Cutaneous penetration of soft nanoparticles via photodamaged skin: Lipid-based and polymer-based nanocarriers for drug delivery.

Photoaging is recognized as the factor damaging skin-barrier function. The aim of this study was to examine the impact of ultraviolet (UV) irradiation on the cutaneous penetration of soft nanoparticles, including nanostructured lipid carriers (NLCs) and poly(lactic-co-glycolic acid) polymer nanoparticles (PNs). In vitro cutaneous permeation of retinoic acid (RA) carried by nanoparticles was evaluated. In vivo nude mouse skin distribution of topically applied nanoparticles was observed by fluorescence and confocal microscopies. The association of nanoparticles with cultured keratinocytes was measured by flow cytometry and fluorescence microscopy. The average diameter and surface charge were 236nm and -32mV for NLCs, and 207nm and -12mV for PNs. The ultrastructural images of skin demonstrated that the application of UV produced a loss of Odland bodies and desmosomes, the organelles regulating skin-barrier function. UVA exposure increased skin deposition of RA regardless of nanoparticle formulation. UVB did not alter RA deposition from nanoparticles as compared to the non-treated group. Exposure to UVA promoted RA delivery into hair follicles from NLCs and PNs by 4.2- and 4.9-fold, respectively. The in vivo skin distribution also showed a large accumulation of Nile red-loaded nanoparticles in follicles after UVA treatment. The soft nanoparticles were observed deep in the dermis. PNs with higher lipophilicity showed a greater association with keratinocytes compared to NLCs. The cell association of PNs was increased by UVA application, whereas the association between NLCs and keratinocytes was reduced two times by UVA. It was concluded that both follicles and intercellular spaces were the main pathways for nanoparticle diffusion into photodamaged skin.

Morphological abnormalities in mitochondria of the skin of patients with sporadic amyotrophic lateral sclerosis.

OBJECTIVES: Mitochondrial dysfunction has been reported in the central nervous system, hepatocytes and peripheral blood lymphocytes from patients with sporadic amyotrophic lateral sclerosis (SALS). However, the status of skin mitochondria has not been reported, in spite of the fact that SALS patients present skin abnormalities. The objective of the present study was to compare mitochondrial ultrastructural parameters in keratinocytes from patients with SALS and healthy controls. METHODS: Our study was based on the analysis of 112 skin mitochondria from 5 SALS patients and 99 organelles from 4 control subjects by electron microscopy. RESULTS: Computerized image analysis showed that mitochondrial major axis length, area and perimeter of the organelle were significantly smaller in SALS respect of healthy control subjects. Morphologically, SALS mitochondria presented cristolysis and breakage of the outer membrane. CONCLUSIONS: Mitochondrial dysfunction in the skin may possibly reflect changes occurring in mitochondria of the central nervous system. The analysis of mitochondrial morphology in this tissue may be of value to follow disease progression and, eventually, the effectiveness of current therapies for SALS.

Comparison of scanning acoustic microscopy and histology images in characterizing surface irregularities among engineered human oral mucosal tissues.

Acoustic microscopy was used to monitor an ex vivo produced oral mucosal equivalent (EVPOME) developed on acellular cadaveric dermis (AlloDerm®). As seeded cells adhered and grew, they filled in and smoothed out the surface irregularities, followed by the production of a keratinized protective outermost layer. If noninvasive in vitro ultrasonic monitoring of these cellular changes could be developed, then tissue cultivation could be adjusted in-process to account for biologic variations in the development of these stratified cell layers. Cultured keratinocytes (from freshly obtained oral mucosa) were harvested and seeded onto AlloDerm® coated with human type IV collagen and cultured 11 days. EVPOMEs were imaged on the 11th day post-seeding using a scanning acoustic microscope (SAM) that consists of a single-element transducer: 61 MHz center frequency, 32 MHz bandwidth, 1.52 f-number. The specimen surface was determined by thresholding the magnitude of the signal at the first axial incidence of a value safely above noise: 20-40 dB above the signal for the water and 2-dimensional (2-D) ultrasonic images were created using confocal image reconstruction. A known area from each micrograph was divided into 12-40 even segments and examined for surface irregularities. These irregularities were quantified and one-way analysis of variance (ANOVA) and linear regression analysis were performed to correlate the surface profiles for both the AlloDerm® and EVPOME specimens imaged by SAM. Histology micrographs of the AlloDerm® and EVPOME specimens were also prepared and examined for surface irregularities. Unseeded AlloDerm® averaged seven to nine surface changes per 400 µm. The number of changes in surface irregularities decreased to two to three per 400 µm on the mature EVPOMEs. The numbers of surface irregularities between the unseeded AlloDerm® vs. developing EVPOME are similar for both histology and SAM 2-D B-scan images. For the EVPOME 2-D B-scan micrographs produced by SAM, the decrease in surface irregularities is indicative of the stratified epithelium formed by seeded oral keratinocytes; verified in the histology images between the AlloDerm® and EVPOME. A near 1:1 linear correlation shows the similarities between the two imaging modalities. SAM demonstrates its ability to discern the cell development and differentiation occurring on the EVPOME devices. Unlike histology, SAM measurements are noninvasive and can be used to monitor tissue graft development without damaging any cells/tissues.

Acoustic microscopy analyses to determine good vs. failed tissue engineered oral mucosa under normal or thermally stressed culture conditions.

This study uses scanning acoustic microscopy (SAM) ultrasonic profilometry to determine acceptable vs. failed tissue engineered oral mucosa. Specifically, ex vivo-produced oral mucosal equivalents (EVPOMEs) under normal or thermally stressed culture conditions were scanned with the SAM operator blinded to the culture conditions. As seeded cells proliferate, they fill in and smooth out the surface irregularities; they then stratify and produce a keratinized protective upper layer. Some of these transformations could alter backscatter of ultrasonic signals and in the case of the thermally stressed cells, produce backscatter similar to an unseeded device. If non-invasive ultrasonic monitoring could be developed, then tissue cultivation could be adjusted to measure biological variations in the stratified surface. To create an EVPOME device, oral mucosa keratinocytes were seeded onto acellular cadaveric dermis. Two sets of EVPOMEs were cultured: one at physiological temperature 37 °C and the other at 43 °C. The specimens were imaged with SAM consisting of a single-element transducer: 61 MHz center frequency, 32 MHz bandwidth, 1.52 f#. Profilometry for the stressed and unseeded specimens showed higher surface irregularities compared to unstressed specimens. Elevated thermal stress retards cellular differentiation, increasing root mean square values; these results show that SAM can potentially monitor cell/tissue development.

Acceleration of permeability barrier recovery by exposure of skin to 10-30 kHz sound.

BACKGROUND: Previous reports show that ultrasound can influence human brain electrical activity and systemic hormone levels in various parts of the body, other than the ear, so there may be an unknown ultrasound-responsive system in humans. OBJECTIVES: In the present study, we examined the effects of sound on skin permeability barrier homeostasis. METHODS: We broke the skin barrier of hairless mice by tape stripping, and then exposed the skin to sound for 1 h to evaluate the effect on barrier recovery rate. RESULTS: Exposure of skin to sound at frequencies of 10, 20 and 30 kHz for 1 h accelerated barrier recovery, and 20 kHz sound induced the fastest recovery. Application of 5 kHz sound had no effect on barrier recovery rate. Significant acceleration was observed even when the sound source was located 3 cm away from the skin surface. The recovery rate depended on the sound pressure. An electron-microscopic study indicated that lamellar body secretion between stratum corneum and stratum granulosum was increased by exposure to sound at 20 kHz. CONCLUSIONS: These results suggest that epidermal keratinocytes might be influenced by ultrasound in a manner that results in modulation of epidermal permeability barrier homeostasis.

Low-frequency low-intensity ultrasounds do not influence the survival and immune functions of cultured keratinocytes and dendritic cells.

Low-frequency ultrasounds (US) are used to enhance drug transdermal transport. Although this phenomenon has been extensively analyzed, information on US effects on the single skin cell components is limited. Here, we investigated the possible effects of low-frequency US on viability and immune functions of cultured human keratinocytes and dendritic cells (DC), skin cells involved in the regulation of many immune-mediated dermatoses. We demonstrated that US, employed at low-frequency (42 KHz) and low-intensity (0.15 W/cm(2)) values known to enhance drug and water transdermal transport, did not affect extracellular-signal-regulated-kinase (ERK)1/2 activation, cell viability, or expression of adhesion molecules in cultured keratinocytes. Moreover, US at these work frequency and intensity did not influence the keratinocyte expression and release of immunomodulatory molecules. Similarly, cultured DC treated with low-frequency low-intensity US were viable, and did not show an altered membrane phenotype, cytokine profile, nor antigen presentation ability. However, intensity enhancement of low-frequency US to 5 W/cm(2) determined an increase of the apoptotic rate of both keratinocytes and DC as well as keratinocyte CXCL8 release and ERK1/2 activation, and DC CD40 expression. Our study sustains the employment of low-frequency and low-intensity US for treatment of those immune skin disorders, where keratinocytes and DC have a pathogenetic role.

Improved alkaline comet assay protocol for adherent HaCaT keratinocytes to study UVA-induced DNA damage.

The comet assay is one of the well-accepted tests to measure radiation-induced DNA damage. The most commonly used protocols require single-cell suspensions that are embedded in agarose in order to perform electrophoresis. For adherently growing cells such as human HaCaT skin keratinocytes this method bears several problems. We show that trypsinization required for maintaining single-cell suspensions is prolonged after UV radiation and thereby reduces cell viability and allows partial repair, with the consequence of reduced damage detection after irradiation. Therefore, we here introduce a modified version of the comet assay where HaCaT cells are seeded onto comet slides 24h before the assay and overlaid with agarose immediately after irradiation. Using this modification we are now able to reproducibly measure high DNA-damage levels (13-fold increase compared with controls) following irradiation with 60J/cm(2) UVA as well as a dose-dependent increase of DNA damage after 10, 20 and 60J/cm(2) UVA. Thus, by maintaining the cells in their natural configuration, i.e. adherently growing, we exclude several artefacts that are likely to influence the damage responses. These include: (i) trypsinization-dependent changes in cell morphology and polarity (clear lateral, i.e. adherent, and apical side of keratinocytes) which are likely of consequence for the gene-expression pattern, (ii) trypsin- and dislodgement-induced damage reducing cell viability, and (iii) the time delay between damage induction and damage evaluation to unpredictable results due to partial repair. Since these advantages pertain to all adherently growing cells, this improved protocol is not restricted to HaCaT cells but offers great potential also with all non-haematopoietic cells for obtaining accurate results and for studying repair processes in a highly reproducible manner.

Weak force stalls protrusion at the leading edge of the lamellipodium.

Protrusion, the first step of cell migration, is driven by actin polymerization coupled to adhesion at the cell's leading edge. Polymerization and adhesive forces have been estimated, but the net protrusion force has not been measured accurately. We arrest the leading edge of a moving fish keratocyte with a hydrodynamic load generated by a fluid flow from a micropipette. The flow arrests protrusion locally as the cell approaches the pipette, causing an arc-shaped indentation and upward folding of the leading edge. The effect of the flow is reversible upon pipette removal and dependent on the flow direction, suggesting that it is a direct effect of the external force rather than a regulated cellular response. Modeling of the fluid flow gives a surprisingly low value for the arresting force of just a few piconewtons per micrometer. Enhanced phase contrast, fluorescence, and interference reflection microscopy suggest that the flow does not abolish actin polymerization and does not disrupt the adhesions formed before the arrest but rather interferes with weak nascent adhesions at the very front of the cell. We conclude that a weak external force is sufficient to reorient the growing actin network at the leading edge and to stall the protrusion.

Intraepithelial anchoring fibril components.

Cultured human keratinocytes and cultured human cervical carcinoma cells (ME-180) contained intracellular pools of antigens that reacted with the anchoring fibril antibodies AF1 and AF2. In keratinocytes, these antigens formed a basement membrane-like structure near the apical portions of the cells. Using flow cytometric techniques, pretreatment of the ME-180 cells with acetone revealed large intracellular pools of antigen. The intracellular epitope was calcium sensitive. Some forms of recessive dystrophic epidermolysis bullosa have retention of intracellular portions of the anchoring fibril suggesting a relation of the intracellular anchoring fibril antigens to that disease.