hidrogel de lidocaÍna e prilocaÍna encapsulados em nanocÁpsulas de...
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UNIVERSIDADE ESTADUAL DE CAMPINAS
FACULDADE DE ODONTOLOGIA DE PIRACICABA
BRUNO VILELA MUNIZ
HIDROGEL DE LIDOCAÍNA E PRILOCAÍNA
ENCAPSULADOS EM NANOCÁPSULAS DE
POLI(ÉPSILON-CAPROLACTONA) PARA ANESTESIA
TÓPICA ORAL .
PIRACICABA
2018
BRUNO VILELA MUNIZ
HIDROGEL DE LIDOCAÍNA E PRILOCAÍNA
ENCAPSULADOS EM NANOCÁPSULAS DE
POLI(ÉPSILON-CAPROLACTONA) PARA ANESTESIA
TÓPICA ORAL .
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.
Orientadora: Prof.ª. Drª. Michelle Franz Montan Braga Leite
Co-orientadora: Prof.ª. Drª. Maria Cristina Volpato
ESTE EXEMPLAR CORRESPONDE À
VERSÃO FINAL DA TESE DEFENDIDA
PELO ALUNO BRUNO VILELA MUNIZ, E
ORIENTADO PELA PROFª. DRª.
MICHELLE FRANZ MONTAN BRAGA
LEITE E CO-ORIENTADO PELA PROF.ª.
DRª. MARIA CRISTINA VOLPATO.
PIRACICABA
2018
AGRADECIMENTOS
À Universidade Estadual de Campinas (UNICAMP), na pessoa do Magnífico
Reitor Prof. Dr. Marcelo Knobel.
À Faculdade de Odontologia de Piracicaba (FOP), na pessoa de seu diretor Prof.
Dr. Guilherme Elias Peçanha Henriques.
Ao Departamento de Ciências Fisiológicas da FOP-UNICAMP, na pessoa de seu
Chefe Prof. Dr. Francisco Carlos Groppo.
À Coordenadoria Pós-Graduação (CPG) da FOP-UNICAMP, na pessoa de sua
coordenadora Profa. Dra. Cinthia Maria Tabchoury.
Ao Programa de Pós-Graduação em Odontologia (PPGO) da FOP-UNICAMP, na
pessoa de seu coordenador Prof. Dr. Marcelo de Castro Meneghim.
À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pela
bolsa de doutorado concedida.
À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), pelo auxílio
financeiro (2012/06974-4) para a realização deste trabalho.
À professora Michelle Franz Montan Braga Leite, minha orientadora, que ensinou
as responsabilidades, os percalços e atalhos para coordenar um laboratório de pesquisa sem
nunca o deixar parar. Nossas reuniões sempre foram pautadas pelo respeito, compreensão,
atenção, exigência e excelência na orientação. Devo agradecer imensamente pelo apoio,
especialmente nos momentos difíceis, e dizer, sem nenhuma ressalva, que levo para a vida
inteira uma grande amiga.
À minha co-orientadora, Profª. Drª. Maria Cristina Volpato, pela retidão, prontidão
e eterna orientação.
Ao Prof. Dr. Leonardo Fernandes Fraceto e Profa. Eneida de Paula pela parceria e
oportunidade de colaboração e ensinamentos envolvendo a caracterização das formulações
testadas no presente trabalho e disponibilidade em ajudar sempre que solicitado.
À Dra. Lígia Nunes de Morais Ribeiro, pela ajuda com os testes de validação,
interpretação dos resultados e disponibilidade e prontidão de sempre!
Aos professores da Área de Farmacologia, Anestesiologia, e Terapêutica, Prof. Dr.
Francisco Carlos Groppo, Prof. Dr. Pedro Luiz Rosalen, Prof. Dr. Eduardo Dias de Andrade e
Prof. Dr. José Ranali, pelos ensinamentos e conversas sobre os mais diversos assuntos.
Aos técnicos dos laboratórios de Farmacologia e Fisiologia Eliane Melo (Ely), José
Carlos Gregório (Zé) e Fábio Padilha (Fabinho) pela bela amizade, conversas entusiasmadas,
solicitude e orientações.
À Maria Elisa dos Santos, pela alegria e carinho no exercício de suas funções,
sempre solicita para atender, com gentileza e dedicação a todos.
À Sra. Érica Alessandra Sinhoreti, à Sra. Ana Paula Carone e à Sra. Raquel
Quintana Sachi, membros da CPG da FOP-UNICAMP, e à Srta. Elisa dos Santos, secretária do
PPGO da FOP-UNICAMP pela cordialidade, solicitude e presteza de seus serviços.
Aos professores das bancas de qualificação primeira e segunda fase, Prof. Dr. Pedro
Luiz Rosalen, Profª Drª Karina Cogo, Prof. Dr. Cleiton Pita, Profª Dr.ª Laura Oliveira e Profª
Drª Nathalie Melo.
À Cristália Produtos Químicos Farmacêuticos LTDA pela doação dos anestésicos
locais Lidocaína e Prilocaína utilizados nessa tese.
AGRADECIMENTOS ESPECIAIS
Aos meus pais queridos, Edson e Val, pelo amor incondicional, que é combustível
para que eu possa seguir meus projetos, pela preocupação, pelo zelo, incentivo e orgulho. Pela
atuação basilar nas decisões mais importantes da minha vida. Vocês me incentivaram a estar
aqui e buscar sempre crescimento pessoal e profissional.
A minha joia, o ser único em minha vida, minha irmã Mariane, que de alguma
maneira foi o grande propulsor em minha vida. Me ensinando a viver desde o primeiro minuto
e norteando meus ideais.
Aos meus familiares: tios Jussara, Gera, Val, Leandro, Gilberto, Ana e, em especial,
Irene; primos Maurício, Augusto, Fabinho e Fernando, que sempre me apoiaram e contribuíram
na minha formação, e propiciaram belos momentos juntos.
Em especial aos meus avós Meiri e Marcos, por sempre confiarem e alimentarem o
meu desejo pelo conhecimento, sem nunca perder o bom humor e alegria.
A minha noiva Larissa Motta, pelo apoio, incentivo, carinho, amor e confiança. Um
alento a todas as minhas preocupações.
Aos meus amigos de mestrado, doutorado e demais professores da farmacologia:
Ana Paula Bentes, Bruno Bueno, Bruna Benso, Carina Denny, Irlan Almeida (Iran), Karina
Cogo, Luciano Serpe, Marcelo Franchin, Marcos Cunha, Laila Facin, Jonny Burga, Lívia
Galvão, Luciana Berto, Paula Sampaio, Sérgio Rochelle, Talita Graziano e Verônica Freitas,
pelo companheirismo e a certeza de levar essas amizades pelo resto da vida.
Em especial aos meus amigos mais do que especiais, Jaiza, Stephany, Camilinha,
Cleiton, Luiz Eduardo, Diogo (Gazé), Augusto (Gutão), Felipe (Anacleto), Klinger com quem
sempre pude contar em todos os momentos e contribuíram enormemente com o
desenvolvimento desse projeto (contribuição laboral ou moral), sem nunca perder o bom humor.
Aos meus amigos da terrinha, Thiago, Luis Gustavo, Israel, Higor e Marcão, por
compartilharem das minhas insanidades.
Agradeço também as pessoas, amigos e família que não foram citados aqui, mas
estiveram na torcida pela realização e sucesso deste trabalho, obrigada!
À terrinha, Itararé - SP, onde cresci e aprendi a ser feliz.
RESUMO
Anestésicos tópicos são comumente aplicados na mucosa oral para redução ou prevenção da
dor durante a realização de procedimentos médicos ou odontológicos, porém não há um
anestésico tópico ideal para uso em mucosa oral com eficácia garantida na maioria dos casos,
em baixas concentrações e com baixo tempo de latência. O objetivo do presente trabalho foi
desenvolver e avaliar hidrogéis à base de Carbopol® Ultrez 10 contendo os anestésicos locais
lidocaína e prilocaína (5%) encapsulados (CNLP) em nanocápsulas de poli (ε-caprolactona)
(PCL) ou não (CLP) para uso tópico na mucosa oral. As nanocápsulas em suspensão foram
caracterizadas em termos de tamanho, índice de polidispersão (PDI), carga superficial
(potencial zeta-ZP), análise de rastreamento da partícula (NTA), estabilidade físico-química,
análise estrutural, eficiência de encapsulação (EE%) e cinética de liberação in vitro. As
formulações semissólidas foram submetidas à análise reológica, propriedades mecânicas
(dureza, compressibilidade, coesividade e adesividade), estabilidade acelerada, cinética
liberação in vitro, avaliação da capacidade mucoadesiva in vitro (força de destacamento)
citotoxicidade em cultura de células HaCat e FGH, e capacidade de permeação in vitro através
do epitélio de mucosa jugal e palatina. As formulações foram avaliadas quanto à eficácia
anestésica tópica em ratos Wistar, em modelo de tail-flick. As nanocápsulas desenvolvidas
apresentaram tamanho de aproximadamente de 400 nm, PDI ≤ 0.262, ZP -20 a -25 mV,
contendo 2.9x1012 part./mL avaliado pelo método NTA, com estabilidade físico-química
durante 180 dias. As nanocápsulas apresentaram altos níveis de encapsulação tanto da lidocaína
quanto da prilocaína (83% e 72%, respectivamente) e apresentaram cinética de liberação com
modelagem matemática semi-empírica (Korsmeyer-Peppas, R2 ≥ 0,985; LDC n = 0.82, PLC n
= 0.72). As formulações de hidrogéis apresentaram fluxo pseudoplástico não-newtoniano,
característicos de géis, além de boas propriedades mecânicas, capacidade mucoadesiva e estável
por 6 meses. As formulações apresentaram menor citotoxicidade nas células avaliadas em
relação à formulação comercial (EMLA®), e apresentaram menor fluxo de permeação dos
anestésicos locais através dos epitélios de mucosa oral. Além disso, a formulação de anestésicos
locais encapsulados apresentou melhor eficácia anestésica tópica em relação ao EMLA. Em
conclusão, o presente estudo desenvolveu uma formulação semissólida para liberação
sustentada de anestésicos locais para uso em mucosa oral com boas propriedades mecânicas,
estável, mucoadesivo e com boa eficácia e duração anestésica in vivo, sendo um potencial
candidato para futuros testes em humanos. Esses dados abrem perspectivas para uma melhora
na qualidade da anestesia tópica.
Palavras-chave: Lidocaína, Prilocaína, Nanocápsulas de poli(ε-caprolactona), Hidrogel,
Mucosa oral, Anestesia tópica, Odontologia
ABSTRACT
Topical anesthetics are commonly applied to the oral mucosa for reduction or prevention of
pain during medical or dental procedures, but there is no topical anesthetic ideal for use in oral
mucosa with efficacy guaranteed in most cases in low concentrations and with low time latency.
The objective of the present work was to develop and evaluate Carbopol-based hydrogels
containing local anesthetics lidocaine and prilocaine (5%) encapsulated (CNLP) or not (CLP)
in poly (ε-caprolactone) (PCL) nanocapsules for topical use in Oral mucosa. The suspension
nanocapsules were characterized in terms of size, polydispersity index (PDI), surface charge
(zeta-ZP potential), particle tracking analysis (NTA), physicochemical stability, structural
analysis, encapsulation efficiency) and in vitro release kinetics. Semi-solid formulations were
submitted to rheological analysis, mechanical properties (hardness, compressibility,
cohesiveness and adhesiveness), accelerated stability, in vitro release kinetics, in vitro
mucoadhesive capacity (posting strength) cytotoxicity in HaCat and HGF cell culture, and
capacity of in vitro permeation through the epithelium of jugal mucosa and palatine. The
formulations were evaluated for topical anesthetic efficacy in rats. The nanocapsules developed
had a size of approximately 400 nm, PDI ≤ 0.262, ZP -20 a -25 mV, containing 2.9x1012
part./mL evaluated by the NTA method, with physico-chemical stability for 180 days. The
nanocapsules had high levels of encapsulation of both lidocaine and prilocaine (83% and 72%,
respectively) and presented a semi-empirical release mechanism (Korsmeyer-Peppas, R2 ≥
0.985). The hydrogel formulations showed a non-Newtonian pseudoplastic flow, characteristic
of a semisolid pharmaceutical form, besides good mechanical properties, mucoadhesive
capacity and stable for 6 months. The formulations had lower cytotoxicity in the cells evaluated
in relation to the commercial formulation (EMLA®), and presented lower permeation flux of
the local anesthetics through oral mucosa epithelia. In addition, the formulation of encapsulated
local anesthetics presented better anesthetic efficacy than EMLA. In conclusion, the present
study developed a semi-solid formulation for sustained release of local anesthetics for use in
oral mucosa with good mechanical properties, stable, mucoadhesive and with good efficacy and
in vivo anesthetic duration, being a potential candidate for future human trials. These data open
perspectives for an improvement in the quality of topical anesthesia.
Keywords: Lidocaine; prilocaine; polymeric nanocapsules; hydrogel; oral mucosa; topical
anesthesia; local anesthetics
SUMÁRIO
1 INTRODUÇÃO ................................................................................................................ 12
2 ARTIGO: Hybrid Hydrogel and Polymeric nanocapsules co-loaded with lidocaine
and prilocaine aiming topical intraoral anesthesia ............................................................. 16
3 CONCLUSÃO .................................................................................................................. 55
REFERËNCIAS ..................................................................................................................... 56
ANEXOS ................................................................................................................................. 60
Anexo 1 - Certificado de aprovação do estudo pela CEUA.................................................60
Anexo 2 - Comprovante de submissão do trabalho..............................................................61
12
1 INTRODUÇÃO
Anestésicos tópicos são comumente aplicados na mucosa oral para redução ou
prevenção da dor durante a realização de diversos procedimentos na área da saúde como injeção
da solução de anestésico local, colocação de bandas para realização de isolamento absoluto,
extração simples de dentes decíduos, procedimentos cirúrgicos realizados na mucosa oral
(como biópsia ou drenagem de abcesso por exemplo), intubação endotraqueal, e endoscopia
digestiva (Lee, 2016).
A lidocaína, anestésico local do tipo amino-amida, foi disponibilizada comercialmente
a partir de 1948, e desde então, é o anestésico local padrão ouro para comparação nessa classe
de fármacos devido a sua boa eficácia, reduzida alergenicidade, baixo custo e boa segurança
clínica (Moore & Hersh, 2010; Ogle & Mahjoubi, 2012). Para uso tópico, a lidocaína está
disponível comercialmente nas concentrações de 2, 4, 5 e 10% em forma de pomadas, geleia,
creme e sprays para uso em mucosas (Lee, 2016).
Desenvolvido em 1980, e aprovado nos EUA pelo FDA (Food and Drug
Administration), em 1992, o EMLA® (mistura eutética de lidocaína a 2,5% e prilocaína a 2,5%,
AstraZeneca) vem sendo testado para uso em mucosa oral (Svensson et al., 1992; Donaldson &
Meechan, 1995; Vickers et al., 1997; McMillan et al., 2000; Sohmer et al., 2004; Abu Al-Melh
et al., 2005; Al-Melh & Andersson, 2007; Al-Melh & Andersson, 2008; Franz-Montan et al.,
2015).
Embora seja utilizada na cavidade oral, segundo a bula do fabricante, a formulação
comercial EMLA® foi desenvolvida para uso dermatológico e apresenta indicação apenas para
mucosa genital, porém não para outros tipos de mucosa, como a oral por exemplo. Nesse tipo
de mucosa, apresenta eficácia em reduzir a dor à punção, mas não a dor decorrente da injeção
da solução anestésica em mucosa palatina (Franz-Montan et al., 2012). A formulação também
apresenta algumas características organolépticas como gosto amargo (pH = 9.0), sensação de
queimação durante a aplicação e baixa viscosidade, que podem dificultar sua aceitação pelos
pacientes (Meechan & Donaldson, 1994). Sendo assim, não há um anestésico tópico ideal para
uso em mucosa oral com eficácia garantida na maioria dos casos, em baixas concentrações e
com baixo tempo de latência (Meechan, 2002; Franz-Montan et al., 2012; Franz- Montan et al.,
2017).
Os sistemas de liberação de fármacos (drug delivery systems) tem como objetivos
principais melhorar a biodisponibilidade, reduzir a toxicidade e, dessa maneira, aumentar o
índice terapêutico dos fármacos. Assim, os anestésicos locais (AL) são alvos de pesquisas
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visando a encapsulação, associação ou complexação dos mesmos em sistemas de liberação
sustentada, baseados em ciclodextrinas, lipossomas e biopolímeros (micro ou nanopartículas
poliméricas), buscando a melhora na eficácia e diminuição da toxicidade (de Araújo et al., 2013;
Pitorre et al., 2017).
As micro e nanopartículas poliméricas tem se tornado um dos sistemas de liberação de
fármacos mais estudados devido à elevada estabilidade em temperatura ambiente e em
temperatura corporal e pela possiblidade de uso por diversas vias (oral, tópica intraoral,
dérmica, ocular e parenteral) (Soppimath et al., 2001; Schaffazick et al., 2003; Andrade et al.,
2014; Shah et al., 2014; Negi et al., 2015; Basha et al., 2015; Ribeiro et al., 2016; Manaia et al.,
2017; Pham et al., 2018).
As nanopartículas poliméricas são esferas coloidais sólidas, que variam de 10 a 1000
nm, e podem ser classificadas, dependendo da composição e organização estrutural, em
nanoesferas ou nanocápsulas. As nanoesferas são compostas por matriz polimérica densa e as
nanocápsulas por núcleo oleoso envolto por uma parede polimérica. O fármaco pode estar
dissolvido na matriz polimérica, no núcleo ou adsorvido à parede polimérica (Schaffazick et
al., 2003; Singh & Lillard, 2009; Mora-Huertas et al., 2010).
O grupo de pesquisa ao qual este projeto está vinculado, caracterizou a encapsulação de
benzocaína, lidocaína e articaína em nanocápsulas sintetizadas com diferentes polímeros, como
o poli(D,L-lactídeo-co-glicolídeo), poli(L-lactídeo) e o poli(épsilon-caprolactona), e
demonstrou que essas associações apresentaram boa estabilidade, liberação sustentada in vitro
e melhora da eficácia anestésica in vivo (de Melo et al., 2011; Moraes et al., 2011; De Melo et
al., 2012; Ramos Campos et al., 2013; Silva de Melo et al., 2014; Melo et al., 2018).
A nanocápsula de poli(épsilon-caprolactona) é considerada um sistema de liberação
promissor tendo em vista que o polímero é biodegradável e biocompatível, utilizado para
encapsulação de diversos fármacos (Woodruff & Hutmacher, 2010). Além disso, podem ser
empregadas tanto para formulações líquidas, quanto semissólidas como os hidrogéis (Woodruff
& Hutmacher et al., 2010; Pohlmann et al., 2013; Melo et al.,2018).
Um hidrogel pode ser definido como uma preparação semissólida formada por uma rede
tridimensional obtida a partir de ligações cruzadas de um polímero ou copolímero em um
veículo líquido aquoso, no qual adquire consistência viscosa, impedindo a sedimentação de
partículas nele incorporado (Kopecek, 2007; Annabi et al., 2014; Sahoo et al., 2014; Sharpe et
al., 2014).
Existem diversos polímeros utilizados para formar hidrogéis, sendo os polímeros
carboxivinílicos ou carbômeros (carbopols) um dos mais utilizados. Os carbopols são
14
considerados materiais com baixo potencial tóxico e irritante, sem nenhuma evidência de
hipersensibilidade à sua estrutura (Sahoo et al., 2014). Esse tipo de polímero aniônico deve
sofrer um processo de neutralização durante o seu preparo com uma base, a fim de se adquirir
a viscosidade desejável (da Silva et al., 2008).
Sendo assim, uma tendência atual é a criação de sistemas híbridos compostos por
exemplo de um hidrogel e um sistema nanoparticulado, como as nanopartículas poliméricas.
Um sistema híbrido é constituído de dois materiais distintos formando uma única formulação
com propriedades físico-químicas e biológicas únicas inalcançável pelos componentes quando
separados (Merino et al., 2015; Gao et al., 2016). Essa dupla interface 3D (Figura 1) atrai
atenção para enfrentar desafios biológicos e médicos, permitindo a criação de materiais com
propriedades superiores de liberação sustentada e direcionada do fármaco no local do
tratamento (Gao et al., 2016).
Figura 1. Ilustração esquemática das nanopartículas inseridas na rede de um hidrogel.
Nosso grupo de pesquisa demonstrou a eficácia clínica de diversas formulações à base
de Carbopol® Ultrez10 contendo AL como benzocaína, lidocaína e ropivacaína encapsulados
em lipossomas convencionais como anestésico tópico prévio à anestesia local em mucosa oral
em voluntários sadios (Franz-Montan et al., 2007; Franz-Montan et al., 2010a; Franz-Montan
et al., 2010b; Franz-Montan et al., 2015).
Em decorrência da eficácia demonstrada para o anestésico tópico EMLA® em anestesia
de mucosa oral (McMillan et al., 2000; Al-Melh & Andersson, 2007 e 2008; Franz-Montan et
al., 2015) e da melhor eficácia in vivo de benzocaína associada à nanocápsulas de poli(épsilon-
caprolactona) (De Melo et al., 2012) a hipótese do presente trabalho foi que a utilização desse
sistema de liberação contendo lidocaína e prilocaína associados ao hidrogel à base de Carbopol®
apresentasse boa eficácia anestésica tópica.
Nesse contexto, o objetivo deste trabalho foi avaliar a estabilidade, as características
físico-químicas e perfil de liberação dos anestésicos locais lidocaína (2,5%) e prilocaína (2,5%)
15
em nanocápsulas de poli(épsilon-caprolactona) em suspensão; e avaliar a estabilidade,
citotoxicidade, propriedades mecânicas, reológicas e mucoadesivas, perfil de liberação e de
permeação, e eficácia in vivo desse sistema formulado em um hidrogel à base de Carbopol®
objetivando aplicação tópica em mucosa oral.
16
MANUSCRIPT SUBMITTED TO SCIENTIFIC REPORTS
HYBRID HYDROGEL AND POLYMERIC NANOCAPSULES CO-LOADED
WITH LIDOCAINE AND PRILOCAINE AIMING TOPICAL INTRAORAL
ANESTHESIA
Authors:
Bruno Vilela Muniz¹. MSc, PhD student
Diego Baratelli². MSc
Stephany Di Carla¹. MSc student
Luciano Serpe¹. PhD
Camila Batista da Silva¹. PhD
Viviane Aparecida Guilherme3. PhD
Lígia de Morais Ribeiro Nunes3. PhD
Cintia Maria Saia Cereda3. PhD
Eneida de Paula³. PhD, Full Professor
Maria Cristina Volpato1. PhD, Full Professor
Francisco Carlos Groppo¹. PhD, Full Professor
Leonardo Fernandes Fraceto². PhD, Associate Professor
Michelle Franz-Montan1*. PhD, Assistant Professor
1 Department of Physiological Sciences, Piracicaba Dental School, University of Campinas –
UNICAMP, Piracicaba, São Paulo, Brazil
² São Paulo State University – UNESP, Institute of Science and Technology of Sorocaba,
Department of Environmental Engineering, Sorocaba, São Paulo, Brazil
17
³ Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas
– UNICAMP, Campinas, São Paulo, Brazil
*Corresponding author:
Michelle Franz-Montan
Av. Limeira 901, Bairro Areião, 13414-903, Piracicaba, São Paulo, Brazil
Tel.: 55 19 2106 5306; FAX: 55 19 2106 5306; E-mail: [email protected]
18
Abstract
This study reports the development of nanostructured hydrogels for the sustained release of the
eutectic mixture of lidocaine and prilocaine (both at 2.5%) for oral topical use. The local
anesthetics, free or encapsulated in poly(ε-caprolactone) nanocapsules, were incorporated into
Carbopol® hydrogel. The nanoparticle suspensions were characterized in vitro in terms of
particle size, polydispersity, and surface charge, using dynamic light scattering measurements.
The nanoparticle concentrations were determined by nanoparticle tracking analysis. Evaluation
was made of physicochemical stability, structural features, encapsulation efficiency, and in
vitro release kinetics. The Carbopol® hydrogels were submitted to rheological, accelerated
stability, and in vitro release tests, as well as determination of mechanical and mucoadhesive
properties, in vitro cytotoxicity towards FGH and HaCat cells, and in vitro permeation across
buccal and palatal mucosa. Anesthetic efficacy was evaluated using Wistar rats. Nanocapsules
were successfully developed that presented desirable physicochemical properties and a
sustained release profile. The hydrogel formulations were stable for up to 6 months under
critical conditions and exhibited non-Newtonian pseudoplastic flows, satisfactory
mucoadhesive strength, non-cytotoxicity, and slow permeation across oral mucosa. In vivo
assays revealed higher anesthetic efficacy in tail-flick tests, compared to a commercially
available product. In conclusion, the proposed hydrogel has potential for provision of effective
and longer-lasting superficial anesthesia at oral mucosa during medical and dental procedures.
These results open perspectives for future clinical trials.
Keywords: Local anesthetics, topical anesthesia, oral mucosa, polymeric nanocapsules,
hydrogel, lidocaine, prilocaine.
19
1. Introduction
Topical anesthetics are applied superficially to reduce or control pain in medical and
dental procedures such as local anesthesia injection, placement of orthodontic bands, simple
extraction of primary teeth, rubber-dam clamp placement, biopsies, abscess incision,
endotracheal intubation, and endoscopy (Lee, 2016). Nevertheless, variables such as the class
and concentration of the anesthetic agent, pH, additives, contact time at the mucosa, duration
of action, and site of application influence the success of superficial anesthesia (Meechan,
2008).
Lidocaine (LDC) and prilocaine (PLC) are amine-amide local anesthetics (LAs) widely
used in biomedical procedures worldwide (Lee, 2016). When these LAs are combined, they
form an eutectic mixture that is commercially available as EMLA®, a topical formulation
originally designed for dermal use, with proven effectiveness inside the oral cavity
(Daneshkazemi et al., 2016). However, the formulation also presents organoleptic
characteristics such as a bitter taste (pH = 9.0) and a burning sensation during application, which
can hinder its acceptance by patients (Meechan & Donaldson, 1994). The advantages and
disadvantages of EMLA® led to the development of new formulations containing the eutectic
mixture of LDC and PLC in drug delivery systems such as mucoadhesive films and
nanostructured lipid carriers, aiming at topical oral application (Couto et al., 2017; Ribeiro et
al., 2016).
Several approaches have been adopted for optimization of the effective loading of LAs
into polymeric nanoparticles. The encapsulation of benzocaine, lidocaine, and articaine in
polymeric nanoparticles composed of poly(lactide-co-glycolide), poly-L-lactide, and poly-ε-
caprolactone resulted in long-term stability, sustained release, and increased anesthetic efficacy
in vivo (de Melo et al., 2011; Moraes et al., 2011; de Melo et al., 2012; Ramos Campos et al.,
2013; Silva de Melo et al., 2014; Melo et al., 2018).
Polymeric nanocapsules consist of an oily core and an ultrathin polymeric wall,
providing particles smaller than 1 μm (Fessi et al., 1989). Poly-ε-caprolactone (PCL) is among
the biodegradable polymers most widely employed for the preparation of nanocapsules, due to
its desirable properties for incorporation in semisolid drug delivery systems, such as
hydrophobicity and biocompatibility (Woodruff & Hutmacher, 2010).
Hydrogels are three-dimensional polymer networks cross-linked by physical or
chemical agents, which can absorb large amounts of biological fluids. Their useful properties
include biocompatibility, flexibility, and suitable rheological behavior, enabling their use in a
wide range of applications including wound healing and topical delivery of active molecules at
20
the skin and mucosa (Alves et al., 2011; Singh & Lee, 2014). Carbopol® is an acrylic acid
copolymer employed as a matrix in several semisolid formulations, with interesting properties
such as mucoadhesion, which promotes adherence to the mucus layer, leading to prolonged
residence times of incorporated drugs (Netsomboon & Bernkop-Schnürch, 2016; Singh & Lee,
2014). Carbopol® formulations containing benzocaine (Franz-Montan et al., 2010), lidocaine
(Franz-Montan et al., 2015), or ropivacaine (Franz-Montan et al., 2007, 2010, 2012)
encapsulated in liposomes were shown to be effective in promoting topical anesthesia in the
human oral mucosa.
The objective of the present study was to evaluate the performance of a hybrid system
based on poly(ε-caprolactone) nanocapsules in Carbopol® hydrogel, which was developed for
the topical oral delivery of 5% lidocaine-prilocaine.
2. Results and Discussion
2.1. Characterization of poly(ε-caprolactone) nanocapsules
The encapsulation of different LAs using other polymeric nanocapsules has been
described previously (Campos et al., 2013; Melo et al., 2014; Moraes et al., 2011; You et al.,
2017), with the aim of prolonging analgesia and minimizing side effects (de Paula et al., 2012).
Particle size and polydispersity index (PDI) are critical parameters of nanostructured systems
that influence drug encapsulation efficiency, formulation stability, and release behavior
(Mohanraj & Chen, 2007). The surface charge of nanoparticles (zeta potential, ZP) can be used
as a predictive index of particle stability, as well as to elucidate the location of the active
molecules in the nanocapsule (Berbel Manaia et al., 2017). The nanoparticle concentration is
another parameter that affects the stability and biological activity of drug delivery systems
(Ribeiro et al., 2018).
The results obtained for particle size, PDI, ZP, nanoparticle concentration, and pH,
according to time, for nanocapsules with and without LAs, are provided in Figure S1
(Supplementary Material). For the freshly prepared samples (Day 0), the analyzed parameters
of the suspensions containing LDC+PLC were similar to those reported elsewhere for poly(ε-
caprolactone) nanocapsules containing LAs (Melo et al., 2018).
The nanocarrier showed high encapsulation capacities for both LDC (83±3%) and PLC
(72±3%), indicating its superior performance, compared to polymeric nanospheres that usually
present lower encapsulation efficiencies due to the absence of the oily inner core (Jones &
Grainger, 2009). The higher EE% for LDC than for PLC could be explained by the higher
octanol/water partition coefficient of LDC (P = 304), compared to PLC (P = 129), for the
21
molecules in their neutral forms (Strichartz et al., 1990). Hence, PLC would be more likely to
interact with the aqueous phase and the polymer matrix of the nanocapsules, while LDC would
exhibit better interaction with the oily nucleus (de Melo et al., 2011), due to its greater
hydrophobicity.
The stability of the formulation during 180 days of storage at room temperature (25 C)
is also shown in Figure S1. Changes in the mean diameter of the nanocapsules were detected
after 90 days of storage (p < 0.05). The PDI values confirmed that the monodisperse size
distribution was maintained until the end of the experiment, as required for this type of
formulation.
Another factor indicating the stability of the system was the high ZP modulus, reflecting
repulsion among the nanocapsules in suspension (Mohanraj & Chen, 2006). The nanoparticles
containing LDC and PLC presented satisfactory ZP values throughout the storage period, with
continued inter-particle repulsion that could be attributed to the presence of PVA, which could
form a barrier around the nanocapsules, resulting in steric stabilization (Ramos Campos et al.,
2013).
Nanoparticle tracking analysis (NTA) is a technique for monitoring the physicochemical
stability of nanoparticulate systems that can provide unique information concerning the particle
concentration (Ribeiro et al., 2018). In the present case, there were no significant changes in
nanoparticle concentration (p > 0.05) throughout the study period, corroborating the other
stability parameters determined by DLS, hence confirming the stability of the system.
The pH decreased over time (p < 0.01), as reported previously (Ramos Campos et al.,
2013), indicating degradation of the polymers and formation of acidic products by hydrolysis
of the polymer chains, suggesting a possible lack of stability and breakdown of the
nanocapsules. However, considering the observed stability of the particle size distribution, the
polymer hydrolysis did not affect the structural properties of the nanoparticles. The morphology
of the nanocapsules was confirmed by transmission electron microscopy (Figure 1).
22
Figure 1. Transmission electron micrographs of PCL with (a, b) and without (c, d) LDC+PLC,
at two different magnifications (10,000x and 60,000x).
The micrographs revealed some differences between the nanocapsules with and without
LDC+PLC. The spherical core-shell structure for the nanocapsules with the LAs
(PCL/LDC+PLC) (Figures 1a and 1b) suggested that the LDC and PLC bases were incorporated
into the hydrophobic core, as previously reported for lidocaine-loaded poly(ε-caprolactone)-
poly(ethylene glycol) nanoparticles (Yin et al., 2009).
The ATR-FTIR technique was used to investigate the interactions among the
nanocapsules and the LAs. The FTIR-ATR spectra (Figure 2a) of the poly(ε-caprolactone)
nanocapsules (denoted NP) and the LAs showed typical bands of the molecules (Gehrcke et al.,
2018; Ribeiro et al., 2016). In the spectrum of the NP formulation, a band at 1580-1450 cm-1
could be attributed to C=C vibrations, while a band at 1735-1650 cm-1 corresponded to C=O
and a band at 3000-2800 cm-1 was related to C-H of saturated carbons (Campos et al., 2013; de
Melo et al., 2014). The spectroscopic profile of the polymeric nanoparticles remained similar
after encapsulation of PLC+LDC. The bands in the region 2950-3305 cm-1 for the pure LAs
completely disappeared in the spectrum of the NP/LDC+PLC formulation, indicating
dissolution of the compounds in the nanocapsule core and the maintenance of particle integrity.
23
4000 3500 3000 2500 2000 1500 1000 500
LDC
3248 3019
2965 2925
2800
16571499
15
95
1374
1209
10
67
98
8
760
PLC3303
3233
1646
14
91 748
1610
1524
1138
941
30
35
2965
2878
2830
1374
1280
731847
1113
1253
1378
1469
17512870
29443442
NP
1171
756
1113
11621245
1303
13
781461
15
14
15
931693
17432870
2944
Tra
nsm
ita
nce
(a
.u.)
NP/LDC-PLC
3467
0 50 100 150 200 250
69 °C
LDC
43 °C
PLC
NP-LDC/PLC
44 °C
184 °C
H
ea
t F
low
(W
/g)
Temperature (°C)
NP
53 °C
191 °C
EXO up
Figure 2. FTIR-ATR spectra (a) and DSC analysis (b) of the poly(ε-caprolactone) nanocapsules
(NP and NP/LDC-PLC), lidocaine (LDC), and prilocaine (PLC).
DSC is a thermoanalytical technique that can provide information concerning
polymorphic changes in materials (Knopp et al., 2016). The calorimetry results (Figure 2b)
b
a
24
revealed the endothermic peaks corresponding to the melting points of the substances at 43 C
(PLC), 69 C (LDC), and 53 C (PCL) (Ribeiro et al., 2016; Youm et al., 2012). The PCL/LDC-
PLC formulation showed a lower melting point (44 C), compared to PCL, confirming the
interaction among the components.
Figure S2 (Supplementary Material) shows the in vitro release of LDC and PLC from
the PCL/LDC+PLC formulation, compared to the LAs free in solution. A burst release effect
was observed for the LAs in solution, with 50% release of the drugs within the first 30 min.
Complete release of the free LAs occurred within 4 h, while the release of the encapsulated
drugs did not reach 100%, even after 18 h. The faster release of PLC was in agreement with its
lower encapsulation efficiency, compared to LDC.
As shown in Table S1 (Supplementary Material), the mathematical model that provided
the best description of LDC and PLC release from the nanocapsules was the semi-empirical
Korsmeyer-Peppas model, which is commonly applied to nanostructured systems. This
mathematical model has been used previously to describe the release of LDC and PLC loaded
in polymeric nanospheres (Ramos-Campos et al., 2013). Based on the values of the release
exponent (n), the release of the LAs from the nanocapsules could be characterized as being due
to non-Fickian anomalous transport (0.45 < n < 0.89) (Peppas & Sahlin, 1989). Hence, there
was more than one diffusion mechanism involved, with rapid diffusion of non-encapsulated
LAs and slow diffusion of encapsulated LAs, with the latter requiring the compounds to cross
the physical polymer barrier in order to be released (Ferrero et al., 2010).
2.2. Characterization of the hydrogels
2.2.1. Rheological studies
Rheological analysis can be used to assist in elucidating the potential of innovative
pharmaceutical formulations (Lee et al., 2009). Figure 3 shows the rheological behaviors (flow
curve profiles) of Carbopol® hydrogels containing LDC and PLC (5%), free or associated with
the poly(ε-caprolactone) nanocapsules (CLP and CNLP, respectively), in comparison to
EMLA®.
25
Figure 3. Rheological profiles showing the shear stress as a function of shear rate for the
Carbopol® hydrogels containing LDC and PLC (5%), free or associated with the poly(ε-
caprolactone) nanocapsules (CLP and CNLP, respectively), in comparison with the commercial
formulation composed of the eutectic mixture of LDC and PLC (EMLA®). The inset shows the
hysteresis areas. For each formulation, the upper and lower curves correspond to ascending and
descending measurements, respectively. The SD values were below 5%.
For all formulations, the rheograms reflected non-Newtonian pseudoplastic flows. The
viscosity depends on the shear rate (Lippacher et al., 2004). In other words, when the shear rate
increases with increasing shear stress (Lee et al., 2009), typical properties of semi-solid
formulations are observed (Ourique et al., 2011).
Thixotropy is a rheological indicator of viscosity. This parameter can be calculated by
measuring the area contained in the hysteresis loop of the rheological curves, as shown in Figure
3 (Lee et al., 2009). A larger hysteresis area was found for the non-encapsulated LDC-PLC
Carbopol® hydrogel, suggesting delayed recovery of its structure (Gaspar & Campos, 2003).
An intermediate hysteresis area value is desirable in order to improve the shelf-life and provide
good spreadability of semisolid formulations, as observed for the CNLP hydrogel and EMLA®.
Knowledge of thixotropic properties can help in increasing the retention times of topically
applied formulations, leading to better therapeutic efficacy (Lee et al., 2009).
26
2.2.2. Mechanical and mucoadhesive properties
The mechanical properties obtained by texture profile analysis (TPA) for all the
hydrogels are shown in Table 1.
Table 1. Mean (±SD) values obtained for the mechanical properties and mucoadhesive
strengths of Carbopol® hydrogels containing LDC and PLC (5%), free or encapsulated in
poly(ε-caprolactone) nanocapsules (CLP and CNLP, respectively), in comparison with
EMLA®.
Mechanical properties Mucoadhesive strength
Formulation
Hard.
(N)
Comp.
(N.mm)
Cohes. Adhes. (N.mm) Detachment force (N)
CNLP 0.383±0.028a 1.390±0.168a 0.694±0.016a 0.165±0.021a 0.024±0.005a,b
CLP 0.186±0.011b 0.727±0.040b 0.747±0.027b 0.123±0.006b 0.022± 0.004a
EMLA® 0.133±0.007c 0.500±0.030c 0.852±0.016c 0.173±0.006a 0.032±0.004b Hard.: hardness; Comp.: compressibility; Cohes.: cohesiveness; Adhes.: adhesiveness. Different letters indicate
significant differences among the hydrogels for each parameter evaluated (ANOVA/Tukey-Kramer test, p < 0.05).
Each parameter was analyzed separately (n = 5).
An ideal semisolid formulation for use on the oral mucosa should have properties that
enable easy application and spreading, determined by hardness and compressibility,
respectively. Its permanence at the desired site without disintegrating can be predicted by higher
values of adhesiveness and cohesiveness (Calixto et al., 2018; Cubayachi et al., 2015).
The TPA results indicated that the CNLP hydrogel possessed good mechanical
properties (Eiras et al., 2017; Jones et al., 2002). The presence of the poly(ε-caprolactone)
nanocapsules increased hardness (p < 0.05) and compressibility (p < 0.05), with the values
obtained being within the range reported to be ideal for topical oral application (Jones et al.,
2000). The cohesiveness and adhesiveness values were also satisfactory and were comparable
to those reported for other semisolid formulations designed for topical application to the oral
mucosa (Cubayachi et al., 2015; Karavana et al., 2012).
The mucoadhesive strength, evaluated in terms of the detachment force, represents the
force required to detach the hydrogel from the mucosal surface. The detachment force obtained
for CNLP was similar to that for the commercial formulation (p > 0.05) and higher than for
CLP (p < 0.05) (Table 1). These results indicated that the presence of the nanocapsules did not
affect the mucoadhesive properties of Carbopol®, in agreement with the findings of Frank et al.
(2017), who also observed that the presence of poly(ε-caprolactone) nanocapsules did not alter
the mucoadhesive capacities of carboxymethylcellulose and chitosan gels.
27
2.2.3. Accelerated stability study
Accelerated stability testing was used to observe possible physicochemical changes
(weight loss, LA dosage, and pH) that could occur during storage of the hydrogels as a result
of degradation of components of the formulation. The study was performed at constant
controlled temperature (40 ± 2 oC) and humidity (75 ± 5% RH), for up to 6 months, according
to the ICH procedure (ICH Expert Working Group, 2003). As can be seen in Table S2
(Supplementary Material), storage did not affect the physicochemical properties of the CNLP
formulation (p > 0.05), indicative of good stability.
2.2.4. In vitro release kinetics and mathematical modeling
The profiles of LDC and PLC release from the hydrogels are shown in Figures S3(a)
and S3(b), respectively. The release kinetics data, as evaluated by the determination coefficient
values of the applied mathematical models, are shown in Table S3 (Supplementary Material).
The Weibull mathematical model provided the best description of the release
mechanism (R2 ≥ 0.946). This model consists of an empirical equation that is often used to
describe the process of drug delivery using spherical media (Zhou et al., 2014). Based on the
“b” value, the release of LDC in the absence of nanocapsules (CLP, b = 0.95) was according to
a non-Fickian process (0.75 < b < 1) involving a combination of more than one release
mechanism. This indicated that the local anesthetic was associated with the Carbopol® structure
and depended on relaxation of the structure for its release and subsequent diffusion to the
external environment. In the case of PLC (CLP, b = 0.13), the compound showed a disordered
distribution in the spaces between the three-dimensional structures of the gel (b < 0.35). In the
presence of the nanocapsules (CNLP), the release of LDC (b = 0.72) changed to Fickian release
(0.69 < b < 0.75), indicating that it occurred by diffusion between the gel layers. However, in
this formulation, the release of PLC (b = 0.93) changed to non-Fickian transport (0.75 < b < 1),
indicating the existence of two release mechanisms, with swelling and relaxation of the
nanocapsule polymers, followed by subsequent diffusion from the hydrogel to the external
medium (Papadopoulou et al., 2006).
2.2.5. In vitro permeation studies
The parameter values for permeation of LDC and PLC from the different hydrogel
formulations across oral epithelia (palatal = keratinized model; buccal = non-keratinized model)
are shown in Table 2.
28
Table 2. Mean (±SD) values of the steady-state flux (Jss) and lag time for permeation of
lidocaine (LDC) and prilocaine (PLC), free or associated with poly(ε-caprolactone)
nanocapsules (CLP and CNLP, respectively), from Carbopol® hydrogels across porcine buccal
and palatal mucosal epithelium, in comparison to EMLA®.
Epithelium
Local
anesthetic Formulation Jss (µg.cm−2.h−1) Lag time (h) R2
Buccal mucosa
LDC
CNLP 160.88±31.20a 0.57±0.11a 0.993±0.005
CLP 248.03±14.60b 0.34±0.20a.b 0.997±0.002
EMLA® 280.32±44.43b 0.09±0.04b 0.997±0.002
PLC
CNLP 172.24±20.99c 0.50±0.15 0.993±0.008
CLP 168.14±20.40c 0.28±0.09 0.997±0.003
EMLA® 283.27±43.46d 0.27±0.14 0.995±0.002
Palatal mucosa
LDC
CNLP 119.63±8.83a 0.00±0.00 0.991±0.003
CLP 207.18±25.79b 0.21±0.21a 0.996±0.003
EMLA® 316.82±16.93c 0.00±0.00 0.994±0.003
PLC
CNLP 92.10±8.75d 0.00±0.00 0.994±0.002
CLP 118.06±13.05e 0.17±0.05 # 0.995±0.006
EMLA® 338.33±16.3f 0.00±0.00 0.995±0.004
PLC: prilocaine; LDC: lidocaine; Jss: steady state flux; R2: coefficient of determination of the linear regression
model. Different letters indicate a significant difference (p < 0.05, ANOVA/Tukey test). Each permeation
parameter was analyzed separately for each local anesthetic. Mean ± SD (n = 6).
Due to its similarity with human tissues in terms of structure, lipids, and permeability,
porcine buccal mucosa is frequently used for in vitro drug permeation studies (Diaz-Del
Consuelo et al., 2005). The palatal mucosa was selected because of its keratinized layer, which
provides a more effective permeation barrier and simulates the application site used in clinical
studies (Franz-Montan et al., 2016).
The fluxes of the two LAs were significantly lower (p < 0.05) for CNLP than for the
commercial EMLA® formulation. This could be explained by the high degree of encapsulation
of the drugs in the PCL nanocapsules (Table 2), which decreased the supply of the free drugs
crossing the barrier. In addition, the permeation fluxes were influenced by the nature and
composition of the formulations tested, which altered the solubility and partitioning of the
drugs, compared to the commercial formulation, which is a cream (de Araujo et al., 2010; Franz-
Montan et al., 2015; Moghadam et al., 2013).
Considering the fluxes of the LAs across the buccal mucosa, LDC showed a significant
effect of encapsulation, since both CLP and EMLA® presented higher LDC fluxes (p > 0.05),
compared to CNLP, suggesting that the high encapsulation degree of LDC (83%) reduced its
rate of transfer across the barrier, as reported elsewhere (Maestrelli et al., 2009; Puglia et al.,
2011). On the other hand, the PLC flux was only influenced by the composition of the
29
formulations, as evidenced by the lower fluxes observed for both CNLP and CLP (p < 0.05),
compared to EMLA®.
For the palatal mucosa, both LAs presented fluxes in the decreasing order EMLA® >
CLP > CNLP (p < 0.05) (Table 2), suggesting that for the keratinized barrier model, the
composition and nature of the formulation significantly influenced transport of the drugs
through the barrier. The higher rate of permeation of EMLA® across the keratinized barrier
could be explained by the presence of polyoxyl hydrogenated castor oil surfactant in the
formulation (EMLA® cream, 5 mg/g, AstraZeneca), which was found by Moghadam et al.
(2013) to disorganize the lipid barrier of the stratum corneum, hence facilitating drug transfer.
The lag times obtained for the LAs with the buccal mucosa (Table 2) showed no
difference between LDC in CNLP and in CLP (p > 0.05). However, both hydrogels showed
slower transfer of LDC, compared to EMLA® (p < 0.05). On the other hand, the transfer of PLC
showed no influence of formulation or encapsulation (p > 0.05). Considering the palatal mucosa
(Table 2), both LDC and PLC presented a longer lag time when present in the CLP formulation
(p < 0.01), while their permeation was immediate when present in the CNLP and EMLA®
formulations. This feature could result in faster onset of topical anesthesia in keratinized
mucosa. In previous work, permeation parameters determined in vitro were correlated to the
efficacy (in humans) of topical anesthetics present in a liposomal Carbopol® system (Franz-
Montan et al., 2013; 2015).
2.3. In vitro cytotoxicity assays
Biological studies are essential for provision of detailed information about the contact
of drugs with the oral mucosa surface. In viability tests, FGH and HaCat cells are considered
important cellular models for the testing of potential irritants (Benavides et al., 2004). The
results of the cell viability tests are shown in Figures 4(a) and 4(b).
30
G e l c o n c e n t r a t io n ( m g /m L )
Ce
ll v
iab
ilit
y (
%)
0 2 0 4 0 6 0 8 0 1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
E M L A
C a r b o p o l
C a rb o p o l N a n o
C L P
C N L P
G e l c o n c e n t r a t io n ( m g /m L )
Ce
ll v
iab
ilit
y (
%)
0 2 0 4 0 6 0 8 0 1 0 0
0
2 0
4 0
6 0
8 0
1 0 0 E M L A
C a r b o p o l
C a rb o p o l N a n o
C L P
C N L P
Figure 4. Cell viability determined using the MTT test after exposure of (a) FGH and (b) HaCat
cells to the hydrogel formulations (n = 9).
The presence of the PCL nanocapsules (CNLP) did not alter the cellular toxicity,
compared to the toxicity found for the free drugs (CLP). Considering the FGH cells, 2 h of
exposure resulted in no statistically significant effects on cell viability for any of the hydrogel
formulations. These results were in agreement with previous work concerning evaluation of the
side effects of local anesthetic formulations (Blumenthal et al., 2006).
Protective effects (higher than 60%) were observed for the hydrogel formulations based
on PCL/LDC+PLC, for both HaCat and FGH cells, up to the maximum concentrations
evaluated. Similar behavior has been reported for PCL nanocapsule formulations containing
lidocaine, compared to free lidocaine (2%) (Ramos Campos et al., 2013). This is a highly
desirable feature of nanostructured formulations used as drug delivery systems. Finally,
PCL/LDC+PLC or LDC+PLC incorporated into the Carbopol® hydrogel presented lower
cytotoxicity than the commercially available formulation (EMLA®), representing an additional
advantage of this new drug delivery system intended for topical application to the oral mucosa.
b
a
31
2.4. Evaluation of in vivo anesthetic efficacy
The tail-flick test is an in vivo model commonly reported in the literature as being
effective for evaluation of the antinociceptive activity of topical formulations (de Araujo et al.,
2010), so it was therefore selected here for determination of the effectiveness of the hydrogels.
The anesthetic efficacy results are shown in Figure 5.
Figure 5. Analgesia time courses, durations, areas under the efficacy curves (AUC5-90), and
effect ratios for the tail-flick tests employing the different hydrogels. The data are shown as a
percentage of the maximum possible effect (%MPE). ** p < 0.01; a CNLP versus EMLA®;
Kruskal-Wallis/Dunn test. Median (minimum-maximum) (n = 6). The effect ratios were
calculated by dividing the AUC for CNLP or CLP by the AUC for EMLA®.
The CNLP formulation showed significant improvements in the duration of anesthesia
(p < 0.01) and the maximum possible effect (p < 0.01), compared to the commercial formulation
(Figure 6). Considering the AUC5-90, CNLP presented a 2.43-fold higher anesthetic efficacy,
compared to the commercial formulation. Sharma et al. (2017) found that the eutectic LDC-
PLC (5%) mixture associated with a thermoresponsive mixed micellar nanogel, used for topical
anesthesia, presented a 1.25-fold greater effect, relative to EMLA®, supporting the present
findings. Puglia et al. (2011) reported lower permeation flux and higher anesthetic activity for
0
20
40
60
80
100
120
0 20 40 60 80 100
(%M
PE
)
Time (min)
EMLA
CLP
CNLP
Formulations Analgesia
Duration
(%EMP) Effect Fold
(min) AUC5-90 EMLA CLP
CNLP 60 (60-75) a** 4266.5 (3545.8-5875.0)a** 2.43 1.18
CLP 45 (30-75) 3600.9 (1614.8-5375.0) 2.05 1
EMLA 30 (15-45) 1758.1 (1562.5-2068.5) 1 0.49
32
an LDC formulation associated with nanostructured lipid carriers, compared to the non-
encapsulated drug.
Considering that the non-encapsulated formulations showed faster initial action, as
observed for the maximum possible effect at the first evaluation time (Figure 7), the improved
anesthetic efficacy of the CNLP hydrogel was probably due to reduced loss of the LAs to the
systemic circulation. Consequently, the encapsulation of the drugs acted to increase the
residence time of effective amounts of the compounds at the free nerve endings.
Conclusions
Lidocaine (2.5%) and prilocaine (2.5%) were successfully loaded into poly(ε-
caprolactone) nanocapsules, resulting in a hydrogel with desirable characteristics including
stability and a satisfactory permeability profile. The new formulation showed improved
mechanical, rheological, and mucoadhesive properties, together with lower cytotoxicity and
higher in vivo anesthetic efficacy, compared to the commercial product. The formulation
developed in this work has the potential to provide effective and longer-lasting superficial
anesthesia at the oral mucosa during medical and dental procedures. These results open
perspectives for future clinical trials.
33
Materials and Methods
2.5. Chemical agents
Local anesthetics in the base form (LDC and PLC) and capric/caprylic triglyceride
(Myritol® 318) were kindly donated by Cristália (Itapira, SP, Brazil) and Chemspecs (São
Paulo, SP, Brazil), respectively. Carbopol® Ultrez 10 was purchased from Lubrizol (San Diego,
CA, USA); Nipagin from Chemco (Hortolândia, SP, Brazil); acetone and chloroform from
Synth (Labsynth, Diadema, SP, Brazil); poly(vinyl alcohol) (PVA, MW 30,000-70,000),
polycaprolactone (PCL, MW 70,000-90,000), triethanolamine, glycerine, phosphoric acid,
ammonium hydroxide, uranyl acetate, polysorbate 80 (Tween 80), and MTT Assays from
Sigma-Aldrich (Merck KGaA, Darmstadt, Germany); acetonitrile from J.T. Baker
(Phillipsburg, NJ, USA); anhydrous dibasic sodium phosphate, anhydrous monobasic sodium
phosphate, anhydrous monobasic potassium phosphate, sodium chloride, and potassium
chloride from Dinâmica (Dinâmica Química Contemporânea, Diadema, SP, Brazil). Ultrapure
water from a Milli-Q system (Millipore, Bedford, MA, USA) was used to prepare all solutions.
The commercially available topical anesthetic product composed of the eutectic mixture of
LDC and PLC (5%) (EMLA® cream, AstraZeneca, Brazil) was used as the positive control in
some experiments, as detailed below.
2.6. Analytical procedures
Simultaneous quantification of LDC and PLC was performed using an HPLC system
(Thermo Electron Corporation, Waltham, MA, USA) equipped with an automatic sampler
(Surveyor Autosampler Plus Lite, Thermo Electron Corporation) and a Surveyor UV-VIS Plus
detector (Thermo Electron Corporation). ChromeQuest 5.0 software (Thermo Fisher Scientific,
Waltham, MA, USA) was used for data collection. The local anesthetics were separated on a
Phenomenex Gemini C18 reversed-phase column (5 µm, 150 x 4.60 mm). The mobile phase
consisted of a mixture of acetonitrile:buffer (25 mM NH4OH, pH 7.0, adjusted with H3PO4) at
a ratio of 40:60 (v:v), pumped at 1.2 mL/min. The injection volume was 200 µL. Detection of
the LAs was performed at a wavelength of 220 nm (Franz-Montan et al., 2015).
2.7. Preparation of poly(ε-caprolactone) (PCL) nanocapsules containing 2.5%
lidocaine (LDC) and 2.5% prilocaine (PLC)
Polymeric nanocapsules composed of poly(ε-caprolactone) (PCL) and containing the
eutectic mixture of LDC (2.5%) and PLC (2.5%) (PCL/LDC+PLC) were prepared using an oil-
in-water emulsion/solvent evaporation method (Melo et al., 2018). Briefly, LDC (250 mg), PLC
34
(250 mg), and capric/caprylic triglycerides (200 mg) were dissolved in acetone (10 mL) and
mixed with chloroform (20 mL) containing the polymer (400 mg), followed by ultrasonication
(1 min, 90 W). The aqueous phase (50 mL) containing PVA (175 mg) was then added to the
pre-emulsion obtained and the mixture was ultrasonicated (8 min, 90 W). The resulting
emulsion was rotary-evaporated to 5 mL, in order to remove the solvents. The resultant
PCL/LDC+PLC samples were stored in a dryer at 4 °C before use.
2.8. Preparation of porcine oral mucosa
Porcine oral mucosa was prepared according to the methodology described previously
(Franz-Montan et al., 2016). Pig maxillae (from 5-months-old Landrace pigs weighing around
75-80 kg) were obtained in a local slaughterhouse, immediately after slaughter, stored in ice-
cold isotonic phosphate buffer (pH 7.4), and transported to the laboratory within 1 h.
For the in vitro permeation studies, samples of palatal (keratinized mucosa model) and
buccal (from the cheek region, non-keratinized mucosa model) mucosa were separated from
the underlying tissue using a scalpel and were washed with saline. Intact mucosa was immersed
in deionized water at 60 °C for 2 min. The epithelium was carefully separated from the
connective tissue and was used immediately (Franz-Montan et al., 2016).
In the mucoadhesive strength experiment, porcine buccal mucosa from the cheek was
used. The cheek was removed with a scalpel and washed in distilled water. The underlying
muscle layer was smoothed and retained, in order to support the mucosa. The tissues were kept
in isotonic phosphate buffer (pH 7.4) and were rapidly used in the experiments (Cubayachi et
al., 2015).
All the experiments involving porcine oral mucosa were performed with mucosa from
at least three different animals.
2.9. Characterization of poly(ε-caprolactone) nanocapsules
2.9.1. Determination of particle size, polydispersity, and surface charge
The nanoparticle average diameter (nm) and polydispersity index (PDI), determined by
dynamic light scattering (DLS), together with the surface charge (zeta potential, ZP), were
measured using a Zetasizer ZS-90 particle analyzer (Malvern Instruments, Malvern, UK). The
PCL suspensions (with and without LDC+PLC) were analyzed in triplicate at 25 °C, on three
different days, using a scattering angle of 90° (Melo et al., 2018).
2.9.2. Nanoparticle concentration
35
The nanoparticle concentration was determined by nanoparticle tracking analysis
(NTA), using an LM20 instrument (NanoSight, Amesbury, UK) equipped with a 532 nm laser.
The nanoparticle concentration (particles/mL) was obtained in real time, at room temperature,
based on light scattering and the individual Brownian motion tracks of the nanoparticles
(Ribeiro et al., 2018). Using a sterile syringe, the formulation was injected into the sample
chamber until the liquid filled the tip of the syringe. The measurements (n = 3) were performed
over a period of 6 months.
2.9.3. Physicochemical stability of nanoparticles in suspension
The stabilities of the formulations containing PLC and LDC were evaluated by
measuring the nanoparticle size, PDI, ZP, concentration, and pH after 7, 15, 30, 60, 90, 120,
150, and 180 days of storage at room temperature (25 C) (Mora-Huertas et al., 2010).
2.9.4. Structural analyses
2.9.4.1. Attenuated total reflectance-Fourier transform infrared (FTIR)
analysis
Infrared analyses (ATR-FTIR) were performed of the PCL nanocapsules with and
without LDC+PLC. LDC and PLC were also analyzed in their free forms. The spectra were
obtained using FTIR spectrophotometers (Bruker IFS 66 v/S or Perkin Elmer Spectrum 65
instruments) fitted with ATR cells and operated in reflectance mode, in the range 4500-500 cm-
1, with steps of 2 cm-1.
2.9.4.2. Differential scanning calorimetry (DSC) analysis
The same formulations were also submitted to DSC measurements performed using a
TA Q20 calorimeter equipped with a cooling system. After calibrating the equipment with
indium, 5 mg portions of the samples were placed in aluminum pans and the thermal profiles
were obtained in the temperature range from 0 to 250 °C, at a heating rate of 10 °C/min, under
a flow of nitrogen.
2.9.5. Determination of nanoparticle morphology
The morphologies of the PCL nanoparticles with and without LDC+PLC were evaluated
by transmission electron microscopy (TEM), using an EM-900 instrument (Carl Zeiss, Jena,
Germany) operated at 80 kV. Briefly, the samples were diluted, deposited onto copper grids
36
coated with carbon film, contrasted using uranyl acetate (2%), dried at room temperature, and
analyzed.
2.9.6. Encapsulation efficiency (EE%)
The encapsulation efficiencies of LDC and PLC in PCL were determined by the method
combining ultrafiltration and centrifugation (Melo et al., 2018). The EE% values were
determined from the difference between the total and free LDC and PLC concentrations,
measured in the suspension and ultrafiltrate, respectively. The PCL/LDC+PLC samples were
centrifuged for 15 min, at 28000 g, in regenerated cellulose Microcon ultrafiltration units with
a molecular exclusion pore size of 30 kDa (Millipore, Billerica, Massachusetts), followed by
quantification of the compounds using HPLC. The total amounts of LDC+PLC were
determined following dilution of the suspensions with acetonitrile to solubilize the polymers
and completely release the LAs.
2.9.7. In vitro release kinetics
The profiles of in vitro release of PLC and LDC from the PCL nanoparticles were
investigated using a two-compartment system consisting of donor (1 mL) and acceptor (80 mL)
compartments, separated by a cellulose membrane with a molecular exclusion pore size of 10
kDa (Melo et al., 2018). The system was maintained under sink conditions, with constant
magnetic stirring (300 rpm) at room temperature (25 C). Aliquots of 300 μL were periodically
withdrawn from the acceptor compartment, over a total period of 1400 min. The samples were
analyzed by HPLC. The experiment was performed in triplicate. The data were treated by the
application of different mathematical models in order to select the best model (defined by the
highest R2 value) to describe the LA release mechanism. The software used was KinetDS 3.0
(Jagiellonian University Medical College, Kraków, Poland).
2.10. Hydrogel preparation
The Carbopol® (carboxyvinyl derivative) hydrogel base (C) was prepared according to
the technique described previously (Silva et al., 2008), using the following components:
Carbopol® (2%, gelling agent); Propylene glycol (5%, solvent and wetting); Methylparaben
(0.2%, preservative); Glycerin (8%, wetting and emollient agent); Deionized water (solvent);
Triethanolamine (pH 7,0, alkalinizing agent). For preparation of the Carbopol® hydrogel with
or without 2.5% LDC and 2.5% PLC associated with the poly(ε-caprolactone) nanocapsules,
the resulting hydrogel (placebo) was immediately mixed with nanocapsule suspensions with
37
(CNLP) or without (CN) the local anesthetics (50:50, v/v) at the desired final drug
concentration (5% w/w LDC+PLC). Carbopol® hydrogel containing free 2.5% LDC and 2.5%
PLC (CLP) was prepared by dissolving appropriate amounts of the local anesthetics (5% w/w
LDC+PLC) in the propylene glycol during hydrogel preparation.
2.11. Characterization of the hydrogel formulations
2.11.1. Rheological measurements
Rheological measurements were performed using a Haake RheoStress 1 rheometer
(Thermo-Haake, Germany) with plate-plate geometry (plate diameter of 20 mm). The hydrogel
formulations were submitted to continuous variation of shear rate from 0 to 300 s-1, and the
resulting shear stress was measured. The tests were performed in triplicate (n = 3) at a constant
temperature (25 ± 1 oC) maintained by a thermostatically-controlled water bath. The rheological
behaviors of the hydrogel formulations were evaluated from curves obtained by plotting the
shear stress (Pa) as a function of shear rate (s-1) (Melo et al., 2018).
2.11.2. Mechanical properties of the hydrogels
The mechanical properties (hardness, compressibility, cohesiveness, and adhesiveness)
of the hydrogel formulations were determined using a texture analyzer (Model TA-XT Plus,
Stable Micro Systems) operated in texture profile analysis (TPA) mode. A 10 mL beaker was
filled with approximately 10 g of each formulation (to a fixed height) and the samples were left
in a water bath at 37 °C for 24 h for removal of air bubbles. The analytical probe (10 mm
diameter) was compressed twice into each sample, to a depth of 5 mm, at a rate of 2.0 mm.s-1,
with a delay period of 15 s between the end of the first compression and the beginning of the
second compression.
2.11.3. Accelerated stability study
The hydrogels were placed in a stability chamber with controlled temperature and
humidity (40 °C and 75% relative humidity). The parameters evaluated were the anesthetic
dosage, pH, and weight loss (ICH Expert Working Group, 2003).
Determination of the dosages of lidocaine and prilocaine in the formulations
The dosages of the LAs in the hydrogels were measured by diluting appropriate amounts
of the semisolid formulations, followed by HPLC analyses, as described previously.
pH analysis
38
The pH values of the hydrogel formulations were determined by potentiometric
measurements using an Analyzer Model 300M pH meter equipped with an electrode for
semisolids.
Weight loss test
The percentage weight loss throughout the study was calculated using the following
equation:
% weight loss = [(MWI-MWS)×3]/MWI x 100
where, MWI is the mean initial weight, MWS is the mean weight at subsequent times (3 or 6
months), and 3 is the correction factor to 75% RH (ICH Expert Working Group, 2003).
2.11.4. In vitro release kinetics of the hydrogels and mathematical modeling
The release of the LAs from the hydrogels (CLP and CNLP) was evaluated using a
Franz-type vertical diffusion system (Manual Transdermal System, Hanson Research
Corporation, Chatsworth, CA, USA) with permeation area of 1.77 cm2 and a 7 mL volume
acceptor. The hydrogels (CNLP and CLP) were applied under infinite dose conditions (using
300 mg) over a dialysis membrane with a molecular exclusion pore size of 1,000 Da. The
system was maintained at 37 °C, under magnetic stirring (300 rpm) (Melo et al., 2018). Aliquots
of 300 µL were withdrawn from the acceptor compartment for HPLC analysis, with the volume
being maintained constant by addition of the same volume of fresh buffer solution. Samples
were collected at predetermined intervals during a period of 24 h, in triplicate.
KinetDS 3.0 software was used to analyze the release curves and the best kinetic model
was selected based on the coefficient of determination (R2).
2.11.5. In vitro permeation studies
In vitro permeation assays were performed with porcine buccal and palatal mucosal
epithelium, using the same Franz-type vertical diffusion system described above (Franz-
Montan et al., 2016) .
The epithelium was positioned on a 0.45 μm cellulose filter with the connective side of
the tissue facing the filter, due to its fragility, hence reducing the release of impurities into the
acceptor compartment, without altering the permeation of the LAs (Franz-Montan et al., 2016).
The hydrogels (CNLP and CLP), epithelium, and membrane filter were clamped
between the donor and acceptor compartments. The acceptor compartment was filled with
PBS:alcohol (70:30, v/v) that had been filtered and degassed. The acceptor medium was
39
selected according to the solubility of the LAs, in order to maintain sink conditions whereby
the concentrations of the drugs in this compartment never reached 10% of their solubility (LDC
= 17.95 mg/mL; PLC = 21.89 mg/mL).
The experiment was performed at 37 °C during 5 h, under magnetic stirring (350 rpm).
At predetermined intervals, 300 μL volumes of the samples were collected and the same
volumes of fresh acceptor medium were added. The samples were analyzed by HPLC, as
described previously.
For each cell, the cumulative amounts of the LAs transported across the mucosal
epithelium, per unit of area, was plotted against time. The steady-state fluxes (Jss) of the LAs
were calculated from the slopes of the linear portions of the curves. The lag times were obtained
from the intercepts on the time axis. All experiments were conducted six times (Franz-Montan
et al., 2016).
2.12. In vitro evaluation of mucoadhesive strength
The mucoadhesive strengths of the formulations were evaluated by measuring the force
required to detach them from pig buccal mucosa, using the same texture analyzer described
above, in accordance with Cubayachi et al. (2015). The buccal mucosa was positioned
horizontally at the lower end of the TPA probe and the formulation was placed at the upper end.
Prior to the mucoadhesion testing, the buccal mucosa was hydrated with 50 µL of artificial
saliva for 5 min. The analytical probe was then lowered until it made contact with the mucosa
surface. The rupture tensile strength was determined by applying a compressive force of 0.5 N
for 30 s and then moving the probe at a constant speed of 1.0 mm.s-1. The force required to
detach the formulation from the mucosa surface was determined from the curve of force plotted
against distance. All the measurements were performed at ambient temperature (21 ± 1 °C), in
quintuplicate.
2.13. In vitro cytotoxicity assays
The cytotoxicities of the hydrogel formulations were evaluated by the MTT (a yellow
tetrazole) reduction test with human epithelial cells (HaCaT) and human gingival fibroblasts
(FGH). Viable cells were exposed for 2 h to the hydrogel formulations (CNLP and CLP)
containing the local anesthetics at different concentrations (ranging from 0.312 to 5 mg/mL of
LDC and PLC). The formulations contained from 6.25 to 100 mg/mL of hydrogel. The same
amounts of hydrogel (6.25 to 100 mg/mL) free of local anesthetics (CN and C) were diluted
and used as placebos. The percentages of viable cells were determined after incubation in the
40
presence of MTT for 3 h at 37 °C, with measurement of the amount of MTT converted to
insoluble formazan by mitochondrial dehydrogenases (Ferreira et al., 2017; Oliveira et al.,
2014). A 100 μL volume of ethanol was added to each well in order to dissolve the formazan
crystals, resulting in a purple solution.
The fraction of viable cells was obtained by quantification of the original formazan
using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA) operated at a
wavelength of 570 nm, with conversion of the value to the percentage of viable cells.
2.14. In vivo anesthetic efficacy evaluation
The experiments were conducted after approval by the Animal Ethics Committee of
UNICAMP (protocol #2850-1) and in accordance with the Principles of Laboratory Animal
Care (NIH publication #85-23, revised in 1985). Male adult Wistar rats (200-250 g) obtained
from the Multidisciplinary Center of Biological Investigation of Laboratory Animals (CEMIB-
UNICAMP) were kept in cages under light/dark cycles of 12 h, at 25 ± 2 °C, and were provided
with water and food ad libitum for at least 7 days before the experiments. The Wistar rats were
divided into groups of 6 animals and each animal was used only once in the experiment.
The topical anesthetic efficacies of the hydrogels containing LDC and PLC were
assessed using the tail-flick test, as previously described by Grant et al. (1993) and modified by
De Araujo et al. (2010). Briefly, the animals were positioned in an acrylic confinement
chamber, while maintaining free the distal portion of the tail (5 cm). The time required for tail
removal (latency) following exposure to the heat produced by an incandescent lamp (55 °C)
was considered as the aversive response, generating a baseline that was recorded for each
animal prior to the start of the experiment. In order to avoid injury due to thermal insult, a
maximum time of 15 s (cut-off value) was established for contact with the heat source.
Approximately 0.5 g portions of the hydrogels and the commercial formulation (EMLA®) were
applied 2 cm from the tail base, with the aid of Micropore™ protective tape, for 2 min. The
formulations were then removed and nociceptive stimulus was applied to the same region.
Measurements were performed immediately after formulation removal and then every 15 min
until the animal returned to its baseline pain response. After the tail-flick test, the animals were
sacrificed by deep general anesthesia. Analgesia was defined as an at least 50% increase in the
time required for tail removal, compared to the observed baseline value. Calculation of the
maximum possible effect (%MPE) was performed by subtracting the baseline from the latency
time and dividing the resulting value by the cut-off minus the baseline. The value obtained was
multiplied by 100.
41
2.15. Data analysis
The results from the suspension and hydrogel characterizations, the in vitro permeation
tests, and the mucoadhesion tests were statistically evaluated using the Student’s t-test, or by
analysis of variance (ANOVA) followed by Tukey’s post-hoc test. These analyses were
performed with Biostat 5.0 for Windows® software (Instituto Mamirauá, Belém, PA, Brazil).
The results of the in vitro cytotoxicity assays and the in vivo tests were analyzed using the
Kruskal-Wallis test followed by the Dunn post-hoc test. These analyses were performed with
GraphPad Prism 6.0 software (GraphPad, San Diego, CA). Statistical significance was defined
as p < 0.05.
Acknowledgments
Financial support was provided by the São Paulo State Research Foundation (FAPESP,
grants #2012/06974-4 and #2013/22326-5). B.V.M. received a doctorate scholarship from
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil). The authors
are grateful to the PhD students Camila Cubayachi for help with the mucoadhesion studies and
Ana Laís Nascimento Vieira for assistance during the accelerated hydrogel stability tests.
42
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49
Supplementary Material
HYBRID HYDROGEL AND POLYMERIC NANOCAPSULES CO-LOADED WITH
LIDOCAINE AND PRILOCAINE AIMING TOPICAL INTRAORAL ANESTHESIA
Authors:
Bruno Vilela Muniz¹. MSc, PhD student
Diego Baratelli². MSc
Stephany Di Carla¹. MSc student
Luciano Serpe¹. PhD
Camila Batista da Silva¹. PhD
Viviane Aparecida Guilherme3. PhD
Lígia de Morais Ribeiro Nunes3. PhD
Cintia Maria Saia Cereda3. PhD
Eneida de Paula³. PhD, Full Professor
Maria Cristina Volpato1. PhD, Full Professor
Francisco Carlos Groppo¹. PhD, Full Professor
Leonardo Fernandes Fraceto². PhD, Associate Professor
Michelle Franz-Montan1*. PhD, Assistant Professor
50
Figure S1. Mean (±SD) size (nm), PDI, pH, surface charge (ZP), and nanoparticle
concentration (.1012 part./mL) of poly(ε-caprolactone) nanocapsules (PCL) and nanocapsules
containing lidocaine-prilocaine (PCL/LDC+PLC), according to time, during 180 days of
storage at 25 oC. Comparison between suspensions with or without LAs (PCL/LDC+PLC vs.
PCL) for each period: Student’s t-test (p < 0.01). Variation of formulation parameters over time
(relative to Day 0): ANOVA/Tukey’s test (p < 0.05; n = 3).
51
Figure S2. Percentages of (a) lidocaine (LDC) and (b) prilocaine (PLC) released from free
solution and poly(ε-caprolactone) nanocapsules (PCL/LDC+PLC), at 25 ºC (n = 3). SD values
were below 5%.
b
a
52
Figure S3. Mean (SD) percentages of (a) lidocaine and (b) prilocaine released from Carbopol®
hydrogels containing LDC and PLC (5%), free or associated with poly(ε-caprolactone)
nanocapsules (CLP and CNLP, respectively), according to time, at 37 ºC (n = 3).
a
b
53
Table S1. Correlation coefficient values obtained for lidocaine (LDC) and prilocaine (PLC),
using different mathematical models applied to analyze the kinetics of release from the
nanocapsules.
Release kinetics Korsmeyer-Peppas Weibull Zero
order
R2 k n R2 R2
Lidocaine PCL/LDC+PLC 0.99 0.003 0.82 0.89 0.70
Prilocaine PCL/LDC+PLC 0.98 0.007 0.72 0.88 0.66
Table S2. Mean (±SD) local anesthetic content (dosage), pH, and weight variation of the
different Carbopol® hydrogels containing LDC and PLC (5%), free or associated with poly(ε-
caprolactone) nanocapsules (CLP and CNLP, respectively), compared to EMLA®, during 6
months storage at 40 ± 2 °C and 75% RH.
Hydrogel
formulation
Time
(months)
Dosage pH ΔP
LDC PLC
CNLP
T0 2.77 ± 0.09 2.02 ± 0.04 7.324 ± 0.24 ------
T3 1.91 ± 0.03a 1.88 ± 0.03 7.100 ± 0.26 0.11 ± 0.03
T6 2.44 ± 0.37 1.87 ± 0.30 7.310 ± 0.09 0.14 ± 0.04
CLP
T0 2.77 ± 0.07 2.50 ± 0.09 7.482 ± 0.07 ------
T3 2.34 ± 0.02ª 3.17 ± 0.05 4.831 ± 0.11ª 0.09 ± 0.01
T6 2.69 ± 0.02b 2.20 ± 0.09ª 8.405± 0.21a,b 0.01 ± 0.01*
EMLA®
T0 3.15 ± 0.01 2.49 ±0.02 8.945 ± 0.26 ------
T3 2.29 ± 0.05a 2.30 ± 0.04 9.024 ± 0.29 0.27 ± 0.44
T6 2.84 ± 0.35b 2.5 ± 0.30 9.423 ± 0.26 0.01 ± 0.03
T0: time zero; T3: 3 months; T6: 6 months; ΔP: weight variation for T3 and T6, relative to T0 and T3, respectively;
*p < 0.01. Different letters indicate statistically significant differences among the times (p < 0.05): a relative to T0; b relative to T3 (ANOVA/Tukey’s test; n = 3).
54
Table S3. Release kinetics mechanisms for lidocaine and prilocaine, free or associated with
poly(ε-caprolactone) nanocapsules (CLP and CNLP, respectively), contained in Carbopol®
hydrogels, as evaluated by the determination coefficient values (R2) obtained following
application of different mathematical models.
Release
kinetics
Weibull Korsmeyer-
Peppas
Zero
order
R2 K b R2 R2
Lidocaine CNLP 0.96 0.49 0.72 0.89 0.61
CLP 0.95 0.72 0.95 0.93 0.68
Prilocaine CNLP 0.94 0.50 0.93 0.72 0.60
CLP 0.94 0.54 0.13 0.74 0.65
55
3 CONCLUSÃO
O encapsulamento da lidocaína (2,5%) e da prilocaína (2,5%) em nanocápsulas de
poli(ε-caprolactona) foi estável e apresentou boas características físico-químicas, estabilidade
e liberação sustentada, sendo possível a incorporação dessa suspensão em uma formulação
semi-sólida. O hidrogel de Carbopol® associado às nanocápsulas apresentou boas propriedades
mecânicas, reológicas e mucoadesivas esperadas para uma formulação tópica, além de boa
estabilidade, baixa citotoxicidade, com bom perfil de permeação e melhor eficácia anestésica
in vivo quando comparada com uma formulação comercial. Esses resultados promissores abrem
perspectivas para testes futuros em humanos, permitindo seu uso em diversas condições na área
de saúde.
56
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ANEXOS
Anexo 1: Certificado de aprovação do estudo pela Comissão de Ética no Uso de Animais
61
Anexo 2: Comprovante de submissão do trabalho