Penetration and distribution of three lipophilic probes in vitro in human skin focusing on the hair follicle.

Fluorescent model substances of increasing lipophilicity (Oregon Green) 488, Bodipy, FL C5 and Bodipy 564/570 C5) were selected to enable the visualization in the skin using confocal laser scanning microscopy. After measuring the penetration for 18 h, the nonfixed human scalp skin was imaged from the bottom parallel to the stratum corneum and in a cross-section view perpendicular to the skin surface. The images were evaluated by calculating relative accumulation values for different penetrants. The studies indicate that the penetrated amount is highest for Bodipy FL C5 (medium lipophilicity) and lowest for Bodipy 564/570 C5 (high lipophilicity) whereas Bodipy 564/570 C5 (high lipophilicity) reveals the highest relative accumulation in parts of the hair follicle compared to Oregon Green 488 (low lipophilicity). The addition of 30% (v/v) ethanol to the donor phase of substance with a low lipophilicity increases the follicular delivery. From our results we conclude that delivery to the hair follicle can be improved by increasing the drugs lipophilicity and optimizing the composition of the donor phase. However, no conclusion can be drawn about the actual route of transport to the hair follicle.     

Safety aspects of non-ionic surfactant vesicles: a toxicity study related to the physicochemical characteristics of non-ionic surfactants.

Two different toxicity models were used to assess the relationship between the physicochemical properties of non-ionic surfactant vesicles (NSVs), and the safety of these vesicles for topical drug administration. The vesicles used in this study consisted of polyoxyethylene alkyl ethers (CnEOm) in which the number of C atoms (n) varied between 12 and 18 and the number of oxyethylene units (m) between 3 and 7. The physicochemical properties of the vesicles are described in terms of hydrophilic-lipophilic balance (HLB) values, and critical micelle concentrations (CMC), and the rigidity of the bilayers as determined by the gel-liquid transition temperatures and the cholesterol content of the bilayers. The first toxicity model, comprising the measurement of the ciliary beat frequency, is a tool to assess the safety of intranasally applied formulations. Studies using this ciliotoxicity model revealed that by increasing the length of the alkyl chain of the surfactant, a decrease in toxicity was observed. The opposite correlation was found if the length of the polyoxyethylene headgroup was increased. Furthermore, it was observed that gel-state vesicles produce less of an effect on the ciliary beat frequency than liquid state vesicles. The second toxicity model, comprising the determination of cell proliferation of human keratinocytes, is a method to assess skin irritancy. In contrast to the ciliotoxicity model the length of the polyoxyethylene headgroup and of the alkyl chains did not seem to have an effect on the safety of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Permeant lipophilicity and vehicle composition influence accumulation of dyes in hair follicles of human skin

In skin and hair research drug targeting to the hair follicle is of great interest. Therefore the influence of permeant lipophilicity and vehicle composition on local accumulation has been examined using confocal laser scanning microscopy (CLSM). Formulations saturated with either Oregon Green^® 488, Bodipy^® FL C₅ or Bodipy^® 564/570 C₅ were prepared. The dyes were applied in citric acid buffer, 8% (w/v) surfactants in citric acid buffer or 8% (w/v) surfactants/20% (w/v) propylene glycol in citric acid buffer. Flow-through diffusion experiments were performed with fresh human scalp skin, after which the skin was imaged using CLSM. Diffusion studies showed for Oregon Green^® 488 (low lipophilicity) a higher flux when applied in citric acid buffer compared to surfactants. In contrast the fluxes of the more lipophilic dyes (Bodipy^® FL C₅ and Bodipy^® 564/570 C₅) are highest when applied in surfactants/propylene glycol. CLSM studies revealed that follicular accumulation increased with (i) a lipophilic dye and (ii) application of lipophilic dyes in surfactants–propylene glycol. Therefore we conclude that targeting to the hair follicle can be increased by the use of lipophilic drugs in combination with surfactant solutions and propylene glycol.     

Cationic Liposomes Loaded with a Synthetic Long Peptide and Poly(I:C): a Defined Adjuvanted Vaccine for Induction of Antigen-Specific T Cell Cytotoxicity

For effective cancer immunotherapy by vaccination, co-delivery of tumour antigens and adjuvants to dendritic cells and subsequent activation of antigen-specific cytotoxic T cells (CTLs) is crucial. In this study, a synthetic long peptide (SLP) harbouring the model CTL epitope SIINFEKL was encapsulated with the TLR3 ligand poly(inosinic-polycytidylic acid) (poly(I:C)) in cationic liposomes consisting of DOTAP and DOPC. The obtained particles were down-sized to about 140 nm (measured by dynamic light scattering) and had a positive zeta-potential of about 26 mV (according to laser Doppler electrophoresis). SLP loading efficiency was about 40% as determined by HPLC. Poly(I:C) loading efficiency was about 50%, as assessed from the fluorescence intensity of fluorescently labelled poly(I:C). Immunogenicity of the liposomal SLP vaccine was evaluated in vitro by its capacity to activate dendritic cells (DCs) and present the processed SLP to SIINFEKL-specific T cells. The effectiveness of the vaccine to activate CD8⁺ T cells was analysed in vivo after intradermal and subcutaneous immunisation in mice, by measuring antigen-specific T cells in blood and spleens and assessing their functionality by cytokine production and in vivo cytotoxicity. The liposomal formulation efficiently delivered the SLP to DCs in vitro and induced a functional CD8⁺ T cell immune response in vivo to the CTL epitope present in the SLP. The SLP-specific CD8⁺ T cell frequency induced by the poly(I:C)-adjuvanted liposomal SLP formulation showed an at least 25 fold increase over the T cell frequency induced by the poly(I:C)-adjuvanted soluble SLP. In conclusion, cationic liposomes loaded with SLP and poly(I:C) have potential as a powerful therapeutic cancer vaccine formulation.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-014-9686-4) contains supplementary material, which is available to authorized users.KEY WORDS: cancer immunotherapy, cationic liposomes, CTL epitope, peptide antigen, Poly(I:C)     

Prenatal and early postnatal fatty acid status and neurodevelopmental outcome

The present review addresses the effect of pre- and postnatal supplementation of nutrition with long-chain polyunsaturated fatty acids (LCPUFA) on neurodevelopmental outcome. The few studies which addressed the effect of prenatal LCPUFA status or prenatal LCPUFA supplementation suggest that a better prenatal arachidonic acid (AA) and doxosahexaenoic acid (DHA) status might be related to a better neurodevelopmental outcome until at least 18 months of age. A review of the few randomized controlled trials on formula supplementation with LCPUFA in preterm infants did not provide evidence for a significant beneficial effect of LCPUFA on developmental outcome. A review of the trials on formula supplementation with LCPUFA in term infants revealed that supplementation with LCPUFA, in particularly supplementation with >or=0.30% DHA, has a beneficial effect on neurodevelopmental outcome until 4 months. The studies could not demonstrate a consistent positive effect beyond that age. It was concluded that the relatively subtle effects of LCPUFA supplementation on neurodevelopmental outcome do not only depend on dosage but also on the gestational period during which the nutritional components are supplied: supplementation prior to term seems to have more effect than that after term.     

Lipid and ultrastructural characterization of reconstructed skin models

The study aimed at evaluating tissue architecture and quality of the permeability barrier in commercially available reconstructed human skin models; EpiDerm, SkinEthic and Episkin in comparison to native tissue. For this purpose, tissue architecture was examined by electron microscopy and epidermal lipid composition was analyzed by HPTLC. Stratum corneum lipid organization was investigated by electron microscopy in combination with RuO(4) post-fixation and by SAXD. Ultrastructurally, the overall tissue architecture showed high similarities with native epidermis. In the stratum corneum extracellular space, lipid lamellae consisting of multiple alternating electron-dense and electron-lucent bands were present. This regular pattern was not seen throughout the whole stratum corneum probably due to the observed irregular lamellar body extrusion in some areas. Lipid analyses revealed the presence of all major epidermal lipid classes. Compared with native epidermis the content of polar ceramides 5 and 6 was lower, ceramide 7 was absent, and the content of free fatty acids was very low. These differences in lipid composition may account for differences observed in SAXD pattern of Episkin and EpiDerm penetration models. In the latter only the long-distance periodicity unit of about 12 nm was observed and the short periodicity unit was missing. In conclusion, all three skin models provide a promising means for studying the effects of topically applied chemicals, although the observed deviations in tissue homeostasis and barrier properties need to be optimized.     

Quantitative assessment of the transport of elastic and rigid vesicle components and a model drug from these vesicle formulations into human skin in vivo

The aim of this study was to quantitatively assess the distribution profiles of elastic and rigid vesicle material in human skin in vivo. Furthermore, the distribution profiles of the model drug ketorolac applied in these vesicle formulations was investigated. A deuterium-labelled phospholipid was incorporated into these vesicles to serve as a marker for the vesicle material. The vesicles were loaded with ketorolac at saturated concentrations. Vesicle solutions were applied non-occlusively onto the skin and the treated site was sequentially tape-stripped. Tape-strips were analyzed for vesicle material using attenuated total reflectance-Fourier transform infrared spectroscopy and for ketorolac by extraction of the tape-strips followed by high pressure liquid chromatography. Distribution profiles in the stratum corneum (SC) were obtained for the elastic and rigid vesicle material and for the ketorolac. These profiles have suggested that elastic vesicle material can rapidly enter the deeper layers of the SC and can reach almost the SC-viable epidermal junction. Rigid vesicle material, however, did not penetrate deep into the SC. Furthermore, the elastic vesicles were better than the rigid vesicles in the enhancement of ketorolac transport into human SC. The distribution profile of ketorolac in the deeper SC layers was, however, different from that of the vesicle material. This suggests that once the elastic vesicles partition into the SC, the ketorolac is released from the vesicles. The elastic vesicles are superior to the rigid vesicles both in terms of vesicular transport into the SC and in terms of therapeutic potential as a skin delivery vehicle.     

Exclusive breastfeeding of healthy term infants for at least 6 weeks improves neurological condition

To investigate the minimal duration of exclusive breastfeeding for optimal neurological outcome, we assessed the quality of general movements (GM) at 3 mo of 147 breastfed healthy term infants that were followed from birth. The quality of GM is a sensitive marker of neurological condition. The quality of GM was classified as normal-optimal, normal-suboptimal, mildly abnormal and definitely abnormal. Information on social and pre- and perinatal conditions and the duration of breastfeeding was collected prospectively. Logistical regression analyses were used to adjust for confounders. There was a positive association between breastfeeding duration and movement quality, with a saturation effect at the age of approximately 6 wk. In the group of infants breastfed for < or = 6 wk (n = 55), 18% exhibited normal-optimal GM, 47% normal-suboptimal GM, and 47% mildly abnormal GM. In contrast, in the group of infants breastfed for > 6 wk (n = 92), 43% exhibited normal-optimal GM, 45% normal-suboptimal GM, and 12% mildly abnormal GM. Exclusive breastfeeding for >6 wk was therefore associated with markedly less abnormal and more normal-optimal GM. Thus, we conclude that breastfeeding for > 6 wk might improve the neurological condition in infants.     

Elasticity of vesicles affects hairless mouse skin structure and permeability.

One of the possibilities for increasing the penetration rate of drugs through the skin is the use of vesicular systems. Currently, special attention is paid to the elastic properties of liquid-state vesicles, which are supposed to have superior properties compared to gel-state vesicles with respect to skin interactions. In this study, the effects of vesicles on hairless mouse skin, both in vivo and in vitro, were studied in relation to the composition of vesicles. The interactions of elastic vesicles containing the single chain surfactant octaoxyethylene laurate-ester (PEG-8-L) and sucrose laurate-ester (L-595) with hairless mouse skin were studied, in vivo, after non-occlusive application for 1, 3 and 6 h. The skin ultrastructure was examined by ruthenium tetroxide electron microscopy (TEM) and histology. The extent, to which vesicle constituents penetrated into the stratum corneum, was quantified by thin layer chromatography (TLC). The interactions of the elastic vesicles containing PEG-8-L and L-595 surfactants were compared with those observed after treatment with rigid vesicles containing the surfactant sucrose stearate-ester (Wasag-7). Furthermore, skin permeability experiments were carried out to investigate the effect of treatment with PEG-8-L micelles, elastic vesicles (containing PEG-8-L and L-595 surfactants) or rigid Wasag-7 vesicles on the 3H(2)O transport through hairless mouse skin, in vitro, after non-occlusive application. Treatment of hairless mouse skin with the elastic vesicles affected the ultrastructure of the stratum corneum: distinct regions with lamellar stacks derived from the vesicles were observed in intercellular spaces of the stratum corneum. These stacks disrupted the organization of skin bilayers leading to an increased skin permeability, whereas no changes in the ultrastructure of the underlying viable epidermis were observed. Treatment with rigid Wasag-7 vesicles did not affect the skin ultrastructure or skin permeability. TLC measurements showed that after 1 h of non-occlusive application of elastic or rigid vesicles, a six-fold increased amount of elastic vesicle material was present within the stratum corneum compared to rigid vesicle material. After 3 and 6 h of application the amount of PEG-8-L vesicle material in SC decreased to approximately three- and two-fold, respectively, compared to Wasag-7 vesicle material. Pretreatment of the hairless mouse skin with the elastic vesicles containing 70 mol% PEG-8-L increased the diffusion of 3H(2)O with an optimum application dose of 2.5 mg lipids/cm(2) compared to PBS pretreatment. No significant difference in the enhancement of the 3H(2)O-diffusion was observed between PEG-8-L micelles or elastic vesicles containing 30 or 70 mol% PEG-8-L. Pretreatment with the rigid Wasag-7 vesicles decreased the diffusion rate of 3H(2)O, most probably by the formation of a lipid layer on the skin surface. The effect of the elastic vesicles on the skin permeability is supported by the ultrastructural changes observed by TEM in the intercellular lipid domains. The elastic vesicles containing 70 mol% PEG-8-L disorganize the lipid bilayers thereby creating or modifying pathways for possible drug penetration.