Toxicological aspects of long-term treatment of keratinocytes with ZnO and TiO2 nanoparticles.

Sunscreens containing ZnO and TiO(2) nanoparticles (NPs) are increasingly applied to skin over long time periods to reduce the risk of skin cancer. However, long-term toxicological studies of NPs are very sparse. The in vitro toxicity of ZnO and TiO(2) NPs on keratinocytes over short- and long-term applications is reported. The effects studied are intracellular formation of radicals, alterations in cell morphology, mitochondrial activity, and cell-cycle distribution. Cellular response depends on the type of NP, concentration, and exposure time. ZnO NPs have more pronounced adverse effects on keratinocytes than TiO(2). TiO(2) has no effect on cell viability up to 100 µg mL(-1), whereas ZnO reduces viability above 15 µg mL(-1) after short-term exposure. Prolonged exposure to ZnO NPs at 10 µg mL(-1) results in decreased mitochondrial activity, loss of normal cell morphology, and disturbances in cell-cycle distribution. From this point of view TiO(2) has no harmful effect. More nanotubular intercellular structures are observed in keratinocytes exposed to either type of NP than in untreated cells. This observation may indicate cellular transformation from normal to tumor cells due to NP treatment. Transmission electron microscopy images show NPs in vesicles within the cell cytoplasm, particularly in early and late endosomes and amphisomes. Contrary to insoluble TiO(2), partially soluble ZnO stimulates generation of reactive oxygen species to swamp the cell redox defense system thus initiating the death processes, seen also in cell-cycle distribution and fluorescence imaging. Long-term exposure to NPs has adverse effects on human keratinocytes in vitro, which indicates a potential health risk.

Current view on nanosized solid lipid carriers for drug delivery to the skin.

Drug delivery into the skin is problematic because, as a natural barrier, it has a very low permeation rate. Several approaches have been made to increase the latter. Current advances in materials science and nanotechnology promise the development of new generations of drug carriers for therapeutic, diagnostic and protective purposes. We have reviewed various lipid drug delivery systems to the skin, with the main focus on solid lipid nanoparticles (SLNs). Further the article concerns possible variants in formulation of SLNs, the selection of lipid compounds and safe stabilizers, and the influence of the physicochemical properties of the drugs on distribution after loading into SLNs. In the last part the advantages of delivery into keratinocytes by SLNs over that of the free drug is addressed, focusing on decreased cytotoxicity of the incorporated drug in SLNs and controlled drug delivery to the subcellular compartments. This knowledge is important in the design and production of advanced solid lipid drug carriers. Additionally, the need for novel approaches or devices to improving permeation is also discussed in the context of moving the nanocarriers to targets deeper in the skin.

Improvements of cellular stress response on resveratrol in liposomes.

Resveratrol (RSV) has proven potential in prophylaxis and treatment of various disorders mediated by free radicals and oxidative stress. RSV solubility, stability, and cytotoxicity must be regulated for satisfactory bioavailability. Here, RSV was loaded into liposomes, characterized by PCS and TEM and evaluated on HEK293 cell line by metabolic activity assay, electron paramagnetic resonance, and fluorescence microscopy. RSV at 10 microM induced changes in cell metabolic activity and significantly improved antioxidative capacity. At 100 microM it showed concentration-dependent cytotoxicity. Oligolamellar liposomes with mean diameter 84 nm, polydispersity index 0.2, and zeta potential -40 mV showed high entrapment of RSV and rapid cellular internalization. Cell stress caused by UV-B irradiation diminished cell metabolic activity by 50%. RSV loaded into them showed no cytotoxicity at 100 microM and stimulated cellular metabolic and antioxidant activity levels to eliminate the harmful effect of the stress. Localization of RSV within liposomal bilayer is crucial for stimulation of cell-defense system, prevention of RSV cytotoxicity, and its long-term stability. In summary, evidence of different metabolic activity using free RSV and LIP-RSV is presented indicating that liposome-mediated uptake of RSV is more effective for improvement of the cell-stress response.

The combinations of polymorphisms in vitamin D receptor, osteoprotegerin and tumour necrosis factor superfamily member 11 genes are associated with bone mineral density.

1alpha,25-dihydroxyvitamin D(3) upregulates tumour necrosis factor superfamily member 11 (TNFSF11) that codes for the receptor activator of nuclear factor kappaB ligand (RANKL), and downregulates osteoprotegerin (OPG) expression. We have analyzed the individual effects of polymorphisms in the vitamin D receptor gene (VDR), OPG and TNFSF11, and searched for interactions between them. Six hundred and forty one subjects were evaluated: 239 osteoporotic and 228 non-osteoporotic post-menopausal, 57 pre-menopausal women and 117 elderly men. The subjects were genotyped for BsmI, FokI and Cdx2 in VDR, K3N in OPG and -290C>T, -643C>T and -693G>C in TNFSF11 gene. Bone mineral density (BMD) and biochemical markers were measured. In the osteoporotic women, femoral neck BMD (BMD-fn) showed associations with BsmI(VDR) and Cdx2(VDR) (P=0.015 and 0.047 respectively), and lumbar spine BMD (BMD-ls) with K3N(OPG) and -290C>T(TNFSF11) (P=0.021 and 0.017). No association with BMD was found in the non-osteoporotic women. In the pre-menopausal women, the Cdx2(VDR) polymorphism was associated with BMD-fn and total hip BMD (P=0.011 and 0.011). In elderly men, FokI(VDR) was associated with BMD-fn and BMD-ls (P=0.040 and 0.036). Interestingly, the -290C>T(TNFSF11)-K3N(OPG) combination was associated with BMD-th (P=0.041) in the osteoporotic women. In the non-osteoporotic women, the combination K3N(OPG)-Cdx2(VDR) was associated with BMD-ls, BMD-th and BMD-fn (P=0.032, 0.049 and 0.022), and the combination -290C>T(TNFSF11)-K3N(OPG) with BMD-fn (P=0.029). For the first time, the presence of gene-gene interactions between VDR, OPG and TNFSF11 genes was studied. Our results strongly suggest further confirmation of their combined influence on larger cohorts.

Surface active stabilizer tyloxapol in colloidal dispersions exerts cytostatic effects and apoptotic dismissal of cells.

Solid lipid nanoparticles (SLN) have been praised for their advantageous drug delivery properties such as biocompatibility, controlled release and passive drug targeting. However, the cytotoxicity of SLN and their ingredients, especially over a longer time period, has not been investigated in detail. We examined the critical issues regarding the use of a surface active stabilizer Tyloxapol (Tyl) for the preparation of solid lipid particles (SLP) and their effects on cellular functions and viability. SLP composed of behenate, phospholipids and a stabilizer, Tyloxapol or Lutrol (Lut), were prepared by the lipid melt method, labeled with a fluorescent dye and tested on Jurkat or HEK293 cells. The nano-sized particles were rapidly internalized and exhibited cytoplasmic localization. Incubation of cells with SLP-Tyl resulted in a dose- and time-dependent cytostatic effect, and also caused moderate and delayed cytotoxicity. Tyloxapol solution or SLP-Tyl dispersion caused the detachment of HEK293 cells, a decrease in cell proliferation and alterations in cellular morphology. Cell cycle analysis revealed that, while the unfavourable effects of SLP-Tyl and Tyloxapol solution are similar initially, longer incubation results in partial recovery of cells incubated with the dispersion of SLP-Tyl, whereas the presence of Tyloxapol solution induces apoptotic cell death. These findings indicate that Tyloxapol is an unfavourable stabilizer of SLP used for intracellular delivery and reinforce the role of stabilizers in a design of SLP with minimal cytotoxic properties.

Influence of nanosized delivery systems with benzyl nicotinate and penetration enhancers on skin oxygenation.

Many novel nanosized delivery systems have been designed for topical application of drugs since they can overcome the skin barrier and improve drug bioavailability. The increased absorption is often a consequence of a reversibly disrupted barrier function of the skin by the vehicle itself or by specific ingredients that act as penetration enhancers. This paper reports the effects of two nanosized systems (microemulsion and liposomes), in the presence and absence of penetration enhancers (PE), on the topical delivery of a lipophilic drug in vivo and compares that to classical hydrogel formulation. A vasodilator benzyl nicotinate (BN), which increases the blood flow of the skin, was incorporated into the formulations, and skin oxygenation was followed by electron paramagnetic resonance oximetry. It was found that microemulsions and liposomes (with or without PE) accelerate the rate of BN action when compared to hydrogel. However, incorporation of PE in microemulsion also improves the effectiveness of BN action. To understand why PE enhances the action of BN, its effect on the structure of the stratum corneum was investigated in vitro. The increased fluidity of the stratum corneum lipids provides an explanation for the greater penetration of BN into the skin when the drug and PE are together incorporated into the appropriate formulation.