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 ß-cyclodextrin (ß-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.

Enzyme-assisted purification of denatured atelocollagen, using pronase as scavenging agent.

Collagen liquid-crystal characteristics can be exploited in advanced applications, like microelectromechanical systems (MEMS), collagen-silica biohybrids or intelligent substrates for tissue engineering. Such applications necessarily require high purity of the collagen colloidal solutions, in terms of macromolecular unitary composition, which is equivalent to almost zero poydispersity. In this work a protocol for the enzyme-assisted removal of non-triple helical polypeptide entities in atelocollagen solutions was conceived, using pronase as scavenger, in the presence of poly(ethyleneglycol) (PEG 10000) as a crowding agent, trimethylamine-N-oxide dihydrate, (TMAO) as kosmotropic agent and anhydrous sodium sulfate as anti-chaotrope salt. Unusually high enzyme concentration (3 : 100 w/w 5 U/mg Pronase : total dry protein) imposes the triple-helix integrity protection, which was achieved by means of protective adjuvants. The adjuvant agents mixture comprising the crowder, the kosmotrope and the anti-chaotropic salt, was formulated according to the Design of Experiments (DOE) principles. The crowding agent represents the key factor in modulating pronase hydrolytic action upon atelocollagen substrate. The optimal adjuvant mixture tested in order to confirm the model validity had the composition: 0.675 PEG, 0.200 TMAO, 0.125 Na2SO4 (mass fractions). The proposed protocol is suitable for purifying medium and large quantities of atelocollagen previously solubilized through an alkali-enzyme technique.

[Preprocessing of collagenous sources with the aim of extracting minimally denatured collagen]. / Prelucrarea preliminara a surselor colagenice in vederea extractiei colagenului minimal denaturat.

UNLABELLED: Biomedical applications of collagen are conditioned by its use in a state very close to the native state, as it concerns conformation and reactivity. Minimal denaturation implies that both the collagen structure and the side amino acid groups are kept intact. The bulk of alterations take place in phases preceding the extraction itself. MATERIAL AND METHOD: The authors have established an optimal protocol for preprocessing of young bovine hides, i.e. to obtain a maximum solubilisation yield when a standard proteases-based solubilisation technique was applied. RESULTS: The sequence: unhairing--loosing--cleaning--labilisation of corium tissue was taken in account. The effect of the treating composition upon the deamination degree of the extracted collagen was determined through a binary gradient HPLC technique, on a ProPac WCX-10 analytical column. The optimal range of loosing--cleaning recipe components was found to be between 18 ... 27% dithiotreitol (DDT), 12 ... 20% N-ethyl-acetamide (NEM) and 30 ... 53% iodide-acetamide (IDAM).

[Study on an injectable biomimetic hydroxyapatite--atelocollagen composite]. / Studiu asupra unui compozit biomimetic hidroxiapatita--atelocolagen.

The paper presents a procedure for preparing mineralized collagen nanofibrils, mixed with deficient nanoparticulate calcium hydroxyapatite in a ratio close to that found in living bone structures. Such a composition might represent an injectable osteoconductive constituent, used in surgical kits for bone reconstruction. The FT-IR spectra of the prepared composite reveals a weak absorption at 871.82 cm(-1), indicating the nucleation of hydroxy-apatite at the acidic atelocollagen functions. More than that, a 45 cm(-1) ipsochromic shift of carboxylic band suggest a strong ionic interaction with hydroxyapatite. The X-Ray spectra show a high resemblance between the synthetic composite and the Fluka hydroxyapatite, but only for 2theta angles higher than 30 degrees. Below that value the protein presence induce crystalline disorders.