most severe constraints in producing them is related to the strict reproducibility of their molecular characteristics, which is ensured through the synthesis. In this work, we report an original route to obtain polycationic nanoentities with low variability, which are able to act as cooperating carriers for dsDNA complexation and transport. The carriers are synthesized by rigorous conjugation of β-cyclodextrin (β-CD) with precise ratios of 2 kDa branched poly(ethyleneimine) (b-PEI) and 0.75 kDa poly(ethylene glycol) (PEG). Low cytotoxicity was the key parameter of the carrier design, besides the highest possible transfection ability, and both of these features were proven by HeLa cell culture assays. A reporter gene which induces the expression of green fluorescent protein (GFP), inserted in a plasmid, was used to perform the necessary quantitative measurements. In silico molecular modelling guided the carrier design and confirmed the functional mimicry of histones in the tight and compact nucleosome-like spiral packaging of dsDNA. The carrier molecules, synthesized with high reproducibility, are expected to be feasible for application in gene transfection.

Isocyanate-Functionalized Collagen Hydrolysates as Pretanning Agents for Organic Wet-White Leather

The present paper aims to synthesize and test a pretanning agent based on isocyanatefunctionalized collagen hydrolysates of low molecular mass (0.9 ÷ 3.6 kDa; polypeptides that include 8 to 32 amino acids), obtained starting from hides wet wastes, and used without further purification. Raw colloidal suspension resulted after hydrolysis was centrifuged to separate insoluble particles, and then was repeatedly filtered to retain coarse particulate and fatty maters. The clarified solution was concentrated by vaporization up to 35 % w/v dry matter, mixed with 10 % v/v dimethyl sulfoxide (DMSO), and maturated overnight under efficient mixing, in a hermetically closed reaction vessel, at ambient temperature. Using a solution of diisocyanate in DMSO, the molecular mass of the product was further increased by cross-linking, in parallel with the functionalization in the virtue of a small amount of free isocyanate groups that remain unreacted. The functionalized collagen hydrolysate was characterized by chemical (total nitrogen content, the amount of free carboxyl, amino, and isocyanate groups) and instrumental methods (infrared spectroscopy). The fraction of increased molecular mass after cross-linking was determined by comparative dialysis through membranes with 3.5 and 12 kDa. Pretanning ability of the functionalized hydrolysates was estimated by gelatin precipitation, and tested on sheep pelts. An increase of 6 oC was measured for the shrinkage temperature. Pretanned pelts were gradually dehydrated (preventing the local drying) and drum-tumbled, and then was treated once again with the same functionalized product, in a short concentrated float. A supplemental increase of shrinkage temperature with 8 oC was measured.