Les travaux du laboratoire ont montré que les filaments intermédiaires, qui forment un des trois éléments essentiels du cytosquelette, peuvent lier la tubuline sur des sites spécifiques appelés TBS (Tubulin-Binding Site). Certains peptides correspondant à ces séquences sont capables d'inhiber in vitro la polymérisation des microtubules (MT). Les travaux présentés dans cette thèse consistent à poursuivre la caractérisation structurale et fonctionnelle de ces peptides. Ainsi, il a été possible de montrer qu'un peptide issu de la vimentine, Vim-TBS.58-81, est capable de rentrer dans les cellules de glioblastome humain T98G et de se localiser au niveau nucléaire. Une fois couplé à un peptide proapoptotique agissant au niveau nucléaire, il est capable d'inhiber la prolifération de ces cellules. Un autre peptide issu de la sous-unité légère des neurofilaments, NFL-TBS.40-63, est capable de rentrer dans toutes les lignées de gliome testées jusque-là, de déstabiliser leur réseau de MT et d'inhiber leur prolifération et leur migration sans affecter les cellules saines du cerveau (astrocytes et neurones). Injecté par stéréotaxie en intra tumoral chez des rats porteurs d'un gliome F98, ce peptide ralentit la croissance de la tumeur et reste localisé dans le tissu tumoral. Une analyse structure/fonction de ce peptide a mis en évidence des structures secondaires de type feuillet β et hélice α. Après greffage à la surface de nanocapsules lipidiques (NCL), ce peptide permet également l'amélioration de leur entrée dans les cellules de gliome in vitro et in vivo. Enfin, des NCL contenant du Paclitaxel ou du Ferrociphénol et recouvertes de peptide NFL-TBS.40-63 se sont révélées plus efficaces dans l'inhibition de la croissance tumorale dans un modèle de souris porteuse d'un gliome GL261 et dans un modèle de rat porteur d'un gliome 9L respectivement. L'ensemble de ces travaux révèle de nouvelles fonctions de ciblage et de pénétration cellulaire pour des peptides issus de filaments intermédiaires.

This thesis details the development of ultra-short, single-walled carbon nanotubes (US-tubes) for use as nanocapsules to contain and deliver medical agents for both imaging (Gd3+ ) and therapeutic (211 At) purposes. In particular, Gd3+ -loaded US-tubes, known as gadonanotubes, operate as high-performance MRI contrast agents with relaxivities (image enhancement efficacy) a factor of 40-100 greater than current clinical contrast agents. Furthermore, gadonanotube relaxivities are highly pH-dependent, with image intensity nearly tripling from pH 7.5 to 6.8. Coupled with their high efficacies and targeting potential, these agents are promising candidates for next-generation targeted imaging probes for the early detection of cancer. Single gadonanotubes have also been encapsulated in a polymer shell for use as an intravenous MRI contrast agent. In addition, a new functionalization scheme has been developed to covalently attach a variety of amino acids in high quantity to the outer surface of the gadonanotubes and to attach a small peptide sequence for targeting breast cancer cells. The gadonanotubes have also been used as magnetic cell labeling agents, while also demonstrating efficacy in vivo as contrast agents. In addition to functioning as an imaging agent platform, the US-tubes have demonstrated efficacy as nanocapsules for radiotherapeutic agents. Astatine-211 (At-211), an α-emitting radionuclide, can be loaded inside the US-tube with excellent containment stability for the targeted delivery of an α-radiotherapeutic agent to micrometastatic and single-cell cancers. The loading levels for At-211 are comparable to, or better than, other known compounds. At-211, existing as the mixed-halogen compound 211 AtCl, is retained in the US-tube nanocapsules due to van der Waals forces between the 211AtCl and the interior sidewalls of the nanotube. Finally, the US-tubes have been shown to induce few health risks in mammalian experiments. Acute toxicity tests were conducted on mice with both raw and purified full-length carbon nanotubes (SWNTs), as well as US-tubes, using large doses (up to 1 g/kg of bodyweight). Even at these large doses, no animal death was recorded, although in a few cases behavioral changes were observed. Nanotubes were observed to be eliminated from the liver and kidneys through the urine and feces. It is believed that any toxicity at high doses can be attenuated (and prevented) by properly formulating the administered dose.

Η Πακλιταξέλη αποτελεί ένα από τα πιο ευρέως διαδεδομένα χημειοθεραπευτικά φάρμακα και ενδείκνυται κυρίως σε καρκίνο του πνεύμονα, του μαστού, των ωοθηκών, καθώς και σε σάρκωμα Kaposi ασθενών με AIDS. Ωστόσο, η χρήση της συνδέεται με αρκετές παρενέργειες, γεγονός που επέβαλε την τροποποίησή της σε Συστήματα Ελεγχόμενης Χορήγησης (DDS). Τα συγκεκριμένα συστήματα επιτρέπουν την εκλεκτική μεταφορά του φαρμάκου στον καρκινικό ιστό με μηχανισμούς παθητικής ή ενεργητικής στόχευσης, καθώς και τον έλεγχο του φαρμακοκινητικού προφίλ. Ιδιαίτερα ελπιδοφόρα φαίνεται να είναι η ενσωμάτωση της PTX σε πολυμερικές νανοκάψουλες. Η δομή πυρήνα - κελύφους των νανοκαψακίων καλύπτει τη χορήγηση μίας μεγάλης ποικιλίας ενεργών συστατικών, με το ενδιαφέρον να εστιάζεται σε αντινεοπλασματικά, αντιφλεγμονώδη, αντιικά και ανοσοκατασταλτικά. Η εισαγωγή των φαρμάκων στα νανοκαψάκια μπορεί να οδηγεί σε αύξηση της αποτελεσματικότητας και σταθερότητας των δραστικών ουσιών, καλύτερη in vivo συμπεριφορά και υψηλότερη ενδοκυτταρική πρόσληψη. Ορισμένες μελέτες προτείνουν την εγκαψακίωση νανοσωματιδίων οξειδίου του σιδήρου (SPIONs) σε συστήματα χορήγησης φαρμάκων, τα οποία έχουν αποδειχθεί πολύτιμα εργαλεία τόσο στον τομέα της διάγνωσης (μαγνητική τομογραφία πυρηνικού συντονισμού), όσο και της θεραπείας. Με εφαρμογή εξωτερικού εναλλασσόμενου μαγνητικού πεδίου καθίσταται εφικτή η συσσώρευση των μαγνητικών νανοφορέων στον ιστό - στόχο (μαγνητική στόχευση), ενώ η θεραπεία επέρχεται μέσω επαγωγής μαγνητικής υπερθερμίας. Η εν λόγω μεταπτυχιακή διατριβή πραγματεύεται την εγκαψακίωση της ΡΤΧ και των SPIONs σε πολυμερικές νανοκάψουλες, παρασκευασμένες από το συμπολυμερές πολυ(γαλακτικού οξέος) – πολυ(αιθυλενογλυκόλης) (PLA-PEG). Το PLA αποτελεί ένα βιοαποικοδομήσιμο πολυμερές, το οποίο δημιουργεί έναν πυρήνα, ικανό να εγκαψακιώσει και να προστατέψει υδρόφοβες ουσίες. Από την άλλη μεριά, το PEG είναι ένα υδρόφιλο, βιοσυμβατό πολυμερές, το οποίο προσδίδει στερεοχημική σταθεροποίηση στα συστήματα και παρατείνει το χρόνο κυκλοφορίας τους. Πιο συγκεκριμένα, παρασκευάστηκαν μαγνητικά νανοκαψάκια με συμπολυμερή PLA-PEG, διαφορετικού μοριακού βάρους, με τη μέθοδο της καθίζησης (nanoprecipitation). Οι μαγνητικοί νανοκρυσταλλίτες συντέθηκαν με την τεχνική της θερμικής αποικοδόμησης, ενώ ως πρόδρομη ένωση χρησιμοποιήθηκε ελαϊκός σίδηρος [Fe(Olate)3]. Διερευνήθηκαν διάφορες παράμετροι σχετικά με την σύνθεση (ποσότητα SPIONs, αναλογία πολυμερούς/ελαίου, ποσότητα φαρμάκου), έτσι ώστε να δημιουργηθούν βέλτιστοι νανοφορείς. Επιπλέον, εξετάστηκε η σταθερότητα των διασπορών τόσο στο χρόνο, όσο και στην παρουσία ηλεκτρολυτών (NaCl) με μέτρηση της υδροδυναμικής διαμέτρου και του επιφανειακού φορτίου, χρησιμοποιώντας την τεχνική της δυναμικής σκέδασης φωτός (DLS). Ακολούθησε μορφολογική εκτίμηση των νανοκαψακίων με τη βοήθεια ηλεκτρονικής μικροσκοπίας διάδοσης (ΤΕΜ) και δομικός χαρακτηρισμός τους μέσω φασματοσκοπίας FTIR. Σε επόμενο στάδιο, μετρήθηκε το ποσοστό φόρτωσης της ΡΤΧ με χρήση υγρής χρωματογραφίας (HPLC) και πραγματοποιήθηκαν μελέτες αποδέσμευσης, παρουσία και μη εξωτερικού εναλλασσόμενου μαγνητικού πεδίου, σε διάλυμα φωσφορικών (pH =7.4) στους 37 oC για 24 h. Τέλος, εξετάστηκε η κυτταροτοξικότητα των «φορτωμένων» (με ΡΤΧ) μαγνητικών νανοκαψακίων και συγκρίθηκε με εκείνη των «κενών» νανοφορέων, αλλά και του φαρμάκου. Ο έλεγχος αυτός πραγματοποιήθηκε με χρώση καρκινικών κυττάρων (σειρά Α549) από ιωδιούχο προπίδιο (ΡΙ) και μέτρηση του φθορισμού των νεκρών κυττάρων από κυτταρομετρία ροής. Τα αποτελέσματα ήταν ιδιαιτέρως ικανοποιητικά, καθώς οι νανοφορείς παρουσιάζουν σταθερότητα στο χρόνο (έλεγχος περιόδου ενός μηνός), αλλά και παρουσία ηλεκτρολυτών. Το ποσοστό φόρτωσης της ΡΤΧ υπολογίστηκε ~1.2 %, ενώ η απόδοση εγκαψακίωσης κυμαινόταν από 15 - 25 %. Επίσης, παρουσίασαν υψηλή απόδοση εγκαψακίωσης SPIONs (~65 %), εξαρτώμενη από την πολυμερική σύνθεση των νανοφορέων. Οι μελέτες απελευθέρωσης παρουσίασαν παρατεταμένη αποδέσμευση της πακλιταξέλης, η οποία αποκρίνεται σε παρουσία εξωτερικού μαγνητικού πεδίου. Τέλος, τα «κενά» (χωρίς φάρμακο) μαγνητικά νανοκαψάκια δεν παρουσίασαν κυτταροτοξικότητα, σε αντίθεση με τα «φορτωμένα» με φάρμακο, τα οποία προκάλεσαν μεγαλύτερη τοξικότητα σε καρκινικά κύτταρα και από το ελεύθερο φάρμακο. Τα παραπάνω αποτελέσματα δικαιολογούν την περαιτέρω διερεύνηση της πιθανής χρησιμοποίησης των PLA-PEG μαγνητικών νανοκαψακίων ως φορείς στοχευμένης χορήγησης της Πακλιταξέλης. / Paclitaxel is one of the most widely used chemotherapeutic drugs and is particularly suitable to lung, breast, ovarian cancer and AIDS - related Kaposi sarcoma. However, its use is associated with several side effects, certain of them attributable to the formulation used clinically today, which imposed its formulation in Targetable Drug Delivery Systems (TDDS). These systems allow selective delivery of drug in the tumor tissue by passive or active targeting mechanisms and control of the pharmacokinetic profile. Particularly promising appears to be the encapsulation of PTX in polymeric nanocapsules. The core - shell structure of the nanocapsules covers the delivery of a wide variety of active ingredients, with interest focused on anti-neoplastic, anti-inflammatory, antiviral and immunosuppressive. The nanoencapsulation of drugs can increase the efficiency and the stability of active substances, while allowing better in vivo behavior and higher intracellular uptake. Some studies suggest the encapsulation of iron oxide nanoparticles (SPIONs) in such systems, which have been proven valuable tools in both diagnosis (through nuclear magnetic resonance imaging) and therapy. The application of an external alternating magnetic field permits the accumulation of magnetic nanocarrier in target-tissue (magnetic targeting), while the treatment occurs by inducing magnetic hyperthermia. This thesis discusses the encapsulation of PTX and SPIONs in polymeric nanocapsules, prepared from biocompatible poly (lactic acid) - poly (ethylene glycol) (PLA-PEG) copolymers. The PLA is a biodegradable polymer, which generates a core capable to encapsulate and protect hydrophobic substances. On the other hand, the PEG is a hydrophilic, biocompatible polymer, which gives steric stabilization systems and prolong their circulation time after intravenous administration. Specifically, magnetic nanocapsules were synthesized with varying molecular weight of copolymers PLA-PEG through the nanoprecipitation method, while the magnetic nanoparticles were composed through thermal decomposition and oleate iron [Fe (Olate) 3] was used as a precursor. The optimum parameters were determined, conferring to the nanocapsules high stability. In addition, they were characterized with regard to their size and zeta potential at different salt concentrations (up to 2M NaCl) using dynamic light scattering (DLS). Then, the morphological characteristics of nanocapsules were evaluated by transmission electron microscopy (TEM) and their structural characterization by spectroscopy FTIR. In the next step, the Paclitaxel loading was determined with liquid chromatography (HPLC) and release studies were conducted in phosphate buffer (pH = 7.4) at 37 oC for 24 h, including triggered drug-release by application of AC magnetic field. Finally, the cytotoxicity of drug-loaded (with PTX) magnetic nanocapsules was tested and compared to that of blank (without drug) nanocarriers and free drug. For these studies the cancer cell line A549 was used and after their incubation with the nanocapsules or the free drug, the dead cells were stained by propidium iodide (PI) and measured by flow cytometry. The obtained results were particularly satisfactory, as nanocarriers show stability over time (up to one month) and at high salt concentrations, much higher than the physiologic one. The loading of PTX was measured to be 1.2%, for a theoretical loading of 1.9 %, and the yield of encapsulation ranged from 15-25%. The nanocapsules also showed high encapsulation efficiency for SPIONs (~ 65%), depending on the polymeric composition of nanocarriers. Drug release studies showed that the nanocapsules exhibited controlled release properties. Furthermore, the release appeared to respond to the presence of external magnetic field. Finally, the «blank» magnetic nanocapsules did not exhibit cytotoxicity, whereas the «loaded» nanocarriers caused higher toxicity than the free drug. These results justify further investigation of the PLA-PEG magnetic nanocapsules as drug delivery systems of Paclitaxel.

Objetivos: Desenvolver, caracterizar e avaliar a eficácia in vivo e o perfil farmacocinético de suspensões de nanocápsulas (NC) poliméricas contendo quinina (QN). Metodologia: As NC-QN foram preparadas através de nanoprecipitação com diferentes concentrações de QN: 2 (NC2-QN), 3 (NC3-QN) e 4 mg/mL (NC4-QN). A NC4-QN também foi revestida com quitosana. Todas as formulações foram caracterizadas através de taxa de encapsulação, teor, diâmetro, índice de polidispersão, potencial zeta e pH, sendo a estabilidade avaliada por 30 dias. A avaliação da eficácia foi realizada com diferentes doses de cada formulação, em modelo de malária experimental em ratos Wistar infectados com Plasmodium berguei. A dose de NC-QN com a qual se obteve 100 % de cura dos animais foi selecionada para a avaliação farmacocinética. Nesses experimentos, os animais sadios ou infectados receberam a QN livre ou a NC2-QN, 25 mg/kg, iv bolus. As amostras de plasma coletadas em tempos pré-determinados foram quantificadas por CLAE com método validado para a determinação da QN. Os protocolos com animais foram aprovados pelo Comitê de Ética em Pesquisa da UFRGS (#2005477). Resultados e discussão: As suspensões coloidais preparadas com diferentes concentrações de QN apresentaram diâmetro adequado, população modispersa, potencial zeta diferente de zero, doseamento e taxa de encapsulação superior a 90 %. Somente a NC2-QN, na dose de 75 mg/kg/dia, q8h, curou todos os animais. Nas doses de 30 e 60 mg/kg/dia, q8h, 28,6 e 85,7% dos animais sobreviveram, respectivamente, com a NC2-QN. Houve uma diminuição significativa no t½ das NC2-QN (32,9 ± 8,9 min) em relação ao fármaco livre (69,8 ± 44,6 min), no grupo de animais infectados (α = 0,05) em função de uma tendência de aumento do CLtotal (7,1 ± 3,3 versus 9,9 ± 2,1 L/h/kg) do fármaco nanoencapsulado. Conclusões: A nanoencapsulação da QN reduziu a dose efetiva do fármaco no modelo animal avaliado em 30 % e aumentou a sobrevivência dos animais infectados em cerca de 60 %, apresentando-se como uma alternativa potencial para o tratamento da malária. / Objectives: The aims of this study were to develop and characterize polymeric nanocapsules (NC) containing quinine (QN) and to evaluate their efficacy in vivo as well as the pharmacokinetic profile of the nanoencapsulated drug. Methodology: NC-QN were prepared by nanoprecipitation with different drug concentration 2 (NC2- QN), 3 (NC3-QN) and 4 mg/mL (NC4-QN). All formulations were characterized in terms of encapsulation efficacy, drug loading, zeta potential, particle size, polydispersion index, and pH. The stability nanocapsules suspensions were evaluated during 30 days. An experimental malaria model with Plasmodium berghei was used to evaluate NC-QN efficacy in Wistar rats. Different doses were tested for each formulation. The pharmacokinetic evaluation was performed with the dose of NC–QN which presented 100 % efficacy in malaria model. The NC2-QN or QN free were administrated by i.v. route (25 mg/kg) to health and infected rats. Blood samples were collected at pre-determinated time points and quantified by an HPLC validated method. Animal protocols were approved by UFRGS Ethics in Research Committee (# 2005477). Results and discussion: All suspensions presented adequate particle size, monodisperse population, negative zeta potential, drug content and encapsulation efficiency higher than 90 %. The formulation NC2-QN (75 mg/kg/day) administrated q8h daily during 7-9 days post-infection cured all infected rats. For NC2-QN, 30 and 60 mg/kg/day, q8h, 28.6 and 85.7 % of survival were observed, respectively. NC2-QN presented significant decrease in QN t½ compared to the free drug (32.9 ± 8.9 min and 69.8 ± 44.6 min, respectively), in infected rats (α = 0,05). This occurs due to the tendency of increase in CLtotal (7.1 ± 3.3 to 9.9 ± 2.1 L/h/kg, for free QN and NC2-QN, respectively). CLtotal increased in the encapsulated group. Conclusion: Nanoencapsulation reduced QN effective dose in 30 % and increased in 60 % the survival of the infected animals. Theses results indicate that NC-QN is a potential strategy to be investigated for malaria treatment.

Objetivos: Avaliar a farmacocinética do éster etílico de indometacina (IndOEt) em ratos Wistar, após sua administração oral (p.o) e intravenosa (i.v.) nas formas livre ou nanoencapsulada, e monitorar a formação de indometacina (IndOH) in vivo. Metodologia: Os protocolos experimentais foram aprovados pelo Comitê de Ética em Pesquisa da UFRGS (2005478). Nanocápsulas (NC) contendo IndOEt (IndOEt- NC) foram administradas p.o. (10 mg/kg) e i.v. bolus (5 mg/kg) a ratos machos Wistar (n = 5 a 7/grupo) e as concentrações plasmáticas de IndOEt e IndOH resultantes foram determinadas por CLAE com detecção por UV, empregando-se metodologia validada. Os grupos controle foram tratados com suspensão aquosa de IndOEt (10 mg/kg), com ou sem polissorbato 80, ou com IndOH pelas vias i.v. ou p.o. (10 mg/kg) (n = 7 a 11/grupo). Os perfis plasmáticos individuais foram avaliados por abordagem não-compartimental e compartimental utilizando os softwares Excel® 2003 e Scientist® 2.01, respectivamente, para a determinação dos parâmetros farmacocinéticos, que foram comparados estatisticamente utilizando-se teste “t” de Student ou ANOVA (α = 0,05). A avaliação do local de absorção p.o. de IndOEt-NC foi realizada pela quantificação de IndOEt e IndOH no plasma periférico e da veia porta, homogeneizado de fígado e de parede, bem como conteúdo, de porções do intestino delgado (n= 4/tempo) após a administração oral da formulação. Resultados e Discussão: Após a administração de IndOEt-NC por ambas as vias, apenas a IndOH foi detectada, indicando uma rápida hidrólise do éster. Em todos os casos, o IndOEt não alterou os parâmetros farmacocinéticos da IndOH, exceto a biodisponibilidade. Os perfis plasmáticos de IndOH i.v. foram descritos pelo modelo de 2 compartimentos, e os orais, pelo de 1 compartimento com absorção de primeira ordem. A avaliação da absorção oral de IndOEt-NC evidenciou que o IndOEt é, provavelmente, liberado e hidrolisado ainda na luz do trato gastrintestinal, sendo a IndOH formada in vivo e absorvida. Conclusões: O IndOEt nanoencapsulado ou não, quando administrado pelas vias oral e i.v., é rapidamente convertido a IndOH. A IndOH é, portanto, responsável pela atividade antiedematogênica relatada para o IndOEt-NC. / Objective: To evaluate the pharmacokinetics of either nanoencapsulated (NC) or free indomethacin ethyl ester (IndOEt) after intravenous (i.v.) and oral (p.o.) administration to Wistar rats and to determine the pharmacokinetics of indomethacin (IndOH) formation. Methodology: Animal experiments were approved by UFRGS Ethics in Research Committee (# 2005478). The pharmacokinetics was investigated in male Wistar rats after oral and i.v. administration of IndOEt-NC (10 mg/kg and 5 mg/kg, respectively) (n = 5-7/group) and IndOEt and IndOH plasma concentrations were determined by a validated HPLC with UV detection method. The control groups were treated with IndOEt aqueous suspension (10 mg/kg p.o.) or with IndOH aqueous suspension by the p.o. or i.v. routes (10 mg/kg) (n = 7 -11/group). Noncompartmental and compartmental approaches were used for individual profiles analysis using Excel® 2003 or Scientist® 2.01 softwares, respectively. The site of IndOEt-NC oral absorption was investigated in peripheral and portal vein plasma, hepatic tissue, intestine epithelia, and lumen content (n = 4/time). Results and Discussion: After IndOEt administration by both routes, only IndOH was detected in plasma, suggesting a fast hydrolysis of the ester. The IndOH parameters after administration of IndOEt and IndOH were similar, except for the bioavailability. The pharmacokinetic parameters of IndOH were modeled by two compartment open model after i.v. dosing and by one compartment with first order absorption after oral administration. After oral administration, IndOEt-NC was converted in IndOH in the gastrointestinal tract and then absorbed as such. Conclusions: After i.v. and oral administration, either IndOEt-NC or the free drug is quickly converted in IndOH. After oral administration, IndOEt-NC is released and hydrolyzed to IndOH following its absorption at the gastrointestinal tract. Therefore, the IndOH is responsible for antiendematogenic activity reported for IndOEt-NC.

O objetivo deste trabalho foi desenvolver e caracterizar formulações tópicas semi-sólidas, utilizando como substância ativa a nimesulida, associando-a a sistemas coloidais (nanoesferas, nanocápsulas ou nanoemulsões). Os parâmetros de liberação, penetração transcutânea e retenção cutânea do antiinflamatório veículado aos sistemas nanoestruturados foram estudados através de metodologia “in vitro”, empregando pele humana como membrana. Os métodos de nanoprecipitação e nanodispersão foram utilizados e possibilitaram a obtenção de nanocápsulas, nanoesferas e nanoemulsões de nimesulida. A análise das características físico-químicas dos sistemas estudados demonstrou que os diâmetros médios obtidos foram em torno de 300 nm. As taxas de associação situaram-se próximas a 99 % e os valores de pH entre 5,1-5,3. As suspensões contendo nimesulida foram incorporadas em géis de Carbopol 940®, apresentando leve coloração amarelada e características organolépticas satisfatórias, após a incorporação das mesmas. As taxas de recuperação e os valores de pH para os géis contendo nanocápsulas, nanoesferas e nanoemulsão de nimesulida, mantiveramse estáveis durante o período de armazenagem (120 dias). Todas as formulações de géis foram caracterizadas como sistemas não-newtonianos apresentando comportamento pseudoplástico (shear thinning), sendo os reogramas ajustados pelo modelo de Ostwald (coeficiente de regressão>0,99). Experimentalmente, nenhum fenômeno de tixotropia foi detectado para as formulações testadas e a incorporação dos nanocarreadores não modificou o tipo de fluxo apresentado por estes sistemas. Os diferentes sistemas nanocarreadores incorporados nos géis hidrofílicos, foram investigados em função do potencial de liberação do fármaco na pele, usando célula de difusão tipo Franz e técnica de tape stripping em pele humana. O fármaco foi detectado no estrato córneo para o gel contendo nanocápsulas (GNM-NC) e para o gel contendo nanoesferas (GNM-NS) de nimesulida, não havendo diferença significativa entre estes dois valores. Por outro lado, o fármaco não foi detectado no estrato córneo para o gel contendo nanoemulsão de nimesulida, sendo permeado diretamente para a derme. A presença da nimesulida na epiderme/derme foi significativamente maior para o gel contendo nanocápsulas (GNM-NC) do que para o gel contendo nanoesferas (GNM-NS) ou nanoemulsão (GNM-NE). Os géis contendo nimesulida incorporada em nanocarreadores (GNM-NC, GNM-NS e GNM-NE) foram capazes de promover a penetração do fármaco no estrato córneo e/ou na camada de pele viável, com relação ao fármaco livre na mesma concentração. Estes estudos comparativos demonstraram a influência da presença do polímero e do tipo de nanocarreador na penetração do fármaco em pele humana. / The objective of this work was to develop and characterize semisolid topical formulations containing nimesulide-loaded nanospheres, nanocapsules or nanoemulsion. In order to study the in vitro percutaneous penetration of nimesulide from semi-solid topical formulations containing nanocarriers, an in vitro methodology using human skin was employed. The nanoprecipitation and the spontaneous emulsification methods were used to prepare the colloidal suspensions and nanoemulsion. For all formulation the diameters are in the sub 300 nm ranges. The encapsulation efficiencies were close to 99 % in all cases and pH values ranged between 5.1 and 5.3. Each drug-loaded nanocarrier formulation was incorporated in Carbopol 940® gels. The semisolid dosage forms showed yellowish, glossy and homogeneous aspect after the incorporation of the colloidal suspensions and nanoemulsion. The recovery of nimesulide and the pH values for the gels containing nanoemulsion, nanospheres or nanocapsules remained constant during storage (120 days). For all formulations, the rheograms exhibited a non-Newtonian behavior presenting pseudoplastic characteristics and shear thinning. The rheograms were adjusted to Ostwald’s model showing regression coefficients higher than 0.9900. None thixotropic phenomenon was experimentally detected under the test conditions for all formulations. The ability of delivering the drug into the human skin was investigated using stripping technique and Franz-type diffusion cells. The gel containing nanocapsules (GNM-NC) and the gel containing nanospheres (GNM-NS) released the nimesulide in the same extension into the stratum corneum (SC). On the other hand, for the gel containing nanoemulsion (GNM-NE), the nimesulide was not quantified in SC, but it has been directly permeated for the dermis. The penetration of the nimesulide using the gel containing nanocapsules (GNM-NC) was larger in the deeper skin than using the gel containing nanospheres (GNM-NS) or the one containing nanoemulsion (GNM-NE). The gels containing nanocarriers (GNM-NC, GNM-NS and GNM-NE) were able to release the drug in the viable layer of the skin, comparing to a non-particulated nimesulide-loaded formulation at the same concentration. For the first time, this comparative study showed the influence of the presence of the polymer and of the type of nanocarrier on the permeation of a drug through the human skin.

Atualmente, existem diversos produtos cosméticos de base nanotecnológica disponíveis no mercado nacional, incluindo protetores solares, produtos para a prevenção do envelhecimento cutâneo, ou ainda, produtos que veiculam uma ampla gama de componentes. Em contrapartida ao grande interesse industrial, várias organizações não governamentais têm expressado a sua preocupação acerca das incertezas do impacto dos novos nanomateriais sobre a saúde humana e sobre as possíveis aplicações e implicações sociais dessa nova tecnologia. Além disso, tem sido amplamente questionada a adequação dos atuais sistemas regulatórios governamentais no que diz respeito aos produtos oriundos da nanotecnologia. De fato, mundialmente, não existem requisitos específicos para o registro de produtos cosméticos de base nanotecnológica, sendo a análise realizada, de maneira geral, caso a caso. Dentro desse contexto, esse estudo traçou uma descrição do perfil da produção industrial de produtos cosméticos de base nanotecnológica no Brasil, buscando avaliar a disponibilidade dos mesmos no mercado nacional e a sua segurança de uso com base na análise das matérias primas constituintes dos produtos, determinação do seu diâmetro de partícula e dados disponíveis na literatura. Em uma última etapa, foram propostos alguns parâmetros para a regulação de produtos cosméticos de base nanotecnológica no Brasil, em especial, no que diz respeito à classificação dos produtos e a rotulagem dos mesmos. / There are a number of nanocosmetic products in the domestic market today, including sunblock, products for prevention against skin aging, or even products which circulate in a wide range components. Running counter to major industrial interests, many non-governmental organizations have expressed their concern regarding the uncertainties of the impact new nanomaterials may have on human health as well as the possible applications and social implications that this new technology may bring. Moreover, the adaptation of current governmental regulations has widely come under scrutiny regarding products stemming from nanotechnology. In fact, worldwide, there are no specific requirements to register nanotechological cosmetic products, as each is generally analyzed on a case by case basis. Within this context, this study traced the profile of the industrial production of nanotechnological cosmetic products in Brazil, with the aim of assessing the availability of these products in the domestic market and their safe usage based on the analysis of the raw materials which make up the products, the determining of particle diameter, and the data available in the literature. In the final stage, some parameters for the regulation of nanotechnological cosmetic products in Brazil are proposed, especially as regards the classification and labeling of products.

Objetivos: Desenvolver e caracterizar hidrogéis de quitosana contendo capsaicinóides nanoencapsulados, bem como analisar o efeito das nanocápsulas e do veículo nos perfis de liberação dos capsaicinóides. Metodologia: As nanocápsulas foram obtidas (método de deposição interfacial do polímero préformado) utilizando Eudragit® RS100 e triglicerídeos do ácido cáprico/caprílico como parede polimérica e núcleo oleoso, respectivamente, e foram caracterizadas. Para determinação do teor e da taxa de associação da capsaicina e dihidrocapsaicina às nanocápsulas utilizou-se metodologia validada por CLAE-UV. As nanocápsulas contendo capsaicinóides foram incorporadas em hidrogel de quitosana. Para tanto, determinaram-se previamente as características reológicas e o hidrogel ideal para uso tópico. Através dos estudos de liberação in vitro, utilizando método de saco de diálise, foram obtidos os perfis de liberação dos fármacos a partir das nanocápsulas e destas incorporadas no veículo semissólido. Resultados e Discussão: As nanocápsulas apresentaram diâmetro médio de partícula de 153 ± 7nm (PDI < 0,2), potencial zeta de + 9,62 ± 1,48 mV, pH de 5,7 ± 0,1 e uma leve tendência à sedimentação, típica de suspensões. A nanoencapsulação dos capsaicinóides não provocou alterações nas características da formulação. O teor e a taxa de associação foram próximos a 100% para ambos capsaicinóides, considerando concentração de 0,5mg/mL de capsaicinoides na formulação. Foi possível obter um hidrogel de quitosana contendo nanocápsulas adequado para uso tópico. Previamente, observou-se uma grande influência da concentração de quitosana na consistência dos hidrogéis e um comportamento reológico pseudoplástico que não foi alterado pela adição das nanocápsulas. Observou-se uma liberação controlada devido à nanoencapsulação, de modo que dihidrocapsaicina foi liberada mais lentamente do que a capsaicina. Pode-se observar um retardo na liberação da capsaicina, quando as nanocápsulas foram incorporadas no hidrogel. Conclusões: Nanocápsulas estáveis e capazes de controlar a liberação dos capsaicinóides foram obtidas, sendo que tanto o núcleo oleoso quanto o polímero são responsáveis pelo controle da liberação. A formulação semissólida contendo nanocápsulas, obtida a partir do polímero quitosana, manteve a liberação controlada observada e apresentou adequadas características para aplicação tópica. / Purpose: To develop and characterize chitosan hydrogels containing nanoencapsulated capsaicin and dihydrocapsaicin, as well as to analyze the effect of the nanocapsulation and the vehicle on the in vitro release of the capsaicinoids. Methodology: Nanocapsules were obtained (interfacial deposition of the pre-formed polymer method) using Eudragit® RS100 and capric/caprylic triglycerids as the polymeric shell and oily core, respectively, and they were characterized. An HPLCUV validated method was used to determine the drug content and drug encapsulation efficiency. The nanocapsules containing capsaicinoids were incorporated in a chitosan hydrogel. Previously, the rheological properties and the most suitable hydrogel aiming the topical delivery were determined. By means of in vitro release experiments (dialyses bags), the release profiles of the capsaicinoids from the nanocapsules and the gel containing nanocapsules were obtained. Results and Discussion: The nanocapsule formulation presented an average diameter of 153 ± 7nm (PDI < 0.2), the zeta potential was +9.62 ± 1.48 mV, and the pH 5.7± 0.1. A slight tendency to sedimentation was observed, typical of suspensions. The drug encapsulation did not alter the formulation properties. The drug content and encapsulation efficiency were nearly 100% for both capsaicinoids, considering a drug amout of 0.5mg/mL of capsaicinoids in the formulation. A chitosan hydrogel containing nanocapsules were successfully obtanined. The chitosan concentration significantly influenced the consistence of the formulations, and the pseudoplastic behavior did not change after the nanocapsule incorporation. controlled release was observed due to nanoencapsulation, in a way that capsaicin released faster than dihydrocapsaicin. The release of capsaicin from the hydrogel containing nanocapsules presented a delay in comparison with the release from the nanocapsule suspension. Conclusions: Stable nanocapsules capable of controlling the capsaicinoids release were obtained. Both the oily core and the polymeric shell are responsible for the controlled release. The semi-solid formulation containing nanocapsules obtained with the polymer chitosan maintained the controlled release and presented suitable properties for cutaneous applications.

Nanocápsulas poliméricas têm sido propostas como carreadores para filtros solares com o objetivo de prolongar o tempo de resistência desses no estrato córneo. O presente trabalho teve por objetivo avaliar comparativamente a permeação cutânea in vitro da benzofenona-3 (BZ3) a partir de nanocápsulas poliméricas incorporadas em diferentes formulações semi-sólidas. Primeiramente, suspensões de nanocápsulas contendo concentrações crescentes de BZ3 foram preparadas, caracterizadas físico-quimicamente e sua estabilidade foi determinada. A suspensão contendo 5 mg/mL de BZ3 apresentou diâmetro médio de 247 ± 4 nm, potencial zeta de -9,50 ± 1,02 mV, pH de 6,6 ± 0,1, teor e taxa de associação próximos a 100 % e foi selecionada para a continuidade do estudo. Na segunda etapa do trabalho a suspensão de nanocápsulas contendo 5 mg/mL de BZ3 foi incorporada em diferentes formulações semi-sólidas, hidrogel (HGNCBZ3), creme-gel (CGNCBZ3) e emulsão água em silicone (SNCBZ3). Estas formulações semi-sólidas foram caracterizadas segundo teor, pH, diâmetro médio das nanocápsulas incorporadas e comportamento reológico. As formulações semi-sólidas apresentaram pH adequado para aplicação cutânea, teores experimentais próximos aos valores teóricos e comportamento reológico não-newtoniano pseudoplástico. A permeação cutânea do filtro solar foi avaliada através de células de difusão de Franz e as formulações contendo BZ3 nanoencapsulada apresentaram maior quantidade e tempo de resistência do filtro no estrato córneo, comparando com as formulações contendo o ativo livre. A SNCBZ3 apresentou maior quantidade da BZ3 no estrato córneo quando comparada ao HGNCBZ3 e ao CGNCBZ3. Em uma última etapa do trabalho avaliou-se a fotoestabilidade da BZ3 nanoencapsulada e livre frente à irradiação por UVA e concluiu-se que a nanoencapsulação aumenta a fotoestabilidade da BZ3. Também foi avaliada a alergenicidade do filtro solar químico e observou-se que, nem o filtro nanoencapsulado ou livre, nem as nanocápsulas sem o ativo, apresentaram sensibilização cutânea. Em conclusão, o conjunto dos resultados obtidos demonstra que estas formulações são sistemas promissores para a aplicação cutânea de filtros solares. / Polymeric nanocapsules have been proposed as carriers for sunscreens in order to prolong the residence time of these substances in the stratum corneum. The aim of this work was to evaluate in vitro the skin permeation of benzophenone-3 (BZ3) from polymeric nanocapsules incorporated in different semi-solid formulations. Nanocapsule suspensions containing different concentrations of BZ3 were prepared, physico-chemically characterized and their stability was determinated. The suspension containing 5 mg/mL of the BZ3 presented average size of 247 ± 4 nm, zeta potential of - 9,50 ± 1,02 mV and pH values of 6,6 ± 0,1. This sample showed an amount of BZ3 and drug entrapment of 100 %. The nanocapsule suspension containing 5 mg/mL was incorporated in different semi-solid formulations, hydrogel (HGNCBZ3), cream-gel (CGNCBZ3) and emulsion water in silicon (SNCBZ3). The amount of BZ3, pH, average size and rheological properties of these formulations were evaluated. The formulations presented pH adequate for skin application and content of BZ3 close to 100 %. All semi-solid formulations showed pseudoplastic reological behavior. The skin permeation of BZ3 was evaluated by Franz diffusion cells and the formulation containing nanoencapsulated BZ3 showed an increase in the residence time of sunscreen in the stratum corneum and high amount of BZ3 compared to the formulations containing the free sunscreen. SNCBZ3 presented greater BZ3 quantity in the stratum corneum compared to HGNCBZ3 and CGNCBZ3. Finally, the photostability of free and nanoencapsulated BZ3 under UVA irradiation was evaluated and the results indicated that the nanoencapsulation increased the BZ3 photostability. The allergenicity of free and nanoencapsulated BZ3 was also determinated. Nanoencapsulated or free BZ3, and empty nanocapsules, did not present skin sensibilization. In conclusion, the results indicated that these formulations are promising delivery systems for the cutaneous applications of sunscreens.

As nanocápsulas poliméricas têm sido alvo de grande interesse por permitirem alterações no tempo de permanência das substâncias no estrato córneo da pele. O objetivo deste trabalho foi avaliar a influência da carga de superfície positiva das nanocápsulas revestidas com quitosana sobre as características físico-químicas e avaliar a penetração cutânea in vitro da benzofenona-3 a partir destas nanocápsulas incorporadas em hidrogéis. As nanocápsulas preparadas com Span 60® (NC-S: 234 nm; NC-S-B3: 260 nm; NC-Q-S-B3: 295 nm) apresentaram diâmetros médios maiores do que as preparadas com Epikuron 170® (NC-E: 174 nm; NC-E-B3: 175; NC-Q-E-B3: 202 nm), e todas apresentaram baixos valores de polidispersão (< 0,2), o que foi confirmado por de microscopia eletrônica de transmissão. O revestimento de quitosana conduziu a um potencial de superfície positivo (NC-Q-S-B3: 21 mV, NC-Q-E-B3: 21 mV), enquanto nas nanocápsulas não revestidas esses foram negativos (NC-S-B3: -12 mV, NC-E-B3: -8 mV). O teor de benzofenona-3 e as taxas de associação foram de aproximadamente 100 % para todas as amostras após preparação e após 30 dias de armazenamento. Análises em Turbiscan LAb® mostraram que as nanocápsulas sem revestimento de quitosana preparadas com Span 60® (NC-S-B3) apresentaram tendência à coalescência. As nanocápsulas incorporadas em hidrogéis de Natrosol® em total substituição à água não alteraram o comportamento pseudoplástico originalmente conferido ao Natrosol®. Análises em Turbiscan LAb® dos hidrogéis mostraram que não há tendência à desestabilização para todas as formulações. Estudos de penetração cutânea usando células de difusão de Franz demonstraram que as nanocápsulas contendo benzofenona-3 revestidas com quitosana foram eficazes em prolongar o tempo de residência do filtro solar sobre o estrato córneo da pele e, também, em reduzir a sua chegada ao meio receptor das células de Franz. Em conclusão, o conjunto de resultados indica que as nanocápsulas contendo benzofenona-3 e revestidas com quitosana são sistemas promissores para aplicação cutânea da benzofenona-3. / Polymeric nanocapsules have been considered to be of great interest because of their ability to promote variation in time-residence of substances on the skin stratum corneum. The aim of this work was to evaluate either the positive charged surface, due to chitosan coating, plays any influence in the nanocapsules physico-chemical properties and to evaluate the in vitro skin penetration of benzophenone-3 from these nanocapsules after incorporation in hydrogels. The nanocapsules prepared with Span 60® (NC-S: 234 nm; NC-S-B3: 260 nm; NC-Q-S-B3: 295 nm) presented higher average diameter than the ones prepared with Epikuron 170® (NC-E: 174 nm; NC-EB3: 175; NC-Q-E-B3: 202 nm), and all of them presented low polidispersity values (under 0,2), results that were confirmed by transmission electronic microscopy. The chitosan coating led to a positive zeta potential (NC-Q-S-B3: 21 mV, NC-Q-E-B3: 21 mV) while the uncoated nanocapsules presented negative zeta potential (NC-S-B3: - 12 mV, NC-E-B3: -8 mV). All the samples showed an amount of benzophenone-3 of 100 % after preparation and 30 days of storage. The drug entrapment was 100 % for all formulations. Turbiscan LAb® analyses demonstrated that the uncoated nanocapsules, prepared with Span 60®, have the tendency to coalescence. The incorporation of the nanocapsules in Natrosol® hydrogels, by substitution of the water for the nanocapsules suspension, did not cause any alteration in the originally observed pseudoplastic reological behavior of Natrosol®. Turbiscan LAb® analyses have also shown no tendency to destabilization for all hydrogel formulations. Skin penetration studies using Franz diffusion cells, have shown that the chitosan coated nanocapsules containing benzophenone-3 were considered to be efficient in prolonging the time-residence of the sunscreen in the stratum corneum and also in reducing its presence in the receptor medium of the Franz cells. In conclusion, the results indicate that the chitosan coated nanocapsules containing benzophenone-3 are promising systems for cutaneous application of the referred sunscreen.