Skin Delivery of Kojic Acid-Loaded Nanotechnology-Based Drug Delivery Systems for the Treatment of Skin Aging

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mucoadhesive beads of gellan gum/pectin intended to controlled delivery of drugs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Therapeutic Delivery of miR-200c Enhances Radiosensitivity in Lung Cancer

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mucoadhesive Amphiphilic Methacrylic Copolymer-Functionalized Poly(epsilon-caprolactone) Nanocapsules for Nose-to-Brain Delivery of Olanzapine

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Development and characterization of a biocompatible soybean oil-based microemulsion for the delivery of poorly water-soluble drugs

The aim of this work was the development and characterization of a biocompatible microemulsion (ME) containing soybean oil (O), phosphatidylcholine/sodium oleate/Eumulgin®HRE40 as the surfactant mixture (S) and water or buffer solution as the aqueous phase (W), for oral delivery of the poorly water-soluble drugs sulfamerazine (SMR) and indomethacin (INM). A wide range of combinations to obtain clear oil-in-water (o/w) ME was observed from pseudo-ternary phase diagrams, which was greater after the incorporation of both drugs, suggesting that they acted as stabilizers. Drug partition studies indicated a lower affinity of the drugs for the oil domain when they were ionized and with increased temperature, explained by the fact that both drugs were introduced inside the oil domain, determined by nuclear magnetic resonance. High concentrations of SMR and INM were able to be incorporated (22.0 and 62.3 mg/mL, respectively). The ME obtained presented an average droplet size of 100 nm and a negative surface charge. A significant increase in the release of SMR was observed with the ME with the highest percentage of O, because of the solubilizing properties of the ME. Also, a small retention effect was observed for INM, which may be explained by the differences in the partitioning properties of the drugs. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3535-3543, 2015.

Development and in vitro skin delivery of dexamethasone acetate-loaded surfactant-based systems

Topical corticosteroids, e.g., dexamethasone acetate (DMA), are extensively used to treat cutaneous inflammatory disorders even though their use is correlated with potential local and systemic side effects. The objective of this study was to develop and test the topical delivery of DMA-loaded surfactant based systems in vitro; these studies could guarantee a suitable delivery and therapeutic efficacy, as well as minimize DMA's side effects. A phase diagram was constructed using polyoxypropylene (5) polyoxyethylene (20) cetyl alcohol as the surfactant (S), isopropyl myristate as the oil phase (O) and water (W). The systems were characterized using polarization light microscopy (PLM), as well as rheological and small angle X-ray scattering (SAXS) measurements. Depending on the concentration of the constituents, it was possible to obtain microemulsions (MEs) and liquid crystalline mesophases (lamellar and hexagonal). These types of arrangement were verified using PLM measurements. The SAXS results revealed that increasing the W/S ratio led to ME, as well as lamellar (LAM) and hexagonal (HEX) arrangements. The MEs displayed typical Newtonian behavior while the LAM and HEX phases exhibited pseudoplasticity and plasticity, respectively. The MEs displayed excellent drug solubilization that was approximately 10-fold higher than was observed with the individual components. The in vitro cutaneous permeation studies using pig ear skin and analysis of the mechanical parameters (hardness, compressibility, cohesiveness and adhesiveness) were carried out with a HEX phase and O/W emulsion. The HEX phase achieved better drug permeation and retention in the skin while its mechanical properties were suitable for skin administration. PPG-5-CETETH-20-based systems may be a promising platform delivering DMA and other topical corticosteroids through the skin.

Development and analysis of formulations to the delivery of caffeine for the treatment of gynoid lipodystrophy

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Liquid Crystal Nanodispersions Enable the Cutaneous Delivery of Photosensitizer for Topical PDT: Fluorescence Microscopy Study of Skin Penetration

Topical photodynamic therapy (PDT) has been applied to almost all types of nonmelanoma skin cancer and numerous superficial benign skin disorders. Strategies to improve the accumulation of photosensitizer in the skin have been studied in recent years. Although the hydrophilic phthalocyanine zinc compound, zinc phthalocyanine tetrasulfonate (ZnPcSO4) has shown high photodynamic efficiency and reduced phototoxic side effects in the treatment of brain tumors and eye conditions, its use in topical skin treatment is currently limited by its poor skin penetration. In this study, nanodispersions of monoolein (MO)-based liquid crystalline phases were studied for their ability to increase ZnPcSO4 uptake by the skin. Lamellar, hexagonal and cubic crystalline phases were prepared and identified by polarizing light microscopy, and the nanodispersions were analyzed by dynamic light scattering. In vitro skin penetration studies were performed using a Franz's cell apparatus, and the skin uptake was evaluated in vivo in hairless mice. Aqueous dispersions of cubic and hexagonal phases showed particles of nanometer size, approximately 224 +/- 10 nm and 188 +/- 10 nm, respectively. In vitro skin retention experiments revealed higher fluorescence from the ZnPcSO4 in deeper skin layers when this photosensitizer was loaded in the hexagonal nanodispersion system when compared to both the cubic phase nanoparticles and the bulk crystalline phases (lamellar, cubic and hexagonal). The hexagonal nanodispersion showed a similar penetration behavior in animal tests. These results are important findings, suggesting the development of MO liquid crystal nanodispersions as potential delivery systems to enhance the efficacy of topical PDT.

In Vitro Characterization of Chitosan Gels for Buccal Delivery of Celecoxib: Influence of a Penetration Enhancer

Celecoxib (Cx) shows high efficacy in the treatment of osteoarthritis and rheumatoid arthritis as a result of its high specificity for COX-2, without gastrolesivity or interference with platelet function at therapeutic concentrations. Besides of anti-inflammatory effects, Cx also has a potential role for oral cancer chemoprevention. For these conditions, oral administration in long-term treatment is a concern due to its systemic side effects. However, local application at the site of injury (e.g., buccal inflammation conditions or chemoprevention of oral cancer) is a promising way to reduce its toxicity. In this study, the in vitro characterization of mucoadhesive chitosan (CHT) gels associated to AzoneA (R) was assessed to explore the potential buccal mucosal administration of Cx in this tissue. Rheological properties of gels were analyzed by a rheometer with cone-plate geometry. In vitro Cx release and permeability studies used artificial membranes and pig cheek mucosa, respectively. Mucoadhesion were measured with a universal test machine. CHT gels (3.0%) containing 2.0% or 3.0% Az showed more appropriate characteristics compared to the others: pH values, rheology, higher amount of Cx retained in the mucosa, and minimal permeation through mucosa, besides the highest mucoadhesion values, ideal for buccal application. Moreover, the flux (J) and amounts of drug released decreased with increased CHT and Az concentrations. CHT gels (3.0%) associated with 2.0% or 3.0% Az may be considered potential delivery systems for buccal administration of Cx.

Stratum corneum lipids liposomes for the topical delivery of 5-aminolevulinic acid in photodynamic therapy of skin cancer: preparation and in vitro permeation study

Abstract Background Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) is a skin cancer therapy that still has limitations due to the low penetration of this drug into the skin. We have proposed in this work a delivery system for 5-ALA based on liposomes having lipid composition similar to the mammalian stratum corneum (SCLLs) in order to optimize its skin delivery in Photodynamic Therapy (PDT) of skin cancers. Methods SCLLs were obtained by reverse phase evaporation technique and size distribution of the vesicles was determinated by photon correlation spectroscopy. In vitro permeation profile was characterized using hairless mouse skin mounted in modified Franz diffusion cell. Results Size exclusion chromatography on gel filtration confirmed vesicle formation. SCLLs obtained by presented a degree of encapsulation of 5-ALA around 5.7%. A distribution of vesicle size centering at around 500 nm and 400 nm respectively for SCLLs and SCLLs containing 5-ALA was found. In vitro 5-ALA permeation study showed that SCLLs preparations presented higher skin retention significantly (p < 0.05) on the epidermis without SC + dermis, with a decreasing of skin permeation compared to aqueous solution. Conclusions The in vitro delivery performance provided by SCLLs lead to consider this systems adequate for the 5-ALA-PDT of skin cancer, since SCLLs have delivered 5-ALA to the target skin layers (viable epidermis + dermis) to be treated by topical PDT of skin cancer.

Cationic nanoparticles for delivery of amphotericin B: preparation, characterization and activity in vitro

Abstract Background Particulate systems are well known to be able to deliver drugs with high efficiency and fewer adverse side effects, possibly by endocytosis of the drug carriers. On the other hand, cationic compounds and assemblies exhibit a general antimicrobial action. In this work, cationic nanoparticles built from drug, cationic lipid and polyelectrolytes are shown to be excellent and active carriers of amphotericin B against C. albicans. Results Assemblies of amphotericin B and cationic lipid at extreme drug to lipid molar ratios were wrapped by polyelectrolytes forming cationic nanoparticles of high colloid stability and fungicidal activity against Candida albicans. Experimental strategy involved dynamic light scattering for particle sizing, zeta-potential analysis, colloid stability, determination of AmB aggregation state by optical spectra and determination of activity against Candida albicans in vitro from cfu countings. Conclusion Novel and effective cationic particles delivered amphotericin B to C. albicans in vitro with optimal efficiency seldom achieved from drug, cationic lipid or cationic polyelectrolyte in separate. The multiple assembly of antibiotic, cationic lipid and cationic polyelctrolyte, consecutively nanostructured in each particle produced a strategical and effective attack against the fungus cells.

Evaluation of pyroptosis in macrophages using cytosolic delivery of purified flagellin

Pyroptosis is a molecularly controlled form of cell death that exhibits some features of apoptosis as well of necrosis. Pyroptosis is induced by inflammasome-activated caspase-1 or caspase-11 (caspase-4 in humans), as a result of distinct pathogenic or damage stimuli. Although pyroptosis displays some morphological and biochemical features of apoptosis, it has an inflammatory outcome due to the loss of plasma membrane integrity and the consequent release of intracellular contents, reminiscent to necrosis. Here, we use cytosolic delivery of purified flagellin as an experimental tool to trigger pyroptosis and describe potential methods to study this form of cell death. Finally, we discuss the advantages and limitations of these methods

The EU and Dispute Settlement within the WTO: a strategy for the protection of the Union’s autonomy in the domestic regulation of goods?

Oggetto della ricerca è l’accertamento dell’esistenza, nonché la definizione, della strategia dall’UE in materia di controversie commerciali aventi ad oggetto l’interpretazione e l’applicazione di norme facenti capo agli Accordi OMC in materia di misure sanitarie e di barriere tecniche al commercio. Nella prima parte della tesi, si ricostruiscono gli obbiettivi perseguiti dall’UE in materia di controversie SPS e TBT. In questo contesto, un’importanza di primo piano è attribuita alla difesa dell’autonomia regolamentare dell’Unione. Ad essa si riconduce la prassi UE finalizzata a prevenire il sorgere di controversie sul piano bilaterale attraverso la conclusione di accordi di mutuo riconoscimento, la cui portata ella sottolinea essere tuttavia limitata. L’analisi di cinque controversie sorte in ambito OMC di cui l’Unione è o è stata parte convenuta e che si fondano su presunte o accertate violazioni delle norme facenti capo ai due accordi menzionati consente di classificare gli argomenti giuridici avanzati dall’Unione nel contesto di tali controversie. Nella seconda parte della ricerca, la candidata identifica i mezzi a servizio della strategia UE, in primo luogo, attraverso l’analisi del quadro giuridico relativo alla partecipazione dell’Unione e degli Stati Membri al sistema OMC di risoluzione delle controversie; in secondo luogo, attraverso lo studio, da un lato, dello status delle norme OMC nell’ordinamento UE e, dall’altro, degli effetti delle pronunce dell’Organo di Risoluzione delle Controversie e della questione della responsabilità dell’Unione per violazione del diritto OMC. Sulla base del lavoro di ricerca svolto, si conclude che una strategia dell’UE esiste nella misura in cui l’Unione persegue l’obbiettivo di preservare la propria autonomia regolamentare attraverso, anche se non esclusivamente, gli strumenti afferenti all’ordine giuridico interno analizzati nella seconda parte. La candidata conclude altresì che la riforma del diritto delle relazioni esterne operata dal Trattato di Lisbona può indurre un cambiamento di tale strategia.

Spectroscopic studies on Cyclodextrin and Metal Organic Framework based potential nanovectors for delivery of Anticancer and Antiviral drugs

The aim of this work is to contribute to the development of new multifunctional nanocarriers for improved encapsulation and delivery of anticancer and antiviral drugs. The work focused on water soluble and biocompatible oligosaccharides, the cyclodextrins (CyDs), and a new family of nanostructured, biodegradable carrier materials made of porous metal-organic frameworks (nanoMOFs). The drugs of choice were the anticancer doxorubicin (DOX), azidothymidine (AZT) and its phosphate derivatives and artemisinin (ART). DOX possesses a pharmacological drawback due to its self-aggregation tendency in water. The non covalent binding of DOX to a series of CyD derivatives, such as g-CyD, an epichlorohydrin crosslinked b-CyD polymer (pb-CyD) and a citric acid crosslinked g-CyD polymer (pg-CyD) was studied by UV visible absorption, circular dichroism and fluorescence. Multivariate global analysis of multiwavelength data from spectroscopic titrations allowed identification and characterization of the stable complexes. pg-CyD proved to be the best carrier showing both high association constants and ability to monomerize DOX. AZT is an important antiretroviral drug. The active form is AZT-triphosphate (AZT-TP), formed in metabolic paths of low efficiency. Direct administration of AZT-TP is limited by its poor stability in biological media. So the development of suitable carriers is highly important. In this context we studied the binding of some phosphorilated derivatives to nanoMOFs by spectroscopic methods. The results obtained with iron(III)-trimesate nanoMOFs allowed to prove that the binding of these drugs mainly occurs by strong iono-covalent bonds to iron(III) centers. On the basis of these and other results obtained in partner laboratories, it was possible to propose this highly versatile and “green” carrier system for delivery of phosphorylated nucleoside analogues. The interaction of DOX with nanoMOFs was also studied. Finally the binding of the antimalarial drug, artemisinin (ART) with two cyclodextrin-based carriers,the pb-CyD and a light responsive bis(b-CyD) host, was also studied.

Role of αvβ3 – Integrin and TLR2 in the innate response to Herpes Simplex Virus infection and Delivery of retargeted oncolytic Herpes Simplex via carrier cells

In the first part of my thesis I studied the mechanism of initiation of the innate response to HSV-1. Innate immune response is the first line of defense set up by the cell to counteract pathogens infection and it is elicited by the activation of a number of membrane or intracellular receptors and sensors, collectively indicated as PRRs, Patter Recognition Receptors. We reported that the HSV pathogen-associated molecular patterns (PAMP) that activate Toll-like receptor 2 (TLR2) and lead to the initiation of innate response are the virion glycoproteins gH/gL and gB, which constitute the conserved fusion core apparatus across the Herpesvirus. Specifically gH/gL is sufficient to initiate a signaling cascade which leads to NF-κB activation. Then, by gain and loss-of-function approaches, we found that αvβ3-integrin is a sensor of and plays a crucial role in the innate defense against HSV-1. We showed that αvβ3-integrin signals through a pathway that concurs with TLR2, affects activation/induction of interferons type 1, NF-κB, and a polarized set of cytokines and receptors. Thus, we demonstrated that gH/gL is sufficient to induce IFN1 and NF-κB via this pathway. From these data, we proposed that αvβ3-integrin is considered a class of non-TLR pattern recognition receptors. In the second part of my thesis I studied the capacity of human mesenchymal stromal cells isolated by fetal membranes (FM-hMSCs) to be used as carrier cells for the delivery of retargeted R-LM249 virus. The use of systemically administrated carrier cells to deliver oncolytic viruses to tumoral targets is a promising strategy in oncolytic virotherapy. We observed that FM-hMSCs can be infected by R-LM249 and we optimized the infection condition; then we demonstrate that stromal cells sustain the replication of retargeted R-LM249 and spread it to target tumoral cells. From these preliminary data FM-hMSCs resulted suitable to be used as carrier cells