CLEITON PITA DOS SANTOS
CARACTERIZAÇÃO, BIOCOMPATIBILIDADE, PERFIL DE
PERMEAÇÃO E EFICÁCIA ANESTÉSICA DE UMA
FORMULAÇÃO DE LIDOCAÍNA ASSOCIADA A
NANOCÁPSULAS DE POLI(EPSILON-CAPROLACTONA).
CHARACTERIZATION, BIOCOMPATIBILITY, PERMEATION
PROFILE AND ANESTHETIC EFFICACY OF A
FORMULATION OF LIDOCAINE-LOADED
POLY(EPSILON-CAPROLACTONE) NANOCAPSULES.
Piracicaba 2015
UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ODONTOLOGIA DE PIRACICABA
CLEITON PITA DOS SANTOS
CARACTERIZAÇÃO, BIOCOMPATIBILIDADE, PERFIL DE PERMEAÇÃO E EFICÁCIA ANESTÉSICA DE UMA FORMULAÇÃO DE LIDOCAÍNA ASSOCIADA À
NANOCÁPSULAS DE POLI(EPSILON-CAPROLACTONA).
CHARACTERIZATION, BIOCOMPATIBILITY, PERMEATION PROFILE AND ANESTHETIC EFFICACY OF A FORMULATION OF LIDOCAINE-LOADED
POLY(EPSILON-CAPROLACTONE) NANOCAPSULES.
Tese apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutor 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-orientadora: Profa. Dra. Michelle Franz Montan Braga Leite
Este exemplar corresponde à versão final da tese defendida por Cleiton Pita dos Santos e orientada pela Prof(a). Dr(a). Maria Cristina Volpato.
PIRACICABA 2015
DEDICATÓRIA
Dedico este trabalho aos meus pais, os quais são meu exemplo de educação, dedicação e trabalho. Muito obrigado por todo carinho, confiança e amor que sempre demonstraram através de apoio nas palavras e ações. Amo muito vocês.
AGRADECIMENTOS
À Universidade Estadual de Campinas e à Faculdade de Odontologia de Piracicaba (FOP/UNICAMP), por intermédio do Reitor Prof. Dr. José Tadeu Jorge e do diretor Prof. Dr. Guilherme Elias Pessanha Henriques, pelo apoio no desenvolvimento deste trabalho.
À Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP, pelo apoio financeiro concedido sob as formas de bolsa de doutorado (Processo #2012/02590-7), auxílio projeto de pesquisa (Processo #2012/07310-2) e jovem pesquisador (#2012/06974-4).
À Coordenadoria de Pós-Graduação da FOP/UNICAMP, na pessoa da Profa. Dra. Cínthia Pereira Machado Tabchoury, coordenadora dos cursos de pós-graduação da FOP/UNICAMP.
À Cristália Produtos Químicos e Farmacêuticos Ltda e à Chem Specs Comercio e Representações Ltda, pela doação de lidocaína e Myritol 318, respectivamente.
À minha orientadora Profa. Dra. Maria Cristina Volpato pelo conhecimento, dedicação e experiência que passa aos seus orientados. Obrigado por todo cuidado, minuciosidade e ensinamentos que foram muito além da experiência didática, serei eternamente grato por tudo.
À minha co-orientadora Profa. Dra. Michele Franz-Montan Braga Leite, que mostrou ser um exemplo de inovação, sucesso e determinação. É uma grande satisfação fazer parte do seu trabalho e ser seu aluno.
Aos docentes da Área de Farmacologia, Anestesiologia e Terapêutica Profs. Drs. Francisco Carlos Groppo, Eduardo Dias de Andrade, Pedro Luiz Rosalen e José Ranali, juntamente com a Profa. Carina Denny, vocês são grandes mestres e contribuem com sua didática e personalidade para formar alunos mais completos e preparados.
Ao Prof. Dr. Leonardo Fernandes Fraceto e a sempre solícita Profa. Dra. Nathalie Ferreira Silva de Melo do Laboratório de Química Ambiental da Unesp Sorocaba, que participaram de forma ativa neste trabalho, muito obrigado pela parceria e apoio de vocês.
À minha banca de pré-qualificação e qualificação: Prof. Dr. Marcelo Rocha Marques, Prof. Dr. Gilson César Nobre Franco, Profª. Drª. Karina Cogo Müller e Profª. Drª. Janaina de Cassia Orlandi Sardi, aos quais agradeço imensamente a disponibilidade em participarem desta avaliação.
Aos professores Viviane Fusco Seixas, Vanessa Rocha Lima Shcaira, José Ranali, Bruno Bueno Silva, Cristiane de Cássia Bergamaschi, Carina Denny e Pedro Luiz Rosalen, que aceitaram ser banca desta defesa de tese e se empenharam na avaliação deste trabalho.
Aos profissionais do departamento de Ciências Fisiológicas da FOP/UNICAMP, especialmente da Área de Farmacologia, Anestesiologia e Terapêutica: Eliane Melo Franco, Maria Elisa dos Santos e José Carlos Gregório e do biotério Wanderlei o meu muito obrigado pela contribuição de vocês.
Aos Profs. Drs. Ricardo Della Colleta e Edgar Graner e ao biólogo Fábio Haach Téo, da área de Patologia Oral do departamento de Diagnóstico Oral, da FOP/UNICAMP, pelo auxílio.
Aos meus amigos e companheiros de mestrado e doutorado: Ana Paula Bentes, Camila Batista da Silva, Paula Sampaio, Bruno Nani, Josy, Salete, Bruna Benso, Lívia, Aline, Irlan, Jonny Burga, Luciano Serpe, Laila Marcos, Bigode, Burns, Marcelo, Gazé e Luiz. Obrigado pelas conversas, momentos de alegria e companheirismo.
Agradecimento especial aos amigos: Luiz Eduardo, Camila Batista e Jonny Burga, que estiveram mais próximos e souberam ouvir e dar conselhos em momentos difíceis. Eu admiro muito vocês.
À Juliana Públio a minha grande inspiração, exemplo de dedicação e superação. Obrigado por estar ao meu lado, por ser paciente e acreditar em um futuro cada vez melhor.
Aos meus pais José e Alzenes, os quais são exemplos de humildade, simplicidade e dedicação aos filhos. Muitíssimo obrigado por todo carinho e amor.
A todas as pessoas que participaram, contribuindo para realização deste trabalho, direta ou indiretamente.
RESUMO
A lidocaína é o anestésico local padrão ouro, apresentando latência curta e duração moderada quando associada a vasoconstritor. Entretanto, sem vasoconstritor promove anestesia de curta duração na polpa dental e, quando utilizado como anestésico tópico no palato não bloqueia a dor da injeção. Os sistemas de liberação de medicamentos, como as nanoparticulas, tem se mostrado eficazes em diminuir a toxicidade e aumentar a duração da anestesia e a permeação desses fármacos. Desta forma, este estudo teve como objetivos caracterizar uma formulação de lidocaína associada a nanocápsulas de poli(epsilon-caprolactona) (LDC-Nano), avaliar seu efeito citotóxico, a capacidade atravessar a mucosa e a eficácia anestésica em tecido inflamado, comparando com as formulações de lidocaína (LDC) e lidocaína associada à epinefrina (LDC-Epi). Foram avaliados por 120 dias o pH das suspensões, e o tamanho e índice de polidispersão das nanopartículas (técnica de espalhamento da luz dinâmica). Os termogramas das formulações (LDC-Nano e nanocápsulas sem anestésico - Nano), dos componentes (LDC, poli(epsilon-caprolactona) - PCL) e a mistura física destes foram avaliados por calorimetria diferencial de varredura. A eficiência de encapsulação foi analisada por ultrafiltração-centrifugação. A citotoxicidade das formulações foi determinada pelo teste MTT em células HaCaT. A capacidade de permeação da formulação foi determinada in vitro em célula de difusão vertical tipo Franz através de epitélio de esôfago de porco. A eficácia anestésica foi testada in vivo em modelo de inflamação (ferida cirúrgica na pata de ratos) com o analgesímetro de von Frey. Os resultados foram avaliados pelos testes t (diâmetro e polidispersão), regressão não linear (MTT), t com correção de Welch (permeação), ANOVA e comparações múltiplas de Holm-Sidak (duração da anestesia) e Log-Rank Mantel-Cox (sucesso da anestesia), com significância de 5%. As formulações LDC-Nano e Nano apresentaram, respectivamente, tamanho médio de 557,8 ± 22,7 nm e 530,5 ± 9 nm, índice de polidispersão de 0,08 ± 0,01 e 0,16 ± 0,02 e pH 8,1 ± 0,21 e 6,3 ± 0,21, permanecendo estáveis por 120 dias. A eficiência de encapsulação foi de 51, 8 ± 1,87%. O teste de calorimetria mostrou haver interação entre lidocaína e nanocápsulas. A DL50 da LDC-Nano e LDC foi respectivamente 0,47% e 0,48% e ambas diferiram da LDC-Epi (0,57%) (p <0,0001). A LDC-Nano aumentou o fluxo
(p<0,0001) e a permeabilidade (p=0,0002) através do epitélio esofágico em relação à LDC. No teste in vivo LDC-Nano aumentou o tempo de anestesia (43 ± 8 min) (p=0,0003) em relação a LDC (24 ± 11 min), porém LDC-Epi apresentou duração maior (118 ± 10 min) (p<0,0001). Concluindo, LDC-Nano apresentou boas características físico-químicas e estabilidade, sem alterar a citotoxidade da lidocaína. Embora a encapsulação tenha aumentado o tempo de anestesia da lidocaína em tecido inflamado, seu efeito foi inferior ao promovido pela associação com epinefrina. O aumento na permeação com a lidocaína encapsulada coloca em perspectiva um possível uso tópico para essa formulação.
Palavras-chave: lidocaína, nanocápsulas de poli(epsilon-caprolactona), sistema de liberação de medicamentos, permeação de drogas, eficácia anestésica, citotoxicidade.
ABSTRACT
Lidocaine is the gold standard local anesthetic, and presents short onset and moderate anesthesia duration when associated to a vasoconstrictor. However, without vasoconstrictor, promotes short duration pulpal anesthesia and, as topical anesthetic, does not block the injection pain. Drug release systems, such as nanoparticles have shown efficacy in decreasing toxicity, and improving anesthesia duration and drug permeation. Therefore, the objectives of this study were to characterize a formulation of lidocaine encapsulated in poly(epsilon-caprolactone) nanocapsules (LDC-Nano) and to evaluate its cytotoxicity and permeation through mucosa and the anesthetic efficacy in inflamed tissue, comparing to the obtained with lidocaine (LDC) and lidocaine with epinephrine (LDC-Epi). The pH of the suspensions, and nanoparticles size and polidispersivity were evaluated for 120 days (dynamic light scattering). The thermograms of the formulations (LDC-Nano, nanocapsules without anesthetic – Nano), components of the formulations (LDC, poly(epsilon-caprolactone) – PCL) and the physical mixture of them were evaluated by differential scanning calorimetry. Encapsulation efficiency was analyzed by ultrafiltration-centrifugation. The cytotoxicity of the formulations to HaCaT cells were determined by MTT. Permeation profiles were evaluated in vitro across pig esophageal epithelium in Franz-type vertical diffusion cells. The anesthetic efficacy was evaluated in vivo in a model of inflammation (surgical wound in the paw of rats) with von Frey anesthesiometer. Data were analyzed by t test (diameter and polydispersivity), nonlinear fit analysis (MTT assay), unpaired t test with Welch correction (in vitro permeation assay), ANOVA and Holm-Sidak's multiple comparisons test (anesthesia duration) and Log-Rank Mantel-Cox test (anesthesia success), with 5% significance. LDC-Nano and Nano presented, respectively, mean diameter nanocapsules of 557.8 ± 22.7 nm and 530.5 ± 9 nm, polidispersivity of 0.08 ± 0.01 and 0.16 ± 0.02 and pH 8.1 ± 0.21 and 6.3 ± 0.21 and remained stable for 120 days. Encapsulation efficiency was 51.8 ± 1.87%. Differential scanning calorimetry showed interaction between lidocaine and Nano. The LD50 of LDC-Nano and LDC was respectively 0.47% and 0.48% and both differed from LDC-Epi (p <0.0001). LDC-Nano increased steady-state flux (p<0.0001) and permeability (p=0.0002) across esophageal epithelium in relation to LDC. In the in vivo essay LDC-Nano
increased anesthesia duration (43 ± 8 min) (p=0.0003) in relation to LDC (24 ± 11min), however LDC-Epi provided a higher duration (118 ± 10 min) (p<0.0001). In conclusion, LDC-Nano presented good physicochemical characteristics and stability, with similar toxicity in relation to lidocaine. It increased anesthesia duration in inflamed tissue, although to a lower degree than that provided by epinephrine. The improvement in permeation opens the perspective of its use as a topical anesthetic.
Key-words: lidocaine, poly(epsilon-caprolactone) nanocapsules, drug release systems, drug permeation, anesthetic efficacy, cytotoxicity.
SUMÁRIO
Página 1. INTRODUÇÃO... ... ... ... 13 2. ARTIGO Abstract…………... Introduction……… Materials and Methods………. Results and Discussion……… Conclusions………... Acknowledgments ……… References …...……… 17 18 18 26 36 37 37 3. DISCUSSÃO……… 43 4. CONCLUSÂO………... 46 5. REFERÊNCIAS... 47 ANEXOSAnexo 1. Aprovação da Comissão de Ética no Uso de Animais... Anexo 2. Submissão à European Journal of Pharmaceutics
and Biopharmaceutics………
52
1. INTRODUÇÃO
Os anestésicos locais constituem o grupo de fármacos mais utilizados para o controle da dor em procedimentos odontológicos e grande parte das intervenções médicas cirúrgicas. O bloqueio reversível da entrada de íons sódio no axônio, impedindo a despolarização e condução do impulso elétrico caracteriza a ação dos mesmos, permitindo a realização de procedimentos na ausência de percepção dolorosa e, ainda, o controle da dor no período pós-operatório (Malamed, 2013; Mohan et al., 2011).
Lidocaína é o anestésico local mais utilizado e considerado como padrão ouro de comparação. A lidocaína foi introduzida comercialmente em 1944 e graças a sua boa eficácia, segurança clínica, reduzida alergenicidade, início de ação rápido, duração da atividade anestésica moderada e baixo custo, é usada para anestesia local em odontologia e medicina, em técnicas infiltrativas, bloqueios nervosos periféricos, anestesia epidural, subaracnóide e regional venosa (De Jong, 1994; Lauretti, 2008; Moore & Hersh, 2010). Este anestésico está também disponível em formulações de uso tópico, com o propósito de diminuir a dor à punção e reduzir o desconforto em procedimentos de intubação consciente e endoscopia (Manica, 2008).
Quimicamente a lidocaína é uma aminoamida (2-dietilamino-2’,6-acetoxilidida). Como os demais anestésicos locais, a lidocaína é utilizada na forma de sal ácido (cloridrato), o qual apresenta alta solubilidade e estabilidade adequada para uso clínico. É metabolizada no fígado pelas oxidases e amidases microssomais,em xilidina e monoetilglicina xilidida (MEGX), a qual é metabolizada em xilidina e em glicina xilidida (GX). A meia-vida plasmática da lidocaína é relativamente curta, 1,6 h, entretanto a GX pode permanecer por mais tempo em circulação (até 2 dias após injeção de lidocaína em bolus). O metabólito final excretado pelos rins é a p-hidroxi-xilidina. Tanto a MEGX quando a GX são importantes do ponto de vista toxicológico, pois a primeira apresenta atividade cardiovascular (embora menor que a da lidocaína) e potencial de induzir convulsões, como a lidocaína, enquanto que a GX pode potencializar a ação convulsivante da lidocaína e da MEGX (De Jong, 1994).
No Brasil, a lidocaína é comercializada em concentrações de 4% a 10% para uso em mucosas e em concentrações de 2% e 3% para soluções injetáveis na odontologia (Dicionário..., 2013). Com relação às soluções injetáveis, as formulações contendo vasoconstritor (epinefrina, norepinefrina e fenilefrina) apresentam aumento considerável da duração da anestesia, permitindo aumento da duração da anestesia pulpar para cerca de 60 minutos, em comparação à lidocaína sem aditivos que provê 5 a 10 minutos de anestesia pulpar (Malamed, 2013).
Especificamente para a anestesia tópica odontológica, foi demonstrado que a lidocaína a 5% não reduz de forma significativa a dor à injeção da solução anestésica em técnica intraligamentar (Meechan & Thomason, 1999) e na mucosa palatina (Bhalla et al., 2009). Neste último local, mesmo a associação de lidocaína 2,5% com prilocaína 2,5%, constituindo a mistura eutética de lidocaína e prilocaína (EMLA® AstraZeneca), não é capaz de eliminar a dor à injeção anestésica no palato (Franz-Montan et al., 2012). Como na odontologia, em medicina também há dificuldade em se conseguir uma formulação anestésica tópica eficaz, conforme relatado por Jiang et al. (2011) ao estudar formulações para uso previamente à intubação em pacientes conscientes.
A fim de promover maior eficácia terapêutica no uso de anestésicos tópicos, várias alternativas têm sido estudadas, como aumento da concentração do sal anestésico (Fukayama et al., 2002), utilização de ultrassom (fonoforese) (Packer et al., 2013), iontoforese e pré-tratamento da mucosa com microagulhas (Nayak & Das, 2013), inserção do anestésico em adesivos (Nakamura et al., 2013) e associação a carreadores (de Paula et al., 2012; Franz-Montan et al., 2015).
A associação a carreadores tem se revelado uma proposta eficaz para aumentar a eficácia terapêutica e a segurança clínica de várias classes de fármacos, incluindo anestésico locais, tanto para uso tópico, quanto sistêmico (de Paula, et. al., 2012; Grillo et al., 2010; Mora-Huertas et al., 2010; de Melo et al., 2012; Ramos Campos, et. al, 2013a). Dentre os carreadores, os mais estudados são os lipossomas, as ciclodextrinas e as nanopartículas poliméricas.
As nanopartículas poliméricas constituem bons carreadores pois apresentam estabilidade e permitem fácil manipulação. Esta última característica permite alteração de sua composição possibilitando a incorporação de diversos tipos de fármacos, além do controle da degradação da nanopartícula e da liberação
do fármaco. Além disso, apresentam tamanho entre 10 e 1000 nm, podendo ser administradas inclusive por via intravenosa (Schaffazick et al., 2003; Mohanrag & Chen, 2006; Mora-Huertas et al., 2010).
As nanopartículas são constituídas por polímeros biodegradáveis e podem ser classificadas em nanoesferas ou nanocápsulas, de acordo com a composição e organização estrutural (Schaffazick et al., 2003; Mora-Huertas et al., 2010). As nanoesferas são constituídas por uma matriz polimérica densa e não possuem óleo em sua composição. Nas nanoesferas os fármacos podem apresentar-se imersos na matriz polimérica ou adsorvidos à superfície destas. As nanocápsulas por sua vez são vesículas constituídas por um núcleo oleoso envolto por uma parede polimérica, permitindo a encapsulação do fármaco no núcleo, adsorção do mesmo à superfície da nanocápsula ou, ainda, embebimento do
fármaco na membrana polimérica (Schaffazick et al., 2003; Mohanrag & Chen, 2006;
Mora-Huertas et al., 2010).
O aumento da duração anestésica proporcionado pela associação de anestésicos locais a nanopartículas tem sido demonstrado em vários estudos. A encapsulação de benzocaína em nanocápsulas de poli(lactideo-co-glicolideo) (De Melo et al., 2011) e poli(L-lactideo) (De Melo et al., 2012) proporcionou aumento da atividade anestésica, motora e sensitiva, em bloqueio do nervo ciático de camundongos. Da mesma forma, a encapsulação da bupivacaína em nanopartículas de alginato/quitosana e alginato/bis(2-etilexil) sulfosuccinato também aumentou a duração do bloqueio sensorial e motor no mesmo modelo (Grillo et al., 2010).
Com relação à lidocaína, foi relatado aumento de 4 vezes na permeação em pele de ratos quando a mesma foi encapsulada em lipossomas associados a quitosana conjugada com peptídeos (Wang et al, 2013). Em outro estudo, em pele de cobaios, Pathak & Nagarsenker (2009) observaram que a associação da lidocaína a nanopartículas lipídicas sólidas promove permeação sustentada em pele de cobaios, de forma bifásica, com permeação de 50% da lidocaína nas primeiras 6 a 8 h e o restante sendo permeado em 24 h, enquanto que para a formulação comercial de lidocaína a permeação total ocorre em 6 a 8 h. No mesmo estudo foi demonstrado que a formulação de lidocaína em nanopartículas lipídicas sólidas promove latência moderada e longa duração de ação (24 h) em comparação com a
solução de lidocaína, que promove início rápido de ação, porém com menor duração de anestesia (1 h), em modelo de “pinprick” no dorso de cobaios.
Estudando outras composições de nanopartículas lipídicas sólidas, Leng et al. (2012) também observaram boa estabilidade e aumento da duração da anestesia epidural em ratos, variando de 4 h, 8 h e 12 h, para nanopartículas compostas por ácido esteárico, monoestearina e palmitoesterato de glicerol, respectivamente, em comparação à formulação de lidocaína sem aditivos, que promoveu apenas 2 h de duração.
Recentemente Ramos Campos et al. (2013b) desenvolveram uma preparação de articaína base em nanocápsulas de poli(epsilon-caprolactona), obtendo encapsulação de 79,6%. Usando o mesmo componente, poli(epsilon-caprolactona), porém na forma de nanoesferas, Ramos Campos et al. (2013a) obtiveram 93% de encapsulação para a lidocaína, com aumento do bloqueio sensitivo do nervo ciático de ratos para 420 minutos, em comparação ao tempo de anestesia promovido pela lidocaína sem aditivos (240 minutos). Nesse estudo também foi observado efeito protetor (diminuição da toxicidade) da lidocaína em fibroblastos 3T3, quando a mesma estava encapsulada nas nanoesferas.
A partir destes resultados, neste estudo propôs-se preparar e caracterizar uma formulação de lidocaína em nanocápsulas de poli(epsilon-caprolactona), avaliando sua toxicidade in vitro, bem como sua atividade anestésica e permeação em mucosa, com vistas a sua possível aplicação futura para obtenção de anestesia em tecidos inflamados e para anestesia tópica.
2. ARTIGO
Submetido à European Journal of Pharmacy and Biopharmaceutics
Lidocaine-loaded poly(epsilon-caprolactone) nanocapsules: characterization,
in vitro cytotoxicity and mucosal permeation, and in vivo anesthetic efficacy
Cleiton Pita dos Santos, Michelle Franz-Montan, Francisco Carlos Groppo, Leonardo Fernandes Fraceto, Nathalie Ferreira Silva de Melo, Camila Batista da Silva, Luiz Eduardo Nunes Ferreira, Luciano Serpe, Jonny Burga Sanchez, Maria Cristina Volpato
ABSTRACT
Lidocaine is the gold standard local anaesthetic, presenting short onset and moderate anaesthesia duration. However, without vasoconstrictor, it promotes short anaesthesia duration and does not block the injection pain as topical anaesthetic. Drug release systems, such as nanoparticles, have shown efficacy in decreasing toxicity, improving both anaesthesia duration and drug permeation. We characterized a formulation of lidocaine encapsulated in poly(epsilon-caprolactone) nanocapsules (LDC-Nano) and evaluated its cytotoxicity in HaCaT cells (MTT), permeation across pig oesophageal epithelium (Franz-type vertical diffusion cells), and the anaesthetic efficacy in the hind paw incision model in rats (von Frey anaesthesiometer). LDC-Nano was compared to lidocaine (LDC) and lidocaine associated to epinephrine (LDC-Epi). LDC-Nano presented good physicochemical characteristics and remained stable for 120 days. Lidocaine toxicity on HaCaT cells was unchanged by encapsulation, while epinephrine decreased it. Both encapsulation and epinephrine increased anaesthesia duration, however epinephrine was more efficient. The permeation assay showed encapsulation increasing the steady-state flux and permeability coefficient of lidocaine across oesophageal epithelium. Encapsulation into nanocapsules improved lidocaine permeation, encouraging future studies as a topical anaesthetic.
Keywords: Lidocaine; Poly(epsilon-caprolactone) nanocapsules; Cytotoxicity; Permeation; Anaesthetic efficacy
INTRODUCTION
Local anaesthetics are essential for the pain control in many procedures in medicine and dentistry. Lidocaine is a gold standard local anaesthetic and it has been widely used due to the fast onset and good anaesthetic efficacy [1]. However, it presents some limitations. Pulpal anaesthesia duration is limited to 10 minutes when lidocaine is used as plain solution, and the association to a vasoconstrictor is essential for the dental treatment [1]. When used as a topical anaesthetic, it allows a pain-free puncture, but it does not provide a pain-free injection during local anaesthesia, especially in the palate, like other local anaesthetics [2, 3].
Many delivery systems have been studied in order to reduce toxicity and to improve biodisponibility and clinical efficacy of drugs. Among these systems, polymeric nanoparticles have been shown to effectively enhance local anaesthetic efficacy and reduce cytotoxicity [4, 5, 6].
Polymeric nanoparticles are classified as nanospheres and nanocapsules. The former are composed by dense polymeric matrix, where the drug molecules can be dispersed; the nanocapsules presents a lipophilic or hydrophilic nucleus surrounded by a polymeric matrix. Drugs may be encapsulated in the nucleus, imbedded in the polymeric capsule or adsorbed to the surface. Both nanoparticles enable high drug encapsulation efficiency [7, 8].
In fact, encapsulation efficiency was 79.6% for articaine in poly(epsilon-caprolactone) nanocapsules [9], and 93% for lidocaine in poly(epsilon-poly(epsilon-caprolactone) nanospheres [6], decreasing cytotoxicity, and allowing almost two fold increase in local anaesthesia duration [6]. However, some of the encapsulated formulations may present moderate or prolonged onset of action, as observed for lidocaine in solid lipid nanoparticles [10].
Based in the good results (reduced toxicity and improved anaesthesia duration) obtained with articaine encapsulated in poly(epsilon-caprolactone) nanocapsules [9], we aimed to characterize a formulation of lidocaine loaded into poly(epsilon-caprolactone) nanocapsules, evaluating its cytotoxicity, permeability
across pig oesophageal epithelium, and anaesthetic efficacy in a postoperative pain model in rats.
MATERIALS AND METHODS Materials
The following materials were used: lidocaine hydrochloride (donated by Cristália Produtos Químicos e Farmacêuticos Ltda, Itapira, Brazil), Myritol oil (donated by Chem Specs Comercio e Representaçoes Ltda, Sao Paulo, Brazil), poly(epsilon- caprolactone), polyvinyl alcohol (PVA), phosphate-buffered saline (PBS), MTT-(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-odium bromide), and epinephrine bitartrate salt (Sigma–Aldrich, St. Louis, Missouri), chloroform, ammonium acetate, and cetone (Labsynth, Diadema, Brazil), Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum, penicillin, streptomycin sulfate (Vitrocell, Campinas, Brazil), trypsin (Life Tech Brasil Com. Ind. Prod. Bio Ltda, Itapevi, Brazil), sodium chloride 0.9% (Ind. Equiplex farm., Brazil), isofluorine (Isoforine, Cristália Chemicals and Pharmaceuticals Ltd., Itapira, SP, Brazil) filter units of regenerated cellulose with a molecular exclusion pore size of 30 kDa (Microcon, Millipore, Billerica, Massachusetts), and 6-0 nylon suture (Brasuture Ind Imp Exp Ltda, São Sebastião da Grama, SP, Brazil). The solvents used in the chromatographic analyses were HPLC-grade acetonitrile (JT Baker, Phillipsburg, New Jersey), and deionized water (Milli-Q system, Millipore, Billerica, Massachusetts). The permeation experiments were performed by using Franz diffusion cells (Manual Transdermal System, Hanson Research Corporation, Chatsworth, CA, USA), and pig esophagi (donated by Angelelli Fridge Ltda, Piracicaba, Brazil).
Animals
Thirty-two male Wistar rats (250 ± 50 g) were obtained from CEMIB/
University of Campinas, Brazil (credited by ICLAS—International Council for
Laboratory Animal Science). The rats were maintained at 22 ± 1 oC room
temperature and 12 h light/dark cycle, with free access to water and food. The protocol of this study was approved by Ethics Committee on Animal Experimentation
of the University of Campinas – CEUA/UNICAMP (#2636-1), and conducted in
Preparation of lidocaine encapsulated in poly(epsilon-caprolactone) nanocapsules
Lidocaine base (C14H22N2O) was prepared from lidocaine hydrochloride (C14H23ClN2O.H2O) according to Larsen et al. (2002)[11]. Briefly, 50 mg/mL of lidocaine hydrochloride was dissolved in Milli-Q water and the pH was adjusted to 9 with a 5 M calcium hydroxide solution. The aqueous phase was removed in a separation funnel with ethyl acetate (10 mL), in triplicate. Following, anhydrous sodium sulphate was added to the organic phase to eliminate the remaining humidity, followed by new filtering and evaporation of the solvent at 35 oC.
Nanocapsules of poly(epsilon-caprolactone) containing lidocaine were prepared by emulsification/diffusion method (oil / water). Two solutions, one containing lidocaine base dissolved in 10 mL acetone, and another containing 400 mg poly(epsilon-caprolactone), 200 mg Myritol 318 and 20 mL chlorophormium were mixed and sonicated for 1 minute at 100 W [6, 12, 13]. 50 mL of an aqueous solution containing 150 mg of PVA was added to the preemulsion, and the mixture was sonicated during 8 min. The organic solvent was then evaporated, and 10 mL of an aqueous formulation of 20 mg/mL lidocaine encapsulated in poly(epsilon-caprolactone) nanocapsules (83% encapsulation efficiency) was obtained. In order to obtain a formulation with approximately 50% encapsulation, 298 mg of lidocaine hydrochloride was added to lidocaine encapsulated formulation at a final volume of 16.6 mL.
Lidocaine analysis
Lidocaine was quantified by high-performance liquid chromatography (HPLC) in a Thermo Finnigan Surveyor HPLC System with an UV-Vis Plus Detector, Autosampler Plus Lite and a quaternary LC Pump Plus monitored by ChromQuest 5.0 Chromatography Data System (Thermo Fisher Scientific Inc). The chromatographic conditions comprised a C18 reversed-phase column (5 μm, 150 x 4.60 mm, Gemini, Phenomenex), a mobile phase with the mixture of acetonitrile and buffer (25 mM NH4OH, adjusted to pH 7.0 with H3PO4) 60:40 (v:v), a flow rate of 1.2 mL.min-1, injection volume of 20 µL, and detection wavelength of 220 nm, at room temperature (adapted from Franz-Montan et al., 2015)[14].
Previously the lidocaine quantification, the method was evaluated to confirm specificity, linearity, accuracy, precision, detection limit (DL), and quantification limit (QL) [15].
The specificity of the analytical method was confirmed by injecting the lidocaine formulations, blank nanocapsules, and phosphate buffer. Linearity was obtained with eight concentrations ranging from 0.1 to 200 µg/mL, which were analysed in three consecutive days, in triplicate. The linear regression of analytical curves (peak area versus concentration of the drug) were used to define linearity (y = 27807x + 9734.3, R² = 0.9999).
Intraday and between-days precision and accuracy were determined by three lidocaine concentrations (5, 50, and 200 µg/mL) in triplicate, on three consecutive days. The method presented precision (RSD) between 0.19 and 2.96%, and accuracy between 97.15 and 104.2% for the intra- and inter-day evaluations, respectively. The limits of detection (LD = 0.31 μg/mL) and quantification (LQ = 1.04 μg/mL) were calculated based on the standard deviation of the response and the slope [15].
Characterization, stability and encapsulation efficiency of lidocaine encapsulated in poly(epsilon-caprolactone) nanocapsules
Physicochemical stability of lidocaine encapsulated in poly(epsilon-caprolactone) nanocapsules was evaluated by measuring the nanocapsules diameter and pH of the formulation after 0, 30, 60, and 120 days. During this period, the formulation was stored in amber flasks at room temperature [16, 17]. pH was evaluated by using a calibrated potentiometer (Tecnal). Nanocapsules diameter were analysed by dynamic light scattering with a ZetaSizer Nano ZS 90 analyser (Malvern Instruments, Malvern, UK), at 25 oC, and 90o angle. Previously to the analysis, the formulation was diluted (1:100, v/v) in deionized water and the nanoparticle size distribution was determined (polydispersivity index).
Encapsulation efficiency was evaluated by using a previously reported ultrafiltration-centrifugation method [6, 17, 18]. Briefly, after centrifugation of the formulation in regenerated cellulose filter units with a molecular exclusion pore size of 30 kDa, the free fraction of nonencapsulated lidocaine formulation was quantified by using HPLC. The analysis were conducted in triplicate. Encapsulation efficiency
(%) was indirectly determined by subtracting the amount of lidocaine nonencapsulated from the total amount (100%) of lidocaine in the formulation.
Differential Scanning Calorimetry (DSC)
DSC analysis were conducted by using a TA Q20 calorimeter coupled with a cooling system. Indium element was used for calibration, and samples were analysed by heating them from -10 oC to 300 oC, at a rate of 10 oC·min-1, under nitrogen flow. Five milligrams of each sample were placed in aluminium pans and submitted to the analysis; an empty pan was used as reference [19]. The following samples were analysed: lidocaine (LDC), poly(epsilon-caprolactone) (PCL), physical mixture of lidocaine and caprolactone) (PM LDC + PCL), caprolactone) nanocapsules (Nano), and lidocaine encapsulated in poly(epsilon-caprolactone) nanocapsules (LDC-Nano).
In Vitro cytotoxicity (MTT)
Cell culture
HaCaT cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium) with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin, and incubated in 5% CO2 at 37 oC. The medium was renewed every 2 days until the monolayer cultures reached 70 to 80% of confluence. Then, the cells were washed with PBS, followed by incubation with trypsin for 5 minutes. The trypsin was deactivated by addition of DMEM supplemented with fetal bovine serum and the cell suspension centrifuged at 3000 rpm, for 5 min at 20 oC. The resulting supernatant was removed and medium added, and mixed for resuspension of cells, which were used for MTT assay.
The formulations tested were: lidocaine (LDC), lidocaine encapsulated in poly(epsilon-caprolactone) nanocapsules (LDC-Nano) and lidocaine with epinephrine (LDC-Epi). LDC-Epi was prepared by adding epinephrine (1:100.000 final concentration) to a lidocaine solution immediately before its use.
MTT cell viability assay
MTT assay uses colourimetry to evaluate the in vitro viable cells. The test is based on the reduction of tetrazolium (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) - MTT into insoluble formazan by mitochondrial dehydrogenases, which occurs only in living cells.
A concentration of 5 × 104 HaCaT cells with 200 µl culture medium per well was seeded into 96-well plates and incubated in 5% CO2, at 37 oC, for 30 h. Afterwards, cells were exposed for 1 hour to LDC, LDC-Nano and LDC-Epi, with
lidocaine concentrations ranging from 0.08 % to 1 %. In addition, control formulations
were also used (Nano, Epi, and DMEM. Cell viability was determined after incubation
with MTT (0.3 mg/mL) for 3 h at 37 oC. The formazan crystals formed were dissolved in ethanol and the absorbance was measured in a spectrophotometer (ASYS UVM 340 Biochrom LTDA, Cambridge, England) at 570 nm wavelength, at room temperature [20]. The assays were performed in triplicate.
In vitro permeation assays across pig esophageal epithelium
Tissue preparation and selection
Esophagi were removed from pig immediately after death at a local slaughterhouse (Angelelli Fridge LTDA, Piracicaba, Brazil), and transported in iced isotonic phosphate buffer (pH 7.4). Esophagi were cut longitudinally and rinsed with saline. The mucosa was detached from the muscular layer and immersed in water bath (60 oC, 60 seconds) to separate the epithelium from the connective tissue [21]. Permeation study was performed only with fresh tissues from at least three animals in eight replicates.
Previously, the tissue was positioned between donor and receptor chambers of the Franz cells filled with phosphate buffer in order to verify the epithelium integrity. Resistivity (r) was measured by using alternated current of 100 mV (RMS) potency and 10 Hz frequency applied by a signal generator equipment (33210A, Agilent Technologies, Barueri, SP, Brazil) attached to Ag/AgCl electrodes [22]. The electrical current was measured at the receptor compartment by a multimeter, and resistivity (r) of the tissue was calculated considering the Ohm’s law (Eq. 1):
Eq. (1)
Where P corresponds the potency of the alternated current (100 mV); I is the intensity of the electrical current measured in µA; and A is the membrane area (1.77 cm2).
Only tissues with electrical resistance higher than 3 KΩ/cm2
were considered adequate for the permeation test [22].
In vitro permeation assay
In vitro permeation of 2% lidocaine solution (LDC) or 2% lidocaine
associated with poly(epsilon-caprolactone) nanocapsules (LDC-Nano) across pig esophagus epithelium was performed in Franz type vertical diffusion cells with permeation area of 1.77 cm2 and receptor compartment of 7 mL in volume (Manual Transdermal System, Hanson Research Corporation, Chatsworth, CA, USA) under constant magnetic stirring (400 rpm). The experiment was carried out at 37 °C during 5 hours.
The selected epithelium were placed between donor and receptor chambers over a cellulose filter (0.45 µm pore size, Millipore) with the connective side facing the filter, to avoid any damage to the barrier [14, 21, 23].
Both receptor and donor compartments were filled with filtered/degassed phosphate buffer (pH 7.4), and were allowed to equilibrate for 60 minutes. Thereafter, the donor chamber buffer was replaced by 1 mL of either LDC or LDC-Nano.
Samples of 300 µL were periodically withdrawn (0, 15, 30, 45, 60, 90, 120, 150, 180, 240 and 300 minutes) from the acceptor chamber and immediately replaced with fresh buffer, which was considered for the dilution effect. The samples were analysed by HPLC as previously described.
The steady-state flux (Jss) and lag time (tlag) were obtained by the linear portion of the cumulative amount of lidocaine permeated per cm2 of esophagus epithelium (Q) versus time.
Anaesthetic efficacy - Hind paw incision model in rats
Anaesthetic efficacy was tested in a model of surgical hypernociception development in rats, which was first described by Vandermeulen et al. (1994) [24] and Brennan et al. (1996) [25], and modified by Grant et al. (1997) [26]. Briefly, the rats were placed in cages composed by plastic dividers positioned over a wired mesh floor platform for acclimatization during 30 minutes. Bellow the platform, a mirror allowed visualization of rat paws and the application of force with a von Frey
anaesthesiometer (Insight Equipment Ltda, Ribeirão Preto, Brazil). After
acclimatization, increasing forces ranging from 0.0073 N to 0.456 N were applied each five minutes to the right hind paw to establish a baseline, considering the mean of three measurements. After this procedure, the animals were anaesthetized with isoflurane and an incision of 1 cm long x 3 mm depth was made in the plantar surface of the right paw, followed by three sutures. Twenty-four hours later, the animals were placed in the wired mesh floor cage for 30 minutes and increasing forces were applied laterally to the wound. The animals presenting at least 20% decrease in the force necessary to elicit paw withdrawal were considered hyperalgesic, and were submitted to the anaesthetic efficacy test. These animals
received laterally to the wound an infiltration of 0.1 mL of 2% LDC, 2% LDC-Epi and
2% LDC-Nano (n= 8 rats/group) or their respective controls (NaCl or Nano, n = 4 rats/group). Five minutes after infiltration and each five minutes thereafter the animals were submitted to the forces (0.0073 N to 0.456 N). The absence of paw withdrawal reflex to the maximal force application was considered as paw anaesthesia [26].
Statistical analysis
Data were tested for normal distribution by using Shapiro-Wilks test and compared by t test (diameter and polydispersivity), nonlinear fit analysis (MTT assay), unpaired t test with Welch correction (in vitro permeation assay), ANOVA and Holm-Sidak's multiple comparisons test (anaesthesia duration) and Log-Rank Mantel-Cox test (anaesthesia success) by using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, California, USA). Statistical significance was set at 5%.
RESULTS AND DISCUSSION
Characterization, Stability, and Encapsulation Efficiency
The encapsulation of lidocaine in poly(epsilon-caproplactone) nanocapsules did not change the size of nanoparticles (p=0.13) (Table 1; Fig.1); the polydispersivity was lowered when lidocaine was encapsulated (p=0.008).
Table 1. Characterization parameters (mean ± standard deviation) of nanocapsules of poly(epsilon-caprolactone) (Nano) and of poly(epsilon-caprolactone) nanocapsules loaded with lidocaine (LDC-Nano).
Formulations Mean diameter (nm) Polydispersivity pH Encapsulation efficiency (%) Nano 530.5 ± 9 0.16 ± 0.02 6.3 ± 0.21 - LDC-Nano 557.8 ± 22.7* 0.08 ± 0.01** 8.1 ± 0.21 51. 8 ± 1.87 * p = 0.13; ** p = 0.008; t test.
Figure 1. Size distribution of poly(epsilon-caprolactone) nanocapsules loaded with lidocaine (LDC-Nano) (blue) and poly(epsilon-caprolactone) nanocapsules (Nano) (orange) obtained with Photon Correlation Spectroscopy technique.
Concerning the period of observation, LDC-Nano presented good stability (Fig. 2). Diameter size (Fig. 2A) of LDC-Nano was compatible with the ones reported in the literature for lidocaine encapsulated in poly(epsilon-caproplactone) nanospheres [6], and articaine encapsulated in poly(epsilon-caproplactone) nanocapsules [9]. In fact, small variations were observed in diameter size of many polymeric nanoparticles [5, 6, 16, 27, 28].
The polydispersivity index (Fig. 2B) also showed small variations during 120 days, and all the measures were under 0.2, showing good homogeneity and stability [6,7]. Similar results were reported for polymeric nanoparticles [4, 16 28].
Along with diameter size and polydispersitivity, the analysis of pH also provides information about nanoparticle stability. Large changes in these parameters indicate aggregate formation and loss of stability [18]. The pH decrease may be related to hydrolysis of poly(epsilon-caproplactone) nanocapsules, increasing the concentration of terminal carboxylic groups promoted by relaxation of polymeric chains, as previously reported [6, 18]. In the present study, a small decrease in pH (Fig. 2C) was observed in the first period of observation, but it remained stable in the last two periods (60 and 120 days). This finding is similar to ones reported for poly(epsilon-caproplactone) nanospheres loaded with lidocaine [6].
Figure 2. Mean diameter (A), polidispersivity index (B), and pH (C) of lidocaine encapsulated in poly(epsilon-caprolactone) nanocapsules evaluated during 120 days of storage at ambient temperature.
Differential Scanning Calorimetry (DSC)
Figure 3 shows an endothermic peak of 68 oC for LDC, which corresponds to the LDC melting temperature (Tm). Another 240 oC peak indicates the thermal degradation of LDC. PCL presented a Tm of 61.2 oC due to its crystalline phase fusion, which is comparable to the findings of literature [29]. Nano showed an endothermic peak around 54.5 oC, indicating that its preparation process increased the polymeric crystal heterogeneity, which produced less perfect structures with lower Tm. LDC-Nano Tm changed from 54.5 ºC to 52.8 ºC, suggesting that the interaction between both LDC and Nano altered the compound crystalline reticulum. No melting-endothermic peak of lidocaine in LDC-Nano was observed, meaning that lidocaine could be dispersed inside or adsorbed to the surface of nanocapsules [30].
0 50 100 150 200 250 300 350 LDC Temperature (o C) Heat flux (W / g) PCL PM LDC + PCL Nano LDC-Nano
Figure 3. DSC thermograms of lidocaine (LDC), poly(epsilon-caprolactone) (PCL), physical mixture of lidocaine and poly(epsilon-caprolactone) (PM LDC + PCL), poly(epsilon-caprolactone) nanocapsules (Nano), and lidocaine encapsulated in poly(epsilon-caprolactone) nanocapsules (LDC-Nano).
In vitro cytotoxicity (MTT)
Lidocaine toxicity has been studied in many cell types, showing a wide range of results. The 50% cell lethality concentration for lidocaine has been reported as 0.4% for SH-SY5Y (human neuroblastoma) cells after 10 min treatment [31], 3.56% for human oral mucosa fibroblasts after 1 h exposure [32], and 0.09% for SH-SY5Y cells after 20 min treatment [33]. Lidocaine at 1.6 mg/mL (0.16%) induced 70% of adipose cells death after 24 h exposure [34]. These results show that lidocaine can promote tissue damage even at clinically used concentrations, usually 2% in infiltration solutions, reaching 10% in topical formulations [1].
Another important factor is the exposure period to the local anaesthetics. Although tissue toxicity of local anaesthetics may be reduced by dilution in tissue
fluids and absorption into the blood circulation, topical application may expose cells to local anaesthetic for a considerable length of time (1 h). Therefore, in the present study the exposure period was 1 hour. After this period, controls showed HaCaT viability of 95.7% (Nano) and 94.8% (epinephrine). LDC-Nano did not change lidocaine toxicity to HaCaT cells (LD50: LDC = 0.48%; LDC-Nano = 0.47%; p=0.54) (Fig. 4), showing that this formulation is biocompatible. The association of epinephrine decreased lidocaine toxicity (LD50 LDC-Epi = 0.58%; p <0.0001). These concentrations are very close and within the range in which lidocaine is clinically used. To our knowledge, there are no studies on the effect of epinephrine added to local anaesthetics over the viability of HaCaT cells.
Controversial results have been published concerning the effects of epinephrine associated to local anaesthetics on cellular viability. Jacobs et al. (2011) [35] reported no change in chondrocytes bioavailability when the cells were exposed to 2% lidocaine with or without epinephrine (added to the medium) for 24 h. However, Braun et al. (2013) [36] showed increased death of synoviocytes exposed to a 24 h infusion with bupivacaine associated to epinephrine when compared to the plain bupivacaine formulation. In addition, the effect may be concentration dependent [37].
Specifically for drug carriers, a protective effect has been shown on the toxicity of local anaesthetics. Ramos Campos et al. (2013a) [6] observed lower toxic effect of lidocaine encapsulated in poly(epsilon-caprolactone) nanospheres than plain lidocaine on 3T3 cells. Similar protection were provided by encapsulation of bupivacaine in alginate/chitosan and alginate/sodium bis(2-ethylhexyl) sulfosuccinate [4]. The protective effect observed in these studies may be related to the percentage of local anaesthetic encapsulation, which were 93.3% [6] and 75.6% to 85.7% [4]. The higher percentage of drug encapsulation in these studies in relation to ours (51.8%) could provide lower free local anaesthetic concentration to interact with cells. The differences observed between these results and the ones obtained in the present study could be due to the different cell line used.
Figure 4. HaCaT cells viability (%, Mean ± SEM), determined by MTT assay, after 1 hour exposure to different concentrations of lidocaine (LDC), nanocapsules poly(epsilon-caprolactone) loaded with lidocaine (LDC-Nano) and lidocaine with epinephrine (LDC-Epi) (triplicate). (non-linear fit analysis; * p<0.0001 in relation to LDC and LDC-Nano).
In vitro permeation assay across pig oesophageal epithelium
Oral mucosa is recognized as an efficient barrier when it is not disrupted or damaged [38]. This barrier is associated to the outermost layers of epithelium, which is the structure that limits drug permeation [39]. The presence of amorphous material with short stack lipid lamellae in the intercellular region derived from the membrane coating granules is responsible for the barrier characteristics [40].
The oral cavity has been used for drug delivery for both local (topical) and systemic (transmucosal) effects due to low cost, easy administration, avoidance of presystemic elimination [40, 41], and increased permeability in comparison to the skin [42]. Permeation studies aiming at oral application use isolated buccal or
oesophageal epithelium as barriers. In the present study, pig oesophageal epithelium was chosen, since pig tissues are the most used due to their similar histologic structure, permeability, and lipid composition [38, 43, 44]. In addition, both pig oesophagus and buccal epithelia presents similar permeability [21].
In order to improve the buccal permeation of topical formulations, enhancers, such as nanoparticles, has gained special attention [40]. In the present study, we demonstrated the efficacy of poly(epsilon-caprolactone) nanocapsules to increase lidocaine permeation across the pig oesophagus epithelium. Figure 5 shows the permeation profile of LDC and LDC-Nano (the linear intervals between 0.25 and 5 h; regression coefficients higher than 0.97). The polymeric nanoparticles used here increased the lidocaine flux by 7.46 times (p<0.0001) and the permeated amount of lidocaine by 6.61 times (p=0.0002) (Table 2). Besides the improved permeation, a lack of lag time was also observed, characterizing an immediate transport of the drug. According to Figueiras et al. (2010) [45] this fact can enable a fast onset of pharmacological effect.
Figure 5. Permeation profiles of lidocaine obtained with 2% lidocaine solution (LDC) or 2% lidocaine encapsulate in poly(epsilon-caprolactone) nanocapsules (LDC-Nano) across pig oesophageal epithelium after 5 h experiment applied under infinite dose conditions (mean±SD, n=6).
Table 2. Mean values (±SD) of the steady-state flux (Jss), cumulative amount of lidocaine permeated per cm2 of oesophageal epithelium after 5 hours (Q5h), enhancement ratio (ER) and coefficient of determination (R2) of the linear portion of the curve for both formulations (LDC and LDC-Nano).
Formulation Jss (μg.cm-2
.h-1) ERa Q5h (μg.cm-2) ERb R2
LDC 41.53 ± 9.04 1.0 224.80 ± 40.46 1.0 0.98 ± 0.003 LDC-Nano 309.71 ± 55.41*** 7.46 1485.76 ± 394.11** 6.61 0.993 ± 0.003 a
Enhancement ratio between the steady-state flux of LDC-Nanoin comparison to LDC
b
Enhancement ratio between the cumulative amount of lidocaine permeated per cm2 of oesophageal epithelium after 5 hours of LDC-Nanoin comparison to LDC
*** p < 0.0001 ** p = 0.0002; Unpaired t test with Welch correction. Each parameter was analysed separately.
Similarly, it was demonstrated that polymeric nanoparticles were able to increase flux rates of ibuprofen across pig skin when compared to the drug in buffer solution [46]. The permeation enhancement effect was also observed when lidocaine and benzocaine were encapsulated in phosphatidylcholine-based liposomes. Previous studies showed increased permeation profile, flux and permeability coefficients for encapsulated local anaesthetics when compared with the non-encapsulated commercial formulations (Xylocaina® and Benzotop®, respectively) [14, 23]. These studies also showed a strong and moderate correlation between the in
vitro flux and the in vivo topical anaesthetic efficacy (in healthy volunteers) of both
benzocaine and lidocaine after application at non-keratinized or keratinized oral mucosa [14, 23], suggesting that the in vitro permeation flux is a helpful tool to predict effectiveness of topical anaesthetics.
It is possible to assume that a high in vitro permeation flux of a local anaesthetic could result in a good topical anaesthetic effect at oral mucosa, irrespectively of keratinization. Thus, the high flux observed for lidocaine associated with polymeric nanocapsules opens perspectives for future in vivo efficacy studies in humans.
Anaesthetic efficacy - Hind paw incision model in rats
This model has been used to study pain/inflammation since it correlates to the postoperative pain in humans [47, 48]. In the present study, all the animals developed hypernociception, showing the efficacy of the model. At the first evaluation period, 5 min after the injection, the withdrawal response was absent in all the animals (Fig. 6), indicating paw anaesthesia and a fast onset.
Figure 6. Anaesthesia success after infiltration of 2% lidocaine (LDC), nanocapsules poly(epsilon-caprolactone) loaded with 2% lidocaine (LDC-Nano), and 2% lidocaine with epinephrine (LDC-Epi) in rat paws submitted to hyperalgesia development. (Log-Rank Mantel-Cox test; n= 8; * p<0.0001 in relation to the other formulations; # p=0.0008 in relation to LDC).
The encapsulation in poly(epsilon-caprolactone) nanocapsules increased anaesthetic success (p=0.0008) and duration (p=0.0003) of lidocaine (Figs. 6 and 7). Anaesthesia duration with Lido-Nano was almost two-fold higher than that observed with lidocaine (43±8 min and 24±11 min, respectively). Using another model (paw pressure after sciatic nerve block in mice), Ramos Campos et al (2013a)
[6] also observed increased analgesia, ranging from 240 min to 420 min after injection of lidocaine and lidocaine encapsulated in poly(epsilon-caprolactone) nanospheres, respectively. The differences in anaesthesia duration observed between these studies may be related to the type of local anaesthetic injection and tissue condition. Block injections usually provide extended periods of anaesthesia than infiltration [1]. In addition, inflammation diminishes anaesthetic efficacy due to the increased tissue vascularity, lower tissue pH, and peripheral sensitization of nerve fibres [1, 47, 49).
Although the encapsulation in nanocapsules has promoted a significant increase in the anaesthesia duration, the association with epinephrine was more efficient (p<0.0001) than both formulations, encapsulated and plain lidocaine, leading to 118 (±10 min) of anaesthesia. The main reason for this increased efficacy may be related to the potent vasoconstriction promoted by epinephrine, contrasting and overlaying the increased vascularity promoted by inflammation. In fact, Khoshbaten & Ferrell (1990) [50] reported increased vasoconstrictor effect of epinephrine, probably due to the postjunctional -adrenoceptor sensitization after induction of inflammation in rabbit knee. In addition, it was demonstrated by Liu et al. (1995) [51] that anaesthesia duration after subcutaneous infiltration of 1% lidocaine can be increased by 100%, even using very low epinephrine concentration, such as 1:3,200,000.
Therefore, concerning inflamed tissues, which pose a challenge to adequate anaesthesia, the encapsulation of lidocaine in nanocapsules was not superior to the existing alternative, i.e., the association with epinephrine.
Figure 7. Anaesthesia duration (mean and standard deviation) after infiltration of 2% lidocaine (LDC), nanocapsules poly(epsilon-caprolactone) loaded with 2% lidocaine (LDC-Nano), and 2% lidocaine with epinephrine (LDC-Epi) in rat paws submitted to hyperalgesia development. (Anova, Holm-Sidak's multiple comparisons test; n= 8; * p<0.0001 in relation to the other formulations; # p=0.0003 in relation to LDC).
CONCLUSIONS
We successfully presented a lidocaine encapsulated in poly(epsilon-caprolactone) nanocapsules suspension with good physicochemical characteristics, and similar in vitro cytotoxicity with lidocaine. This formulation also increased the anaesthetic efficacy when compared to lidocaine. The improved permeation parameters suggests a potential application as a topical formulation, encouraging future clinical studies in vivo.
ACKNOWLEDGMENTS
This study was financially supported by São Paulo Research Foundation (FAPESP), grants # 2012/02590-7, 2012/06974-4 and 2012/07310-2. The authors thank to Cristália Produtos Químicos e Farmacêuticos Ltda (Itapira, Brazil) and Chem Specs Comercio e Representações Ltda, Sao Paulo, Brazil) for Lidocaine and Myritol Oil 318 donation, respectively.
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