Coating gold nanorods with self-assembling peptide amphiphiles promotes stability and facilitates in vivo two-photon imaging.

Gold nanorods (GNRs) are versatile asymmetric nanoparticles with unique optical properties. These properties make GNRs ideal agents for applications such as photothermal cancer therapy, biosensing, and in vivo imaging. However, as-synthesised GNRs need to be modified with a biocompatible stabilising coating in order to be employed in these fields as the ligands used to stabilise GNRs during synthesis are toxic. An issue is that GNR performance in the aforementioned techniques can be affected by these modified coatings. For example if coatings are too thick then GNR entry into cells, or their sensitivity in sensing applications, can be compromised. Here we show that thiolated peptide amphiphiles (PAs) can act as GNR stabilisers and provide a thin and highly-stable coating under physiologically relevant conditions. Additionally, all tested PAs formed highly ordered (51.8-58.8% ß-content), and dense (2.62-3.87 peptides per nm2) monolayers on the GNR surface. Moreover, the PA-coated GNRs demonstrated no cytotoxicity in vitro and, via injection in zebrafish embryos, the behavior and cellular interactions of such PA-coated GNRs were visualised in vivo, in real time, with two-photon (2P) microscopy.

The importance of ceramide headgroup for lipid localisation in skin lipid models.

The stratum corneum's lipid matrix is a critical for the skin's barrier function and is primarily composed of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The lipids form a long periodicity phase (LPP), a unique trilayer unit cell structure. An enzyme driven pathway is implemented to synthesize these key lipids. If these enzymes are down- or upregulated as in inflammatory diseases, the final lipid composition is affected often altering the barrier function. In this study, we mimicked down regulation of enzymes involved in the synthesis of the sphingosine and CER amide bond. In a LPP lipid model, we substituted CER N-(tetracosanoyl)-sphingosine (CER NS) with either i) FFA C24 and free sphingosine, to simulate the loss of the CER amide bond, or ii) with FFA C24 and C18 to simulate the loss of the sphingosine headgroup. Our study shows the lipids in the LPP would not phase separate until at least 25% of the CER NS is substituted keeping the lateral packing and conformational ordering unaltered. Neutron diffraction studies showed that free sphingosine chains localized at the outer layers of the unit cell, while the remaining CER NS head group was concentrated in the inner headgroup layers. However, when FFA C18 was inserted, CER NS was dispersed throughout the LPP, resulting in an even distribution between the inner and outer water layers. The presented results highlight the importance of the CER NS headgroup structure and its interaction in combination with the carbon chain invariability for optimal lipid arrangement.

Effects of ozone on stratum corneum lipid integrity and assembly.

The stratum corneum (SC) acts as the main barrier of the skin against exogenous substances (e.g. air pollutants) and against the loss of endogenous substances such as water. The SC consists of keratin-rich dead cells surrounded by crystalline lamellar lipid regions. The main lipid classes are ceramides (CERs), free fatty acids (FFAs), and cholesterol (CHOL). Tropospheric ozone (O3) is a potent oxidant compound that reacts instantly with biological molecules such as lipids and proteins. Although it has been reported that O3 induces biological responses at the cellular level, to the best of our knowledge, there is no information related to the damages O3 can cause at the level of the SC extracellular lipid matrix. The aim of our work was to investigate which SC lipid subclasses are prone to oxidation when exposed to O3 and how the changes in chemical structures affect the lipid organization in a stratum corneum substitute (SCS) membrane. Ultimately, the barrier properties of the SCS were examined. Our studies revealed that O3 induces chemical modifications of the unsaturated bonds in CERs and CHOL. The appearance of carbonyl groups at the headgroup level and the removal of the linoleate moiety of omega­O­acylceramides (CER EOS) impact the lamellar organization of the lipid assembly and to a lesser extent the lateral packing of the lipids. Unexpectedly, these changes improved the barrier function of the SCS.

Multitargeted Approach for the Optimization of Morphogenesis and Barrier Formation in Human Skin Equivalents.

In vitro skin tissue engineering is challenging due to the manifold differences between the in vivo and in vitro conditions. Yet, three-dimensional (3D) human skin equivalents (HSEs) are able to mimic native human skin in many fundamental aspects. However, the epidermal lipid barrier formation, which is essential for the functionality of the skin barrier, remains compromised. Recently, HSEs with an improved lipid barrier formation were generated by (i) incorporating chitosan in the dermal collagen matrix, (ii) reducing the external oxygen level to 3%, and (iii) inhibiting the liver X receptor (LXR). In this study, we aimed to determine the synergic effects in full-thickness models (FTMs) with combinations of these factors as single-, double-, and triple-targeted optimization approaches. The collagen-chitosan FTM supplemented with the LXR inhibitor showed improved epidermal morphogenesis, an enhanced lipid composition, and a better lipid organization. Importantly, barrier functionality was improved in the corresponding approach. In conclusion, our leading optimization approach substantially improved the epidermal morphogenesis, barrier formation, and functionality in the FTM, which therefore better resembled native human skin.

The Importance of Free Fatty Chain Length on the Lipid Organization in the Long Periodicity Phase.

The skin's barrier ability is an essential function for terrestrial survival, which is controlled by intercellular lipids within the stratum corneum (SC) layer. In this barrier, free fatty acids (FFAs) are an important lipid class. As seen in inflammatory skin diseases, when the lipid chain length is reduced, a reduction in the barrier's performance is observed. In this study, we have investigated the contributing effects of various FFA chain lengths on the lamellar phase, lateral packing. The repeat distance of the lamellar phase increased with FFA chain length (C20-C28), while shorter FFAs (C16 to C18) had the opposite behaviour. While the lateral packing was affected, the orthorhombic to hexagonal to fluid phase transitions were not affected by the FFA chain length. Porcine SC lipid composition mimicking model was then used to investigate the proportional effect of shorter FFA C16, up to 50% content of the total FFA mixture. At this level, no difference in the overall lamellar phases and lateral packing was observed, while a significant increase in the water permeability was detected. Our results demonstrate a FFA C16 threshold that must be exceeded before the structure and barrier function of the long periodicity phase (LPP) is affected. These results are important to understand the lipid behaviour in this unique LPP structure as well as for the understanding, treatment, and development of inflammatory skin conditions.

Arrangement of Ceramides in the Skin: Sphingosine Chains Localize at a Single Position in Stratum Corneum Lipid Matrix Models.

Understanding the structure of the stratum corneum (SC) is essential to understand the skin barrier process. The long periodicity phase (LPP) is a unique trilayer lamellar structure located in the SC. Adjustments in the composition of the lipid matrix, as in many skin abnormalities, can have severe effects on the lipid organization and barrier function. Although the location of individual lipid subclasses has been identified, the lipid conformation at these locations remains uncertain. Contrast variation experiments via small-angle neutron diffraction were used to investigate the conformation of ceramide (CER) N-(tetracosanoyl)-sphingosine (NS) within both simplistic and porcine mimicking LPP models. To identify the lipid conformation of the twin chain CER NS, the chains were individually deuterated, and their scattering length profiles were calculated to identify their locations in the LPP unit cell. In the repeating trilayer unit of the LPP, the acyl chain of CER NS was located in the central and outer layers, while the sphingosine chain was located exclusively in the middle of the outer layers. Thus, for the CER NS with the acyl chain in the central layer, this demonstrates an extended conformation. Electron density distribution profiles identified that the lipid structure remains consistent regardless of the lipid's lateral packing phase, this may be partially due to the anchoring of the extended CER NS. The presented results provide a more detailed insight on the internal arrangement of the LPP lipids and how they are expected to be arranged in healthy skin.

One Peptide for Them All: Gold Nanoparticles of Different Sizes Are Stabilized by a Common Peptide Amphiphile.

The functionalization of gold nanoparticles (GNPs) with peptidic moieties can prevent their aggregation and facilitate their use for applications both in vitro and in vivo. To date, no peptide-based coating has been shown to stabilize GNPs larger than 30 nm in diameter; such particles are of interest for applications including vaccine development, drug delivery, and sensing. Here, GNPs with diameters of 20, 40, and 100 nm are functionalized with peptide amphiphiles. Using a combination of transmission electron microscopy, UV-vis spectroscopy, and dynamic light scattering, we show that GNPs up to 100 nm in size can be stabilized by these molecules. Moreover, we demonstrate that these peptide amphiphiles form curvature-dependent, ordered structures on the surface of the GNPs and that the GNPs remain disperse at high-salt concentrations and in the presence of competing thiol-containing molecules. These results represent the development of a peptide amphiphile-based coating system for GNPs which has the potential to be beneficial for a wide range of biological applications, in addition to image enhancement and catalysis.

Hyperalphalipoproteinemic scavenger receptor BI knockout mice exhibit a disrupted epidermal lipid barrier.

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesteryl esters (CE) from high-density lipoproteins (HDL). An impaired SR-BI function leads to hyperalphalipoproteinemia with elevated levels of cholesterol transported in the HDL fraction. Accumulation of cholesterol in apolipoprotein B (apoB)-containing lipoproteins has been shown to alter skin lipid composition and barrier function in mice. To investigate whether these hypercholesterolemic effects on the skin also occur in hyperalphalipoproteinemia, we compared skins of wild-type and SR-BI knockout (SR-BI-/-) mice. SR-BI deficiency did not affect the epidermal cholesterol content and induced only minor changes in the ceramide subclasses. The epidermal free fatty acid (FFA) pool was, however, enriched in short and unsaturated chains. Plasma CE levels strongly correlated with epidermal FFA C18:1 content. The increase in epidermal FFA coincided with downregulation of cholesterol and FFA synthesis genes, suggesting a compensatory response to increased flux of plasma cholesterol and FFAs into the skin. Importantly, the SR-BI-/- epidermal lipid barrier showed increased permeability to ethyl-paraminobenzoic acid, indicating an impairment of the barrier function. In conclusion, increased HDL-cholesterol levels in SR-BI-/- mice can alter the epidermal lipid composition and lipid barrier function similarly as observed in hypercholesterolemia due to elevated levels of apoB-containing lipoproteins.

Hypercholesterolemia in young adult APOE-/- mice alters epidermal lipid composition and impairs barrier function.

Long-term exposure to hypercholesterolemia induces the development of skin xanthoma's characterized by the accumulation of lipid-laden foam cells in humans and in mice. Early skin changes in response to hypercholesterolemia are however unknown. In this study, we investigated the skin lipid composition and associated barrier function in young adult low-density lipoprotein receptor knockout (LDLR-/-) and apolipoprotein E knockout (APOE-/-) mice, two commonly used hypercholesterolemic mouse models characterized by the accumulation of apolipoprotein B containing lipoproteins. No differences were observed on cholesterol content in the epidermis in LDLR-/- mice nor in the more extremely hypercholesterolemic APOE-/- mice. Interestingly, the free fatty acid profile in the APOE-/- epidermis shifted towards shorter and unsaturated chains. Genes involved in the synthesis of cholesterol and fatty acids were downregulated in APOE-/- skin suggesting a compensation for the higher influx of plasma lipids, most probably as cholesteryl esters. Importantly, in vivo transepidermal water loss and permeability studies with murine lipid model membranes revealed that the lipid composition of the APOE-/- skin resulted in a reduced skin barrier function. In conclusion, severe hypercholesterolemia associated with increased apolipoprotein B containing lipoproteins affects the epidermal lipid composition and its protective barrier.

Preferential arrangement of lipids in the long-periodicity phase of a stratum corneum matrix model.

The lipid matrix of the stratum corneum, the outermost skin layer, consists primarily of ceramides, cholesterol, and FFAs. These lipids form a trilayer long-periodicity phase (LPP) that is unique to this barrier. Knowledge about the LPP is essential in understanding the barrier function. Previous studies of LPP lipid models have identified the position of the major lipid classes and suggested that a large fraction of FFAs and the ceramide acyl chain are present in the central region. However, the precise arrangement, such as lipid subclass mixing (isolated or mixed) and ceramide conformation (extended or hairpin), remains unknown. Here, we deuterated FFAs and the ceramide acyl chain to study CD2 and CH2 interactions with Fourier-transform infrared spectroscopy. The ceramide and FFAs of various chain lengths were not in separate domains but had mixed together. The larger number of CD2-CD2 lipid chain interactions in the LPP than in a symmetrical bilayer structure implied that the ceramide had primarily adopted an extended conformation. Shorter FFAs were present in the central region of the LPP. This model explores the biophysical properties of the stratum corneum's LPP to improve the understanding of the barrier function of this layer.

Topically Applied Ceramides Interact with the Stratum Corneum Lipid Matrix in Compromised Ex Vivo Skin.

PURPOSE: To determine whether formulations containing ceramides (including a ceramide with a long hydroxyl acyl chain linked to a linoleate, CER EOS) and fatty acids are able to repair the skin barrier by normalizing the lipid organization in stratum corneum (SC). METHODS: The formulations were applied on a skin barrier repair model consisting of ex vivo human skin from which SC was removed by stripping. The effect of formulations on the lipid organization and conformational ordering in the regenerated SC were analyzed using Fourier transform infrared spectroscopy and small angle X-ray diffraction. RESULTS: Application of the formulation containing only one ceramide on regenerating SC resulted in a higher fraction of lipids adopting an orthorhombic organization. A similar fraction of lipids forming an orthorhombic organization was observed after application of a formulation containing two ceramides and a fatty acid on regenerating SC. No effects on the lamellar lipid organization were observed. CONCLUSIONS: Application of a formulation containing either a single ceramide or two ceramides and a fatty acid on regenerating SC, resulted in a denser lateral lipid packing of the SC lipids in compromised skin. The strongest effect was observed after application of a formulation containing a single ceramide.

Solid and fluid segments within the same molecule of stratum corneum ceramide lipid.

The outer layer of the skin, stratum corneum (SC) is an efficient transport barrier and it tolerates mechanical deformation. At physiological conditions, the majority of SC lipids are solid, while the presence of a small amount of fluid lipids is considered crucial for SC barrier and material properties. Here we use solid-state and diffusion nuclear magnetic resonance to characterize the composition and molecular dynamics of the fluid lipid fraction in SC model lipids, focusing on the role of the essential SC lipid CER EOS, which is a ceramide esterified omega-hydroxy sphingosine linoleate with very long chain. We show that both rigid and mobile structures are present within the same CER EOS molecule, and that the linoleate segments undergo fast isotropic reorientation while exhibiting extraordinarily slow self-diffusion. The characterization of this unusual self-assembly in SC lipids provides deepened insight into the molecular arrangement in the SC extracellular lipid matrix and the role of CER EOS linoleate in the healthy and diseased skin.

Degree of Skin Barrier Disruption Affects Lipid Organization in Regenerated Stratum Corneum.

Previously, a skin barrier repair model was developed to examine the effect of formulations on the lipid properties of compromised skin. In this model, the lipid organization mimics that of several skin diseases with impaired skin barrier and less dense lateral lipid organization. In addition, parakeratosis was occasionally observed. The present study investigated whether the extent of initial barrier disruption affects lipid organization and parakeratosis in regenerated stratum corneum. After barrier disruption and stratum corneum regeneration the fraction of lipids adopting a less dense lateral organization gradually increased with increasing degree of barrier disruption. Only when 75% of the stratum corneum was removed, were parakeratosis and a change in lamellar organization observed. This demonstrates the possibility of using the skin barrier repair model to study the effects of formulations on compromised skin in which the presence of parakeratosis and lipid organization can be modified by the extent of barrier disruption.

Free fatty acids chain length distribution affects the permeability of skin lipid model membranes.

The lipid matrix in the stratum corneum (SC) plays an important role in the barrier function of the skin. The main lipid classes in this lipid matrix are ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The aim of this study was to determine whether a variation in CER subclass composition and chain length distribution of FFAs affect the permeability of this matrix. To examine this, we make use of lipid model membranes, referred to as stratum corneum substitute (SCS). We prepared SCS containing i) single CER subclass with either a single FFA or a mixture of FFAs and CHOL, or ii) a mixture of various CER subclasses with either a single FFA or a mixture of FFAs and CHOL. In vitro permeation studies were performed using ethyl-p-aminobenzoic acid (E-PABA) as a model drug. The flux of E-PABA across the SCS containing the mixture of FFAs was higher than that across the SCS containing a single FA with a chain length of 24 C atoms (FA C24), while the E-PABA flux was not effected by the CER composition. To select the underlying factors for the changes in permeability, the SCSs were examined by Fourier transform infrared spectroscopy (FTIR) and Small angle X-ray scattering (SAXS). All lipid models demonstrated a similar phase behavior. However, when focusing on the conformational ordering of the individual FFA chains, the shorter chain FFA (with a chain length of 16, 18 or 20 C atoms forming only 11m/m% of the total FFA level) had a higher conformational disordering, while the conformational ordering of the chains of the CER and FA C24 and FA C22 hardly did not change irrespective of the composition of the SCS. In conclusion, the conformational mobility of the short chain FFAs present only at low levels in the model SC lipid membranes has a great impact on the permeability of E-PABA.

Skin lipids: localization of ceramide and fatty acid in the unit cell of the long periodicity phase.

The lipid matrix of the skin's stratum corneum plays a key role in the barrier function, which protects the body from desiccation. The lipids that make up this matrix consist of ceramides, cholesterol, and free fatty acids, and can form two coexisting crystalline lamellar phases: the long periodicity phase (LPP) and the short periodicity phase (SPP). To fully understand the skin barrier function, information on the molecular arrangement of the lipids in the unit cell of these lamellar phases is very desirable. To determine this arrangement in previous studies, we examined the molecular arrangement of the SPP. In this study, neutron diffraction studies were performed to obtain information on the molecular arrangement of the LPP. The diffraction pattern reveals nine diffraction orders attributed to the LPP with a repeating unit of 129.4 ± 0.5 Å. Using D2O/H2O contrast variation, the scattering length density profiles were calculated for protiated samples and samples that included either the perdeuterated acyl chain of the most abundant ceramide or the most abundant perdeuterated fatty acid. Both perdeuterated chains are predominantly located in the central part of the unit cell with substantial interdigitation of the acyl chains in the unit cell center. However, a fraction of the perdeuterated chains is also located near the border of the unit cell with their acyl chains directing toward the center. This arrangement of lipids in the LPP unit cell corresponds with the location of their lipid headgroups at the border and also inside of the unit cell at a well-defined position (±21 Å from the unit cell center), indicative of a three-layer lipid arrangement within the 129.4 ± 0.5 Å repeating unit.

Modulation of stratum corneum lipid composition and organization of human skin equivalents by specific medium supplements.

Our in-house human skin equivalents contain all stratum corneum (SC) barrier lipid classes, but have a reduced level of free fatty acids (FAs), of which a part is mono-unsaturated. These differences lead to an altered SC lipid organization and thereby a reduced barrier function compared to human skin. In this study, we aimed to improve the SC FA composition and, consequently, the SC lipid organization of the Leiden epidermal model (LEM) by specific medium supplements. The standard FA mixture (consisting of palmitic, linoleic and arachidonic acids) supplemented to the medium was modified, by replacing protonated palmitic acid with deuterated palmitic acid or by the addition of deuterated arachidic acid to the mixture, to determine whether FAs are taken up from the medium and are incorporated into SC of LEM. Furthermore, supplementation of the total FA mixture or that of palmitic acid alone was increased four times to examine whether this improves the SC FA composition and lipid organization of LEM. The results demonstrate that the deuterated FAs are taken up into LEMs and are subsequently elongated and incorporated in their SC. However, a fourfold increase in palmitic acid supplementation does not change the SC FA composition or lipid organization of LEM. Increasing the concentration of the total FA mixture in the medium resulted in a decreased level of very long chain FAs and an increased level of mono-unsaturated FAs, which lead to deteriorated SC lipid properties. These results indicate that SC lipid properties can be modulated by specific medium supplements.

Monounsaturated fatty acids reduce the barrier of stratum corneum lipid membranes by enhancing the formation of a hexagonal lateral packing.

The effectiveness of the skin barrier underlies the outer layer of the skin: the stratum corneum (SC). However, in several skin diseases this barrier is impaired. In two inflammatory skin diseases, atopic eczema and Netherton syndrome, an increased level of monounsaturated fatty acids (MUFAs) has been observed as opposed to healthy skin. In the present study, we aimed to investigate the effect of MUFAs on the lipid organization and skin lipid barrier using an in vitro model membrane system, the stratum corneum substitute (SCS), mimicking the SC lipid composition and organization. To achieve our goal, the SCS has been prepared with increasing levels of MUFAs using various chain length. Permeation studies and trans-epidermal water loss measurements show that an increment of MUFAs reduces the lipid barrier in the SCS. The increased level of unsaturation exerts its effect by reducing the packing density in the lipid organization, while the lamellar phases are not affected. Our findings indicate that increased levels of MUFAs may contribute to the impaired skin barrier in diseased skin.

The effect of the chain length distribution of free fatty acids on the mixing properties of stratum corneum model membranes.

The stratum corneum (SC) plays a fundamental role in the barrier function of the skin. The SC consists of corneocytes embedded in a lipid matrix. The main lipid classes in the lipid matrix are ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The aim of this study was to examine the effect of the chain length of FFAs on the thermotropic phase behavior and mixing properties of SC lipids. Fourier transform infrared spectroscopy and Raman imaging spectroscopy were used to study the mixing properties using either protonated or deuterated FFAs. We selected SC model lipid mixtures containing only a single CER, CHOL and either a single FFA or a mixture of FFAs mimicking the FFA SC composition. The single CER consists of a sphingoid base with 18 carbon atoms and an acyl chain with a chain length of 24 carbon atoms. When using lignoceric acid (24 carbon atoms) or a mixture of FFAs, the CER and FFAs participated in mixed crystals, but hydration of the mixtures induced a slight phase separation between CER and FFA. The mixed crystalline structures did not phase separate during storage even up to a time period of 3months. When using palmitic acid (16 carbon atoms), a slight phase separation was observed between FFA and CER. This phase separation was clearly enhanced during hydration and storage. In conclusion, the thermotropic phase behavior and the mixing properties of the SC lipid mixtures were shown to strongly depend on the chain length and chain length distribution of FFAs, while hydration enhanced the phase separation.

In vitro model systems for studying the impact of organic chemicals on the skin barrier lipids.

This paper describes two synthetic lipid models designed to replace human stratum corneum (SC) in studies of the impact of volatile organic chemicals on the molecular organization of the skin barrier lipids. The models built upon previously developed self-assembled lipid membranes which have composition and 3D organization similar to those of the lipid matrix in SC. In one model the target chemicals were incorporated in the lipids before their self-assembly, and in the other one they were applied on top of a preformed lipid membrane. The chemicals could be incorporated within the model membranes in quantities close to those reached within human SC upon heavy surface loading. The dose-dependent effects of the chemicals on the lateral molecular organization in the models were qualitatively identical to those observed by infrared spectroscopy in human SC. The models facilitated the interpretation of X-ray diffraction profiles used to determine the nature of the interactions between the chemicals and the lipid lamellae and the position of the exogenous molecules within the unit cell of the lipid phases. These model systems are suitable for in vitro studies in the areas of skin biophysics, dermatology, transdermal drug delivery, and risk assessment.

Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients.

A hallmark of atopic eczema (AE) is skin barrier dysfunction. Lipids in the stratum corneum (SC), primarily ceramides, fatty acids, and cholesterol, are crucial for the barrier function, but their role in relation to AE is indistinct. Filaggrin is an epithelial barrier protein with a central role in the pathogenesis of AE. Nevertheless, the precise causes of AE-associated barrier dysfunction are largely unknown. In this study, a comprehensive analysis of ceramide composition and lipid organization in nonlesional SC of AE patients and control subjects was performed by means of mass spectrometry, infrared spectroscopy, and X-ray diffraction. In addition, the skin barrier and clinical state of the disease were examined. The level of ceramides with an extreme short chain length is drastically increased in SC of AE patients, which leads to an aberrant lipid organization and a decreased skin barrier function. Changes in SC lipid properties correlate with disease severity but are independent of filaggrin mutations. We demonstrate for the first time that changes in ceramide chain length and lipid organization are directly correlated with the skin barrier defects in nonlesional skin of AE patients. We envisage that these insights will provide a new therapeutic entry in therapy and prevention of AE.