Maria Vitória Lopes Badra Bentley

A vehicle for photodynamic therapy of skin cancer : influence of dimethylsulphoxide on 5-aminolevulinic acid in vitro cutaneous permeation and in vivo protoporphyrin IX accumulation determined by confocal microscopy

Topical application of 5-aminolevulinic acid (5-ALA) followed by light irradiation is a new concept of photodynamic therapy (PDT) of skin cancers. 5-ALA is a prodrug that can be converted by the heme biosynthetic pathway into protoporphyrin IX, an effective photosensitizer. In the present work we propose the enhancement of 5-ALA-induced protoporphyrin IX accumulation by dimethylsulphoxide (DMSO) and ethylenediamine-tetraacetic acid disodium salt (EDTA). The presence of 20% DMSO (w/w) in oil-in-water emulsions increased the in vitro permeation of 5-ALA through hairless mouse skin. In vivo studies demonstrated a significant increase in the amount of protoporphyrin IX extracted from healthy hairless mouse skin after 3 h treatment with an oil-in-water emulsion containing 10% 5-ALA (w/w), 3% EDTA (w/w) and 20% DMSO (w/w). By confocal scanning laser microscopy imaging, an observed increase in red fluorescence, at 476 nm excitation and emission detected longer than 590 nm, in skin that had received this treatment, was attributed to protoporphyrin IX accumulation. Although no effect of EDTA on short-term protoporphyrin IX accumulation in skin was detected, this chelator could protect 5-ALA from decomposition during prolonged topical administration. The results obtained indicate that association of 5-ALA, EDTA and 20% DMSO may enhance the delivery of 5-ALA to the skin in the topical PDT.

In vitro skin permeation and retention of 5-aminolevulinic acid ester derivatives for photodynamic therapy

In photodynamic therapy (PDT), 5-aminiolevulinic acid (5-ALA) applied topically is converted, via the heme cycle, into protoporphyrin IX (PpIX), a photosensitizing agent, which upon excitation with light can induce tumor destruction. Due to its hydrophilic and zwitterionic characteristics, 5-ALA has limited penetration into the skin. More lipophilic 5-ALA ester derivatives are expected to cross stratum corneum more easily than 5-ALA. According to the determination of the partition coefficients of 5-ALA methyl, n-butyl, n-hexyl and n-octyl esters, these compounds showed an increased affinity to the SC, with 5-ALA hexyl ester and 5-ALA-octyl ester having the highest partition coefficients. Our in vitro skin permeation studies demonstrated an increased permeated amount for hexyl-ALA after 6 h of incubation, compared to other esters and 5-ALA. After 6 h, more 5-ALA-hexyl ester and -octyl ester were retained at viable epidermis and dermis than 5-ALA. According to these results, and considering that the conversion of 5-ALA into PpIX occurs preferentially in epidermis, it can be supposed that topical use of ester derivatives with longer chains (C(6) or C(8)) is an interesting proposal to optimize topical 5-ALA-PDT

Potential use of gelcasting hydroxyapatite porous ceramic as an implantable drug delivery system.

Hydroxyapatite (HA) ceramic in a porous configuration is suggested as a drug release system. A new technique for the production of this material, based on the foaming of suspensions and in situ polymerization (gelcasting method), resulted in a material whose characteristics are likely to make it useful as an implantable drug delivery system. Three batches of HA ceramic with different porosities were characterized by X-ray diffraction and scanning electron microscopy (SEM). Pore size and shape as well as density were determined. In vitro experiments were performed in order to evaluate the dissolution behavior of cisplatin in the system. X-ray diffraction analysis showed that the final product consisted of a single phase, indicating that the sintering process had not affected the structure of the HA. Energy dispersive X-ray analysis (EDX) showed absence of impurities. Pore diameters were in the range 15--34 microm. SEM showed that the material presented a highly interconnected spheroidal porous network with open micropores and closed macropores. In vitro experiments showed significant differences in the release rate of cisplatin between three different porosities.

Delivery Systems and Local Administration Routes for Therapeutic siRNA

ABSTRACTWith the increasing number of studies proposing new and optimal delivery strategies for the efficacious silencing of gene-related diseases by the local administration of siRNAs, the present review aims to provide a broad overview of the most important and latest developments of non-viral siRNA delivery systems for local administration. Moreover, the main disease targets for the local delivery of siRNA to specific tissues or organs, including the skin, the lung, the eye, the nervous system, the digestive system and the vagina, were explored.

A delivery system to avoid self-aggregation and to improve in vitro and in vivo skin delivery of a phthalocyanine derivative used in the photodynamic therapy.

The hydrophilic character and aggregation phenomena exhibited by the photosensitizer zinc phthalocyanine tetrasulfonate (ZnPcSO(4)) make it difficult for this compound to penetrate the skin, and reduce the compounds photodynamic efficacy. A microemulsion (ME) was developed to increase the skin penetration of ZnPcSO(4) while avoiding its aggregation. Ternary phase diagrams composed of surfactants (Span® 80/Tween® 80), canola oil and a propylene glycol (PG)/water mixture (3:1) were constructed as a basis for choosing an adequate ME preparation. Rheological, electrical conductivity, dynamic light scattering and zeta potential studies were carried out to characterize the ME formulations. Monomerization of ZnPcSO(4) in the ME was determined photometrically and fluorometrically. In vitro skin penetration and retention of the compound in the skin were measured using porcine ear skin mounted on a diffusion cell apparatus. The in vivo accumulation 6h after ZnPcSO(4) application was determined fluorometrically in hairless mice skin. Confocal laser scanning microscopy was used to visualize ZnPcSO(4) distribution in the skin. A ME composed of canola oil:surfactant:PG-water at 38:47:15 (w/w/w) was chosen for ZnPcSO(4.) This was oil-in-water with internal phase diameter of 15.7±0.15nm. Spectroscopic techniques confirmed that the ME was able to keep ZnPcSO(4) in its monomeric form. In the in vitro penetration of ZnPcSO(4) in the stratum corneum (SC) and in epidermis (without stratum corneum) with dermis ([E+D]) was 33.0- and 28.0-fold higher, respectively compared to the control solution of the drug. In vivo studies, confirmed that when the ME was used as carrier, ZnPcSO(4) concentrations in the SC and [E+D] were about 1.6- and 5.6-fold higher, respectively, than controls. Visualization of ZnPcSO(4) skin penetration by confocal laser scanning microscopy confirmed that the ME increased both penetration and biodistribution of this photosensitizer in the skin.

Sustained Release Carriers Used to Delivery Bone Morphogenetic Proteins in the Bone Healing Process

Bone morphogenetic proteins (BMPs) are multi‐functional growth factors belonging to the transforming growth factor β superfamily, especially BMP‐2, induce bone formation in vivo, and clinical application in repair of bone fractures and defects is expected. However, appropriate systems to delivery BMPs for practical use need to be developed with the objective to heal cartilage and bone‐related diseases in medical, dental and veterinary practice. Thus, the aim of this article was to present an overview of the principals carriers used to delivery BMPs and alternative delivery systems for these proteins.

Long-term follow-up of topical 5-aminolaevulinic acid photodynamic therapy diode laser single session for non-melanoma skin cancer

Photodynamic therapy (PDT) is based on the association of a light source and light sensitive agents in order to cause the selective death of tumor cells. To evaluate topical 5-aminolaevulinic acid (5-ALA) and diode laser photodynamic single session therapy single session for non-melanoma skin cancer (NMSC), a long-term follow-up was performed. Nineteen Bowens disease (BD) and 15 basal cell carcinoma (BCC) lesions were submitted to 6-h topical and occlusive 20% 5-ALA plus DMSO and EDTA, and later were exposed to 630 nm diode laser, 100 or 300 J cm(-2) dose. At 3 months tumor-free rate was 91.2% (31/34) whereas at 60 months, 57.7% (15/26), slightly higher in BCC (63.6%; 7/11). The relation between the reduction of the clinical response and the increase of tumor dimension observed at 18 months was lost at 60 months. The sBCC recurrence was earlier compared to the nBCC one. ALA-PDT offered important advantages: it is minimally invasive, an option for patients under risk of surgical complications; clinical feasibility; treatment of multiple lesions in only one session or lesions in poor healing sites and superior esthetical results. However, the recurrence rate increase after ALA-PDT diode laser single session can be observed at long-term follow-up, and the repetitive sessions, an additional advantage of the method, is strongly recommended. The clinical response and recurrence time seem to be related to the laser light dose and NMSC types/sub-types, thickness and dimension, which must be considered for the choice of the ALA-PDT.

Liquid crystalline phase nanodispersions enable skin delivery of siRNA.

The ability of small interfering RNAs (siRNAs) to potently but reversibly silence genes in vivo has made them particularly well suited as a new class of drugs that interfere with disease-causing or disease-promoting genes. However, the largest remaining hurdle for the widespread use of this technology in skin is the lack of an effective delivery system. The aim of the present study was to evaluate nanodispersed systems in liquid crystalline phases that deliver siRNA into the skin. The proposed systems present important properties for the delivery of macromolecules in a biological medium, as they are formed by substances that have absorption-enhancing and fusogenic effects; additionally, they facilitate entrapment by cellular membranes due to their nano-scale structure. The cationic polymer polyethylenimine (PEI) or the cationic lipid oleylamine (OAM) were added to monoolein (MO)-based systems in different concentrations, and after dispersion in aqueous medium, liquid crystalline phase nanodispersions were obtained and characterized by their physicochemical properties. Then, in vitro penetration studies using diffusion cell and pig ear skin were carried out to evaluate the effect of the nanodispersions on the skin penetration of siRNA; based on these results, the nanodispersions containing MO/OA/PEI/aqueous phase (8:2:5:85, w/w/w/w) and MO/OA/OAM/aqueous phase (8:2:2:88, w/w/w/w) were selected. These systems were investigated in vivo for skin penetration, skin irritation, and the ability to knockdown glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein levels in animal models. The results showed that the studied nanodispersions may represent a promising new non-viral vehicle and can be considered highly advantageous in the treatment of skin disorders; they were effective in optimizing the skin penetration of siRNA and reducing the levels of the model protein GAPDH without causing skin irritation.

Development and validation of HPLC method for imiquimod determination in skin penetration studies.

A simple, rapid and sensitive analytical procedure for the measurement of imiquimod in skin samples after in vitro penetration studies has been developed and validated. In vitro penetration studies were carried out in Franz diffusion cells with porcine skin. Tape stripping technique was used to separate the stratum corneum (SC) from the viable epidermis and dermis. Imiquimod was extracted from skin samples using a 7:3 (v/v) methanol:acetate buffer (100 mM, pH 4.0) solution and ultrasonication. Imiquimod was analyzed by HPLC using C(8) column and UV detection at 242 nm. The mobile phase used was acetonitrile:acetate buffer (pH 4.0, 100 mM):diethylamine (30:69.85:0.15, v/v) with flow rate 1 mL/min. Imiquimod eluted at 4.1 min and the running time was limited to 6.0 min. The procedure was linear across the following concentration ranges: 100-2500 ng/mL for both SC and tape-stripped skin and 20-800 ng/mL for receptor solution. Intra-day and inter-day accuracy and precision values were lower than 20% at the limit of quantitation. The recovery values ranged from 80 to 100%. The method is adequate to assay imiquimod from skin samples, enabling the determination of the cutaneous penetration profile of imiquimod by in vitro studies.

Topical photodynamic therapy for Bowen's disease of the digit in epidermolysis bullosa

nails. She had no prior manicure treatments to the nails or cuticles. After 3 months of daily isotretinoin her rosacea had cleared but the appearance of her nails caused her significant psychological distress and she elected to discontinue isotretinoin. Trimethoprim 200 mg twice daily was commenced but was discontinued due to nausea. She was recommenced on doxycycline 100 mg daily with 0Æ75% metronidazole gel. Three months after discontinuing isotretinoin the nail dystrophy had grown out 3 mm from the proximal nail fold (Fig. 1b). At 6 months her nails had completely returned to normal (Fig. 1c). The patient’s rosacea remains well controlled 9 months after discontinuing isotretinoin. Review of the images suggested that the patient had elkonyxis rather than median nail dystrophy. A diagnosis of isotretinoin-induced elkonyxis was made. Elkonyxis is an unusual nail dystrophy where the nail develops a pinched-out appearance which begins at the lunula and progressively grows out. This rare nail dystrophy has only previously been reported to occur in association with etretinate treatment. Our patient was initially thought to have isotretinoin-induced median nail dystrophy, a rare complication of isotretinoin treatment. However, median nail dystrophy tends to be a symmetrical nail dystrophy with a longitudinal defect in the nail in the centre or just off-centre of the nail with numerous transverse feathery cracks. There may be an associated enlarged lunula present. If the longitudinal defect is prominent and there are prominent multiple transverse nail defects in a ‘fir tree’ or ‘herring bone’ like appearance, the condition is often termed ‘median canaliform (Heller’s) dystrophy’. Median nail dystrophy is thought most commonly to be due to repeated inadvertent or selfinflicted trauma to the proximal nail fold. The cause for retinoid elkonyxis is unknown. Possible mechanisms would include alteration in the formation of keratins within the nail matrix or altered keratinization of the proximal nail fold and ⁄or cuticle (or alternatively the nail bed) which impairs or impinges on the uninterrupted formation of the nail plate. Close inspection of our sequential images demonstrates loss of cuticle early on (Fig. 1a) but re-establishment of a normal cuticle 3 months after discontinuing isotretinoin (Figs 1b,c). This suggests that abnormalities of the cuticle and proximal nail fold may contribute to the formation of elkonyxis. In our case, the temporal onset of elkonyxis after commencing isotretinoin, resolution following discontinuation of isotretinoin and the absence of trauma or other skin disease affecting the proximal nail fold is suggestive of a causal relationship. It is interesting that isotretinoin 20 mg daily (but not at doses of 20 mg twice and thrice weekly) elicited the development of elkonyxis. This suggests that the development of elkonyxis may in part be dose related in susceptible individuals. Elkonyxis is a rare and unusual type of nail dystrophy previously reported with etretinate treatment. Elkonyxis appears to be an idiosyncratic and possibly dosedependent nail dystrophy rarely associated with retinoid therapy.