universidade federal do rio grande do norte · gian, claudinha, josie, josi e todos pelos quais...

MINISTÉRIO DA EDUCAÇÃO

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

SÍNTESE E AVALIAÇÃO DA ATIVIDADE ANTITUMORAL DE NANOGÉIS DE

FUCANA A DA ALGA MARROM Spatoglossum schröederi (C.Agardh)

Kützing

JAILMA ALMEIDA DE LIMA

NATAL/RN 2014




Kützing

Tese apresentada ao Programa de Pós-

Graduação em Ciências da Saúde da

Universidade Federal do Rio Grande do

Norte como requisito para a obtenção do

título de Doutor em Ciências da Saúde.

Orientador: Prof. Dr. Hugo Alexandre de O. Rocha

NATAL/RN 2014

CATALOGAÇÃO NA FONTE

L732s

Lima, Jailma Almeida de.

Síntese e avaliação da atividade antitumoral de nanogéis de

fucana A da alga marrom Spatoglossum schöederi (C. Agardh)

Kützing / Jailma Almeida de Lima. – Natal, 2014.

106f. : il.

Orientador: Prof. Dr. Hugo Alexandre de O. Rocha.

Tese (Doutorado) – Programa de Pós-Graduação em Ciências

da Saúde. Centro de Ciências da Saúde. Universidade Federal do

Rio Grande do Norte.

1. Fucanas – Tese. 2. Polissacarídeos sulfatados – Tese.

3. Atividade antitumoral – Tese. 4. Nanogéis – Tese. I. Rocha,

Hugo Alexandre de O. II. Título.

RN-UF/BS-CCS CDU: 582.272(043.2)

ii

MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE

CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

Coordenador do Programa de Pós-Graduação em Ciências da Saúde:

Prof. Dr. Eryvaldo Sócrates Tabosa do Egito

iii




Kützing

Aprovada em: 21 / 03 / 2014

Banca Examinadora:

Presidente da Banca:

Prof. Dr. Hugo Alexandre de Oliveira Rocha (UFRN)

Membros da Banca

Profa. Dra Valéria Soraya de Farias Sales (UFRN)

Prof. Dr. Artur da Silva Carriço (UFRN)

Profa. Dra. Valquíria Pereira de Medeiros (UFJF)

Profa. Dra. Norma Maria Barros Benevides (UFC)

iv

Dedico esta obra

A Deus.

Só tenho que agradecer-Te. Obrigada, Senhor, por tudo!

Grande é a sua Bondade e Misericórdia!

A minha Mãe, Francisca (Rosa).

Agradeço-te por tudo, especialmente pelo Amor que sempre me deste de forma

incondicional. EU TE AMO!

v

Dedico esta obra

A Hugo Rocha,

O meu eterno agradecimento por ser um educador nato, e por proporcionar não só a

mim, mas todos ao seu redor, a possibilidade de crescimento e busca por algo melhor.

Obrigada, Hugo, por tudo!

A Família BIOPOL,

Obrigada a todos àqueles que fazem ou que já fizeram parte desta história...

Dayanne (Dayn ou amigan), Mariana, Sara, Karol, Cinthia, Leandro, Diego (Popó),

Ruth, Rafael, Gabriel, Moacir, Raniere, Joanna, Letícia, Kaline, Arthur, Vinicius, Max,

Rony, Marília, Monique, Ajax, Fred, Larisse, Regina, Sarah (pequena), Mônica, Pablo,

Danielle, Almino Afonso, Jéssica, Mariane, Fernanda (Pôia), Leonardo Nobre (Leo),

Profa. Fabiana Lima, Ana Karina, Ana Karinne (Donana), Daniel, Fernando, Ivan,

Nednaldo, Eduardo, Edjane, Valquíria.

vi

Dedico esta obra

A “amigannn” Dayn,

Obrigada pela amizade, pelo companheirismo e por ter me proporcionado fazer parte

de sua família. Obrigada também pela confiança, bem mais que isso, pelo privilégio de

ser madrinha de seu filho Heitor e suplente de Helena!

A NOSSA AMIZADE É

Mais que uma mão estendida,

mais que um belo sorriso,

mais do que a alegria de dividir,

mais do que sonhar os mesmos sonhos

ou doer as mesmas dores,

muito mais do que o silêncio que fala

ou da voz que cala para ouvir

é a amizade, o alimento

que nos sacia a alma

e nos é ofertado por alguém

que crê em nós!

vii

Agradecimentos especiais

À UFRN, à Pós-graduação em Ciências da Saúde e ao Departamento de Bioquímica pela oportunidade de concluir esse curso de Pós-graduação, assim como as agências

Financiadoras CAPES e CNPq.

Agradeço novamente ao meu orientador Prof. Dr. Hugo Rocha pela oportunidade oferecida, pela atenção e auxílio prestados durante a pesquisa.

A todos os professores do CCS (UFRN), aos coordenadores do programa de pós-

graduação (PPGCSa) e às secretárias do programa.

As professoras da banca de qualificação: Profa. Naisandra Bezerra e Profa. Ivonete Araújo

A todos os professores do DBQ (UFRN), em especial, a Profa. Edda Lisboa Leite, pela sua força e por toda a sua contribuição à instituição. Obrigada por sempre ter

participado e me proporcionado grandes ensinamentos, principalmente de vida!

Aos meus amigos de laboratório pela colaboração e ajuda nos meus experimentos e também em tudo que precisei:

A Dayn (“amigann” e comadre) que tanto amo por me compreender e por sempre ter uma palavra amiga. Tenho por ti um enorme carinho. Obrigada por fazer parte de sua vida e de

seus flhos Helena e Heitor e daqui a alguns meses de Heloísa. A Karol, por ser esse doce de pessoa, alguém muito especial para mim! Tenho certeza

que seu futuro será brilhante e, mais que isso, queria agradecer-te por sempre estar perto nas horas mais difíceis com carinho e compreensão! Você sempre deixa o laboratório mais

alegre.

A Cinthia. Você é meu exemplo de transformação! Obrigada por tudo, obrigada por ter confiado a mim a tarefa tão importante de ser madrinha (casamento) e mais que isso, de

poder fazer parte da sua vida e de conviver com seu filho Davi. Obrigada A Ruth (Lut Lut), a “safada” que amo de paixão, a Rafael (super Rafildo), que não é o

ABC, mas é o mais querido, Leandro (Lelê) exemplo de profissional e de amigo.

Agradeço também de forma especial a Mariana e a Sara, por terem sido as primeiras a me incentivarem a vir para a Bioquímica. A Mariana, por me apoiar, por estar comigo em tudo que preciso, por me fazer ver meus erros, por ser essa grande amiga, te adoro! A

Sara, por ser nosso pilar de conhecimento “nosso Google”, aquela que nos socorre sempre e em qualquer tempo, te adoro muito. Muito obrigada, amigas, vocês são demais e

muito importantes para mim, estarão sempre no meu coração!!!

Agradeço muito a todos, todos vocês são mais que especiais nessa trajetória, a vocês o meu eterno agradecimento: Gabriel, Moacir, Joanna, Pablo, Raniere, Fernanda (Pôia), Leonardo Nobre (Leo), Letícia, Kaline, Arthur, Vinicius, Max, Rony, Marília, Monique,

Ajax, Fred, Larisse, Regina, Sarah (Pequena), Mônica, Danielle, Almino Afonso, Jéssica, Profa. Fabiana Lima, Ana Karina, Ana Karinne (Donana), Daniel, Fernando,

Ivan, Diego (Popó) e Valquíria.

Agradeço de forma especial a todos os amigos que fiz aqui no Departamento de

Bioquímica: Adriana Brito, Ana Katarina, Luciana Rabêlo, Jonalson, Anderson (Negão), Paula Ivani (Paulinha), Antônio, Marina, Ingrid, Lívia, Ana Katarina, Jefferson, Rômulo, Ana

Luiza, Paula e Demetrius, Juliana, Roberta, Thuany,

viii

Aos amigos dos laboratórios LAMA e LBMG. Conheci pessoas maravilhosas e que me ajudaram bastante no desenvolvimento de algumas técnicas: Beatriz Mesquita, Susana,

Nilmara, Paula Anastácia, Rita, Mayara, Jana, Felipe, Isabel, Dani (Pôia branca), Leonam

Agradeço a mais nova professora da genética, Susana Moreira, você superou todas as

dificuldades e chegou lá, que bom que ficarás por aqui. Tu és giro!!!

A todos que me ajudaram direta ou indiretamente nesta tese: Danilo Cavalcanti, Karla (Farmácia), Priscyla (UFPE), Guiman, Marina e Haroldo.

A Lurdinha, pessoa batalhadora, de uma humildade e sabedoria enorme. Você é uma pessoa

especial e que ensinou muito, não só a parte laboratorial, mas principalmente sobre a vida. Sou

muito grata pelos seus ensinamentos e pelos momentos “felizes” que compartilhamos. Muito

obrigada por tudo.

Agradeço, de forma especial, àqueles que contribuíram de forma diferencial para minha formação como pessoa: aos meus grandes amigos e amigas Joanna D'arc e sua filha

Joyce (que acompanhamos seu crescimento), Sara, Mariana Santana, Chrístier e Railson, vocês foram uma das melhores conquistas, todos vocês são formidáveis e pessoas muito

especiais pra mim. Muito obrigada por me aturarem e me aceitarem como sou. Muito obrigada do fundo do coração. Agradeço também a Adaíres e a Wanessa por fazerem

parte dessa história.

Agradeço à família de Dayn: Leonardo Oliveira (Leo), Dona Célia, Seu José, França, Drielle, Dmitryev, Dastaev (Patrícia e Monick), Dmetryus, Seu Abelardo, Helena, meu

afihado Heitor, Heloísa e a Dona Ceiça. Muito obrigada pela força e pelo carinho. Também dedico esta tese a vocês!

Muito obrigada a todos que fazem parte da Ong “Vida é Alegria”: Mileide, dona Neide, Sara, Mariana, Adaíres, Ricardo, Narjara, Adriana Sabiana, Adineide, Jobson, Fernando, Gian, Claudinha, Josie, Josi e todos pelos quais tenha esquecido o nome, mas que fazem

parte desse grupo pela oportunidade de ajudar a tantas crianças!

Agradeço a todos os amigos que trabalham comigo no CRI Inês, Josinete, Patrícia, Elias, Mércia, Lúcia, Ana Tereza, Severina, Lidiane, Adriana Pinto,

Sueldo, Cimária e Tarciana por compreenderem e me apoiarem nessa batalha que foi o doutorado.

Agradeço a Tarciana, minha companheira do coração, muito obrigada pelo apoio e por ser tão especial para mim. Agradeço também a todos da sua família: Taísa, Vanessa, Rosa,

Maria, Seu Tarcísio e Antônio.

Agradeço também de forma especial aos grandes amigos Eutália e André. Vocês são dois anjos que Deus colocou no mundo, não tenho palavras para descrever

como sou feliz por ter vcoês como amigos!

Agradeço a minha “mãe” Vivi e a Mari também por, apesar de longe, estarem tão perto de mim.

Como foi difícil escrever essa parte! Por mais que eu possa agradecer, ainda assim seria

muito pouco, não há como agradecer pelas alegrias, pelo apoio, pelo carinho, pelo consolo

nas frustações. São muitas as pessoas a quem gostaria de agradecer, mas poderá ser que

ao longo dos agradecimentos a memória possa esquecer de uma ou outra pessoa, mas

que não as tornam menos importantes!

ix

“Talvez não tenha conseguido fazer o melhor, mas

lutei para que o melhor fosse feito. Não sou o que

deveria ser, mas Graças a Deus, não sou o que era

antes”.

(Marthin Luther King)

x

RESUMO

Fucanas são polissacarídeos sulfatados encontrados em algas marrons e equinodermos. Tem sido demonstrado que uma fucana denominada de fucana A, obtida da alga marrom Spatoglossum schröederi, apresenta uma série de efeitos biológicos, em particular, a atividade antitumoral. Com intuito de se potencializar essa atividade, foram adicionados grupamentos tióis a estrutura da fucana A. Posteriormente, os nanogéis foram sintetizados pela formação de nanocomplexos entre a fucana A tiolada e o polietileno glicol (PEG) em várias relações 2.5, 5.0, 10, 15 e 30. Os nanogéis com as relações de 10 e 15 (FucA:PEG10 e FucA:PEG15) foram os que se apresentaram com os menores tamanhos, mais esféricos, com diâmetro em torno de 186,95 ± 10,62 nm e carga de superfície ligeiramente negativa. Após a síntese dos nanogéis, estes foram submetidos aos ensaios antiproliferativos com células da linhagem tumoral 786-0 nas concentrações 8,0 a 64 µg/mL. As células foram analisadas durante um período de 24, 48 e 72 horas. Os dados mostraram que em todas as concentrações de nanogéis de fucana A, a atividade antiproliferativa foi tempo e dose dependente, o mesmo não sendo observado para a fucana A avaliada isoladamente. O nanogel de FucA:PEG15 também induziu apoptose por mecanismos dependentes e independentes de caspases. Posteriormente, FucA:PEG15 também foi marcado com FITC sendo completamente incorporado pelas células 786-0 após 1 hora. Quando a endocitose celular foi parada, o FucA:PEG15 teve o seu efeito antiproliferativo reduzido. Apesar de FucA:PEG15 não possuir efeito anticoagulante por aPTT e PT (até 100 µg/mL), ele apresesentou efeito antioxidante e angiogênico. Esses dados mostram que o nanogel de fucana A exibe várias efeitos (antiproliferativa, antioxidante e antiangiogênica) e, portanto, o seu potencial para a terapia do câncer deve ser investigada.

Palavras chaves: Polissacarídeos sulfatados, fucanas, nanogéis, atividade

antitumoral, citotoxicidade.

xi

LISTA DE ABREVIATURAS E SIGLAS

µL Microlitros

786-0 Linhagem de células derivadas de adenocarcinoma renal

aPTT Tempo de tromboplastina parcialmente ativada

B16-F10 Linhagem de células de melanoma murino

CAT Capacidade antioxidante total

CO2 Dióxido de carbono

DAPI 4′,6′-diamino-2-fenilindol

DAPI 4',6-diamidino-2-phenylindole

DLS Dynamic light scattering

DMEM Meio de cultura sintético complexo – Dubelcco’s Modified Eagle’s Medium

DMSO Dimetilsulfóxido

DPPH 2,2-difenil-1-picrilhidrazila

ECs Células endoteliais ativadas

EHS Tumor Engelbreth-Holm-Swarm

F0.5 Fração precipitada com 0,5 volumes de acetona







FITC Isotiocianato de fluoresceína

Fuc A Fucana A

g Grama

G0 Fase do ciclo celular em que a célula permanece indefinidamente na intérfase

HeLa Linhagem de células de carcinoma cervical humano

HepG2 Linhagem de Células de hepatocarcinoma humano

HS-5 Linhagem de células estromais da medula óssea humana

kDa Kilodalton

M Molar

MEV Microscópio eletrônico de varredura

mg Miligrama

Mili-Q Água ultrapura

Min. Minutos

mL Mililitros

mM Milimolar

mm Milímetros

MTT 3-(4,5-dimethylthiazol-2-y1)2,5-diphenil tetrazolium bromide)

xii

MW Peso molecular

nm Nanômetros

PA Para análise

Panc-1 Linhagem de células de adenocarcinoma de pâncreas

PBS Solução tampão de salino fosfato

PDA Tampão 1,3 diamino propano acetato

PEG Polietileno glicol

pH Potencial de hidrogênio

PI Iodeto de propídio

PT Tempo de protrombina

RAEC Linhagem de células endoteliais de aorta de coelho

RPMI Meio de cultura sintético complexo criado pelo Roswell Park Memorial Institute

SFB Soro fetal bovino

xiii

LISTA DE FIGURAS Figura 1. Alga marrom S. schröederi (C. Agardh) Kützing. A) em exsicata (Foto:

Nednaldo Dantas) e B) na natureza (Foto: Edjane Barroso)..................... 21

xiv

SUMÁRIO

RESUMO................................................................................................................................... x LISTA DE ABREVIATURAS E SIGLAS..................................................................................... xi LISTA DE FIGURAS.................................................................................................................. xiii 1. INTRODUÇÃO....................................................................................................................... 15 2. JUSTIFICATIVA.................................................................................................................... 18 3. OBJETIVOS........................................................................................................................... 19

3.1. OBJETIVO GERAL................................................................................................. 19 3.2. OBJETIVOS ESPECÍFICOS.................................................................................. 19

4. MÉTODOS............................................................................................................................. 20 4.1. MATERIAIS BIOLÓGICOS..................................................................................... 21

4.1.1. Algas...................................................................................................... 21 4.1.2. Linhagens e culturas celulares........................................................... 22

4.2. EXTRAÇÃO E PURIFICAÇÃO DA FUCANA A...................................................... 22 4.2.1. Obtenção do pó cetônico..................................................................... 22 4.2.2. Proteólise.............................................................................................. 22 4.2.3. Fracionamento do extrato bruto com concentrações crescentes

de acetona............................................................................................. 23

4.2.4. Cromatografia em coluna de troca iônica.......................................... 23 4.3. SÍNTESE E CARACTERIZAÇÃO DOS NANOGÉIS.............................................. 24

4.3.1. Síntese das fucanas tiolada................................................................. 24 4.3.2. Caracterização físico-química dos nanogeis de fucanas................. 24 4.3.3. Transmitância dos nanogéis............................................................... 24 4.3.4. Dynamic Light Scattering (DLS).......................................................... 25 4.3.5. Estabilidade........................................................................................... 25 4.3.6. Microscopia eletrônica de varredura (MEV)....................................... 25 4.3.7. Microscopia confocal........................................................................... 25 4.3.8. Espectroscopia de infravermelho....................................................... 26 4.3.9. Atividade antiproliferativa.................................................................... 26 4.3.10. Conjugação da fucana A com fluoresceína (FITC).......................... 27 4.3.11. Avaliação da viabilidade e morte celular por anexina V-FITC/

iodeto de propídio (PI)....................................................................... 27

4.3.12. Atividade antioxidante....................................................................... 28 4.3.13. Atividade anticoagulante................................................................... 28 4.3.14. Ensaio de formação de tubo de matrigel......................................... 28

4.4. ANÁLISE ESTATÍSTICA........................................................................................ 29

5. ARTIGOS PRODUZIDOS...................................................................................................... 30 5.1. ARTIGO 1 (SUBMETIDO)...................................................................................... 32 5.2. CAPÍTULO DE LIVRO............................................................................................ 55 5.3. ARTIGO 2............................................................................................................... 77 5.4. ARTIGO 3............................................................................................................. 83

6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES...................................................................... 91 7. REFERÊNCIAS..................................................................................................................... 93 8. ANEXOS................................................................................................................................ 96

8.1. NORMAS PARA FORMATAÇÃO DA TESE (CCS)............................................... 97 8.2. NORMAS DA REVISTA PARA SUBMISSÃO (MARINE DRUGS)......................... 100 8.3. DECLARAÇÃO....................................................................................................... 105 8.4. COMITÊ DE ÉTICA................................................................................................ 106

15

Almeida-Lima J. PPGCSA/CCS

1. INTRODUÇÃO

O câncer é um dos problemas mais complexos que os sistemas de saúde

mundial enfrentam e essa doença está prestes a se tornar uma das maiores

causas de mortalidade nas próximas décadas. Segundo a Organização

Mundial de Saúde (WHO) o número de casos de câncer no mundo deverá

aumentar em 75% até 2030. E segundo esta mesma pesquisa, essa taxa pode

ser ainda mais alta e chegar a 90% em países mais pobres [1, 2].

Para o tratamento do câncer, os três principais tratamentos atuais são a

cirurgia, a radioterapia e a quimioterapia, cuja escolha depende do tipo de

tumor e do estágio de seu desenvolvimento [3]. Embora esses tratamentos

sejam de grande valor, podem apresentar desvantagens e limitações, como

complicações pós-cirúrgicas e toxicidade sistêmica. Por essa razão, pesquisas

que buscam métodos alternativos e/ou complementares de tratamento estão

em evidência e visam sempre ser mais eficientes em relação às terapias

convencionais [4].

Atualmente, a nanotecnologia exibe um indispensável papel

especialmente no campo da medicina (nanomedicina) e vem sendo apontada

como uma das grandes promessas do futuro. As abordagens nanoterapêuticas

têm tratado diferentes tipos de câncer e têm tornado possível uma nova era na

quimioterapia, já que vários tipos de nanoestruturas (dendrímeros,

nanossondas magnéticas, nanoesferas, nanopartículas, hidrogéis, lipossomos,

dentre outros) têm sido sintetizados para atingir as células cancerosas, tanto

para o diagnóstico quanto para terapias específicas [5, 6], já que esses

sistemas nanométricos oferecem a vantagem de reduzir ou eliminar efeitos

colaterais da quimioterapia por atuarem diretamente nas células cancerosas e

não permanecerem livres na via sistêmica.

Dentre os nanossistemas sintetizados, os nanogéis atualmente emergem

como um grupo capaz de interagir especificamente com células cancerígenas.

Nanogéis usualmente são definidos como dispersões aquosas de

nanopartículas formadas por polímeros química ou fisicamente interligados.

16


Além disso, eles têm atraído crescente interesse devido o seu potencial como

nanocarreadores de vários compostos, dentre eles os biopolímeros [7, 8].

Entre a numerosa quantidade de biopolímeros que tem sido proposta para

a preparação de nanogéis, polissacarídeos têm inúmeras vantagens sobre os

polímeros sintéticos por serem não tóxicos, biocompatíveis, biodegradáveis e

solúveis em água [9]. Ao longo dos últimos anos, um polímero em especial tem

chamado a atenção na área dos polissacarídeos, é conhecido como fucana.

Fucana é um termo utilizado para denominar uma família de polissacarídeos

sulfatados cujo açúcar mais representativo é a α-L-fucose sulfatada. Elas são

encontradas em algas marrons e em equinodermas (ouriço e pepino do mar)

[10, 11].

Ao longo de algumas décadas nosso grupo de pesquisa (localizado no

Laboratório de Biotecnologia de Polímeros Naturais – BIOPOL – UFRN, sob a

responsabilidade do Prof. Dr. Hugo Rocha) tem intensificado os estudos com

os polissacarídeos extraídos da alga marrom Spatoglossum schröederi

(Dictyotaceae). Essa alga sintetiza três tipos de fucanas e a obtida em maior

quantidade foi nomeada de fucana A [12, 13]. A disponibilidade permanente

desse organismo em grandes quantidades tornou-a uma excelente escolha

para a prospecção de compostos bioativos.

Em estudos anteriores, Barroso e colaboradores trabalhando com a

fucana A da S. schröederi observaram que esse polímero não apresentava

atividade anticoagulante in vitro, porém, demonstrou atividade antitrombótica in

vivo, sendo observado um efeito dose-dependente alcançando a saturação ao

redor de 20 µg/g de peso de rato. A fucana A também apresentou um efeito

tempo-dependente, alcançando a saturação por volta de 16h após a sua

administração [13].

Como essa atividade antitrombótica apresentada pela fucana A foi de

enorme importância farmacológica, testes com esse polímero continuaram a

ser realizados na tentativa de preencher as lacunas que ainda faltam para,

quem sabe, em um futuro próximo, a fucana possa ser utilizada como um

fármaco. Estudos para verificar a toxicidade da fucana A foram investigados.

Testes de toxicidade in vivo com ratos Wistar [14] e in vitro para observar a

17


genotoxicidade [15] foram realizados e não mostraram nenhum efeito danoso

provocado pela fucana A, mesmo utilizando altas concentrações. Ainda neste

mesmo trabalho, a atividade citotóxica da fucana A foi testada contra várias

linhagens tumorais, onde foi observado que esse polímero inibiu a proliferação

celular em torno de 43,7% para as células Panc-1 e HeLa (0,05 a 1 mg/mL) e

que ele não matou células normais.

A citotoxicidade para células tumorais também foi encontrada para a

heparina (polissacarídeo sulfatado de origem animal com estrutura química

semelhante a da fucana). Esse polímero demonstrou atividade antiproliferativa

frente a uma gama de linhagens celulares [16, 17].

Esse grande interesse pelas fucanas de algas pode estar relacionado

com a sua semelhança estrutural com a heparina, o que daria a esse

polissacarídeo atividades semelhantes às deste glicosaminoglicano. Além

disso, por serem de origem vegetal, elas poderiam apresentar menores riscos

de contaminações e são encontradas em abundância na natureza, já que são

recursos naturais renováveis.

No caso da heparina, têm sido desenvolvidos sistemas de nanogéis que

são resistentes ao ambiente extracelular e que não se degradam no interior

celular, promovendo a liberação controlada da heparina no interior das células

tumorais e, por conseguinte, a morte celular por apoptose induzida pela

heparina. Assim, quando Bae e colaboradores, utilizando nanogéis de

heparina, trataram células tumorais B16-F10, observaram que a proliferação

celular foi inibida em cerca de 50%, enquanto que a heparina sozinha inibiu o

crescimento celular em aproximadamente 10% [18].

Inicialmente nanogéis de fucana A da S. schröederi foram produzidos pela

conjugação da fucana a hexadecilamida. O nanogel de fucana A produzido

mostrou tamanho médio de 123 nm, carga negativa e estabilidade química (70

dias). Em seguida, o teste de citotoxicidade desse nanogel foi realizado contra

as linhagens tumorais HepG2, 786-0, HS-5, sendo a 786-0 a que apresentou

maior inibição, com aproximadamente 43,7% (0,05 a 0,5 mg/mL). Entretanto,

esse tipo de nanogel tem algumas limitações, pois tem um ambiente interno

lipofílico, como também há uma modificação estrutural considerável da fucana,

18


o que pode alterar suas atividades [19]. Portanto, outras técnicas de síntese de

nanogéis de fucanas devem ser testadas a fim de se obter nanogéis de

fucanas que apresentem as atividades da fucana A.

19


2. JUSTIFICATIVA

O Estado do Rio Grande do Norte possui uma grande diversidade de

espécies de macroalgas marinhas, organismos estes que são potências

produtores de compostos com grande potencial farmacológico e biotecnológico.

Dentre eles, destacam-se os polissacarídeos sulfatados. Apesar do potencial

dos polissacarídeos sulfatados encontrados em nossa região, esses compostos

ainda não são conhecidos, o que faz com que esses recursos naturais não

sejam aproveitados.

Recentemente, foram encontradas fucanas com alta atividade

antitumoral, sintetizadas por algas do litoral potiguar e o nosso grupo de

pesquisa vem se dedicando a pesquisar as fucanas dessas algas. Mas, apesar

da forte atividade antiproliferativa já encontrada em algumas fucanas de algas

marrons, há um empecilho que dificulta os avanços dos estudos com fucanas

antiproliferativas que é o seu caráter iônico, elas podem assim se ligar a uma

gama de proteínas extracelulares antes de entrarem no interior celular, o que

exige uma elevada concentração de fucanas para que elas possam

desempenhar o seu efeito.

Devido a isso, a síntese de nanogéis de fucana foi o recurso utilizado

neste trabalho para intensificar os estudos de suas atividades biológicas,

especialmente, o seu efeito antitumoral, já que os nanogeís são liberados

(introduzidos) diretamente dentro das células tumorais, sem serem “perdidos

na circulação”. Para tal, uma parceria foi estabelecida com a Universidade do

Minho (Portugal), centro de referência na produção de nanogéis, o que

viabilizou o desenvolvimento desses nanogéis aqui no Brasil.

20


3. OBJETIVOS

3.1. GERAL

Sintetizar um nanogel de fucana A pela adição de grupos tióis e avaliar seu

efeito antiproliferativo e apoptótico frente a linhagem tumoral 786-0, como

também seu efeito antioxidante, anticoagulante e angiogênico.

3.2. ESPECÍFICOS

Extrair os polissacarídeos sulfatados da alga marrom S. schröederi por

fracionamento cetônico;

Obtenção da fucana A por cromatografia de troca iônica da fração

cetônica F0.6v;

Sintetizar nanogéis a partir da fucana A obtida;

Caracterizar físico-quimicamente os nanogéis de fucana A;

Avaliar as atividades antiproliferativa, anticoagulante, antioxidante e

antiangiogênica dos nanogéis produzidos e da fucana A livre;

Avaliar a capacidade dos nanogéis de fucana em induzir apoptose em

células tumorais;

Verificar a sua internalização celular pela conjugação do FITC com os

nanogéis de fucana A.

21


4. MÉTODOS

4.1. MATERIAIS BIOLÓGICOS

4.1.1. Algas

A alga marinha marrom Spatoglossum schröederi (C. Agardh) Kützing

(Figura 1) foi coletada na Praia de Búzios, município de Nísia Floresta (litoral

sul do Rio Grande do Norte), em marés baixas entre 0,0 a 0,2 metros a uma

temperatura situada entre 28-30°C. As algas foram recolhidas quando já

desprendidas do substrato, mas permanecendo flutuando nas águas de maré-

baixa.

As algas foram trazidas ao laboratório no mesmo dia da coleta e

acondicionadas em sacos de polietileno, lavadas em água corrente,

examinadas cuidadosamente para remoção de epífitas, inclusões calcárias e

sais, sendo postas para secar em estufa aerada a 45°C. Em seguida foram

trituradas, pesadas e guardadas em frascos de vidro hermeticamente fechados.

Figura 1 – Alga marrom S. schröederi (C. Agardh) Kützing. A) em exsicata (Foto:

Nednaldo Dantas) e B) na natureza (Foto: Edjane Barroso)

22


4.1.2. Linhagens e culturas celulares

As linhagens celulares de adenocarcinoma renal (786-0) e de endotélio

da aorta de coelho (RAEC) foram mantidas em meio RPMI e HAM-F12,

respectivamente. Todas as células foram cultivadas a 37°C em uma incubadora

umidificada na presença de 5% CO2, com os meios suplementados com 10%

de soro fetal bovino (SFB) e antibióticos (100 U/mL de penicilina e 100 μg/mL

de estreptomicina). As células 786-0 foram doadas pela Profa. Dra. Carmen

Ferreira (Departamento de Bioquímica, UNICAMP, Brasil) e as RAEC pela

Profa. Dra. Helena Nader (Departamento de Bioquímica, UNIFESP, Brasil).

4.2. EXTRAÇÃO E PURIFICAÇÃO DA FUCANA A

4.2.1. Obtenção do pó cetônico

A alga seca e pulverizada foi suspensa em dois volumes de acetona PA

para despigmentação e delipidação do material. Essa solução ficou a

temperatura ambiente durante um período de 24 horas. Posteriormente, a

mistura foi decantada e o resíduo colocado para secar a 45°C sob aeração e

denominado de “pó cetônico”. Esse pó foi utilizado em seguida na proteólise.

4.2.2. Proteólise

Para a realização dessa etapa, foram adicionados dois volumes de NaCl

a 0,25 M ao pó cetônico (100 g) e o pH ajustado para 8,0 com NaOH. A esse

material foi adicionado a enzima proteolítica prozima (15 mg/g de pó cetônico).

Essa suspensão permaneceu em banho-maria a 60°C durante um período de

18h. Depois, foi filtrado e o sobrenadante submetido a uma centrifugação

10.000 x g por 15 minutos a temperatura de 4°C. Após a centrifugação, o

sobrenadante, que contém os polissacarídeos solúveis foi denominado de

extrato bruto de polissacarídeos sendo seco à pressão reduzida, triturado,

pesado e guardado para posteriores análises.

23


4.2.3. Fracionamento do extrato bruto com concentrações crescentes de

acetona

O extrato polissacarídico bruto obtido foi fracionado com volumes

crescentes de acetona, obtendo-se as frações polissacarídicas. Os valores de

acetona adicionados foram determinados pela turvação da solução, que

caracteriza a precipitação de polissacarídeos devido à adição desse solvente

polar. Adicionou-se um volume de acetona, sob agitação leve, necessário para

que se visualizasse uma turvação da solução, essa solução foi mantida em

repouso a 4ºC durante 18h, o precipitado foi coletado por centrifugação a 8.000

x g por 15 minutos a 4ºC e seco a pressão reduzida.

Em seguida, esse procedimento foi repetido até que não se visualizasse

mais a formação de precipitado. As frações obtidas foram denominadas

conforme o volume de acetona no qual foram precipitadas (F0.5, F0.6, F0.7,

F0.9, F1.1, F1.3 e F2.0).

4.2.4. Cromatografia em coluna de troca iônica

A fração cetônica F0.6 (que contém a fucana A) foi submetida à

complexação com a resina de troca iônica Lewatite (10 mg de material para

cada 1,0 mL de resina) e a eluição foi realizada passo a passo utilizando-se

molaridades crescentes de NaCl, como descrito por Dietrich e colaboradores

[20]. Foram coletadas frações, com volume total de três vezes o volume da

resina, para cada molaridade de sal (0.3, 0.5, 0.7, 1.0, 1.5, 2.0, e 3.0 M), as

quais foram separadas pela ausência de positividade para o método de fenol-

ácido sulfúrico [21]. O fluxo de coleta foi de 1 mL/min, sendo o volume de

eluição igual para todas as molaridades coletadas.

As frações eluídas com 1.0 e 1.5 M de NaCl foram precipitadas com 2

volumes de metanol PA a 4°C e deixadas em repouso por 18h, sendo

posteriormente centrifugadas a 10.000 x g, por 15 minutos e secas a pressão

reduzida. Essas duas frações (1.0 e 1.5 M) são consideradas como detentoras

da fucana A [12, 13].

24


4.3. SÍNTESE E CARACTERIZAÇÃO DOS NANOGÉIS 4.3.1. Síntese das fucanas tiolada As fucanas inicialmente foram dissolvidas em tampão citrato (0,1 M, pH

3.0) e postas para reagir com periodato de sódio por 2h a 4°C. Após esse

procedimento, as fucanas, agora tioladas, foram conjugadas com cisteamina

por aminação redutiva. Para tal, após diálise contra água destilada, as fucanas

modificadas reagiram com a cisteamina por duas horas em PBS. Após esse

período, foi acrescido à solução boridreto de sódio 0,1 M lentamente, sendo a

solução agitada por 1h a 4°C. Essa solução foi então dialisada sob atmosfera

de nitrogênio par minimizar a oxidação dos grupos tiois e posteriormente

liofilizada [18].

4.3.2. Caracterização físico-química dos nanogeis de fucanas As fucanas tioladas foram em seguida misturadas com polietileno glicol

(PEG) em diferentes proporções (2.5, 5.0, 10, 15 e 30), o que permitiu

posteriormente escolher qual a melhor proporção para a síntese do nanogel. A

mistura seca de fucana tiolada e PEG foi solubilizada em DMSO e incubada

por 6h a 37°C. A complexação entre a fucana e o PEG ocorreu através de

pontes de hidrogênio. Após esse período, a solução foi sonicada por 3 minutos

gerando pontes dissulfeto entre as moléculas de fucana tioladas. O nanogel

resultante foi exaustivamente dialisado e assim ficando livre das moléculas de

PEG e DMSO residuais.

4.3.3. Transmitância dos nanogéis

A transmitância da solução contendo nanogéis de fucana A foi

preparada usando várias razões em peso de PEG (0, 2,5, 5,0, 10, 15, 30). A

leitura foi medida a 400 nm com o leitor de microplacas Multiskan Ascent

(Thermo Labsystems Franklin, MA, EUA).

25


4.3.4. Dynamic Light Scattering (DLS)

O diâmetro e o potencial zeta dos nanogéis foram medidos por

espectroscopia de correlação de fótons usando o equipamento DLS da

Brookhaven 90 Plus (Brookhaven Instruments Corporation, New York, USA).

Os nanogéis de fucana A foram preparados em água a 25°C a uma

concentração de 1 mg/mL.

4.3.5. Estabilidade

A estabilidade foi avaliada analisando o nanogel, solubilizado em água,

para a sua distribuição de tamanho. A análise foi realizada semanalmente, até

42 dias, conforme descrito anteriormente. A solução nanogel foi mantida a 4°C

durante o estudo e removidos para análise 24 horas antes de cada medição,

sempre realizada a 25°C.

4.3.6. Microscopia eletrônica de varredura (MEV)

Tamanho e forma dos nanogéis A fucana foram avaliados por

microscopia eletrônica de varredura. Para ambas as experiências, 50 µL da

solução de nanogel foi depositado sobre uma superfície limpa de mica, deixado

a secar a 25ºC e, em seguida, observada em microscópio de varredura

Shimadzu, modelo SSX550 (Shimadzu Scientific Equipment, UK).

4.3.7. Microscopia confocal

As células 786-0 (1 × 105) foram colocadas em lamínulas de vidro de 12

mm de diâmetro em placas de 24 wells (Nunc; Naperville, IL, USA). Após 3 dias

em cultura, as células foram lavadas três vezes com PBS (0,1 M, pH 7,4), e,

em seguida, tratadas com a fucana A ou com o nanogel de fucana marcado

com fluoresceína em meio RPMI isento de soro por 15, 30 ou 60 min a 37°C.

Em seguida, as células foram lavadas e fixadas com paraformaldeído a

2% (4°C). Após lavagem com PBS, os núcleos das células foram coradas com

solução de DAPI (50 µg/mL em PBS) durante 30 minutos, sendo lavadas cinco

26


vezes em PBS novamente e em seguida adicionado Fluoromount-G (EM

Sciences; Ft. Washington, WA, EUA) e colocado uma lamínula de vidro e

examinadas com um microscópio confocal ou de fluorescência (Zeiss Axio

Examiner LSM 710, Jena, Alemanha).

4.3.8. Espectroscopia de infravermelho

A espectroscopia de infravermelho foi realizada em espectrômetro

Perkin-Elmer de 4400 a 400 cm-1 no Departamento de Química da

Universidade Federal do Rio grande do Norte. A fucana A e os nanogéis de

fucana A (~5 mg) foram analisados após secagem em aparelho de

Abdenhalden sob a forma de pastilha de KBr contendo P2O5 a 60ºC.

4.3.9. Atividade antiproliferativa

A atividade antiproliferativa dos nanogéis obtidos foi avaliada pelo ensaio

colorimétrico do MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenil tetrazolium

bromide) [22]. Esse método é baseado na redução do MTT a cristais de

formazan pelas células vivas.

Aproximadamente 1 x 104 células das linhagens 786-0 foram colocadas

em placa estéril de 96 wells para um volume final de 100 µL de meio RPMI

suplementado com 10% de soro fetal bovino. Após 24 horas, o meio foi

removido e as células foram carenciadas por 24 horas com meio sem soro.

Posteriormente, o meio foi aspirado e as células foram estimuladas a sair de

G0 pelo acréscimo de meio com SFB 10% na ausência (controle) e na

presença das amostras (8, 16, 32, 48 e 64 µg/mL). Após 24 horas de

tratamento, MTT (1 mg/mL) foi adicionado às células, e incubadas por mais 4

horas. Após esse período, o meio foi aspirado e adicionou-se 100 µL de etanol

PA para dissolver os cristais de formazan formados e precipitados. A

quantificação da absorbância foi feita em leitor de placa de 96 wells em

comprimento de onda de 570 nm. O ensaio foi realizado em triplicata. O cálculo

de inibição da proliferação celular foi realizado em comparação com o controle

contendo células não tratadas com as amostras.

27


4.3.10. Conjugação da fucana A com fluoresceína (FITC)

A fim de visualizar a incorporação celular do nanogel (FucA:PEG15) e da

fucana A, ambas foram marcadas com sal de sódio de fluoresceína (Sigma).

Resumidamente, 5 mg de fucana A ou nanogel foram colocados para reagir

com 1 mg de fluoresceína em solução 0,1 M de PBS (pH 7,0), sendo agitada

durante 1 hora, protegida da luz, a temperatura ambiente. A solução foi

dialisada contra água destilada (MW 12 kDa) e, em seguida, liofilizada.

4.3.11. Avaliação da viabilidade e morte celular por anexina V-FITC/ iodeto

de propídio (PI)

Para avaliar a viabilidade e morte celular, foi utilizado um kit para

detecção de apoptose de Anexina V-FITC/PI, de acordo com as instruções do

fabricante, com pequenas modificações (BD Pharmingen, San Diego, CA). As

células 786-0 foram plaqueadas em uma concentração de 2 x 105 células em

placas de 6 wells. Após 24 horas para adesão das células, o meio foi removido

e as células foram carenciadas por 24 horas com meio sem soro.

Posteriormente, o meio foi aspirado e as células foram estimuladas a sair de

G0 pelo acréscimo de meio com SFB 10% na ausência (controle) e na

presença de nanogel (FucA:PEG15) e fucana A (64 µg/mL). Após o tratamento,

as células foram tripsinizadas, coletadas e lavadas duas vezes com PBS

gelado e ressuspendidas em 100 µL de tampão de ligação. Um total de 5 µL de

Anexina V-FITC e 5 µL de iodeto de propídio (PI) foram adicionados e a mistura

foi incubada por 30 minutos no escuro. Finalmente, 400 µL do tampão de

ligação foram adicionados às células, a suspensão celular foi analisada por

citometria de fluxo. A percentagem de células em apoptose foi determinada a

cada 10.000 eventos e os gráficos representam dados obtidos de três

experimentos separados. Para análise desses dados foi utilizado o programa

FlowJo® Analysis Software versão 9.3.2 (Tree Star Incorporation, OR, EUA).

28


4.3.12. Atividade antioxidante

Quatro testes foram realizados para analisar a atividade antioxidante do

nanogel e da fucana A; foram eles: poder redutor, DPPH, sequestro de radicais

superóxido e capacidade antioxidante total. A metodologia foi seguida de

acordo com o descrito por Costa e colaboradores [23] e Vinayak, Sabu e

Chatterji [24].

4.3.13. Atividade anticoagulante

Os ensaios de tempo de tromboplastina parcial ativada (aPTT) e tempo

de protrombina (PT) foram realizados seguindo o protocolo fornecido pelos

“kits” comerciais adquiridos. Para esses ensaios foi utilizada uma massa de

100 µg para o nanogel (FucA:PEG15) e para a fucana A, sendo considerado o

possuidor de atividade aquela amostra capaz de prolongar em duas vezes o

tempo normal de coagulação. Foram utilizadas como meio de comparação da

atividade anticoagulante, a clexane (heparina de baixo peso molecular). Os

tempos de coagulação foram determinados utilizando-se um coagulômetro

automático.

4.3.14. Ensaio de formação de tubo de matrigel

O ensaio de formação do tubo foi adaptado a partir de Dreyfuss e

colaboradores [25]. A matrigel purificada a partir do tumor EHS foi

descongelada a 4°C e mantidas em gelo, cultivada em placas de 24 wells, e

incubadas a 37°C durante 16 h para gelificação. A ECs (105 células) foi

plaqueada no Matrigel em meio Ham-F12 contendo 10% de SFB em soro

fisiológico (controle) e em diferentes concentrações (12.5, 25, 50, e 100 µg/mL)

de nanogel (FucA:PEG15). As culturas foram mantidas a 3 °C em atmosfera

úmida de CO2 a 2,5% durante 24 horas. Cada tratamento foi realizado em

triplicata. A formação do tubo foi examinada sob um microscópio de luz

invertida em 50 X (ampliação). Três imagens foram tomadas de forma aleatória

em diferentes áreas.

29


4.4. ANÁLISE ESTATÍSTICA

Todos os dados dos experimentos realizados foram expressos como

média ± desvio padrão. Foi utilizado o teste de análise paramétrica de análise

de variância (ANOVA) seguido do teste de Tukey (Nível de significância de

p<0,05) como GraphPad InStat® Software versão 3.05 para Windows 95

(GraphPad Software Incorporation, San Diego, CA, EUA).

30


5. ARTIGOS PRODUZIDOS

5.1. Artigo 1 (SUBMETIDO)

Evaluation of potential antitumor activity of fucan nanogel

Periódico: Marine Drugs

Nanotechnology (The reference number for the article is NANO-102955)

Fator de impacto: 3.978

ISSN: 1660-3397 (Printed version)

ISSN: 1660-3397 (Online version)

Qualis: Medicina II – A2

Indexada: PubMed – indexado por MEDLINE

5.2. Capítulo de livro

Chapter 6 – Application of Marine Polysaccharides in Nanotechnology

Periódico: Marine Medicinal Glycomics

Biotechnology in Agriculture, Industry and Medicine Biochemistry

Research Trends

In: Vitor Hugo Pomin. (Org.). Marine Medicinal Glycomics. 1ed.New York: Nova

Science, 2013, v. 01, p. 65-114.

Binding: ebook

ISBN: 978-1-62618-649-1

5.3. Artigo 2

Evaluation of acute and subchronic toxicity of a non-anticoagulant, but

antithrombotic algal heterofucan from the Spatoglossum schröederi in

Wistar rats

Periódico: Brazilian Journal of Pharmacognosy

Rev. Bras. Farmacogn. Braz. J. Pharmacogn. 21(4): Jul./Aug. 2011


ISSN: 0102-695X (Printed version)

ISSN: 1981-528X (Online version)

Qualis: Medicina II – B3

31


Indexada: SCOPUS, SciELO, EMBASE e GEOBASE

5.4. Artigo 3

Evaluating the possible genotoxic, mutagenic and tumor cell proliferation-

inhibition effects of a non-anticoagulant, but antithrombotic algal

heterofucan

Periódico: Journal of applied toxicology : JAT.

J Appl Toxicol. 2010 Oct;30(7):708-15. doi: 10.1002/jat.1547.


ISSN: 0260-437X (Printed version)

ISSN: 1099-1263 (Online version)

Qualis: Medicina II – B1

Indexada: PubMed – indexado por MEDLINE

32

5.1. ARTIGO 1 (SUBMETIDO)

Mar. Drugs 2014, 12, 1-x manuscripts; doi:10.3390/md120x000x

marine drugs ISSN 1660-3397

www.mdpi.com/journal/marinedrugs

Article

Evaluation of potential antitumor activity of fucan

nanogel

Jailma Almeida-Lima1,2

, Arthur Anthunes Jacome Vidal1, Dayanne Lopes

Gomes1,2

, Ruth Medeiros Oliveira1, Leonardo Thiago Duarte Barreto Nobre

3,

Mariana Santana Santos Pereira Costa1, Nednaldo Dantas-Santos

2, Helena

Bonciani Nader3, Francisco Miguel Gama

4, Edda Lisboa Leite

1, Hugo Alexandre

Oliveira Rocha1,2*

1

Laboratory of Biotechnology of Natural Polymers (BIOPOL), Department of

Biochemistry, Federal University of Rio Grande do Norte (UFRN), Natal-RN 59078-

970, Brazil; E-Mails: [email protected] (J.A.-L.); [email protected]

(A.A.J.V.); [email protected] (D.L.G.); [email protected]

(R.M.O.); [email protected] (M.S.S.P.C); [email protected] (E.L.L);

[email protected] (H.A.O.R) 2

Graduate Program in Health Sciences, Federal University of Rio Grande do Norte

(UFRN), Natal-RN 59078-970, Brazil; E-Mails: [email protected] (N.D.-S.) 3

Department of Molecular Biology, Department of Biochemistry, Federal University

of São Paulo - UNIFESP, São Paulo-SP, 04044-020, Brazil; E-Mails:

[email protected] (L.T.D.B.N); [email protected] (H.B.N) 4 IBB—Institute for Biotechnology and Bioengineering, Centre for Biological

Engineering, Minho University, Braga 4704-553, Portugal; E-Mails:

[email protected] (F.M.G.)

* Author to whom correspondence should be addressed; E-Mail: [email protected];

Tel.: +55-84-3215-3416 (ext. 207); Fax: +55-84-3211-9208.

Received: / Accepted: / Published:

OPEN ACCESS

33

Abstract: Fucan is a term that defines a family of homo- and

heteropolysaccharides containing sulfated L-Fucose. In this work a

heterofucan (Fucan A) from seaweed Spatoglossum schröederi was

thiolated and treated by ultrasonication, giving rise to intermolecular

disulfide bonds and to the formation of fucan A nanogels. Fucan A nanogel

decorated with polyethylene glycol (FucA:PEG15) was stable for over one

month and showed an average diameter of 187 ± 11 nm in aqueous solution

and a zeta potential of -23.1 ± 2.0 mV, as measured by dynamic light

scattering. This nanogel inhibits the proliferation of 786-0 renal

adenocarcinoma cells in a dose-dependent way. Flow cytometric analysis

showed that FucA:PEG15 induces apoptosis through caspase and caspase-

independent mechanisms. The nanogel labeled with FITC was completely

taken up by 786-0 cells after 1 hour. When we stopped the cell endocytosis,

the FucA:PEG15 antiproliferative effect was abolished. In additon,

FucA:PEG15 has antioxidant activity and also exhibits antiangiogenic

activity . These data show that fucan A nanogel exhibits several activities

(antiproliferative, antioxidant, and antiangiogenic) and therefore its potential

for cancer therapy should be further investigated.

Keywords: fucan; sulfated polysaccharide; brown seaweed; nanogel;

cytotoxicity

1. Introduction

The term cancer is used to refer to more than one hundred different types of diseases

that have in common the characteristic uncontrolled proliferation of anaplastic cells.

These cells, at some point, will invade other tissues and organs. It is estimated that more

than 21 million people will contract cancer and 13 million deaths are expected by 2030.

Although cancer accounts for around 13% of all deaths in the world, more than 30%

could be prevented by modifying or avoiding key risk factors [1].

The use of chemotherapy for the treatment of cancer raises concerns about the

debilitating side effects of this form of treatment; on the other hand, poor cellular

internalization and insufficient intracellular drug release reduces its efficacy [2].

The development of nanomedicine aims to solve the issues associated with the

systemic administration of toxic pharmaceuticals. Thus, chemotherapeutic agents have

been encapsulated, conjugated, entrapped, or loaded into nanoformulations, resulting in

the site-specific drug delivery, thereby reducing the systemic toxicity [3].

Among the available nanosystems, nanogels are particularly attractive since they are

easy to produce, are affordable, and may effectively incorporate a variety of drugs,

including biopharmaceuticals [4]. Nanogels are composed of cross-linked three-

dimensional polymer chain networks that are formed via covalent linkages or self-

34

assembly processes. An increased interest has been witnessed recently for smart

nanogels allowing site-specific and controlled drug release, paving the way for the

development of improved cancer therapeutic formulations [5].

Among the numerous polymers that have been proposed for the preparation of

nanogels, polysaccharides have a number of advantages over the synthetic polymers,

which were initially employed in the field of pharmaceutics [6]. Fucans are

polysaccharides containing substantial amounts of L-fucose and sulfate ester groups,

expressed by brown seaweed [7] and some marine invertebrates (sea urchins and sea

cucumbers) [8]. Algal fucans showed several biological/pharmacological activities such

as antithrombotic, antiviral, anticoagulant, antioxidant, and anti-inflammatory

[9,10,11,12]. In addition, fucan anti-cancer activities have been reported frequently in

recent years, and the potential mechanisms of action were also investigated

[13,14,15,16].

Among the seaweed fucans, we can highlight those extracted from Spatoglossum

schröederi (Dictyotaceae). This brown seaweed is found along almost the entire

Brazilian coast (about 8000 km). The permanent availability of this organism in large

amounts makes it an excellent choice for prospecting bioactive compounds. It

synthesizes three fucans and the one obtained in the larger quantity was named fucan A.

We showed that fucan A is not toxic in vivo [17], but it demonstrated antiproliferative

activity against several tumor cell lines [18]. However, this activity was observed only

for high concentrations, probably because fucan A, due its ionic nature, binds onto a

great variety of proteins, becoming unavailable to perform the bioactivity of interest.

Another kind of sulfated polysaccharide known as heparin binds several proteins [19]

including extracellular matrix proteins [20]. In order to diminish this inactivating

effect, Bae and colleagues synthesized a heparin nanogel cross-linked with disulfide

linkages. When B16-F10 tumor cells were treated with heparin nanogel, their

proliferation was inhibited by about 50%, whereas free heparin alone inhibited the cell

growth to a much smaller extent (~10%) [21].

Previously, we chemically modified fucan A by grafting hexadecylamine to the

hydrophilic backbone. The obtained amphiphilic material self-assembled into fucan A

nanogel, which showed antiproliferative activity against human renal tumor cells (786-0

cells) [22]. In this study fucan A was covalently linked to thiol groups and then cross-

linked with disulfide linkages to produce fucan A nanogel for efficient cellular uptake.

This way, a fully hydrophilic nanogel is obtained. In addition, we investigated the

antiproliferative effect of the newly developed fucan A nanogels using 786 tumor cell

lines. The data obtained indicated that the fucan A nanogel exhibits high stability and is

a more powerful inhibitor of cell growth than free fucan A.

2. Results and Discussion

2.1. Synthesis of the fucan A nanogels

In the present work, fucan A was chemically modified with thiol groups and then

cross-linked with disulfide linkages to produce reducible fucan A nanogels. For this

purpose, the carboxyl groups of fucan glucuronic acids were oxidized and conjugated to

35

cysteamine by reductive amination, yielding thiolated fucan A. Then, the nanogels

were synthesized by a method that comprises two steps: Thiolated fucan was initially

co-dissolved in DMSO with PEG, enabling the interaction between the two polymers,

presumably by hydrogen bonding, resulting in the spontaneous formation of nanosized

complex particles in the DMSO phase. In the second step, thiolated fucan A monomers

within the inner core of the complexes were covalently linked together by disulfide

linkages. This was achieved by ultrasonic treatment, which promotes the formation of

free radicals, which in turn accelerate the oxidation reaction of the thiol groups of the

cysteamine linked to fucan A. The crosslinked fucan nanogels were finally obtained by

withdrawing the DMSO and free PEG through exhaustive dialysis (Figure 1A).

Figure 1. A) Scheme of the synthesis of fucan A nanogels. B)

Transmittance of solution containing nanogels of fucan A prepared using

various weight ratios of PEG (0, 2.5, 5.0, 10, 15, 30) at 400 nm.

The effect of the fucan:PEG ratio on the size of nanogels was investigated. The fucan

A concentration was kept constant and PEG content increased at the ratio of 2.5, 5.0,

10, 15, and 30 times the concentration of fucan A; hence, the obtained samples were

called FucA:PEG2.5, FucA:PEG5.0, FucA:PEG10, FucA:PEG15, and FucA:PEG30,

respectively. We can observe (Figure 1B) that in the absence of PEG the transmittance

value was kept at 100%, i.e., similar to the control of dissolved fucan A. However, with

increasing PEG content, the transmittance gradually decreases to about 60%, for the

higher ratio of FucA:PEG 30, indicating the formation of colloidal particles, the

nanogels, as shown in the following section.

36

2.2. Characterization of the nanogels

The average hydrodynamic diameters of nanogels were measured at a concentration

of 1 mg/mL in water by dynamic light scattering (DLS). The effect of various fucan

A:PEG weight ratios on nanogel size, polydispersity (PDI), and zeta potential was

analyzed. As we can see in Table 1, a weight ratio of FucA:PEG of 30, originates a very

large nanogel (917.18 ± 83.12 nm), whereas the others showed a size ranging from

186.95 ± 10.62 to 301.44 ± 2.26. Bae and colleagues [21], using the same method to

obtain heparin nanogels, elected a weight ratio of heparin:PEG of 15 as the best one

because it would favor stronger interactions between PEG and heparin, leading to the

formation of compact nanogels. This observation is in agreement with ours, since

FucA:PEG15 showed the smaller size (186.95 ± 10.62 nm) among the tested

combinations. However, the size of FucA:PEG15 colloidal micelles is in the same order

of magnitude of other nanogels obtained with fucan A using a different synthetic route

[22], and of others obtained using different acidic polysaccharides and the same

synthesis approach. For instance, heparin nanogels resulted in a stable structure with an

average diameter of 248.7 ± 26.8 nm [21] and acetylated hyaluronic acid gave rise to

nanogels ranging from 275 ± 4 to 447 ± 8 nm [23].

The size of nanomaterials is an extremely important factor determining its fate in

vivo—biodistribution and pharmacokinetics—since it affects namely the phagocytosis

and ability to cross biological barriers. According to Dong and Mumper [24],

nanoparticles with a size around 220 nm are ideal targets for passive targeting of tumors

since the majority of solid tumors exhibit vascular pore cut-offs between 380 and 780

nm. Thus, using the size as a parameter of choice, all fucan A nanogels, except

FucA:PEG30, have potential application against tumor cells. However, other parameters

should be evaluated to confirm this statement.

Table 1. Physicochemical characteristics of nanogels obtained using

different ratios of the polyethylene glycol (PEG) determined by DLS.

Samples Diameter

(nm)

Polydispersity

(PDI)

Conductance

(µS)

Zeta

Potencial

(mV)

FucA:PEG2.5 301.44 ± 2.26 0.69 ± 0.03 193 -28.12 ± 1.55

FucA:PEG5.0 297.15 ± 30.97 0.48 ± 0.09 254 -28.61 ± 0.15

FucA:PEG10 277.10 ± 17.41 0.49 ± 0.03 548 -26.11 ± 0.05

FucA:PEG15 186.95 ± 10.62 0.54 ± 0.06 347 -23.08 ± 1.96

FucA:PEG30 917.18 ± 83.12 0.84 ± 0.09 339 -22.18 ± 0.13

The zeta potential was negative for all nanogels, ranging from -22.18 ± 0.13 to -

28.61 ± 0.15 mV. These results can be explained by the negative charge of fucan A due

to the presence of ionized carboxyl and sulfated groups. This is in good agreement with

37

the zeta potential values found in previous studies for nanoparticles prepared with other

fucans [25], and with chitosan with a blend dextran sulfate [26].

Furthermore, nanogel, as evidenced by the polydispersity index (PDI), ranged

between 0.48 ± 0.09 to 0.84 ± 0.09. The PDI is dimensionless and scaled such that

values smaller than 0.05 are rarely seen other than with highly monodisperse standards.

Values greater than 0.7 are indicative of samples with a very broad size distribution and

probably not suitable for analysis using the dynamic light scattering (DLS) technique

[27,28]. Thus, the best nanogels regarding the heterogeneity of size distribution were

FucA:PEG5.0, FucA:PEG10, and FucA:PEG15.

Size and surface morphology of fucan A nanogels were evaluated by SEM. Figure 2

shows that not all formulations presented a spherical shape, in the case of FucA:PEG2.5

nanoparticles were not detected, while in the case of FucA:PEG30 particles with a

fibrous shape were observed. It can be speculated that some phase transition may have

occurred during the drying of the samples, since in the case of FucA:PEG2.5 nanosized

particles were detected by DLS. Nonetheless, these results demonstrate the critical

relevance of the balance of fucan A vs PEG, suggesting that the arrangement of the two

polymers determines the organization of the nanogel. On the other hand, FucA:PEG5.0,

FucA:PEG10, and FucA:PEG15 showed a spherical shape with an average diameter of

113.33 ± 0.23 nm, 101.85 ± 0.43 nm, and 98.25 ± 1.69 nm, respectively (Figure 2F).

The diameter measured by DLS was slightly larger than the one obtained from SEM,

presumably due to the swelling of fucan A nanogels in water (taking into account that

carbohydrate polymers are highly hygroscopic), since the samples needed to be dried

for analysis by SEM, as noted by other authors [21,23].

Figure 2. SEM photograph of nanogels with various weight ratios of

fucan A:PEG (A) 2.5, (B) 5, (C) 10, (D) 15, (E) 30, and F) average

diameter of the nanogels. The diameters of particles in SEM images were

measured by comparing them with the size bar.

38

2.3. FTIR of the fucan A nanogels

FTIR spectra of fucan A, FucA:PEG10, and FucA:PEG15 are depicted in Figure 3.

Characteristic sulfate absorptions were identified in the FTIR spectra: bands around

1265 cm−1

for asymmetric S=O stretching vibration and around 1041 cm−1

for

symmetric C–O vibration associated with a C–O–SO3 group [18]. The bands at 813–850

were caused by the bending vibration of C–O–S. At 3200–3500 cm−1

, fucan A and

fucan A nanogels showed bands from the stretching vibration of O–H [9]. The band

around 2900 cm−1

corresponds to stretching vibrations of CH2, which is higher in fucan

nanogel spectra due to the presence of stretching vibrations of CH2 in cysteamine

residues [29], as further confirmed by the band at 1468 cm−1

, featuring a higher

intensity in fucan nanogel spectra. This band corresponds to C–H symmetric

deformation vibration [30]. A band around 1410 cm−1

was identified in all spectra and

was assigned to symmetric vibration of COO− of glucuronic acid. The presence of

glucuronic acid was also confirmed by the antisymmetric stretching vibration of COO−

at 1618 cm−1

[31], which overlaps with the vibration of water. The H2O molecule has

strong IR absorbance with three prominent bands around 3400 (O–H stretching), 2151

(water association), and 1618 cm−1

(H-O-H bending) [32] in the fucan A spectrum.

However, the band intensity decreases in fucan nanogels. The absorption band due to S–

H stretching vibrations of the thiol group was also observed at 2600–2550 cm−1

[33].

Figure 3. Infrared of fucan A and nanogels FucA:PEG10, FucA:PEG15,

Fuc:PEG30.

2.4. Nanogel Stability

The nanogels FucA:PEG10 and FucA:PEG15 showed the smallest particle size and

best morphological features, and therefore were chosen for further characterization. In

order to investigate the stability of the macromolecular association over time, we used

39

DLS. As presented in Figure 4, the fucan A nanogels were highly stable in aqueous

solution, suggesting that their structural integrity was preserved.

Figure 4. Stability of fucan A nanogels FucA:PEG10 and FucA:PEG15

accessed by DLS. The nanogels were stored at 4 °C for up to 42 days and

analyzed in DLS at temperature 25°C.

2.5. Cytotoxicity assay

The cytotoxic of FucA:PEG10, FucA:PEG15, and Fucan A upon 786-0 cells was

investigated for 24 h using a colorimetric MTT-based assay (Figure 5). Fucan A

displayed a dose-dependent inhibitory effect, which was quite expressive (by about 3

fold) in the case of nanogels. FucA:PEG10 and FucA:PEG15 also showed a dose-

dependent effect, reaching saturation at around 0.04 and 0.06 mg/mL respectively. In

addition, the data also indicate that FucA:PEG15 is slightly more efficient as an

antiproliferative compound than FucA:PEG10.

Dantas-Santos et al. [22], using fucan A nanogels grafting hexadecylamine,

evaluated the cell viability of various types of tumor cells. The 786-0 cell line was the

more susceptible one (inhibition of ~40%). However, this inhibition was obtained only

for a concentration as high as 500 µg/mL, ten times higher than the required using

FucA:PEG15.

40

Figure 5. Inhibition of proliferation of 786 cells incubated with Fucan A,

FucA:PEG10, or FucA:PEG15 nanogels at various concentrations for 24

hours. Data are expressed as means ± standard deviation. a,b,c,d,e The

different letters indicate significant difference between the concentrations

of the same compound (p < 0.05).

2.6. Apoptotic effect of fucan A nanogels

Since FucA:PEG15 was the most potent cell growth inhibitor, it was chosen for

mechanistic studies, namely to determine whether cell death for apoptosis was

responsible for the observed effect. For this purpose, 786-0 cells were treated with

FucA:PEG15 (64 µg/mL) for 24 hours, followed by flow cytometric analysis.

Cell apoptosis features the exposure of phosphatidylserine on the external side of the

cell membrane, which can be recognized by annexin V. On the other hand, necrotic

cells can be identified using propidium iodide (PI), which stains only necrotic cells

bearing a compromised, porous cell membrane. Generally, cells stained with annexin V

are indicative of early apotosis and stained cells with PI, indicative of necrosis, while

double labeling is indicative of late apoptosis [34].

In Figure 6 we can see the results of flow cytometry analysis of cells cultivated in the

presence and absence of nanogels (control group). For the control group (Figure 6A),

91.4% of the cells are negative for annexin V and PI. However, after the FucA:PEG15

treatment, this number dropped to 59.0%, whereas the percentage of cells stained with

annexin V increased from 7.8 to 39%. Furthermore, the percentage of cells stained with

PI did not change (Figure 6B). These data indicate that FucA:PEG15 inhibits

proliferation by inducing apoptosis.

41

In order to determine the role of caspases in the FucA:PEG15 nanogel-induced

apoptosis, 786-0 cells were incubated with ZVAD-FMK. As can be seen in Figure 6B,

in the presence of this pan-caspase inhibitor, the percentage of cells positive for annexin

decrease from 39 to 27%. Additionally, in the presence of E64 (cysteine peptidase

inhibitors, mainly cathepsin and calpains specific) the effect also decreased to about

10% of inhibition (Figure 6D). These data indicate that FucA:PEG15 has a complex

mechanism of apoptosis induction.

Several fucans have been shown to induce apoptosis in different types of tumor cells,

an event often related to caspase activation [35,36,37]; however, other proteins involved

in cell survival pathways may be affected by the presence of fucans in the culture

medium, such as proteins from ERK1/2MAPK pathway [16,38] and PI3K/AKT

pathway [13], apoptosis-inducing factor (AIF) [10], JNK/c-Jun/AP-1 pathways, and

death receptor-mediated and mitochondria-mediated apoptotic pathways [14].

The analysis of the aforementioned data suggests that the mechanism of apoptosis

induction by fucans is very complex, in agreement with the results obtained in this work

using the fucan A nanogel. Further work will be dedicated to identifying mainly the cell

proteins involved in the FucA:PEG15 mechanism of action to induce apoptosis.

Figure 6. Cytometry with cells 786-0. A) Control (Anexin + PI); B)

FucA:PEG15; C) FucA:PEG15 + ZVAD and D) FucA:PEG15 +E64 all

with the same concentration (64 µg/mL).

42

2.7. FucA:PEG15 intracellular uptake

The intracellular uptake of nanogels was investigated by confocal microscopy and

flow cytometry. For this purpose, the nanogel was conjugated with FITC and used as

fluorescence probe, allowing the observation of its interaction with the 786-0 cells.

The antiproliferative effect (around 40% for a concentration of 0.06 mg/mL) was not

affected by the FITC labeling. It is important to mention that FITC alone has no activity

on 786-0 cell proliferation (data not shown). The confocal observation of 786-0 cells

after incubation with FucA:PEG15+FITC for 1 h allowed the detection of internalized

fucan nanogel (Figure 7B). In addition, the nanogel is observed in the perinuclear space

(Figure 7C). The free FITC was also assayed under the same conditions and did not

show any binding to cells or extracellular matrix (data not shown).

The internalization kinetics was analyzed by flow cytometry (Figure 7). As can be

seen in Figure 7D, 15 min suffice for the cells to become labeled with

FucA:PEG15+FITC. Furthermore, the incubation of the cells in the presence of an

excess of fucan A (ten times more) prevented the entry of the nanogel into the cell,

probably because the same cell receptor/endocytic pathway was used.

Lira and colleagues [25] assigned a similar antiproliferative activity against J774

macrophages and NIH-3T3 fibroblasts to fucan nanoparticles. These authors also

showed that the fucan nanogels need to be internalized in order to exert their

antiproliferative activity. In order to confirm this hypothesis, the cells were exposed to

FucA:PEG15 nanogels (from 0.01 to 0.1 mg/mL) for 2 hours at 4 °C (condition in

which the endocytosis is substantially inhibited). After washing and addition of medium

without nanogel, the cell proliferation was measured after 24 hours. In another set of

experiments the 786-0 cells were incubated with FucA:PEG15 nanogels (from 0.01 to

0.1 mg/mL) for 6, 12, 18, and 24 hours at 4 °C. In both set of experiments,

FucA:PEG15 nanogels did not demonstrate an antiproliferative effect (data not shown).

Figure 7. Confocal microscopy and flow cytometry of 786-0 cells treated

with nanogel or Fucan A. A) nuclei stained with DAPI; B) region of

cytoplasm stained with FITC and C) overlap of the images A and B. The

cells used for confocal microscopy were treated only with FucA:PEG15

nanogels. Flow cytometry D) FucA:PEG15 at the same concentration

(100 µg/mL) at different times 15, 30 min, and 1h; E) FucA:PEG15

(1000 µg/mL), FucA:PEG15+FITC (100 µg/mL) + Fucan A (1000

µg/mL) and FucA:PEG15 (100 µg/mL).

43

2.8. Antioxidant activity of the nanogel

Antioxidants are substances that are useful for fighting cancer and other processes

that potentially lead to various diseases; antioxidants act by preventing the onset of

cancer during carcinogenesis, and they are generally beneficial to cells [39].

In this work, the antioxidant activity was evaluated in different assays: reducing

power, DPPH radical-scavenging assay, superoxide radicals, and total antioxidant

capacity (TCA). The results are shown in Table 2. The FucA:PEG15 nanogel and fucan

A did not show antioxidant activity in power reducing, superoxide anion scavenging,

and DPPH radical-scavenging assays. However, in the total antioxidant activity assay,

both the nanogel and fucan A were positive.

Table 2. Antioxidants activities with nanogel and fucan A.

Antioxidant activity Fucan A FucA:PEG15

Reducing power (%) nd nd

DPPH (%) nd nd

Superoxide radical (%) 0.0 1.6

TCA (mg/g ascorbic acid equivalents) 26.20a 36.31

b

nd - not detected. a,b Different letters indicate significant difference between

the samples of the same concentration (p < 0.05).

44

2.9. Anticoagulant activity of the nanogel

Fucans from various seaweeds, including different Dictyotales [31,40], possess

anticoagulant activity. In this way, we evaluated the anticoagulant activity of

FucA:PEG15 using the PT and aPTT test. But in all conditions evaluated (from 10 to

100 µg/mL), FucA:PEG15 showed no anticoagulant activity (data not shown).

2.10. FucA:PEG15 inhibits the angiogenesis

Using the matrigel assay, we investigated whether FucA:PEG15 can inhibit

angiogenesis in vitro. This matrigel is composed mainly of laminin, TGF-β, and

entactin, and has been used to study angiogenesis in vivo and in vitro [41]. As shown in

Figure 8 (B, C, and D), the nanogels inhibit the formation of capillary-like tubes by

endothelial cells. The FucA:PEG15 significantly inhibited capillary tube formation with

increasing concentration of nanogel, 12.5 µg/mL (Figure 8B) to 100 µg/mL (Figure

8D), compared to the control group (Figure 8A). In addition, the nanogels did not show

any cytotoxic effect against RAEC cells, even when they were incubated on the matrigel

(data not shown).

Angiogenesis is one of the most important events for the maintenance and growth of

tumors. The newly formed vessels are responsible for delivering oxygen and nutrients to

the growing tumor. Various drugs have been developed for use as antiangiogenic agents

and thus combat the development of tumors [42]. The fucan nanogels studied here

exhibit cytotoxic activity against tumor cells, antioxidant activity, and antiangiogenic

activity, properties that have been extensively researched in compounds used in the

treatment of tumors. In addition, nanogels here are particles with potential for drug

carriers, including anticancer.

45

Figure 8. Effects of nanogel on capillary tube formation of rabbit aortic

endothelial cells (RAEC). The cells were non-treated (A-control group)

and treated with different concentrations of FucA:PEG15 (B- 12.5

μg/mL, C- 50 μg/mL, and D- 100 μg/mL). 50x magnification.

3. Experimental Section

3.1. Materials

Poly (ethylene glycol (PEG, Mw 4000)), MTT (3-(4,5-dimethylthiazol-2-yl)-2-5-

diphenyltetrazoliumbromide), and cellulose acetate dialysis bags, with 6,000 or 12,000

MWCO, were purchased from Sigma Chemical Company (St. Louis, MO, USA). L-

glutamine, sodium bicarbonate, penicillin, streptomycin, sodium pyruvate, and

phosphate buffered saline (PBS) were purchased from Invitrogen Corporation

(Burlington, ON, USA). The 786-0 renal adenocarcinoma (ATCC CRL-1932) and

RAW 264 monocyte macrophage (ATCC TIB-71) cell lines were donated by Dr.

Carmen Ferreira (Department of Biochemistry, UNICAMP, Brazil). Cell culture

medium components (RPMI-1640 Medium or DMEM), trypsin, and fetal calf serum

(FCS) were obtained from Cultilab (Campinas, SP, Brazil). Rabbit aortic endothelial

cells (RAEC) were maintained at 37 °C under stress of 2.5% CO2, in HAM-F12

medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine

serum. All other solvents and chemicals were of analytical grade. The fucan A from

46

Spatoglossum schröederi was obtained as described by Almeida-Lima and colleagues

[18].

3.2. Synthesis of fucan A nanogels

Fucan A (10 mg, 50 µmol) dissolved in 0.1 M citrate buffer (pH 3.0) was reacted

with sodium periodate (112 mg, 523 µmol) for 2 h at 4 ºC. After dialysis against

distilled water (Mw cutoff of 6 kDa), the oxidized fucan A was reacted for 2 h with

cysteamine hydrochloride (60 mg, 523 µmol) in 0.1 M phosphate-buffered saline (PBS,

pH 7.0). Then, 10 ml of 0.1 M NaBH4 solution was slowly added, and the mixture was

stirred for 1 h at 4 ºC. The solution was dialyzed and then lyophilized. Thiolated fucan

A was solubilized in DMSO. Five milligrams of thiolated fucan was mixed with PEG in

distilled water at different weight ratios of PEG to thiolated fucan A, and then

lyophilized. The dried mixture of thiolated fucan A and PEG was solubilized in 10 mL

of DMSO with incubation for 4 h at 37 ºC. The solution was sonicated for 3 min using a

Sonicator Sonics Vibra-Cell (20 kHz, output control 3) to facilitate the oxidation

reaction of thiol groups. The resultant fucan A nanogels were purified by extensive

dialysis against distilled water (Mw cutoff of 12 kDa). The degree of thiolation was

estimated as approximately 24%, by comparing the relative peak intensity ratio found in

2600–2550 cm−1

at nanogels and fucan A spectrum, which correspond to S–H stretching

vibrations of the thiol group, as shown in the results section.

3.3. Transmittance of hydrogels

The transmittance of the solution containing nanogels of fucan A prepared using

various weight ratios of PEG (0, 2.5, 5.0, 10, 15, 30) was measured at 400 nm with a

Multiskan Ascent Microplate Reader (Thermo Labsystems, Franklin, MA, USA)

3.4. Dynamic light scattering (DLS)

The effective hydrodynamic diameter and zeta-potential of nanozymes was measured

by photon correlation spectroscopy using Brookhaven 90 Plus Nanoparticle Size

Analyzer (Brookhaven Instruments Corporation, New York, USA). Fucan A nanogel

dispersions were prepared in water at 25 °C at a concentration of 1 mg/ml. The zeta

potential was obtained by using a disposable capillary cell in automatic mode on the

same instrument.

3.5. Scanning electron microscopy (SEM)

Size and shape of the fucan A nanogels were evaluated by scanning electron

microscopy (SEM). For both experiments, 50 µL of the nanogel solution was deposited

onto a clean mica surface, allowed to dry at 25 ºC, and then observed in a Shimadzu

electron microscope, model SSX550 (Shimadzu Scientific Equipment, UK).

47

3.6. Fourier Transform Spectra (FT-IR)

All samples (5mg) were mixed thoroughly with dry potassium bromide. A pellet was

prepared, and the infrared spectrum was measured on a Thermo Nicolet Nexus

spectrometer instrument (model Nexus 470 FT-IR).

3.7. Stability

The stability was evaluated analyzing the nanogel, solubilized in water, for its size

distribution, as described above. The analysis was performed weekly, up to 42 days, as

described above. The nanogel solution was kept at 4 °C during the study and removed

for analysis about 24 hours before each measurement, always performed at 25 °C.

3.8. MTT Cytotoxicity assay

The cells were grown in 25 cm2 flasks in DMEM medium. For the experiments, the

cells were seeded into 96-well plates at a density of 5 × 103 cell/well and allowed to

attach overnight in 100 μL medium. The fucan A nanogels and the non-modified

polysaccharide were added at a final concentration of 8.0, 16, 32, 48 and 64 μg/mL, for

24, 48, and 72 h at 37 ºC and 5% CO2. In some tests, the RAEC cells were grown on

matrigel in 24-well plate at a density of 5 × 104 cell/well in 500 μL medium.

After incubation, traces of samples were removed by washing the cells twice with

100 μL PBS; then, MTT (1mg/mL) dissolved in 100 μL of fresh medium was added and

incubated for 4 h at 37 ºC, 5% CO2. The medium was aspirated, and the MTT-formazan

product was dissolved in 100 μL of ethanol and estimated by measuring the absorbance

at 570 nm in a Multiskan Ascent Microplate Reader (Thermo Labsystems, Franklin,

MA, USA). All concentrations were tested in triplicates and the experiment was

repeated at least three times. The percentage of cell proliferation inhibition was

calculated as follows:

%Inhibition =

3.9. Conjugation of fucan with fluorescein

In order to visualize cellular uptake, fluorescein-labeled fucan A and the nanogel

(FucA:PEG15) were prepared by conjugating fluorescein sodium salt (Sigma). In brief,

5 mg of fucan A or nanogel were reacted with 1 mg of fluorescein in 0.1 M PBS

solution (pH 7.0) and stirred for 1 hour protected from light, at room temperature. The

solution was dialyzed against deionized water (Mw cutoff of 12 kDa) and then

lyophilized.

48

3.10. Analysis by Flow Citometry

The number of apoptotic cell deaths induced by FucA:PEG15 nanogels was

measured by flow cytometry using Annexin V-FITC Apoptosis Detection Kit (BD

Biosciences, San Diego, CA, USA). The 786-0 cells were placed in a 6-well plate (2 ×

105 cells/mL) and after 48 h stimulated to enter G0 using a medium without serum for

24 h. Next, the medium was replaced by DMEM supplemented with 10% FBS, in the

presence of fucan A (64 µg/mL) or FucA:PEG15 (64 µg/mL). A negative control was

prepared without the presence of polysaccharide. The cell effect of FucA:PEG15

incubated with pan-caspase inhibitor ZVAD-FMK (carbobenzóxy valyl-alanyl-aspartyl-

[O-methyl]-fluorometilcetone) or the inhibitor cysteine proteinase (E-64) was also

tested. After 24 h the cells were harvested, and after centrifugation the cell pellets were

washed twice with cold PBS and suspended in 50 μL of 1 × Anexinn-V buffer. Cells

were then incubated with 5 μL of annexin V-FITC and 2 μL of PI at room temperature

for 15 min in the dark. After incubation, 300 μL of 1 × binding buffer (10 mM

HEPES/NaOH, 140 m M NaCl, 2.5 mM CaCl2, pH 7.4) was added to each tube. The

cells were immediately analyzed by Facscanto II flow cytometry (BD Biosciences, San

Diego, CA, USA) in FL1 channel (excitation at 488 nm and emission at 530 nm) and

FL3 (excitation at 650 and emission at 630 nm). A total of 40.000 events were acquired.

For data analysis, FlowJo® Analysis Software version 9.3.2 was used.

3.11. Confocal analysis

The 786-0 cells (1 × 105) were placed on 12 mm-diameter glass cover slips in 24-

well cluster plates (Nunc; Naperville, IL, USA). After 3 days in culture, the cells were

washed three times with PBS (0.1 M pH 7.4), and then treated with the fluorescein-

labeled fucan A or nanogel FucA:PEG15 (64 μg/mL) in serum-free RPMI medium for

15, 30, or 60 min at 37 °C. Afterwards, the cells were washed and fixed with 2%

paraformaldehyde (4 °C). Then, after washing with PBS, the cell nuclei were stained

with DAPI solution (50 μg/mL in PBS) for 30 min and washed five times in PBS, once

in water, and glass cover was mounted in Fluoromount-G (E.M. Sciences; Ft.

Washington, WA, USA) and examined with a confocal or fluorescence microscope

(Zeiss Axio Examiner LSM 710, Jena, Germany).

3.12. Antioxidant activity

Four assays were performed to analyze the antioxidant activity of the fucan A and

nanogel obtained: reducing power, DPPH, superoxide radical scavenging, and total

antioxidant capacity as previously described [43,44].

3.13. Anticoagulant activity

49

Both the protrombin time (PT) and activated partial thromboplastin time (aPTT)

coagulation assays were performed with a coagulometer as described earlier [43] and

measured using citrate-treated normal human plasma. All assays were performed in

duplicate and repeated at least three times on different days (n = 6).

3.14. Matrigel tube formation assay

The tube formation assay was adapted from Dreyfuss et al. [45]. Matrigel purified

from EHS tumor was thawed at 4 °C on ice and plated on the bottom of 24 well-plates,

and incubated at 37 °C for 16 h for gelification. ECs (105 cells) were seeded on Matrigel

in F12 medium containing 10% FBS and different amounts (12.5, 25, 50, and 100

µg/mL) of FucA:PEG15 nanogel or saline (control). The cultures were maintained at 37

°C in a 2.5% CO2 humidified atmosphere for 24 h. Each treatment was performed in

triplicate. Tube formation was examined under an inverted light microscope at 50x

magnification. Three images were randomly taken in different areas.

3.15. Statistical analysis

All data are presented as the average ± SEM. Tests for significant differences

between the groups were done using one-way ANOVA with multiple comparisons

(Kruskal-Wallis) using GraphPad Prism 4.0 (GraphPad software, San Diego, EUA).

4. Conclusions

Several polysaccharide-derived nanoparticles have been reported as inert vehicles

used to carry drugs or other molecules. Here we synthesize a fucan-derived nanogel

(FucA:PEG15), which showed cancer-specific antiproliferative activity, antioxidant and

angiogenic. All these activities are indicated as being important for anti-cancer drugs.

Thus, FucA:PEG15 is therefore more than an inert vehicle and its activity could be

potentially applied for cancer cell treatment associated with various anti-cancer drugs.

Further work is being carried out on the incorporation of drugs into the FucA:PEG15, as

well as on the mechanisms of its antiproliferative activity. These results will be reported

in the near future.

Acknowledgments

Research was supported by the Ministério de Ciência, Tecnologia e Informação

(MCTI), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),

Fundação de Apoio a Pesquisa do Estado do Rio Grande do Norte (FAPERN),

Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), and Conselho

Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil. Hugo AO

50

Rocha and Helena B Nader are CNPq fellowship-honored researchers. Ruth M Oliveira,

Leonardo TDB Nobre had a Ph. D. scholarship from CNPq; and Jailma Almeida-Lima

and Nednaldo Dantas-Santos had a Ph. D. scholarship from CAPES. This research was

submitted to the Graduate Program in Health Sciences at the Federal University of Rio

Grande do Norte as part of the D.Sc. thesis of Jailma Almeida-Lima. Miguel Gama

thanks CAPES for support through the program “Ciência sem fronteiras”.

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5.2. CAPÍTULO DE LIVRO

59

Chapter 6

APPLICATION OF MARINE POLYSACCHARIDES IN

NANOTECHNOLOGY

Raniere Fagundes Melo-Silveira, Jailma Almeida-Lima, and Hugo

Alexandre Oliveira Rocha* Laboratório de Biotecnologia de Polímeros Naturais (BIOPOL), Departamento de Bioquímica,

Universidade Federal do Rio Grande do Norte (UFRN), Natal/RN, Brazil

ABSTRACT

Scientific knowledge in the field of nanotechnology has been expanded rapidly over the past

two decades. The potential application of this new technology in several areas of science has

attracted attention to the development of synthesis and application of nanosize materials.

Nanostructures based on natural polysaccharides have been of particular interest in light of their

good biocompatibility, biodegradability, reduced toxic side effects and improved therapeutic

effects. Another advantage of using polysaccharides is that these molecules contain reactive

groups which can be used to introduce different chemical ligands. Marine polysaccharides such

as alginates, chitosans, carrageenans and fucoidans are examples of polymers that have been

studied for nanoparticles syntheses and that have shown promising results in several

applications. Alginate and chitosan nanostructures have been recently proposed as a system for

sustained release of several drugs, construction of biosensors such as immunosensor for the

detection of Escherichia coli in food specimens. Chitosan nanoparticles have been used to

remove heavy metals in a water treatment process; chitosan/alginate nanocomposites was

reported to be a good candidate for oral delivery of bioactive peptides; carrageenan and fucan

nanoparticles showed great spread ability and water-holding capability, a relevant method to

modulate interactions of the nanoparticles with several cells including tumor cells. The present

paper will reviewed recent progress in marine polysaccharide nanotechnology that presents itself

as the vanguard of the development of intelligent systems for biotechnology.

* E-mail address: [email protected]

60

1. INTRODUCTION

The term nanotechnology was proposed by Norio Tanaguchio (University of Tokyo) in 1974

when he referred to the ability of certain materials to form stable structures at nanometer scales.

Currently the term nanotechnology can be defined as the design and manufacture of materials,

device and systems with control at nanometer dimensions. But other more elaborate definitions can

be found. However, one should always bear in mind that the essence of nanotechnology is the size

control at the nanometer scale.

Nanotechnology represents a milestone in the sciences because of the possibility of producing

materials with nanometer-scale physical, chemical and biological exceptional. In this context

appears nanomedicine, which is the application of nanotechnology to medicine. This is a relatively

new term that has emerged in the last decades of the last century. This term covers procedures for

diagnosis, prevention and treatment of disorders and sequels, as well as preserving and improving

human health using molecular tools and molecular knowledge of the human body. Currently

nanomedicine exploits carefully structured nanoparticles, such as dendrimers, carbon fullerenes

(buckyballs), magnetic nanoprobes and nanoshells to reach specific organ and tissue. These

nanoparticles can serve both for diagnosis and for specific therapies. It is believed that with the

development of nanotechnology in the coming years, more complex nanodevices will be produced to

have a broader use of nanoparticles in medicine [1].

There are several classes of chemical compounds which may be used in applications

nanomedicine, one of which is represented by polysaccharides. Polysaccharides are natural polymers

consisting of one or different kinds of monosaccharides. It constitutes a very diverse and highly

versatile class of materials that have the most varied possible applications [2]. The polysaccharides

can be found in several sources, present in almost all taxa (Table 1).

Table 1. Usual sources of some polysaccharides

Organism Polysaccharide Source

Seaweeds

Alginate/Fucans/Fu

coidans Brown algae

Agarans/Carrageen

ans/ Agar-Agar/

Agarose

Red algae

Animals

Hyaluronic Acid Bovine vitreous humor,

gallinaceous crest

Heparin Bovine lung and porcine

intestines

Quitin/Chitosan Carapaces of crustaceous

Fungi Glucans Pleurotus ostreatus, Agaricus

blazei

Bacterias

Xantan

Dextran

Gelan

Xanthomonas ssp

Leuconostoc spp

Sphingmonas elodea

Plants

Cellulose Eucalyptus, pine trees and

other plants

Xilan Several sources

Starch Corn, wheat, potato and

portions of other plants

Inuline Several sources

Guar gum Cyamopsis tetragonolobus

and Cyamopsis Psoraloides

Gum karaya Sterculia urens, S. tomentosa

and other species of Sterculia

61

Gum arabic Acacia senegal and other

species of Acacias

Nanostructured systems have an important role in future therapies because they are widely

described in the literature as systems which cross the barrier-acting gastrointestinal epithelial

absorption. In addition, nanoparticles are able to control and direct the release of bioactive

substances. In nanomedicine, a drug (or a synthetic biopolymer) may be dissolved, incorporated,

encapsulated and adsorbed or bound to nanoparticles in order to improve their pharmacokinetic

profile, increase treatment efficiency, reduce adverse effects of preferential accumulation at specific

sites, so that there is low concentrations in healthy tissues; increase the chemical and conformational

stability of a variety of therapeutic agents such as small molecules, peptides and oligonucleotides.

The history of polysaccharides in nanotechnology begins with cellulose. Subsequently,

nanoparticles of other polysaccharides, such as hyaluronic acid, inulin, dextran and were being

described. Based on this, the synthesis of polysaccharide nanoparticles have been the focus of

relevant studies, compared to several other distribution systems, they show exceptional stability, and

respond very efficiently to biological systems in addition to providing low cytotoxicity and an

inexpensive obtaining process, thus opening a range of opportunities for the development of a

variety of biomedical applications, like as cell imaging, drug loading (Figure 1). Regarding the

polysaccharides of marine origin, the study of their use in nanotechnology has increased in recent

years and gained prominence in the scenario of cutting-edge research for numerous areas of science

[3]. Therefore, efforts were concentrated in this chapter in order to review information about the use

of polysaccharides of marine origin: carrageenan, fucoidans, quiosanas and alginic acids as raw

material for the production of different types of nanoparticles and their applicability.

Figure 1. The schematic represents the versatility of nanoparticles polysaccharides. Different regions of the

nanoparticles could act distinct roles in biomedical applications.

2. CARRAGEENAN

2.1. Chemical Structure

Carrageenan are a group of natural polysaccharides extracted from red seaweed (Rhodophyceae)

having particularity of forming colloids and gels in aqueous media at very low concentrations. These

gels are thermoreversible transparent and exhibit a wide range of textures, ranging from very elastic

and cohesive gel to firm and brittle, depending on the properties of the carrageenan used.

62

The carrageenan are known since the eighteenth century, but only in the fourteenth century they

started being extracted from red seaweed Chondrus crispus and used as an emulsifier and gelling

agent in homemade food by Irish population of the city of "Carrageen", where the name carrageenan

originated. Today carrageenan is one of the most used hydrocolloid worldwide, already surpassing

other polysaccharides such as alginates, agar.

The production of carrageenan was originally dependent on the natural deposits, especially C.

crispus (popularly known as "Irish Moss"), with a limited resource base. Since the early 70s of the

twentieth century, the industry has been rapidly expanding the availability and possibility of

cultivation of algae producing carrageenan in other hot water countries, with low labor cost.

Currently, most of the algae used for the production of carrageenan is from cultivations, although

there is still some demand for "Irish Moss," originating from natural deposits in Europe and Canada

and certain other types of algae yet uncultivated of South America. Current carrageenan production

is estimated at $ 240 million annually. Global demand has been growing around 5% annually over

the past 30 years, with prices ranging from $ 10 to $ 30 a pound, depending on their specifications

and quality. Europe has the largest market for carrageenan (55%). However, growth of its use is

strongly expected in regions such as Central America and South and Southeast Asia, where

consumption of carrageenan is expected to increase by 50% in the coming years.

The primary structure of carrageenan is a linear structure that is based on alternating copolymers

connections with β-1,3-D-galactose and α-1,4-D-galactose, with variable degrees of sulfation. The

units are joined together by alternating glycosidic bonds α-1, 3 and β-1, 4 which form the

disaccharide repeating unit of carrageenan. In some carrageenan a α-galactose can be dehydrated

giving rise to 3,6-Anhydro-α-D-galactose, there is a possibility of monosaccharides hydroxyls to be

substituted by methyl or pyruvate. These structural variables lead to the theoretical possibility of 42

different carrageenans. However, only 15 structures have been identified to date (Table 2).

Table 2. Disaccharide repeating structures of carrageenan a

Family Greek

symbol

1,3-linked 1,4-linked

Kappa Kappa (κ ) β-D-galactose 4-

sulfate

3,6-anhydro-α-D-galactose

Iota (ι) β-D-galactose 4-

sulfate


2-sulfate

Mu (μ) β-D-galactose 4-

sulfate

α -D-galactose 6-sulfate

Nu (ν) β-D-galactose 4-

sulfate

α -D-galactose 2,6-di-

sulfate

Omicron

(ο)

β-D-galactose 4-

sulfate


Beta Beta (β) β-D-galactose 3,6-anhydro-α-D-galactose

Gamma (γ) β-D-galactose α -D-galactose 6-sulfate

Omega (ω) β-D-galactose 6-

sulfate


Psi (ψ) β-D-galactose 6-

sulfate


Lambda Delta (δ) β-D-galactose α -D-galactose 2,6-di-

sulfate

Alfa (α) β-D-galactose 3,6-anhydro-α-D-galactose

2-sulfate

Lambda (λ) β-D-galactose 2- α -D-galactose 2,6-di-

63

sulfate sulfate

Theta (θ) β-D-galactose 2-

sulfate


2-sulfate

Xi (ξ) β-D-galactose 2-

sulfate


Pi (π) β-D-galactose P,2-

sulfate


aAdapted from [4], P: pyruvate acetal

Due to their half-ester sulfate moieties, the carrageenan are anionic polymers strongly. The most

common types of carrageenan are traditionally called kappa (κ), iota (ι) and lambda (λ) [5]. They

differ only in the number of sulfate groups per disaccharide: κ have one, have two γ and λ is three

(Figure 2). The κ-carrageenan has a negative charge per disaccharide unit and presents the best

gelling properties among the three most common types of carrageenan [6].

Figure 2. Structural units of the three main types of carrageenans: (A) κ-carrageenan, (B) ι-carrageenan and (C) λ-

carrageenan. The disaccharides of κ-, ι- and λ-carrageenan are depicted, showing the β-1→4 and the α-1→3 bonds.

2.2. Nanoparticles of Carrageenan

In many types of nanoparticles, the carrageenan are not used as the main raw material, but as

polymers which cover the surface of the nanoparticle by giving it different characteristics from

inside, as well as protecting them from the external environment.

Colloidal nanocapsules (NC) have shown great potential as releasing peptides, lipophilic drugs

and vaccines in various types of mucus. NCs are composed of central oil by a surfactant and a

hydrophilic compound covering the entire surface of the NCs. During NCs formation or in a

subsequent incubation step, a water soluble polymer bearing an opposite electrical charge to that of

the surfactant can be incorporated by the ionic coat interaction, effectively yielding a stable colloidal

core-shell nanocapsule structure. One of the most promising hydrophilic polymers that have been

used in the production of NCs is carrageenan, mainly κ-carrageenan. These polysaccharides in NCs

are used at very low concentrations and the concentration that is used influences the size of the NCs.

64

Moreover, the negative charges of carrageenan stabilize the positive charge of the surfactant used

which can reduce cytotoxicity of surfactant related to its property to create pores in cells.

Metallic nanoparticles have a wide range of applications, but its low stability and size

distribution and heterogeneous form in aqueous represent a major problem for their use. These

problems can be minimized by coating the nanoparticles with polysaccharides. Daniel-da-Silva and

coworkers (2007) have synthesized supramagnetic nanoparticles of Fe3O4 in the presence of three

different types of carrageenan through a co-precipitation method [7]. Besides avoiding spontaneous

agglomeration of the nanoparticles, the carrageenan promoted the formation of smaller particles.

These authors also showed that the size and shape of the nanoparticles were influenced by the type

of carrageenan (kappa, iota, or lambda) as the concentration of polysaccharide used. Those particles

formed with iota-carrageenan were the most stable. Potential applications of such nanoparticles

include separation and cell labeling, contrast enhancement in magnetic resonance imaging (MRI),

controlled delivery of drugs and treatment of cancers by hyperthermia.

During the production of this chapter there were no examples found in the literature of

nanoparticles synthesized exclusively from carrageenan. However, nanoparticles synthesized from

carrageenan associations with other polysaccharides have been found, which gives the particle very

interesting characteristics obtained for drug delivery. Like for example, carrageenan and chitosan

nanoparticles. These nanoparticles are produced under hydrophilic conditions by a very mild process

of ionic interaction between the amino groups of chitosan positively charged and negatively charged

sulfate groups of the carrageenan. This procedure avoids the use of organic solvents and other

aggressive conditions which may be detrimental to the integrity of the drug to be released. Studies

show that the carrageenan-chitosan nanoparticle have an associated load capacity ranging from 4%

to 17% and excellent capacity to provide a controlled release of drug over a prolonged period of 3

weeks. In addition, these nanoparticles have shown no cytotoxic behavior in biological assays in

vitro when using L929 fibroblasts, which is critical of the biocompatibility of these carriers. The

development of carrageenan-chitosan nanoparticles has shown promising properties for use not only

as releasing agents, but also in other fields, such as tissue engineering and regenerative medicine [9].

3. FUCOIDAN


Fucoidan are heteropolysaccharides having in its constitution sulfated L-fucose therefore can

also be called heterofucan. Xylose, galactose, glucose, mannose, glucuronic acid are the other

monosaccharides that are commonly found in the constitution of fucoidan addition of fucose. There

are still some fucoidan showing fucose residues substituted by acetyls groups.

The first description of the purification of a fucoidan date of year 1913 and refers to acidic

polysaccharides extracted from brown seaweed Laminaria digitata, Fucus vesiculosus and

Ascophyllum nodosum. However, only in 1931 it was demonstrated the presence of sulfate groups in

fucose monomers. The understanding of these chemical structural compounds only intensified in the

50’s of last century.

Literature data show that fucoidan are structurally very complex molecules. They can have

different types and amounts of monosaccharides which may be interconnected by different types of

glycosidic bonds, although the connections 1-3 and 1-4 being the most common. Furthermore, most

of them are branched. This structural complexity is compounded by the fact that the amount and

location of sulfate substitution is different in each molecule, and may also occur in other

monosaccharides in addition to fucose. In some cases, there is the possibility that some of fucose

residues are acetylated.

The fucoidan are only synthesized by brown algae. However, because of structural complexity

as described in the previous paragraph, each algae synthesizes one or more type of fucoidan, which

is structurally different from that synthesized by any other brown algae, fact that causes the variety

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of fucoidan (Table 3) found in nature to be high in comparison with other molecules such as

proteins, despite fucoidan are only synthesized by brown algae.

Table 3. Structural properties of some different fucoidan sources

This structural complexity partly justifies the diversity of biological activities and

pharmacological properties attributed to fucoidan such as antitumor, antiviral, immunomodulatory,

anticoagulant, anti-inflammatory, and antioxidant, [10, 22-25].

3.2. Fucoidans Nanoparticles and Its Application in Medicine

There is an increasing need to develop a reliable methodology and "Eco" for synthesizing metal

nanoparticles which can be used for many applications. Natural compounds such as fucoidans are

one means that can be used for this purpose and various metal ions can be used, such as iron, silver

and gold, among others.

66

Soisuwan et al (2010) using fucoidans from Cladosiphon okamuranus and Kjellamaniella

crassifolia were able to synthesize first gold nanoparticles coated with fucoidan [26]. The data

showed that the particles had an average size of 10 nm and that linear fucoidan C.okamuranus

nanoparticles were produced with less dispersion in size than the fucoidan K. crassifolia. In that

same year silver nanoparticles coated with fucoidan were also synthesized for the first time. In this

case the authors took advantage of the reductive capacity of fucoidan from Fucus vesiculosus and

managed to produce nanoparticles in an "environmentally friendly" way. The data show that the

particle size and its distribution were dependent on the concentration of fucoidan and silver used

during the synthesis process [27]. These nanoparticles are being tested as antimicrobials and as

components in sensors.

The fucoidan, as with the carrageenan are also used in the synthesis of nanoparticles associated

with other biopolymers or synthetic polymers. Nanoparticles formed by the combination of chitosan

(positive charges) and fucoidan (negative charge) from F. vesiculosus seaweed were prepared in

order to be resistant to the gastrointestinal environment. Currently, the oral route is considered the

most convenient and comfortable when administering medications to patients. However, the

significantly low pH gastric environment of the stomach causes deterioration of many drugs prior to

absorption. Therefore, conveyor systems resistant to gastrointestinal environment have been studied.

The data show that the best nanoparticles were obtained with the 1:1 ratio of fucoidan and chitosan.

This particle has been able to incorporate curcumin (an antitumor drug) and release it in greater

quantity only at pH above 6.0 [28].

Fucoidans-based nanoparticles have also been synthesized. Lira and colleagues (2011) have

synthesized nanoparticles with fucoidan extracted from seaweed Sargassum cymosum. These

particles showed low cytotoxicity against J774 macrophage and 3T3 fibroblasts. Confocal

microscopy studies showed that these nanoparticles are internalized by cells. The data also showed

that the interactions of nanoparticles with macrophages can be modulated by the introduction of

fucoidan in the center of the nanoparticles and future studies elucidate the mechanisms of uptake and

intracellular pathways of its trajectory [29].

Nanoparticles were synthesized with a fucoidan from the Spatoglossum schroederi seaweed.

The synthesized nanogels of about 123 nm showed high stability in size and conformation for 70

days. These particles inhibited the proliferation of human tumor cells in liver, kidney and bone tissue

being able to promote the activation of caspases and affect the distribution of these cells in different

cell cycle phases [30].

4. CHITOSANS


Chitosan (QS) is a linear polysaccharide derived from chitin, compound found in the

exoskeleton of some insects, fungi and marine invertebrates, such as shrimp and crab. The chemical

structure of chitosan molecule is very similar to cellulose and chitin. Chitosan is a linear random

polymer structure composed of backbone of β(1-4)-2 acetoamida-2-deoxy-D-glucopyranose and 2-

amino-2-deoxy-D-glucopyranose. The amino groups are protonated and this polysaccharide showed

positively charged at physiological fluids. The process of deacetylation of chitin to chitosan

formation is not complete and the degree of deacetylation of chitin, a polymer defines or chitosan,

the amount of acetoaminated groups in the chitosan is extremely low (degree of acetylation of less

than 0.35).

Chitin was discovered at the beginning of the 19th

century by French scientist Henri Braconnot.

But the QS was only discovered in the 80’s of the 20th

century. Commercially chitosan is obtained

from the deacetylation of chitin extracted from crab and shrimp shells.

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4.2. Chitosan nanoparticles (NP-QS)

Many of the polymers used today in many areas, are synthetic materials and their

biocompatibility and biodegradability are very limited. Moreover, the QS is one of the most

abundant natural polysaccharide and well known for its biodegradability and non-toxic properties.

Also, the QS has unique characteristics as a dependent pH behavior, dependent conformational

variability of the environment in which is found, mucus adherence and ease of overcoming the

epithelial junctions [31]. These characteristics makes the use of QS already performed in various

fields like biomaterials, pharmaceuticals and cosmetics, sequestration of metal ions, agriculture,

foodstuff and treatments (clarification, flocculation, etc., due to their effective interaction with other

polyelectrolytes) [32]. However, due to the property of QS, the biotechnological potential can be

much broader and its applications can be extended to other fields of human activity. A clear example

of the search for new applications for the QS is a quantity of scientific articles that focused primarily

nanotechnology and QS. Currently, there are approximately 10,000 articles related to QS

nanoparticles, accumulating more than 119,000 citations; these data show the great interest by the

scientific community in developing QS nanoparticles. In 2011, more than 1400 articles have been

published relating to nanotechnology chitosans (Figure 3) [31].

Figure 3. Scientific impact of NP-QS. The graphs represent annual perspective of (a) numbers of publications by year

related to the topic, and (b) shows the number of citations referring to articles published related to the topic. Boolean

search defined: Topic = (chitosan) AND Topic =(nanoparticle* OR nanosphere* OR complex* OR nanocarrier*)

AND Topic = (chitosan* OR nanoparticles* OR nanomedicine*); Languages = (ENGLISH); Timespan = All Years.

Databases = SCI-EXPANDED, SSCI, NCBI, SCIENCEDIRECT. Retrieved on October, 18th, 2012.

Among this substantial amount of scientific articles relating nanotechnology and QS, there is a

great emphasis on those who are targeted for nanomedicine, especially for applications related to

transporting molecules as drugs, proteins, hormones, nucleic acids and hydrophobic molecules,

besides its use in tissue engineering and diagnosis of disease assays. However, you can find articles

evaluating the potential of QS in less usual areas for polysaccharides such as in the production of

biosensors.

The drug delivery systems (DDS - Drug Delivery Systems) are being intensely investigated in

recent years due to its great potential to enhance the therapeutic index of small molecules. Among

these systems major advances have been achieved in the area: i-Vaccination; ii- Transmucosal

Peptide/ Protein Delivery; iii-Controlled Release Drug and iv- Gene Therapy.

Nanocarriers drug to be used as DDS has as an important feature the ability to form a porous

structure that allows the input and specially the output of drugs, and this characteristic is easily

obtained with the chitosan nanoparticles (NP-QS) including when using simple techniques, low cost,

as boiling internal process [33]. Moreover, NP-QS has other important features that drive their DDS

studies as they exhibit biodegradability, low cytotoxicity, antibacterial activity, permit the addition

covalently easily, other molecules its structure and mucus adherence. These NP-QS properties

provided the development of numerous oral, ocular, nasal, vaginal, intra-vesicular systems for the

drift, targeting and controlled release of drugs and other molecules in the body of animals, including

humans [34, 35].

68

For the production of NP-QS many techniques can be used, as nanocomplexity, formation of

ianotrophic icing mixtures, layer-layer capsulation, water-oil emulsification, membrane

emulsification, compression, casting, adsorption processes. This QS plasticity may be posted on

different techniques for producing nanoparticles partly explains the large number of articles on NP-

QS related carriers of drugs. Another important factor is that QS can be modified chemically to

improve its solubility in certain solvents and encourage interaction with specific drugs or other

molecules such as nucleic acids and proteins, enhancing the development of NPs appropriate to the

compound to be loaded [36]. Furthermore, in the development of NP-QS, QS can be combined with

a variety of other materials such as alginates, fucoidan, carrageenan, hidroxihapatitas, hialunonic

acid, calcium phosphate, poly (ɛ-caprolactone (PCL), peptides and factors growth, so that it can be

modular action of polymers that comprise the nanoparticle and thus uses them in release systems.

As early as the mid 90’s of the last century it was possible to identify several data showing the

use of QS NP-carriers of drugs and other molecules such as, for example, a system combining

nanocapsules polycaprolactone as carriers and advantages of mucus adhesive chitosan and cationic

PLL (poly-L-lysine) was prepared with the aim of increasing viability of ocular drugs. Although

both the PLL as chitosan show a similarity in positively charged surface, only chitosan nanocapsules

covered with increased ocular penetration of indomethacin in relation with the not covered

nanocapsules [37].

During the last two decades studies on QS nanoparticles were intensified which greatly

increased the knowledge about the synthesis methodologies and their mechanisms of action. At the

beginning of the century, Mitra and colleagues proposed the use of chitosan nanoparticles to

improve the therapeutic efficacy of doxorubicin (DXR), a widely used drug in the treatment of solid

tumors, but has undesirable side effects such as cardiotoxicity. DXR was conjugated to dextran (to

facilitate the incorporation of the drug nanoparticles and QS incorporated into the chitosan

nanoparticles (100 ± 10 nm in diameter) and were then evaluated in vivo in Balb c mice with tumor

[38]. Use of chitosan nanoparticles as carriers increased the survival of the animals compared to the

free drug [38]. In the following years, some work showed the development of knowledge in the

production methodology of NP-QS and incorporation of different molecules to the nanoparticles

produced. As the work of Ma and colleagues who demonstrated the importance of pH on the

incorporation efficiency of NP-QS insulin, indicating that the next at pH 6 the amount of insulin

incorporated fold [39]. After, Sarmento and colleagues in an assay using rodents, showed that blood

glucose levels were significantly diminished when nanoparticles of chitosan/alginate load with

insulin were orally administered [40].

Banerjee and colleagues in 2002 report the importance of amine groups in the size of chitosan

nanoparticles. The QS-NP had a size of ± 30 nm in diameter when ± 10% of amino groups were

linked (using glutaraldehyde as an agent for the cross-linked) and upper diameter of 110 nm where

virtually all groups were linked [41]. The loads on positive QS proved sums to the properties

displayed by NP-QS, as observed by El-Shabouri (2002), who found that the incorporation of

cyclosporin A (Cly-A) in NP-QS proved very effective when compared to glycocholate

nanoparticles (SGC); the author has shown that positively charged nanoparticles improved the

viability of oral Cly-A by approximately 73%, since the NP-SGC decreased by about 36% [42].

More recently, Sonaje and colleagues have proposed that NP-QS containing drugs can adhere and

passed through intestinal mucus; the infiltrated NP-QS become unstable and disintegrate near the

epithelial cell surface due the microenvironment pH and thus release the loaded drug [43]. Thus, the

released drug could then enter the systemic circulation due to the QS-mediated TJ opening (Figure

4), which could explain the effect of NP-QS to improve the drug oral viability.

69

Figure 4. NP-QS intestinal absorption and drugs release. Nanoparticles are capable of passing through the

mucosal barrier produced by epithelial cells and release the drug in the intracellular space because of their pH-

induced structural change.

Other studies suggested the use of NP-QS for topical use in genetic immunization. To propose

this, Cui and Mumper (2001) incorporated a plasmid (pDNA) containing luciferase and specific IgG

antigen in NP-QS and subsequently administered this nanoparticle topically. Data showed a

significant luciferase expression in the skin epidermis of mice that underwent topical application of

NP-QS loaded with DNA, indicating a significant efficiency in the introduction of pDNA in the

nucleus of epithelial cells when carried by NP-QS. Moreover, there is a significant serum IgG titer to

the antigen specific for β-galactosidase expression after 28 days of application of NP-QS/pDNA

[44].

One of the qualities of a system for controlled release of drugs is the possibility of continuous

analysis of drug delivery to their specific location. Using the principle of electron transfer and

energy transfer fluorescent resonance (FRET), new models of chitosan nanoparticles have been

developed to monitor the release of drugs. By associating within the nanoparticle drug interacts with

the particle preventing the release of fluorescent light freedom to be determined, the drug in the

nanoparticle site in the body returns to the initial state and emits fluorescent light indicating the

release of the drug [45].

Magnetic/luminescent QS Nanogels were synthesized by direct icing of chitosan, fluorescent

particles (CdTe quantum dots) and iron oxide supermagnetic nanogels inside and subsequently

loaded with insulin. The incorporation of these nanoparticles by the cells seems to be mediated by

insulin receptors and the high incorporation efficiency of these chitosan nanogels directed to the

liver cells and a high viability of these cells after using nanogel (above 80%) of NP-QS is a potential

tool for targeted drug delivery, cell imaging research and delivery of targeted diet supplements [46].

The use of chitosan nanoparticles as carriers of small molecules systems makes them a potential

tool for immunization against various microorganisms. Intranasal administration of nanoparticles

(NPs) of poly gama-glutamate/chitosan recombinants loaded with antigens of influenza virus

hemagglutinin (rHA) or inactivated virus is able to reach the mucous membrane, such as mucosal

adjuvant induces a high degree of protective immunity mucus of the respiratory tract. Intranasal

administration of rHA or inactivated virus antigen loaded NPs were able to protect mice when a

lethal dose of highly pathogenic influenza (H5N1 virus) was injected in animals [47]. Other studies

have demonstrated that NP-QS alone already present as agents that can combat micro-organisms, as

70

demonstrated by Shiet and coworkers (2006) [48]. These authors showed that NP-QS had

bactericidal activity against Staphylococcus aureus and Staphylococcus epidermidis.

Chitosan nanoparticles can also act as biosensors aiding in the diagnosis of various diseases.

Carbon nanotubes were used as biosensors; its multiple walls modified by adding magnetic

nanoparticles of chitosan and improved the accuracy and reproducibility sensitivity quantification of

human serum albumin from hospital samples [49]. NP-QS can act as diagnostic imaging when

directed specifically to certain targets in the body, such as tumor cells. A new magnetic nanoparticle

formed from the "layer-layer" technique (shown) the combined use of iron oxide nanoparticles (core

of the nanoparticle), followed with layers of chitosan, fluorescent particles (CdTe quantum dots), a

new layer chitosan, folate layer (targeting cellular receptors for folate) and adiamicine (an

antitumour), that nanoparticles were effective in releasing the drug targeting to tumor cells, and are

easily detected because of the localized presence of fluorescent molecules [46].

Besides the use in biomedical systems, chitosan nanoparticles can be developed and adapted for

use as environmental clean-up. NP-SQ has been developed for the removal of heavy metals and dyes

from aqueous environments. Metals such as mercury, copper and zinc were efficiently removed (to

98% removal of mercury) from aqueous solutions using a magnetic chitosan resin [50]. In a study

using magnetic nanoparticles coated with chitosan-β-cyclodextrin (CDC) it was possible to remove

approximately 90% of methyl blue dye aqueous solution. The CDC grafted onto the Fe3O4

nanoparticles contributes to an improvement in adsorption capacity due to the presence of several

hydroxyl groups, carboxyl groups, amino groups and formation of inclusion complexes to absorb the

dye. Adsorption shown to be dependent on pH and temperature and with magnetic chitosan

nanoparticle was stable and easily recovered and reused [51].

5. ALGINATES


Alginate is a term used to describe salts of alginic acid, but is commonly used for all derivatives

of alginic acid and including itself. In brown algae, alginates promote the required flexibility for the

growth conditions of the algae in the marine environment. Naturally, they form salts with gelatinous

characteristics when combined with the minerals in sea water.

Alginates (ALG) are unbranched polysaccharides consisting of four links between 14 β-D-

mannuronic acid (M) and its C-5 epimer α-L-glucuronic acid (G); these monomers may be present in

homopolymeric blocks of consecutive M (M blocks) or G (G blocks) or alternating M and G

monomers (MG blocks). This natural polymer is an important component of brown algae cell wall,

and also an exopolysaccharide of bacteria such as Pseudomonas aeruginosa.

Alginates are the most abundant heteropolysaccharides of brown algae comes to constitute about

40-60% of the dry weight of certain species of seaweed. Most are commercially extracted from the

brown algae Macrocystis pyrifera (Pacific Coast in America) and Ascophyllum nodosum (Europe).

But other species also deserve mention, as Laminaria digitata, Laminaria hyperborea, and several

species of Sargassum.

In 1883, Dr. E. C. C. Standford, Scottish scientist, was the first to isolate and use the name

alginic acid, since then, many researchers have been devoted to the study of these molecules with

multifunctional and comprehensive biotechnological potential. The industrial exploitation of algal

alginates is directly linked to their properties: ability to form gel, change the viscosity of the

solutions; films form (calcium or sodium salts) or fiber (calcium salt)

ALG have been found in many types of applications in the biomedical and engineering due to its

favorable characteristics such as biocompatibility and easy icing using simple techniques. Alginates

are particularly attractive for use as wound healing, drug delivery systems and other small

molecules, and tissue engineering, since the gels formed from such polymers hostage a similar

structure to the extracellular matrix in tissues and also may be manipulated to play many important

71

roles. Among the pharmacological properties of alginates, it stands out its anti-ulcer because the

alginate forms a protective layer of the gastric wall preventing the action of stomach acids in their

walls. There is also its application as a material which coats capsules and controlled release of drugs

in the treatment of skin ulcers, acting as a barrier between the organ and the immune system, among

others.

5.2. Nanoparticles of Alginates

The proportion of MG blocks in the alginate molecule appears to influence the properties of the

polymer for forming magnetic nanoparticles of iron. Alginate rich in fatty guluronic ratio (M / L <1),

such as those found in species Sargassum retains large amount of iron in the loop formed, and thus

form larger amount of nanoparticles of iron oxide compared to other alginates [52].

Beyond the easy synthesis methodology nanoparticles ALG, the polymers can also form meshes

micrometric that facilitate the production of other types of nanoparticles, as demonstrated by Wang

and coworkers, in 2012 [56]. They have proposed a new method for the production of barium sulfate

nanoparticles (NP-BaSO4) by using microgels Ba-ALG, the mesh alginate served as a template

lightweight and porous prevented precipitation and aggregation of nanoparticles BaSO4. Due to the

NP-opaque characteristics BaSO4, these structures can act as radiopaque agents in the area of

diagnostic imaging [53].

Great efforts have been directed to the application of nanoparticles with carrier agents, following

the trend in the area of controlled release of drugs and other molecules knowledge on NP-ALG as

carriers, which has increased significantly in the last decade. In 2002, González-Rodríguez and

colleagues used a system of nanoparticles of alginate/chitosan for controlled release of diclofenac

sodium. The nanoparticles were synthesized through an icing method using calcium ion (Ca2 +) and

Aluminum (Al3 +) and loaded with sodium diclofenac. It was demonstrated that drug release was

dependent of pH where NPs at pH acid drug release did not occur; moreover, around pH 6.4-7.2 was

completely released [54]. In the following year, and Robinson published a study which showed the

importance of the proportions of components (such as alginate, PLL , chitosan and calcium chloride)

in the synthesis of nanoparticles alginate, being such concentrations critical for the formation of

nanospheres or microspheres. A mass ratio of less than 0.2 between calcium chloride and alginate

was essential for the formation of nanospheres as well as adding another cathonic product as

chitosan and PLL [55].

Over the last decade more data were generated and different methodologies were used to

develop ALG nanoparticles. Recent works have shown that the NP-ALG may be used as efficient

delivery systems and release of drugs and other small molecules. NP-ALG (ALG-PDEA) (alginic

acid / poly [2 - (diethylamino) ethyl methacrylate]) were synthesized and loaded with doxorubicin

(DOX) and evaluated for antitumor activity in vitro and in vivo through cell and animal culture

model. The results of speed and amount of in vitro release of DOX from ALG-PDEA increase when

the pH of the medium decreases. Furthermore, in vivo results showed that NPs were efficiently

incorporated into tumor cells, benefiting from the increased permeability of solid tumors and

retaining (EPR effect). In addition, NPs loaded significantly suppress the tumor as compared to free

DOX and bio-distribution studies showed that DOX was increased at the tumor site and longer blood

circulation in animals, and still lesser concentration of drug in heart and lung, thereby reducing the

inherent toxicity of DOX [59]. Other data from the same research group using a new method of NP-

ALG synthesized using alginate (negatively charged) and 2,2 - (ethylenedioxy) bis (ethylamine)

(positively charged and then the addition interlinking calcium agent (cross-link) and then loaded

with DOX. These 100 nm nanoparticles showed zeta potential of-30mV, were to be incorporated

within the tumor cells, increased drug bio-distribution and accumulation at the tumor site. Moreover,

the data showed interlinking agent (Ca2 +) in stabilization of NPs in an environment at physiological

pH [56].

72

The ALG can also be associated with other polysaccharides of marine origin for the preparation

of nanoparticles. NPs sodium alginate / chitosan were loaded with 5-fluorouracil (5-FU) and

evaluated as drug delivery systems for ophthalmic use. In this work it became evident that the molar

relationship between chitosan and alginate are fundamental to the size of the nanoparticle and the

drug encapsulation efficiency of the NP. The in vivo results showed an increase in the effective

concentration of 5-FU in the aqueous humor as compared with a solution of 5-FU revealing an

effective topical application to ocular NP-ALG combined with chitosan [57].

With the increased number of work and knowledge of nanoparticles of alginate, new

applications have been proposed for the NP-ALG. Silver nanoparticles / alginate (AgNP/ALG) were

synthesized and used for drinking water disinfection. AgNP/ALG were synthesized using three

different couplers as filter material packed columns for simultaneous filtration and disinfection as an

alternative for the treatment of drinking water. When compared to silver particles, the AgNP/ALG

able to eliminate satisfactorily E. Coli and produce a smaller amount of silver deposit, being thereby

more effective and less toxic than the silver-only [58].

CONCLUSION

Marine polysaccharide nanomedicine technology has reached considerable maturity in the last

20 years. There is now an extensive body of intellectual property related to polysaccharides based

nanomedicines, and compelling evidence for the potential of marine polysaccharides nanoparticules

for many challenging drug delivery and tissue engineering applications. Currently, there is a broad

wisdom network about nanoparticles from marine polysaccharide and compelling evidence for the

potential nanocarriers to drug delivery, gene silencing or immunomodulatory peptides release and

even other applications, like image diagnostic. The structural diversity of polysaccharides allows

chemical modifications and the nanoproducts physicochemical features can be shaped to connect

with different cellular targets and also improve the bio-compatibly which improves use efficiency of

a particular drug and also dramatically reduces the side effects. Despite promising in vitro results

have been achieved over the last ten years, and optimistic data has been developed in human trials

exhaustive in vivo researches are necessary to address challenges in drug human administration and

then could be used in wide scale for therapeutic or even preventive measures. However, many

authors are confident that in short time based-marine polysaccharides nanoproducts will enter the

market.

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5.3. ARTIGO 2

83

5.4. ARTIGO 3

91


6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES

O grupo em que está inserido este trabalho tem estudado há algumas

décadas as atividades biológicas/farmacológicas presentes em fucanas em

diversas espécies de algas, como os polissacarídeos de alga. Este projeto de

pesquisa inicialmente teve como objetivo inovar nas pesquisas com

polissacarídeos e desenvolver uma nova linha de pesquisa na área da

nanomedicina propondo-se a sintetizar nanogéis de fucana, além da inserção

de novas técnicas as metodologias já utilizadas no laboratório (BIOPOL).

Os estudos mais avançados se concentraram em torno da fucana A

obtidas da alga S. schröederi. Esse polímero em pesquisas anteriores tem

apresentado uma excelente atividade biológica, especialmente pela alta

atividade antitumoral. Além disso, o material biológico a ser utilizado é de fácil

obtenção nas quantidades desejadas para o desenvolvimento deste trabalho, o

que não foi empecilho para o pleno trabalho das atividades propostas no início

da pesquisa. O laboratório também fez colaboração com o Prof. Miguel Gama,

da Universidade do Minho (Portugal), que tem experiência com nanopartículas

sintetizadas a partir de polissacarídeos, e que ajudou o nosso grupo na

caracterização dos nanogéis obtidos. Além disso, tivemos também a

colaboração da Profa. Helena Nader da UNIFESP, que se comprometeu a nos

auxiliar nos experimentos ou na utilização de equipamentos de não

disponibilidade aqui. Outros departamentos da UFRN das mais diversas áreas

(Física, Química, Engenharia de Materiais, Farmácia, Genética e Instituto do

Cérebro) também foram utilizados para a utilização de equipamentos como

também para o desenvolvimento de técnicas novas.

A produção de nanogéis de fucana foi desafiadora, já que fomos o

primeiro grupo a iniciar as pesquisas com nanogéis no Departamento de

Bioquímica da UFRN, mas foi compensadora, pois com o desenvolvimento

desse projeto pode-se iniciar uma fase nova na pesquisa na tentativa de

elucidar cada vez mais os mecanismos de ação pelos quais ela age, e

consequentemente, a longo prazo começar a fazer o registro de patentes.

Ao longo do desenvolvimento desta pesquisa, o cronograma de

atividades seguiu dentro dos padrões esperados e todas as dificuldades

92


encontradas foram superadas, através da ajuda dos colaboradores de diversos

centros de pesquisa, e inclusive do próprio laboratório onde se realizou a

pesquisa (BIOPOL/UFRN), com o auxílio de um aluno da iniciação científica

(Arthur Vidal), quando tive oportunidade de orientá-lo durante parte dos

experimentos.

Também considero importante ressaltar que durante processo de

formação participei de alguns Congressos, Simpósios e Encontros científicos

internacionais, nacionais e regionais, como autora ou em coautoria de

aproximadamente 20 resumos científicos. Destaco a minha participação no 4º.

International Symposium in Biochemistry of Macromolecules and Biotechnology

em 2012, quando um dos trabalhos em que sou coautora recebeu a Menção

Honrosa Prêmio Marcionilo Lins. Também destaco alguns eventos mais

relevantes: XLII Annual Meeting of the Brazilian Society for Biochemistry and

Molecular Biology – SBBq (2013) e 62ª Reunião Anual da SBPC (2010).

Até o momento tenho 13 artigos publicados em revista científicas, sendo

dois como primeira autora e os demais dividindo coautoria. As revistas foram:

Holos (IFRN), Publica (UFRGN) Revista Brasileira de Farmacognosia,

Toxicology Letters, Journal of Applied Toxicology, Biomedicine &

Pharmacotherapy, Carbohydrate Polymers, Marine Drugs, Molecules e Journal

of Applied Phycology. Além disso, também fiz parte na elaboração do capítulo

de livro na Marine Medicinal Glycomic e de uma banca examinadora de

trabalho de conclusão de curso (monografia).

O desenvolvimento deste estudo me proporcionou a oportunidade de

interagir com diversos ramos da pesquisa científica, bem como a aprendizagem

de algumas técnicas, o que conferiu um caráter interdisciplinar ao trabalho.

Como projeto para o futuro, planejamos continuar com os estudos na mesma

linha de pesquisa, em um futuro pós-doutorado.

93


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ANEXOS

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8.1. NORMAS PARA FORMATAÇÃO DA TESE (CCS)

98


99


100


8.2. NORMAS DA REVISTA PARA SUBMISSÃO (MARINE DRUGS)

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Abstract and Keywords: The abstract should be prepared as one paragraph of about

200 words. For research articles, abstracts should give a pertinent overview of the work,

its purpose, the main methods or treatments applied; summarize the article's findings or

facts and indicate the authors' conclusions or interpretation. As such, the abstract aims

at being an objective representation of the article and must not contain results or data

which are not presented and substantiated in the main text. Note that abstracts serve

two main purposes: on one hand abstracts are used by potential readers to assess the

relevancy of an article for their own work. On the other hand, abstracts are used by

indexing databases to catalog articles appropriately. Also, three to 10 pertinent

keywords need to be added after the abstract. We recommend that the abstract and the

keyword list use words that are specific to the article yet reasonably common within the

subject discipline.

Abstract Graphic: Authors are encouraged to provide a self-explanatory graphical

abstract of the paper to be used along with the abstract on the Table of Contents and

search results. The graphic should not exceed 550 pixels width/height and can be

provided as a PDF, JPG, PNG or GIF file. The minimum text size in the graphic should

be 12 pt.

Units: SI units (International System of Units) should be used for this journal. Imperial,

US customary and other units should be converted to SI units whenever possible before

submission of a manuscript to the journal.

Figures, Schemes and Tables: Authors are encouraged to prepare figures and

schemes in color. Full color graphics will be published free of charge. We kindly request

authors to provide figures and schemes at a sufficiently high resolution (min. 600 pixels

width, 300 ppi). Figures and schemes must be numbered (Figure 1, Scheme I, Figure 2,

Scheme II, etc.) and a explanatory title must be added. Tables should be inserted into

the main text, and numbers and titles for all tables supplied. All table columns should

have an explanatory heading. To facilitate the copy-editing of larger tables, smaller

fonts may be used, but in no case should these be less than 10 pt in size. Authors

should use the Table option of MS Word to create tables, rather than tabs, as tab


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delimited columns are often difficult to format for the final PDF output. Please supply

captions for all figures, schemes and tables. The captions should be prepared as a

separate paragraph of the main text and placed in the main text before a table, a figure

or a scheme.

Equations: If you are using Word, please use either the Microsoft Equation Editor or

the MathType add-on in your paper. It should be editable, not in the format of a picture.

Chemical Structures and Reaction Schemes: Chemical structures and reaction

schemes should be drawn using an appropriate software package designed for this

purpose. As a guideline, these should be drawn to a scale such that all the details and

text are clearly legible when placed in the manuscript (i.e. text should be no smaller that

8-9 pt.). To facilitate editing we recommend the use of any of the software packages

widely available for this purpose: MDL® Isis/Draw, ACD/ChemSketch®, CS

ChemDraw®, ChemWindow®, etc. Free versions of some of these products are

available for personal or academic use from the respective publishers. If another less

common structure drawing software is used, authors should ensure the figures are

saved in a file format compatible with of one of these products.

Physical and Spectroscopic Data: Physical and spectroscopic data as well as tables

for NMR data should be prepared according to the ACS's Preparation and Submission

of Manuscripts standard (page 4).

Experimental Data: To allow for correct abstracting of the manuscripts all compounds

should be mentioned by correct chemical name, followed by any numerals used to refer

to them in the paper. The use of the IUPAC nomenclature conventions is preferred,

although alternate naming systems (for example CAS rules) may be used provided that

a single consistent naming system is used throughout a manuscript. For authors

perhaps unfamiliar with chemical nomenclature in English we recommend the use of

compound naming software such as AutoNom. Full experimental details must be

provided, or, in the case of many compounds prepared by a similar method, a

representative typical procedure should be given. The general style used in the Journal

of Organic Chemistry is preferred. Complete characterization data must be given for all

new compounds. For papers mentioning large numbers of compounds a tabular format

is acceptable. For known compounds appropriate literature references must be given.

Conflicts of Interest: Authors must identify and declare any personal circumstances or

interest that may be perceived as inappropriately influencing the representation or

interpretation of reported research results. If there is no conflict of interest, please state

"The authors declare no conflict of interest." This should be conveyed in a separate

"Conflicts of Interest" statement immediately preceding the "References" section of the

manuscript. Financial support for the study must be fully disclosed under the

"Acknowledgments" section.

Acknowledgments: Please clearly indicate grants that you have received in support of

your research work (including funds for covering the costs to publish in open access).

Note that some funders will not refund article processing charges (APC) if the funder

and grant number are not clearly identified in the paper. The Acknowledgments section

is placed just before the References section.

References: Please ensure that a comprehensive list of all relevant references is

provided at the end of the manuscript, and that all references are cited within the paper

and numbered consecutively throughout the paper (including citations in tables and

legends). References should preferably be prepared with a bibliography software

package, such as Zotero, EndNote orReferenceManager. If references are prepared

manually they must be checked for integrity and correctness.

Reference Formatting: All the references mentioned in the text should be listed

separately and as the last section at the end of the manuscript, and be numbered

consecutively throughout the paper. Do not repeat references in the references list.

http://pubs.acs.org/paragonplus/submission/jnprdf/jnprdf_authguide.pdf

http://pubs.acs.org/paragonplus/submission/jnprdf/jnprdf_authguide.pdf

http://www.zotero.org/

http://endnote.com/

http://www.refman.com/

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Reference numbers should be placed in square brackets [ ], and placed before the

punctuation; for example [4] or [1-3]. For embedded citations in the text with pagination,

use both parentheses and brackets to indicate the reference number and page

numbers; for example [5] (p. 10). or [6] (pp. 101–105). Include the full title for cited

articles. See the Reference Preparation Guide for more detailed information.

Supplementary Material and Research Data: authors are encouraged to make their

experimental and research data openly available. Large datasets and files should be

deposited to specialized data repositories. Small datasets, spreadsheets, images, video

sequences, conference slides, software source code, etc. can be included with the

submission and published as supplementary material. Please read the information

about Supplementary Material and Data Deposit beneath for additional information and

instructions.

Authorship and Authors Contributions

For research articles with more than one author, authors are asked to prepare a short, one

paragraph statement giving the individual contribution of each co-author to the reported

research and writing of the paper. The paragraph should be titled "Author Contributions" and

placed in the paper after the "Acknowledgement" section and before the "Conflicts of Interest"

statement.

Only major contributors should be listed as authors. Those with small or technical contributions

can be mentioned in the Acknowledgements section. Authors themselves are responsible for

the correct identification and attribution of authorship. According to the COPE standard, to

which this journal adheres, "all authors should agree to be listed and should approve the

submitted and accepted versions of the publication. Any change to the author list should be

approved by all authors including any who have been removed from the list. The corresponding

author should act as a point of contact between the editor and the other authors and should

keep co-authors informed and involve them in major decisions about the publication (e.g.

responding to reviewers’ comments)." [1]

1. Wager, E.; Kleinert, S. Responsible research publication: international standards for

authors. A position statement developed at the 2nd World Conference on Research

Integrity, Singapore, July 22-24, 2010. In Promoting Research Integrity in a Global

Environment; Mayer, T., Steneck, N., eds.; Imperial College Press / World Scientific

Publishing: Singapore; Chapter 50, pp. 309-16.

Correct Identification of Components of Natural Products

The correct identification of the various components of extracts from natural sources is of key

importance, and as publishers we are keenly aware of our responsibility to the scientific

community in this area. Consequently, for papers on this topic, we have adopted the

recommendations of the Working Group on Methods of Analysis of the International

Organization of the Flavour Industry (IOFI), as published in Flavour Fragr. J. 2006, 21, 185.

These recommendations may be summarized as follows:

Any identification of a natural compound must pass scrutiny by the latest forms of available

analytical techniques. This implies that its identity must be confirmed by at least two different

methods, for example, comparison of chromatographic and spectroscopic data (including mass,

IR and NMR spectra) with those of an authentic sample, either isolated or synthesized. For

papers claiming the first discovery of a given compound from a natural source, the authors must

http://www.mdpi.com/authors/references

http://www.mdpi.com/journal/marinedrugs/instructions#supplements

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provide full data obtained by their own measurements of both the unknown and an authentic

sample, whose source must be fully documented. Authors should also consider very carefully

potential sources of artifacts and contaminants resulting from any extraction procedure or

sample handling.

Ethics Approval of Research

Manuscripts containing original descriptions of research conducted in humans or experimental

animals must contain details of approval by a properly constituted research ethics committee.

As a minimum, the project identification code, date of approval and name of the ethics

committee or institutional review board should be cited in the Experimental section.

Potential Conflicts of Interest

It is the authors' responsibility to identify and declare any personal circumstances or interests

that may be perceived as inappropriately influencing the representation or interpretation of

clinical research. If there is no conflict, please state "The authors declare no conflict of interest.".

This should be conveyed in a separate "Conflicts of Interest" statement preceding the

"Acknowledgments" and "References" sections at the end of the manuscript. Financial support

for the study must be fully disclosed under the "Acknowledgments" section.

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8.3. DECLARAÇÃO

DECLARAÇÃO DE CORREÇÃO DE PORTUGUÊS

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8.4. COMITÊ DE ÉTICA

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