Anticancer activity of anandamide in human cutaneous melanoma cells

Cannabinoids are implicated in the control of cell proliferation, but little is known about the role of the endocannabinoid system in human malignant melanoma. This study was aimed at characterizing the in vitro antitumor activity of anandamide (AEA) in A375 melanoma cells. The mRNA expression of genes that code for proteins involved in the metabolism and in the mechanism of AEA action was assessed by RT-PCR. Cell viability was tested using WST-1 assay and the apoptotic cell death was determined by measuring caspase 3/7 activities. A375 cells express high levels of fatty acid amide hydrolase (FAAH), cyclooxygenase (COX)-2, cannabinoid receptor 1 (CB1), transient receptor potential cation channel subfamily V member 1 (TRPV1) and G-protein-coupled receptor 55 (GPR55) genes. AEA induced a concentration-dependent cytotoxicity with an IC50 of 5.8±0.7 µM and such an effect was associated to a caspase-dependent apoptotic pathway. AEA cytotoxicity was potentiated by FAAH inhibition (2-fold increase, p<0.05) and mitigated by COX-2 or lipoxygenase (LOX) inhibition (5- and 3-fold decrease, respectively; p<0.01). Blocking CB1 receptors partially decreased AEA cytotoxicity, whereas selective antagonism on the TRPV1 barely affected the mechanism of AEA action. Finally, methyl-β-cyclodextrin, a membrane cholesterol depletory, completely reversed the cytotoxicity induced by the selective GPR55 agonist, O-1602, and AEA. Overall, these findings demonstrate that AEA induces cytotoxicity against human melanoma cells in the micromolar range of concentrations through a complex mechanism, which involves COX-2 and LOX-derived product synthesis and CB1 activation. Lipid raft modulation, probably linked to GPR55 activation, might also have a role.

The inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored by in vitro photorepair of cyclobutane pyrimidine dimers

Exposing skin to UVB (280–320 nm) radiation suppresses contact hypersensitivity by a mechanism that involves an alteration in the activity of cutaneous antigen-presenting cells (APC). UV-induced DNA damage appears to be an important molecular trigger for this effect. The specific target cells in the skin that sustain DNA damage relevant to the immunosuppressive effect have yet to be identified. We tested the hypothesis that UV-induced DNA damage in the cutaneous APC was responsible for their impaired ability to present antigen after in vivo UV irradiation. Cutaneous APC were collected from the draining lymph nodes of UVB-irradiated, hapten-sensitized mice and incubated in vitro with liposomes containing a photolyase (Photosomes; Applied Genetics, Freeport, NY), which, upon absorption of photoreactivating light, splits UV-induced cyclobutane pyrimidine dimers. Photosome treatment followed by photoreactivating light reduced the number of dimer-containing APC, restored the in vivo antigen-presenting activity of the draining lymph node cells, and blocked the induction of suppressor T cells. Neither Photosomes nor photoreactivating light alone, nor photoreactivating light given before Photosomes, restored APC activity, and Photosome treatment did not reverse the impairment of APC function when isopsoralen plus UVA (320–400 nm) radiation was used instead of UVB. These controls indicate that the restoration of APC function matched the requirements of Photosome-mediated DNA repair for dimers and post-treatment photoreactivating light. These results provide compelling evidence that it is UV-induced DNA damage in cutaneous APC that leads to reduced immune function.

Enzyme-catalyzed synthesis of poly[(R)-(-)-3-hydroxybutyrate]: formation of macroscopic granules in vitro.

A combined chemical and enzymatic procedure has been developed to synthesize macroscopic poly[(R)-(-)-3-hydroxybutyrate] (PHB) granules in vitro. The granules form in a matter of minutes when purified polyhydroxyalkanoate (PHA) synthase from Alcaligenes eutrophus is exposed to synthetically prepared (R)-3-hydroxybutyryl coenzyme A, thereby establishing the minimal requirements for PHB granule formation. The artificial granules are spherical with diameters of up to 3 microns and significantly larger than their native counterparts (0.5 micron). The isolated PHB was characterized by 1H and 13C NMR, gel-permeation chromatography, and chemical analysis. The in vitro polymerization system yields PHB with a molecular mass > 10 x 10(6) Da, exceeding by an order of magnitude the mass of PHAs typically extracted from microorganisms. We also demonstrate that the molecular mass of the polymer can be controlled by the initial PHA synthase concentration. Preliminary kinetic analysis of de novo granule formation confirms earlier findings of a lag time for the enzyme but suggests the involvement of an additional granule assembly step. Minimal requirements for substrate recognition were investigated. Since substrate analogs lacking the adenosine 3',5'-bisphosphate moiety of (R)-3-hydroxybutyryl coenzyme A were not accepted by the PHA synthase, we provide evidence that this structural element of the substrate is essential for catalysis.

Interazione di nanoparticelle metalliche con menbrane biologiche: risultati di studi in vitro su cute, mucosa del cavo orale e meningi ed implicazioni per la salute

L’utilizzo di nanomateriali, ovvero una nuova classe di sostanze composte da particelle ultrafini con dimensioni comprese fra 1 e 100 nm (American Society for Testing Materials - ASTM), è in costante aumento a livello globale. La particolarità di tali sostanze è rappresentata da un alto rapporto tra la superficie e il volume delle particelle, che determina caratteristiche chimico-fisiche completamente differenti rispetto alle omologhe macrosostanze di riferimento. Tali caratteristiche sono tali da imporre una loro classificazione come nuovi agenti chimici (Royal Society & Royal Academy of Engineering report 2004). Gli impieghi attuali dei nanomateriali risultano in continua evoluzione, spaziando in diversi ambiti, dall’industria farmaceutica e cosmetica, all’industria tessile, elettronica, aerospaziale ed informatica. Diversi sono anche gli impieghi in campo biomedico; tra questi la diagnostica e la farmacoterapia. È quindi prevedibile che in futuro una quota sempre maggiore di lavoratori e consumatori risulteranno esposti a tali sostanze. Allo stato attuale non vi è una completa conoscenza degli effetti tossicologici ed ambientali di queste sostanze, pertanto, al fine di un loro utilizzo in totale sicurezza, risulta necessario capirne meglio l’impatto sulla salute, le vie di penetrazione nel corpo umano e il rischio per i lavoratori conseguente al loro utilizzo o lavorazione. La cute rappresenta la prima barriera nei confronti delle sostanze tossiche che possono entrare in contatto con l’organismo umano. Successivamente agli anni ‘60, quando si riteneva che la cute rappresentasse una barriera totalmente impermeabile, è stato dimostrato come essa presenti differenti gradi di permeabilità nei confronti di alcuni xenobiotici, dipendente dalle caratteristiche delle sostanze in esame, dal sito anatomico di penetrazione, dal grado di integrità della barriera stessa e dall’eventuale presenza di patologie della cute. La mucosa del cavo orale funge da primo filtro nei confronti delle sostanze che entrano in contatto con il tratto digestivo e può venir coinvolta in contaminazioni di superficie determinate da esposizioni occupazionali e/o ambientali. È noto che, rispetto alla cute, presenti una permeabilità all’acqua quattro volte maggiore, e, per tale motivo, è stata studiata come via di somministrazione di farmaci, ma, ad oggi, pochi sono gli studi che ne hanno valutato le caratteristiche di permeazione nei confronti delle nanoparticelle (NPs). Una terza importante barriera biologica è quella che ricopre il sistema nervoso centrale, essa è rappresentata da tre foglietti di tessuto connettivo, che assieme costituiscono le meningi. Questi tre foglietti rivestono completamente l’encefalo permettendone un isolamento, tradizionalmente ritenuto completo, nei confronti degli xenobiotici. L’unica via di assorbimento diretto, in questo contesto, è rappresentata dalla via intranasale. Essa permette un passaggio diretto di sostanze dall’epitelio olfattivo all’encefalo, eludendo la selettiva barriera emato-encefalica. Negli ultimi anni la letteratura scientifica si è arricchita di studi che hanno indagato le caratteristiche di assorbimento di farmaci attraverso questa via, ma pochissimi sono gli studi che hanno indagato la possibile penetrazione di nanoparticelle attraverso questa via, e nessuno, in particolar modo, ha indagato le caratteristiche di permeazione delle meningi. L’attività di ricerca svolta nell’ambito del presente dottorato ha avuto per finalità l’indagine delle caratteristiche di permeabilità e di assorbimento della cute, della mucosa del cavo orale e delle meningi nei confronti di alcune nanoparticelle, scelte fra quelle più rappresentative in relazione alla diffusione d’utilizzo a livello globale. I risultati degli esperimenti condotti hanno dimostrato, in vitro, che l’esposizione cutanea a Pt, Rh, Co3O4 e Ni NPs determinano permeazione in tracce dei medesimi metalli attraverso la cute, mentre per le TiO2 NPs tale permeazione non è stata dimostrata. È stato riscontrato, inoltre, che la mucosa del cavo orale e le meningi sono permeabili nei confronti dell’Ag in forma nanoparticellare.

Inter- and intralaboratory variation of in vitro diffusion cell measurements: An international multicenter study using quasi-standardized methods and materials

In vitro measurements of skin absorption are an increasingly important aspect of regulatory studies, product support claims, and formulation screening. However, such measurements are significantly affected by skin variability. The purpose of this study was to determine inter- and intralaboratory variation in diffusion cell measurements caused by factors other than skin. This was attained through the use of an artificial (silicone rubber) rate-limiting membrane and the provision of materials including a standard penetrant, methyl paraben (MP), and a minimally prescriptive protocol to each of the 18 participating laboratories. Standardized calculations of MP flux were determined from the data submitted by each laboratory by applying a predefined mathematical model. This was deemed necessary to eliminate any interlaboratory variation caused by different methods of flux calculations. Average fluxes of MP calculated and reported by each laboratory (60 +/- 27 mug cm(-2) h(-1), n = 25, range 27-101) were in agreement with the standardized calculations of MP flux (60 +/- 21 mug cm(-2) h(-1), range 19-120). The coefficient of variation between laboratories was approximately 35% and was manifest as a fourfold difference between the lowest and highest average flux values and a sixfold difference between the lowest and highest individual flux values. Intra-laboratory variation was lower, averaging 10% for five individuals using the same equipment within a single laboratory. Further studies should be performed to clarify the exact components responsible for nonskin-related variability in diffusion cell measurements. It is clear that further developments of in vitro methodologies for measuring skin absorption are required. (C) 2005 Wiley-Liss, Inc.

Percutaneous absorption of steroids: Determination of in vitro permeability and tissue reservoir characteristics in human skin layers

The skin localization of steroids following topical application is largely unknown. We determined the distribution of five steroids in human skin using excised epidermal, dermal, and full-thickness membranes in vitro. There was no significant difference in steroid maximum flux through epidermal and full-thickness membranes, other than significantly lower fluxes for the most polar steroid, aldosterone. Hydrocortisone had the highest dermal diffusivity and dermal penetration, and the accumulation of hydrocortisone and corticosterone was higher than that of the other steroids. Slower penetration and higher accumulation in the viable epidermis of progesterone in full-thickness skin were consistent with dermal penetration limitation effects associated with high lipophilicity. Copyright (c) 2006 S. Karger AG, Basel

Vehicle effects on the in vitro penetration of testosterone through equine skin

The effects of three vehicles, phosphate-buffered saline (PBS), ethanol (50% in PBS w/w) and propylene glycol (50% in PBS w/w) on in vitro transdermal penetration of testosterone was investigated in the horse. Skin was harvested from the thorax of five Thoroughbred horses after euthanasia and stored at -20 degrees C until required. The skin was then defrosted and placed into Franz-type diffusion cells, which were maintained at approximately 32 degrees C by a water bath. Saturated solutions of testosterone, containing trace amounts of radiolabelled [C-14]testosterone, in each vehicle were applied to the outer (stratum corneum) surface of each skin sample and aliquots of receptor fluid were collected at 0, 2, 4, 8, 16, 20, 22 and 24 h and analysed for testosterone by scintillation counting. The maximum flux (J(max)) of testosterone was significantly higher for all sites when testosterone was dissolved in a vehicle containing 50% ethanol or 50% propylene glycol, compared to PBS. In contrast, higher residues of testosterone were found remaining within the skin when PBS was used as a vehicle. This study shows that variability in clinical response to testosterone could be expected with formulation design.

The effects of vehicle and region of application on in vitro penetration of testosterone through canine skin

The effects of the vehicles phosphate-buffered saline (PBS), ethanol (EtOH; 50% in PBS w/w) and propylene glycol (PG; 50% in PBS w/w) and the region of administration on in vitro transdermal penetration of testosterone was investigated in the dog. Skin was harvested from the thorax, neck (dorsal part) and groin regions of greyhounds after euthanasia and stored at -20 degrees C until required. The skin was then de-frosted and placed into Franz-type diffusion cells which were maintained at approximately 32 degrees C by a water-bath. Saturated solutions of testosterone, containing trace amounts of radiolabelled (C-14) testosterone, in each vehicle were applied to the outer (stratum corneum) surface of each skin sample and aliquots of receptor fluid were collected at 0, 2, 4, 8, 16, 20, 22 and 24 h and analysed for testosterone by scintillation counting. The maximum flux (J(max)) of testosterone was significantly higher for all sites when dissolved in a vehicle containing 50% EtOH or 50% PG, compared to PBS. In contrast, higher residues of testosterone were found remaining within the skin when PBS was used as a vehicle. This study shows that variability in percutaneous penetration of testosterone could be expected with formulation design and site of application. (C) 2004 Elsevier Ltd. All rights reserved.

Pre-formulation and systematic evaluation of amino acid assisted permeability of insulin across in vitro buccal cell layers

The aim of this work was to investigate alternative safe and effective permeation enhancers for buccal peptide delivery. Basic amino acids improved insulin solubility in water while 200 and 400 µg/mL lysine significantly increased insulin solubility in HBSS. Permeability data showed a significant improvement in insulin permeation especially for 10 µg/mL of lysine (p < 0.05) and 10 µg/mL histidine (p < 0.001), 100 µg/mL of glutamic acid (p < 0.05) and 200 µg/mL of glutamic acid and aspartic acid (p < 0.001) without affecting cell integrity; in contrast to sodium deoxycholate which enhanced insulin permeability but was toxic to the cells. It was hypothesized that both amino acids and insulin were ionised at buccal cavity pH and able to form stable ion pairs which penetrated the cells as one entity; while possibly triggering amino acid nutrient transporters on cell surfaces. Evidence of these transport mechanisms was seen with reduction of insulin transport at suboptimal temperatures as well as with basal-to-apical vectoral transport, and confocal imaging of transcellular insulin transport. These results obtained for insulin is the first indication of a possible amino acid mediated transport of insulin via formation of insulin-amino acid neutral complexes by the ion pairing mechanism.

Evaluation of polycaprolactone scaffold degradation for 6 months in vitro and in vivo

The use of polycaprolactone (PCL) as a biomaterial, especially in the fields of drug delivery and tissue engineering, has enjoyed significant growth. Understanding how such a device or scaffold eventually degrades in vivo is paramount as the defect site regenerates and remodels. Degradation studies of three-dimensional PCL and PCL-based composite scaffolds were conducted in vitro (in phosphate buffered saline) and in vivo (rabbit model). Results up to 6 months are reported. All samples recorded virtually no molecular weight changes after 6 months, with a maximum mass loss of only about 7% from the PCL-composite scaffolds degraded in vivo, and a minimum of 1% from PCL scaffolds. Overall, crystallinity increased slightly because of the effects of polymer recrystallization. This was also a contributory factor for the observed stiffness increment in some of the samples, while only the PCL-composite scaffold registered a decrease. Histological examination of the in vivo samples revealed good biocompatibility, with no adverse host tissue reactions up to 6 months. Preliminary results of medical-grade PCL scaffolds, which were implanted for 2 years in a critical-sized rabbit calvarial defect site, are also reported here and support our scaffold design goal for gradual and late molecular weight decreases combined with excellent long-term biocompatibility and bone regeneration. (C) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 90A: 906-919, 2009

Evaluation of the in vitro bioactivity of bioceramics

Two common methods have been used to evaluate the in vitro bioactivity of bioceramics for the application of bone repair. One is to evaluate the ability of apatite formation by soaking ceramics in simulated body fluids (SBF); the other method is to evaluate the effect of ceramics on osteogenic differentiation using cell experiments. Both methods have their own drawbacks in evaluating the in vitro bioactivity of bioceramics. In this commentary paper we review the application of both methods in bioactivity of bioceramics and conclude that (i) SBF method is an efficient method to investigate the in vitro bioactivity of silicate-based bioceramics, (ii) cellular bioactivity of bioceramics should be investigated by evaluating their stimulatory ability using standard bioceramics as controls; and (iii) the combination of these two methods to evaluate the in vitro bioactivity of bioceramics can improve the screening efficiency for the selection of bioactive ceramics for bone regeneration.

Mathematical models for describing the shape of the in vitro unstretched human crystalline lens

We developed orthogonal least-squares techniques for fitting crystalline lens shapes, and used the bootstrap method to determine uncertainties associated with the estimated vertex radii of curvature and asphericities of five different models. Three existing models were investigated including one that uses two separate conics for the anterior and posterior surfaces, and two whole lens models based on a modulated hyperbolic cosine function and on a generalized conic function. Two new models were proposed including one that uses two interdependent conics and a polynomial based whole lens model. The models were used to describe the in vitro shape for a data set of twenty human lenses with ages 7–82 years. The two-conic-surface model (7 mm zone diameter) and the interdependent surfaces model had significantly lower merit functions than the other three models for the data set, indicating that most likely they can describe human lens shape over a wide age range better than the other models (although with the two-conic-surfaces model being unable to describe the lens equatorial region). Considerable differences were found between some models regarding estimates of radii of curvature and surface asphericities. The hyperbolic cosine model and the new polynomial based whole lens model had the best precision in determining the radii of curvature and surface asphericities across the five considered models. Most models found significant increase in anterior, but not posterior, radius of curvature with age. Most models found a wide scatter of asphericities, but with the asphericities usually being positive and not significantly related to age. As the interdependent surfaces model had lower merit function than three whole lens models, there is further scope to develop an accurate model of the complete shape of human lenses of all ages. The results highlight the continued difficulty in selecting an appropriate model for the crystalline lens shape.