Filip Kiekens

Drying techniques of probiotic bacteria as an important step towards the development of novel pharmabiotics.

The increasing knowledge about the human microbiome leads to the awareness of how important probiotics can be for our health. Although further substantiation is required, it appears that several pathologies could be treated or prevented by the administration of pharmaceutical formulations containing such live health-beneficial bacteria. These pharmabiotics need to provide their effects until the end of shelf life, which can be optimally achieved by drying them before further formulation. However, drying processes, including spray-, freeze-, vacuum- and fluidized bed drying, induce stress on probiotics, thus decreasing their viability. Several protection strategies can be envisaged to enhance their viability, including addition of protective agents, controlling the process parameters and prestressing the probiotics prior to drying. Moreover, probiotic viability needs to be maintained during long-term storage. Overall, lower storage temperature and low moisture content result in good survival rates. Attention should also be given to the rehydration conditions of the dried probiotics, as this can exert an important effect on their revival. By describing not only the characteristics, but also the viability results obtained by the most relevant drying techniques in the probiotic industry, we hope to facilitate the deliberate choice of drying process and protection strategy for specific probiotic and pharmabiotic applications.

Mixed Hemi/Ad-Micelle Sodium Dodecyl Sulfate-Coated Magnetic Iron Oxide Nanoparticles for the Efficient Removal and Trace Determination of Rhodamine-B and Rhodamine-6G

Mixed hemi/ad-micelle sodium dodecyl sulfate (SDS)-coated magnetic iron oxide nanoparticles (MHAMS-MIONPs) were used as an efficient adsorbent for both removal and preconcentration of two important carcinogenic xanthine dyes named rhodamine-B (RB) and rhodamine-6G (RG). To gain insight in the configuration of SDS molecules on the surface of MIONPs, zeta potential measurements were performed in different [SDS]/[MIONP] ratios. Zeta potential data indicated that mixed hemi/ad-micelle MHAM was formed in [SDS]/[MIONP] ratios over the range of 1.1 to 7.3. Parameters affecting the adsorption of dyes were optimized as removal efficiency by one variable at-a-time and response surface methodology; the obtained removal efficiencies were ∼100%. Adsorption kinetic and equilibrium studies, under the optimum condition (pH = 2; amount of MIONPs = 87.15 mg; [SDS]/[MIONP] ratio = 2.9), showed that adsorption of both dyes are based on the pseudo-second-order and the Langmuir isotherm models, respectively. The maximum adsorption capacities for RB and RG were 385 and 323 mg g(-1), respectively. MHAMS-MIONPs were also applied for extraction of RB and RG. Under optimum conditions (pH = 2; amount of damped MHAMS-MIONPs = 90 mg; eluent solvent volume = 2.6 mL of 3% acetic acid in acetonitrile), extraction recoveries for 0.5 mg L(-1) of RB and RG were 98% and 99%, with preconcentration factors of 327 and 330, respectively. Limit of detection obtained for rhodamine dyes were <0.7 ng mL(-1). Finally, MHAMS-MIONPs were successfully applied for both removal and trace determination of RB and RG in environmental and wastewater samples.

Mixed hemi/ad-micelles coated magnetic nanoparticles for the entrapment of hemoglobin at the surface of a screen-printed carbon electrode and its direct electrochemistry and electrocatalysis

An efficient procedure for the physical entrapment of proteins within a biocompatible matrix and their immobilization on electrode surfaces is of utmost importance in the fabrication of biosensors. In this work, the magnetic entrapment of hemoglobin (Hb) at the surface of a screen-printed carbon electrode (SPCE), through mixed hemi/ad-micelles (MHAM) array of positively charged surfactant supported iron oxide magnetic nanoparticles (Mag-NPs), is reported. The Hb/MHAM@Mag-NPs biocomposite is captured at SPCE by a super magnet (Hb/MHAM@Mag-NPs/SPCE). To gain insight in the configuration of the mixed hemi/ad-micelles of CTAB at Mag-NPs, zeta-potential measurements were performed. The entrapment of Hb at MHAM@Mag-NPs was confirmed by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and Fourier transform infrared spectroscopy (FT-IR). Direct electron transfer of the Hb intercalated into the composite film showed a pair of well-defined quasi-reversible redox peak at formal potential of -0.255 V vs. Ag/AgCl corresponding to heme Fe(III)/Fe(II) redox couple. It shows that the MHAM@Mag-NPs composite could increase the adsorption ability for Hb, thus provides a facile direct electron transfer between the Hb and the substrate. The proposed biosensor showed excellent electrocatalytic activity to the H2O2 reduction in the wide concentration range from 5.0 to 300.0 µM obtained by amperometric measurement. The Michaelis-Menten constant (Km) value of Hb at the modified electrode is 55.4 µM, showing its high affinity. Magnetic entrapment offers a promising design for fast, convenient and effective immobilization of protein within a few minutes for determination of the target molecule in low sample volume at disposable cost-effective SPCE.

Ordered mesoporous silica to enhance the bioavailability of poorly water-soluble drugs: Proof of concept in man

Formulating poorly water soluble drugs using ordered mesoporous silica materials is an emerging approach to tackle solubility-related bioavailability problems. The current study was conducted to assess the bioavailability-enhancing potential of ordered mesoporous silica in man. In this open-label, randomized, two-way cross-over study, 12 overnight fasted healthy volunteers received a single dose of fenofibrate formulated with ordered mesoporous silica or a marketed product based on micronized fenofibrate. Plasma concentrations of fenofibric acid, the pharmacologically active metabolite of fenofibrate, were monitored up to 96h post-dose. The rate (Cmax/dose increased by 77%; tmax reduced by 0.75h) and extent of absorption (AUC0-24h/dose increased by 54%) of fenofibrate were significantly enhanced following administration of the ordered mesoporous silica based formulation. The results of this study serve as a proof of concept in man for this novel formulation approach.

Formation of aerobic granular sludge during the treatment of petrochemical wastewater

In this study, petrochemical wastewater from the port of Antwerp was used for the development of aerobic granular sludge. Two different reactor setups were used, (1) a completely aerated sequencing batch reactor (SBRae) with a feast/famine regime and (2) a sequencing batch reactor operated with an anaerobic feast/aerobic famine strategy (SBRan). The seed sludge showed poor settling characteristics with a sludge volume index (SVI) of 285mL.gMLSS-1 and a median particle size by volume of 86.0µm±1.9µm. In both reactors, granulation was reached after 30days with a SVI of 71mL.gMLSS-1 and median granule size of 264.7µm in SBRan and a SVI of 56mL.gMLSS-1 and median granule size of 307.4µm in SBRae. The chemical oxygen demand (COD) and dissolved organic carbon (DOC) removal was similar in both reactors and above 95%. The anaerobic DOC uptake increased from 0.13% to 43.2% in 60days in SBRan.

Evaluation of a newly developed HPMC ophthalmic insert with sustainehttp://10.10.23.106:8080/TDXPSLIVEGANG/gateway/elsevierjournal/index.jsp#d release properties as a carrier for thermolabile therapeutics

A novel drug delivery system (DDS) with sustained release properties was developed to allow ocular protein delivery. The DDS developed is aimed at overcoming stability issues during preparation such as denaturation of proteins caused by shear forces applied or due to elevated temperatures and air entrapment potentially causing oxidation of the molecule. The rod-shaped HPMC inserts were loaded with lysozyme and several HPMC types were studied and compared. An aqueous colloidal HPMC solution (hydrogel) was prepared and subsequently dried at 150°C to dehydrate the polymer solution. This partially dehydrated polymer cylinder was loaded with an aqueous glycerol/lysozyme solution at 2°C. A 2(4) full factorial design was set up to evaluate the effect of the different preparation parameters on water uptake and release properties. As a result, four out of sixteen formulations revealed homogenous distribution for lysozyme in both duplicates. The change in water uptake over time was dependent on the type of HPMC polymer used but not between the chosen HPMC percentages. After 240min, 50% of lysozyme loaded was released depending on the chosen formulation. Lysozyme molecules exhibit slower release from a K100M matrix compared to E10M inserts, albeit the overall effect is relatively limited.

Interplay between Lactobacillus rhamnosus GG and Candida and the involvement of exopolysaccharides

A number of clinical studies have shown protective effects of lactobacilli against Candida species in the gastrointestinal tract, the urogenital tract and the oral cavity, while others did not show clear effects. Evidence on the mode of action of lactobacilli against Candida is also still lacking. In this study, the anti‐Candida activity of the model probiotic strain Lactobacillus rhamnosus GG was explored in different assays to determine molecular interactions. We found that L. rhamnosus GG was able to interfere with Candida growth, morphogenesis and adhesion. These three aspects of Candidas physiology are all crucial to its opportunistic pathogenesis. In follow‐up assays, we compared the activity of L. rhamnosus GG wild‐type with its exopolysaccharide (EPS)‐deficient mutant and purified EPS to evaluate the involvement of this outer carbohydrate layer. Our data demonstrate that purified EPS can both interfere with hyphal formation and adhesion to epithelial cells, which indicates that EPS is part of a combined molecular mechanism underlying the antihyphal and anti‐adhesion mechanisms of L. rhamnosus GG.

Optimisation of HPMC ophthalmic inserts with sustained release properties as a carrier for thermolabile therapeutics

A methodology was developed and optimised for the preparation of a new drug delivery system (DDS) with sustained release properties to allow ocular protein delivery and to limit destructive production steps during manufacturing. Elevated temperatures, shear forces and an oxidative environment should be avoided in order to prevent denaturation or oxidation of proteins. An aqueous HPMC solution was prepared using heat and casted into small semi-rod-shaped PVC blisters. The polymer solution was allowed to cool down and was partially dehydrated at room temperature. A drug solution containing glycerol, drug and water was subsequently added to rehydrate the partially dehydrated polymer matrix at a temperature of 2°C. Several parameters of the production process were varied to determine their influence on the release kinetics from HPMC inserts from three different molecules of different molecular weight. This production method was further optimised in order to shorten the rehydration time from weeks to days, while eliminating heat and shear forces on the selected drug molecules sodium fluorescein, lysozyme and albumin. Slow release kinetics were achieved for sodium fluorescein and lysozyme as model drug molecules. The higher molecular weight of albumin prevented a good penetration into the insert during the rehydration process resulting in predominantly burst release. The biocompatibility of a viscous HPMC solution was evaluated on SV40-human corneal epithelial cells with PrestoBlue® and no cytotoxic effects were observed.

Comparative studies on osmosis based encapsulation of sodium diclofenac in porcine and outdated human erythrocyte ghosts

The objective of our study was to develop controlled drug delivery system based on erythrocyte ghosts for amphiphilic compound sodium diclofenac considering the differences between erythrocytes derived from two readily available materials - porcine slaughterhouse and outdated transfusion human blood. Starting erythrocytes, empty erythrocyte ghosts and diclofenac loaded ghosts were compared in terms of the encapsulation efficiency, drug releasing profiles, size distribution, surface charge, conductivity, surface roughness and morphology. The encapsulation of sodium diclofenac was performed by an osmosis based process - gradual hemolysis. During this process sodium diclofenac exerted mild and delayed antihemolytic effect and increased potassium efflux in porcine but not in outdated human erythrocytes. FTIR spectra revealed lack of any membrane lipid disorder and chemical reaction with sodium diclofenac in encapsulated ghosts. Outdated human erythrocyte ghosts with detected nanoscale damages and reduced ability to shrink had encapsulation efficiency of only 8%. On the other hand, porcine erythrocyte ghosts had encapsulation efficiency of 37% and relatively slow drug release rate. More preserved structure and functional properties of porcine erythrocytes related to their superior encapsulation and release performances, define them as more appropriate for the usage in sodium diclofenac encapsulation process.

Formation of aerobic granular sludge and the influence of the pH on sludge characteristics in a SBR fed with brewery/bottling plant wastewater

A laboratory-scale sequencing batch reactor (SBR) was operated for 450 days to assess aerobic granule formation when treating brewery/bottling plant wastewater by consistent application of a feast/famine regime. The experiment was divided into three major periods according to the different operational conditions: (I) no pH control and strong fluctuations in organic loading rate (OLR) (1.18 ± 0.25 kgCOD·(m3·day)-1), (II) pH control and aeration control strategy to reduce OLR fluctuations (1.45 ± 0.65 kgCOD·(m3·day)-1) and (III) no pH control and stable OLR (1.42 ± 0.18 kgCOD·(m3·day)-1). Aerobic granule formation was successful after 80 days and maintained during the subsequent 380 days. The aerobic granular sludge was characterized by SVI5 and SVI30 values below 60 mL.g-1 and dominated by granular, dense structures. An oxygen uptake rate based aeration control strategy insured endogenous respiration at the end of the aerobic phase, resulting in stable SBR operation when the influent composition fluctuated. The quantitative polymerase chain reaction results show no significant enrichment of Accumulibacter or Competibacter during the granulation process. The 16S rRNA sequencing results indicate enrichment of other, possibly important species during aerobic granule formation while treating brewery wastewaters.