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Purpose Several active compounds are sensitive to light, especially to the ultraviolet radiation (UV-R) leading to their degradation or modification, with lost or decrease of their biological activity. The aim of this study was to perform a systematic review regarding photostabilization strategies used on health products and perform a critical appraisal of their effectiveness. Results The bibliographic search identified 2261 results and merely 40 studies met the selection criteria. Of these, 85% referred to encapsulation strategies, 10% to antioxidants and 5% to the use of solar filters. Cyclodextrins (CD’s) were the most used encapsulation systems (32.5%) followed by liposomes and lipid nanoparticles (each 17.5%), microparticles (15%) and polymeric nanoparticles (10%). The most effective were found to be liposomes and lipid nanoparticles. However, the different methodological conditions used limit the true relevance of this finding. Conclusions A gold standard strategy suitable for all compounds cannot be proposed. Instead, case-by-case evaluation, supported on the photodegradation mechanism is recommended. Systematic studies that compare different photostabilization strategies undertaken with the same irradiation conditions are also needed.

Avobenzone is one of the most common UVA-filters in sunscreens, and is known to be photounstable. Some of the strategies used to stabilize this filter present some drawbacks like photosensitization reactions. Antioxidants are widely used as cosmetic ingredients that prevent photoageing and complement the photoprotection offered by the UV-filters preventing or reducing photogenerated reactive species. The purpose of this work was to study the effect of antioxidants in the photostabilization of avobenzone. The filter dissolved in dimethyl sulfoxide or incorporated in a sunscreen formulation was irradiated with simulated solar radiation (750 W/m(2)). The tested antioxidants were vitamin C, vitamin E, and ubiquinone. The area under the curve of the absorption spectrum for UVA range and the sun protection factor (SPF) were calculated. Vitamin E (1:2), vitamin C (1:0.5) and ubiquinone (1:0.5) were the more effective concentrations increasing the photostability of avobenzone. In sunscreen formulations, the most effective photostabilizer was ubiquinone which also promoted an increase in SPF. This knowledge is important to improve effectiveness of sunscreen formulation. Antioxidants can be valuable ingredients for sunscreens with a triple activity of filter stabilization, SPF boosting and photoageing prevention.

Study of excipients incompatibility with drugs in an early phase of pharmaceutical development is still a persistent difficulty within the pharmaceutical industry. We examine here the compatibility between an experimental drug (nebicapone) and common excipients using differential scanning calorimetry (DSC), high sensitivity DSC (HSDSC) and a conventional heat stress test. The results obtained indicate that nebicapone may be compatible with lactose monohydrate and sodium croscarmellose but is incompatible with magnesium stearate. This study concludes that HSDSC, in stepwise isothermal mode, may be used as a potential tool for detecting excipient incompatibilities.

Enchytraeids are important members of the soil fauna living in the true soil layer instead of the humus like most earthworms, resulting in a different interaction with chemicals in soil. It is well known that the detectable concentration of contaminants in the soil cannot fully predict a biological effect; therefore bioaccumulation studies are of great importance. In this study the bioaccumulation pattern of the pesticide lindane ([(14)C]gamma-HCH) in Enchytraeus albidus was analyzed in two different soil types, an artificial Organisation for Economic Co-operation and Development (OECD) soil and a natural agricultural soil. The effect of aging on bioaccumulation and bioavailability was also analyzed. Aging experiments consisted of having the same procedures at different times after soil contamination: Immediately after contamination and 1, 2, 4, 6, and 10 months after that. Major differences occurred within the first month. Considering the overall data (10 months), it is possible to observe that in OECD soil there is a correlation between concentration in soil and in the organisms, but that was not observed for the natural soil. The decrease patterns for concentration in soil and in watery soil extracts were different: monophasic behavior occurred in OECD soil, and a biphasic pattern was measured in natural soil. It was observed that the bioavailable ( i.e., water-extractable) fraction of lindane in the natural soil decreased faster than the total soil concentration. In natural soil the decrease of concentration appeared faster than in OECD soil; this may be related to the lower amount of organic matter content.

To study the accumulation and the histological effects in mice organs caused by hexavalent chromium, one of the corrosion products released from AISI 316L stainless steel implants, mice groups were subcutaneously injected with a metallic solution of chromium during a certain period of time. Similar injections were made with HBSS (Hank's Balanced Salt Solution) in other groups to be used as controls. The levels of chromium found in the liver, kidney and spleen of the control and the treated animals were obtained by atomic absorption spectrometry (AAS) and were compared to those obtained by AdSV (adsorptive stripping voltammetry) to test the accuracy of the results. During the experimental period, the liver and spleen showed a progressive and significant accumulation of chromium whereas in the kidney the significant accumulation found after the first week practically remained unchanged during the four weeks. Apparently, the histological analysis of these tissues did not evidence any relevant morphological alteration induced by the chromium accumulations during the four weeks of treatment.

The purpose of this study was to investigate the effects of 316L stainless steel (SS) corrosion products on the in vitro biomineralization process, because tissue necrosis, bone loss, impaired bone mineralization, and loosening of orthopedic implants are associated with ions and debris resulting from biodegradation. Rat bone marrow cells were cultured in experimental conditions that favored the proliferation and differentiation of osteoblastic cells and were exposed to SS corrosion products obtained by electrochemical means for periods ranging from 1 to 21 days. Quantification of total and ionized Ca and P, as well as Fe, Cr, and Ni, ions in the culture media of control and metal added cultures during the incubation period was performed to study the influence of corrosion products on the Ca and P consumption that occurs during the mineralization process. Control cultures and metal effects on cultures were evaluated concerning DNA content, enzymatic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and alkaline phosphatase (ALP) activity. Histochemical detection of ALP, Ca, and phosphate deposition, and examination of the cultures by scanning and transmission electron microscopy (SEM and TEM) were also performed. The presence of SS corrosion products resulted in impairment of the normal behavior of rat bone marrow cultures. Levels of Cr and Ni in the medium of cultures exposed to 316L SS corrosion products decreased throughout the incubation period, suggesting a regular deposition of these species; these results were supported by TEM observation of the cultures. Cultures exposed to the corrosion products presented lower DNA content, MTT reduction, and ALP activity and failed to form mineralized areas. These cultures showed negative staining on histochemical reactions for the identification of calcium and phosphate deposition and SEM and TEM examination did not show mineral globular structures or mineralization foci, respectively, which is characteristic of cultures grown in control conditions. These results suggest that metal ions associated with 316L SS are toxic to osteogenic cells, affecting their proliferation and differentiation.

The toxic effects caused by nickel (Ni) per si were explored by performing in vivo studies on mice following subcutaneous administration of a metallic solution of nickel at 1, 2, 3, and 4 weeks to evaluate the side effects of this metal ion when released from stainless steel implants. Other groups were similarly injected with HBSS and used as controls. Accumulation of Ni ions on liver, spleen, and kidney was assessed by an electrochemical method, adsorptive stripping voltammetry (AdSV) using microelectrodes, and atomic absorption spectrometry (AAS). Alterations of those organs induced by Ni ions were studied, showing that several histological changes had been induced. Chemical analysis and histological features indicate that Ni is partially accumulated in the study organs.

Summary The toxic effects caused by iron in kidney was performed on experimental studies with mice following administration of a metallic solution of this specie to simulate the iron corrosion products of a metallic implant. To quantify the total levels of iron present in this organ, an electrochemical method was chosen based on the application of square wave voltammetry using adsorptive collection of iron-catechol on a mercury film microelectrode (MFM). The optimal working conditions to produce a very stable and reproducible iron peak in the digested kidney samples were found to be pH=7.2 provided by 8.0 mmol/L PIPES buffer, catechol concentration of 3.0×10 -4 mol/L, deposition potential -1.80 V and deposition time 20 s. These results were compared with those obtained by atomic absorption spectrometry (AAS) indicating a good performance of the electrochemical method used. The analytical results show an increase of iron concentration with treatment time, which indicates that this metal ion is partially accumulated in the kidney. This accumulation induces with time some morphological alterations as evidenced by the histological analysis.

The cytocompatibility of stainless steel 316L (SS 316L) corrosion products was investigated with particular focus on the dose- and time-effect of electrochemically dissolved SS and the corresponding separate metal ions on osteogenic bone marrow derived cells. Type AISI 316L stainless steel (Fe 63.9%, Cr 18.0%, Ni 12.5%, Mo 2.8%, Si 1.2%, Mn 1.6% and C 0.025%, weight for weight) was anodically dissolved in Hank's Balanced Salt Solution (HBSS) and diluted to the following concentrations: 500 microg ml(-1) of Fe, 122 microg ml(-1) of Cr and 101 microg ml(-1) of Ni, as estimated by atomic absorption spectrometry. Similarly, salt solutions containing 50 microg ml(-1) of Fe (FeCl3 x 6H2O), 122 microg ml(-1) of Cr (CrCl3 x 6H2O) or 101 microg ml(-1) of Ni (NiNO3) were prepared. All solutions were diluted 1:10(3), 1:10(4) and 1:10(5) and their effects on cell proliferation and function of rabbit bone marrow cells were studied up to 28 days of culture. Bone marrow cells (second subculture) were cultured in alpha-Minimal Essential Medium (alpha-MEM) supplemented with 10% fetal bovine serum 10(-8) mol l(-1) dexamethasone, 2.52 x 10(-4) mol l(-1) ascorbic acid and 10(-2) mol l(-1) beta-glycerophosphate. The osteoblast response to the presence of metal ions was evaluated by biochemical assays (enzymatic reduction of MTT for evaluation of cell viability/proliferation, and estimation of alkaline phosphatase (ALP) activity) and histochemical assays (identification of ALP positive cells and calcium and phosphates deposits). Results suggest a decrease in the expression of the osteoblast phenotype in the presence of ion and alloy solutions. Stainless steel corrosion products elicited slight effects but the corresponding metal ions produced pronounced effects on the osteoblast phenotype, namely an alteration in the levels and temporal expression of ALP and lower and retarded tissue mineralization ability.

Chromium is the metallic species of most concern with the use of stainless steel implants. To study the distribution of chromium in mice organs with time, several animals were subcutaneously injected with 2.70 × 10−3 mol/L of chromium solution during a certain period of time. Square wave voltammetry using adsorptive collection of Cr3+ -DTPA complex on a mercury film microelectrode (MFM) was used for quantitation. Optimized conditions included DTPA concentration of 2.0 × 10−2 mol/L, sodium acetate 4.0 × 10−2 mol/L, sodium nitrate 0.40 mol/L, potassium permanganate 1.0 × 10−3 mol/L, pH ca. 6.1, deposition potential of −1.15 V, deposition time 20 s, frequency 100 Hz, step 2.5 mV and an amplitude of 20 mV. The detection limit of the method was 1.54 × 10−8 mol/L after deposition of 20s. Other species presented in the biological material such as calcium and iron do not interfere with the chromium determinations. The accuracy of the proposed method was checked by comparing the results with those obtained by atomic absorption spectrometry (AAS) yielding good agreement. The results showed an increase of chromium levels with time in liver, kidney and spleen, indicating that this metal is partially accumulated in the studied organs.

The apparent volume and compressibility index of commonly used excipients were evaluated according to European Pharmacopeia monograph (seventh edition) in order to study the influence of the procedure conditions. The results suggested that the leveling of the powder inside the cylinder ought to be avoided.

Alternative vectors to deliver viable cells of probiotics, to those conferring limited resistance to gastrointestinal conditions, still need to be sought. Therefore the main goal of the study was to develop tablets able to protect entrapped probiotic bacteria from gastric acidity, thus providing an easily manufacturing scale-up dosage form to deliver probiotics to the vicinity of the human colon. Whey protein concentrate microparticles with Lactobacillus paracasei L26 were produced by spray-drying and incorporated in tablets with cellulose acetate phthalate and sodium croscarmellose. The viability of L. paracasei L.26 throughout tableting as well as its gastric resistance and release from the tablets were evaluated. Storage stability of L. paracasei L26 tablets was also performed by evaluation of viable cells throughout 60 days at 23°C and 33% relative humidity. A decrease of approximately one logarithmic cycle was observed after the acid stage and the release of L. paracasei L26 from the tablets occurred only after 4 h in the conditions tested. Microencapsulated L. paracasei L26 in tablets revealed some susceptibility to the storage conditions tested since the number of viable cells decreased 2 log cycles after 60 days of storage. However, the viability of L. paracasei L26 after 45 days of storage did not reveal significant susceptibility upon exposure to simulated gastrointestinal conditions. The developed probiotic tablets revealed to be potential vectors for delivering viable cells of L. paracasei L26 and probably other probiotics to persons/patients who might benefit from probiotic therapy. The authors gratefully acknowledge Formulab and DSM for providing the whey protein concentrate and probiotic strain, respectively. This work was funded by FEDER under the Operational Program for Competitiveness Factors— COMPETE and by National funds via FCT—Fundação para a Ciência e a Tecnologia within the framework of project PROBIOCAPS—references PTDC/AGR-ALI/71051/2006 andFCOMP-01-0124-FEDER-008792,andthroughindividual research grants (SFRH/BPD/73781/2010, SFRH/BD/77647/ 2011 and SFRH/BPD/65410/2009) by FCT under QREN– POPH funds, co-financed by the European Social Fund and Portuguese National Funds from MCTES. published

Fibroblasts from normal human skin were cultured for a period of 21 days in the absence or in the presence of metal ions. The effects of stainless steel (SS) corrosion products were compared to the effects of iron, chromium and nickel ions used either separately (Fe, Cr, or Ni solutions) or combined (Fe+Cr+Ni solution). At several periods of time (4, 7, 14 and 21 days) the cell cultures were analysed for the following parameters: (a) metal ion accumulation by atomic absorption spectrometry; (b) cell morphology and viability by the neutral red assay; (c) cell proliferation by DNA assessment, and enzyme activity by both (d) MTT reduction and (e) acid phosphatase activity. Results showed that SS-corrosion products and the corresponding metal ions combined at the same concentrations, Fe+Cr+Ni solution, had opposite effects on fibroblast cultures. In fact, SS-corrosion products caused no apparent effects on cell morphology nor on cell proliferation whereas Fe+Cr+Ni solution stimulated both neutral red uptake and cell proliferation. The enzymatic assays showed that SS-corrosion products caused inhibition of both MTT reduction and acid phosphatase activity in contrast to Fe+Cr+Ni solution which stimulated their activity. Furthermore, in all biological parameters studied, a strong association was observed between the effects of Fe+Cr+Ni solution and Cr alone, suggesting that Cr was the metal ion mostly involved in the stimulatory effects of the combined solution.

Stainless steel (SS) corrosion products were obtained by electrochemical dissolution of SS type AISI 316L. Mice were injected subcutaneously with 0.5 ml of SS solution (containing 283 μg Fe, 69.3 μg Cr and 57 μg Ni) each 72 h, for 10 days or 14 days. After the treatment time, livers were removed and were analysed for: (a) liver wet weight; (b) contents in Fe, Cr and Ni; (c) histological and ultrastructural alterations. Results showed that the percentage of liver weight per animal body weight was significantly higher (p < 0.05) in SS-injected animals than in the control animals. The atomic absorption spectrometry analysis of dry livers showed that chromium, but not iron or nickel, had a significant increase (p < 0.05) in SS-treated mice compared to the control animals. No histopathological differences between 10 and 14 days of SS-injection could be detected, however, massive hepatic degeneration was observed in both groups when compared to the control. These histological changes in SS-treated mice were confirmed at the ultrastructural level, as hepatocytes exhibited an augmentation of vacuoles in their cytoplasm. These actual liver morphological alterations suggest that the hepatocyte function may be hampered, which constitutes a matter of some concern since liver is a blood filtering organ.

Stainless steel is a metallic biomaterial commonly used in orthopaedic surgery. In this study we looked at the effects of stainless steel corrosion products on spleen, in order to evaluate their potential immunotoxicological effects. For this purpose stainless steel, type AISI 316L, was electrochemicallydissolved in a physiological salt solution. The final solution, containing 490 μg/ml Fe, 224 μg/ml Cr and 150 μg/ml Ni, was injected subcutaneously in mice. After several periods of time (4, 10 and 14 days) spleens were removed and analysed for: (a) their contents in Fe, Cr and Ni, (b) histological alterations, (c) enumeration of cellular populations. Results showed that stainless steel corrosion products accumulated in spleen, caused histological alterations, and induced changes in cellular populations. When compared to chromium and nickel, iron was the metal ion preferentially accumulated in spleen. The increase of the multinucleated giant cell population was accompanied by depletion of lymphocyte populations. This study indicates that stainless steel corrosion products can spread systemically and accumulate in spleen inducing histological and cellular alterations which may give rise to immunotoxicological consequences.

Special attention has been focused on the toxicity of some metallic species released from implanted materials, which accumulate in vital organs over long periods of time. A set of experiments with mice was designed to investigate the individual effects caused by iron in the liver and spleen. Histological features of these organs were evaluated and slight morphological changes were observed during the treatment time suggesting a negative correlation with the duration of the iron treatment. In addition, to associate the histological changes in the organs with iron accumulation an electrochemical method, adsorptive stripping voltammetry, was chosen to quantify the iron levels in these mentioned organs. The accuracy of the proposed method was checked by atomic absorption spectrometry. Both organs showed elevated concentrations of iron, nearly twofold, 7 days after iron administration compared to control organs.

Summary A catalytic cathodic stripping voltammetric procedure for the determination of total chromium in osteoblast-like cell culture medium using a mercury film microelectrode (MFM) was optimised. The method is based on the pre-concentration of the Cr(III)-diethylenetriaminepentaacetic acid (DTPA) complex by adsorption at the potential of -1.00 V ( vs. Ag/AgCl) in the presence of 10×10 -3 mol/L DTPA, 0.70 mol/L sodium nitrate, 0.04 mol/L sodium acetate and 1.0×10 -3 mol/L potassium permanganate at pH 5.9–6.0. The limit of detection obtained for a 40 s collection time was 2.80×10 -10 mol/L of chromium. The results achieved by stripping voltammetry using the MFM were compared to those obtained by atomic absorption spectrometry (AAS) to ensure the reliability of the electrochemical method. This procedure proved to be an alternative to AAS and valuable in biocompatibility studies performed in vitro using osteoblast-like cells.

Rat bone marrow cells were cultured in experimental conditions that favour the proliferation and differentiation of osteoblastic cells (i.e., 2.52×10 -4 mol l -1 ascorbic acid, 10 -2 mol l -1 β -glycerophosphate and 10 -8 mol l -1 dexamethasone) in the absence and in the presence of stainless-steel corrosion products, for a period of 18 days. An AISI 316L stainless-steel slurry (SS) was obtained by electrochemical means and the concentrations of the major metal ions, determined by atomic absorption spectrometry, were 8.78×10 -3 mol l -1 of Fe, 4.31×10 -3 mol l -1 of Cr and 2.56×10 -3 mol l -1 of Ni. Bone marrow cells were exposed to 0.01, 0.1 and 1% of the SS and at the end of the incubation period, control and treated cultures were evaluated by histochemical assays for the identification of the presence of alkaline phosphatase and also calcium and phosphate deposition. Cultures were further observed by scanning electron microscopy. Levels of total and ionised calcium and phosphorus in the culture media collected from control and metal exposed cell cultures were also quantified. Histochemical staining showed that control cultures presented a strong reaction for the presence of alkaline phosphatase and exhibited formation of calcium and phosphates deposits. The presence of 0.01% SS caused no detectable biological effects in these cultures, 0.1% SS impaired osteoblastic behaviour and, 1% SS resulted in cell death. In the absence of bone cells, levels of total and ionised calcium and phosphorus in the control and metal added culture medium were similar throughout the incubation period. A significant decrease in the levels of ionised calcium and phosphorus were observed in the culture medium of control cultures and also in cultures exposed to 0.01% SS after two weeks of incubation, an event related with the formation of mineral calcium phosphate deposits in these cultures. In cultures grown in the presence of 0.1 and 1% SS corrosion products, levels of calcium and phosphorus were similar to those observed in the absence of cells. Results showed that stainless-steel corrosion products above certain concentrations may disturb the normal behaviour of osteoblast-like rat bone marrow cell cultures.

In vitro studies to determine Fe3+ levels in mice liver samples were performed using platinum microelectrodes and atomic absorption spectrophotometry. Microelectrodes have been shown to be useful for quantitative analysis of metal ions released during the biodegradation process that occurs on implanted metallic biomaterials.

It is shown that it is possible to use microelectrodes to develop a number of strategies for the measurement of silver electronucleation kinetics at the molecular level. The direct measurement of individual events and therefore stochastic deterministic growth of the centres allows determination of an accurate value of the arrival time of the first nucleus. The distribution of these arrival times follows the simplest model of a pure birth process with identical rate constants for all the aggregation steps.

Mercury film microelectrodes were used to determine iron in osteoblast-like cell culture medium by performing adsorptive cathodic stripping voltammetry in the presence of catechol. The optimized analytical conditions are a catechol concentration of 5.00 × 10−4 mol L−1, pH 8.0 and a collection potential of −1.80 V which eliminates the copper interference. The reoxidation period was limited to the time between switching to −0.10 V and scanning up to the metal peak (ca. −0.40 V). The limit of detection obtained for a 30 s collection time was 1.93 × 10−8 mol L−1 iron. The results achieved by this procedure and using the square-wave voltammetry technique are accurate and precise. Biological assays were performed to evaluate the mineralization process.

The effect of the ionic strength and composition of solutions in the direct potentiometric determination of total and ionized calcium content using a calcium selective electrode was investigated. In the presence of a constant complexation buffer (CCB), the addition of sodium chloride and sodium bicarbonate causes a decrease in the limit of detection of the potentiometric response. When the procedure was tested in osteoblast-like cell culture medium no electrode interferences or method interferences were observed by the presence of proteins, i.e., the total and ionized calcium in this medium can be measured by potentiometric means.