Assembly of a Tripeptide and Anti-Inflammatory Drugs into Supramolecular Hydrogels for Sustained Release

5siSupramolecular hydrogels offer interesting opportunities for co-assembly with drugs towards sustained release over time, which could be achieved given that the drug participates in the hydrogel nanostructure, and it is not simply physically entrapped within the gel matrix. dLeu-Phe-Phe is an attractive building block of biomaterials in light of the peptide’s inherent biocompatibility and biodegradability. This study evaluates the assembly of the tripeptide in the presence of either of the anti-inflammatory drugs ketoprofen or naproxen at levels analogous to commercial gel formulations. Fourier-transformed infrared (FT-IR), circular dichroism, Thioflavin T fluorescence, transmission electron microscopy (TEM), and oscillatory rheometry are used. Drug release over time is monitored by means of reverse-phase high performance liquid chromatography, and shows different kinetics for the two drugs.openopenKurbasic, Marina; Romano, Chiara Damiana; Garcia Fernandez, Ana Maria; Kralj, Slavko; Marchesan, SilviaKurbasic, Marina; Romano, CHIARA DAMIANA; GARCIA FERNANDEZ, ANA MARIA; Kralj, Slavko; Marchesan, Silvi

Higher and lower supramolecular orders for the design of self-assembled heterochiral tripeptide hydrogel biomaterials

The self-assembly behaviour of the eight stereoisomers of Val\u2013Phe\u2013Phe tripeptides under physiological conditions is assessed by several spectroscopy and microscopy techniques. We report the first examples of self-organised hydrogels from tripeptides in the L\u2013D\u2013L or D\u2013L\u2013D configuration, besides the expected gels with the D\u2013L\u2013L or L\u2013D\u2013D configuration, thus widening the scope for using amino acid chirality as a tool to drive self-assembly. Importantly, the positions of D- and L-amino acids in the gelling tripeptides determine a higher or lower supramolecular order, which translates into macroscopic gels with different rheological properties and thermal behaviours. The more durable hydrogels perform well in cytotoxicity assays, and also as peptides in solution. An appropriate design of the chirality of self-assembling sequences thus allows for the fine-tuning of the properties of the gel biomaterials. In conclusion, this study adds key details of supramolecular organization that will assist in the ex novo design of assembling chiral small molecules for their use as biomaterials

Dentin permeability of the apical third in different groups of teeth

This ex vivo study evaluated dentin permeability of the root canal in the apical third of different human groups of teeth. Eighty teeth were used, 8 from each dental group: maxillary and mandibular central incisors, lateral incisors and canines, maxillary first premolars (buccal and palatal roots), mandibular first premolars, and maxillary and mandibular second premolars, totalizing 88 roots that were distributed in 11 groups. The root canals were instrumented, irrigated with 1% NaOCl and 15% EDTA. Roots were immersed in 10% copper sulfate for 30 min and then in 1% rubeanic acid alcohol solution for the same period; this chemical reaction reveals dentin permeability by the formation of copper rubeanate, which is a dark-colored compound. Semi-serial 100-µm-thick cross-sections were obtained from the apical third of the roots. Five sections of each apical third were washed, dehydrated, cleared and mounted on glass slides for examination under optical microscopy. The percentage of copper ion infiltration and the amount of tubular dentin were quantified by morphometric analysis. The penetration of copper ions in the apical third ranged from 4.60 to 16.66%. The mandibular central and lateral incisors presented the highest dentin permeability (16.66%), while the maxillary canines and mandibular second and first premolars presented the lowest dentin permeability (4.60%, 4.80% and 5.71%, respectively; p<0.001). The other teeth presented intermediate permeability. In conclusion, dye penetration into dentin tubules at the apical region is strongly dependent on the group of teeth evaluated.Este estudo ex vivo avaliou a permeabilidade da dentina do canal radicular do terço apical de diferentes grupos de dentes humanos. Foram utilizados 80 dentes, sendo 8 de cada grupo dental superior e inferior: incisivos centrais, incisivos laterais, caninos, primeiros pré-molares superiores (raízes vestibulares e palatinas), primeiros pré-molares inferiores, segundos pré-molares superiores e inferiores, totalizando 88 raízes, as quais foram distribuídas em 11 grupos. Os canais foram instrumentados, irrigados com NaOCl a 1% e EDTA a 15%. As raízes foram imersas em sulfato de cobre a 10% por 30 min e acido rubeânico a 1% pelo mesmo período. Esta reação química revela a permeabilidade da dentina por meio da formação de um complexo escurecido denominado rubeanato de cobre. Hemi-secções de 100 µm de espessura foram obtidas do terço apical da raiz. Cinco secções do terço apical foram lavadas, desidratadas, diafanizadas e montadas em lâminas para análise em microscopia óptica. A porcentagem de infiltração de íons cobre e a quantidade de dentina tubular foram quantificadas por meio de análise morfométrica. A penetração de íons cobre no terço apical da raiz variou de 4,60% a 16,66%. Os incisivos centrais e laterais apresentaram a maior permeabilidade dentinária (16,66%), e os caninos superiores e segundos e primeiros pré-molares inferiores as menores (4,60%, 4,80% e 5,71%, respectivamente; p<0,001). Os outros dentes apresentaram permeabilidade intermediaria. Conclui-se que a penetração de corante nos túbulos dentinários da região apical é extremamente dependente do grupo de dentes avaliado.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

Design of a hydrophobic tripeptide that self-assembles into amphiphilic superstructures forming a hydrogel biomaterial

We report the rational design of a heterochiral hydrophobic tripeptide self-assembling into amphiphilic D-superstructures that yield a self-supportive hydrogel at physiological pH. The material endures cell culture conditions and sustains fibroblast proliferation. Tripeptide superstructures are thoroughly analysed by several techniques

Functional analysis of yeast gene families involved in metabolism of vitamins B1 and B6

In order to clarify their physiological functions, we have undertaken a characterization of the three-membered gene families SNZ13 and SNO13. In media lacking vitamin B6, SNZ1 and SNO1 were both required for growth in certain conditions, but neither SNZ2, SNZ3, SNO2 nor SNO3 were required. Copies 2 and 3 of the gene products have, in spite of their extremely close sequence similarity, slightly different functions in the cell. We have also found that copies 2 and 3 are activated by the lack of thiamine and that the Snz proteins physically interact with the thiamine biosynthesis Thi5 protein family. Whereas copy 1 is required for conditions in which B6 is essential for growth, copies 2 and 3 seem more related with B1 biosynthesis during the exponential phase

Prevalence of foodborne pathogens in rural pigs and in derived cold pork meats - preliminary report

The rural breeding of one or two pigs and their domestic slaughtering is a significance reality in the Veneto Region, as a consequence of an ancient tradition still surviving in this countryside. In the eastern part of the Venice Province, about 2,500 rural pigs are bred and slaughtered ever year in the period between November and February. Many data are available on mdustnal breeding and processing, whereas very little is known about the prevalence of foodborne pathogens both in live ammals and in denved food, mainly sausage, salami and cold pork meats

Morphologic and morphometric analysis of the root canal apical third cleaning after biomechanical preparation using 3.3% Ricinus communis detergent and 1% NaOCl as irrigating solutions

This study evaluated, by morphologic and morphometric analyses, the cleaning of apical third of root canals instrumented with nickel-titanium rotary files using different irrigating solutions. Twenty-seven single-rooted mandibular premolars were assigned to three groups (n=9), according to the irrigating solution used: Group I, distilled and deionized water; Group II, 1% NaOCl; and Group III, 3.3% Ricinus communis detergent. Biomechanical preparation was performed with Protaper Plusâ nickel-titanium files as follows: S1, SX and S2 at the cervical and middle thirds, and 25/02, 25/04, 25/06, 30/02, 30/04 and 30/06 to complete the instrumentation, operating at 1 mm from the root apex. Irrigation was done at each file change with 2 mL of irrigating solution, totalizing 20 mL for each tooth. After biomechanical preparation, the apical thirds were serially sectioned and histologically processed. The cross-sections were examined by an optical microscope (X40) connected to a computer. The images were captured and analyzed using a computer software and submitted to morphometric analysis with aid of a grid. The percentage of debris remaining at the apical third was calculated. Data were submitted to statistical analysis by ANOVA and Tukey test. The results showed no statistically significant difference (p>;0.01) between the groups irrigated with 1% NaOCl and 3.3% Ricinus communis detergent, which presented the lowest percentage of debris at the apical third, 8.49% and 10.11%, respectively. The group irrigated with distilled and deionized water had the highest percentage of debris (15.58%) and was statistically different from the other groups (pEstudou-se, por meio de análise morfológica e morfométrica, a capacidade de limpeza promovida pela instrumentação rotatória com limas Ni-Ti e irrigação com diferentes soluções. Vinte e sete pré-molares inferiores foram distribuídos em três grupos (n=9), de acordo com a solução irrigante utilizada: Grupo I, água destilada e deionizada; Grupo II, NaOCl a 1% e Grupo III, detergente derivado do óleo de mamona (Ricinus communis) a 3,3%. O preparo biomecânico foi realizado com sistema rotatório de Ni-Ti Protaper Plus, obedecendo a seguinte ordem de uso dos instrumentos: S1, SX e S2 para o terço cervical e médio, seguida do 25/02, 25/04, 25/06, 30/02, 30/04 e 35/02, completando a instrumentação todos operando 1 mm aquém do ápice. A irrigação foi realizada a cada troca de instrumento com 2 mL de solução irrigante, totalizando um volume de 20 mL para cada dente. Após o preparo biomecânico, os terços apicais dos dentes foram submetidos ao processamento histológico. Os espécimes foram analisados em microscópio óptico (40X) conectado a um computador. As imagens foram capturadas e analisadas utilizando-se softwares específicos e submetidas à análise morfométrica por meio de uma grade de integração. A porcentagem de debris presente no terço apical dos canais foi calculada. Os dados foram submetidos à análise estatística por meio de ANOVA e teste de Tukey. Os resultados do presente estudo evidenciaram que não houve diferença estatisticamente significante (p>;0,01) entre os grupos irrigados com NaOCl a 1% e detergente de mamona a 3,3%, que apresentaram menor porcentagem de debris no terço apical, 8,49% e 10,11% respectivamente. O grupo irrigado com água destilada e deionizada apresentou a maior porcentagem de debris (15,58%), sendo estatisticamente diferente (

Nanoscale Assembly of Functional Peptides with Divergent Programming Elements

Self-assembling peptides are being applied both in the biomedical area and as building blocks in nanotechnology. Their applications are closely linked to their modes of self-assembly, which determine the functional nanostructures that they form. This work brings together two structural elements that direct nanoscale self-association in divergent directions: proline as a β-breaker and the β-structure-associated diphenylalanine motif, into a single tripeptide sequence. Amino acid chirality was found to resolve the tension inherent to these conflicting self-assembly instructions. Stereoconfiguration determined the ability of each of the eight possible Pro-Phe-Phe stereoisomers to self-associate into diverse nanostructures, including nanoparticles, nanotapes, or fibrils, which yielded hydrogels with gel-to-sol transition at a physiologically relevant temperature. Three single-crystal structures and all-atom molecular dynamics simulations elucidated the ability of each peptide to establish key interactions to form long-range assemblies (i,e., stacks leading to gelling fibrils), medium-range assemblies (i.e., stacks yielding nanotapes), or short-range assemblies (i.e., dimers or trimers that further associated into nanoparticles). Importantly, diphenylalanine is known to serve as a binding site for pathological amyloids, potentially allowing these heterochiral systems to influence the fibrillization of other biologically relevant peptides. To probe this hypothesis, all eight Pro-Phe-Phe stereoisomers were tested in vitro on the Alzheimer's disease-associated Aβ(1-42) peptide. Indeed, one nonfibril-forming stereoisomer effectively inhibited Aβ fibrillization through multivalent binding between diphenylalanine motifs. This work thus defined heterochirality as a useful feature to strategically develop future therapeutics to interfere with pathological processes, with the additional value of resistance to protease-mediated degradation and biocompatibility

Heterochirality and Halogenation Control Phe-Phe Hierarchical Assembly

Diphenylalanine is an amyloidogenic building block that can form a versatile array of supramolecular materials. Its shortcomings, however, include the uncontrolled hierarchical assembly into microtubes of heterogeneous size distribution and well-known cytotoxicity. This study rationalized heterochirality as a successful strategy to address both of these pitfalls and it provided an unprotected heterochiral dipeptide that self-organized into a homogeneous and optically clear hydrogel with excellent ability to sustain fibroblast cell proliferation and viability. Substitution of one l-amino acid with its d-enantiomer preserved the ability of the dipeptide to self-organize into nanotubes, as shown by single-crystal XRD analysis, whereby the pattern of electrostatic and hydrogen bonding interactions of the backbone was unaltered. The effect of heterochirality was manifested in subtle changes in the positioning of the aromatic side chains, which resulted in weaker intermolecular interactions between nanotubes. As a result, d-Phe-l-Phe self-organized into homogeneous nanofibrils with a diameter of 4 nm, corresponding to two layers of peptides around a water channel, and yielded a transparent hydrogel. In contrast with homochiral Phe-Phe stereoisomer, it formed stable hydrogels thermoreversibly. d-Phe-l-Phe displayed no amyloid toxicity in cell cultures with fibroblast cells proliferating in high numbers and viability on this biomaterial, marking it as a preferred substrate over tissue-culture plastic. Halogenation also enabled the tailoring of d-Phe-l-Phe self-organization. Fluorination allowed analogous supramolecular packing as confirmed by XRD, thus nanotube formation, and gave intermediate levels of bundling. In contrast, iodination was the most effective strategy to augment the stability of the resulting hydrogel, although at the expense of optical transparency and biocompatibility. Interestingly, iodine presence hindered the supramolecular packing into nanotubes, resulting instead into amphipathic layers of stacked peptides without the occurrence of halogen bonding. By unravelling fine details to control these materials at the meso- A nd macro-scale, this study significantly advanced our understanding of these systems