Improved triamcinolone acetonide-eluting contact lenses based on cyclodextrins and high hydrostatic pressure assisted complexation.

Contact lenses (CLs) constitute an advantageous platform for the topical release of corticosteroids due to their prolonged contact with the eye. However, the lipophilic nature of corticosteroids hampers CLs' ability to release therapeutic amounts. Two approaches to improve loading and release of triamcinolone acetonide (TA) from poly(2-hydroxyethyl methacrylate)-based hydrogels were investigated: adding 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) to the monomers solution before polymerization (HEMA/i-CD) and an hydrogels' post-treatment with HP-ß-CD (HEMA/p-CD). The effect of HP-ß-CD and sterilization by high hydrostatic pressure (HHP) on the hydrogel properties (water content, oxygen and ion permeability, roughness, transmittance, and stiffness) was evaluated. The HEMA/i-CD hydrogels had stronger affinity for TA, sustaining its release for one day. HHP sterilization promoted the formation of cyclodextrin-TA complexes within the hydrogels, improving their drug-loading capacity ¼60 %. Cytotoxicity and irritability tests confirmed the safety of the therapeutic CLs. TA released from the hydrogels permeated through ocular tissues ex vivo and showed anti-inflammatory activity. Finally, a previously validated mathematical model was used to estimate the ability of the TA-loaded CLs to deliver therapeutic drug concentrations to the posterior part of the eye. Overall, HP-ß-CD-containing CLs are promising candidates for the topical ocular application of TA as an alternative delivery system to intraocular injections.

Dexamethasone phosphate and penetratin co-eluting contact lenses: a strategy to enhance ocular drug permeability.

Contact lenses (CLs) have been suggested as drug delivery platforms capable of increasing the drug residence time on the cornea and therefore its bioavailability. However, when targeting the posterior segment of the eye, the drug released from CLs still encounters the barrier effect of the ocular tissues, which considerably reduces the efficacy of administration. This work aims at the development of CLs able to simultaneously deliver an anti-inflammatory drug (dexamethasone sodium phosphate) and a cell-penetrating peptide (penetratin), the latter acting as a drug carrier across the tissues. Hydroxyethyl methacrylate (HEMA)-based hydrogels were functionalized with acrylic acid (AAc) and/or aminopropyl methacrylamide (APMA) to serve as CL materials with increased affinity for the drug and peptide. APMA-functionalized hydrogels sustained the dual release for 8 h, which is compatible with the wearing time of daily CLs. Hydrogels demonstrated suitable light transmittance, swelling capacity and in vitro biocompatibility. The anti-inflammatory activity of the drug was not compromised by the presence of the peptide nor by sterilization. The ocular distribution of the drug after 6 h of CL wearing was evaluated in vivo in rabbits and revealed that the amount of drug in the cornea and aqueous humor significantly increased when the drug was co-delivered with penetratin.

Antiseptic-Loaded Casein Hydrogels for Wound Dressings.

Chronic wound treatment accounts for a substantial percentage of the medical expenses worldwide. Improving and developing novel wound care systems can potentially help to handle this problem. Wound dressings loaded with antiseptics may be an important tool for wound care, as they inhibit bacterial growth at the wound site. The goal of the present work was to investigate the potential of using casein hydrogel dressings loaded with two antiseptic drugs, Octiset® or polyhexanide, to treat chronic wounds. Casein-based hydrogels are inexpensive and have several properties that make them suitable for biomedical applications. Two types of casein were used: casein sodium salt and acid casein, with the formulations being labelled CS and C, respectively. The hydrogels were characterised with respect to their physical properties (swelling capacity, water content, morphology, mechanical resistance, and stability), before and after sterilisation, and they showed adequate values for the intended application. The hydrogels of both formulations were able to sustain controlled drug-release for, at least, 48 h. They were demonstrated to be non-irritant, highly haemocompatible, and non-cytotoxic, and revealed good antimicrobial properties against Staphylococcus aureus and Pseudomonas aeruginosa. Steam-heat sterilisation did not compromise the material's properties. The in vivo performance of C hydrogel loaded with Octiset® was evaluated in a case study with a dog. The efficient recovery of the wounds confirms its potential as an alternative for wound treatment. To our knowledge, this is the first time that wound dressings loaded with Octiset®, one of the most efficient drugs for wound treatment, were prepared and tested.

Effects of Non-Conventional Sterilisation Methods on PBO-Reinforced PVA Hydrogels for Cartilage Replacement.

Articular cartilage (AC) degradation is a recurrent pathology that affects millions of people worldwide. Polyvinyl alcohol (PVA) hydrogels have been widely explored for AC replacement. However, their mechanical performance is generally inadequate, and these materials need to be reinforced. Moreover, to be used in a clinical setting, such materials must undergo effective sterilisation. In this work, a PVA hydrogel reinforced with poly(p-phenylene-2,6-benzobisoxazole) (PBO) nanofibres was submitted to three non-conventional sterilisation methods: microwave (MW), high hydrostatic pressure (HHP), and plasma (PM), in order to evaluate their impact on the properties of the material. Sterilisation was achieved in all cases. Properties such as water content and hydrophilicity were not affected. FTIR analysis indicated some changes in crystallinity and/or crosslinking in all cases. MW was revealed to be the most suitable method, since, unlike to PM and HHP, it led to a general improvement of the materials' properties: increasing the hardness, stiffness (both in tensile and compression), and shear modulus, and also leading to a decrease in the coefficient of friction against porcine cartilage. Furthermore, the samples remained non-irritant and non-cytotoxic. Moreover, this method allows terminal sterilisation in a short time (3 min) and using accessible equipment.

Oral Health Status of Adult Dysphagic Patients That Undergo Endoscopic Gastrostomy for Long Term Enteral Feeding.

BACKGROUND: Endoscopic Gastrostomy (PEG) is required to maintain a feeding route when neurological or cancer dysphagia impact oral intake. This study aimed to evaluate the oral health and oral changes of PEG-patients without oral feeding for three months. METHODS: Prospective observational study, with a PEG-patients convenience sample. Data were obtained before PEG (T0) and 3 months after gastrostomy (T1). Initial oral hygiene habits were collected through a questionnaire. Intra-oral evaluation was performed using: Plaque Index (IP), Gingival Index (IG), Decayed, Missing and Filled Teeth Index (DMF), Community Periodontal Index (CPI), and Attachment Loss (AL). T0 and T1 were compared to evaluate oral health evolution. RESULTS: Thirty-nine patients aged 65.3 ± 17.4 years were included. Initial (T0) oral health was worse than expectable. Between assessments period, oral indexes suffered a general deterioration with statistical relevance to the DMF. The frequency of deep periodontal pockets and attachment loss remained stable. CONCLUSIONS: PEG-patients presented poor oral health and insufficient oral hygiene habits, even before gastrostomy. After three months of PEG feeding, oral health suffered a general deterioration. This outcome was probably associated with the absence of oral feeding activity, which is beneficial to oral homeostasis, and further reduced oral hygiene. Improved oral daily care and dental appointments should become part of the PEG-patients follow-up.

White wine grape pomace as a suitable carbon source for lipid and carotenoid production by fructophilic Rhodorotula babjevae.

AIM: We aim to explore the non-structural sugars from white wine grape pomace (WWGP) as the input carbon source for the co-production of multiple high-value products by the non-fastidious yeast Rhodotorula babjevae to create a sustainable and economically appealing process. METHODS AND RESULTS: Water extraction of unfermented, soluble sugars from WWGP yielded extracts with similar amounts of glucose and fructose, which were used to prepare a growth medium. Rhodorotula babjevae multiplied as fast on WWGP-based medium as on a reference medium but achieved higher cell dry weight (CDW) and lower intracellular triacylglycerol accumulation (22.5% vs. 28.6%) in WWGP-based medium. In addition, R. babjevae produced mannitol and arabitol and carotenoids and secreted polyol esters of fatty acids, a rare type of glycolipid as confirmed by Fourier transform-infrared, nuclear magnetic resonance and high-performance liquid chromatography analyses. Remarkably, R. babjevae consumed simultaneously both fructose and glucose when on WWGP-based medium and left glucose practically untouched in the reference medium, evidencing a fructophilic character. CONCLUSIONS: Rhodorotula babjevae, a metabolic versatile yeast, proliferated on a minimally processed extract and successfully converted glucose and fructose into high-value products. SIGNIFICANCE AND IMPACT OF STUDY: Different chemicals with market potential can be produced through the valorization of abundant waste feedstocks generated by the wine industry to which R. babjevae can contribute.

Drug-Loaded Hydrogels for Intraocular Lenses with Prophylactic Action against Pseudophakic Cystoid Macular Edema.

Pseudophakic cystoid macular edema (PCME), caused by chronic inflammation, is the most common cause of visual impairment in the medium-term after cataract surgery. Therefore, the prophylactic topical administration of combined steroidal and non-steroidal anti-inflammatory drugs is commonly done. Drug-eluting intraocular lenses (IOLs) gained interest as an efficient way to overcome the compliance issues related to the use of ocular drops without the need for additional surgical steps. The incorporation of functional monomers and molecular imprinting were herein applied to design hydrogels suitable as IOLs and able to co-deliver steroidal (dexamethasone sodium phosphate) and non-steroidal (bromfenac sodium) drugs. The incorporation of N-(2-aminopropyl) methacrylamide (APMA) increased the drug uptake and improved the in vitro release kinetics. Imprinting with bromfenac resulted in a decreased drug release due to permanent drug bonding, while imprinting with dexamethasone increased the amount of dexamethasone released after dual-drug loading. The application of a mathematical model to predict the in vivo drug release behavior suggests the feasibility of achieving therapeutic drug concentrations of bromfenac and dexamethasone in the aqueous humor for about 2 and 8 weeks, respectively, which is compatible with the current topical prophylaxis after cataract surgery.

Polymerizable Matrix Metalloproteinases' Inhibitors with Potential Application for Dental Restorations.

Collagen cleavage by matrix metalloproteinase (MMP) is considered a major cause of dental resins long term failure. Most MMP inhibitors display significant toxicity and are unsuitable for dental resins' applications. Here we report a study of a new class of inhibitors that display the unique property of being co-polymerizable with other vinyl compounds present in commercial dental resins, limiting their release and potential toxicity. Computational affinity towards the active site of different MMP-1; -2; -8; -9 and -13 of several compounds showed interesting properties and were synthesized. These free compounds were tested concerning their toxicity upon contact with two different cell types, with no substantial decrease in cell viability at high concentrations. Even so, compound's safety can be further improved upon copolymerization with commercial dental resins, limiting their release.

Imprinted hydrogels with LbL coating for dual drug release from soft contact lenses materials.

A combined strategy to control the release of two drugs, one anti-inflammatory (diclofenac sodium, DCF) and one antibiotic (moxifloxacin hydrochloride, MXF), from a soft contact lens (SCL) material, was assessed. The material was a silicone-based hydrogel, which was modified by molecular imprinting with MXF and coated by the layer-by-layer (LbL) method using natural polyelectrolytes: alginate (ALG), poly-l-lysine (PLL) and hyaluronate (HA), crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Imprinting was used to increase the amount of MXF loaded and to sustain its release, while the LbL coating acted as a diffusion barrier for DCF and improved the surface properties. The drugs were loaded by soaking in a DCF + MXF dual solution. High hydrostatic pressure (HHP) was successfully applied in the sterilization of the drug-loaded hydrogels. The transmittance, refractive index, wettability and ionic permeability of the hydrogels remained within the required levels for SCLs application. The concentrations of the released DCF and MXF stayed above the IC50 and the MIC (for S. aureus and S. epidermidis) values, for 9 and 10 days, respectively. No ocular irritancy was detected by the HET-CAM test. NIH/3T3 cell viability demonstrated that the drug-loaded hydrogels were not toxic, and cell adhesion was reduced.

Moxifloxacin-imprinted silicone-based hydrogels as contact lens materials for extended drug release.

Contact lenses may act as drug release platforms for the treatment of ocular infections, but there is still the need for extending their typical release periods and enhancing ocular bioavailability. The present study aimed to develop a molecularly imprinted silicone-based hydrogel to be used in the manufacturing of contact lenses that can be loaded efficiently and be able to release the antibiotic moxifloxacin hydrochloride (MXF) in a sustained way. A set of hydrogels was prepared by the molecular imprinting method using acrylic acid (AA) as the functional monomer for the specific recognition of MXF. The modified hydrogels loaded a higher amount of MXF, which was released for a longer time. In vitro experiments, using a microfluidic cell to mimic the ocular surface fluid turnover, showed that the imprinted hydrogel TRIS(300)-I prepared with the highest content in AA led to MXF concentrations in the release medium which were effective against S. aureus and S. epidermidis for about 2 weeks. Furthermore, some important properties such as water uptake, wettability, transmittance, ionic permeability, and Young´s modulus of the modified hydrogel remained within the range of values recommended for contact lenses. No cytotoxicity and no potential ocular irritancy effect were detected. Such hydrogel seems to be a promising alternative to the current options for the treatment of ocular infections.

Diclofenac sustained release from sterilised soft contact lens materials using an optimised layer-by-layer coating.

A layer-by-layer (LbL) coating was designed using ionic polysaccharides (chitosan, sodium alginate, sodium hyaluronate) and genipin (crosslinker), to sustain the release of diclofenac sodium salt (DCF) from soft contact lens (SCL) materials. The coating was hydrophilic, biocompatible, non-toxic, reduced bacterial growth and had minor effects on the physical properties of the material, such as wettability, ionic permeability, refractive index and transmittance, which remained within the recommended values for SCLs. The coating was applied on a silicone-based hydrogel and on commercial SofLens and Purevision SCLs. The coating attenuated the initial drug burst and extended the therapeutic period for, at least, two weeks. Relevantly, the problems of sterilizing drug loaded SCLs coated with biopolymers, using classic methods that involve high temperature or radiation, were successfully solved through high hydrostatic pressure (HHP) sterilization.

Challenges in Matrix Metalloproteinases Inhibition.

Matrix metalloproteinases are enzymes that degrade the extracellular matrix. They have different substrates but similar structural organization. Matrix metalloproteinases are involved in many physiological and pathological processes and there is a need to develop inhibitors for these enzymes in order to modulate the degradation of the extracellular matrix (ECM). There exist two classes of inhibitors: endogenous and synthetics. The development of synthetic inhibitors remains a great challenge due to the low selectivity and specificity, side effects in clinical trials, and instability. An extensive review of currently reported synthetic inhibitors and description of their properties is presented.

A New Pathway for Mannitol Metabolism in Yeasts Suggests a Link to the Evolution of Alcoholic Fermentation.

The yeasts belonging to the Wickerhamiella and Starmerella genera (W/S clade) share a distinctive evolutionary history marked by loss and subsequent reinstatement of alcoholic fermentation mediated by horizontal gene transfer events. Species in this clade also share unusual features of metabolism, namely the preference for fructose over glucose as carbon source, a rare trait known as fructophily. Here we show that fructose may be the preferred sugar in W/S-clade species because, unlike glucose, it can be converted directly to mannitol in a reaction with impact on redox balance. According to our results, mannitol is excreted to the growth medium in appreciable amounts along with other fermentation products such as glycerol and ethanol but unlike the latter metabolites mannitol production increases with temperature. We used comparative genomics to find genes involved in mannitol metabolism and established the mannitol biosynthesis pathway in W/S-clade species Starmerella bombicola using molecular genetics tools. Surprisingly, mannitol production seems to be so important that St. bombicola (and other W/S-clade species) deploys a novel pathway to mediate the conversion of glucose to fructose, thereby allowing cells to produce mannitol even when glucose is the sole carbon source. Using targeted mutations and 13C-labeled glucose followed by NMR analysis of end-products, we showed that the novel mannitol biosynthesis pathway involves fructose-6-phosphate as an intermediate, implying a key role for a yet unknown fructose-6-P phosphatase. We hypothesize that mannitol production contributed to mitigate the negative effects on redox balance of the ancient loss of alcoholic fermentation in the W/S clade. Presently, mannitol also seems to play a role in stress protection.

Evidence for loss and reacquisition of alcoholic fermentation in a fructophilic yeast lineage.

Fructophily is a rare trait that consists of the preference for fructose over other carbon sources. Here, we show that in a yeast lineage (the Wickerhamiella/Starmerella, W/S clade) comprised of fructophilic species thriving in the high-sugar floral niche, the acquisition of fructophily is concurrent with a wider remodeling of central carbon metabolism. Coupling comparative genomics with biochemical and genetic approaches, we gathered ample evidence for the loss of alcoholic fermentation in an ancestor of the W/S clade and subsequent reinstatement through either horizontal acquisition of homologous bacterial genes or modification of a pre-existing yeast gene. An enzyme required for sucrose assimilation was also acquired from bacteria, suggesting that the genetic novelties identified in the W/S clade may be related to adaptation to the high-sugar environment. This work shows how even central carbon metabolism can be remodeled by a surge of HGT events.

Stepwise Functional Evolution in a Fungal Sugar Transporter Family.

Sugar transport is of the utmost importance for most cells and is central to a wide range of applied fields. However, despite the straightforward in silico assignment of many novel transporters, including sugar porters, to existing families, their exact biological role and evolutionary trajectory often remain unclear, mainly because biochemical characterization of membrane proteins is inherently challenging, but also owing to their uncommonly turbulent evolutionary histories. In addition, many important shifts in membrane carrier function are apparently ancient, which further limits our ability to reconstruct evolutionary trajectories in a reliable manner. Here, we circumvented some of these obstacles by examining the relatively recent emergence of a unique family of fungal sugar facilitators, related to drug antiporters. The former transporters, named Ffz, were previously shown to be required for fructophilic metabolism in yeasts. We first exploited the wealth of fungal genomic data available to define a comprehensive but well-delimited family of Ffz-like transporters, showing that they are only present in Dikarya. Subsequently, a combination of phylogenetic analyses and in vivo functional characterization was used to retrace important changes in function, while highlighting the evolutionary events that are most likely to have determined extant distribution of the gene, such as horizontal gene transfers (HGTs). One such HGT event is proposed to have set the stage for the onset of fructophilic metabolism in yeasts, a trait that according to our results may be the metabolic hallmark of close to 100 yeast species that thrive in sugar rich environments.

Prevalence of yeast other than Candida albicans in denture wearers.

PURPOSE: The isolation of yeast species other than Candida albicans from the oral mucosa has been increasing in frequency, suggesting that those may constitute emerging potential oral colonizers. The purpose of this work was to determine whether yeast species other than C. albicans are associated with factors related to wearing of dental prostheses. MATERIALS AND METHODS: tRNA-PCR fingerprinting and sequencing of the 26S rDNA D1/D2 domain were used to identify all yeasts isolated from CHROMagar™ Candida cultures of oral swabs collected from 178 patients. RESULTS: Besides C. albicans, 13 other species were identified, corresponding to 34% of the yeast isolates. The majority of the non-C. albicans species were not detected as single colonizers but rather in co-colonization with one or two other yeasts, often with C. albicans. No significant associations were found with non-C. albicans species. On the contrary, the best-fitted logistic regression model predicts that either wearing a denture (adjusted odds = 4.6) or insufficient oral hygiene (adjusted odds = 2.3) are risks for colonization by yeast, in general. CONCLUSIONS: The colonization with non-C. albicans species and co-colonization were not independently associated with any of the analyzed host-related factors. In particular, neither wearing a removable denture nor being elderly were significant predictors.

Fsy1, the sole hexose-proton transporter characterized in Saccharomyces yeasts, exhibits a variable fructose:H(+) stoichiometry.

In the model yeast Saccharomyces cerevisiae, hexose uptake is mediated exclusively by a family of facilitators (Hxt, hexose transporters). Some other Saccharomyces species (e.g. Saccharomyces bayanus and Saccharomyces pastorianus) possess, in addition, a specific fructose transporter (Fsy1, fructose symporter) that has been previously described to function as a proton symporter. In the present work, we compared growth of a yeast strain in which FSY1 occurs naturally in anaerobic, fructose- and glucose-limited chemostat cultures. Especially at low specific growth rates, fructose-proton symport was shown to have a strong impact on the biomass yield on sugar. We subsequently employed energized hybrid plasma membrane vesicles to confirm previous observations concerning the mode of operation and specificity of Fsy1 mediated transport. Surprisingly, these experiments suggested that the carrier exhibits an unusual fructose:H(+) stoichiometry of 1:2. This energetically expensive mode of operation was also found consistently in vivo, in shake flask and in chemostat cultures, and both when Fsy1 is the sole transporter and when the Hxt carriers are present. However, it is observed only when Fsy1 is operating at higher glycolytic fluxes, a situation that is normally prevented by downregulation of the gene. Taken together, our results suggest the possibility that fructose symport with more than one proton may constitute an energetically unfavorable mode of operation of the Fsy1 transporter that, in growing cultures, is prevented by transcriptional regulation.

FSY1, a horizontally transferred gene in the Saccharomyces cerevisiae EC1118 wine yeast strain, encodes a high-affinity fructose/H+ symporter.

Transport of glucose and fructose in the yeast Saccharomyces cerevisiae plays a crucial role in controlling the rate of wine fermentation. In S. cerevisiae, hexoses are transported by facilitated diffusion via hexose carriers (Hxt), which prefer glucose to fructose. However, utilization of fructose by wine yeast is critically important at the end of fermentation. Here, we report the characterization of a fructose transporter recently identified by sequencing the genome of the commercial wine yeast strain EC1118 and found in many other wine yeasts. This transporter is designated Fsy1p because of its homology with the Saccharomyces pastorianus fructose/H(+) symporter Fsy1p. A strain obtained by transformation of the V5 hxt1-7Δ mutant with FSY1 grew well on fructose, but to a much lesser extent on glucose as the sole carbon source. Sugar uptake and symport experiments showed that FSY1 encodes a proton-coupled symporter with high affinity for fructose (K(m) 0.24±0.04mM). Using real-time RT-PCR, we also investigated the expression pattern of FSY1 in EC1118 growing on various carbon sources. FSY1 was repressed by high concentrations of glucose or fructose and was highly expressed on ethanol as the sole carbon source. The characteristics of this transporter indicate that its acquisition could confer a significant advantage to S. cerevisiae during the wine fermentation process. This transporter is a good example of acquisition of a new function in yeast by horizontal gene transfer.

Maltotriose utilization by industrial Saccharomyces strains: characterization of a new member of the alpha-glucoside transporter family.

Maltotriose utilization by Saccharomyces cerevisiae and closely related yeasts is important to industrial processes based on starch hydrolysates, where the trisaccharide is present in significant concentrations and often is not completely consumed. We undertook an integrated study to better understand maltotriose metabolism in a mixture with glucose and maltose. Physiological data obtained for a particularly fast-growing distiller's strain (PYCC 5297) showed that, in contrast to what has been previously reported for other strains, maltotriose is essentially fermented. The respiratory quotient was, however, considerably higher for maltotriose (0.36) than for maltose (0.16) or glucose (0.11). To assess the role of transport in the sequential utilization of maltose and maltotriose, we investigated the presence of genes involved in maltotriose uptake in the type strain of Saccharomyces carlsbergensis (PYCC 4457). To this end, a previously constructed genomic library was used to identify maltotriose transporter genes by functional complementation of a strain devoid of known maltose transporters. One gene, clearly belonging to the MAL transporter family, was repeatedly isolated from the library. Sequence comparison showed that the novel gene (designated MTY1) shares 90% and 54% identity with MAL31 and AGT1, respectively. However, expression of Mty1p restores growth of the S. cerevisiae receptor strain on both maltose and maltotriose, whereas the closely related Mal31p supports growth on maltose only and Agt1p supports growth on a wider range of substrates, including maltose and maltotriose. Interestingly, Mty1p displays higher affinity for maltotriose than for maltose, a new feature among all the alpha-glucoside transporters described so far.