i
CAMILA BATISTA DA SILVA DE ARAUJO CANDIDO
BIOCOMPATIBILIDADE, PERFIL DE PERMEAÇÃO E
EFICÁCIA ANESTÉSICA DE FORMULAÇÕES DE
ARTICAÍNA ASSOCIADA À NANOCÁPSULAS DE POLI
(EPSLON-CAPROLACTONA).
BIOCOMPATIBILITY, PERMEATION PROFILE AND
ANESTHETIC EFFICACY OF ARTICAINE-LOADED
POLY(EPSILON-CAPROLACTONE) NANOCAPULES
FORMULATIONS.
Piracicaba 2015
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UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ODONTOLOGIA DE PIRACICABA
CAMILA BATISTA DA SILVA DE ARAUJO CANDIDO
BIOCOMPATIBILIDADE, PERFIL DE PERMEAÇÃO E EFICÁCIA ANESTÉSICA DE FORMULAÇÕES DE ARTICAÍNA ASSOCIADA À NANOCÁPSULAS DE POLI
(EPSLON-CAPROLACTONA).
BIOCOMPATIBILITY, PERMEATION PROFILE AND ANESTHETIC EFFICACY OF ARTICAINE-LOADED POLY(EPSILON-CAPROLACTONE) NANOCAPULES
FORMULATIONS.
Tese apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutora em Odontologia, na Área de Farmacologia, Anestesiologia e Terapêutica.
Thesis presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Dentistry, in Pharmacology, Anesthesiology and Therapeutics Area.
Orientadora: Profa. Dra. Maria Cristina Volpato
Co-orientador: Profa. Dra. Michelle Franz-Montan Braga Leite
Este exemplar corresponde à versão final da tese defendida por Camila Batista da Silva de Araujo Candido e orientada pela Profa. Dra. Maria Cristina Volpato.
_______________________ Assinatura da Orientadora
PIRACICABA 2015
vii RESUMO
Articaína é um anestésico local que apresenta boa difusão após infiltração na maxila e mandíbula e rápida biotransformação, mas está associada a maior incidência de parestesia que a lidocaína. Sistemas de liberação como as nanocápsulas têm sido usados para melhorar a disponibilidade e diminuir a toxicidade de fármacos. Os objetivos deste trabalho foram avaliar uma formulação de articaína em nanocápsulas de poli(ɛ-caprolactona) quanto à toxicidade (viabilidade em queratinócitos humanos), capacidade de permeação (através de epitélio de mucosa de esôfago de porco in vitro) e eficácia anestésica (modelo de dor pós-operatória em ratos). As formulações anestésicas testadas foram: articaína (ATC), articaína com epinefrina 1:200.000 (ATCepi), articaína associada a
nanocápsulas de poli(ɛ-caprolactona) (ATCnano), articaína associada a
nanocápsulas de poli(ɛ-caprolactona) com epinefrina 1:200.000 (ATCnanoepi), com
os respectivos controles. As células foram expostas a várias concentrações das formulações durante 1 h e 24 h para os testes de MTT e microscopia de fluorescência. O estudo de permeação foi realizado em célula de difusão vertical tipo Franz com epitélio de de esôfago de porco, sendo avaliados o fluxo e o coeficiente de permeação das formulações ATC e ATCnano. A eficácia anestésica
foi avaliada 24 h após desenvolvimento de hipernocicepção na pata traseira de ratos. Os animais receberam 0,1 mL de cada uma das formulações de articaína ou o respectivo controle lateralmente à ferida. A anestesia foi avaliada com o analgesímetro de von Frey. Os resultados foram avaliados por regressão não-linear (teste MTT), Mann-Whitney (fluxo e coeficiente de permeação) e Log-Rank, ANOVA e teste t LSD (sucesso e duração anestésica) (a=5%). A encapsulação de articaína não alterou o tamanho das nanopartículas e o índice de polidispersão, que se manteve baixo (0,11 ± 0,04). No teste de MTT, em ambos os tempos avaliados, a utilização de aditivos diminuiu a toxicidade da articaína (p<0,0001). No estudo de permeação em epitélio de mucosa, ATCnano apresentou maior fluxo
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(p=0,0007) e coeficiente de permeação (p=0,0004) que ATC. No modelo de dor pós-operatória em ratos a duração e sucesso da articaína 2% e 4%, associadas à epinefrina foram maiores (p<0,05) que os das demais formulações e não diferiram entre si (p>0,05). Conclui-se que a encapsulação e a adição de epinefrina (isolados ou em associação) diminuem a toxicidade da articaína e a encapsulação aumenta a permeação, sinalizando um possível uso da articaína em anestesia tópica. Entretanto, a encapsulação não melhora a eficácia anestésica da articaína em tecidos inflamados, mesmo com adição de epinefrina. A adição de epinefrina é essencial para o aumento do sucesso e duração da anestesia promovida pela articaína. Pela semelhança na eficácia anestésica, a articaína, associada à epinefrina, poderia ser utilizada em tecidos inflamados na menor concentração testada, 2%.
Palavras-chave: Articaína. Epinefrina. Nanocápsulas. Citotoxicidade. Anestesia local.
ix ABSTRACT
Articaine is a local anesthetic thar presents good diffusion after maxillary and mandibular infiltration, and fast biotransformation. However, it is associated to higher paresthesia prevalence than lidocaine. Delivery systems, such as nanocapsules, have been used to improve availability and to reduce toxicity of drugs. The aims of this study were to evaluate an articaine-loaded poli(ɛ-caprolactone) formulation in relation to toxicity (in vitro viability of human keratinocyte cells), in vitro permeation capacity (through the mucosal epithelium of pig esophagus), and anesthetic efficacy (postoperative pain model in rats). The formulations tested were: articaine (ATC), articaine with 1:200,000 epinephrine (ATCepi), articaine loaded-nanocapsules of poly(ε-caprolactone) (ATCnano),
articaine loaded-nanocapsules of poly(ε-caprolactone) with 1:200,000 epinephrine (ATCnanoepi) and the respective controls. HaCaT cells were exposed to formulations
at various concentrations for 1 h and 24 h for MTT and fluorescence microscopy tests. Permeation profiles (flux and permeation coefficient) of ATC and ATCnano
across pig esophageal epithelium were performed in Franz-type vertical diffusion cells. Anesthetic efficacy was evaluated 24 h after development of hypernociception in the hind paw of rats. The animals received, laterally to the wound, 0.1 mL of articaine or control formulations. Local anesthesia was evaluated with von Frey anesthesiometer. Results were evaluated by non-linear regression analysis (MTT test), Mann-Whitney (flux and permeation coefficient) and Log-Rank, ANOVA and t LSD tests (anesthesia success and duration) (a=5%). Encapsulation of articaine did not change nanoparticles size and polidispersion index, which remained at low levels (0.11 ± 0.04). Encapsulation and epinephrine lowered articaine toxicity (p<0.0001) on HaCaT cells. In the permeation study ATCnano presented higher flux (p=0.0007) and permeation coefficient (p=0.0004)
than ATC. In the postoperative pain model in rats 2% and 4% articaine (both associated to epinephrine did not differ (p>0.05) and showed higher anesthesia
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success and duration (p<0.05) than the other articaine formulations. In conclusion, articaine encapsulation and epinephrine addition (isolated or associated) lower articaine toxicity and the encapsulation increases permeation and could provide a future use of articaine for topical anesthesia. However, encapsulation does not improve articaine anesthetic efficacy in inflamed tissues, even with epinephrine addition. Epinephrine addition to articaine is essential to improve its anesthetic success and duration. Due to the similar anesthetic efficacy, when associated to epinephrine, articaine could be used in inflamed tissues at the lower concentration tested, 2%,
xi SUMÁRIO
DEDICATÓRIA ... xiii
AGRADECIMENTOS ... xv
INTRODUÇÃO ... 1
CAPÍTULO 1: CYTOTOXICITY, PERMEATION PROFILE AND ANESTHETIC EFFICACY OF ARTICAINE-LOADED POLY(EPSILON-CAPROLACTONE) NANOCAPULES. ... 6 CONSIDERAÇÕES ... 44 CONCLUSÃO ... 46 REFERÊNCIAS ... 47 ANEXO 1 ... 51 ANEXO 2 ... 52
xiii DEDICATÓRIA
Dedico este trabalho àqueles que compartilharam meus ideais, acreditaram em meus sonhos e não mediram esforços para que eu os realizasse, me incentivando a seguir em frente e a superar todos os desafios: Minha família.
xv AGRADECIMENTOS
Ao meu querido Deus por ter iluminado meu caminho durante esta jornada, ter me abençoado e dado saúde para alcançar meus objetivos.
À Fundação de Amparo a Pesquisa do Estado de São Paulo, FAPESP, pela bolsa de doutorado concedida (Processo #2012/02539-1), auxílio (Processo #2012/07310-2) e jovem pesquisador (#2012/06974-4).
À Universidade Estadual de Campinas, por meio do Reitor Prof. Dr. José Tadeu Jorge, à Faculdade de Odontologia de Piracicaba (FOP/UNICAMP), por meio do diretor Prof. Dr. Guilherme Elias Pessanha Henriques, ao Instituto de Biologia (IB/UNICAMP), por meio do diretor Prof. Dr. Alexandre Leite Rodrigues de Oliveira, pela oportunidade de realização deste trabalho.
À Profa. Dra. Cínthia Pereira Machado Tabchoury, coordenadora dos cursos de pós-graduação da FOP/UNICAMP e à Profa. Profa. Dra. Juliana Trindade Clemente Napimoga coordenadora do Programa de Pós-Graduação em Odontologia da FOP/UNICAMP pelo empenho dedicado aos alunos de pós-graduação.
À minha orientadora Profª. Drª. Maria Cristina Volpato, meus sinceros agradecimentos pela paciência e inteligência ao me orientar. És modelo de professora e amiga e por ti tenho muito carinho, respeito e admiração. Que eu seja, ao longo da minha carreira de docente, pelo menos dez por cento do que você é.
À minha co-orientadora Profª. Drª Michelle Franz Montan Braga Leite pela atenção e confiança depositadas a mim em todos os trabalhos. És uma amiga muito querida, um exemplo a ser seguido por todos nós e merece o meu muito obrigada para sempre. Que nossas parcerias sejam maiores que apenas profissionais.
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Ao Prof. Dr. Francisco Carlos Groppo, meu primeiro orientador de iniciação científica, pela análise estatística dos resultados e contribuição para a publicação do mesmo. Tenho muito orgulho de ser sua aluna.
À Universidade Estadual Paulista “Júlio de Mesquita Filho” por meio do Reitor Prof. Dr. Julio Cezar Durigan, Câmpus de Sorocaba, por meio do diretor Prof. Dr. André Henrique Rosa, pela chance de parceria deste trabalho.
Ao Prof. Dr. Leonardo Fernandes Fraceto e a Drª. Nathalie Ferreira Silva de Melo pelo preparo das formulações nanocápsulas de poli(ɛ-caprolactona), amizade e disponibilidade em ajudar sempre que solicitado.
Aos Profs. Drs. Edgar Graner e Ricardo Della Colleta e ao técnico Fábio Haach Téo do Departamento de Diagnóstico Oral, área de Patologia Oral, por permitir o uso do citômetro de fluxo e auxílio nas análises realizadas.
Aos amigos e professores do Departamento de Morfologia, área de Histologia e Embriologia da FOP/UNICAMP pelos ensinamentos e disponibilidade em auxiliar nas metodologias deste trabalho.
Ao setor de transporte da FOP/UNICAMP, pelas idas e vindas para Campinas.
Aos amigos do laboratório de Biomembranas do Instituto de Biologia da UNICAMP, por terem me acolhido durante minha estada em Campinas.
À Profª Drª Eneida de Paula e ao técnico Márcio Paschoal, pela ajuda, disponibilidade e gentileza nas consultorias.
Aos funcionários do CEMIB-Unicamp e do Frigorífico Angelelli de Piracicaba pelo cuidado e envio de peças fundamentais desta pesquisa. Obrigada! Aos funcionários do biotério da FOP/UNICAMP, que foram indispensáveis para um ambiente de trabalho adequado.
Aos professores da Área de Farmacologia, Anestesiologia, e Terapêutica, Profs. Drs. Eduardo Dias de Andrade, Pedro Luiz Rosalen e José Ranali, pelo incentivo e ensinamentos durante todos esses anos.
Aos professores das bancas de qualificação primeira e segunda fase, Profas. Dras. Ana Paula de Souza Pardo e Karina Cogo Müller e Prof. Dr. Sidney
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Figueroba Raimundo, obrigada pelas contribuições e sugestões para a finalização deste trabalho.
Aos professores titulares e suplentes da banca de defesa desta tese, Profas Dras. Carina Denny, Cristiane de Cássia Bergamaschi, Daniele Ribeiro de Araujo, Vanessa Rocha Lima Shcaira, Juliana Cama Ramacciato e Profs. Drs. José Ranali e Bruno Bueno Silva pela disponibilidade de avaliação deste trabalho.
Aos funcionários da Farmacologia, Maria Elisa dos Santos, Eliane Melo Franco de Souza e José Carlos Gregório pelas gentilezas, ajudas no laboratório, bom humor e abraços essenciais no dia a dia.
Aos amigos da Farmacologia que passaram por aqui e ainda estão presentes, obrigada por tudo. Incentivos e companheirismo durante esses anos foram essenciais. Em especial, aos meus anjos da guarda: Bruno Muniz, Cleiton Pita, Luciano Serpe, Luiz Eduardo Ferreira, e por toda a dedicação a mim e a esse trabalho. Certamente este não seria realizado sem vocês e meus dias na cultura, biotério e laboratório seriam mais sem graça e sem cor. Muito obrigada!!
Às minhas irmãs: Bruna Fronza, Carolina Bosso, Lívia Galvão e Priscila Campioni, e aos irmãos adotivos: Andreia Scriboni, Bruna Corrêia, Pedro Freitas, Thayla Hellen e Valéria Bisinoto um profundo agradecimento pela convivência e amizade. Sentirei muitas saudades de cada jantar, risadas, choros, incentivos. Aprendi muito com cada um de vocês e os guardarei pra sempre no meu coração. Aos meus amigos do colegial, cursinho e graduação que já não divido meu dia a dia, mas que fazem parte da minha vida e da minha história e que se mantiveram na torcida pelo meu sucesso.
Aos meus pais, Sandra Batista e Carlos Alberto agradeço por fazerem de suas vidas um grande exemplo para mim. Cada resposta minha tem uma marca da educação que vocês me deram. Obrigada por terem acreditado em mim. Ao meu irmão: Lucas Batista, por ser um ótimo irmão. Compreensível e amigo assim como minha vovó Dirce que indiscutivelmente é uma avó maravilhosa e que merece muitos beijos e muitos “obrigada vovó” por ter me colocado no colo de Deus todas às vezes que precisei.
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Ao meu marido: Lucas Candido, pelo amor, paciência, amizade e carinho dedicado a mim nesses 9 anos juntos. Você foi mais que essencial para essa vitória e continuará sendo o meu porto seguro por muitos anos. Como sempre digo, com você do meu lado eu posso seguir tranquila, sem pressa pra voar.
Aos meus avós Maria Lucinda e João Batista (in memorian) por terem sido tão especiais na minha vida. Gostaria que vocês estivessem aqui para assistir a conquista desse título
À Kika, minha amada filhinha canina que chegou para me incentivar a voltar a sorrir, fazendo sempre muita festa nos meus retornos e me fazendo sentir especial. Aos meus eternos amores de quatro patas Belly e Gary (in memorian) obrigado pelo carinho, companheirismo e ternura dedicados a mim. Muitas saudades de vocês.
Enfim, sou grata às pessoas queridas, amigos e família que não foram citados aqui, mas estiveram na torcida pela realização e sucesso deste trabalho, obrigada!
1 INTRODUÇÃO
Anestésicos locais são fármacos amplamente utilizadas para controle da dor em procedimentos clínicos nas áreas médica e odontológica, por sua ação de bloqueio dos canais de sódio, inibindo assim a condução de impulsos nervosos para o sistema nervoso central (Malamed, 2013). Dentre os anestésicos locais as amidas são as mais utilizadas, destacando-se a lidocaína, bupivacaína, ropivacaína, mepivacaína, prilocaína e articaína.
Dentre estes, a articaína é um dos mais novos, tendo sido sintetizada em 1969 e comercializada na Alemanha a partir de 1976 (Malamed, 2013). Difere das demais amidas por duas características marcantes. A presença do anel tiofeno (Figura 1), região hidrofóbica da molécula, pode conferir maior difusibilidade à articaína em relação à lidocaína e prilocaína, o que pode ser visto após infiltração na região posterior da mandíbula (Robertson et al., 2007; Meechan, 2011; Nydegger et al., 2014). Entretanto, conforme relatado por Skjevik et al., (2011), o anel tiofeno apresenta menor hidrofobicidade que o anel benzênico, presente nos demais anestésicos. Segundo esses autores a maior difusibilidade seria devido à formação de ponte de hidrogênio intramolecular entre o nitrogênio da amina secundária e o oxigênio do grupo carbonil, conforme observado em estudo de simulação de dinâmica molecular.
O grupo éster, ligado ao anel tiofeno, permite biotransformação mais rápida da articaína em comparação com as demais amidas, iniciando-se no plasma, pela ação das esterases plasmáticas. Devido à rápida biotransformação, a articaína apresenta menor meia-vida plasmática (20 a 44 min) em relação à lidocaína (padrão ouro de comparação dos anestésicos locais do tipo amida) e prilocaína (96 min) (de Jong, 1994; Oertel et al., 1997; Hersh et al., 2006).
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Figura 1 - Estrutura química do cloridrato de articaína. Fonte: Paxton & Thome, 2010
Por suas características favoráveis de difusibilidade, com rápido início de ação e duração adequada, a articaína tem sido muito utilizada na odontologia, sendo o anestésico mais usado na Alemanha e Canadá (Malamed, 2005) e o segundo mais vendido nos Estados Unidos, ultrapassando a lidocaína, que é considerada o padrão ouro de comparação (Malamed, 2013). É comercializada na concentração de 4% associada à epinefrina 1:100.000 ou 1:200.000, não havendo diferença na eficácia anestésica dessas duas formulações (Tófoli et al., 2003). A articaína não é utilizada como anestésico tópico por não apresentar eficácia por essa via em concentrações clinicamente aceitáveis (Malamed, 2013).
Apesar de apresentar excelente difusibilidade, a taxa de sucesso após infiltração de articaína com epinefrina na região posterior da mandíbula para tratamento de dentes inferiores posteriores com pulpite irreversível sintomática não é clinicamente aceitável (40%) (Monteiro et al., 2015). Mesmo em dentes hígidos, a taxa de sucesso relatada é baixa (55% a 64%) em relação à lidocaína (Kanaa et al., 2006; Nydegger, et al., 2014). Apenas um estudo relatou taxa de 87% de sucesso no primeiro molar inferior após infiltração de articaína com epinefrina (Robertson et al., 2007).
Os anestésicos locais apresentam um longo histórico de segurança no uso clínico, entretanto, efeitos adversos ainda são relatados, tanto locais, quanto sistêmicos (Baker & Mulhall, 2012; Malamed, 2013). Especificamente para a
3
articaína, têm sido publicados relatos de parestesia e sensibilidade pós-operatória (Gaffen & Haas, 2009; Hillerup et al., 2011). Uma análise recente dos relatos de efeitos adversos decorrentes do uso de anestésicos locais registrados no órgão responsável pelo controle de alimentos e medicamentos dos Estados Unidos - FDA (Food and Drug Administration) mostrou que esses efeitos estão mais relacionados à parestesia e ao uso dos anestésicos articaína e prilocaína, provavelmente devido à alta concentração disponível nos tubetes anestésicos, 3 e 4% para a prilocaína e 4% para articaína (Piccinni et al, 2014).
Uma possibilidade para a redução da toxicidade, efeitos indesejáveis e melhora na biodisponibilidade desses fármacos é o uso de sistemas de liberação de medicamentos (de Paula et al., 2010). Nos últimos anos, pesquisas envolvendo anestésicos locais associados a moléculas bioativas e/ou sistemas de liberação sustentada tiveram um crescimento significativo. Estudos de eficácia anestésica e toxicidade em modelos in vitro e in vivo, para formulações injetáveis, e estudos de permeação através de barreiras biológicas como pele e mucosa, para formulações tópicas, têm sido conduzidos (de Araujo et al., 2008; Wei et al., 2012; De Melo et al., 2012; Franz-Montan et al., 2013).
Dentre os carreadores de fármacos mais estudados destacam-se os lipossomas, as ciclodextrinas e as nanopartículas poliméricas. Estas últimas consistem de vesículas com diâmetro menor que 1 μm, classificadas em nanoesferas ou nanocápsulas, de acordo com sua composição e organização estrutural. As nanoesferas são compostas por matriz densa polimérica, enquanto que as nanocápsulas apresentam um núcleo oleoso recoberto por uma cápsula polimérica. Nessas nanopartículas o fármaco pode estar disperso no núcleo ou na matriz ou ainda, adsorvido na superfície da partícula (Schaffazick et al, 2003; de Paula et al., 2012).
A partir de 1993 começaram a surgir patentes de preparações de anestésicos locais em nanopartículas poliméricas (de Paula et al., 2010). Dois
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estudos recentes mostraram boa estabilidade, eficiência de encapsulação e liberação sustentada de lidocaína base em nanoesferas de poli(ε-caprolactona) (Ramos Campos et al., 2013a) e de cloridrato de articaína em nanocápsulas de poli(etilenoglicol)-poli(ε-caprolactona) (Silva de Melo et al., 2014a). Neste último estudo a porcentagem de encapsulação da articaína atingiu 60,5%, sendo liberado 50% do fármaco em 150 minutos. O teste de biocompatibilidade in vitro mostrou ainda que a formulação de articaína encapsulada promoveu menor citotoxicidade que a solução de articaína em fibroblastos de camundongo 3T3.
Para a articaína na forma neutra (base) foi também conseguida uma preparação em nanocápsulas de poli(ε-caprolactona) com tamanho adequado, índice de polidispersão menor que 0,2, indicando baixa variação de tamanho, e alta capacidade de encapsulação (79,6%) (Ramos Campos et al., 2013b).
Tem sido demonstrado ainda que sistemas carreadores de medicamentoss conseguem modificar o perfil de permeação dos mesmos atuando como facilitadores. Este aumento de permeação foi demonstrado na pele de animais para a ropivacaína encapsulada em nanopartículas lipídicas (Zhai et al., 2014), para o ibuprofeno em nanopartículas lipídicas sólidas e carreadores de lipídeos nanoestruturados (Abdel-Mottaleb et al., 2011). Além destes, também foi demonstrado aumento de permeação em epitélio de mucosa palatina e esofágica de porco da lidocaína 5% e da benzocaína 10% encapsuladas em lipossomas, em comparação com as preparações comerciais de lidocaína 5% e benzocaína 20%, respectivamente (Franz-Montan et al., 2015).
Para a articaína na forma neutra, foi demonstrado que a encapsulação em nanocápsulas de poli(ε-caprolactona), em formulação de gel de Aristoflex® AVC (copolímero de acriloildimetiltaurato de amônio/vinilpirrolidona, usado como geilificante), promove difusão inicial rápida, seguida de um período de difusão mais lenta da articaína em membrana de acetato de celulose. Esse efeito torna
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essa formulação bastante promissora para uso tópico, se comprovado em ensaios de permeação in vitro em pele e mucosas e in vivo (Silva de Melo, 2014b).
A partir desses achados, propôs-se neste trabalho estudar uma formulação de articaína em nanocápsulas de poli(ε-caprolactona), avaliando: viabilidade celular em queratinócitos humanos, capacidade de permeação através de epitélio de mucosa de esôfago de porco e eficácia anestésica em modelo de dor pós-operatória em ratos.
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CAPÍTULO 1: Cytotoxicity, permeation profile and anesthetic efficacy of
articaine-loaded poly(epsilon-caprolactone) nanocapules.
Camila Batista da Silva de Araujo Candido, Michelle Franz-Montan, Francisco Carlos Groppo, Leonardo Fernandes Fraceto, Nathalie Ferreira Silva de Melo, Luiz Eduardo Nunes Ferreira, Bruno Vilela Muniz, Cleiton Pita dos Santos, Luciano Serpe, Maria Cristina Volpato
ABSTRACT
Purpose To evaluate the cytotoxicity, permeation capacity and anesthetic efficacy
of an articaine-loaded poli(ɛ-caprolactone) formulation (ATCnano).
Methods Mean diameter (MD), polydispersion index (PI), and encapsulation
efficiency (%EE) of ATCnano were evaluated. Cellular viability (fluorescence
microscopy and MTT test) was evaluated after 1 h and 24 h expose of HaCaT cells to: Articaine (ATC), ATCnano, Articaine with epinephrine (ATCepi) and ATC in
nanocapsules with epinephrine (ATCnanoepi). ATC and ATCnano permeation profiles
across pig esophageal epithelium were performed in Franz-type vertical diffusion cells. Anesthetic efficacy was evaluated with von Frey anesthesiometer in a postoperative pain model in rats; formulations were compared to 4% ATCepi
(commercially available formulation).
Results Articaine encapsulation did not alter MD and PI. Articaine %EE was
51.3%. Epinephrine and nanocapsules decreased articaine toxicity (P<0.0001). Encapsulation increased articaine flux (P=0.0007) and permeation coefficient (P=0.0004). 2% ATCepi and 4% ATCepi did not differ from each other (P>0.05),
providing higher anesthesia success and duration (P<0.05) than 2% ATC, 2% ATCnano and 2% ATCnanoepi.
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Conclusions Additives attenuated articaine toxicity to HaCaT cells. ATCnano is a
promising formulation for topical anesthesia, however, it is not effective for pain control in inflamed tissue.
Key words: Articaine; poly(ε-caprolactone) nanocapsules; Citotoxity assay,
8 INTRODUCTION
Articaine is an amide local anesthetic containing a thiophene ring and an ester radical in its chemical structure (Malamed, 2013). It is widely used in dentistry due to the fast onset, appropriate duration of anesthesia, good diffusibility and low plasma half-life compared with other anesthetics of the amide group (Paxton & Thome, 2010). However, reports of paresthesia and transitory post-operative sensitivity have been published, and these findings may be related to the concentration in which the articaine is used, i.e. 4% (Hillerup et al., 2011).
In the last decade, studies involving local anesthetics associated with bioactive molecules and / or sustained release systems increased significantly in order to reduce drug toxicity and improve bioavailability (de Araujo et al., 2008; Cereda et al., 2012; Silva de Melo et al., 2014a).
Among these systems, polymeric nanoparticles have attracted great interest. They consist of vesicles of less than 1 μm diameter, which can be classified as nanospheres or nanocapsules according to the composition and structural organization. Nanospheres are composed of a dense matrix, while nanocapsules consist of a polymeric coat surrounding an oily nucleous. The drug can be dissolved in the nucleous, dispersed in the matrix or adsorbed to the particle surface (Schaffazick et al., 2003; de Paula et al., 2012).
Recently it was shown that poly(ε-caprolactone) nanospheres containing lidocaine presents lower toxicity and improved anesthetic efficiency in comparison with plain lidocaine (Ramos Campos et al., 2013a). Likewise, formulations of articaine-loaded poly(ethylene glycol)-poly(ɛ-caprolactone) and alginate/chitosan showed satisfactory encapsulation efficiencies and lower toxicity when compared with articaine (Silva de Melo et al., 2014a).
Based on these results, the aim of the present study was, to assess the
9
and the anesthetic efficacy in a postoperative pain model, in rats, of an articaine-loaded poly(ε-caprolactone) nanocapsules formulation. As articaine is clinically available associated to epinephrine, the combination of articaine with epinephrine and articaine-loaded poly(ε-caprolactone) nanocapsules with epinephrine were also evaluated.
10 METHODS
Materials
Articaine hydrochloride was donated by DFL Ind Com Ltda (Rio de Janeiro, Brazil). Nanocapsules were prepared with Myritol oil (donated by Chem Specs Comercio e Representaçoes Ltda, Sao Paulo, Brazil), poly(ε-caprolactone) (PCL), and polyvinyl alcohol (PVA) (supplied by Sigma–Aldrich, St. Louis, Missouri), chloroform, ammonium acetate, and cetone (supplied by Labsynth (Diadema, Brazil). The epinephrine bitartrate salt was purchased from Sigma– Aldrich (St. Louis, Missouri) and deionized water obtained with Milli-Q system (Millipore, Billerica, Massachusetts). For cell cultures Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum, penicillin, and streptomycin sulfate were purchased from Vitrocell (Campinas, Brazil), PBS (phosphate-buffered saline) from Sigma-Aldrich (St. Louis, MO] and trypsin from Life Tech Brasil Com. Ind. Prod. Bio Ltda (Itapevi, Brazil). MTT-(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-odium bromide) was purchased from Sigma–Aldrich (St. Louis, Missouri) and the LIVE / DEAD® assay reagents (calcein/AM and ethidium homodimer-1) were obtained from Invitrogen (Carlsbad, CA). The solvents employed in the chromatographic analyses were high performance liquid chromatography (HPLC) grade acetonitrile (JT Baker, Phillipsburg, New Jersey). The permeation experiments were performed by using Franz diffusion cells (Manual Transdermal System, Hanson Research Corporation, Chatsworth, CA, USA); Angelelli Fridge Ltda (Piracicaba, Brazil) donated the pig esophagi. The in vivo assay was performed by using von Frey anesthesiometer (Insight Equipment Ltda, Ribeirão Preto, Brazil), sodium chloride 0.9% (Ind. Equiplex farm., Brazil), isofluorine (Isoforine, Cristália Chemicals and Pharmaceuticals Ltd., Itapira, SP, Brazil) and 6-0 nylon suture (Brasuture Ind Imp Exp Ltda, São Sebastião da Grama, SP, Brazil).
11 Animals
Fifty-four adult male Wistar SPF rats (250-300g) were obtained from the Multidisciplinary Center for Biological Investigation of the University of Campinas (CEMIB/Unicamp, Campinas, SP, Brazil). The animals were maintained in a 12-hour day/night cycle, at 22ºC and were given free access to water and food during the study.
Anesthetic efficacy study in rats was approved by Ethics Committee on Animal Experimentation of the University of Campinas (CEEA-Unicamp/Protocol Number 2638-1) and conducted in accordance with the "Ethical Guidelines for Investigations of Experimental Pain in Conscious Animals," as issued by the International Association for the Study of Pain (Zimmermann, 1983).
Preparation of formulations
A previous study showed a 79.6% encapsulation efficiency of articaine encapsulated in poly(ε-caprolactone) nanocapsules (Ramos Campos et al., 2013b). However, in an in vivo pilot study (data not shown), this formulation provided anesthesia onset unsuitable for clinical use (around 30 minutes) after infraorbital nerve block in rats. Therefore, the percentage of encapsulation was decreased to provide a faster onset. This was confirmed in pilot study, in which all animals presented lip anesthesia at the first anesthesia assessment (5 min after the injection) with around 50% encapsulation of articaine in poly(ε-caprolactone) nanocapsules.
Articaine base (in the neutral form) was obtained as previously described (Silva de Melo 2014b). Briefly articaine hydrochloride was dissolved in water, pH adjusted to 8.5 and aqueous phase extracted with ethyl acetate. The organic phase was dried, filtered and evaporated. The oil obtained was crystallized at -18 oC.
12
Formulations of nanocapsules were prepared by oil-in-water emulsion/solvent evaporation method (Zhou et al., 2010). Two solutions, one containing 400 mg of PCL and 200 mg of Myritol 318 in 20 mL of chloroform and another with 200 mgarticaine base dissolved in 10 mL of acetone, were mixed and sonicated for 1 min at 100 W. This preemulsion was added to 50 mL of aqueous solution containing 150 mg of PVA surfactant, and sonicated for 8 min to form the emulsion. In a rotary evaporator, the organic solvent was removed, and the emulsion was concentrated to a final volume of 10mL, containing 20 mg/mL articaine (Ramos Campos et al., 2013b). Then the volume was adjusted to 18.178 mL and 13.516 mg of articaine hydrochloride were added to allow an encapsulation efficiency of approximately 50%.
Articaine analysis
Samples were analyzed by high-performance liquid chromatography (HPLC- Varian ProStar 325 instrument, Agilent Technologies, Lake Forest, California with an isocratic PS 210 pump, a UV–vis detector and an automatic injector), according to a previous validated method (Silva de Melo et al., 2014a). Galaxy Workstation Software 410 (Agilent Technologies, Lake Forest, California) was used for data collection.
Briefly, the chromatographic conditions consisted of a C18 reversed-phase column (5 μm , 110 Å, 150 x 4.6 mm, Phenomenex Gemini), a mobile reversed-phase of monobasic sodium phosphate (0.02 mol/L, pH 3.0, adjusted with phosphoric acid) and acetonitrile (88:12, v/v), at a flow rate of 1.5 mL/min and controlled temperature of 35 °C. The mobile phase was filtered and degassed. The injection volume was 100 μL and the detector wavelength was set at 274 nm. In these conditions the limit of detection and quantification were 0.007 and 0.023 μg/mL, respectively (R² = 0.9992).
13
Characterization and encapsulation efficiency of articaine loaded-nanocapsules of poly(ε-caprolactone)
The mean diameter (MD), polydispersion index (PI) and pH of articaine loaded-nanocapsules of poly(ε-caprolactone) were evaluated. The MD and PI were evaluated by dynamic light scattering with a ZetaSizer Nano ZS 90 instrument (Malvern Instruments, UK). The measurements were performed at 25 ºC, with a fixed angle of 90º, in diluted samples (about 1.0x10-3 mol/L) in triplicates (Ramos Campos et al., 2013b).
The pH values of the nanocapsules aqueous suspensions were evaluated in triplicates by using a previously calibrated pH meter (Thermo Electron Orion® Model 290A+, Sigma). The results of stability for this formulation were presented in other study (Silva de Melo 2014b).
The encapsulation efficiency (%EE) of articaine was calculated by subtracting the non-encapsulated (free) local anesthetic from the total local anesthetic present in the initial solution. The amount of non-encapsulated articaine was determined by ultrafiltration in filter units with a pore size of 10 kDa (Microcon - Millipore) and centrifugation (14,000 g for 25 minutes), followed by HPLC quantification, as previously described (articaine analysis) (Silva de Melo et al., 2014a). All analyzes were performed in triplicate.
Cell Viability
Cell Culture
Human Keratinocytes (HaCaT) provided by Rio de Janeiro Cell Bank (Rio de Janeiro, Brazil) were grown in 75 cm² TPP® bottles (Techno Plastic Products AG, Trasadingen, Switzerland) containing Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 ºC, in a humid atmosphere of 5% CO2
14
The formulations tested and the respective controls were: articaine (ATC), articaine with 1: 200,000 epinephrine (ATCepi), articaine
loaded-nanocapsules of poly(ε-caprolactone) (ATCnano), articaine loaded-nanocapsules of
poly(ε-caprolactone) with 1: 200,000 epinephrine (ATCnanoepi), DMEM, DMEM with
epinephrine, DMEM with poly(ε-caprolactone) nanocapsules, and DMEM with poly(ε-caprolactone) nanocapsules and epinephrine. DMEM values were used as reference control (100% of cell viability).
Epinephrine (to a final concentration of 1:200,000) was added to formulations immediately before the experiment. Likewise, all formulations were diluted in DMEM immediately before its use.
MTT-Colorimetric Assay
After semi confluence, approximately 1x104 cells were inoculated into 96-well plates, and incubated for 48 hours in 37 ºC, in a humid atmosphere with 5% CO2. Subsequently, cells were exposed to ATC, ATCepi, ATCnano and ATCnanoepi
at concentrations in the range of 0.06% – 1% for 1 h or 24 h. The IC50 (50%
inhibitory concentration of cells) was determined to all formulations and exposure periods.
Cell viability was evaluated after incubation with MTT (0.3 mg/mL) for 3 h at 37 ºC. Mitochondrial dehydrogenases of viable cells promote reduction of tetrazolium bromide – MTT into insoluble formazan. The formazan crystals formed were dissolved in absolute ethanol at room temperature and the optical density of the colored complex formed was measured using a spectrophotometer (ASYS UVM 340 Biochrom LTDA, Cambridge, England) at 570 nm wavelength (Soares et al., 2014). The cell viability values were obtained by conversion of absorbance readings (A570 nm) into percentage of viable cells according to the equation below:
15
Fluorescence Imaging of Cell Viability
The fluorescence imaging of cell viability was performed as previously described, using the LIVE / DEAD® assay reagents ethidium homodimer-1 (EthD-1) and Calcein/AM (Perez-Castro et al., 2009) EthD-1 has affinity to cells with damaged membranes (dead cells) and binds to nucleic acids producing a red fluorescence, visualized using the filter set BP 546/12 (Ex)/LP 590 (Em) with a beam splitter FT 580. Calcein/AM penetrates living cells and is cleaved by cellular esterases, generating green fluorescence, which is visualized using the filter set BP 450–490 (Ex)/BP 515–565 (Em) (Perez-Castro et al., 2009).
When semi confluence was obtained, approximately 1x105 cells were inoculated into 24-well plates (TPP® Techno Plastic Products AG, Trasadingen, Switzerland), and incubated for 48 hours in 37 ◦C, under a humid atmosphere with 5% CO2. Subsequently, cells were exposed to ATC, ATCepi, ATCnano and ATCnanoepi
at the 50% inhibitory concentration obtained in the MTT assay. Methanol at 70% was used as positive control of cell death. After exposure to the formulations for 1 h or 24 h, the cells were washed twice with PBS and 300 μL of a LIVE / DEAD® solution (diluted in sterile PBS [phosphate-buffered saline - Sigma-Aldrich; St. Louis, MO] to a final concentration of 2 mM calcein/AM and 4 mM of EthD-1) was added to each well containing HaCaT cells. The plates were then incubated at room temperature for 30-45 minutes. The fluorescence images were taken on an inverted microscope (Zeiss Axiovert 40 CFL 10) coupled to MEC camera AxioCam (Carl Zeiss, Germany).
In Vitro Permeation Test
Pig esophagi were obtained from a licensed local slaughterhouse (Angelelli Fridge LTDA, Piracicaba, Brazil) and transported to the laboratory in isotonic phosphate buffer (PBS), pH 7.4. The ends were discarded and the esophageal mucosa was carefully separated from outer muscle layer with a scalpel (Diaz Del Consuelo et al., 2005). After immersion of mucosal tissue in distilled
16
water bath at 60 °C for two minutes, the epithelium was gently separated from the connective tissue and kept on isotonic saline.
Permeation experiments were performed using Franz-type vertical diffusion cells with an available diffusion area of 1.77 cm2. Fresh esophageal epithelium was placed over a 0.45 μm regenerate cellulose filter (Millipore, Saint-Quentin Yvelines, France) with the connective side facing the membrane, and it was mounted between the donor and receptor compartments. Sodium phosphate buffer was used as the receptor medium (receiver compartment of 7.0 mL in volume), maintained at 37 ± 1 ºC, and magnetically stirred at 400 rpm. Epithelium barrier integrity was checked and only undamaged specimens presenting at least 3 KΩ/cm2
of tissue resistivity were used (Cubayachi 2014).
Initially, 1.0 mL of the buffer solution was added to the donor compartment and the mounted cells were allowed to equilibrate for 60 min in a water bath at 37 ºC. Following equilibration period, the buffer solution was substituted by 1.0 mL of the formulation test (ATC or ATCnano) applied at the
epithelial surface in infinite dose condition, and the glass chamber was properly closed. The experiments were conducted in Sink conditions. Samples of 300 μL were periodically removed (15, 30, 45, 60, 90, 120, 150, 180 minutes) from the receptor chamber, and immediately replaced with fresh buffer solution (n=9). Articaine concentration was determined by HPLC as previously described.
The cumulative amount of ATC permeated across pig esophageal epithelium was plotted as a function of time. The steady-state flux (Jss) was
obtained from the slope of the linear portion of the curve, and the lag-time was obtained from the interception of this straight line to the “x” axis. Equation 2 was applied to calculate the permeability coefficient (KP) of the drug (de Araujo et al.,
17
Eq. (2)
where Jss (mg.cm-2.h-1) is ATC flux across the epithelium, KP (cm.h-1) is
the permeability coefficient and Cd is the ATC concentration in the donor
compartment (mg.cm-3).
In vivo assay – Local anesthesia in postoperative pain model
The following formulations were evaluated: 2% articaine loaded-nanocapsules of poly(ε-caprolactone) (2% ATCnano), 2% articaine
loaded-nanocapsules of poly(ε-caprolactone) with 1:200,000 epinephrine (2% ATCnanoepi),
2% articaine (2% ATC), 2% articaine with 1:200,000 epinephrine (2% ATCepi), 4%
articaine with 1:200,000 epinephrine (4% ATCepi), and controls (nanocapsules of
poly(ε-caprolactone) (nano), nanocapsules of poly(ε-caprolactone) with 1:200,000 epinephrine (nanoepi), 0.9% sodium chloride solution, 0.9% sodium chloride
solution with 1:200,000 epinephrine.
This experiment consists of paw withdrawal response to force application (von Frey anesthesiometer) after hypernociception development (postoperative pain) (Grant et al., 2007). Briefly, rats were put in cages divided in specific compartments (23 x 20 x 18 cm - width x depth x height) with a wire mesh floor (coupled with an underneath mirror) for 15-30 minutes for acclimatization. After this period, progressively force ranging from 0.0073 N to 0.456 N was applied each five minutes, with an electronic von Frey anesthesiometer (Insight Equipment Ltda, Ribeirão Preto, Brazil) to the plantar surface of right hind paw to establish the baseline response, which was calculated as a mean of three measurements (Grant et al., 2007).
Following this procedure, under isoflurane anesthesia, an incision (1 cm long x 3 mm deep) closed by three sutures was performed in the plantar surface of
18
the right hind paw of the rats. Twenty-four hours after recovering the animals were put in the same cage for acclimatization, and submitted to force application laterally to the wound, as previously described. Animals presenting 20% reduction in the force to elicit paw withdrawal were considered as presenting hyperalgesic pain and were submitted to local anesthetic testing(Grant et al., 2007).
These rats were randomly divided into 9 groups of 6 animals each and received 0.1 mL injection of articaine formulations or controls at the side of the incision. Five minutes after the injection, force (von Frey) was applied laterally to the wound each 10 minutes. Local anesthesia was considered a success when the animal did not withdraw the paw after 0.456 N force application; anesthesia duration was the period of time from the formulation injection and the last time in which the animal did not withdraw the paw. The formulations were coded by one researcher and the experiments were performed by other investigators blinded for the formulations injected.
Statistical analyzes
The results were submitted to nonlinear fit analysis (MTT assay),
Mann-Whitney test (mean diameter and in vitro permeation test), ANOVA and Student-Newman-Keuls test (anesthesia duration), t test (polydispersion index), and Log-Rank test (anesthesia success) with the use of GraphPad Instat (GraphPad Software, Inc., La Jolla, California, USA). The significance level was set at 5%.
19 RESULTS
Characterization of articaine loaded-nanocapsules of poly(ε-caprolactone)
The characteristics of articaine loaded-nanocapsules of poly(ε-caprolactone) and empty poly(ε-poly(ε-caprolactone) nanocapsules formulations are presented in Table I. The size distribution of the nanocapsules formulations is shown in Figure 1. Diameter of nanocapsules (P=0.1) and polydispersion index (P=0.4) did no change after articaine loading nanocapsules of poly(ε-caprolactone).
Table I. Mean diameter (median and interquartile range, in nm),
polydispersion Index (mean and standard deviation), encapsulation efficiency and pH obtained for nanocapsules formulations.
Parameter Articaine loaded-nanocapsules of poly(ε-caprolactone) poly(ε-caprolactone) nanocapsules Mean diameter (nm) * Polydispersion index # pH Encapsulation efficiency (%) 452.90 ± 28.00 0.11 ± 0.04 6.93 ± 0.06 51.33 ± 3.06 436.00 ± 16.05 0.10 ± 0.02 5.55 ± 0.01 ---- * (P=0.1; Mann-Whitney test); # (P=0.4; t test)
20
Figure 1. Size distribution of articaine loaded-nanocapsules of
poly(ε-caprolactone) (A) and poly(ε-poly(ε-caprolactone) nanocapsules (B), obtained using the Photon Correlation Spectroscopy technique.
Cell Viability - MTT-Colorimetric Assay
After 1-hour exposure of HaCaT cells to formulations, articaine (ATC) was the most toxic formulation and the addition of epinephrine (ATCepi) or the
encapsulation in nanocapsules (ATCnano) decreased its toxicity. ATCnanoepi
presented higher IC50 value, showing less toxicity to HaCaT cells. Thus, there was
an increased protective effect when nanocapsules and epinephrine were associated to articaine, reducing its toxicity to HaCaT cells. Figure 2 shows the viability of HaCaT cells after 1-hour exposure to the formulations. Non-linear fit analysis showed significant differences (P<0.0001) among all curves. IC50 values
21
and 95% confidence interval after 1h exposure of HaCaT cells to articaine formulations are shown in Table II (P<0.0001). Cell viability after 1 h exposure to control formulations were as follows: DMEM with epinephrine 98.50%, DMEM with poly(ε-caprolactone) nanocapsules 97.30%, and DMEM with poly(ε-caprolactone) nanocapsules and epinephrine 97.30%.
-2.0 -1.5 -1.0 -0.5 0.0 0 20 40 60 80 100
Articaine concentration [log %]
Ce
ll
v
ia
b
ili
ty
(%
)
ATC ATC epi ATC nano ATC nano epi50
Figure 2. Viability of HaCaT cells (%) after 1-hour exposure to articaine formulations in various concentrations. (ATC: articaine; ATCepi: articaine
with 1:200,000 epinephrine; ATCnano: articaine loaded-nanocapsules of
poly(ε-caprolactone); ATCnanoepi: articaine loaded-nanocapsules of poly(ε-caprolactone)
22
Table II: IC50 (50% inhibitory concentration of cells) and 95% confidence
interval (%) after 1 hour exposure of HaCaT cells to articaine formulations.
Formulations IC50 (%) IC50 95%Confidence Interval (%) ATC ATCepi ATCnano ATCnanoepi 0.3597 0.5786 0.7692 0.9582 0.3034 to 0.4265 0.5469 to 0.6122 0.7272 to 0.8137 0.8538 to 1.075
P<0.0001 for all formulations.
The results of HaCaT viability after 24 h exposure to articaine formulations are shown in Figure 3. Non-linear fit analysis showed significant differences (P<0.0001) among all curves. These results were similar to that observed after 1 h exposure to articaine formulations. ATC was the most toxic and ATCnanoepi the less toxic formulations to HaCaT cells. The addition of epinephrine
or encapsulation in nanocapules decreased articaine toxicity. The increasing order of cytotoxicity was: ATCnano epi< ATCepi< ATCnano< ATC. Table III shows IC50
values and 95% confidence interval after 24h exposure of HaCaT cells to articaine formulations (P<0.0001). Cell viability after 24 h exposure to control formulations were as follows: DMEM with epinephrine 95.60%, DMEM with poly(ε-caprolactone) nanocapsules 97.80%, and DMEM with poly(ε-caprolactone) nanocapsules and epinephrine 93.00%.
23 -3 -2 -1 0 0 20 40 60 80 100
Articaine concentration [log %]
Ce
ll
v
ia
b
ili
ty
(%
)
ATC ATC epi ATC nano ATC nano epi 50Figure 3. Viability of HaCaT cells (%) after 24 hours of exposure to articaine formulations in various concentrations. (ATC: articaine; ATCepi:
articaine with 1:200,000 epinephrine; ATCnano: articaine loaded-nanocapsules of
poly(ε-caprolactone); ATCnanoepi: articaine loaded-nanocapsules of
poly(ε-caprolactone) with 1:200,000 epinephrine). (Non-linear fit analysis; P<0.0001).
Table III. IC50 (50% inhibitory concentration of cells) and 95%
confidence interval (%) after 24 hours exposure of HaCaT cells to articaine formulations.
Formulations IC50 (%) IC50 95% Confidence Interval (%) ATC ATCepi ATCnano ATCnanoepi 0.1176 0.2467 0.1639 0.3487 0.1082 a 0.1277 0.1770 a 0.3439 0.1490 a 0.1803 0.3220 a 0.3776
24 Cell Viability - The LIVE / DEAD® assay
HaCaT cells were exposed for 1 h and 24 h to formulations at the IC50
concentration obtained in the MTT assay.
The results are shown in Figures 4 and 5. Figures 4A and 5A show untreated cells (negative controls) which are colored in green, indicating live cells. The Figures 4B and 5B show cells treated with 70% methanol (positive control); the red color indicate dead cells. The spaces observed in the plates indicate dead cells that were not adherent. Figures 4C to 4F show cell viability after 1 h exposure and Figures 5C to 5F after 24 h exposure to the formulations. The results of the LIVE / DEAD® assay are in agreement with that obtained with MTT assay. In addition, cells submitted to 1 h treatment (Figure 4) present larger size than that submitted to 24 h treatment (Figure 5). Differences in cell distribution can also be seen between Figures 4 and 5.
25
Figure 4: Fluorescent images showing viability of HaCaT cells treated
with articaine formulations at the IC50 concentration for 1 hour at 37 °C: (A) Live
cells (negative control) (B) dead cells (positive control) – treated with 70% methanol (C) ATC (D) ATCepi (E) ATCnano (F) ATCnanoepi. Each image is composed
by two superimposed images taken through different filters for green (calcein) and red (homodimer-1) fluorescence. All images have the same magnification (10x).
C D
E F
26
Figure 5: Fluorescent images showing viability of HaCaT cells treated
with articaine formulations at the IC50 concentration for 24 hours at 37 °C: (A) Live
cells (negative control) (B) dead cells (positive control) – treated with 70% methanol (C) ATC (D) ATCepi (E) ATCnano (F) ATCnanoepi. Each image is composed
by two superimposed images taken through different filters for green (calcein) and red (homodimer-1) fluorescence. All images have the same magnification (10x).
C D
E F
27
In vitro permeation study
Figure 6 illustrates the permeation profiles obtained for ATC
formulations (ATC and ATCnano) across pig esophageal epithelium. Permeation of
ATCnano formulation is increased when compared with ATC formulation.
Single curves of cumulative amount of ATC permeated across epithelium versus time were obtained, and the linear portion was used to calculate permeation parameters (Jss and lag-time). The linear interval was typically between
0.5–3 h, and regression coefficient for all individual curves were higher than 0.9985 (data not shown). Permeability coefficient was calculated according to Eq. 2. The respective permeation parameters are observed in Table IV.
Figure 6. Permeation profiles of articaine across fresh pig esophageal
epithelium from formulations applied in infinite dose conditions (mean ± SD, n=9). Black circle: ATCnano – articaine loaded-nanocapsules of poly(ε-caprolactone) and
28
Table IV: Median values (± interquartile range) of permeation parameters
(steady-state flux- Jss, Lag-time and permeability coefficient - KP) of ATC formulations
(ATC and ATCnano) across fresh pig esophageal epithelium(n=9).
Formulation Jss (μg.cm-2.h-1) Lag time (h) KP (.10-3cm.h-1)
ATC nano 191.96 ± 50.58** 0.000±0.000 6.81±1.80** ATC 22.75±12.48 0.000±0.000 1.44±1.81 ** P<0.001; Mann-Whitney test. Each permeation parameter was analyzed separately.
ATCnano presented a significant higher flux (P=0.0007) and permeation
coefficient (P=0.0004) when compared with ATC. These results are in accordance with permeation profile shown in Figure 6.
In vivo essay – Local anesthesia in postoperative pain model
All animals presented a 20% reduction in the withdrawal response to force application after incision/suture and were included in the experiment. Results of anesthesia success are presented in Figure 7. Control formulations did not showed local anesthesia (not shown). 2% ATCepi and 4% ATCepi did not differ from
each other (P=0.59), and both provided higher anesthesia success (P<0.01) than 2% ATC, 2% ATCnano and 2% ATCnanoepi. 2% ATCnanoepi provided higher anesthesia
success than 2% ATCnano (P=0.034).
As shown in Figure 8, 4% ATCepi (125 ± 28.80 min) and 2% ATCepi
(106.66 ± 39.83 min) provided longer anesthesia duration (P<0.05) than 2% ATC (25 ± 24.28 min), 2% ATCnano (20 ± 15.49 min), and 2% ATCnanoepi (46.66 ± 18.61
29
0
20
40
60
80
100 120 140 160 180 200
0
20
40
60
80
100
Time (min)
A
n
e
s
th
e
ti
z
e
d
a
n
im
a
ls
(
%
)
2% ATC 2% ATCnano 2% ATCepi * 2% ATCnanoepi# 4% ATCepi *Figure 7. Anesthesia success (%) in inflamed tissue after subcutaneous
injection of: 2% articaine (2% ATC), 2% articaine loaded-nanocapsules of poly(ε-caprolactone) (2% ATCnano), 2% articaine with 1:200,000 epinephrine (2% ATCepi),
2% articaine loaded-nanocapsules of poly(ε-caprolactone) with 1:200,000 epinephrine (2% ATCnanoepi), and 4% articaine with 1:200,000 epinephrine (4%
ATCepi) (n= 6 rats/group; Log-Rank; * P<0,01 in comparison to 2% ATC, 2%
30
0
50
100
150
200
2% ATC 2% ATCnano 2% ATCnanoepi 2% ATCepi 4% ATCepiAnesthesia duration (min)
a a
b a
b
Figure 8. Anesthesia duration (mean and standard deviation, in
minutes) after subcutaneous injection of: 2% articaine (2% ATC), 2% articaine loaded-nanocapsules of poly(ε-caprolactone) (2% ATCnano), 2% articaine with
1:200,000 epinephrine (2% ATCepi), 2% articaine loaded-nanocapsules of
poly(ε-caprolactone) with 1:200,000 epinephrine (2% ATCnanoepi), and 4% articaine with
1:200,000 epinephrine (4% ATCepi) (n = 6 rats/group; ANOVA and LSD t test;
31 DISCUSSION
Articaine presents interesting properties, among them high diffusibility. The formation of an intramolecular hydrogen bond between the secondary amine nitrogen and the carbonyl oxygen of the methoxycarbonyl substituent of articaine molecule is the responsible for the increased ability to diffuse across the cortical mandibular bone and reach teeth roots. Skjevik et al., (2011), As related by these authors, the tiophene ring presents lower hydrophobicity then benzene ring and therefore could not be responsible for the better diffusion shown by articaine in relation to other local anesthetics.
Other properties included lower plasma half-life compared with other anesthetics of the amide group and appropriate interaction with lipid membranes (Paxton & Thome 2010; Lygre et al., 2009). Along with these properties special attention has been focused on local adverse effects such as paresthesia(Paxton & Thome 2010; Hillerup et al., 2011). The present study addressed the effects of articaine encapsulation in poly(ε-caprolactone) nanocapsules on cell viability, mucosal epithelium permeation and anesthetic efficacy.
Characterization of articaine loaded-nanocapsules of poly(-caprolactone)
As observed in the results, mean diameter size and polydispersion index did not change after incorporation of articaine into nanocapsules; polydispersion index remained at low values, showing good homogeneity of the system.
Mean diameter size observed for articaine in poly(ε-caprolactone) nanocapsules (450.07 ± 27.98) were in the range observed for articaine encapsulated in poly(ethylene glycol)-poly(ε-caprolactone) (569.2 ± 30.5 nm), articaine in alginate/chitosan nanospheres (342.40 ± 20.50 nm) and lidocaine in poly (ε-caprolactone) nanospheres (449.60 ± 0.50 nm) (Silva de Mello et al., 2014a; Ramos Campos et al., 2013a), which are consistent with values observed for colloidal suspensions (Guterres et al., 1995). Accordingly, the polydispersion
32
index (0.11 ± 0.04) was in the range observed for articaine encapsulated in poly(ethylene glycol)-poly(ε-caprolactone) (0.144 ± 0.025), articaine in alginate/chitosan nanospheres (0.236 ± 0.030) and lidocaine in poly ( ε-caprolactone) nanospheres (0.072) (Silva de Mello et al., 2014a; Ramos Campos et al., 2013a) and indicates homogeneity of the nanoparticles.
Cell viability
Local anesthetics have been tested in different kind of cells, as neuroblastoma, mesenchymal stem, keratinocytes and fibroblasts(Silva de Melo et al., 2014a; Perez-Castro et al., 2009; Rahnama et al., 2013; Kontargiris et al., 2012). The immortalized human keratinocytes (HaCaT) have a good proliferative capacity in culture, being used in in vitro studies of cytotoxicity (Maupas et al., 2011; Boonkaew et al., 2014) and other cellular responses to stimulus (Portugal-Cohen et al., 2010; Jiao et al., 2013).
In the present study, the encapsulation of articaine in nanocapsules of poly(ε-caprolactone) decreased articaine toxicity of HaCaT cells exposed for 1 hour and 24 hours to the formulations. Reduction of articaine toxicity have also been reported after its incorporation into poly(ethyleneglycol)-poly(ε-caprolactone) nanocapsules an into alginate / chitosan nanospheres. After exposure to plain articaine, Balb-c 3T3 cells showed a 50% reduction in viability while the exposure of these cells to articaine-loaded poly(ethyleneglycol)-poly(ε-caprolactone) nanocapsules and articaine-loaded alginate / chitosan nanospheres, resulted in a viability loss of 20% and 30%, respectively (Silva de Melo et al., 2014a). Similarly to the observed for articaine, the incorporation of bupivacaine into alginate/chitosan nanoparticles reduced its toxicity to 3T3 fibroblasts(Grillo et al., 2010).
The results obtained in the present study could be in part explained by the fact that formulations of articaine-loaded poly(ε-caprolactone) nanocapsules presented nearly 50% of the drug free to interact with HaCaT cells, while the remaining were encapsulated and gradually released. Therefore, cells were
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exposed to the full articaine concentration for a shorter period when the formulations containing articaine encapsulated were used.
In addition, the combination of articaine-loaded poly(ε-caprolactone) nanocapsules with epinephrine promoted a further reduction in cytotoxicity. This effect was observed for all formulations containing this vasoconstrictor in both time periods of exposure, 1 h and 24 h. The protective effect of epinephrine was also demonstrated in human articular chondrocytes exposed to 1% lidocaine with epinephrine in comparison to 1% lidocaine (Jacobs et al., 2011). According to these authors, a possible explanation for the protective effect provided by epinephrine could be due to its interaction with the local anesthetic and with the chondrocytes. However, no mechanism on this occurrence was reported.
In the present study, a possible interaction could have occurred between the negative charges of the external surface of the poli(Epsilon-caprolactone) nanocapsules (zeta potential of -12.8 for nanocapsules and -6.7 for nanocapsules containing articaine) and the secondary amine nitrogen of epinephrine molecule (pka 8.55, Tuckerman et al., 1959), which could be positively charged at physiological pH (7.4). This interaction could prevent articaine release from nanocapsules; therefore, a lower concentration of the anesthetic would be available to interact with HaCaT cells, lowering toxicity. However, this hypothesis cannot be asserted, since the release kinetics of articaine from nanocapsules has not been evaluated in the presence of epinephrine.
All anesthetic formulations tested in the present study showed increased cytotoxicity (lower IC50) with longer treatment period. The same was observed for
Schwann cells after 4, 24, 48 and 72 hours exposure to lidocaine, mepivacaine, chloroprocaine, ropivacaine, and bupivacaine (Yang et al., 2011) and for human articular chondrocytes exposed to lidocaine for 15, 30 and 60 min (Jacobs et al., 2011).
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The LIVE / DEAD® assay confirmed the findings of cytotoxicity obtained with MTT-Assay. ATCnanoepi showed increased population of live cells
(green collored), being the least toxic to HaCaT cells in both exposure periods.
In vitro permeation study
Pig esophageal epithelium has been used as permeation barrier model in transmucosal studies aiming at oral delivery due to its similarities to the human oral mucosa, such as histological organization, permeability and lipid composition (Diaz Del Consuelo et al., 2005; Franz-Montan et al.,2013). Therefore, this model was chosen to evaluate the effects of encapsulation on articaine permeation.
Drug delivery systems such as liposomes, nanoparticles, and nanocapsules are considered promising drug delivery carriers in topical application (Cevc 2004). Several studies demonstrated that these carriers are able to modify drug permeation profile as they can produce a permeation-enhancing effect ( Montan et al., 2013; Zhai et al., 2014; Abdel-Mottaleb et al., 2011; Franz-Montan et al., 2015). Our results showed that articaine encapsulation improved permeation profile. These findings are in agreement with the ones reported for skin and mucosal epithelium. Considering dermal application, lipid nanoparticles demonstrated to increase permeation profile of ropivacaine across full thickness dorsal mice skin (Zhai et al., 2014) and solid lipid nanoparticles and nanostructured lipid carriers were able to increase flux rate of ibuprofen across full-thickness pig ear skin(Abdel-Mottaleb et al., 2011).
Recently it has been shown that liposomal encapsulation increases the permeability coefficient and flux through pig palatal and esophagus epithelium of 5% lidocaine and 10% benzocaine in comparison to the commercially available 5% lidocaine ointment and 20% benzocaine gel, respectively (Franz-Montan et al., 2013; Franz-Montan et al., 2015). Similarly, in the present study, articaine associated to polymeric nanocapsules presented a higher flux and permeability
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coefficient across pig esophagus epithelium when compared with articaine solution.
As there is a correlation between flux and anesthetic efficacy, permeation is a valuable tool to predict anesthetic efficacy during preclinical tests (Franz-Montan et al., 2013). Even though articaine is not used for topical anesthesia as it is not effective in clinically accepted concentrations (Malamed, 2013), the higher flux and permeability coefficient observed for ATC with poly(ε-caprolactone) nanocapsules suggests the possibility for its future application in topical anesthesia at oral mucosa and in vivo studies must be carried on to confirm this hypothesis.
In vivo essay – Local anesthesia in postoperative pain model
Local anesthesia for posterior mandibular teeth usually is achieved with inferior alveolar nerve block, as infiltration does not provide acceptable anesthetic success levels. However, articaine with epinephrine has been shown to provide higher anesthesia success than lidocaine with epinephrine in healthy mandibular posterior teeth, as well in teeth with symptomatic pulpitis (hyperalgesic teeth) (Ashraf et al., 2013; Nydegger et al., 2014). Although presenting a higher success rate, is too low for clinical use (Nydegger et al., 2014). Therefore, a formulation providing slow release could improve articaine behavior.
Our results showed that hyperalgesia due to the surgical wound did not affect the onset of anesthesia after articaine formulations infiltration, as 100% of the animals showed local anesthesia (absence of paw withdrawal) in the first assessment period (5 min). These results show that sufficient amount of articaine was available to block nerve ending fibers at this period after injection. However, anesthesia duration differed considerably among groups.
Formulations of articaine at 2% and 4% associated to epinephrine did not differ regarding duration and anesthesia success. This result confirms the ones
