Skin Of Wistar Rats Research Articles

Eucalyptus globulus essential oil increased neovascularization and vascular endothelial growth factor expression in male wistar rats exposed to UVB

Background: Eucalyptus globulus essential oil contained 1,8-cineol, which may accelerate wound healing. This study aimed to prove Eucalyptus globulus essential oil's effect on increasing neovascularization and Vascular Endothelial Growth Factor (VEGF) expression in Wistar rats skin exposed to ultraviolet B (UVB).
 Methods: A Post-test only controls group study design was applied on 36 male Wistar rats, aged 3-4 months old, weighing 150-200 grams. The animal subject was exposed to UVB (65 mJ/cm2, 65 seconds for seven days), injured on their back using a punch biopsy with a diameter of 10 mm, and divided into two groups. For seven days, on the wound, the control group was given 2 cc of 0.9% NaCl twice a day, while the treatment group was given Eucalyptus globulus essential oil (2 cc). Both groups were given oral Amoxicillin three times a day. The skin tissue was taken on day eight to assess neovascularization and VEGF expression.
 Results: The results show that neovascularization in the treatment group was higher than in the control group (p < 0.05). Meanwhile, the median VEGF expression in the treatment group was also higher than in the control group (p < 0.05).
 Conclusions: Topical administration of Eucalyptus globulus essential oil increased neovascularization and VEGF expression in the skin of male Wistar rats injured and exposed to UVB. Further research is needed to assess the effectiveness of the essential oil in enhancing neovascularization and VEGF expression in humans with multiple exposures at different doses to optimize the wound-healing effect.

Hydrogel composite containing azelaic acid and tea tree essential oil as a therapeutic strategy for Propionibacterium and testosterone-induced acne

Azelaic acid (AzA) is a USFDA bioactive prescribed against acne vulgaris. It possesses delivery challenges like poor aqueous solubility, low skin-penetrability, and dose-dependent side effects, which could be overcome by its synergistic combination with tea tree oil (TTO) as a microemulsion (ME)-based hydrogel composite. AzA-TTO ME was prepared to employ pseudo-ternary phase diagram construction. The best AzA-TTO ME was of uniform size (polydispersity index < 0.7), nano-range (~357.4 ± 2% nm), transmittance (> 90%), and negative zeta potential (−1.42 ± 0.25% mV) values. ME hydrogel composite with optimum rheological and textural attributes showed better permeation, retention, and skin-compliant characteristics, vis-a-vis marketed formulation (Aziderm™) when evaluated in Wistar rat skin. In vitro antibacterial efficacy in bacterial strains, i.e., Staphylococcus aureus, Propionibacterium acne, and Staphylococcus epidermidis, was evaluated employing agar well plate diffusion and broth dilution assay. ME hydrogel has shown an increase in zone of inhibition by two folds and a decrease in minimum inhibitory concentration (MIC) by eightfold against P. acnes vis-a-vis AzA. Finally, ME hydrogel composite exhibited a better reduction in the papule density (93.75 ± 1.64%) in comparison to Aziderm™ 72.69 ± 4.67%) on acne as developed in rats by inducing testosterone. Thus, the developed AzA-TTO ME hydrogel composite promises an efficacious and comparatively safer drug delivery system for the topical therapy of acne vulgaris.Graphical abstract

UPLC-MS/MS Method Validation for Estimation of Resveratrol in Rat Skin from Liposphere Gel Formulation and Its Application to Dermatokinetic Studies in Rats.

For the quantification of resveratrol (RV) in the Wistar rat skin, an ultra-performance liquid chromatography-mass spectrometric (UPLC-MS/MS) method was developed and validated on ACQUITY UPLC BEH C18 column (1.7 μm). The mobile phase ratio of methanol (A) and 2% formic acid (B) (ratio 10: 90% v/v, 80: 20 v/v) at isocratic elution with flow rate 0.3 mL/min, and run time 3 min was used for analysis. In addition, the use of multiple reaction monitoring (MRM)/ES+ mode to detect the analytes and to track parents to daughter ion transition of 229.17 > 107.04 m/z (time scan 3 min, retention time 1.48) for RV and curcumin as an internal standard shows 369.16 > 176.93m/z (scan time is 2.80 min, retention time is 1.11), respectively. Linearity was observed in the range of 2.5 to 2,000 ng/mL (R2 = 0.987). Precision and accuracy on rat skin were within the acceptability range (RE%: ±15; RSD%: ±15). Moreover, it showed a good percentage recovery and found within acceptance limit 90-110%. Lower limit of detection and quantitation for the method observed to be 2.5 and 20 ng.mL-1, respectively. Method application indicated successful determination of dermatokinetics parameters of RV from lipospheres gel and suspension in the rats.

Improved Efficacy and Stability of Silymarin Loaded Nanocochleates Over Liposomes for the Treatment of Skin Diseases

Aim: Silymarin, a complex polyphenolic component mixture with anti-oxidant, anti-inflammatory, and membrane-stabilizing property is being investigated in several dermatological conditions. Present research aims to evaluate potential of silymarin loaded nanocochleates and liposomal topical application for treating chronic skin diseases.&#x0D; Study Design: Silymarin loaded liposomes and nanocochleates were formulated and optimized using Design Expert software. Different invitro and exvivo tests were performed to compare their performance.&#x0D; Place and Duration of Study: The study was conducted in Smt. Kashibai Navale College of Pharmacy, Pune, India, between January 2019 till February 2020.&#x0D; Methodology: Liposomes were prepared using ethanol injection method and further treated with calcium chloride to form nanocochleates by trapping method. Design of experiments (32 Factorial Design) was used for optimization of nanocochleates. Cell line studies (HaCaT cell lines) and short term stability studies were performed to compare the efficacy and stability respectively.&#x0D; Results: Particle size, entrapment efficiency and drug deposition in Wistar Rat Skin was found to be statistically significant for nanocochleates over liposomes proving superiority of cochleates. Both the carriers sustained release of silymarin for 24h. Antimicrobial efficacy of nanocochleates against E.coli and S.aureus was significant. Inhibition of hyper proliferation of HaCaT cell lines (key mechanism by which most of the antipsoriatic drugs act) demonstrated the superiority of nanocochleates over liposomes.The nanocochleates also displayed better stability compared to liposomes due to decreased entrapment efficacy and leakage of drug.&#x0D; Conclusion: Silymarin loaded Nanocochleates could prove as a promising topical drug delivery system for the treatment of chronic skin diseases like psoriasis.

Protective Effects of Acehnese Traditionally Fermented Coconut Oil (Pliek U Oil)1 and its Residue (Pliek U) in Ointment against UV Light Exposure: Studies on Male Wistar Rat Skin (Rattus novergicus)

A protective effect of Acehnese traditionally fermented coconut oil (Pliek U oil) and its residue (Pliek U) have been studied against UVB radiation. The research was performed by preparing the active ingredient in ointment formulation and protective test in male Wistar rat skin (Rattus novergicus). Four different extraction methods were done to produce four active ingredients, that are Pliek U oil (PUO), ethanolic Pliek U oil extract (EPUOE), ethanolic Pliek U extract (EPUE), and ethanolic hexane residue Pliek U extract (EHRPUE). The protective effect was determined based on the erythema index (EI), melanin index (MI), and level of protein oxidation parameters. The protective level from each active ingredient was studied by PCA analysis. The result showed that the active ingredient could inhibit the redness and protein oxidation in skin tissue. PCA results show that concentration (20 v. 40%) does not affect the protection level of the active ingredients of Pliek U oil, but it does affect the one of Pliek U. Therefore, these active ingredients are potential for sunscreen application.

Effect of snail mucus on angiogenesis during wound healing.

Background: Angiogenesis is the process through which new blood vessels are formed from existing ones. This process plays an important role in supplying the oxygen and nutrients needed for cellular metabolism and eliminating cell debris during wound healing. Snail mucus can bind to several factors that stimulate angiogenesis, including vascular endothelial growth factor, platelet-derived growth factor, and fibroblast growth factor. The aim of this study is to observe changes in angiogenesis during the healing of wounds topically applied with snail mucus. Methods: Punch biopsy was performed on the back of male Wistar rats to obtain four wounds, and different concentrations of snail mucus were applied to each of these wounds. The animals were sacrificed on days 2, 4, and 7 to observe the extent of angiogenesis during wound healing by microscopy. Results: Two-way ANOVA showed differences in number of blood vessels formed (p = 0.00) and day of observation (p = 0.00) between groups. Post hoc Tukey's HSD test showed that 24% snail mucus treatment does not significantly affect wound healing (p = 0.488); by contrast, treatment with 48% and 96% snail mucus demonstrated significant effects on angiogenesis (p = 0.01). Spearman's test showed interactive effects between snail mucus concentration and day of observation on the extent of angiogenesis (p = 0.001, R = 0.946). Conclusion: Topical application of snail mucus gel can increase angiogenesis during wound healing in Wistar rat skin.

Enhanced Transdermal Delivery of Bisoprolol Hemifumarate via Combined Effect of Iontophoresis and Chemical Enhancers: Ex Vivo Permeation/In Vivo Pharmacokinetic Studies.

Bisoprolol hemifumarate (BH) is an antihypertensive drug that is used as first-line treatment for chronic hypertension and angina pectoris. Our study was performed to enhance the transdermal delivery of BH, a hydrophilic drug active with high molecular weight, through differently prepared hydrogels. The synergistic effect of permeation enhancers and iontophoresis was investigated via both ex vivo and in vivo permeation studies. Ex vivo iontophoretic permeation studies were performed by using male albino Wistar rat skin. Cellosolve® hydrogel (F7) showed a 1.5-fold increase in Q180, Jss, and FER compared to F5 (lacking permeation enhancer). BH pharmacokinetic data were studied in human volunteers, following transdermal delivery of F7, using Phoresor® Unit II iontophoresis device, compared to conventional oral tablets. F7 showed 1.9- and 2-fold higher values of Cmax and AUC0–40, respectively compared to Concor® tablets, as well as a smaller Tmax (2.00 ± 2.00 h). The relative bioavailability of F7 was found to be 201.44%, relative to Concor® tablets, demonstrating the significantly enhanced transdermal permeation of BH from the selected hydrogel by iontophoresis, in human volunteers. Finally, results showed the successful utility of permeation enhancers combined with iontophoresis in significantly enhanced transdermal permeation of BH, despite its large molecular weight and hydrophilic nature. Therefore, this strategy could be employed as a successful alternative route of administration to conventional oral tablets.

EVALUATION OF ANTIOXIDANT AND WOUND HEALING ACTIVITIES OF Spirulina sp. EXTRACT

Spirulina spp. is an alga from phylum Cyanobacteria that have multi-benefit due to their potential compounds like protein, carbohydrate, lipid, mineral, pigment, and phenolic compounds. Several types of research already describe Spirulina spp. capacity as an antioxidant, anti-inflammation, and antimicrobial agent, thus expand its application as a food source, cosmeceutical, and pharmaceutical compound. This study observed and evaluated Spirulina spp. extract as a potential source for cosmeceutical compound antioxidant and wound healing activities. Spirulina spp. was extracted with a variable solvent like water, ethanol, acetic acid, and buffer for 1 and 3 hours by the maceration method. Spirulina extract which give the highest antioxidants activity, was evaluated for wound healing, toxicity, and irritation. The result showed that Spirulina spp. water extracted for one hour using the maceration method have the highest antioxidant capacity with IC50 45.21 mg/mL, AEAC 0.12 mg Vit. C/g sample, phycocyanin content 6.478 µg/ml, and chlorophyll content 2.642 µg/ml. Treatment with 1% Spirulina extracts exhibits a higher wound healing effect rather than 10% Spirulina spp. extract, control group, and povidone-iodine 10%, on Wistar rat skin. Spirulina spp. water extract showed capacity as a whitening agent with inhibitory activity on tyrosinase were IC50 0.51 mg/mL. Spirulina spp. water extract had no irritation and toxicity effect on rabbit skin and mouse, respectively. This study showed that Spirulina spp. water extract is a potential source for antioxidant activity, wound healing capacity, and whitening agent; without toxicity and irritation effect. The result showed that Spirulina spp. extract could be potential value in cosmeceutical application.

MORPHOFUNCTIONAL REBUILDING OF FIBROUS STRUCTURES OF RAT'S SKIN DERMIS UNDER 3D-SCAFFOLD IMPLANTATION BASED ON POLYPROLACTONE

The use of various scaffolds makes it possible to model the future fibrous framework of the newly formed regenerate, and also serves as a substrate for the colonization of the cellular component. The development of tissue engineering in regenerative medicine requires an understanding of more precise mechanisms of the formation of the connective frame at the site of the defect. The aim of this study was to study the morphofunctional rearrangement of the fibrous structures of the dermis of the skin of rats, in response to for the implantation of a 3D-scaffold based on polyprolactone. The study was carried out on the skin of male Wistar rats at different times after implantation of a 3D-scaffold based on polyprolactone. The biomaterial with the implantable scaffold was studied using scanning electron microscopy. As a result of the study, it was revealed that active collagenogenesis is determined around the structures of the 3D-scaffold based on polyprolactone. By the 14th day, a large number of circularly directed collagen fibers are visualized, which are the basis for further building a connective tissue capsule around the fibers. Conclusions are drawn about the effectiveness of using a 3D-scaffold based on polyprolactone. This implant can also be effective for the colonization of allogeneic cultured fibroblasts and the creation of biomedical tissue engineering products on its basis.

Effect of snail mucus on angiogenesis during wound healing

Background: Angiogenesis is the process through which new blood vessels are formed from existing ones. This process plays an important role in supplying the oxygen and nutrients needed for cellular metabolism and eliminating cell debris during wound healing. Snail mucus can bind to several factors that stimulate angiogenesis, including vascular endothelial growth factor, platelet-derived growth factor, and fibroblast growth factor. The aim of this study is to observe changes in angiogenesis during the healing of wounds topically applied with snail mucus. Methods: Punch biopsy was performed on the back of male Wistar rats to obtain four wounds, and different concentrations of snail mucus were applied to each of these wounds. The animals were sacrificed on days 2, 4, and 7 to observe the extent of angiogenesis during wound healing by microscopy. Results: Two-way ANOVA showed differences in number of blood vessels formed (p = 0.00) and day of observation (p = 0.00) between groups. Post hoc Tukey’s HSD test showed that 24% snail mucus treatment does not significantly affect wound healing (p = 0.488); by contrast, treatment with 48% and 96% snail mucus demonstrated significant effects on angiogenesis (p = 0.01). Spearman’s test showed interactive effects between snail mucus concentration and day of observation on the extent of angiogenesis (p = 0.001, R = 0.946). Conclusion: Topical application of snail mucus gel can increase angiogenesis during wound healing in Wistar rat skin.

Anti-Inflammatory Activity of Human Lens Crystallin Derived Peptide.

Inflammation has become the culmination point for several chronic diseases like skin diseases, asthma, neurological disorders, cancer, and cardiovascular disorders. Mini αA-crystallin peptide, identified from a highly conserved region of human lens protein αA-crystallin, is known to have a chaperone-like function; hence, it has generated interest in exploring the anti-inflammatory potential of the peptide. The objective of the study was to evaluate the anti-inflammatory potential of mini αA chaperones using in vitro, ex-vivo, and in vivo models. The peptide was tested for its phosphodiestarase4 B inhibition, anti-inflammatory and free radical scavenging abilities in HaCaT cells. Carbopol gel formulations with varying concentrations of mini αA-crystallin peptide and diclofenac sodium were prepared and optimized. Skin permeation studies of prepared formulations were carried out on excised abdominal skin of Wistar rat using a vertical type diffusion cell. Carrageenan induced rat paw oedema model was used for determining the anti-inflammatory potential of the peptide in prepared gel formulation with or without diclofenac sodium. The peptide exhibited appreciable free radical scavenging and weak PDE4B inhibition. Gel formulation with 1% Tween-80, 1% carbopol, and 10% ethanol showed better permeation compared to other formulations. The in vitro skin permeation studies revealed good improvement in permeation characteristics of diclofenac and peptide from the gels. The peptide was retained within the skin tissue, which is an ideal requirement for the delivery of an anti-inflammatory topical formulation. In preclinical anti-inflammatory studies, gel formulation containing mini αA-peptide and diclofenac sodium showed a significant decrease in paw volume compared to other combinations tested. The study revealed the additive effect in anti-inflammatory activity by combining mini-αA peptide and diclofenac sodium which effectively reduced the inflammation.

Photoprotective activity of geopropolis produced by Melipona subnitida (Apidae, Meliponinae) in the semiarid of the Brazilian Northeast

Although geopropolis has been the subject of many chemical and pharmacological studies, there are few studies investigating the photoprotective activity of formulations containing propolis. Thus, we investigated in vivo the photoprotective efficacy of the cream containing geopropolis extract by macroscopic and histological evaluation of the skin of Wistar rats subjected to ultraviolet radiation (UVB). We also evaluated the chemical composition of hydroethanolic extract using the HPLC-DADESI-MS/MS technique, as well as antioxidant activity by the photocolorimetric method of free radical DPPH (2.2-diphenyl-1-picrylhydrazine) and cytotoxic activity by the in vitro MTT quantitative method [brometo de 3- (4.5dimetiltiazol-2-il)-2.5-difeniltetrazolio]. The extract had a varied chemical composition, 29 different phenolic compounds being detected, distributed between phenols and flavonoids, the latter being represented by chalcones, flavones and flavonols. The highest percentages of DPPH inhibition e o baixo valor de IC50 indicaram que o extrato apresentou alta atividade antioxidante. The hydroethanolic extract did not exert cytotoxic effects since high percentages of viability of L929 fibroblasts were observed after incubation for 72 hours at different concentrations of the extract. On skin submitted to cream application containing of geopropolis extract and the irradiation with UVB did not occur macroscopic and histological lesions. Thus, we concluded that the cream containing of geopropolis extract produced by Melipona subnitida was able to protect the skin from lesions induced by UVB irradiation, thus demonstrating photoprotective effect.

Formulation development and evaluation of drug-in-adhesive-type transdermal patch of metoclopramide HCl

The objective was to design and develop a drug-in-adhesive-type transdermal patch formulation of metoclopramide HCl, which is used as an effective antiemetic, dopaminergic antagonist agent. In DIA patches, drug is directly embedded in the polymeric adhesive layer and permeates through the skin to provide systemic effect, avoiding gastrointestinal tract complications, including the first-pass metabolism. Six different patch formulations were prepared by a solvent evaporation method using varying metoclopramide HCl concentrations using acrylate polymer DURO-TAK® 387/2510 for adhesive support. In vitro permeation profiles and parameters were obtained by using the skin of hairless albino Wistar rats. The influence of drug content on the rate of permeation was investigated, and patch formulation was optimized based on Q12, flux, and lag time. The effect of permeation enhancers (EO and PG) on the permeation rate of metoclopramide HCl was also studied. The skin irritation study was performed to observe the hypersensitivity against the patch component. Stability studies of the optimized formulation of a transdermal patch for three months at three different temperatures were investigated. The transdermal patch formulation containing 10% w/w metoclopramide HCl was capable of effectively delivering metoclopramide HCl for systemic effect without any skin irritation. It has Q12 of 3.892 ± 0.0043 mg/cm2, flux rate of 0.2501 mg/cm2 h, and the coefficient of permeability of 6.25E−02 cm/h with a lag time of 0.442 h. The stability data revealed that the optimized patch formulation could be stored at 4 °C in the refrigerator with a shelf life of 3.53 months. This newly developed metoclopramide transdermal DIA patch is an effective alternative to oral delivery with desirable antiemetic activity.

Employing optical microscopy technique to determine the tissue action of Hamamelis virginianaL. (Hamamelidaceae), Maytenus ilicifoliaMart. ex Reissek (Celastraceae) and Casearia sylvestris Sw. (Salicaceae) tinctures on skin-fixed histological sections of Wistar rats

Objective: In this study, we aimed to determine the action of the tinctures of Hamamelis virginiana, Maytenus ilicifolia, and Casearia sylvestris on tissues. For this, we investigated the histological sections of fixed skin tissue of Wistar rats for the changes in collagen and elastic fibers, epithelial cells, conjunctive cells, epidermal attachments, pigments, and granules using the optical microscopy technique. Since in the literature and published articles, the use of in vivo models, such as Wistar rats, is predominant to evaluate the healing action of herbal medicines. Methods: The tinctures of Hamamelis virginiana, Maytenus ilicifolia, and Casearia sylvestris, and ethanol 70% (blank) were used. The tinctures were obtained at 10% (w/v) through percolation using 70% ethanol (v/v) as the extraction liquid. This study was conducted in duplicate for each tincture and different incubation times (4, 24, and 48 h) at 37 °C in an oven. The slides used in this study (Wistar rat skin) were previously processed at the histology laboratory since the waste material was donated to conduct this experiment. The Research Ethics Committee approved the use of animals of the Life Sciences Center of the Pontifical Catholic University of Campinas (PUC-CAMPINAS) under the protocol approval number 367/08. Before initiating the staining process, the slides were removed from the oven, and the tincture deposited on the slides was removed. Weigert staining was performed subsequently. For semi-quantitative analysis, the histological sections were carefully observed, and the number of collagen and elastic fibers was evaluated based on the following scale: (+) normal fiber presence, (−) slight decrease, (− −) moderate decrease, and (− − −) intense decrease. The images were digitally captured to obtain the results using a photomicroscope. Results: The degradation of collagen fibers was best evidenced upon using Hamamelis virginiana tincture, which is concordant with the existing reports in the literature on its healing action via the precipitation of dermal proteins. Maytenus ilicifolia and Casearia sylvestris tinctures exhibited low proteolytic capacity as they only caused degradation of elastic fibers that are more delicate and very different from collagen in their constitution. Therefore, the application of the latter two as healing agents (which acts through precipitating proteins) is limited. Conclusions: This experimental histological model using the optical microscopy technique exhibits the advantage of agility and high efficiency as a simple and powerful approach.

Перестройка структурных компонентов дермы крысы в условиях дерматотензии

Introduction. Skin expansion is known to be the most effective way to obtain skin for alloplastic closing of large defects, and it has proven to be successfully used in various fields of surgery, cosmetology, and traumatology. At the same time, the issues of skin flap restructuring when it is stretched and possible range of its application are still relevant. In this regard, the aim was to study the adaptive skin rearrangements of the fibrous skeleton in a murine skin expansion model. Materials and methods. We used the skin of mature male Wistar rats (N=30) after a 2-week stretching. We utilized a complex morphological light and electron microscopy approach as well as an immunohistochemi-cal method to determine the types of collagen and to study the alterations in skin samples. Results. We revealed pronounced reactive changes in the skin structural components in the skin expansion area. On day 14, we observed a decrease in the epidermal cell layer thickness as well as stretching, partial disorganization, and damage of dermal fibrous structure. At the same time, cellular mechanisms of regen-eration and activation of collagen synthesis were launched, the morphological substrate of which was (1) a significant 2.7-fold increase in the number density of fibroblasts, which produce substances for building fibrous structures, (2) a five-fold increase in macrophages, which are a catalyst for the fibroblast functional activity and (3) a collagen fiber types’ redistribution s with Type III collagen structures’ predominance. Conclusion. The results indicate, first of all, the restructuring of the dermal fibrous component, which im-plies reparative and restorative processes. These must be taken into account in clinical practice to achieve not only an esthetic effect but also the subsequent adequate functioning and vital activity of the skin flap. Keywords: skin expansion, fibrous dermis, remodeling, skin, hyperextension

The effects of Brazilian chitosan-based biomaterials on wound healing in rats

The use of natural supplies is a resource to mimic an original extracellular matrix that allows for migration, proliferation, and cellular organization. Chitosan from Brazilian Atlantic Ocean had low protein, minerals percentage and excellent antibacterial activity. The aim of this study was to evaluate and to compare the effectiveness of different types of acids as solvents with Brazilian chitosan-membrane in the healing process of skin lesions. Experimental full-thickness 2 × 2 cm wounds were created on the dorsum skin of Wistar rats. The applied different treatments were saline, collagenase®, microcrystalline chitosan salt membrane (MCSM), microcrystalline chitosan acetic acid membrane (MCAAM), and microcrystalline chitosan hydrochloric acid membrane (MCHAM). The wound repairs were measured morphologically and histologically on days 0, 3, 7, 10, and 14. The exudate formation and the final wound contractions were similar in all of the groups. There were mild exudations in the groups with chitosan-membranes, despite the formation of crust under the membrane. This configured a serum hematic aspect, but there was no impact on the healing process. The MCHAM group had more favorable aspects that histologically showed the healing phases. A significant migration of neutrophils and macrophages seen by myeloperoxidade and Beta-N-Acetylglucosaminidase activities was evident in the chitosan groups, MCHAM and MCSM, respectively. Furthermore, the MCHAM group created its histological arrangement in a dense and more consistent manner.

MAL-associated methyl nicotinate for topical PDT improvement

Photosensitization of all tissue in sufficient quantity to generate damage is one of the limiting factors for Photodynamic Therapy (PDT) efficiency. Methyl nicotinate (MN) is a thermogenic and vasodilating substance that facilitates the topical tissue penetration of some compounds. The topical MAL (methyl aminolevulinate) PDT is commonly used as a precursor of protoporphyrin IX (PpIX). This study investigates the safety of topical use in NM, as well as its ability to improve the efficiency of topical PDT. For this, we investigate the cytotoxicity of MN, as well as its actions in increasing cellular metabolism and vasodilation. Besides, its ability to optimize the formation of PpIX in the tissue when associated with MAL cream was investigated, besides assessing the severity of necrosis obtained by treatments. The cytotoxicity of MN was tested for concentrations of 0, 0.1, 0.25, 0.5, 0.75 and 1% in cell culture. For the concentration of 0.5%, the cellular metabolism was evaluated using confocal microscopy to calculate the redox rate. In the Chorioallantoic Membrane Model, vasodilation was evaluated for concentrations of 0.5 and 1% MN during 1 h of incubation. In the animal model, the healthy skin of Wistar rat was used to evaluate the production of PpIX in the tissue and the degree of necrosis obtained by Photodynamic therapy when using NM associated with methyl aminolevulinate. It was observed the non-cytotoxicity in vitro of MN in the concentration used (0.5%) and its ability to increase cellular metabolism. In a chorioallantoic model, the MN vasodilation power was demonstrated for different caliber of vessels. In vivo studies are showing that the incorporation of MN in the MAL cream increases the amount of PpIX produced in the tissue causing a higher effect on the epidermis after PDT. This improvement of the protocol may make the procedure more effective both in the destruction of tumor tissue and in the treatment of deeper cells decreasing possible recurrence, in addition to allowing improvements in the protocol, such as reducing the cream's incubation time.

Cyclooxygenase-2 Modulates Glycosaminoglycan Production in the Skin During Salt Overload.

Sodium (Na+) can accumulate in the skin tissue, sequestered by negatively charged glycosaminoglycans (GAGs). During dietary salt overload, the amount and charge density of dermal GAG molecules – e.g., hyaluronic acid (HA) and chondroitin sulfate (CS) – increases; however, the regulation of the process is unknown. Previously, it has been demonstrated that the level of cyclooxygenase-2 (COX-2) activity and the content of prostaglandin E2 (PGE2) are elevated in the skin due to high-salt consumption. A link between the COX-2/PGE2 system and GAG synthesis was also suggested. We hypothesized that in dermal fibroblasts (DFs) high-sodium concentration activates the COX-2/PGE2 pathway and also that PGE2 increases the production of HA. Our further aim was to demonstrate that the elevation of the GAG content is ceased by COX-2 inhibition in a salt overloaded animal model. For this, we investigated the messenger RNA (mRNA) expression of COX-2 and HA synthase 2 enzymes as well as the PGE2 and HA production of DFs by real-time reverse transcription PCR (qRT-PCR) and ELISA, respectively. The results showed that both high-sodium concentration and PGE2 treatment increases HA content of the media. Sodium excess activates the COX-2/PGE2 pathway in DFs, and COX-2 inhibition decreases the synthesis of HA. In the animal experiment, the HA- and CS disaccharide content in the skin of male Wistar rats was measured using high performance liquid chromatography-mass spectrometry (HPLC-MS). In the skin of rats receiving high-salt diet, the content of both HA‐ and monosulfated-CS disaccharides increased, whereas COX-2 inhibition blocked this overproduction. In conclusion, high-salt environment could induce GAG production of DFs in a COX-2/PGE2-dependent manner. Moreover, the COX-2 inhibition resulted in a decreased skin GAG content of the salt overloaded rats. These data revealed a new DF-mediated regulation of GAG synthesis in the skin during salt overload.

QUALITY BY DESIGN APPROACH FOR DEVELOPMENT AND OPTIMIZATION OF NITRENDIPINE LOADED NIOSOMAL GEL FOR ACCENTUATED TRANSDERMAL DELIVERY

Objective: The purpose of the present investigation was to develop and optimize nitrendipine loaded niosomal gel for transdermal delivery using quality by design approach.&#x0D; Methods: Niosomal formulations were developed by application of the thin-film hydration method using different ratios of span 60, cholesterol, temperature, and optimized by three factors-three levels Box-Behnken statistical design. The independent variables were non-ionic surfactant, cholesterol, and temperature, while vesicle size, polydispersity index, and entrapment efficiency were dependent variables. The nitrendipine loaded optimized formulation was incorporated into gel and evaluated for in vitro release, ex-vivo skin permeation, confocal laser scanning microscopy, and histopathological studies.&#x0D; Results: The optimized formulation showed the vesicular size of 226.1±4.36 nm, polydispersity index of 0.282±0.012, and entrapment efficiency of 95.34±3.18% with spherical morphology. The optimized niosomal gel formulation showed transdermal flux 127.60 µg/cm2h through albino Wistar rat skin. Niosomal gel was proved significantly superior by confocal laser scanning microscopy for satisfactory permeation and distribution of gel, deep into the rat skin. Furthermore, dermal safety was confirmed by histopathological studies for transdermal application.&#x0D; Conclusion: It was concluded that the developed niosomal gel overcomes the limitation of low penetration through rat skin and could be a potential nano vesicular system for transdermal delivery.

The effects of iontophoresis and electroporation on transdermal delivery of indomethacin evaluated in vitro and in vivo

The objective of this study was to evaluate the transdermal delivery of indomethacin in vitro and in vivo enhanced by iontophoresis and/or electroporation through several barriers. Excised Wistar rat skin, pig skin, human epidermal membrane and cellulose membrane were tested for the in vitro indomethacin permeation, whereas drug concentration of in the plasma was analyzed to monitor the percutaneous absorption in Wistar rat in vivo. Iontophoresis enhanced the in vitro transdermal delivery of indomethacin as compared to the passive diffusion. Pulsing of high voltages, as known as electroporation, followed by iontophoresis did result in a synergistic effect in contrast to iontophoresis or electroporation alone. The in vitro result indicated that higher drug permeation could be induced during and after the application of iontophoresis. In some case, the permeation could benefit from the electroporation in term of cumulative amount within 12 hr. Furthermore, the combination of electroporation and iontophoresis resulted in a higher permeation amount than iontophoresis or electroporation alone did. Both iontophoresis and the combination protocol could reduce interspecies difference in vitro. The in vivo results were similar to that of in vitro ones. Iontophoresis and electroporation/iontophoresis resulted in higher AUC (area under curve) of blood concentration profiles of indomethacin. In addition, the combination protocol significantly induced higher transepidermal water loss (TEWL). It is concluded that the in vivo permeation results did correspond to the enhancement efficiency of the in vitro experiments.