Publications by authors named "Maria Vitória Lopes Badra Bentley"

40 Publications

Ex vivo model of human skin (hOSEC) for assessing the dermatokinetics of the anti-melanoma drug Dacarbazine.

Eur J Pharm Sci 2021 May 19;160:105769. Epub 2021 Feb 19.

Division of Dermatology - Wound Healing & Hansen's Disease Lab, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil. Electronic address:

Alternative models to replace animals in experimental studies remain a challenge in testing the effectiveness of dermatologic and cosmetic drugs. We proposed a model of human organotypic skin explant culture (hOSEC) to assess the profile of cutaneous drug skin distribution, adopting dacarbazine as a model, and respective new methodologies for dermatokinetic analysis. The viability tests were evaluated in primary keratinocytes and fibroblasts, and skin by MTT and TTC assays, respectively. Then, dacarbazine was applied to the culture medium, and the hOSEC method was applied to verify the dynamics of skin distribution of dacarbazine and determine its dermatokinetic profile. The results of cell and tissue viability showed that both were considered viable. The dermatokinetic results indicated that dacarbazine can be absorbed through the skin, reaching a concentration of 36.36 µg/mL (18,18%) of the initial dose (200 µg/mL) after 12 h in culture. Histological data showed that the skin maintained its structure throughout the tested time that the hOSEC method was applied. No apoptotic cells were observed in the epidermal and dermal layers. No visible changes in the dermo-epidermal junction and no inflammatory processes with the recruitment of defense cells were observed. Hence, these findings suggest that the hOSEC concept as an alternative ex vivo model for assessing the dynamics of skin distribution of drugs, such as dacarbazine, and determining their respective dermatokinetic profiles.
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http://dx.doi.org/10.1016/j.ejps.2021.105769DOI Listing
May 2021

Cytotoxic and chemosensitizing effects of glycoalkaloidic extract on 2D and 3D models using RT4 and patient derived xenografts bladder cancer cells.

Mater Sci Eng C Mater Biol Appl 2021 Feb 27;119:111460. Epub 2020 Aug 27.

College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA.

Glycoalkaloids have been widely demonstrated as potential anticancer agents. However, the chemosensitizing effect of these compounds with traditional chemotherapeutic agents has not been explored yet. In a quest for novel effective therapies to treat bladder cancer (BC), we evaluated the chemosensitizing potential of glycoalkaloidic extract (GE) with cisplatin (cDDP) in RT4 and PDX cells using 2D and 3D cell culture models. Additionally, we also investigated the underlying molecular mechanism behind this effect in RT4 cells. Herein, we observed that PDX cells were highly resistant to cisplatin when compared to RT4 cells. IC values showed at least 2.16-folds and 1.4-folds higher in 3D cultures when compared to 2D monolayers in RT4 cells and PDX cells, respectively. GE + cDDP inhibited colony formation (40%) and migration (28.38%) and induced apoptosis (57%) in RT4 cells. Combination therapy induced apoptosis by down-regulating the expression of Bcl-2 (p < 0.001), Bcl-xL (p < 0.001) and survivin (p < 0.01), and activating the caspase cascade in RT4 cells. Moreover, decreased expression of MMP-2 and 9 (p < 0.01) were observed with combination therapy, implying its effect on cell invasion/migration. Furthermore, we used 3D bioprinting to grow RT4 spheroids using sodium alginate-gelatin as a bioink and evaluated the effect of GE + cDDP on this system. Cell viability assay showed the chemosensitizing effect of GE with cDDP on bio-printed spheroids. In summary, we showed the cytotoxicity effect of GE on BC cells and also demonstrated that GE could sensitize BC cells to chemotherapy.
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http://dx.doi.org/10.1016/j.msec.2020.111460DOI Listing
February 2021

Advances in lyotropic liquid crystal systems for skin drug delivery.

Expert Opin Drug Deliv 2020 12 18;17(12):1781-1805. Epub 2020 Sep 18.

School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo , Ribeirão Preto, SP, Brazil.

Introduction: Lyotropic liquid crystals (LLCs) are organized mesophases with intermediate properties between liquids and solids. The LLC and its liquid crystalline nanoparticles (LCNPs) have attracted great interest from the scientific community in recent years as potential drug delivery systems due to the high internal ordering and symmetry with a wide interfacial area.

Areas Covered: This article aims to gather information and to provide a description of the highly organized structures of LLCs. Updates on production methods and new insights for LCNPs optimization and physico-chemical and morphological caracterization techniques were discussed. We also discussed why these systems proved to be a platform for the design of nanocarrier drug delivery, with an emphasis on topical and transdermal applications.

Expert Opinion: Drug delivery platforms are of particular importance to improve the biopharmaceutical aspects of therapies topically. Although several systems can be used, LLC or LCNPs appear to be favored due to their similarity to the lipid structure of the skin. The highly ordered structure and the possibility of chemical modifications make it possible to obtain better clinical responses. The results of several studies support the innovations in this field and predict that these systems can innovate the market of technologies for the treatment of cutaneous diseases and cosmetology.
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http://dx.doi.org/10.1080/17425247.2020.1819979DOI Listing
December 2020

Liquid crystalline nanodispersion functionalized with cell-penetrating peptides improves skin penetration and anti-inflammatory effect of lipoic acid after in vivo skin exposure to UVB radiation.

Drug Deliv Transl Res 2020 12;10(6):1810-1828

School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Avenida do Café, s/n, Ribeirão Preto, SP, 14040-903, Brazil.

In this study, the development and the performance of a new targeted liquid crystalline nanodispersion (LCN) by the attachment of cell-penetrating peptides (CPP) onto their surfaces to improve skin delivery of lipoic acid (LA) were evaluated. For that, the synthesis and characterization of this new platform as well as its spatiotemporal analysis from in vitro and in vivo topical application were explored and extensively discussed in this paper. The TAT or D4 peptides were chosen as CPP due to specific target strategies by the charge grouping on the skin surface or target the overexpressed epidermal growth factor receptor (EGFR) of cell membrane of keratinocytes, respectively. Thus, the nanoparticle characterization results when taken together suggested that designed LCNs maintained their hexagonal phase structure, nanoscale particle size, and low polydispersity index even after drug, lipopolymers, and peptide additions, which are proved to be favorable for topical skin delivery. There were no statistical differences among the LCNs investigated, except for superficial charge of LCN conjugated with TAT which may have altered the LCN zeta potential due to cationic charge of TAT amino acid sequence compared with D4. The cumulative amounts of LA retained into the skin were determined to be even higher coming from the targeted LCNs. Moreover, the exogenous antioxidant application of the LA from the LCNs can prevent ROS damage, which was demonstrated by this study with the less myeloperoxidase (MPO) activity and decrease in cytokine levels (TNF-alpha and IL-1β) generated by the oxidative stress modulation. Together, the data presented highlights the potential of these targeted LCNs, and overall, opens new frontiers for preclinical trials.
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http://dx.doi.org/10.1007/s13346-020-00840-2DOI Listing
December 2020

Therapeutic applications and delivery systems for triptolide.

Drug Deliv Transl Res 2020 12;10(6):1584-1600

School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil.

Triptolide (TPL) is a natural compound and active component of Tripterygium wilfordii Hook F., an Asian native woody vine widely used for over 200 years in Chinese medicine. Hot water, ethanol-ethyl acetate, and chloroform-methanol extracts are the first reported TPL preparations in the literature, and since then, several studies for application in inflammation processes and cancer are described due to the antitumor, anti-inflammatory, and immunosuppressive characteristics of the molecule. However, physicochemical properties such as poor solubility and bioavailability are the main concerns regarding the TPL safety and efficacy in clinical studies since trials have reported adverse side effects alongside the excellent TPL therapeutic effects. Here, we review the main TPL applications and issues related to the drug usage, and a comprehensive summary of diseases is provided. Special emphasis is given to drug delivery systems designed to overcome the TPL physicochemical characteristics such as poor drug solubility, and how to increase efficacy and obtain a safe drug profile. Graphical abstract.
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http://dx.doi.org/10.1007/s13346-020-00827-zDOI Listing
December 2020

Topical delivery of siRNA into skin using ionic liquids.

J Control Release 2020 07 24;323:475-482. Epub 2020 Apr 24.

John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA; Wyss Institute for Biologically Inspired Engineering, 52 Oxford St, Cambridge, MA 02138, USA. Electronic address:

Skin diseases such as lupus, cancer, psoriasis, and hyperhidrosis can potentially be treated effectively by suppressing allele-specific genes using small interfering RNA (siRNA). Injections of siRNA into skin, though effective, are painful and cover small surface areas and thus are not suitable as a long-term treatment option. Topical delivery of siRNA is an attractive alternative option to mediate RNA interference (RNAi). However, the barrier function of the epidermis impedes effective permeation of siRNA into the skin. Herein, we describe topical delivery of siRNA using ionic liquids (ILs) capable of complexing with siRNA non-covalently and delivering it effectively. Using complementary and synergistic strategies of ionic liquids, we report delivery of effective doses of siRNA into skin. The first strategy involved the use of hydrophobic cations to robe the siRNA and the second strategy involved the use of choline-geranic acid ionic liquid (CAGE) to enhance its dermal penetration. In vitro studies in porcine skin confirmed the synergistic effect of these strategies in enhancing epidermal and dermal penetration. In vivo application of siRNA formulation to SKH-1E hairless mice significantly suppressed GAPDH expression with no clinical evidence of toxicity. This is a simple, personalized, and scalable platform for effective topical delivery of siRNA for treating genetic skin diseases.
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http://dx.doi.org/10.1016/j.jconrel.2020.04.038DOI Listing
July 2020

Microemulsion co-delivering vitamin A and vitamin E as a new platform for topical treatment of acute skin inflammation.

Mater Sci Eng C Mater Biol Appl 2020 May 7;110:110639. Epub 2020 Jan 7.

School of Pharmaceutical Sciences of Ribeirão Preto, Department of Pharmaceutical Sciences, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.

In this study, we developed a water-in-oil microemulsion containing vitamin A (retinol) and vitamin E (α-tocopherol), which serves as a multifunctional nanosystem that co-delivers antioxidants and displayed additive effect against acute skin inflammation. Microemulsion (ME) was prepared by mixing a surfactant blend (Tween 80 and propylene glycol, 5:1) with isopropyl myristate and water (ratio of 50:40:10, respectively). Vitamin A (0.05% w/w concentration) and/or vitamin E (0.1% w/w concentration) were incorporated into the surfactant mixture of ME by stirring with a magnetic stirrer for 30 min. This multifunctional ME displayed physical stability, with low cytotoxicity in 3T3 cell line, as well as cellular internalization into the cytosol. In vivo treatments using ME delivering α-tocopherol reduced dermal expression of TNF-α by 1.3-fold (p < 0.01), when compared to unloaded ME treatment group. When retinol was added into the ME containing α-tocopherol, it further reduced TNF-α expression by 2-fold (p < 0.001), suggesting the additive effect of vitamin E and vitamin A in the treatment against skin inflammation. In conclusion, we successfully developed the use of water-in-oil ME to pack both vitamin E and vitamin A, and demonstrated for the first time its anti-inflammatory potential when applied topically to TPA-induced inflamed skin.
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http://dx.doi.org/10.1016/j.msec.2020.110639DOI Listing
May 2020

Nanotechnology approaches in the current therapy of skin cancer.

Adv Drug Deliv Rev 2020 01 27;153:109-136. Epub 2020 Feb 27.

School of Pharmaceutical Sciences of Ribeirao Preto - University of Sao Paulo, Ribeirao Preto, SP, Brazil. Electronic address:

Skin cancer is a high burden disease with a high impact on global health. Conventional therapies have several drawbacks; thus, the development of effective therapies is required. In this context, nanotechnology approaches are an attractive strategy for cancer therapy because they enable the efficient delivery of drugs and other bioactive molecules to target tissues with low toxic effects. In this review, nanotechnological tools for skin cancer will be summarized and discussed. First, pathology and conventional therapies will be presented, followed by the challenges of skin cancer therapy. Then, the main features of developing efficient nanosystems will be discussed, and next, the most commonly used nanoparticles (NPs) described in the literature for skin cancer therapy will be presented. Subsequently, the use of NPs to deliver chemotherapeutics, immune and vaccine molecules and nucleic acids will be reviewed and discussed as will the combination of physical methods and NPs. Finally, multifunctional delivery systems to codeliver anticancer therapeutic agents containing or not surface functionalization will be summarized.
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http://dx.doi.org/10.1016/j.addr.2020.02.005DOI Listing
January 2020

Nanostructured lipid carrier co-delivering tacrolimus and TNF-α siRNA as an innovate approach to psoriasis.

Drug Deliv Transl Res 2020 06;10(3):646-660

School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Av. do Café s/n, Ribeirao Preto, SP, 14040-903, Brazil.

Since psoriasis is an immuno-mediated skin disease, long-term therapies are necessary for its treatment. In clinical investigations, tacrolimus (TAC), a macrolide immunosuppressive inhibitor of calcineurin, arises as an alternative for the treatment of psoriasis, acting in some cytokines involved in the pathogenesis of the disease. Here, we aim to study the psoriasis treatment with TAC and siRNA for one of most cytokines expressed in psoriasis, the TNF-α. A multifunctional nanostructure lipid carrier (NLC) was developed to co-delivery TAC and siRNA. Results showed that the particle size and zeta potential were around 230 nm and + 10 mV, respectively. The release study demonstrated a controlled release of TAC, and the permeation and retention profile in the skin tissue show to be promising for topical application. The cell viability and uptake in murine fibroblast presented low toxicity associated to uptake of NLC in 4 h, and finally, the in vivo animal model demonstrates the efficiency of the NLC multifunctional, exhibiting a reduction of the cytokine TNF-α expression about 7-fold and presenting a synergic effect between the TAC and TNF-α siRNA. The developed system was successfully to treat in vivo psoriatic animal model induced by imiquimod and the synergic combination was reported here for the first time. Graphical abstract.
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http://dx.doi.org/10.1007/s13346-020-00723-6DOI Listing
June 2020

Advances in Specific Gene Knockdown of Therapeutic Targets by RNA Interference.

Curr Pharm Des 2018 ;24(23):2631

Professor of School of Pharmaceutical Sicences of Ribeirao Preto University of Sao Paulo, Brazil.

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http://dx.doi.org/10.2174/138161282423181024150201DOI Listing
October 2019

Current Non-viral siRNA Delivery Systems as a Promising Treatment of Skin Diseases.

Curr Pharm Des 2018 ;24(23):2644-2663

School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Avenida do Cafe, s/n, 14040903, Ribeirao Preto, SP, Brazil.

Background: Gene therapy is a new approach to discover and treat many diseases. It has attracted considerable attention from researchers in the last decades. The gene therapy through RNA interference has been considered one of the most recent and revolutionary approaches used in individualized therapy. In the last years, we have witnessed the rapid development in the field of the gene silencing and knockdown by topical siRNA. Its application in gene therapy has become an attractive alternative for drug development.

Methods: This article will address topical delivery of siRNA as a promising treatment for skin disorders. An update on the advances in siRNA-based nanocarriers as a powerful therapeutic strategy for several skin diseases will be discussed giving emphasis on in vitro evaluations.

Results: Through the in-depth review of the literature on the use of siRNAs for skin diseases we realize how widespread this use is. We have also realized that nanoparticles as non-viral vectors are increasingly being explored. Skin diseases where the use of siRNA has been explored most are skin cancer (melanoma and nonmelanoma), psoriasis, vitiligo, dermatitis and leprosy. But we also report here other diseases where the use of siRNA has been growing as acne, alopecia areata, cutaneous leishmaniasis, mycoses, herpes, epidermolysis bullosa and oculocutaneous albinism. Also highlighted, the first clinical trial of siRNA for cutaneous diseases, aimed at Pathyounychia Congenita.

Conclusion: The treatment of skin diseases based on topical delivery of siRNA, which act by inhibiting the expression of target transcripts, offers many potential therapeutic advantages for suppressing genes into the skin.
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http://dx.doi.org/10.2174/1381612824666180807120017DOI Listing
October 2019

In Vitro TyRP-1 Knockdown Based on siRNA Carried by Liquid Crystalline Nanodispersions: an Alternative Approach for Topical Treatment of Vitiligo.

Pharm Res 2018 Mar 20;35(5):104. Epub 2018 Mar 20.

School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Avenida do Café, s/n, Ribeirão Preto, SP, 14040-903, Brazil.

Purpose: Vitiligo is a skin disease characterized by depigmentation and the presence of white patches that are associated with the loss of melanocytes. The most common explanation for the cause of this condition is that it is an autoimmune condition. TyRP-1 is involved in melanin pigment synthesis but can also function as a melanocyte differentiation antigen. This protein plays a role in the autoimmune destruction of melanocytes, which results in the depigmentation, characteristic of this disease. In this study, we evaluated liquid crystalline nanodispersions as non-viral vectors to deliver siRNA-TyRP-1 as an alternative for topical treatment of vitiligo.

Methods: Liquid crystalline nanodispersions were obtained and characterized with respect to their physical-chemical parameters including size, PdI and zeta potential, as well as Small Angle X-ray Scattering and complexing to siRNA. The effects of the liquid crystalline nanodispersions on the cellular viability, cell uptake and levels of the knockdown target TyRP-1 were evaluated in melan-A cells after 24 h of treatment.

Results: The liquid crystalline nanodispersions demonstrated adequate physical-chemical parameters including nanometer size and a PdI below 0.38. These systems promoted a high rate of cell uptake and an impressive TyRP-1 target knockdown (> 80%) associated with suitable loading of TyRp-1 siRNA.

Conclusions: We demonstrated that the liquid crystalline nanodispersions showed promising alternative for the topical treatment of vitiligo due to their physical parameters and ability in knockdown the target protein involved with autoimmune destruction of melanocytes.
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http://dx.doi.org/10.1007/s11095-017-2330-0DOI Listing
March 2018

Ketoprofen Microemulsion for Improved Skin Delivery and In Vivo Anti-inflammatory Effect.

AAPS PharmSciTech 2017 Oct 3;18(7):2783-2791. Epub 2017 Apr 3.

Faculdades Integradas Padre Albino-FIPA, Rua dos Estudantes, 255, Catanduva, São Paulo, 15809-144, Brazil.

We have designed a microemulsion (ME) containing Ketoprofen (KET) for anti-inflammatory effect evaluated using the rat paw edema model. The ME was prepared by adding propylene glycol (PG) loaded with 1% KET/water (3:1, w/w), to a mixture of sorbitan monooleate and polysorbate 80 (47.0%) at 3:1 (w/w) and canola oil (38.0%). The physicochemical characterization of KET-loaded ME involved particle size and zeta potential determination, entrapment efficiency, calorimetric analysis, and in vitro drug release. The in vivo anti-inflammatory study employed male Wistar rats. Measurement of the foot volume was performed using a caliper immediately before and 2, 4, and 6 h after injection of Aerosil. KET-loaded ME showed particle size around 20 nm, with zeta potential at -16 mV and entrapment efficiency at 70%. Moreover, KET was converted to the amorphous state when loaded in the formulation and it was shown that the drug was slowly released from the ME. Finally, the in vivo biological activity was similar to that of the commercial gel, but ME better controlled edema at 4 h. These results demonstrated that the ME formulation is an alternative strategy for improving KET skin permeation for anti-inflammatory effect. Furthermore, our findings are promising considering that the developed ME was loaded with only 1% KET, and the formulation was able to keep a similar release profile and in vivo effect compared to the commercial gel with 2.5% KET. Therefore, the KET-loaded developed herein ME is likely to have a decreased side effect compared with that of the commercial gel, but both presented the same efficacy.
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http://dx.doi.org/10.1208/s12249-017-0749-6DOI Listing
October 2017

Development and characterization of novel 1-(1-Naphthyl)piperazine-loaded lipid vesicles for prevention of UV-induced skin inflammation.

Eur J Pharm Biopharm 2016 Jul 27;104:101-9. Epub 2016 Apr 27.

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal.

1-(1-Naphthyl)piperazine (1-NPZ) has shown promising effects by inhibiting UV radiation-induced immunosuppression. Ultradeformable vesicles are recent advantageous systems capable of improving the (trans)dermal drug delivery. The aim of this study was to investigate 1-NPZ-loaded transethosomes (NPZ-TE) and 1-NPZ-loaded vesicles containing dimethyl sulfoxide (NPZ-DM) as novel delivery nanosystems, and to uncover their chemopreventive effect against UV-induced acute inflammation. Their physicochemical properties were evaluated as follows: vesicles size and zeta potential by dynamic and electrophoretic light scattering, respectively; vesicle deformability by pressure driven transport; rheological behavior by measuring viscosity and I-NPZ entrapment yield by HPLC. In vitro topical delivery studies were performed in order to evaluate the permeation profile of both formulations, whereas in vivo studies sought to assess the photoprotective effect of the selected formulation on irradiated hairless mice by measuring myeloperoxidase activity and the secretion of proinflammatory cytokines. Either NPZ-TE or NPZ-DM exhibited positive results in terms of physicochemical properties. In vitro data revealed an improved permeation of 1-NPZ across pig ear skin, especially by NPZ-DM. In vivo studies demonstrated that NPZ-DM exposure was capable of preventing UVB-induced inflammation and blocking mediators of inflammation in mouse skin. The successful results here obtained encourage us to continue these studies for the management of inflammatory skin conditions that may lead to the development of skin cancers.
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http://dx.doi.org/10.1016/j.ejpb.2016.04.023DOI Listing
July 2016

Optimization of protoporphyrin IX skin delivery for topical photodynamic therapy: Nanodispersions of liquid-crystalline phase as nanocarriers.

Eur J Pharm Sci 2016 Feb 3;83:99-108. Epub 2015 Dec 3.

School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café, s/n, 14040-160, Ribeirão Preto, SP, Brazil. Electronic address:

Nanodispersions of liquid-crystalline phases (NLPs) composed of monoolein and oleic acid were chosen as nanocarriers to improve the topical retention of the photosensitizer protoporphyrin IX (PpIX) and thereby optimize photodynamic therapy (PDT) using this photosensitizer. The nanodispersions were characterized by polarized light microscopy, small-angle X-ray diffraction and dynamic light scattering. The stability and encapsulation efficiency (EE%) of the nanodispersions were also evaluated. In vitro and in vivo skin penetration studies were performed to determine the potential of the nanodispersions for cutaneous application. In addition, skin penetration and skin irritancy (in an animal model) after in vivo application were visualized by fluorescence light microscopy. The nanodispersion obtained was characterized as a monodisperse system (~150.0 nm) of hexagonal liquid-crystalline phase, which provided a high encapsulation efficiency of PpIX (~88%) that remained stable over 90 days of investigation. Skin penetration studies demonstrated that the nanodispersion enhanced PpIX skin uptake 11.8- and 3.3-fold (in vitro) and 23.6- and 20.8-fold (in vivo) compared to the PpIX skin uptake of control formulations, respectively. In addition, the hexagonal phase nanodispersion did not cause skin irritation after application for two consecutive days. Overall, the results show that the nanocarrier developed is suitable for use in topical PDT with PpIX.
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http://dx.doi.org/10.1016/j.ejps.2015.12.003DOI Listing
February 2016

Development, characterization, and skin delivery studies of related ultradeformable vesicles: transfersomes, ethosomes, and transethosomes.

Int J Nanomedicine 2015 18;10:5837-51. Epub 2015 Sep 18.

Instituto de Investigação do Medicamento (iMed.ULisboa), Universidade de Lisboa, Lisboa, Portugal.

Ultradeformable vesicles (UDV) have recently become a promising tool for the development of improved and innovative dermal and transdermal therapies. The aim of this work was to study three related UDV: transfersomes, ethosomes, and transethosomes for the incorporation of actives of distinct polarities, namely, vitamin E and caffeine, and to evaluate the effect of the carrier on skin permeation and penetration. These actives were incorporated in UDV formulations further characterized for vesicles imaging by transmission electron microscopy; mean vesicle size and polydispersity index by photon correlation spectroscopy; zeta potential by laser-Doppler anemometry; deformability by pressure-driven transport; and incorporation efficiency (IE) after actives quantification by high-performance liquid chromatography. Topical delivery studies were performed in order to compare UDV formulations regarding the release, skin permeation, and penetration profiles. All UDV formulations showed size values within the expected range, except transethosomes prepared by "transfersomal method", for which size was smaller than 100 nm in contrast to that obtained for vesicles prepared by "ethosomal method". Zeta potential was negative and higher for formulations containing sodium cholate. The IE was much higher for vitamin E- than caffeine-loaded UDV as expected. For flux measurements, the following order was obtained: transethosomes (TE) > ethosomes (E) ≥ transfersomes (T). This result was consistent with the release and skin penetration profiles for Vitamin E-loaded UDV. However, the releasing results were totally the opposite for caffeine-loaded UDV, which might be explained by the solubility and thermodynamic activity of this active in each formulation instead of the UDV deformability attending to the higher non-incorporated fraction of caffeine. Anyway, a high skin penetration and permeation for all caffeine-loaded UDV were obtained. Transethosomes were more deformable than ethosomes and transfersomes due to the presence of both ethanol and surfactant in their composition. All these UDV were suitable for a deeper skin penetration, especially transethosomes.
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http://dx.doi.org/10.2147/IJN.S86186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4583114PMC
August 2016

Quantification of lipoic acid from skin samples by HPLC using ultraviolet, electrochemical and evaporative light scattering detectors.

J Chromatogr B Analyt Technol Biomed Life Sci 2016 Apr 22;1019:66-71. Epub 2015 Jul 22.

School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Avenida do Café s/n, 14040-903, Ribeirão Preto, SP, Brazil. Electronic address:

Lipoic acid (LA) is an endogenous organosulfur compound with potent antioxidant property. LA is often used as a drug for the treatment of skin disorders. For the accomplishment of topical applications of LA appropriate drug quantification methods are essential. Thus far, no HPLC methods have been reported for the measurement of LA extracted from skin. In this article we report on the development and validation of three sensitive and specific HPLC methods for LA and dihydrolipoic acid (DHLA) using ultraviolet (UV), electrochemical (EC) or evaporative light scattering (ELS) detection. These methods demonstrate different linearity ranges. The chromatographic separations were performed by RP-HPLC (250 × 4 mm, 5 μm) with isocratic elution using an acidic mobile phase for the three detection techniques. The lower limits of detection and quantification were 0.04 and 0.08 ng LA, respectively, for HPLC coupled to ELS, an innovative detector for LA with high sensitivity. The extraction of LA from skin samples showed recoveries greater than 71%. The recovered LA concentrations from stratum corneum and epidermis+dermis layers were: 5.41 ± 0.56 and 4.92 ± 0.33 μg/mL, respectively for HPLC/UV and 6.52 ± 0.49 and 5.01 ± 0.41 μg/mL, respectively, for HPLC/EC for the added LA concentration (6.67 μg/mL), and 8.88 ± 0.46 and 8.95 ± 0.08 μg/mL, respectively, for HPLC/ELS for the added LA concentration (10 μg/mL). These three optimized HPLC methods allowed for a simple, rapid and reliable determination of LA in human skin. They should be useful for the development of drug delivery systems for topical applications of LA.
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http://dx.doi.org/10.1016/j.jchromb.2015.07.029DOI Listing
April 2016

Advances in the bioanalytical study of drug delivery across the skin.

Ther Deliv 2015 ;6(5):571-94

School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Avenida do Café, s/n, 14040903, Ribeirão Preto, SP, Brazil.

The study of a drug's dermal penetration profile provides important pharmaceutical data for the rational development of topical and transdermal delivery systems because the skin is a broadly used delivery route for local and systemic drugs and a potential route for gene therapy and vaccines. Monitoring drug penetration across the skin and quantifying its levels in different skin layers have been constant challenges due to the detection limitations of the available techniques, as well as the inherent interference in this tissue. This review explores and discusses several bionalytical methods that are indispensable tools to study drugs across the skin. In addressing the main topic, we structure the review highlighting the skin as an important route of drug administration and its structure, skin membrane models most used and its properties, in vitro and in vivo assays most used in the study of drug delivery to the skin, the techniques for processing the skin for subsequent analysis by bioanalytical methods that have a theoretical and practical approach showing its applicability, limitations and also including examples of its use. This review has a comprehensive approach in order to help researchers design their experiments and update the applicability and advances in this area of expertise.
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http://dx.doi.org/10.4155/tde.15.20DOI Listing
August 2016

An in situ gelling liquid crystalline system based on monoglycerides and polyethylenimine for local delivery of siRNAs.

Eur J Pharm Sci 2015 Jul 25;74:103-17. Epub 2015 Apr 25.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café, s/n, 14040-903 Ribeirão Preto, SP, Brazil. Electronic address:

The development of delivery systems able to complex and release siRNA into the cytosol is essential for therapeutic use of siRNA. Among the delivery systems, local delivery has advantages over systemic administration. In this study, we developed and characterized non-viral carriers to deliver siRNA locally, based on polyethylenimine (PEI) as gene carrier, and a self-assembling drug delivery system that forms a gel in situ. Liquid crystalline formulations composed of monoglycerides (MO), PEI, propylene glycol (PG) and 0.1M Tris buffer pH 6.5 were developed and characterized by polarized light microscopy, Small Angle X-ray Scattering (SAXS), for their ability to form inverted type liquid crystalline phases (LC2) in contact with excess water, water absorption capacity, ability to complex with siRNA and siRNA release. In addition, gel formation in vivo was determined by subcutaneous injection of the formulations in mice. In water excess, precursor fluid formulations rapidly transformed into a viscous liquid crystalline phase. The presence of PEI influences the liquid crystalline structure of the LC2 formed and was crucial for complexing siRNA. The siRNA was released from the crystalline phase complexed with PEI. The release rate was dependent on the rate of water uptake. The formulation containing MO/PEI/PG/Tris buffer at 7.85:0.65:76.5:15 (w/w/w/w) complexed with 10 μM of siRNA, characterized as a mixture of cubic phase (diamond-type) and inverted hexagonal phase (after contact with excess water), showed sustained release for 7 days in vitro. In mice, in situ gel formation occurred after subcutaneous injection of the formulations, and the gels were degraded in 30 days. Initially a mild inflammatory process occurred in the tissue surrounding the gel; but after 14 days the tissue appeared normal. Taken together, this work demonstrates the rational development of an in situ gelling formulation for local release of siRNA.
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http://dx.doi.org/10.1016/j.ejps.2015.04.017DOI Listing
July 2015

In vitro and in vivo topical delivery studies of tretinoin-loaded ultradeformable vesicles.

Eur J Pharm Biopharm 2014 Sep 20;88(1):48-55. Epub 2014 May 20.

Instituto de Investigação do Medicamento (iMed.ULisboa), Universidade de Lisboa, Lisboa, Portugal.

Introduction: Ultradeformable vesicles are highly promising tools to enhance the percutaneous transport of different drugs such as tretinoin across the skin barrier and also to increase the formulation stability at absorption site and reduce the drug induced irritation.

Methods: Topical delivery of tretinoin-loaded ultradeformable vesicles (tretinoin-UDV) was evaluated concerning different studies, such as: the release and permeation profiles (tape stripping); skin penetration (fluorescence analysis); induced electrical changes in skin barrier properties; cytotoxicity (Trypan Blue assay) and skin irritation in in vivo conditions (Draize test). The novel formulation performance was also compared to a commercial tretinoin formulation regarding in vivo studies.

Results: It was obtained a sustained and controlled drug release, as expected for UDV formulation. In addition, a dermal delivery was observed regarding the permeation study since it was not detected any drug amount in the receptor phase after 24h. Nile Red-UDV stained intensively mostly in the stratum corneum, corroborating the tape stripping results. Tretinoin-UDV decreased skin resistance, suggesting its ability to induce skin barrier disruption. Finally, the formulation vehicle (empty UDV) and tretinoin-UDV were not toxic under in vitro and in vivo conditions, at least, at 5×10(-3)mg/mL and 0.5mg/mL of tretinoin, respectively.

Conclusion: Tretinoin-UDV is a promising delivery system for tretinoin dermal delivery without promoting skin irritation (unlike other commercial formulations), which is quite advantageous for therapeutic purpose.
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http://dx.doi.org/10.1016/j.ejpb.2014.05.002DOI Listing
September 2014

Self-assembling gelling formulation based on a crystalline-phase liquid as a non-viral vector for siRNA delivery.

Eur J Pharm Sci 2014 Jul 12;58:72-82. Epub 2014 Apr 12.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café, s/n, 14040-903 Ribeirão Preto, SP, Brazil. Electronic address:

Liquid crystalline systems (LCSs) form interesting drug delivery systems. These include in situ gelling delivery systems, which present several advantages for use as self-assembling systems for local drug delivery. The aim of this study was to develop and characterize in situ gelling delivery systems for local siRNA delivery. The influence of the components that form the systems was investigated, and the systems were characterized by polarized light microscopy, Small Angle X-ray Scattering (SAXS), swelling studies, assays of their ability to form a complex with genes and of the stability of the genes in the system, as well as assays of in situ gelling formation and local toxicity using an animal model. The system containing a mixture of monoglycerides (MO), oleylamine (OAM), propylene glycol (PG) and tris buffer (8.16:0.34:76.5:15, w/w/w/w) was considered the most appropriate for local siRNA delivery purposes. The molecular structure was characterized as hexagonal phase; the swelling studies followed a second order kinetic model and the water absorption was a fast process reaching equilibrium at 2 h. The system formed a complex with siRNA and remained in a stable form. The gel was formed in vivo after subcutaneous administration of a precursor fluid formulation in mice and was biodegradable in 30 days. The inflammatory process that took place was considered normal. Therefore, the developed liquid crystalline delivery system shows the appropriate characteristics for use as a local siRNA delivery method for gene therapy.
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http://dx.doi.org/10.1016/j.ejps.2014.04.001DOI Listing
July 2014

PLGA nanoparticles as delivery systems for protoporphyrin IX in topical PDT: cutaneous penetration of photosensitizer observed by fluorescence microscopy.

J Nanosci Nanotechnol 2013 Oct;13(10):6533-40

School of Pharmacy, Department of Medicine, Federal University of Rio de Janeiro, Av Carlos Chagas Filho 373, 21.941.902, Rio de Janeiro, RJ, Brazil.

Poly(D,L lactic-co-glycolic acid) (PLGA) based nanoparticles (NPs) are proposed for topical delivery of Protoporphyrin IX (PpIX) in Photodynamic Therapy of skin cancers. PpIX loaded into PLGA NPs showed nanometric average diameter (-280 nm), spherical forms and pH - 5.7, conditions suitable for topical application. In vitro release of PpIX from NPs was sustained up to 24 hr with a burst release effect of about 37.0% at 2 hr. Penetration and distribution of PpIX in hairless mice skin was determined by fluorescence microscopy 8 or 24 hrs after application of PpIX-NPs in the animals. At 24 hours, areas located in deeper regions of the skin were found to have greater fluorescence intensity. The finding indicates a localized effect of PpIX-NPs in the epidermis plus dermis--a site of action for topical PDT--and suggests a potential use of PpIX-NPs in PDT associated to skin cancer treatments.
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http://dx.doi.org/10.1166/jnn.2013.7789DOI Listing
October 2013

Lycopene from tomatoes: vesicular nanocarrier formulations for dermal delivery.

J Agric Food Chem 2013 Jul 17;61(30):7284-93. Epub 2013 Jul 17.

Nanomedicine and Drug Delivery Systems group of iMedUL, Faculdade de Farmácia da Universidade de Lisboa, Lisboa, Portugal.

This experimental work aimed to develop a simple, fast, economic, and environmentally friendly process for the extraction of lycopene from tomato and incorporate this lycopene-rich extract into ultradeformable vesicular nanocarriers suitable for topical application. Lycopene extraction was conducted without a cosolvent for 30 min. The extracts were analyzed and incorporated in transfersomes and ethosomes. These formulations were characterized, and the cellular uptake was observed by confocal microscopy. Dermal delivery of lycopene formulations was tested under in vitro and in vivo conditions. Lycopene extraction proved to be quite safe and selective. The vesicular formulation was taken up by the cells, being more concentrated around the nucleus. Epicutaneous application of lycopene formulations decreased the level of anthralin-induced ear swelling by 97 and 87%, in a manner nonstatistically different from the positive control. These results support the idea that the lycopene-rich extract may be a good alternative to the expensive commercial lycopene for incorporation into advanced topical delivery systems.
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http://dx.doi.org/10.1021/jf401368wDOI Listing
July 2013

Delivery systems and local administration routes for therapeutic siRNA.

Pharm Res 2013 Apr 24;30(4):915-31. Epub 2013 Jan 24.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café, s/n, 14040-903, Ribeirão Preto, SP, Brazil.

With 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.
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http://dx.doi.org/10.1007/s11095-013-0971-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7088712PMC
April 2013

Liquid crystalline phase nanodispersions enable skin delivery of siRNA.

Eur J Pharm Biopharm 2013 Jan 23;83(1):16-24. Epub 2012 Sep 23.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.

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.
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http://dx.doi.org/10.1016/j.ejpb.2012.08.011DOI Listing
January 2013

A Validated Reverse Phase HPLC Analytical Method for Quantitation of Glycoalkaloids in Solanum lycocarpum and Its Extracts.

J Anal Methods Chem 2012 27;2012:947836. Epub 2012 Mar 27.

Laboratório de Farmacognosia, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café, s/n, 14040-903 Ribeirão Preto, SP, Brazil.

Solanum lycocarpum (Solanaceae) is native to the Brazilian Cerrado. Fruits of this species contain the glycoalkaloids solasonine (SN) and solamargine (SM), which display antiparasitic and anticancer properties. A method has been developed for the extraction and HPLC-UV analysis of the SN and SM in different parts of S. lycocarpum, mainly comprising ripe and unripe fruits, leaf, and stem. This analytical method was validated and gave good detection response with linearity over a dynamic range of 0.77-1000.00 μg mL(-1) and recovery in the range of 80.92-91.71%, allowing a reliable quantitation of the target compounds. Unripe fruits displayed higher concentrations of glycoalkaloids (1.04% ± 0.01 of SN and 0.69% ± 0.00 of SM) than the ripe fruits (0.83% ± 0.02 of SN and 0.60% ± 0.01 of SM). Quantitation of glycoalkaloids in the alkaloidic extract gave 45.09% ± 1.14 of SN and 44.37% ± 0.60 of SM, respectively.
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http://dx.doi.org/10.1155/2012/947836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3335309PMC
August 2012

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.

J Control Release 2011 Nov 7;155(3):400-8. Epub 2011 Jul 7.

Universidade de São Paulo, Ribeirão Preto, SP, Brazil.

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 compound's 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.
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http://dx.doi.org/10.1016/j.jconrel.2011.06.034DOI Listing
November 2011

In vitro evaluation of the antioxidant activity of liposomal flavonols by the HRP-H2O2-luminol system.

J Microencapsul 2011 ;28(4):258-67

Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Monte Alegre, Ribeirão Preto, SP, Brazil.

Considering that antioxidant flavonols have been reported to be beneficial to human health, but that their low water solubility and bioavailability limit their administration through systemic route, the development of suitable flavonol-carriers is of great importance for clinical therapeutics. The aim of this study was to prepare liposomes containing flavonols or not and evaluate their antioxidant activity. Vesicles were obtained by ethanol injection method and characterized in terms of entrapment efficiency, size and zeta potential. Inhibitory activity of liposomal flavonols on reactive oxygen species generation was assessed in vitro using luminol-H(2)O(2)-horseradish peroxidase technique. Antioxidant activity of liposomal flavonols is dependent on concentration and chemical structure of active compound. Quercetin and myricetin are the most active flavonols (IC(50) = 0.6-0.9 µmol/L), followed by kaempferol (IC(50) = 3.0-4.5 µmol/L) and galangin (IC(50) = 4.0-7.0 µmol/L). Our results suggest that antioxidant-loaded liposomes may be promising tools for therapy of diseases where oxidative stress is involved.
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http://dx.doi.org/10.3109/02652048.2011.559283DOI Listing
August 2011

Analysis of liquid crystalline nanoparticles by small angle X-ray diffraction: evaluation of drug and pharmaceutical additives influence on the internal structure.

J Pharm Sci 2011 Jul 18;100(7):2849-57. Epub 2011 Feb 18.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av do Café, s/n, 14040-903, Ribeirão Preto, SP, Brazil.

The goal of this work was to study the liquid crystalline structure of a nanodispersion delivery system intended to be used in photodynamic therapy after loading with photosensitizers (PSs) and additives such as preservatives and thickening polymers. Polarized light microscopy and light scattering were performed on a standard nanodispersion in order to determine the anisotropy of the liquid crystalline structure and the mean diameter of the nanoparticles, respectively. Small angle X-ray diffraction (SAXRD) was used to verify the influence of drug loading and additives on the liquid crystalline structure of the nanodispersions. The samples, before and after the addition of PSs and additives, were stable over 90 days, as verified by dynamic light scattering. SAXRD revealed that despite the alteration observed in some of the samples analyzed in the presence of photosensitizing drugs and additives, the hexagonal phase still remained in the crystalline phase.
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http://dx.doi.org/10.1002/jps.22522DOI Listing
July 2011

Celecoxib determination in different layers of skin by a newly developed and validated HPLC-UV method.

Biomed Chromatogr 2011 Nov 10;25(11):1237-44. Epub 2011 Feb 10.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café, s/n, Ribeirão Preto, SP, Brazil.

A simple, rapid and sensitive analytical procedure for the measurement of celecoxib (CXB) levels in skin samples after in vitro penetration studies was developed and validated. In vitro permeability studies in porcine skin were performed for quantification of CXB at different layers of skin, the stratum corneum (SC) and epidermis plus dermis (EP + D) as well as in the acceptor solution (AS) to assess CXB permeation through skin. CXB was quantified by HPLC using a C18 column and UV detection at 251 nm. The mobile phase was methanol-water 72:28 (v/v) and the flow-rate was 0.8 mL/min. The CXB retention time was 5 min. The assay was linear for CBX in the concentration range of 0.1-3.0 μg/mL in the AS (drug permeated through skin) and 5.0-50.0 μg/mL for drug retained in SC and [EP + D] in vitro. The linear correlation coefficients for the different calibration curves were equal or greater than 0.99. Intra- and inter-assay variabilities were below 8.0%. Extraction of CXB from skin samples showed recoveries higher than 95.0% after 15 min of ultrasonic sound and centrifugation at 2500 rpm for 3 min. The method was considered appropriate for the assay of CXB in skin samples, after in vitro cutaneous penetration studies.
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http://dx.doi.org/10.1002/bmc.1596DOI Listing
November 2011