LUCAS ALVES MOURA
PREPARO, CARACTERIZAÇÃO E AVALIAÇÃO DO USO DE NANOESFERAS DE PLGA CONTENDO DOXICICLINA ASSOCIADO AO DEBRIDAMENTO PERIODONTAL NO TRATAMENTO DA PERIODONTITE CRÔNICA AVANÇADA
PREPARATION, CHARACTERIZATION AND EVALUATION OF THE ADJUNCTIVE USE OF PLGA NANOSPHERES LOADING DOXYCYCLINE TO PERIODONTAL DEBRIDEMENT ON TREATMENT OF ADVANCED CHRONIC
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 Clínica Odontológica, na Área de Periodontia. Thesis presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Dental Clinics, in Periodontics Area.
Orientador: Prof. Dr. Antonio Wilson Sallum
Este exemplar corresponde à versão final da tese
defendida por Lucas Alves Moura e orientada pelo Prof. Dr. Antonio Wilson Sallum.
____________________________________ Assinatura do Orientador
Universidade Estadual de Campinas Faculdade de Odontologia de Piracicaba
LUCAS ALVES MOURA
PREPARO, CARACTERIZAÇÃO E AVALIAÇÃO DO USO DE NANOESFERAS DE PLGA CONTENDO DOXICICLINA ASSOCIADO AO DEBRIDAMENTO PERIODONTAL NO TRATAMENTO DA PERIODONTITE CRÔNICA AVANÇADA
PREPARATION, CHARACTERIZATION AND EVALUATION OF THE ADJUNCTIVE USE OF PLGA NANOSPHERES LOADING DOXYCYCLINE TO PERIODONTAL DEBRIDEMENT ON TREATMENT OF ADVANCED CHRONIC
Ficha catalográfica
Universidade Estadual de Campinas
Biblioteca da Faculdade de Odontologia de Piracicaba Heloisa Maria Ceccotti - CRB 8/6403
Moura, Lucas Alves,
M865p MouPreparo, caracterização e avaliação do uso de nanoesferas de PLGA contendo doxiciclina associado ao debridamento periodontal no tratamento da periodontite crônica avançada / Lucas Alves Moura. – Piracicaba, SP : [s.n.], 2015.
MouOrientador: Antonio Wilson Sallum.
MouTese (doutorado) – Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba.
Mou1. Periodontite crônica. 2. Nanoesferas. 3. Sistemas de liberação de medicamentos. 4. Doxiciclina. 5. Plásticos biodegradáveis. I. Sallum, Antonio Wilson. II. Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba. III. Título.
Informações para Biblioteca Digital
Título em outro idioma: Preparation, characterization ans evaluation of the adjunctive use of
PLGA nanospheres loading doxycycline to periodontal debridement on treatment of advanced chronic periodontitis
Palavras-chave em inglês:
Chronic periodontitis Nanospheres
Drug delivery systems Doxycycline
Biodegradable plastics
Área de concentração: Periodontia Titulação: Doutor em Clínica Odontológica Banca examinadora:
Antonio Wilson Sallum [Orientador] Daiane Cristina Peruzzo
Mauro Pedrine Santamaria Karina Gonzales Silverio Ruiz Renato Corrêa Viana Casarin
Data de defesa: 04-02-2015
Programa de Pós-Graduação: Clínica Odontológica
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RESUMO
O objetivo deste estudo foi avaliar o efeito da associação da administração local de nanoesferas contendo doxiciclina (DOX) com o debridamento periodontal no tratamento da periodontite crônica avançada. As nanoesferas foram preparadas pelo método da dupla emulsão (W/O/W), e caracterizadas quanto à morfologia através da microscopia eletrônica de varredura (MEV), e quanto à interação polímero (PLGA) e DOX através da espectroscopia infravermelha da transformada de Fourier (FTIR). A avaliação da liberação controlada de DOX foi feita através da cromatografia líquida de alta eficiência (HPLC), a partir do fluido gengival sulcular (GCF) coletado nos períodos: 2, 5, 7, 10, 15 e 20 dias após aplicação das nanoesferas. Foi realizado um ensaio clínico randomizado controlado duplo cego, incluindo 30 pacientes diagnosticados com periodontite crônica que apresentavam sete sítios com sangramento à sondagem e profundidade de sondagem (PS) ≥ 5 mm, sendo 2 dentes com PS ≥ 7 mm. Os pacientes foram aleatoriamente divididos em 2 grupos e receberam os seguintes tratamentos: debridamento periodontal por 45 minutos associado à administração local de nanoesferas contendo doxiciclina nos sítios com PS ≥ 5 mm (DB+DOX) ou debridamento periodontal por 45 minutos associado à aplicação de nanoesferas vazias (DB). Foram avaliados os seguintes parâmetros clínicos: Índice de Placa (IP), Sangramento à Sondagem (SS), Profundidade de Sondagem (PS) e Nível de Inserção Clínica (NIC), no baseline, 3 e 6 meses após a terapia. A avaliação microbiológica foi feita por meio da reação de cadeia de polimerase – tempo real (“real time” - PCR) para detecção das bactérias: Porphyromonas gingivalis,
Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans e Prevotella intermedia, provenientes do biofilme subgengival coletado no baseline, 1, 3 e 6
meses após o tratamento. Os resultados foram comparados por meio do teste de análise de variância com medidas repetidas, com nível de significância de 5%. Foram obtidas nanoesferas variando de 700 nm a 4 um. Através do FTIR observou-se boa interação da DOX com o PLGA, sem alterações das propriedades químicas de nenhum dos compostos. Após a aplicação das nanoesferas observou-se uma liberação de DOX constante no GCF até o vigésimo dia pós-tratamento, acima da concentração inibitória mínima. No grupo DB+DOX houve redução significativa da PS e ganho de inserção clínica nas bolsas profundas e moderadas em relação ao baseline e entre grupos. Observou-se redução significativa dos níveis bacterianos pós-tratamento, sendo o grupo DB+DOX mais eficaz em manter os níveis mais baixos após 6 meses do tratamento. Dentro das limitações deste trabalho, os resultados sugerem que as nanoesferas de PLGA são efetivas como sistema de liberação controlada local de DOX e quando associadas ao debridamento periodontal podem promover resultados superiores em relação à terapia mecânica isoladamente.
Palavras-chave: Periodontite Crônica. Nanoesferas. Sistemas de Liberação Controlada de Medicamentos. Doxiciclina. Plásticos Biodegradáveis.
ABSTRACT
The aim of this study was to assess the effect of the association of local administration of nanospheres loading doxycycline (DOX) with the periodontal debridement treating advanced chronic periodontitis. Nanospheres were made by double-emulsion method (W/O/W) and characterized regarding morphology, by scaning eléctron microscopy (SEM); and drug/polymer interaction, by Fourier transform infrared spectroscopy (FTIR). The DOX controlled release was assessed by high performance liquid chromatography (HPLC) of gingival crevicular fluid (GCF) samples collected 2, 5, 7, 10, 15 and 20 days after treatment. It was performed randomized double-blinded clinical trial, with thirty patients diagnosed with chronic periodontitis presenting at least seven sites with bleeding on probing and probing depth (PD) ≥ 5 mm, and 2 sites with PD ≥ 7 mm. The patients were randomly allocated in two groups receiving interventions as followed: 45 minutes periodontal debridement + subgengival nanospheres loading DOX (DB+DOX); and 45 minutes periodontal debridement + subgengival void nanospheres (DB). Plaque index (IP), bleeding on probing (BP), probing depth (PD) and clinical attachment level (CAL) were evaluated on baseline, 3, and 6 months after therapy. Real-time polymerase chain reaction (real-time PCR) was used to quantify Porphyromonas gingivalis, Tannerella forsythia,
Treponema denticola, Aggregatibacter actinomycetemcomitans and Prevotella intermedia
from subgingival biofilm samples collected on baseline, 1, 3 and 6 months after treatment. The results were compared by variance analyses test for repeated measures, with significance of 5%. Nanospheres varying from 700 nm to 4 um were obtained. There was a good interaction between DOX and PLGA, with no chemical properties alterations. After local administration of the nanospheres, it was observed constant DOX release in the GCF until 20th day post-treatment, with concentration above the minimum inhibitory concentration. DB+DOX group showed significant PD reduction and CAL gain in deep and moderates pockets comparing to baseline and between groups. It was observed significant reduction of bacteria levels along follow up period, and DB+DOX group was more eficiente in keeping lower levels of bacteria after 6 months from treatment. Within limitations of this study, the results can suggest that PLGA nanospheres are effective carriers for controlled release of DOX and when used adjunctively to periodontal debridement, improved results can be achieved compared to mechanical therapy alone.
Keywords: Chronic Periodontitis. Nanospheres. Drug Delivery Systems. Doxycycline. Biodegradables Plastics.
SUMÁRIO !
DEDICATÓRIA xiii
AGRADECIMENTOS xv
LISTA DE FIGURAS xxi
LISTA DE TABELAS xxiii
LISTA DE ABREVIATURAS E SIGLAS xxv
INTRODUÇÃO 1
CAPÍTULO 1: Characterization and Pharmacokinetic profile of controlled released doxycycline by PLGA nanospheres adjunct to non-surgical
periodontal therapy 4
CAPÍTULO 2: Clinical and microbiological evaluation of locally delivered doxycycline by PLGA nanospheres adjunct to nonsurgical periodontal
therapy: a randomized double blinded clinical trial 24
CONCLUSÃO 52
REFERÊNCIAS* 53
ANEXO 1 55
DEDICATÓRIA
Dedico este trabalho ao meu filho Miguel Ramos Moura, aos meus pais Antonio Carlos de Souza Moura e Regina das Graças Bastos Alves Moura, à minha irmã Carolina Alves Moura e à minha companheira, motivadora e crítica Rayane Ramos Araújo.
AGRADECIMENTOS
Este trabalho não poderia ter sido concluído sem a ajuda de certas pessoas e instituições a quem presto minha homenagem:
À Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas; ao Laboratório de Biomateriais da Pontifícia Universidade Católica de São Paulo; ao Departamento de Microbiologia e Imunologia da Universidade de West Virginia por me acolherem e cederem espaço, material e condições para a execução de todos os experimentos realizados deste trabalho.
Ao meu orientador, Prof. Dr. Antonio Wilson Sallum, por ter acreditado e me motivado a perseguir o meu objetivo. Muito obrigado pela amizade e confiança. Pelo seu jeito estimulador e inspirador sendo exemplo de um grande educador.
Aos professores da Área de Periodontia, Prof. Dr. Marcio Zaffalon Casati, Prof. Dr. Francisco Humberto Nociti Junior, Profa. Dra. Karina Gonzalez Silvério Ruiz e em especial ao Prof. Dr. Enilson Antonio Sallum, que por meio de aulas, seminários, conversas colaboraram com idéias, sugestões e transmitiram conhecimento durante estes anos de convívio, assim como pela confiança a mim cedida.
À Profa. Dra. Eliana Aparecida de Rezende Duek, que me apresentou ao mundo dos biomateriais dando início a todo este trabalho realizado.
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Aos professores e grandes amigos da minha banca de defesa: Profa. Dra Daiane Peruzzo, Prof. Dr. Renato Corrêa Viana Casarin, Prof. Dr. Mauro Pedrine Santamaria e Profa. Dra. Karina Gonzalez Silvério Ruiz.
Aos professores que muito colaboraram na banca de qualificação: Prof. Dr. Marcio Zaffalon Casati, Profa. Dra. Denise Carleto Andia e Prof. Dr. Rafael Nobrega Stipp.
Aos professores suplente da banca de defesa: Prof. Dr. Francisco Humberto Nociti Junior, Profa. Dra. Fernanda Vieira Ribeiro e Profa. Dra. Eliana Aparecida de Rezende Duek
Aos meus queridos amigos do Pará: Armando Koichiro Kaieda, Miki Taketomi Saito, Leonardo Soriano de Mello Santos, Camila Lima, Patricia do Socorro Feio e Isabela Marques.
Aos meus amigos de clínica e pesquisa da FOP: Lucas Araújo Queiroz, Maria Fernanda Peres, Hugo Felipe do Vale, Renato Viana Casarin, Fernanda Vieira Ribeiro, Tiago Taiete, Maria Alice Gatti Palma, Isabela Lima França e Mayra Laino Albiero.
Aos amigos do Labiomat: Vinicius Melo, André Dutra, Isabela Alves, Talita Bianchi Aiello, Daniel Mistura e Bruna Antunes Más.
Aqueles que me suportaram e dividiram morada comigo em Piracicaba e se tornaram grandes amigos e companheiros Mauro Pedrine Santamaria, Adriano Lima, Alan Rodrigo M. Palialol e Anderson Catelan.
Aos amigos de laboratório, nos Estados Unidos: Rosana Schafer, Vitaly S. Cuff, Adam P. Irvin e Brock Karolcick.
À Marília Batista e ao João Paulo Menck Sangiorgio que me ajudaram com as análises estatísticas deste trabalho.
Às estimadas Regina Caetano, Eliete Marim e Mari Fugolin, por sempre estarem a dispostas a me ajudar ou orientar em questões burocráticas e afins.
Aos funcionários da FOP/Unicamp que direta ou indiretamente tornaram possíveis esses anos de formação.
Aos pacientes que participaram deste trabalho e confiaram em mim.
À linda cidade de Piracicaba que tão bem me acolheu, foi meu lar e berço do meu mais valioso tesouro.
E não menos importante, agradeço à minha família, nas pessoas de meus pais Antonio Carlos e Regina Moura, por alimentarem um sonho que se tornou um objetivo e hoje se mostra realizado. Assim como agradeço ao companheirismo, apoio e paciência do meu amor, Rayane Ramos Araújo, para que neste último ano chegássemos a este ponto.
A ciência se compõe de erros que, por sua vez, são os passos até a verdade.
LISTA DE FIGURAS !
CAPÍTULO 1
Figure 1. PLGA nanospheres loaded in the tip for local administration (A); subgingival administration of nanospheres into the periodontal pocket after mechanical debridement
(B); and gingival aspect immediately after the tip removal (C). 20
Figure 2. SEM of nanospheres, after liophilization, presenting regular spherical
morphology and no signs of degradation, such as irregular pores. 21
Figure 3. FTIR of PLGA nanospheres; DOX and PLGA nanospheres loading DOX, showing the interaction between the polymer and the drug. Bands at 1745 and 1451 cm-1
refers to DOX and at 1745 and 1082 cm-1 refers to PLGA. 22
Figure 4. Nanospheres were loaded into the selected periodontal pockets and collected GCF samples were analysed for the DOX concentration (ug/ml) released by HPLC in different time intervals (2, 5, 7, 10, 15 and 20 days) – Different letters indicate statistical
difference by ANOVA/Tukey test (p<0.05). 23
CAPÍTULO 2
Figure 1. CONSORT flowchart of the study. 47
Figure 2. Test group: A) probing depth at baseline; B) Administration of PLGA nanospheres loading DOX into periodontal pockets with designated tip;
C) probing depth at 6 months. 47
Figure 3. Means values of PD reduction (mm) for moderate and deep sites in different time intervals. (*) indicate statistical
intergroup difference (Student’s t-test, p<0.05). 48
Figure 4. Means values of CAL gain (mm) for moderate and deep sites in different time intervals. (*) indicate statistical intergroup
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Figure 5. Mean percentage of sites presenting PD reduction and CAL gain ≥ 2 mm at 6 months. (*) indicate statistical
intergroup difference (Student’s t-test, p<0.05). 49
Figure 6. Amounts (log10) of periodontal pathogens in subgingival biofilm at different intervals for test (DB+DOX) and control (DB) groups in deep periodontal
pockets. (A) P. gingivalis; (B) T. forsythia; (C) T. denticola; (D) A. Actinomycetemcomitans;
(E) P. Intermedia. (*) indicate statistical intergroup difference (Student’s t-test, p<0.05). 50 Figure 7. Amounts (log10) of periodontal pathogens in subgingival biofilm at
different intervals for test (DB+DOX) and control (DB) groups in moderate periodontal pockets. (A) P. gingivalis; (B) T. forsythia; (C) T. denticola; (D) A. Actinomycetemcomitans;
(E) P. Intermedia. (*) indicate statistical intergroup difference (Student’s t-test, p<0.05). 51
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LISTA DE TABELAS ! CAPÍTULO 2 Table!1.!qPCR!primers!for!detecting!bacteria.! 44! Table!2.!qPCR!conditions!for!detecting!DNA.! 44! Table!3.!Demographic!distribution!at!baseline!of!the!studied!population! 44! Table!4.!Distribution!of!dental!plaque!and!bleeding!on!probing!in!different!! time!intervals.! 45! Table!5.!Means,!mm!(SD),!of!PD!and!CAL!at!different!time!intervals.! 45! Table!6.!!Means!of!the!amount!(log10!±!SD)!of!periodontal!pathogens!in!! subgingival!biofilm!at!different!time!intervals!for!DB+DOX!and!DB!groups!! in!deep!pockets.! 46! Table!7.!!Means!of!the!amount!(log10!±!SD)!of!periodontal!pathogens!! in!subgingival!biofilm!at!different!time!intervals!DB+DOX!and!DB!groups!! in!moderate!pockets.! 46! ! ! !
LISTA DE ABREVIATURAS E SIGLAS
CIM – Concentraçao inibitória mínima DB – Periodontal debridement
DCM – Dichloromethane DOX – Doxiciclina/doxycycline
FTIR – Fourier transform infrared spectroscopy GCF – Gingival crevicular fluid
HPLC – High performance liquid chromatography KBr – Potassium bromide (brometo de potássio) MMP – Metaloproteinase de matriz
MIC – Minimum inhibitory concentration NaCl – Sodium chloride (cloreto de sódio) PLGA – poliácido lático-co-ácido glicólico PMN – Polymorphonuclear cells
PVA – Polyvynil Alcohol
SEM – Scaning electron microscopy TGF – Tumor growth factor
! 1! INTRODUÇÃO
A terapia periodontal visa a biocompatibilização da superfície radicular por meio da remoção do biofilme dental, possibilitando o restabelecimento da saúde periodontal (Apatzidou and Kinane 2010).
A raspagem e o alisamento radicular são mecanismos eficazes no tratamento da doença periodontal, entretanto, em alguns casos podem não ser capazes de restabelecer ou manter a saúde periodontal de maneira previsível. Um dos fatores que poderia contribuir para isso seria a persistência ou a recolonização de microrganismos nos sítios tratados (Drisko and Periodontol 1998), já que no esquema convencional de raspagem e alisamento radicular por sextantes ou quadrantes, bolsas não tratadas podem servir como fonte de periodontopatógenos para recolonização (Apatzidou and Kinane 2004; Quirynen et al. 2006).
A preocupação com a recolonização da bolsa periodontal deu origem ao conceito de tratamento da boca toda feito em uma única sessão (Quirynen et al. 1995). Esta abordagem consiste em instrumentação mecânica em 2 sessões dentro de 24 horas, irrigação subgengival com clorexidina 1%, escovação da língua por 1 minuto com gel de clorexidina 1% e uso de enxaguatório de clorexidina 0,12% por 2 meses (Bollen et al. 1996).
Nesse contexto, foi observada a eficácia clínica do debridamento periodontal, que consiste em uma instrumentação subgengival mais conservadora, visando à desorganização do biofilme e remoção das toxinas bacterianas aderidas superficialmente ao cemento radicular (Wennström et al. 2001). Esta abordagem
pode ser realizada em tempo reduzido, em uma única sessão, o que para o paciente significa menos desconforto (Cardaropoli et al. 2003).
Del Peloso Ribeiro et al. (2008) constataram que o debridamento periodontal proporcionou resultados clínicos, microbiológicos e imunológicos semelhantes aos obtidos com a raspagem e alisamento radicular evidenciando a eficácia desta abordagem no tratamento da periodontite crônica avançada.
Com o melhor conhecimento da etiologia da doença periodontal, o uso de agentes antimicrobianos, tanto sistêmicos como locais, tem sido sugerido como coadjuvantes do tratamento mecânico (Soskolne et al. 1998). Essa abordagem pode ser eficaz principalmente em áreas inacessíveis à instrumentação mecânica (Rams and Slots 1996).
A utilização de sistemas de liberação controlada de drogas visa fornecer uma concentração adequada de antimicrobiano que penetre e permaneça no sítio periodontal por períodos de tempo prolongados. A liberação prolongada de um agente antimicrobiano, substancialmente acima de sua concentração inibitória mínima (CIM) (normalmente com concentração de 80 a 100 vezes maior que a CIM) é de particular importância devido à organização da microbiota subgengival como um biofilme (Salvi et al. 2002). Além disso, pode-se citar a redução da toxicidade, uma vez que os efeitos colaterais gerados pelo sistema convencional de administração do medicamento podem ser minimizados pelas pequenas quantidades do princípio ativo que o sistema de liberação controlada aplica, além do aumento da substantividade do fármaco no local de ação desejado (Ravi Kumar 2000).
Desta forma a doxiciclina (DOX), uma derivada da tetraciclina, é um antibiótico de amplo espectro contra anaeróbios Gram-positivos e Gram-negativos, incluindo organismos implicados na etiologia da doença periodontal. Ela é capaz de inibir a metaloproteinase de matriz (MMP) que destrói os constituintes do periodonto como colágeno, fibras elásticas, proteoglicanas e fibronectinas (Emingil et al. 2008; Oringer et al. 2002). Wennström et al. (2001) concluíram que a instrumentação subgengival simplificada pode ser indicada se combinada com a aplicação local de DOX.
A propriedade farmacodinâmica do sistema de liberação controlada também tem papel importante em assegurar a liberação controlada do fármaco.
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glicólico (PLGA) é um copolímero biorreabsorvível bem caracterizado e aprovado para uso em humanos pelo US Food and Drug Administration (US FDA) (Mukherjee et al. 2008) e já empregado como veículo para sistemas de liberação controlada (Astete and Sabliov 2006; Misra et al. 2009). O PLGA permite a liberação local controlada da droga eficientemente a partir da hidrólise de sua estrutura química, a qual é clivada em ácido lático e ácido glicólico até piruvato, quando é assimilado pelo ciclo do ácido tricarboxílico, gerando como produtos finais dióxido de carbono e água (Misra et al. 2009).
Tendo em vista a inexistência de estudos que associem a utilização de nanoesferas com DOX ao debridamento mecânico na terapia periodontal de pacientes com periodontite crônica avançada, o objetivo do presente estudo foi preparar, caracterizar e avaliar o efeito do debridamento periodontal associado à administração local de nanoesferas contendo doxiciclina no tratamento da periodontite crônica avançada.
O Capítulo 1 desta tese foi submetido e aceito no Journal of Biomaterials Science: Polymer Edition. E o Capítulo 2 está de acordo para submissão no Journal of Clinical Periodontology.
CAPÍTULO 1: Characterization and pharmacokinetic profile of controlled released doxycycline by PLGA nanospheres adjunct to non-surgical periodontal therapy
Lucas Alves Moura*†; Fernanda Vieira Ribeirov; Talita Bianchi Aiello†; Eliana Ap. De Rezende Duek†; Enilson Antonio Sallum*; Francisco Humberto Nociti Junior*; Márcio Zaffalon Casati*; Antonio Wilson Sallum*.
* Department of Prosthodontics and Periodontics, Division of Periodontics, Piracicaba Dental School, State University of Campinas (UNICAMP), Piracicaba, São Paulo, Brazil
† Laboratory of Biomaterials, Pontifical Catholic University of Sao Paulo, Sorocaba, Brazil.
v Department of Periodontics, School of Dentistry, Paulista University São Paulo, São Paulo, Brazil.
ABSTRACT
The aim of this pilot study was to assess the release of locally-delivered doxycycline by Poly (L-lactide-co-glycolide) (PLGA) nanospheres in the periodontal pocket of patients with chronic periodontitis, treated by non-surgical periodontal therapy. Nineteen sites of non-adjacent teeth of four different patients were evaluated. 5 mg of PLGA nanospheres loaded with 20% doxycycline hyclate (DOX) were administered per periodontal site. To quantify DOX released into the periodontal pocket, gingival crevicular fluid (GCF) was collected from the sites on days 2, 5, 7, 10, 15, and 20 after DOX application, and high performance liquid chromatography (HPLC) was performed. Data were statistically assessed by ANOVA/Tukey test. At days 2, 5, and 7, the DOX concentration was stably sustained (23.33 ± 1.38; 23.4 ± 1.82; 22.75 ± 1.33 µg/mL, respectively), with no
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concentration (19.69 ± 4.70 µg/mL) was observed only on day 20. The DOX delivery system developed demonstrated a successful sustained release after local administration, as an adjunct to non-surgical periodontal therapy.
Keyword: Delivery system, nanospheres, doxycycline, PLGA, FTIR, non-surgical periodontal therapy.
INTRODUCTION
One of the ultimate goals of periodontal therapy is to establish and maintain adequate infection control in the dentogingival area 1. To this end, non-surgical mechanical treatment has been widely used to treat periodontitis and has presented successful outcomes 2. Nevertheless, there are some conditions that may negatively affect mechanical treatment outcomes; for example, occasionally, the pathogen may have the capacity to invade adjacent tissues 3 and sometimes dentists experience difficulty in accessing deep intrabony defects, furcation defects, or deep pockets with periodontal instruments 4–6. In this context, different antibiotic therapies, which may be systemic or locally delivered, have been introduced as an adjunctive to periodontal pockets for non-surgical treatment 7,8. It has been demonstrated that adjunctive antibiotics to mechanical instrumentation may minimize periodontal pathogens and improve periodontal healing 9–13.
Despite the good outcomes obtained with systemic antibiotics 9,13,14, there are limitations in this antibiotic approach, such as the development of bacterial resistance and poor absorption, which may account for much of the variability in the clinical response to this therapy 14,15. Considering these negative aspects, with regard to the use of systemic antibiotics, the use of local delivery systems in periodontal defects presents advantages; furthermore such methods may allow the establishment of a drug reservoir able to maintain a high antibacterial concentration at the site for prolonged periods of time 16,17.
Use of local anti-infective therapy with pharmacological agents presents wide variations both in the methods of drug delivery and in pharmacological agents used. Drug delivery methods initially included oral rinses, then oral irrigation
devices, followed by subgingival irrigation, using syringes or power irrigation devices 18. Most recently, drug delivery has involved the incorporation of anti-infective drugs into sustained-release vehicles that enable subgingival administration of the drug. These sustained-release vehicles overcome many of the problems inherent in other drug delivery methods, such as rinsing or irrigation 7, in which polymeric micro and nanoparticles act as drug delivery systems and these have been reported as an attractive alternative for progressive and long-term delivery of therapeutic agents 19.
Doxycycline hyclate (DOX), one of the most important antibacterial agents used during periodontal therapy 10,17, is a semisynthetic antibiotic derived from oxytetracycline, which acts by inhibiting bacterial protein synthesis used against a wide range of Gram-positive and Gram-negative bacteria 20–23. Additionally, DOX presents some characteristics of matrix metalloproteinase (MMP) inhibition 24–27, inhibition of peripheral blood polymorphonuclear cell (PMN) degranulation 25,28, increase in the production of tumor growth factor- β1 (TGF- β1) 29, and reduction of collagen breakdown and alveolar bone resorption 12.
Clinical studies have confirmed that the combination of subgingival instrumentation with local doxycycline gel application with a syringe introduced into deep periodontal or peri-implantar sites can be considered as a justified non-surgical treatment of periodontitis and peri-implantitis 17,30–32. Although the antibacterial mechanisms related to DOX 12,25,28 and the clinical outcomes associated with this drug may indicate this agent as an important alternative to adjunctive use during the treatment of periodontal infection, the currently used local delivery systems for DOX present a short sustainable release in the gingival crevicular fluid (GCF) 23,33. Recently, Dawes et al (2009) 34 evaluated the use of poly(L-lactide-co-glycolide) (PLGA) nanospheres as a controlled delivery system of dexamethasone into periodontal pockets and suggested that these could be used to prolong the effect
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The aim of this pilot study was to evaluate the efficiency of locally-delivered doxycycline in GCF by PLGA nanospheres, as an adjunct to non-surgical periodontal therapy.
MATERIALS AND METHODS
Nanosphere formulation
PLGA nanospheres were produced at the Biomaterials Laboratory (Labiomat) of the Faculty of Biological Sciences of Pontifical Catholic University of São Paulo. A protocol adapted from Dawes et al. (2009) 34 and Misra et al. (2009) 35 was adopted. A water-in-oil-in-water (W/O/W) double emulsion solvent evaporation technique was employed to formulate the DOX-loaded PLGA nanospheres. For this method, DOX equivalent to 20% (water in water - w/w) dry weight of the polymer was dissolved in 1 ml of 0.1 mM phosphate buffer, ph 4, to form DOX aqueous solution; 100 mg of PLGA dissolved in 4 ml of dichloromethane (DCM) (Quimex, São Paulo, Brazil) solution was used as the oil phase; 1 ml of the above-prepared DOX aqueous solution was added to 4 ml of DCM solution and emulsified using an ULTRA-TURRAX mixer (T25, IKA, Staufen, Germany) for 4 minutes at 17.5 x 103 RPM. Then, this stable W/O emulsion was slowly added into 200 mL of an aqueous solution containing 2% polyvinyl alcohol (PVA) (Vetec Química Fina Ltda, Rio de Janeiro, Brazil) with 4% of NaCl and emulsified for 6 min at 17.5 x 103 RPM in order to form the W/O/W emulsion at room temperature. Furthermore, solvent removal and hardening of the nanospheres was achieved by continued stirring in a magnetic agitator overnight (Fisatom mod. 752 A, São Paulo, Brazil). Later, the nanospheres were isolated by centrifugation (3500 RPM/3 minutes) and washed with distilled water several times to remove PVA. The nanospheres, thus produced, were frozen at -20ºC for 24 h. Finally, the nanospheres were freeze-dried in an industrial freeze-dryer (GT COM 6011; Fa. Hof Sonderanlagenbau GmbH, Lohra, Germany), performing cycles according to an established generic protocol for unknown samples. After 7 days, the vials were sealed and removed.
Surface morphology of nanospheres was observed by scanning-electron microscope (JEOL JSM-T220A scanning electron microscope, JEOL Ltd, Japan) operating at an accelerating voltage of 10 – 25 kV. A random sample of nanospheres was sputtered with gold to make them conductive and placed on a copper stub prior to the acquisition of scanning-electron microscope images.
Fourier Transform Infrared spectral study (FTIR)
To investigate the possible chemical interactions between the drug and the polymer matrix, Fourier transform infrared spectroscopy (FTIR) spectra were taken on the Perkin Elmer spectrometer (Model Spectrum 1, Perkin Elmer, USA).
Samples were crushed with KBr to produce pellets by applying a pressure of 300 kg/cm2. FTIR spectra of void nanospheres, native DOX and DOX-loaded nanopheres were scanned in the 4000–650 cm-1 range.
Study design
A total of nineteen periodontal pockets with probing depth ≥ 5mm and bleeding on probing, from non-adjacent teeth, of four different patients, were included in the study to receive doxycycline from PLGA nanospheres as an adjunct to non-surgical periodontal therapy.
This study design was approved by the Institutional Review Committee for Human Subjects of the University of Campinas (register number 012/2009). All subjects enrolled in this research signed their Informed Consent; the consent form and protocol was previously approved by the Institutional Committee for Human Research.
Periodontal treatment
Full-mouth mechanical debridement was performed by the same operator (different from the examiner), using an ultrasonic scaler device (CavitronTM, Dentsply, Rio de Janeiro, Brazil), under local anesthesia (Alphacaine – 2% Lidocaine, 1:100.000 Epinephrine, DFL inc, Rio de Janeiro, Brazil), for forty-five minutes (Wennström et
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Sample Collection
Samples of the gingival crevicular fluid (GCF) were collected from sites treated with DOX at 2, 5, 7, 10, 15 and 20 days following periodontal treatment (Straub et al. 2001), by placing filter paper strips (Periopaper, Oraflow, Plainview, NY, USA) into the pocket until a slight resistance was perceived, and then left in place for 30s. Immediately, the volume of the sample was measured with the aid of a calibrated electronic gingival fluid measuring device (Periotron 8000, Oraflow, Plainview, NY, USA). After volume measurements, the strips were placed into empty sterile microcentrifuge tubes and immediately stored at -20°C, according to Vienneau & Kindberg, 1997 36. Strips visibly contaminated by blood were discarded and a new collection was made after 60s. The same examiner that performed all clinical measurements also collected all GCF samples. GCF volume was used to report DOX as a concentration per unit volume (µg/mL).
HPLC Analysis
After collection of the samples in the periods determined, high performance liquid chromatography (HPLC) analysis was performed for determination of the DOX concentration after its release into the GCF in the determined time intervals. The following procedures were used for the analysis, according to Ruz et al. (2004) 37:
Chemicals, reagents and solutions
Doxycycline was provided by Sigma-Aldrich (Sigma-Aldrich Co. LLC Steinheim, Germany). Acetonitrile, methanol, and acetic acid (HPLC grade) were obtained from Merck (Darmstadt, Germany). And Chloroform by Sigma-Aldrich (Sigma-Aldrich Co. LLC Steinheim, Germany).
Standard solutions and samples
Stock solutions of doxycycline from GCF, with a concentration of 320 µg/ml, respectively, were prepared separately by dissolving 8 mg of doxycycline in 25 ml of mobile phase. Finally, 10 standard solutions of doxycycline (1, 5, 16, 32, 80, 120, 160, 240, 280 and 320 µg/mL) were made by further dilution of the solutions with appropriate volumes of mobile phase. Standard and stock solutions of doxycycline were stored at 4ºC.
Apparatus and chromatographic conditions
The apparatus used for the HPLC analysis was a 1525 HPLC system with a 1525 binary HPLC pump and Waters 2487 dual absorbance detector set at 347 nm (Waters Technologies do Brazil LTDA, Barueri, Brazil).
Data acquisition and analysis were performed with a computer using the Breeze 2 software (Waters Technologies do Brazil LTDA, Barueri, Brazil). Separation was carried out at 10ºC on a reversed-phase, 250 mm × 4.6 mm column packed with C18, 5 µm silica reversed-phase particles (Altima) obtained from Alltech (Sedriano, Milan, Italy). This column was preceded by a reversed-phase C18, 5 µm guard column (Kromasil, 20 mm × 4 mm, Symta, Spain). The mobile phase was a mixture of 5% acetic acid–methanol–acetonitrile (55:20:25, v/v/v). Separation was achieved by isocratic solvent elution at a flow-rate of 0.25 ml/min.
Sample preparation
GCF samples were transferred to 13mm ×100mm conic tubes and spiked with 20µl acetonitrile. The tubes were cased, vortex-mixed for 1 min, and centrifuged at 12.85 × g and 4ºC for 10 min. The supernatants were transferred to limited volume autosampler vials, diluted with a mixture of methanol–acetic acid (1:1), cased and placed on the HPLC autosampler. A 50 µl aliquot of the supernatant was injected onto the HPLC column. Subsequently, 3 ml of methanol was added to precipitate the polymer.
Quantitation of doxycycline
Calibration curves were determined by least square linear regression analysis (weighting 1/X2). The peak area ratio of doxycycline versus the corresponding doxycycline concentration was plotted. The linearity of the method was confirmed by comparing the slopes, the intercepts of calibration curves with zero and the correlation coefficients with 1. Moreover, a Student’s t-test was used to compare the back-calculated concentrations with each calibration curve versus the nominal
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Test validation
The method was validated by analysis of DOX quality control samples prepared at four concentrations spanning the calibration range (for GCF: 2, 12, 30 and 70 µg/ml). Four samples of each quality control pool and calibration samples were analyzed on three different days with five samples in each quality control pool. Precision and accuracy was determined.
Statistical analysis
Analysis of variance (ANOVA) was used to detect differences in concentrations of DOX delivered in GCF among evaluated periods. When statistical difference was found, analysis of the difference was determined by post Hoc analysis of Tukey.
RESULTS AND DISCUSSION
Scanning-electron microscopy
The mean size of the spheres was below than 1 µm. They showed round and regular shape with no signs of degradation such as pores (Figure 2).
Fourier Transform Infrared spectral study
FTIR spectroscopy results confirmed the chemical stability of DOX in the nanospheres. Figure 3 shows the FTIR spectra of PLGA nanospheres loading DOX, DOX and PLGA nanospheres. DOX showed characteristic bands due to different functional groups. The band at 3388 cm-1 is due to O-H/N-H stretching vibrations, while those observed at 3000 and 2950 cm-1 are due to the C-H stretching vibrations. Bands at 1745 and 1451 cm-1 are due to primary amide (N-H) bending and aromatic N-H bending vibrations, respectively. Carbonyl (C=O) stretching vibrations are seen at 1383 cm-1, while the bands at 1082 and 846 cm-1 are due to -CH2 bending and C-H bending vibrations, respectively. The bands at 746 and 710 cm-1 belong to C-N stretching vibrations. Those spectroscopies were similar to the ones observed by Misra et al, 2009 35.
Behavior of in vivo release (HPLC)
Assessing the mean of DOX concentration from GCF at 2, 5, 7, 10, 15 and 20 days, it was observed that from day 2 to day 7, the DOX concentrations were
stably sustained (23.33 ± 1.38, 23.4 ± 1.82 and 22.75 ± 1.33 µg/mL, on days 2, 5 and 7, respectively), with no significant differences among the assessment times (p>0.05). At day 10 and day 15, a tendency towards a decrease in DOX concentration was observed (21.74 ± 0.91 and 20.53 ± 4.88 µg/mL, for days 10 and 15, respectively), but statistically significant differences were not noted in comparison to the previously evaluated periods (p>0.05). A significant decrease in the DOX concentration in GCF was observed only on day 20 (19.69 ± 4.70 µg/mL), as compared with the initially (2, 5 and 7 days) assessed periods (p<0.05) (Figure 4).
Considering the lack of studies assessing the concentration of DOX released by nanospheres into periodontal pockets after non-surgical periodontal therapy, the objective of the present study was to evaluate the efficiency of locally delivered DOX by PLGA nanospheres in periodontal pockets of patients with chronic periodontitis, within a 20-day evaluation period, by HPLC. The results of the present study demonstrated a consistent release of DOX by PLGA nanospheres into the periodontal pockets for a duration of at least 15 days, confirming the efficacy of DOX-loading-PLGA nanospheres in maintaining satisfactory levels of local drug delivery for over two weeks at periodontal sites.
Studies using several antimicrobial agents for local administration, such as metronidazole gel, chlorhexidine chip or 10% DOX gel, as an adjunctive to periodontal debridement, have demonstrated that these drug delivery systems may be not so effective 2,23,33. Limitations of these systems include unpredictable penetration in the periodontal pockets by the drug and rapid drug clearance from the pocket resulting in an inadequate time of exposure of subgingival bacteria to the drug 22. However, in addition to prolonged delivery of an antimicrobial agent substantially above its MIC, in vitro, the delivery system needs to act without promoting adverse systemic effects or cytotoxicity 2,38.
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exceeding 19µg/mL, which may classify these nanospheres as a controlled delivery system. Additionally, DOX-loading-PLGA nanospheres presented pharmacokinetic and clinical properties that allowed the delivery of efficacious levels of DOX to the periodontal pockets and the maintenance of these levels for over two weeks. The concentrations and substantivity, observed in this study, were above those obtained for other formulations of locally delivered DOX, such as 10% DOX gel 39, which demonstrated sustained substantivity in the periodontal pockets for a maximum period of 10 days 39,40. Considering these previous findings and taking into account the successful data provided by the present investigation, the use of nanospheres emerges as an important alternative to the drug release and sustaining issue.
Biodegradable polymers have been used for controlled drug delivery as a means of prolonging the action of drugs 7,41,42. In this study, PLGA was used as vehicle, since doxycycline may be encapsulated into bioresorbable nanospheres, and released at a constant rate by diffusion through the polymer 43. The size of the nanospheres, as well as the drug encapsulation efficiency and release rate could be modulated by adjusting the formulation conditions 44. The method for preparing the PLGA nanospheres in the present study allowed a particle size control and favorable distribution without physical aggregation 45. A remarkable fact of this study is that PLGA copolymers were used with free terminal carboxyl groups, which efficiently improve the drug encapsulation and slow down its release rate 46. With the W/O/W technique, the burst effect was followed by a slow release of DOX, leading to a constant release for 2 weeks. Permeability and drug release rates from nanospheres were also controlled by the speed of homogenizing and surfactant used during their production.
The principal advantage of using nanospheres as a drug delivery system is their capacity of sustaining release into the periodontal pocket for longer period than gels and release devices, without promoting any adverse reaction 46–50. As such, this drug delivery system can be used to increase the spectrum of the antimicrobial
activity against the microorganisms causing periodontal diseases and decrease the therapy cost 50.
Innumerous methods have been used for assaying the release of DOX from the GCF 15. The high performance chromatography methods used in the present investigation have been frequently used to determine doxycycline and other tetracycline antibiotics in pharmaceuticals, and biological fluids, such as plasma, serum, urine, blood or saliva because of their sensitivity and selectivity 36,37. Other methods, such as fluorimetric and spectrophotometric techniques have also been used for the determination of doxycycline in pharmaceuticals. However, the specificity of these methods is very low when DOX is measured in biological samples 37.
To further investigate the efficiency of PLGA nanospheres as a controlled delivery system of DOX in periodontal pockets, further studies are required to assess a larger sample of patients and for longer evaluation periods of DOX release. Additionally, evaluations of clinical periodontal parameters and microbiological assays are necessary to determine the clinical relevance of using DOX-loading-PLGA nanospheres as an adjunctive to mechanical periodontal treatment in patients with chronic periodontal disease.
Within the limits of the present study, it may be concluded that the release of DOX in the periodontal pockets by PLGA nanospheres was maintained stable for at least 15 days, classifying these nanospheres as a controlled delivery system. Additionally, DOX-loading-PLGA nanospheres present the pharmacokinetic and clinical properties to deliver DOX to the periodontal pockets adjunct to non-surgical periodontal therapy and to maintain DOX release for over two weeks.
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FIGURES
Figure 1. PLGA nanospheres loaded in the tip for local administration (A); subgingival administration of nanospheres into the periodontal pocket after mechanical debridement (B); and
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Figure 2. SEM of nanospheres, after liophilization, presenting regular spherical morphology and no signs of degradation, such as irregular pores.
Figure 3. FTIR of PLGA nanospheres; DOX and PLGA nanospheres loading DOX, showing the interaction between the polymer and the drug. Bands at 1745 and 1451 cm-1 refers to DOX and at 1745 and 1082 cm-1 refers to PLGA.
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Figure 4. Nanospheres were loaded into the selected periodontal pockets and collected GCF samples were analysed for the DOX concentration (ug/ml) released by HPLC in different time intervals (2, 5, 7, 10, 15 and 20 days) – Different letters indicate statistical difference by ANOVA/Tukey test (p<0.05).
CAPÍTULO 2: Clinical and microbiological evaluation of locally delivered doxycycline by PLGA nanospheres adjunct to nonsurgical periodontal therapy: a randomized double blinded clinical trial
Running
Title: Locally delivered doxycycline adjunct to nonsurgical periodontal therapy. Lucas Alves Moura*†; Isabela Alves†; Eliana Ap. De Rezende Duek†; Christopher F. Cuff! ; Enilson Antonio Sallum*; Francisco Humberto Nociti Junior*; Márcio Zaffalon Casati*; Antonio Wilson Sallum*.
* Department of Prosthodontics and Periodontics, Division of Periodontics, Piracicaba Dental School, State University of Campinas (UNICAMP), Piracicaba, São Paulo, Brazil.
† Laboratory of Biomaterials, Pontifical Catholic University of Sao Paulo, Sorocaba, Brazil.
! Microbiology, Immunology & Cell Biology, School of Medicine, Robert C. Byrd
Health Sciences Center, West Virginia University.
Keywords: Chronic periodontitis; local doxycycline; ultrasonic debridement; PLGA nanospheres; microbiologic assessment.
ABSTRACT
Background: The aim of this clinical study was to asses the effect of ultrasonic periodontal debridement associated to locally delivered doxycycline (20%) by PLGA nanospheres on chronic generalized periodontitis treatment
Methods: Thirty patients with chronic periodontitis and a minimum of seven pockets (>5 mm) in non-molars teeth that bled on probing were selected. Patients were randomly assigned to ultrasonic periodontal debridement followed by local application of doxycycline by PLGA nanospheres (DB+DOX) and periodontal debridement followed by administration
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pockets at baseline, 1, 3 and 6 months. Polimerase chain reaction (PCR) analysis was used to detect the frequency of Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf),
Treponema denticola (Td), Aggregatibacter actinomycetemcomitans (Aa), and Prevotella intermedia (Pi).
Results: At 6 months, no difference was found between groups regarding plaque and bleeding on probing. However, PD reduction was significant for DB+DOX in moderate (2.5 ± 1.3 mm) and deep (4.1 ± 1.1 mm) than for DB (1.1 ± 0.8 mm and 2.7 ± 1.6 mm, respectively); and CAL gain, DB+DOX showed gains (2.82 ± 1.3 mm and 4.73 ± 1.5 mm) versus DB (1.15 ± 1.7 mm and 3.24 ± 2.1 mm) in moderate and deep sites. The proportion of sites showing CAL gain ≥ 2 mm, at 3 months was 100%. There was significant reduction in the bacteria levels for DB+DOX along the follow up and after 6 months comparing to baseline and DB group.
Conclusion: the use of nanospheres loading doxycycline adjunct to periodontal debridement could improve PD reduction and CAL gains followed by reduction of periodontal pathogens levels in chronic periodontitis.
INTRODUCTION
Mechanical therapy by hand instrumentation or ultrasonic debridement, is the most common therapy for periodontitis of varying severity and has well documented efficacy 1, but in not all sites it is effective, maybe because of several subject-related and tooth site-related factors which may compromise the healing of periodontal wound 2.
Occasionally, periodontal pathogens may have the capacity to invade adjacent tissues 3 and sometimes dentists experience difficulty in accessing deep intrabony defects, furcation defects, or deep pockets with periodontal instruments 4–6. In this context, different antibiotic therapies, which may be systemic or locally
delivered, have been introduced adjunct to periodontal pockets for nonsurgical treatment 7,8.
Anti-infective agents have been used in attempt to improve the nonsurgical treatment outcome. Systematic reviews 7,9, described that locally delivered antibiotics adjunct to the mechanical periodontal treatment, significantly improved probing depth (PD) reductions and clinical attachment gains when compared to mechanical treatment alone. It has been demonstrated that adjunctive antibiotics to mechanical instrumentation may minimize periodontal pathogens and improve periodontal healing 10–13.
Polymeric nanoparticles offer an attractive alternative approach for drug delivery due to their biocompatibility, nonimmunogenicity, nontoxicity, biodegradability, simple preparation methods, physicochemical stability and drug-targeting properties 14. Polymeric nanoparticles release the drug efficiently from the polymeric matrix by diffusion, swelling or polymer erosion, or a combination of these processes 15,16. Different synthetic polymers such as the copolymer poly(D,L-lactide-co-glicolide) (PLGA) mostly used in preparation of polymeric nanoparticles 17. This synthetic copolymer is capable of controlled release the therapeutic agent for a longer period of time 18.
In this way, the purpose of the present clinical study was to evaluate whether the association of doxycycline (DOX) encapsulated in PLGA nanospheres to ultrasonic debridment for the treatment of patients with chronic generalized
! 27! METHODS
Study Design
The present study was approved by the Piracicaba Dental School Ethics Committee for Human Research (protocol # 012/2009) as a randomized double blinded parallel clinical study designed to investigate whether a novel local delivery system for doxycycline adjunctive to ultrasonic debridement could provide better results in treating chronic generalized periodontitis.
Population screening and eligibility criteria
Potential patients were selected from those referred to the Graduate Clinic of the Piracicaba Dental School, State University of Campinas. All patients received a complete periodontal examination, including a full-mouth periodontal probing, radiographic examination and complete anamnesis.
The entry criteria were as follows: 1) chronic generalized periodontitis (AAP,1999); 2) non-smokers; 3) at least 7 sites with probing depths >5mm, and 2 sites with probing depth >7mm; 4) at least 20 teeth present; 5) systemically healthy; 6) not received periodontal care 6 months prior to the study; 7) Had not taken medications known to interfere with periodontal health and healing 6 months prior to the study; 8) not pregnant, nor lactant; 9) not sensitive to doxycycline. The CONSORT (Consolidated Standards of Reporting Trials) study flowchart 19 is outlined in Figure 1. All patients received a detailed description of the proposed treatment and gave their informed written consent.
Patients were randomly assigned to the following treatment groups: Test group: DB+DOX - 45 minutes of full-mouth ultrasonic debridement + subgingival application of PLGA nanospheres loading doxycycline 10% (n=15); and Control group: DB - 45 minutes of full-mouth ultrasonic debridement + subgingival application of void PLGA nanospheres (n=15).
The patients were treated by a single-session of periodontal debridement during 45 minutes, using an ultrasonic instrument (Cavitron, Dentsply, Rio de Janeiro, Brazil), under irrigation with sterile saline solution 20. After the debridement, local administration of nanospheres loading doxycycline (DOX) (test) or empty nanospheres (control) was performed (Figure 2).
Both interventions were performed at baseline only and clinical parameters were recorded at baseline, 3 and 6 months after treatment. Microbiological samples, from subgingival biofilm, were collected at baseline, 1, 3 and 6 months after treatment.
Clinical assessments
Clinical parameters were evaluated 20 days following initial visit (baseline), at 3 and 6 months and included: plaque index (PI) and bleeding on probing (BP): percentage of sites presenting visible plaque and bleeding on probing, recorded at six sites per tooth. Full-Mouth Plaque Score and Full-Mouth Bleeding Score (FMBS) were calculated after assessing dichotomously the presence of plaque and BP (from the bottom of the pocket when probing with a manual probe) and
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clinical attachment level (CAL): at six sites per tooth, was also measured with a PCP-15 Periodontal Probe (Hu-Friedy - Chicago, IL, USA). These measures were performed with the use of a plastic stent, presenting edges for the positioning of the stent probe.
A previously calibrated examiner (LAM), masked for the type of treatment, performed all clinical assessments. Duplicate measurements of PD and CAL, taken 1 hour apart, were performed in 10 patients in a pre-study training period. A total of 109 sites were assessed for calibration. Pearson’s correlation test and Student paired t test were applied to verify reproducibility of measurements. The examiner was considered calibrated once positive statistically significant correlation and no statistically significant differences between duplicate measurements were obtained (r = 0.75 for PPD and r = 0.91 for CAL). The differences between the examinations were within 1 mm in 89% of CAL measurements and 93% of PD measurements.
Microbiologic assessment
Sample collection
After removal of supragingival plaque, teeth were isolated using coton rolls. The subgingival plaque samples were obtained using sterile paper points inserted into moderate pocket (5-6mm) and deep pockets (≥7mm) from each patient. The paper point was allowed to remain in position for 45 seconds and was transferred to an Eppendorf tube. The samples were stored at -20ºC.
Bacterial DNA was purified from subgingival biofilm using QIAamp Mini Kit following the manufacturer’s instructions (Qiagen, Germany). The purified DNA was frozen and then lyophilized until further tests.
Standards and positive control
Bacterial strains obtained from American Type Culture Collection (ATCC):
P. intermedia (Pi) (ATCC 25611), P. gingivalis (Pg) (ATCC 33277), A. actinomycetemcomitans (Aa) (ATCC 29522), and from pure culture of T. denticola (Td) and T. forsythensis (Tf) were used as positive controls and standards.
Bacterial DNA was purified using PowerSoil® DNA Isolation Kit (MO BIO Laboratories, Inc). The concentration of DNA was adjusted to 15 ng/ul in water and then six additional 10-fold serial dilutions were made. Three replicate samples of each dilution (5 ul/sample) were then assayed as described below for each specific DNA. The sensitivity of each assay was determined to be the lowest dilution of DNA that produced a statistically significant (p < 0.001) average signal higher than the no template control using a t-test.
PCR detection
Real time polymerase chain reaction (PCR) detection and quantification were based on the amplification of variable regions of the 16S rRNA genes. The primer sequences used in the present study are listed in Table 1.
PCR amplification was performed using 20 ng of DNA in a mixture containing 5ul mastermix (Thermo Scientific Maxima SYBR Green/ROX qPCR Master Mix 2X), 0.3 uM of each primer and completed with RNAase and DNAase free water to a total of 10 ul.
