Ministério Da Educação Universidade Federal Do Rio Grande Do Norte Centro De Ciências Da Saúde Programa De Pós-Graduação Em Ciências Da Saúde

MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

DESENVOLVIMENTO DE NANOEMULSÃO CATIÔNICA PARA INCORPORAR A PEÇONHA DO ESCORPIÃO TITYUS SERRULATUS E AVALIAR EFICÁCIA DO SORO PRODUZIDO A PARTIR DE IMUNIZAÇÃO DE CAMUNDONGOS

ARTHUR SÉRGIO AVELINO DE MEDEIROS

NATAL/RN 2020

ARTHUR SÉRGIO AVELINO DE MEDEIROS

DESENVOLVIMENTO DE NANOEMULSÃO CATIÔNICA PARA INCORPORAR A PEÇONHA DO ESCORPIÃO TITYUS SERRULATUS E AVALIAR EFICÁCIA DO SORO PRODUZIDO A PARTIR DE IMUNIZAÇÃO DE CAMUNDONGOS

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: ARNÓBIO ANTÔNIO DA SILVA JÚNIOR CO-ORIENTADOR: MATHEUS DE FREITAS FERNANDES PEDROSA

NATAL/RN 2020

Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial do Centro Ciências da Saúde - CCS

Medeiros, Arthur Sérgio Avelino de. Desenvolvimento de nanoemulsão catiônica para incorporar a peçonha do escorpião Tityus serrulatus e avaliar eficácia do soro produzido a partir de imunização de camundongos / Arthur Sérgio Avelino de Medeiros. - 2021. 84f.: il. Tese (Doutorado em Ciências da Saúde) - Universidade Federal do Rio Grande do Norte, Centro de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde. Natal, RN, 2020. Orientador: Arnóbio Antônio da Silva Júnior. Coorientador: Matheus de Freitas Fernandes Pedrosa. 1. Escorpiões - Soro antiescorpiônico - Tese. 2. Nanoemulsão - Tese. 3. Adjuvante nanoemulsionado - Tese. 4. Cristal líquido - Tese. 5. Transição de fases - Tese. 6. Imunoadjuvante - Tese. I. Silva Júnior, Arnóbio Antônio da. II. Pedrosa, Matheus de Freitas Fernandes. III. Título.

RN/UF/BS-CCS CDU 595.46

Elaborado por ANA CRISTINA DA SILVA LOPES - CRB-15/263

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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

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ARTHUR SÉRGIO AVELINO DE MEDEIROS

DESENVOLVIMENTO DE NANOEMULSÃO CATIÔNICA PARA INCORPORAR A PEÇONHA DO ESCORPIÃO TITYUS SERRULATUS E AVALIAR EFICÁCIA DO SORO PRODUZIDO A PARTIR DE IMUNIZAÇÃO DE CAMUNDONGOS

Aprovada em 18 / 11 / 2020

Banca examinadora

Arnóbio Antônio da Silva Júnior (Presidente da banca)

Anselmo Gomes de Oliveira (Examinador externo – UNESP)

Bolívar Ponciano Goulart de Lima Damasceno (Examinador externo UEPB)

Carolina Aloisio (Examinador externo – UNC)

Eryvaldo Sócrates Tabosa do Egito (Examinador interno - UFRN)

Matheus de Freitas Fernandes Pedrosa (Examinador interno – UFRN)

DEDICATÓRIA

A formação acadêmica se inicia de um sonho, de um trabalho digno, de uma formação cidadã. Por todas as pessoas especiais que estiveram comigo neste sonho, dedico este trabalho a Deus, à minha mãe Mércia, aos meus tios, aos meus avós, às minhas irmãs Amanda e Alanne, ao meu orientador Arnóbio Silva, e à minha querida esposa Gabriela, por juntos termos trilhados este caminho acadêmico e motivado um ao outro.

RESUMO As picadas de escorpiões estão entre as principais causas de acidentes com animais peçonhentos no Brasil e no mundo. O soro antiescorpiônico contém imunoglobulinas de animais imunizados com a peçonha de escorpiões, que são capazes de proteger pessoas susceptíveis. A produção do soro antiescorpiônico é alvo de melhorias, considerando a evolução de adjuvantes que podem auxiliar nas imunizações e aumentar o estímulo para produção. Sistemas de liberação de fármacos nanoemulsionados são promissores adjuvantes utilizados em muitos imunizantes, e propomos avalia-los na produção do soro antiescorpiônico. Desenvolvemos nanoemulsões, de forma inovadora, para atuar como sistemas de liberação para a peçonha do escorpião Tityus serrulatus e testar seu potencial adjuvante em camundongos Balb/c. No desenvolvimento das formulações, avaliou-se fatores da tecnologia que afetaram positivamente a ação adjuvante. Os resultados mostraram que nanoemulsões obtidas por técnicas de baixa energia apresentaram estabilidade de longo prazo, com perfis de tamanho de gotícula uniforme e reduzido (menor que 200 nm). As formulações desenvolvidas foram estáveis após alterações de pH e salinidade. As nanoemulsões foram obtidas revestidas com o polímero catiônico poli (etilenoimina), sem que também afetasse a estabilidade. As formulações desenvolvidas mostraram ser capazes de carrear os antígenos ligados de forma inespecífica, com as interações entre as nanoemulsões e a peçonha do escorpião demonstradas por espectroscopia FTIR. A peçonha carreada foi menos tóxica que a não carreada em relação à atividade hemolítica, apresentando ainda baixa toxicidade em células de macrófagos (RAW 264.7). A atividade adjuvante foi avaliada pela produção de imunoglobulinas em animais imunizados, testando as formulações desenvolvidas em comparação com o adjuvante convencional hidróxido de alumínio. Animais imunizados com o adjuvante nanoemulsão catiônica produziram comparativamente mais imunoglobulinas que o adjuvante nanoemulsão não-catiônica e que o adjuvante baseado em partículas de alumínio. Palavras chave: Soro antiescorpiônico; nanoemulsão; adjuvante nanoemulsionado; cristal líquido; transição de fases; funcionalização; imunoadjuvante.

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ABSTRACT Scorpion stings are major cause of envenoming by animals in endemic countries, like Brazil. The scorpion antivenom is product that is able to neutralize and to protect sensitive people. However, its production has been subject of improvements taking in account the innovations on the immuno-adjuvant production and the difficult on the venom extraction, from captive animals, and the venom its toxicity. Scorpion venom has a complex composition and an adjuvant for crude venom represents a challenge that can be possibly fulfilled with drug delivery systems based on nanotechnology. In this research, we seek for innovation developing new nanoemulsion formulations to act as drug delivery system for Tityus serrulatus scorpion venom and to evaluate its immunizing properties on Balb/C mice. The nanotechnology development covered features that positively affected the adjuvant activity. We demonstrated that low energy techniques for production of the nanoemulsions allowed long term stability to the formulations with reduced and uniform droplet size (below 200 nm). The developed formulations were also robust to pH and saline variation. The formulations were also able to be covered by PEI and to maintain their stability. The formulations demonstrated capable to nonspecifically adsorb crude venom, as showed through FTIR spectroscopy technique as intermolecular interactions between nanoemulsions and crude venom. Nanoemulsions venom-load also could reduce toxicity related to venom hemolytic activity and to present low toxicity against macrophage cells (RAW 264.7). Adjuvant activity was demonstrated in comparison with aluminum hydroxide adjuvant, which scored for nanotechnology feature of act as crude venom adjuvant more efficiently to induce specific anti-IgG immunoglobulins using PEI-covered nanoemulsions. Key words: Scorpion antivenom; nanotechnology; nanoemulsion adjuvant; liquid crystal; phase transition; surface functionalization; immunoadjuvant.

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LISTA DE ABREVIATURAS E SIGLAS

APC – Células apresentadoras de antígenos (do inglês: antigen presenting cells)

ABS – Absorbância

BCA – Ensaio do ácido bicincronínico (do inglês: Bicinchoninic acid assay)

CL50 – Concentração letal 50

DFPT – Diagrama de fases pseudo-ternário

DMEM – Meio modificado de Dulbecco’s eagle’s (do inglês: Dulbecco's Modified

Eagle Medium)

DLS – Espalhamento dinâmico de luz (do inglês: dynamic light scattering

ELISA – Ensaio de imunoabsorção enzimática (do inglês Enzyme-Linked

Immunosorbent Assay)

FC – Fosfatidilcolina de Soja

FTIR – Infra-vermelho com transformada de Fourier (do inglês: Fourier transform infrared)

HPH – Homogeneização em alta pressão (do inglês: High pressure homogenizer)

MTT – brometo de 3-(4,5-dimetil-2-tiazolil)-2, 5-difenil-2H-tetrazólio

NMP - N-metil-pirrolidona

NE – nanoemulsão

OMS – Organização mundial de saúde

PBS – Solução de tampão fosfato (do inglês: Phosphate buffered saline)

PEI - poli(etilenoimina)

PO188 - Poloxamer 188

PO407 - Poloxamer 407

PRRs – Receptores reconhecedores de padrões (do inglês: Pattern recognition receptors)

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SFB – Soro fetal bovino

SINAN – Sistema nacional de agravos de notificação

SUS – Sistema único de saúde

TIF – Temperatura de inversão de fases

TSV – Peçonha do Tityus serrulatus (do inglês: Tityus serrulatus venom)

T80 – Polisorbato 80

TLR – Receptores toll-like (do inglês: Toll like receptors)

VLP – Partículas semelhantes ao vírus (do inglês: vírus like particles).

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LISTA DE FIGURAS

Figura 1 (4.1) - Representação esquemática da estrutura química dos constituintes das nanoemulsões. Figura 1 (5.1) - Estudos de formulações para a seleção da composição de surfactantes.

Figura 2 (5.1) - Diagrama Pseudo-ternário de fases a 25°C e fotografias de microscopia de luz polarizada das amostras.

Figura 3 (5.1) - Efeito da funcionalização com PEI e incorporação de TsV nas propriedades físico-químicas das formulações.

Figura 4 (5.1) - Efeito das formulações na viabilidade celular em células RAW 264.7 e no efeito hemolítico em hemácias humanas A+.

Figura 5 (5.1) - Avaliação da densidade óptica e do título de anticorpos IgG específicas contra o TsV.

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LISTA DE TABELAS

Tabela 1 (4.2.4) – Composição das formulações Tabela 1 (5.1) - Composição das formulações.

Tabela 2 (5.1) - Medidas de tamanho de gotícula e potencial zeta para formulações com e sem TsV armazenadas a 4°C por seis semanas.

Tabela 1 (5.2) - Neutralização e imunoproteção de soro antiescorpiônico contra peçonha de escorpião de acordo com o adjuvante utilizado para produção.

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SUMÁRIO

1 INTRODUÇÃO ...... 15 2 JUSTIFICATIVA ...... 20 3 OBJETIVOS ...... 21 2.1 Objetivo geral ...... 21 2.2 Objetivos específicos ...... 21 4 MATERIAIS E MÉTODOS...... 22 4.1 Materiais ...... 22 4.2 Métodos ...... 23 4.2.1 Seleção da composição de surfactantes e co-solvente das emulsões ...... 23 4.2.2 Diagrama de fases pseudo-ternário (DFPT) ...... 24 4.2.3 Medidas de tamanho de gotícula, potencial zeta e identificação de cristais líquidos ...... 25 4.2.4 Preparação de nanoemulsões catiônicas revestidas com poli(etilenoimina) (PEI) ...... 25 4.2.5 Avaliação da estabilidade das amostras frente a variações de pH e composição salina ...... 26 4.2.6 Obtenção de nanoemulsões-TSV adsorvidas ...... 26 4.2.7 Espectroscopia de infra-vermelho com transformada de Fourier (FTIR) ...... 27 4.2.8 Viabilidade celular e teste de hemólise ...... 28 4.2.8.1 Cultura de células ...... 28 4.2.8.2 Teste de redução do MTT ...... 28 4.2.8.3 Teste de hemólise ...... 29 4.2.9 Imunização de camundongos ...... 29 4.2.9.1 Animais ...... 29 4.2.9.2 Protocolo de imunização ...... 30 4.2.9.3 Titulação de IgG por ELISA ...... 30 5 ARTIGOS PRODUZIDOS ...... 32 5.1 Produção de artigo científico ...... 33 5.2 Produção de artigo científico ...... 59 6 CONCLUSÕES ...... 78

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7 COMENTÁRIOS, CRÍTICAS E CONCLUSÕES ...... 79 8 REFERÊNCIAS ...... 80

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1 INTRODUÇÃO

Picadas de escorpiões representam um grave problema de saúde pública no Brasil. Esses acidentes são tratados pelo sistema único de saúde (SUS), sendo quantificados através do Sistema Nacional de Agravos de Notificação (SINAN). Acidentes com escorpiões são ainda mais numerosos quando comparados com o número de acidentes com outros animais peçonhentos, como pode-se perceber nos dados públicos do SINAN 1. A alta prevalência dos acidentes com escorpiões se deve ao fato do país ser uma região endêmica para esses animais, a reprodução por partenogênese facilitar sua proliferação, dentre outros 2. Considerando as espécies de escorpiões no Brasil, aquelas que apresentam maior risco à saúde pública são Tityus serrulatus e Tityus bahiensis (encontrados nos estados de São Paulo e Minas Gerais), Tityus stigmurus (região nordeste) e Tityus obscurus (região norte) 3. Picadas com Tityus serrulatus são mais numerosas no país, provavelmente devido sua melhor adaptação em ambientes urbanos e no sudeste 4. O tratamento do escorpionismo requer avaliação e triagem por gravidade dos sintomas. Os efeitos da peçonha do escorpião variam de acordo com a espécie do animal, o estado fisiológico e nutricional do paciente, comorbidades e pertencer a grupos de risco, em especial crianças e idosos. A dor local e marcante é presente na maioria das picadas. Os casos mais graves de envenenamento apresentam manifestações sistêmicas das toxinas, como choque, parada cardiorrespiratória, náuseas, vômitos, taquicardia, hiperglicemia e leucocitose 5,6. O manejo clínico e terapêutico é geralmente realizado em centros especializados por todo o país. Alguns casos de acidentes necessitam de tratamento rápido com soro antiescorpiônico específico, que confere imunidade passsiva. A produção do soro é realizada por laboratórios estatais no Brasil, dentre eles a Fundação Ezequiel Dias (FUNED, Belo Horizonte-MG), o instituto Butantan (São Paulo-SP) e o instituto Vital Brazil (Rio de Janeiro-RJ). O soro antiescorpiônico é um produto biotecnológico composto de imunoglobulinas produzidas por animais imunizados (imunidade ativa). A imunização desses animais, em especial cavalos, geralmente ocorre com a peçonha associada a adjuvantes, com ciclos de hiper-imunizações para maximizar o título de anticorpos produzidos. A partir daí, o sangue desses animais é coletado, processado, e a porção do anticorpo capaz de neutralizar a toxina é purificada 7. 16

Nas imunizações realizadas para a produção do soro, diversos adjuvantes podem ser utilizados. Cada adjuvante atua por diferentes mecanismos, podendo ser selecionados de acordo com o estado patológico a ser combatido. Adjuvantes podem ser classificados de acordo com seu mecanismo de ação em: (i) sistemas de liberação ou (ii) potenciadores do sistema imune 8,9. Os sistemas de liberação são carreadores dos antígenos, aumentando o reconhecimento pelas células do sistema imune, podendo ainda criar uma reação local pro-inflamatória e recrutar células do sistema imune inato 10,11. Por exemplo, sais minerais de alumínio são sistemas de liberação particulados amplamente utilizados para diversas vacinas (imunizantes) e inclusive na produção de soro antiescorpiônico 7. Os potenciadores do sistema imune ativam as células do sistema imune inato, interagindo com receptores de reconhecimento de padrões de patógenos (do inglês: PRRs). Essa segunda classe inclui ainda a aplicação das próprias citocinas 8. Os potenciadores do sistema imune podem interagir com diversos receptores, dentre os receptores de superfície tem-se o receptor toll-like e o receptor tipo-C de lecitina (do inglês: CLRs). Os receptores intracelulares incluem o receptor tipo NOD (do inglês: NLRs), que reconhece domínios de nucleotídeos, e os receptores do tipo gene induzível retinoide (do inglês: RLRs) 12. Os mecanismos imunes dos adjuvantes são diversos, podendo ser estudados de acordo com o tipo de células que ativam e as citocinas que são produzidas pelas células ativadas. Imunizações de animais com toxinas de escorpiões associadas com sais de alumínio promovem a produção de citocinas IL-4, IL-5 e IL-6, produzidas por linfócitos T-CD4+. Com isso, linfócitos B são ativados e secretam IgG1, IgG4 e IgE (resposta humoral) 13. A ativação da resposta humoral, com secreção de anticorpos neutralizantes, é o objetivo da hiper imunização de animais para coleta de soro anti- peçonha, mais especificamente a produção de imunoglobulinas IgG. Outros quadros patológicos, no entanto, precisam de uma resposta imunológica celular. A resposta celular é ativada quando a apresentação de antígenos induz linfócitos T-CD4+ a produzir IL-2, IFN-γ e TNF-α, que ativam células para eliminar patógenos com ciclos intracelulares obrigatórios, como linfócitos T-CD8+, macrófagos e opsonização dos patógenos com anticorpos fixadores de complemento (IgG2a e IgG3) 12. O lipídio A monofosforilado, por exemplo, é aprovado em algumas vacinas como adjuvante agonista de receptores toll-like, capaz de induzir produção de IL-12, IFNγ e ativação de linfócitos Th1. Por isso, este adjuvante vem sendo estudado para ser associado com antígenos de patógenos com ciclos intracelulares obrigatórios, potencializar a imunização e a resposta protetora de memória 8. 17

A pesquisa de novos adjuvantes busca respostas imunes para cada patógeno ou antígeno, sendo benéfico a existência de numerosas opções terapêuticas para uma ampliada gama de situações terapêuticas. A nanotecnologia vem há alguns anos sendo aplicada em sistemas de liberação para antígenos como adjuvantes em imunizações. Neste caso, o estímulo imunológico pode advir de uma liberação controlada e direcionada dos nanocarreadores para células apresentadoras de antígenos. Os sistemas de liberação incluem partículas poliméricas, emulsões, virossomas, partículas semelhantes ao vírus (do inglês: VLP), etc. Por exemplo, nanopartículas de PLGA promoveram um aumento de incorporação de antígenos por células apresentadoras de antígenos 14–16, enquanto que a nanoemulsão de esqualeno (MF59) quando utilizada como adjuvante promove ativação de macrófagos, células dendríticas e monócitos 17 (células apresentadoras de antígenos) em vacinas contra o vírus influenza 16,18. Algumas propriedades específicas de nanocarreadores podem influenciar sua ação como adjuvantes. Por exemplo, o potencial hidrofóbico e a carga de adjuvantes particulados podem modular a afinidade de ligação à antígenos, modular sua captação in vivo, tempo de residência e ativação do sistema imune 19–21. Nanoemulsões com carga catiônica têm demonstrado aumento de reconhecimento celular e adesão a mucosas quando utilizadas como adjuvantes em vacinas 22. Nosso grupo de pesquisa demonstrou anteriormente o potencial de nanopartículas catiônicas de quitosana de carrear a peçonha de serpentes e escorpiões, o que promove uma produção de anticorpos específicos contra seus antígenos 23–25. Surge, portanto, a necessidade de se estudar propriedades físico-químicas de um sistema transportador para maximizar o reconhecimento antigênico, de modo a obter vias eficazes de imunização para produção do soro antiescorpiônico. Nanoemulsões vêm sendo cada vez mais testadas como adjuvantes para muitas aplicações e testes clínicos 11,20,26. As nanoemulsões são dispersões nanométricas de um líquido imiscível em outro, geralmente um oleoso e um aquoso estabilizados por tensoativos, em que existem inúmeras possibilidades quanto a sua composição e tipo de compostos ativos que podem transportar 27. O tamanho, uniformidade do tamanho e o tipo de óleo utilizado podem frequentemente alterar sua ação como adjuvante 28. A preparação de nanoemulsões inclui ajuste de composição e técnicas de obtenção. Considerando que nanoemulsões óleo em água (O/A) são geralmente compostas de tensoativos e fase oleosa dispersos em água, a seleção da composição 18 visa uma maior eficiência emulsiva dos tensoativos. Estudos mostram que misturas de tensoativos geralmente fornecem emulsões com menor tamanho de gotícula ou maior quantidade de fase interna dispersa 29. O motivo inclui aumento de moléculas na interface pelo sinergismo dos tensoativos, ao invés de apenas um tipo de tensoativo com solubilidade preferencial por uma das fases 29. Mais moléculas na interface também promovem uma maior pressão nesta região, o que supera a tensão interfacial e gera um estímulo para a diminuição no tamanho de gotícula 30. Além dos tensoativos, a escolha da fase oleosa pode influenciar nas propriedades da emulsão. A utilização de triglicerídeos de cadeia média (TCM) geralmente fornece nanoemulsões com diâmetro de gotícula menor em detrimento a utilização de ácidos graxos de maior cadeia, pois com menor tamanho e maior solubilidade do óleo aumenta a capacidade do sistema em formar nanoemulsões 31–33. Nanoemulsões podem ser preparadas por técnicas diversas, utilizando alta energia ou baixa energia. Dispositivos mecânicos usam transferência de alta energia para dispersar os componentes e reduzir o tamanho de gotículas 34–36. Técnicas de baixa energia promovem auto associação dos componentes, no qual promove-se mudanças de temperatura e hidratação nos tensoativos 37–39. A auto associação pode ser induzida ainda por outros componentes, como co-solventes 40, que culminam em métodos de obtenção mais simples de nanoemulsões, sem a necessidade de equipamentos que promovam um alto cisalhamento. Além do potencial das nanoemulsões como sistemas de liberação, existem inúmeras possibilidades de funcionalizar (adicionar propriedades através de um ligante) estas estruturas com materiais que possam aumentar a ação adjuvante em questão. Diferentes moléculas ligadas à superfície de nanocarreadores podem aumentar a capacidade de transportar fármacos, como proteínas, vencer barreiras biológicas e membranas celulares, dentre outros 41. No sentido de tornar as nanoemulsões catiônicas para potencializar a ação adjuvante 22, a funcionalização com o polímero poli(etilenoimina) (PEI) traz um potencial aumento de ligação com proteínas e outras moléculas por interações eletrostáticas e hidrofóbicas. A PEI é um polímero biocompatível aprovado para terapia gênica em humanos pelo FDA 42, tendo sido estudada por ligar agentes carregados negativamente como o ácido desoxirribonucleico (DNA, do inglês: deoxyribonucleic acid), promovendo transfecção de genes e podendo ainda aumentar a captação celular desses ácidos nucleicos e de fármacos 42–44. 19

Neste sentido, a utilização de adjuvantes nanoemulsionados catiônicos é promissora. A tecnologia demanda pesquisa de materiais e técnicas para obtenção dos carreadores, que podem influenciar em diversas propriedades físico-químicas, como tamanho e carga de partícula 19 21, que por sua vez podem influenciar a ação adjuvante.

2 JUSTIFICATIVA

Os problemas advindos de picadas de escorpiões no Brasil são endêmicos, sendo necessário melhorar a cadeia de produção do soro antiescorpiônico e sua acessibilidade 10,45. A baixa produtividade de imunoglobulinas do soro demonstra a necessidade de mais pesquisas científicas neste campo 46. Propomos o desenvolvimento de um adjuvante que aumente a eficácia da produção do soro e sua resposta terapêutica. Destacamos que diversos sistemas de liberação foram desenvolvidos por nosso grupo de pesquisa e utilizados como adjuvantes para peçonhas, incluindo nanopartículas catiônicas de quitosana 23 e nanopartículas catiônicas de ácido poli-láctico (PLA) 41. Neste sentido, propomos desenvolver e avaliar carreadores nanoemulsionados contendo um polímero catiônico poli-etilenoimina (PEI) para incorporar a peçonha do escorpião. Este sistema inovador pode ser o primeiro a utilizar nanoemulsões como adjuvante na produção de soro contra o veneno do escorpião Tityus serrulatus, endêmico no Brasil.

3 OBJETIVOS

2.1 Objetivo geral

Desenvolver sistema nanoemulsionado catiônico óleo em água como imunoadjuvante da peçonha do escorpião Tityus serrulatus, no intuito de aumentar a produção de soro antiescorpiônico em camundongos.

2.2 Objetivos específicos

A fim de atingir o objetivo geral, foram propostos os seguintes objetivos específicos: a) Obter sistemas nanoemulsionados catiônicos e não catiônicos, determinando a composição adequada das fases aquosa, oleosa, tensoativos e de PEI que possibilitem um tamanho de gotícula na escala nano (abaixo de 200 nm); b) Incorporar as proteínas da peçonha do escorpião Tityus serrulatus nas nanoemulões; c) Avaliar a estabilidade em temperatura ambiente das nanoemulsões obtidas medindo o tamanho de gotícula, potencial zeta e turbidez; d) Avaliar as propriedades espectroscópicas da peçonha e suas interações com as nanoemulsões por infravermelho médio, no intuito de elucidar as interações moleculares; e) Avaliar viabilidade celular das formulações em cultura de células de macrófagos de camundongos e em hemácias humanas; f) Estimular a produção do soro antiescorpiônico imunizando camundongos com as formulações adjuvantes desenvolvidas e avaliar a produção de anticorpos;

4 MATERIAIS E MÉTODOS

4.1 Materiais

A peçonha do escorpião Tityus serrulatus (TsV, do inglês: Tityus serrulatus venom) foi doada pelo instituto Butantan, SP, Brasil. TsV foi extraída de espécimes adultas de escorpiões e então liofilizada e armazenada a –20 °C até o momento do uso. Soluções de TsV foram preparadas com PBS (do inglês: phosphate buffered saline) no momento do uso. A quantidade de TsV foi expressa em concentração de proteínas quantificadas pelo ensaio do ácido bicincronínico (BCA, do inglês: bicinchoninic acid), usando albumina bovina sérica (BSA, do inglês: bovin sérum albumin) como padrão. Os triglicerídeos de cadeia média (TCM) do ácido caprílico/cáprico (Miglyol®812, Sasol®, Hamburgo, Alemanha) foram usados como fase oleosa dos sistemas emulsionados, os quais contêm 55% de triglicerídeos com ácidos graxos C8 e 45% de triglicerídeos com ácidos graxos C10. A fosfatidilcolina de soja (FC) (pureza > 95%) foi comprada da Avanti Polar Lipids® (Alabaster, AL, EUA). O polissorbato 80 (Tween 80®, T80), glicerol, propilenoglicol e octilfenol etoxilado (Triton X-100 ®) foram comprados da Sigma-Aldrich (St. Louis, Missouri, EUA). A N- metil-pirrolidona (NMP) (pureza 99%) foi comprada da Vetec (Duque de Caxias, RJ, Brasil) e usada como co-solvente. Os tensoativos poliméricos poloxâmero 188 (PO188) e poloxâmero 407 (PO407) e o polímero poli(etilenoimina) hiper-ramificado de 25.000 KDa (PEI) foram comprados da Sigma-Aldrich (St. Louis, Missouri, EUA). O tampão PBS (pH = 7,4) foi preparado dissolvendo em água purificada os seguintes constituintes: 137 mM de NaCl, 3 mM de KCl, 15 mM de KH2PO4, 10 mM de Na2HPO4. A água purificada (condutividade de 0,1 μS/cm) foi preparada usando purificador de osmose reversa, modelo OS50LX, Gehaka (SP, Brasil). Na figura abaixo, estão representadas as estruturas químicas dos constituintes presentes nas formulações nanoemulsionadas.

Figura 1. Representação esquemática da estrutura química dos constituintes das nanoemulsões. a) polissorbato 80 (T80) b) fosfatidilcolina (FC), c) ácido cáprico (C10 dos TCM), d) ácido caprílico (C8 dos TCM), e) poli(etilenoimina) ramificada, f) N-metil- pirrolidona.

4.2 Métodos

4.2.1 Testes preliminares: definição de tensoativos e co-solventes em sistemas emulsionados

Testes preliminares foram realizados para obter emulsões com composição variável, em que foram testados diferentes tensoativos para estabilizar o TCM como fase oleosa. Estes testes obtendo emulsões foram necessários para posterior obtenção de nanoemulsões. As emulsões foram obtidas contendo 10% de TCM, 5% das misturas de tensoativos e 85% de água purificada (10:5:85 m/m/m), sendo preparadas por emulsificação com inversão de fases. A água foi vertida sob a fase oleosa, composta da dispersão dos tensoativos e do TCM na temperatura de 70°C ± 2°C (chapa aquecida), sob agitação magnética a 875 rpm. A composição dos tensoativos foi ajustada testando três tensoativos hidrofílicos, polissorbato 80 (T80), poloxamer 188 (PO188), poloxamer 407 (PO407) e o tensoativo hidrofóbico fosfatidilcolina de soja (FC). Os tensoativos também foram testados aos pares, FC:T80, FC:PO188 e FC:PO407 em diversas razões m/m. As formulações obtidas foram observadas por 7 dias em relação a possíveis instabilidades, como cremagem 24 da emulsão e separação de fases. O índice de cremagem foi calculado pela seguinte equação:

IC % = 100 X HU / HT (Eq.1)

Onde IC% representa o percentual do índice de cremagem, HU representa a altura em cm da fase superior da emulsão e HT a altura total em cm da emulsão no tubo de ensaio.

A turbidez das amostras foi medida com diluição prévia em água purificada (1:20 v/v), tomando 1 ml de emulsão e 19 ml de água purificada. O espalhamento de luz foi medido em espectrofotômetro a 860 nm (Thermo Scientific, EvolutionTM 60S, EUA) usando cubeta de vidro de 1 cm3.

A preparação das emulsões também foi testada adicionando 10% de co- solventes, considerando estudos anteriores desenvolvidos em nosso grupo de pesquisa 40, dispersando os co-solventes na fase oleosa a 70 °C antes da inversão de fases. Três co-solventes foram testados, N-metil-pirrolidona (NMP), glicerina e propilenoglicol.

4.2.2 Diagrama de fases pseudo-ternário (DFPT)

Após a definição dos tensoativos nos testes preliminares (FC:T80:NMP 3:1:3 m/m), a razão tensoativo/óleo foi variada e testada em DFPT. Foram testadas razões crescentes tensoativo/óleo de 0,1, 0,2, até a razão de 0,9 m/m. Este ensaio permitiu classificar diferentes regiões do diagrama para obter emulsões, nanoemulsões, cristais líquidos, dentre outros. Para realizar os diagramas, 2 g da mistura FC:T80:NMP e TCM (fase oleosa) foram colocados em um béquer de 50 ml sob agitação magnética (875 rpm) em chapa aquecida a 70°C ± 2°C, adicionando água purificada lentamente sob gotejamento. O aspecto dos sistemas emulsionados obtidos a cada volume adicionado de água (100 µL; 250 µL; 500 µL; 1 mL) foi observado e classificado após 2 min de acomodação. A obtenção do DFPT também foi testada na temperatura de 25°C ± 2°C, vertendo a água purificada por gotejamento (3ml/minuto) sob a fase oleosa em agitação magnética (875 rpm), partindo de 2 g da mistura FC:T80:NMP (6:2:15 m/m/m) juntamente com TCM (fase oleosa) e proporções variáveis de água de acordo com os pontos selecionados para estudo. A proporção de NMP a 25°C foi ajustada de acordo com a solubilidade da FC nesta temperatura. 25

Após verter a fase aquosa, a emulsão permaneceu por 10 minutos sob agitação magnética e foi submetida a agitação em Ultra-turrax a 11000 rpm por 10 min.

4.2.3 Obtenção de nanoemulsões

As nanoemulsões foram obtidas por dois métodos: (i) método da inversão de fases a 25° C com posterior agitação em Ultra-turrax, como mostrado anteriormente no tópico 4.2.2, e (ii) por diluição de cristais líquidos com transição de fases para nanoemulsões. Os cristais líquidos formados também foram obtidos pelo método de inversão de fases a 25° C e posterior agitação em Ultra-turrax. O cristal líquido foi preparado usando tampão PBS pH = 7,4 como fase aquosa. A fase aquosa foi gotejada sob a fase oleosa na velocidade de 3 ml por minuto na temperatura de 25° C ± 2° C sob agitação magnética a 875 rpm por 10 min. Uma agitação adicional foi realizada em equipamento Ultra-turrax® a 11000 rpm por 10 minutos para a obtenção do cristal líquido. Para obter a nanoemulsão (NE), uma solução de tampão PBS (4,54 g, pH = 7,4) foi gotejada sob o cristal líquido (2,0 g) sob agitação magnética a 360 rpm por 10 min a 25° C ± 2° C.

4.2.4 Preparação de nanoemulsões catiônicas revestidas com poli(etilenoimina) (PEI)

A amostra identificada nos diagramas DFPT com aspecto de gel e denominada cristal líquido (sistema anisotrópico) foi selecionada para ser diluída de forma controlada para uma composição identificada no DFPT como nanoemulsão (sistema isotrópico). Para a preparação da nanoemulsão catiônica (NE-PEI) foram pesados 2,0 g do cristal líquido e pesados 4,54 g da solução aquosa contendo PEI. Para a seleção da quantidade de PEI, sua concentração foi variada relativa ao componente de interface majoritário na emulsão, FC, partindo da proporção PEI:FC de 1:1000 a 1:170. Após fixada a condição de trabalho, 4,54 g da solução aquosa contendo PEI (0,029% m/m) foram gotejados sob o cristal líquido na velocidade de 3 ml/min sob agitação magnética a 25°C ± 2°C. As composições das formulações de nanoemulsões e cristal líquido estão mostradas abaixo na Tabela 1.

Tabela 1. Composição das formulações desenvolvidas

Formulações Concentração dos NE não NE-TsV NE Cristal NE-TsV componentes catiônica não catiônica líquido catiônica vazia catiônica vazia TsV (µg/mL) 0.0 0.0 18.0 0.0 18.0 TCM (% w/w) 2.00 0.9 0.9 0.9 0.9 T80 (% w/w) 4.55 2.0 2.0 2.0 2.0 FC (% w/w) 13.65 6.0 6.0 6.0 6.0 NMP (% w/w) 34.2 15 15 15 15 PEI (% w/w) 0.0 0.0 0.0 0.02 0.02 Água (% w/w) 45.6 76.1 76.1 76.08 76.08

4.2.5 Avaliação da estabilidade das amostras frente a variações de pH e composição salina

As amostras obtidas e classificadas com aspecto gel, nanoemulsão (NE) e nanoemulsão contendo PEI (NE-PEI) foram armazenadas hermeticamente seladas em tubos de vidro a 25° C ± 2° C. A intervalos de tempo específicos foram registrados tamanho de gotícula, PDI e potencial zeta. A amostra com aspecto gel identificada como cristal líquido foi preparada usando tampão PBS pH = 7,4 como fase aquosa, assim como descrito no tópico 4.2.1. Para obter a nanoemulsão (NE), uma solução de tampão PBS (4,54 g, pH = 7,4) foi gotejada sob o cristal líquido (2,0 g) sob agitação magnética a 360 rpm por 10 min a 25° C ± 2° C. O sistema NE também foi obtido com ajuste de pH e presença do tampão salino em condição ácida (pH = 5,5) e alcalina (pH = 8,5) para avaliar a robustez de obter a emulsão com essas variáveis, usando HCl 0,1 M e NaOH 0,1 M para os ajustes de pH tanto na obtenção do cristal líquido quanto na obtenção da nanoemulsão. As formulações obtidas com tampão PBS pH = 7,4 foram utilizadas para medir as propriedades físico-química das amostras e para os experimentos em células in vitro e in vivo em camundongos.

4.2.6 Medidas de tamanho de gotícula, potencial zeta e identificação de cristais líquidos

O diâmetro médio de gotícula e a distribuição de tamanho (índice de polidispersão, PDI) foram medidos pela técnica da dispersão dinâmica de luz (do inglês: dynamic light scattering, DLS) usando o equipamento Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Reino Unido), com comprimento de onda de 632 nm, ângulo de dispersão de 173°, a 25° C. Para as medidas de tamanho, as nanoemulsões foram diluídas utilizando 0,1 ml da emulsão e 9,9 ml de água purificada, a fim de atingir uma concentração apropriada de gotículas para a medida (100-500 27 kcps – do inglês: kilo counts per second). Os valores foram expressos como média ± desvio padrão.

Amostras com aspecto gel foram analisadas por microscopia óptica de luz polarizada para observar presença de comportamento anisotrópico. Uma gota de cada amostra foi espalhada em uma lâmina de vidro, coberta com uma lamínula de vidro e posicionada no microscópio. As imagens foram registradas com aumento de 100X e 400X (aumento de lente objetiva X aumento de lente ocular).

4.2.7 Obtenção de nanoemulsões-TSV carreadas

Uma solução estoque de TSV foi preparada a 1 mg/ml em solução de PBS (pH = 7,4), a qual foi analiticamente analisada pelo teor proteico com o Kit do método BCA de acordo com as recomendações do fabricante (Thermo Fisher Scientific, Waltham, Massachusetts, EUA). Neste método, o teor proteico foi determinado pela redução do cobre em meio alcalino, formando um complexo com o BCA que absorve em 560 nm. Alíquotas dessa solução estoque foram adicionadas nas formulações NE (NE-TSV) e NE-PEI (NE-PEI-TSV) para atingir uma concentração final de TSV de 0,018 mg/ml. Esta concentração de TsV foi utilizada para os ensaios de imunização in vivo, considerando concentrações previamente testadas em nosso grupo de pesquisa 47 e baseado na LD50 desta peçonha em camundongos 48. Para atingir o equilíbrio de adsorção, as amostras permaneceram em agitação magnética a 375 rpm por 1 h em banho de gelo. As amostras permaneceram armazenadas a 4° C ± 2° C em refrigerador por seis semanas, sendo observado o aspecto coloidal através de tamanho de gotícula, PDI e potencial zeta.

4.2.8 Espectroscopia de infra-vermelho com transformada de Fourier (FTIR)

O espectro infra-vermelho das amostras foi registrado utilizando um espectrômetro IR Prestige-21 (Shimadzu corporation, Japão). Uma gota das nanoemulsões foi espalhada no suporte para amostras do equipamento e analisada por ATR-FTIR. A faixa estudada foi entre 4000-1000 cm-1 de número de onda, com 16 varreduras e 1 cm-1 de resolução. 28

4.2.9 Viabilidade celular e teste de hemólise

4.2.9.1 Cultura de células

As células de macrófagos RAW 246.7 (ATCC TIB-71) foram generosamente fornecidas pelo Laboratório de Biotecnologia de Polímeros Naturais, Departamento de Bioquímica, Centro de Biociências, Campus Universitário, Universidade Federal do Rio Grande do Norte, Brasil. As culturas de células foram mantidas em meio de cultura Eagle modificado por Dubecco (DMEM, do inglês: Dulbecco′s Modified Eagle′s Medium), suplementado com 10% de soro bovino fetal, 1% de penicilina (m/v), sob condições estéreis a 37°C e 5% de CO2, com atmosfera umidificada.

4.2.9.2 Teste de redução do MTT (brometo de 3-(4,5-dimetil-2-tiazolil)-2, 5- difenil-2H-tetrazólio)

A viabilidade celular foi avaliada em células RAW 264.7 usando o teste MTT 49. As células foram cultivadas em microplacas de 96 poços com densidade de 7 x 103 células/poço e deixadas para aderir na placa em 100 ml de meio DMEM, a 37° C e

5% de CO2 por 12 h. Monocamadas confluentes nas microplacas foram incubadas com diluições seriadas das amostras das nanoemulsões (0,24 - 15,6 mg/ml) com ou sem peçonha do escorpião (18 µg/ml), mantendo a proporção nanoemulsão/peçonha. As amostras de nanoemulsões foram filtradas por filtração esterilizante com filtro de tamanho de poro de 0,22 nm. As placas contendo as nanoemulsões foram incubadas por 24 h a 37° C. Ao final da incubação, o meio de cultura foi aspirado e adicionado 100 ml de solução de MTT (259 mg/ml). Os cristais de formazan foram dissolvidos em 100 ml de etanol e a absorbância foi mensurada a 570 nm usando leitor de microplacas (Biotek®, Winooski, Vermont, EUA). A solução de PBS foi utilizada como controle negativo para 100% de viabilidade celular. Amostras controle e teste foram preparadas e incubadas em triplicata, repetindo a incubação três vezes. Os resultados foram comparados usando o teste estatístico ANOVA duas entradas e o pós-teste de Bonferroni, comparando os diferentes grupos de adjuvantes e também a comparação entre concentrações. As amostras foram consideradas estatisticamente iguais com valor de p > 0,05 e estatisticamente diferentes e representadas por * com valor de p < 0,05, ** para valor de p < 0,01 e *** para p < 0,001. 29

4.2.9.3 Teste de hemólise

Amostras de sangue A+ foram utilizadas após coletadas de doadores saudáveis humanos de banco de sangue. Cada 1 ml de sangue coletado com K3- EDTA (1 mg EDTA: 1 ml sangue) foi imediatamente utilizado para os experimentos. O sangue foi centrifugado a 400 X g por 10 min em centrífuga 5804 R (Eppendorf®, SP, Brasil). A camada de plasma e a série branca foram removidos depois da centrifugação. As hemácias foram novamente suspensas para 20% (v/v) com solução de PBS e centrifugadas a 700 X g para remover a fase aquosa, repetindo o processo mais duas vezes. As amostras dos adjuvantes não diluídos foram filtradas em filtro de 0,22 nm. Cada 1 ml das amostras dos adjuvantes foi colocado em tubos teste cônicos juntamente com 50 µL da suspensão de hemácias (20% v/v), atingindo concentração de 1% de hemácias. As amostras foram incubadas em triplicata por 1 h a 37° C. Todas as amostras dos adjuvantes foram preparadas com PBS para controle isotônico. Após a incubação, as amostras foram centrifugadas a 200 X g em centrífuga 5418 R (Eppendorf®, SP, Brasil) por 10 min, removendo cuidadosamente o sobrenadante para uma placa com 96 poços para medição da absorbância (ABS) em um leitor de microplacas (Biotek®, Winooski, Vermont, EUA) a 540 nm de comprimento de onda. O controle positivo para hemólise (100% de hemólise) foi uma solução a 1% (v/v) do tensoativo octilfenol etoxilado (Triton® X) e para controle negativo as hemácias foram incubadas com PBS. A percentagem de hemólise foi calculada de acordo com a seguinte equação:

Hemólise (%) = (ABS amostras – ABS controle negativo) * 100 / (ABS controle positivo – ABS controle negativo).

(Eq. 2)

Os resultados foram comparados usando o teste estatístico ANOVA uma entrada e o pós teste de Tukey, comparando os diferentes grupos de adjuvantes. As amostras consideradas estatisticamente diferentes foram marcadas com * para p<0,05, ** para p<0,01 e *** para p<0,001.

4.2.10 Imunização de camundongos

4.2.10.1 Animais

Camundongos Balb-C machos e fêmeas foram selecionados para os experimentos com idade entre 6-8 semanas e peso entre 25-35 g. Cada grupo de 30 animais foi dividido e acondicionado em gaiolas de propileno de 30x19x13 cm (1394 cm2) durante toda a fase experimental, com no máximo 5 animais por gaiola e 7 animais por grupo (3-4 animais por gaiola). Os animais foram mantidos em condições controladas de temperatura (22° C ± 2° C) e luminosidade (controle claro e escuro). Os camundongos foram alimentados com ração extrusada padrão (Presence®, adquirida da Agroline, Campo Grande, MS, Brasil) e água ad libitum, exceto no dia anterior à sedação, quando ficaram em jejum alimentar em overnight, sendo transferidos para a sala de experimentação uma hora antes dos testes para acondicionamento.

4.2.10.2 Protocolo de imunização

As amostras utilizadas para as imunizações foram filtradas por filtração esterilizante com filtro de tamanho de poro de 0,22 nm. A imunização foi realizada por injeção subcutânea de 100 µl na região dorsal dos camundongos das amostras dos diversos grupos, incluindo os adjuvantes com TSV (18 µg/ml) e sem TSV (controles); os adjuvantes ficaram sob acondicionamento a 4°C durante as diversas administrações. As imunizações foram realizadas seis vezes, uma vez a cada 7 dias. Os grupos foram divididos de acordo com os adjuvantes, nanoemulsão (NE), nanoemulsão com PEI (NE-PEI), hidróxido de alumínio (AlOH3), e a associação destes com o TSV a 9 e 18 µg/ml, com 6 animais por grupo. Ao fim das seis semanas, os animais foram sedados com cetamina 10% (30 mg/Kg) e xilazina 2% (300 mg/Kg), sendo submetidos a eutanásia com punção sanguínea cardíaca. Após a coleta de sangue, as amostras foram incubadas por 30 min a 37 ° C e por 2 h a 4 ° C para retração do coágulo. Logo em seguida, as amostras foram centrifugadas três vezes a 15000 x g por 5 min a 4° C para remover as hemácias. O soro foi armazenado a -20° C até o momento da titulação da IgG antipeçonha pelo ensaio de imunoabsorção enzimática (ELISA).

4.2.10.3 Titulação de IgG por ELISA

A IgG antipeçonha dos animais imunizados foi titulada por ELISA. Primeiro, uma placa com 96 poços para ELISA foi revestida incubando com uma solução de TSV 1 µg/100 µl por poço overnight a 4° C em câmara úmida. A placa foi então lavada três vezes com PBS e bloqueada com 200 µl por poço de solução de albumina bovina sérica (BSA 5% m/v/PBS) com incubação de 2 h a 4° C em câmara úmida. Posteriormente, a placa foi lavada três vezes com PBS/polissorbato 20 a 0,05%, 31 adicionando em seguida o soro dos animais imunizados contendo os anticorpos primários antipeçonha em diluições seriadas desde 1:100 a 1:25600 com BSA (0,1% m/v) / PBS, incubando por 1 h a 4° C em câmara úmida. Em seguida, a placa foi lavada por três vezes com PBS / polissorbato 20 a 0,05% e então adicionados 100 µl de anticorpos marcados anti-IgG anti-camundongos, incubando por uma hora a 37°C em câmara úmida. Em seguida, a placa foi lavada três vezes com PBS / polissorbato 20 a 0,05% e revelada com 50 µl por poço de substrato para peroxidase. Por fim, a placa foi incubada no escuro por 15 min e foi adicionada a solução de parada contendo 50

µl por poço de H2SO4 4 N. Os resultados da absorbância foram obtidos em um espectrômetro leitor de placas Biotek®, Winooski, Vermont, EUA) a 490 nm. Os títulos foram calculados como a máxima diluição em que a densidade óptica das amostras de animais imunizados com o TSV foi, no mínimo, duas vezes superior a densidade óptica dos respectivos controles com os adjuvantes. Os resultados dos títulos foram comparados com o teste estatístico ANOVA uma entrada e pós-teste de Tukey entre os diferentes grupos. As amostras consideradas estatisticamente diferentes foram marcadas com * para p<0,05, ** para p< 0,01 e *** para p< 0,001.

4.2.11 Aspectos éticos da pesquisa

O uso científico da peçonha TsV foi aprovado pelo conselho de gestão do patrimônio genético (CGen), processo número 010843/2013-2, registrado no sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SisGen, processo N°AAF17D9). Os ensaios de hemólise foram realizados de acordo com a declaração de Helsinki e de acordo com o protocolo de pesquisa aprovado de n° 2.809.485 pelo comitê de ética em pesquisa com humanos do hospital Onofre Lopes da Universidade Federal do Rio Grande do Norte (HUOL/UFRN). O protocolo experimental dos ensaios envolvendo o uso de camundongos foi aprovado pela Comissão de Ética no Uso de Animais (CEUA) da Universidade Federal do Rio Grande do Norte, com o número 027.040/2017. Foram seguidas ainda diretrizes gerais do Conselho Nacional de Controle de Experimentação Animal (CONCEA) e das diretrizes internacionais para a investigação biomédica envolvendo animais do Conselho de Organizações Internacionais de Ciências Médicas (CIOMS).

5 ARTIGOS PRODUZIDOS

5.1 - O artigo ‘’Self-assembled cationic-covered nanoemulsion as a novel biocompatible immunoadjuvant for antiserum production against Tityus serrulatus scorpion venom’’ foi publicado no ano de 2020 com autoria principal, no periódico Pharmaceutics, ISSN: 1999-4923, que possui fator de impacto 4,421 com Qualis A1 da CAPES para área Medicina II.

5.2 - O artigo ‘’Adjuvant use and immunization protocols for anti-venom production against scorpion venoms’’ foi redigido com autoria própria, com proposta de submissão para o periódico Toxins, ISSN: 2072-6651, que possui fator de impacto 3,76 e Qualis A2 da CAPES para área Medicina II.

5.1 Produção de artigo científico

Article

Self-Assembled Cationic-Covered Nanoemulsion as A Novel Biocompatible Immunoadjuvant for Antiserum Production Against Tityus Serrulatus Scorpion Venom

Arthur Sérgio Avelino de Medeiros 1, Manoela Torres-Rêgo 1,5, Ariane Ferreira Lacerda 1, Hugo Alexandre de Oliveira Rocha 2, Eryvaldo Sócrates Tabosa do Egito 1, Alianda Maira Cornélio 3, Denise V. Tambourgi 4, Matheus de Freitas Fernandes-Pedrosa 1,* and Arnóbio Antônio da Silva-Júnior 1,*

1 Laboratory of Pharmaceutical Technology and Biotechnology, Department of Pharmacy, Federal University of Rio Grande do Norte-UFRN, Natal-RN, 59010-180, Brazil; [email protected] (A.S.A.d.M.); [email protected](M.T.-R.); [email protected] (A.F.L.); [email protected] (E.S.T.d.E.) 2 Department of Biochemistry, Federal University of Rio Grande do Norte-UFRN, Natal-RN, 59010-180, Brazil; [email protected] 3 Department of Morphology, Federal University of Rio Grande do Norte-UFRN, Natal-RN, 59010-180, Brazil; [email protected] 4 Laboratory of Immunochemistry, Butantan Institute, Av. Vital Brasil, 1500, São Paulo-SP, Brazil; [email protected] 5 Graduate Program of Chemistry, Chemistry Institute, Federal University of Rio Grande do Norte, Avenue Senador Salgado Filho, 3000, Lagoa Nova, Natal, 59072-970, Brazil * Correspondence: [email protected] (M.d.F.F.-P.); [email protected] (A.A.d.S.-J.); Tel.: +055-84- 33429820 (M.d.F.F.-P. &. A.A.d.S.-J.); Fax: 055-84-33429833 (M.d.F.F.-P. &. A.A.d.S.-J.)

Received: 5 August 2020; Accepted: 7 September 2020; Published: date

Abstract: This study assesses the efficacy of different nanoemulsion formulations as new and innovative adjuvants for improving the in vivo immunization against the Tityus serrulatus scorpion venom. Nanoemulsions were designed testing key-variables such as surfactants, co-solvents, and the influence of the temperature, which would be able to induce the phase transition from a liquid crystal to a stable nanoemulsion, assessed for four months. Additionally, cationic-covered nanoemulsion with hyper-branched poly(ethyleneimine) was prepared and its performance was compared to the non- cationic ones. The physicochemical properties of the selected nanoemulsions and the interactions among their involved formulation compounds were carefully monitored. The cytotoxicity studies in murine macrophages (RAW 264.7) and red blood cells were used to compare different formulations. Moreover, the performance of the nanoemulsion systems as biocompatible adjuvants was evaluated using mice immunization protocol. The FTIR shifts and the zeta potential changes (from −18.3 ± 1.0 to + 8.4 ± 1.4) corroborated with the expected supramolecular anchoring of venom proteins on the surface of the nanoemulsion droplets. Cell culture assays demonstrated the non-toxicity of the formulations at concentrations less than 1.0 mg/mL, which were able to inhibit the hemolytic effect of the scorpion venom. The cationic-covered nanoemulsion has shown superior adjuvant activity, revealing the highest IgG titer in the immunized animals compared to both the non-cationic counterpart and the traditional aluminum adjuvant. In this approach, we demonstrate the incredible potential application of nanoemulsions as adjuvants, using a nanotechnology platform for antigen delivery system on immune cells. Additionally, the functionalization with hyper-branched poly(ethyleneimine) enhances this recognition and improves its action in immunization.

Pharmaceutics 2020, 12, 927; doi:10.3390/pharmaceutics12100927 www.mdpi.com/journal/pharmaceutics 34

Keywords: Tityus serrulatus antiserum; nanoemulsion adjuvant; liquid crystal; phase transition; surface functionalization; immunoadjuvant

1. Introduction

Scorpion sting is the most common and primary cause of death caused by accidents with venomous animals in tropical and sub-tropical countries 50. In Brazil, the most severe accidents caused by scorpion occurs with the species Tityus serrulatus 51. On the other hand, the antiserum therapy is the only approved and available treatment for accidents with venomous animals such as spiders, snakes, and scorpions. These immunotherapeutic products are produced by the immunization of horses, by injecting the specific venom associated with a suitable traditional adjuvant. However, most of the antibodies presented in these antiserum products are non-specific against the toxins from venom animals, and they may cause side effects and present low potency. Tityus serrulatus venom (TsV) presents a complex and rich composition, which includes oligopeptides, mucopolysaccharides, proteins, nucleotides, amino acids, and a vast variety of peptides. Some of these compounds are capable to modulate ionic channels such as Na+, K+, Ca2+, and Cl-, with enzymes activity, mainly neurotoxins 52,53. The crude venom can be easily dispersed in water or saline solutions, preserving the stability of their ionic peptides, which can be purified and the enzymes can be separated by size exclusion or ion exchange chromatography 54. Thus, the use of adjuvants able to control the release of TsV can offer several benefits in the immunization. The peptides and proteins can be anchored on the surface of particles by electrostatic and hydrophobic interactions 10,55,56. Traditional adjuvants have been used for centuries in vaccines and for anti-serum production for improving the adaptive immune response against antigens of harmful pathogens and toxins. However, although the list of diseases that are included in the immunization programs of the World Health Organization (WHO) is quite extensive 57, the number of approved adjuvants for human use is very limited. The adjuvants most used include aluminum salts, emulsions, liposomes, and agonists for toll- like receptor (TLR) 58,59. Among them, the aluminum-based salt was the pioneer compound used as an adjuvant for human vaccines 26,59. However, it presents major drawbacks such as lack of specificity of its elicited immune response, safety problems, and side effects. These limitations are generally related to the physicochemical or biological properties of the aluminum-based salt adjuvant. Additionally, a suitable antigen-release rate is desired for improving the antigen-specific cellular and humoral response 19. Nanotechnology platforms, such as nanoparticles, nanoemulsions, liposomes, and virus-like particles 19,59, can be perfectly used to solve the drawbacks related to the adjuvants. Nanoemulsions and nanoparticles have proven to enhance antigen recognition by immune cells 14,60. These systems potentially increase the adjuvant efficiency regarding to the expected immune response. Specifically, the nanoemulsions can induce a Th1 response due to the enhanced CpG agonist of toll like receptor (TLR) 60 and glucopyranosyl lipid 61, both agonists adjuvants that induces Th1 responses for influenza and Ebola vaccines, respectively. Particle-based adjuvants can induce the innate immunity activation through patterns receptor recognition (PRRs), which are receptors that recognize the secondary danger- associated signals. This pattern of response was previously demonstrated for aluminum particles and MF59 nanoemulsions 20. Not only the reduced and uniform sized, but also the oil type in nanoemulsions affects adjuvant efficiency 28. The shape of the nanocarriers also affects the recognition by the antigen- presenting cells (APC) and the clearance by the lymph nodes 21. The hydrophobicity and charge of the particle-based adjuvants also modulate the antigen adsorption, controlling its absorption, residence time and the innate immune activation 19–21. Cationic charged nanoemulsions have demonstrated improved cell recognition and mucous adhesion compared to neutral and anionic counterparts. Moreover, the performance of the cationic nanoemulsion can also be improved by changing its composition, specifically its amphiphilic compounds 22. Among the licensed nanotechnology-based vaccines for human use, the MF59 (Novartis) squalene nanoemulsion demonstrates a broad immune response 62. Some previous studies have also reported the use of other 35 nanoemulsion-based adjuvants 20,22,62–64. However, the performance of these nanocarriers has not yet been demonstrated for antiserum production. Considering the association of surfactant with oil in an aqueous environment, different oil in water (O/W) nanocarriers can be prepared, such as for example microemulsions, nanoemulsions, and liquid crystals. Microemulsions and nanoemulsions are isotropic submicron emulsions generally formed by oil droplets stabilized in an aqueous phase by surfactants. The first is a transparent and thermodynamically stable colloidal dispersion due to the large concentration of surfactants, while the second is stabilized by lower concentration of surfactants, forming slight turbid colloidal dispersions and thermodynamically unstable 40,65,66. Liquid crystals (LC) are viscoelastic mesophases formed for arranged surfactants in liquid medium, which can load considerable amount of oil due to their alternated hydrophilic and lipophilic domains [26]. The LC are anisotropic systems, with exception of the cubic phases, which are isotropic and has high viscosity. The self-assembly of surfactants can be induced by changes in the temperature or solubility in the medium, forming thermotropic or lyotropic LCs, respectively 67,68. Nanoemulsions can be prepared using high energy methods, like ultrasonication or high-pressure homogenization 69. Strong disruptive forces are provided from mechanical devices, which break up large droplets into nano-sized oil droplets stabilized in water by suitable amount of surfactants 3435. Besides the several advantages presented by these methods, their scale-up process involves many difficulties to reproduce the high input of energy in the emulsification process. Thus, low energy methods can be a useful alternative to solve this problem and produce ultrafine nanoemulsions. This strategy involves self-assembly of compounds by using phase inversion methods, spontaneous emulsification, and phase transition methods. 70. The phase transition was reported as an interesting approach for preparing nanoemulsions by simple dilution of microemulsions with water altering the surfactant arrangement in the oil/water interface 38, from transitional bicontinuous structures 39. In anterior studies, we have reported a similar strategy to produce biocompatible O/W nanoemulsions from suitable dilutions of lyotropic lamellar LCs 71. According the type of the used surfactant, the phase transition strategy can incorporate concepts of phase inversion temperature (PIT), phase inversion composition (PIC), and catastrophic phase inversion (CPI). The PIT method can generate small and uniform droplets by changes of spontaneous curvature of some non-ionic surfactants, induced by temperate-dependent dehydration. Liquid crystalline or bicontinous structures are formed when a near zero surfactant curvature is observed, which can changed by rapid cooling or heating steps forming kinetically stable nanoemulsions 69,72. A similar transition can be induced by the PIC method, in which the solubility of surfactants and consequently their curvature can be changed by gradual addition of continuous phase, with specific composition 73. The CPI method is similar to PIC method, but the second does not induces changes in the curvature of surfactant, but changing the ratio of dispersed phase until a critical point, in which the dispersed phase became the continuous phase (phase inversion) 27,74. Also, nanoemulsions can be formed by the spontaneous emulsification, which is favored by rapidly diffusion of surfactant and/or co-solvent molecules from dispersed phase to continuous phase, leading to formation of nano-sized droplets as the diffusion occurs 75,76. In the present study, different preparation methods were explored to produce stable nanoemulsions to be used as adjuvants for anti-serum production for the treatment of the Tityus serrulatus scorpion sting. Distinct compositions and formulation approaches, including the cationic nanoemulsions covered with the hyperbranched polyethyleneimine were tested, an FDA approved biocompatible material capable to enhance nucleic acids, genes and drugs delivery 42–44. The physicochemical properties of all formulations were carefully followed aiming to establish a nanotechnology platform for this purpose. Moreover, the in vitro studies in cell cultures and the in vivo experiments with BALB-C mice were performed for different formulations. Different nanocarriers are described in the literature and some of them are commercially available as adjuvants. However, the traditional approved adjuvant aluminum hydroxide Al(OH)3 is the most used for antiserum production against venomous animals, which was selected as the control in this study. 2. Materials and Methods 36

2.1. Materials

The venom from Tityus serrulatus scorpion (TsV) was donated by the Instituto Butantan, SP, Brazil. TsV was extracted from adult specimens of scorpions, then, lyophilized and stored at −20 °C before use. TsV solutions were prepared with PBS at the time of use. The amount of TsV was expressed by protein content, quantified by the bicinchoninic acid assay (BCA) using bovin serum albumin (BSA) as standard. The scientific use of the TsV was approved by the Brazilian Genetic Heritage Management Council (CGEN) (Process number 010843/2013-2) and registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen, Process N°AAF17D9). The medium- chain triglyceride (MCT) (Miglyol®812, Sasol®, Hamburg, Germany) was used as oil phase. The soy phosphatidylcholine (SPC) (purity > 95%) was purchased from Avanti Polar Lipids® (Alabaster, AL, USA). The polysorbate 80 (T80), Tween® 80, glycerol, propylene glycol, and octylphenol ethoxylate (Triton®) were purchased from Sigma-Aldrich Co. (St. Louis, Missouri, USA). N-methyl-pyrrolidone (99%) was purchased from Vetec (Duque de Caxias, RJ, Brazil) and used as co-solvent (CO). Poloxamer 188 and poloxamer 407 were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA.). Poly(ethyleneimine) hyper-branched 25,000 Da (PEI) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). PBS buffer (pH = 7.4) was prepared with the following constituents: 137 mM NaCl, 3 mM KCl, 15 mM KH2PO4, 10 mM Na2HPO4. The purified water (0.1 μS/cm) was prepared using reverse osmosis purification equipment, Model OS50 LX, Gehaka (SP, Brazil). 2.2. Design of Nanoemulsions and Liquid Crystals

2.2.1. Preparation of Emulsions

Initial trials were performed using different surfactants to stabilize the MCT, as the oil phase, in water. Emulsions containing 10% of MCT oil phase, 5% of different surfactant blends and 85% of purified water (10:5:85, w/w/w) were prepared by using the phase inversion emulsification. The aqueous phase was added to the oil phase composed of the surfactants dispersed in the MCT oil in a thermostatic bath stabilized at 70 ± 2 °C, under magnetic stirring at 360 rpm. The composition of the surfactant blend was adjusted using three distinct hydrophilic surfactants, polysorbate 80 (T80), poloxamer 188 (PO188), poloxamer 407 (PO407), and the hydrophobic surfactant, soybean phosphatidylcholine (SPC). The surfactants were also tested in pairs, such as SPC:T80, SPC:PO188, and SPC:407 at several w/w ratios. All formulations were observed for 7 days and any instability, such as creaming, and phase separation, was recorded. The creaming index was calculated according to the following equation:

CI % = 100 X Hu/HT (1) where CI % is the percentage of the creaming index, Hu is the height of the upper phase of the emulsion and HT is the total height of the emulsion placed in the test tube. The turbidity of the diluted samples with purified water (1:20 v/v) was measured at 860 nm, using a 1 cm3 glass cell in a UV/Visible spectrophotometer (Thermo Scientific, EvolutionTM 60S, In the main text, United States). The suitable composition able to induce the formation of stable emulsion with lowest creaming index and highest turbidity of the aqueous phase was selected for assessing the effect of the co-solvent. The addition of 10% w/w of co-solvent at the oil phase was evaluated to prevent phase separation, as we previously studied this effect 40 and considering the co-solvent influence at nanoemulsions formation and stability 77. Three different co-solvents were tested, such as the N-methyl-pyrrolidone (NMP), glycerol, and propylene glycol. 2.2.2. Pseudo-Ternary Phase Diagrams (PTPDs)

The mixture composed of SPC, T80, and NMP (3:1:3 w/w/w) was used as surfactant mixture (SM) selected from initial trials with surfactant blends and co-solvent amount to disperse oil/surfactant mixture. The surfactant mixture was mixed with the oil phase (SM/oil) at a ratio ranging from 0.1 to 0.9 w/w. To build the first PTPD the SM/oil phase was titrated with purified water for the emulsion production, under magnetic stirring at 360 rpm for 10 min, at 70 ± 2 °C. 37

A second PTPD was constructed, in which the mixture composed of SPC, T80, and NMP (6:2:15 w/w/w) was used as the surfactant mixture (SM), and, then, mixed with the SM/oil phase at the ratio ranging from 0.1 to 0.9 w/w. The aqueous phase was dripped (3 mL/min) into the oil phase at 25 ± 2 °C under magnetic stirring at 360 rpm for 10 min. An additional stirring was performed using the ultra- turraxT25 (IKA®, Staufen, Germany) at 11,000 rpm for 10 min. 2.2.3. Droplet Size and Zeta Potential Measurements and Identification of Liquid Crystals

The mean droplet and size distribution (polydispersity index, PdI) were measured by dynamic light scattering (DLS) using Zetasizer Nano (Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, England) at 659 nm with a scattering angle of 90°, at 25 °C. The tested nanoemulsion formulations were diluted at 1:100 (v/v) with purified water to ensure dispersions within suitable experimental range (100– 500 kcps). The zeta potential of the particles was measured by laser Doppler anemometry using the same apparatus. The data were expressed as mean ± standard deviation (SD). 2.2.4. Polarized Light Microscopy (PLM) and Identification of Liquid Crystals

Samples were examined under cross-polarized light microscopy. The Leica DM 750 (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a digital camera was used to analyze several fields of each sample at room temperature. The gel-like samples were selected for the polarized optical microscopy to observe any anisotropic behavior. All samples were placed on a glass slide and covered with a coverslip. Then the slide was placed on the microscope stage and polarized light was passed through the sample and then, the images were captured. The isotropic or anisotropic behavior of the samples was observed in the images taken at 100× and 400× magnification. 2.2.5. Preparation of Cationic Hyper-Branched Poly(Ethyleneimine) (PEI) Covered Nanoemulsions

The gel-like samples identified as liquid crystal (anisotropic system) were subjected to a controlled dilution to obtain stable and small droplet-sized nanoemulsions (isotropic systems). For the cationic- PEI nanoemulsions (NE-PEI), the liquid crystal (2.0 g) was dripped with 4.54 g of PEI aqueous solution (0.029% w/w) at a flow rate of 3 mL/min for a final PEI content of 0.02% w/w. The studied PEI content was varied related to the major interface component, soybean phosphatidylcholine (SPC), with different PEI:SPC ratio ranging from 1:1000 to 1:170 (w/w). The gel-like samples were diluted under magnetic stirring of 375 rpm for 20 min, at 25 ± 2 °C. The distinct composition of nanoemulsions and gel-like samples are presented in Table 1. 2.3. Physical Stability of NE Formulations Against pH and Saline Content

The prepared gel-like samples, the nanoemulsion (NE) and the PEI nanoemulsion (NE-PEI) were stored in hermetically sealed test tubes at 25 °C and the colloidal aspect, the droplet size, and zeta potential were recorded at specific time intervals. The selected gel-like sample identified as liquid crystal was prepared using PBS buffer pH = 7.4 as the aqueous phase. This aqueous phase was dripped (3 mL/min) into the oil phase at 25 ± 2 °C under magnetic stirring at 360 rpm for 10 min. An additional stirring was performed in the ultra-turrax at 11,000 rpm for 10 min. To obtain the NE, the liquid crystal (2.0 g) was dripped with 4.54 g of PBS buffer solution (pH = 7.4) and for NE-PEI the liquid crystal was dripped with PBS buffer solution (pH = 7.4) containing PEI at 0.016 mg/mL under magnetic stirring of 375 rpm for 20 min, at 25 ± 2 °C. In addition, the NE was also prepared with buffer aqueous phase with pH adjustments in acidic condition (pH = 5.5) and alkaline condition (pH = 8.5) in order to evaluate the robustness of the formulation in such conditions using HCl and NaOH 0.1 M for pH adjustments. The stable formulations produced with PBS pH = 7.4 was used for further experiments to evaluate the physical-chemical properties and to perform the in vitro and in vivo experiments. 2.4. Preparation of TsV-Loaded Nanoemulsions

A stock solution of the crude TsV was prepared at 1mg/mL in PBS solution (pH = 7.4), dispersing 5 mg of TsV in 5 mL of PBS, and the protein content was analytically quantified by the BCA protein assay kit as the manufacturer’s recommendations (Thermo Fisher Scientific, Waltham, Massachusetts, USA). In this experiment, the total protein content was determined by copper reduction in alkaline 38 medium, forming a complex with BCA that absorbs at 560 nm. Aliquots of these TsV stock solutions were added to the NE (NE-TsV) or to the NE-PEI (NE-PEI-TsV) to give the final TsV concentration of 0.018 mg/mL. In order to attain adsorption equilibrium, the samples were remained on magnetic stirring at 375 rpm for 1 h, in ice bath. The samples remained stored at 4 °C for six weeks and the colloidal aspect, the droplet size, and the zeta potential were recorded at specific time intervals. 2.5. Fourier Transformed Infrared Spectroscopy (FTIR)

The FTIR spectra of different samples were recorded in an IR Prestige-21 spectrum (Shimadzu ®, Tokyo, Japan). One droplet of the liquid NE was spread and analyzed by ATR-FTIR. The wavenumber range of 4000–1000 cm−1 was analyzed, taking 16 scanning runs with 1 cm−1 of resolution. 2.6. Cell Viability and Hemolysis Tests

2.6.1. Cell Culture

The murine macrophage RAW 246.7 (ATCC TIB-71) cells were gently supplied by the Laboratory of Biotechnology of Natural Biopolymers, Department of Biochemistry, Federal University of Rio Grande do Norte, Brazil. The cell cultures were maintained in Dulbecco’s modified eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, under sterile conditions, at 37 °C, with 5% CO2 and a humidified atmosphere. 2.6.2. MTT Reduction Assay

The cell viability assay was evaluated in the RAW 264.7 cell line using the MTT (3-methyl-[4-5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay reduction assay 49. The cell line was seeded in 96-well microplates at the density of 7 × 103 cells/well with 100 μL of DMEM medium, at 37 °C and 5% CO2, overnight. Confluent cell-monolayers contained in 96-well plates were incubated with serial dilutions of blank (PBS) and TsV-loaded NE samples (15.6 to 0.24 mg/mL of NE). The plates were incubated for 24 h at 37 °C. Thereafter, the growth medium was aspirated, and the plate incubated with 100 µL of MTT solution (259 mg/mL). The purple formazan crystals were dissolved in 100 µL of ethanol and the absorbance measured at 570 nm, using the Epoch 2 microplate spectrophotometer (Biotek®, Winooski, Vermont, USA) 49. The PBS solution was used as a negative control for 100% of cell viability. Control and test samples were assayed in triplicate and the assay was repeated three times. The results were compared using two-way ANOVA statistical analysis and Bonferroni post-test, comparing different groups of adjuvants and, also, their different concentrations. Samples were considered non- statistical different when p > 0.05. The samples considered statistically different was marked with * for p < 0.05, ** for p < 0.01 and *** for p < 0.001. 2.6.3. Hemolysis Test

Freshly collected A+ blood samples from healthy human donors from a blood bank were used, in accordance with the declaration of Helsinki and approved protocol 2.809.485 in the Ethics Committee of Research with Humans of the Onofre Lopes Hospital from the Federal University of Rio Grande do Norte (HUOL/UFRN). Each 1 mL of blood collected in K3EDTA (1.5 mg EDTA: 1 mL blood) was immediately used for the experiments. The blood was centrifuged at 400× g for 10 min in a centrifuge 5804 R model (Eppendorf ®, São Paulo, Brazil). The plasma and white blood cell layers were removed after centrifugation. The red blood cells were then suspended to 20% suspension (v/v) with PBS solution, and centrifuged at 700× g to remove the aqueous phase, repeating the process two more times until clear red blood cell suspension was obtained. Each 1 mL of adjuvant samples were placed in the Eppendorf tubes plus 50 µL of red blood cells suspension 20%(v/v)) for a final concentration of 1% of red blood cells. The samples were incubated in triplicate for 1 h at 37 °C. All the adjuvant samples were solubilized in PBS to isotonic control. After incubation, the samples were, then, centrifuged at 200× g in the Eppendorf centrifuge 5418R model for 10 min and the supernatant was carefully removed to a 96-well plate and analyzed in the Epoch 2 microplate spectrophotometer (Biotek®, Winooski, USA) at 540 nm wavelength. The considered positive control was an octylphenol ethoxylate solution of 1% (v/v) and the 39 negative control was PBS. The hemolysis percentage was calculated according to the following Equation (2):

Hemolysis (%) = (ABSsamples–ABSnegative control) *100/(ABSpositive control–ABSnegative control) (2) The results were compared using one-way ANOVA statistical analysis and Tukey’s multiple comparison test, comparing different groups of adjuvants. The samples considered statistically different was marked with * for p < 0.05, ** for p < 0.01 and *** for p < 0.001. 2.7. In Vivo Immunization Performance

2.7.1. Animals

Male and female BALB/c mice, with 6-8 weeks old and weight of 25–35 g were selected for the experiments. The experimental protocol (approval code 027.040/2017) was in accordance with the Brazilian College of Animal Experimentation (Brazil, 2008), and approved by the Ethics Committee of Animals Use in Research (CEUA) of the Federal University of Rio Grande do Norte (UFRN). Each animal group were divided in 30 × 19 × 13 propylene cages (1394 cm2) during the experiments with maximum 5 animals per cage and 7 animals per group (3–4 animals per cage). The animals were maintained at controlled conditions of temperature (22 ± 2 °C) and luminosity (light and dark control). The animals were feed with free water and food intake (ad libitum), except at the day of sedation, in which they were fasted overnight and transferred for experimentation room one hour before tests for conditioning. 2.7.2. Immunization Protocol

The immunization was performed by subcutaneous injection of 100 µL of scorpion venom at the mice dorsal region with selected adjuvants, previously stored at 4 °C, six times, once a week. The immunizing groups included NE, NE-PEI and Al(OH)3 (0.1% w/v) adjuvants and their associations with TsV at 9 µg/mL and 18 µg/mL, with seven (n = 7) animals per group. At the end of six weeks, the animals were sedated with ketamine 10% w/v (30 mg/kg) and xylazine 2% w/v (300 mg/kg) and then, euthanized with cardiac punction. After blood collection, the samples were stored for 30 min at 37 °C and for 2 h at 4 °C for clot retraction. Immediately afterward, the samples were centrifuged three times at 15,000× g for 5 min at 4 °C to remove the red blood cells using the centrifuge 5804 R model (Eppendorf®, São Paulo, Brazil). The serum was stored at −20 °C until the titration of antivenom IgG by the Enzyme- Linked Immunosorbent Assay test (ELISA). 2.7.3. IgG Titration by ELISA

The antivenom IgG titer of immunized animals was obtained by the ELISA titration. First, a 96 well plate for ELISA was coated with the crude TsV with 1 µg/100 µL per well, incubating overnight for 12 h at 4 °C in a wet chamber. The plate was, then, washed three times with PBS and blocked with 200 µL per well of bovine serum albumin (BSA 5% w/v)/PBS with 2 h incubation at 4 °C in a wet chamber. The plate was washed three times with PBS/polysorbate 20 0.05% and, then, the primary antibody from crude mice serum was added with serial dilutions from 1:100 to 1:25600 with BSA (0.1% w/v)/PBS, incubating for one hour at 4 °C in a wet chamber. The plate was washed three times with PBS/polysorbate 20 0.05% and then, 100 µL of marked anti-mice IgG was added and incubated for one hour at 37 °C in a wet chamber. The plate was washed three times with PBS/polysorbate 20 0.05% and then, revealed with 50 µL/well of peroxidase substrate. The plate was, then, incubated in the dark for 15 min and the stop solution containing 50 µL/well of H2SO4 4N was added. The absorbance results were taken in the Epoch 2 microplate spectrophotometer (Biotek®, Winooski, Vermont, USA) at 490 nm. The titers were calculated as the maximum dilution in which the optical density of samples from immunized animals with TsV was, at least, twice the optical density of animals immunized with adjuvant only. 2.8. Statistical 40

The titer results were compared using one-way ANOVA statistical analysis and Tukey’s multiple comparison test, comparing different groups of adjuvants. The samples considered statistically different was marked with * for p < 0.05, ** for p < 0.01 and *** for p < 0.001. 3. Results

3.1. Design of Nanoemulsions and Liquid Crystal

Several surfactant mixtures were evaluated to better stabilize the emulsion samples. Different compounds were tested. Polysorbate 80, poloxamer 188, and poloxamer 407 as hydrophilic surfactants, and soy phosphatidylcholine (SPC) as hydrophobic surfactant. Figure 1a,b show the creaming index and the turbidity of the emulsions, respectively, for the systems prepared by using the different surfactants pairs. The creaming index increase recorded from different samples represent an instability phenomenon. The turbidity of non-creamed phase (lower phase) was also measured to evaluate the ability of the tested surfactant pair to retain the oil dispersed in the system. With an increasing ratio of hydrophilic surfactants, the nanoemulsions became more unstable, with higher creaming index (Figure 1a) and less turbid aqueous phase (Figure 1b). Based in this initial trials, three systems, using SPC only, SPC:T80 (3:1 w/w) and SPC:PO407 (3:1 w/w) as surfactants, respectively, were selected as the most stable for conducting further studies. The addition of three co-solvents, such as N-methyl-pyrrolidone (NMP), glycerol, and propylene glycol, was tested to improve the stability of the nanoemulsions and, therefore, decrease the creaming phenomenon. However, NMP was the only one capable to dissolve the SPC surfactant. The initial trials of formulations revealed that the use of SPC only, as a surfactant, even associated with NMP was unable to producing stable nanoemulsions and the phase separation occurred rapidly. Figure S1 shows the effect of NMP addition on the creaming index and turbidity of selected nanoemulsions, demonstrating that the mixture of SPC:T80 (3:1 w/w) produced more stable system than SPC:PO407 (3:1), which was selected for further studies using the PPTD (Figure 1c). This assay was conducted under heating (70 ± 2 °C). The NMP was used as co-solvent at the SPC:T80:NMP 3:1:3 (w/w/w) ratio. Some points of o/w nanoemulsions containing considerable amounts of surfactants are observed in the PPDT (Figure 1 c), but no transparent or less turbid liquid crystal was observed. 41

Figure 1. Formulation studies for assessing the surfactant composition: SPC:T80, SPC:PO188, SPC:PO407. (a) Creaming index (CI) and Turbidity of different emulsions; (b) Images of each tested formulation; (c) Pseudo-ternary phase diagram using the SPC:T80 (3:1 w/w).

Consequently, the amount of NMP have to be enhanced in the mixture SPC:T80:NMP 6:2:15 (w/w/w), as a tentative to explore a second PPDT constructed without heating at 25 ± 2 °C (Figure 2a). Among the few points explored to produce O/W systems, it was observed not only emulsions and nanoemulsions, but also a less turbid gel like sample similar to a liquid crystal. The gel-like sample containing 2% w/w of oil phase was identified as an anisotropic phase. The Maltese crosses in Figure 2(bi) suggest a lamellar-like liquid crystal. The amount of used NMP was able to dissolve SPC at 25 oC without heating. The dilution of this sample with purified water formed liquid and translucent colloidal dispersions containing 0.9% w/w of oil phase Figure 2(b-ii). The same occurred when the dilution occurred with PEI aqueous solution Figure 2(b-iii). The completely dark polarized microscopy images confirmed the expected isotropic behavior for the nanoemulsions. It is interesting to observe that the amount of both surfactant and the co-solvent were half-reduced in the NE formulations. 42

Figure 2. Phase diagram constructed at 25 °C, followed by ultra-turrax homogenization (11,000 rpm). (a) Pseudo-ternary phase diagrams (PTPD). (b) Polarized microscopy images (left side—increased 400×) and visual aspect (right side) of (i) gel-like system, (ii) anionic NE and (iii) cationic NE-PEI.

The Table 1 shows the composition of systems presented in the Figure 2. The two formulations (NE and NE-PEI) were selected for TsV loading and further evaluation of their physicochemical properties and the in vitro/in vivo performance.

Table 1. Composition of the formulations.

Formulations Tsv- Ingredient Blank Tsv-Loaded Gel-Like Blank Loaded Amount Cationic Cationic System Anionic NE Anionic NE NE NE TsV (µg/mL) 0.0 0.0 18.0 0.0 18.0 MCT (% w/w) 2.00 0.9 0.9 0.9 0.9 T80 (% w/w) 4.55 2.0 2.0 2.0 2.0 SPC (% w/w) 13.65 6.0 6.0 6.0 6.0 NMP (% w/w) 34.2 15 15 15 15 PEI (% w/w) 0.0 0.0 0.0 0.02 0.02 Water (% w/w) 45.6 76.1 76.1 76.08 76.08 Notes: TsV=Tytyus serrulattus venom; MCT=medium chain triglycerides oil; T80=polysorbate 80; SPC=soy phosphatidylcholine; NMP=N-methyl-pyrrolidone; PEI=poly(ethyleneimine).

3.2. Physicochemical Properties of Anionic and Cationic NE Formulations

The same approach used to obtain the anionic NEs from the liquid crystal was used to produce the cationic NE-PEI. The selected PEI:SPC ratio have achieved a positive zeta potential in the tested formulations (Figure 3a). The experimental data revealed that the PEI:SPC = 1:300 w/w induced cationic- covered NE-PEI with zeta potential of approximately +10 mW with droplet size < 200 nm. Formulations with superior PEI:SPC ratio were not selected because the well-established toxicity of PEI or enhancement of the droplets size. The Figure 3a shows the data from physical stability assay, adequately presented in the Section 3.3. FT-IR spectra recorded for different NE formulations are shown in Figures 3c, 3d, 3e. The cationic covering NE-PEI reduced the intensity of the FTIR bands related to the anionic NE (Figure 3c). This 43 achievement suggests that PEI anchoring perturbs the surface of NEs. The same effect occurs for the TsV-loaded formulations (Figure 3d,e). In addition, it was possible to observe the bathochromic shift of the band recorded in the region of 1634 cm−1 in the NE for 1617 cm−1 after TsV-loading for both NE and NE-PEI. This fact suggests relevant intermolecular interactions among the proteins from TsV with PEI on the surface of NEs. Generally, the amide I C-N and amide II C-N stretch are recorded for proteins at approximately 1600–1700 cm−1 and 1500–1600 cm−1, respectively. 3.3. Physical Stability of NE Formulations Against pH and Saline Content

The NE formulations discussed in this study were designed as biocompatible adjuvants for ready venom-loading by adsorption on surface of droplets, as performed in antiserum production. Thus, the stability of TsV was not considered, but only the ability of nanocarrier resists to possible stress steps involved, such as possible ionic strength or pH changes. Since that SPC is an amphoteric surfactant and that the PEI affects the size of the NEs, the robustness of the NEs to resist pH adjustment and effect of PEI covering on the physical stability were both assessed for four months (Figure 3b). The pH adjustment of the NE formulations was performed using PBS with titrations of different amounts of HCl 0.1 M−1 or NaOH 0.1 M−1 aqueous solutions. The results revealed that the pH variation did not affected the NE stability. Table S1 shows detailed values of size, PdI, and zeta potential measured for studied formulations for entire interval (four months).The presence of PEI promoted an increase in droplet size from 125 to 175 nm, which dropped to 150 nm after the 3rd month. The decrease of droplet size of about 175 nm (after two months) to 150 nm (after three months) can be statistically different, but we have not considered a relevant change able to affect the physical stability of colloidal dispersions and its performance. This achievement is considered a ripening process, mainly considering the PEI equilibrium between the surface/interface of droplets and aqueous phase. After assessing the physical stability of different NE formulations, the TsV-loading was performed for NE and NE-PEI samples. These four formulations remained stored at 4 °C for six weeks and their physicochemical properties were also evaluated at the final interval, as showed in Table 2. The experimental data revealed that both the cationic character and the physical stability were preserved. Indeed, no considerable effect on the droplet size or zeta potential was observed. The PdI values of all nanoemulsions stored at 4 °C (Table 2) are lower than the PdI stored at 25 °C (Supplementary Table 1). This fact may be explained due to the expected changing in the solubility of surfactants at distinct temperatures, which affects the self-assembly behavior. However, the formulations preserved the uniform droplet size when stored at 4 °C. The Figure S2 illustrates some examples. In fact, for the NE- PEI stored for four months, the PdI also ranged from 0.22 to 0.18, values inferior to 0.3. The zeta potential of NE-PEI also slightly decreases over time due to any rearrangement of anchored PEI on the surface of NE, altering the PEI ratio dispersed in the aqueous phase. This achievement corroborates with the observed decrease in droplet size, after four months. 44

Figure 3. Effect of PEI covering and TsV-loading on the physicochemical properties of the NE formulations. (a) Effect of PEI content on the size and zeta potential (ZP); (b) Stability and robustness of NE from pH changes; c, d and e) Effect of the composition on the FTIR spectra of distinct NEs: (c) PEI addition; (d) NE-TsV loading; e) NE-PEI-TsV-loading.

Table 2. Droplet size and zeta potential measurements for different blank and TsV-loaded NEs stored in hermetically sealed vials at 4 °C, for six weeks.

Formulation Mean Size Pdi Zeta Potential (Mv) NE 125.7 ± 0.3 0.13 ± 0.03 −18.3 ± 1.0 TsV-loaded NE 126.3 ± 1.3 0.13 ± 0.01 −13.3 ± 0.6 NE-PEI 165.2 ± 0.5 0.13 ± 0.01 8.4 ± 1.4 TsV-loaded NE-PEI 167.1 ± 0.5 0.13 ± 0.01 5.04 ± 0.09

3.4. In Vitro Cell Viability and Hemolytic Tests

The TsV-loaded NE formulations have a final TsV concentration of 0.018 mg/mL, which make almost impossible to evaluate with precision the in vitro release behavior due to the small amount of venom. Thus, in vitro cell viability and hemolytic tests were performed for evaluating, indirectly, the different behavior profiles of the TsV release and their interactions with the cell in the biological medium. Moreover, comparisons among different TsV containing formulations and TsV alone were performed. The NE, NE-PEI and respective TsV-loaded formulations were subjected to the cell viability tests in the murine RAW 264.7 cell line (Figure 4a). The results revealed a dose dependent cytotoxicity. However, all NE formulations were biocompatible (no cytotoxic) in a concentration equal or less than 0.98 mg/mL, except for the NE-PEI. At high concentration (above 0.98 mg/mL), all formulations induced comparable cytotoxicity. 45

Since that hemolytic effect of TsV is well established in the literature, the ability of NEs to reduce or impairs this effect was also tested (Figure 4b). The hemolysis test confirmed the biocompatibility of both NE and NE-PEI formulations. In addition, comparisons of TsV-loaded NEs formulations with the same concentration of TsV alone (0.018 mg/mL) revealed an extremely low degree of hemolysis from the loaded formulations. This is an important finding, mainly considering the future in vivo use of these formulations.

Figure 4. (a) Cell viability test on RAW 264.7 cells against different formulation and TsV alone. (b) Hemolysis test on human red blood cells against different formulation and TsV alone. The comparisons were performed using the one-way ANOVA followed by the Tukey’s post-test. A p < 0.05 was represented as *, p < 0.01 as ** and p < 0.001 as ***. Note: TsV = 0.018 mg/mL for loaded nanoemulsions and TsV alone.

3.5. Antivenom IgG Titration

Figure 5 shows the antibody titer profile produced by the immunized animals with TsV using different adjuvants. The NE-TsV and NE-PEI-TsV were compared to the traditional aluminum hydroxide (Al(OH)3) adjuvant. Figure 5a shows the optical density, giving a general view of the specific IgG UV-Vis absorption. It is possible to observe that both NE and NE-PEI exhibited superior density of 46 immunoglobulins compared to the Al(OH)3 adjuvant, with greater response for the NE-PEI. The Figure 5b shows the titer measurements and statistics comparisons. The animals immunized with NE-PEI produced statistically higher IgG titer compared to that immunized with both NE and the Al(OH)3.

Figure 5. (a) Evaluation of the optical density profile from antibodies produced by mice immunized subcutaneously for 6 weeks with 0.0018 mg of TsV using the three tested adjuvants, (b) Antivenom IgG titer from mice immunized subcutaneously for 6 weeks with 0.0018 mg of TsV using the three tested adjuvants. The comparisons were performed using the one-way ANOVA followed by Tukey’s post-test. A p < 0.05 was represented as *, p < 0.01 as ** and p < 0.001 as ***.

4. Discussion

Stable self-assembled NEs can be produced by using a fine balance among the aqueous phase, oil phase, and surfactants. In this study, the SPC was mixed with different surfactants in an initial trial to produce a biocompatible blend able to stabilize MCT (oil phase) in water (Figure 1a) and produce a NE system. The systems prepared with polysorbate (T80) and two poloxamers (PO188, PO407) showed the lowest creaming index for mixtures composed of SPC:T80 at 3:1 w/w ratio and SPC:PO407 at 3:1 w/w ratio (Figure 1b). These compositions are interesting and promising for producing pharmaceutical emulsion systems. Indeed, the greater SPC amount (75% w/w) in the surfactant mixture certainly produces more biocompatible emulsion systems 71,78. However, produce self-assembled NEs using SPC is not an easy task. Some properties of the SPC, such as melting point, water solubility, and miscibility with oil phase (MCT) at room temperature make this a practically impossible task when low-energy emulsification methods are used. Thus, the co-solvent addition in the NE formulations have been described as an interesting and promising way to produce small-droplet sized NEs 79. Indeed, the co-solvent reduces the oil/water 47 interfacial tension and decrease the segregation grade among the specific non-polar compounds, improving the diffusion rate of surfactants to the aqueous phase, at the phase inversion moment 71,79,80. Therefore, the N-methyl-pyrrolidone (NMP) was used as a co-solvent in this study due to its ability to dissolve SPC and improve its miscibility in the oily phase of the NEs. NMP has a octanol/water partition coefficient of 1.21, which allows solubilizing several hydrophobic components using this solvent 81. In this study, the addition of NMP allowed to stabilize the NEs produced with SPC:T80 at 3:1 w/w ratio. Consequently, this composition was selected to build a second pseudo-ternary phase diagram (Figure 2a). The mixtures prepared with SPC:PO407 at 3:1 w/w ratio showed instability after the NMP addition. The pure SPC was also tested with NMP, but the instability represented by the phase separation remained as the main barrier. All the achieved NE formulations pointed out in the PPTD exhibited surfactant content superior to 30% w/w (Figure 1c). This amount of surfactant is an important obstacle to produce biocompatible and non-hemolytic NEs 71,79,80. In addition, these formulations showed PdI values > 0.4 (data non showed). In addition, no liquid crystal system was observed on the PPTD for the tested compositions. Some previous trials at our laboratory revealed that the temperature perturbed the droplet size and surfactant arrangement of lipid based dispersed systems (data non showed). Hence, the same experiment with the phase behavior diagram was performed at 25 °C. The second diagram revealed an interesting point, a translucent formulation with high viscosity due to its high surfactant content. (Figure 2a). This feature is uncommon to the NEs, but highly likely to be observed in lyotropic liquid crystal systems 71,79,80. This phenomenon was better characterized using polarized microscopy images (Figure 2b-i), which showed Maltese crosses, a characteristic of lamellar liquid crystal structures 82. In previous studies, our team has already assessed the phase transition of lyotropic lamellar liquid crystals as a strategy to obtain small and uniform droplet sized NEs 40,71. The self-assembly behavior of surfactants in the oil/water interface depends on a fine and limited phase equilibrium. An optimum water addition could induce phase transitions to NEs. The suitable dilution of the gel-like system identified in the second diagram corroborated to this assumption (Figure 2(b-ii)). Furthermore, this method produced small droplet sized NE formulations with remarkable low PdI values (Table 2). The continuous addition of water in the lamellar mesophases can produce intermediate bicontinuous microemulsions with final transition to the NEs 38,40. A similar approach considered a phase transition from cubic liquid crystals to NEs 82,83. The dilution of cubic liquid crystal also produced small droplet-sized NEs compared to other techniques, such as high strength or ultrasonication 82. Unfortunately, these authors diluted the cubic mesophases using the hexadecane as a co-solvent, which is not suitable for application in pharmaceuticals. Thus, the liquid crystal dilution technique presented in this study allows the production of NEs with desirable properties using pharmaceutical grade ingredients. Moreover, the final NEs produced by this approach were subjected to a careful physicochemical characterization for further TsV loading and use of such formulations in in vivo and in vitro studies. The pharmaceutical application of NEs as drug delivery systems relies on their ability for solubilizing drugs in the oil or o/w interface region. The rationale behind this study was to hypothesize that proteins such as the ones found in the TsV could be adsorbed on the NE surface by hydrophobic or electrostatic anchoring. Previous studies have reported the anionic character of TsV at physiological pH and the successful TsV entrapment into the cationic chitosan-based nanoparticles 84. Similar approaches with venom from different scorpion and snake species reported the venom-loading by adsorption on the cationic surface of nanoparticles 23–25. However, the zeta potential of the NE prepared by diluting the liquid crystal identified in the PPTD (Figure 2a-ii) was anionic (Table 2). This limitation was solved by adsorption of the cationic macromolecule PEI. A specific PEI aqueous solution was used in the dilutions, generating NE-PEI systems (Figure 2a-iii). The minimum PEI:SPC ratio able to induce stable cationic NE with a minimal increment on droplet size was carefully assessed (Figure 3a). The PEI:SPC ratio of 1:550 w/w produced NE with zeta potential close to zero, and droplet size of about 150 nm. The chosen PEI:SPC ratio of 1:300 w/w was able to produce translucent NEs (Figure 2b-iii). Similar approaches have demonstrated the use of polymers and macromolecules for stabilizing emulsion systems 85,86, but the high surfactant concentrations remains as the main barrier for their application as parenteral pharmaceutical emulsions. In this study, we found interesting NEs containing of approximately 8% 48 w/w of surfactant mixture (Table 1) in which 75% is SPC, a biocompatible surfactant used in parenteral nutrition formulations. Since that SPC is an amphoteric surfactant and that the PEI arrangement on the surface of NEs can be affected by the pH of medium, the formulations were subjected to a stability test performance using different aqueous media (Figure 3b). The cationic NE-PEI and anionic NEs were stable for the entire period interval of four months for all tested conditions, which included physiological PBS, acid, alkaline, and neutral pH. This assay has shown the robustness of the NE-PEI to be used for protein- loading, which in most cases requires some pH adjustment or dilution of the final formulation. Generally, the addition of saline solutions in the NEs induces droplet size increase because the expected increasing in the Debye length 87. In this study, it was observed a slight increase of about 25nm for the NE systems (Figure 3b). In addition, the NE-PEI sample presented a slight decrease in size and zeta potential after three months (Figure 3b and Supplementary Table 1). However, the PdI also ranged from 0.22 to 0.18. This fact suggests that anchored PEI on the surface of NE droplets is in a constant equilibrium with PEI dispersed in the aqueous phase, as a ripening process. Considering the TsV-loaded NEs, the anchored protein on the NE droplets also may be at balance with PEI-protein aggregates. It is interesting to reinforce that the venom storage as a biologic material is a limitation for long-term storage. Thus, the easily and simple preparing of NE and cationic-covered NE by diluting the LC followed by venom-loading for the intend application is a promising strategy considering the commercially available immunizing protocols. The TsV-loading maintained important physicochemical properties of stable and reproducible colloidal systems as small size and low PdI values (Table 2). Tityus serrulatus venom is a complex mixture of proteins, peptides, polysaccharides, and others. Their supramolecular binding on the surface of the PEI covered NE could allow a slow release of venom enzymes and antigens for a controlled and protective response of the immune system, as similarly reported for cationic nanoparticles 41. In this context, the interactions among the compounds of the formulations were further investigated by FT-IR spectroscopy (Figure 3 c-e). This experiment followed the additional covering of the anionic NE formulation identified in the second PPTD (Figure 2b). Thus, the Figure 3c shows the effect of the PEI covering on the NEs, reducing the intensity of their FT-IR bands. This fact can be better observed for the characteristic C=O stretch at 1634 cm−1 of the carbonyl presented in both the used surfactants and the MCT oil phase. The same effect was observed for the NE-TsV (Figure 3d). In addition, the primary amide bands could be observed in the range of 1600–1700 cm−1, such as that at 1600-1639 cm−1 for the β-sheet, as well as at 1651-1660 for the α-helix portion, and at 1661– 1700 cm−1 for the T-turns 88,89. Considering the TsV-loaded cationic covered NEs in the Figure 3e, the absorption band of the amide I of proteins from TsV can be seen at 1617 cm−1. The equivalent band was also highlighted at 1634 cm−1, the same observed for the anionic NE systems. This phenomenon suggests that a possible hydrophobic interaction with the β-sheet portion of the proteins from the TsV preserved the initial conformation. The hypsochromic shift from 1617 cm−1 to 1634 cm−1 is considered within β- sheet conformation range. In addition, the Figures 3c and 3d show a bathochromic shift of absorption bands from 1692 cm−1 to 1687 cm−1. These results corroborate to the expected T-turns conformation of some proteins. Summarizing, some conformational changes in the β-sheet and T-turns regions occurred after TsV-loading, but those changes remained within the same secondary fold region in the protein structure. Similar achievements were reported in the literature when the lysozyme was adsorbed on the surface of Fe2O3 nanoclusters 88. Thus, the experimental FTIR spectroscopy results provided valuable information to understand the interactions of TsV on the surface of NEs, mainly regarding the followed steps of PEI covering of NEs and additional TsV-loading. The cell viability experiments showed a dose dependent cytotoxic effect of the NE formulations (Figure 4a). This result highlights the statistical differences between venom solution (with extremely low concentration, 0.018mg/mL) with the formulations, which ranges of 0.24 to 15.6 mg/mL, which was 13 to 850 over superior to that used for TsV solution. It was not observed statistical differences between TsV-loaded NE and cationic-covered NE formulations. This fact suggests that the selected PEI:SPC at 1:300 w/w ratio for the NE-PEI did not increased the toxicity of the formulation compared to NE formulation itself. Moreover, at 3.9 mg/mL, the cell survival rate was about 60% and 50% for the NE and the NE-PEI, respectively. The respective TsV-loaded samples showed a trend of superior cell survival rates. This results also reinforces the importance of exploring the concentration range of PEI 49 able to supply an expected cationic charge, with a minimal cytotoxicity effect compared with non- cationic NE formulation. These results are promising for further in vivo evaluation of the selected formulations as adjuvants. We considered that NE formulations discussed in this study are suitable for in vivo application. Several reported studies with nanoemulsions subjected to viability studies revealed to be more cytotoxic or similar than that demonstrated in the present study 90–92. We recognize that it is not a comparison between formulations with only one variable. The cell lines and nanoemulsion composition should be considered as important variables. Since that nanoemulsions present oil droplets covered with surfactants in aqueous media, the cytotoxicity is generally connected with type and concentration of surfactants 93,94. Regarding the TsV activity, some physiological responses have been demonstrated in rats, such as dehydration, hemoconcentration, complement system activation, hepatic and lungs inflammation, and hemolysis 95,96. Thus, a hemolytic assay was conducted for all tested formulations (Figure 4b). Contrary to the cell culture experiments, the TsV have shown a considerably hemolytic effect at the same 0.018 mg/mL concentration previously used for the cell viability experiment. The Ca2+ influx was previously reported as the main mechanism responsible for the venom hemolytic activity, which activates phospholipases that act in the cell membrane 95. However, recent studies demonstrates that TsV does not activate phospholipases 97, suggesting an alternative mechanism that causes hemolysis. Nevertheless, both NE and NE-PEI were able to reduce this hemolytic effect referred to TsV. The experimental data suggests that a possible supramolecular aggregation of TsV on the NE surface avoids this harmful effect, an important feature for further in vivo experiments. Finally, the in vivo potential of the loaded NE and NE-PEI as adjuvants for antiserum production against TsV was evaluated and compared to the Al(OH)3 adjuvant. Figure 5a showed the evaluation of antibody profile for the animals immunized with each sample. Differences can be observed among the optical density observed for the three formulations. As hypothesized, this experiment corroborated to the expected improved adjuvant activity for the TsV-loaded NEs. A superior performance was observed for the NE and NE-PEI compared to the Al(OH)3 traditional adjuvant. (Figure 5a). The Al(OH)3 adjuvant is quite potent and used in considerable number of approved vaccines, which still has an adjuvant to be overcome. In addition, aluminum is the most common formulation for serum production in antivenom protocols. We can observe several studies in the literature in which the response of nanocarrier was not better that aluminum salt or should be associated with it to induce a similar response. In a previous study, we have demonstrated that chitosan NPs was capable to induce a statistically similar IgG titer to Al(OH)3, but not superior 84. Thus, not all nanocarriers are capable of enhance IgG titers, which for antivenon serum are essential for neutralizing the toxins. Nanocarriers as adjuvants may act, in some cases, stimulating the immune response by different mechanism other that immunoglobulins production. Therefore, the adjuvant response depends not only on the use of nanocarriers, but by on their size, shape, hydrophobicity, and charge of particles, which can be tailored. Additionally, Figure 5b shows that the NE-TsV induced a similar IgG titers response to the animals exposed to Al(OH)3. This result demonstrates the superior performance expected for the cationic NE- PEI compared to the anionic NE system. Since that aluminum salts are approved and are one of the most used adjuvants for antiserum production 98,99, the NE-PEI formulation address a promising and novel device to this purpose. In addition, the aluminum generally induces a major production of nonspecific antibodies against the toxins, causing several side effects 100. The larger IgG titers produced by the animals treated with NE-PEI-TsV suggests that this is a promising adjuvant against the harmful scorpion toxins, and, probably, is able to induce a more effective antivenom serum. The superior IgG titers of developed nanocarriers confirms its delivery effectiveness for venom immunogens, possibly increasing cell recognition or even cell uptake of these immunogens. Vaccine field may be benefitted of this type of nanocarriers adjuvants, as literature investigations demonstrated for immunogens and also nanocarrier association with immune agonists adjuvants to potentialize the effect associating two adjuvants mechanisms 14,60,61. The PEI-covered nanoemulsions with superior IgG stimulant production showed that this biocompatible polymer capable of enhance cell uptake of nucleic acids and drugs 42–44 also may enhance protein cell recognition and be applied as vaccine adjuvant. 50

A critical discussion about the anti-serum therapy is necessary to achieve new adjuvants able to induce an efficient immune response with minimal side effects 97. The aluminum salts were introduced as adjuvant in 1920s decade, and this class of compounds are used in most of 140 approved vaccines for the prevention of diseases 97. However, the inflammation problems associated to the aluminum salts as adjuvants are well reported in the literature, which include neurotoxicity. Furthermore, the majority of antibodies in antiserum produced by the immunization of host animals with traditional adjuvants are not capable to neutralize the venom toxins 97. Thus, the NEs platform discussed in this study provide biocompatible formulations able to be used as adjuvant for antiserum productions against TsV, with superior performance compared to the tested aluminum compound. Additionally, the experimental data corroborates that supramolecular aggregation of TsV on the surface of NE did not decrease the recognition of the epitopes. In fact, the increasing polyclonal IgG antibodies titers could improve antivenom serum efficacy and improve the performance of TsV, which could enable a possible lower TsV dose in the final adjuvant formulation. 5. Conclusion

For the first time, different formulations of self-assembled biocompatible NEs were designed as a delivery system for antigens of the Tityus serrulatus scorpion venom. Several NE compositions were carefully assessed using a PPTD approach. The dilution of the gel-like liquid crystal induced the formation of small droplet-sized and stable anionic nanoemulsions. The dilution with a specific and optimized PEI solution produced cationic covered NEs. The in vitro experiments showed that both anionic and cationic NEs were able of TsV-loading, which have shown low cytotoxicity and low hemolytic effect. Finally, the ability of NE systems as adjuvant was tested in Balb-C mice and compared to the traditional Al(OH)3 adjuvant. Possibly the NE systems increases the cell recognition or even cell uptake of these immunogens. The adjuvant performance of the cationic NE formulation was superior to that of the Al(OH)3 adjuvant. In addition, the antivenom IgG titer was considerably enhanced in animals immunized with TsV-loaded cationic-covered NE. In this study, we demonstrated that is possible to modulate the physical chemical properties of self-assembled NEs to induce different performances as adjuvants. As hypothesized, these colloidal nanocarriers are promising adjuvants for antiserum production against the Tityus serrulatus scorpion venom.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Effect of N- methyl-pyrrolidone (NMP) in association with mixtures of soy phosphatidylcholine (SPC) with The polysorbate 80 (T80) or poloxamer 407 (PO407) on the creaming index of emulsions of medium-chain triglyceride (MCT) in water, Figure S2: Droplet size distribution (zeta-sizer images) with respective PdI and zeta potential values assessed for the studied formulations, Table S1: Size, PdI, and Zeta potential measurements for different formulations, at distinct intervals of physical stability of NE formulations against pH and saline content.

Author contributions: A.S.A.M: Investigation, Methodology, Formal analysis, and Writing—Original Draft; M.T.R., A.F.L.: Investigation, Methodology, and Formal analysis; H.A.O.R.: Visualization and methodology; A.M.C: Visualization, writing-original draft; D.V.T: Visualization and methodology; E.S.T.E: Writing—Review & Editing, M.F.F.P and A.A.S.-J.: Conceptualization, Formal analysis; Writing—Review & Editing, Supervision, Project administration, Funding acquisition.

Funding: The Brazilian National Council for Scientific and Technological Development (CNPq) (grant numbers: 308382/2017-0, 436051/2018-4); The Coordination for the Improvement of Graduate Level (CAPES) (PNPD 23038.007487/2011-91, Financial code 001).

Acknowledgments: The authors wish to thank the Butantan Institute, São Paulo, Brazil.

Conflicts of Interest: The author reports no conflicts of interest in this work.

Supplementary Materials 51

Self-Assembled Cationic-Covered Nanoemulsion as A Novel Biocompatible Immunoadjuvant for Antiserum Production Against Tityus Serrulatus Scorpion Venom

Arthur Sérgio Avelino de Medeiros, Manoela Torres-Rêgo, Ariane Ferreira Lacerda, Hugo Alexandre Oliveira Rocha, Eryvaldo Sócrates Tabosa do Egito, Alianda Maira Cornélio, Denise V. Tambourgi, Matheus de Freitas Fernandes-Pedrosa and Arnóbio Antônio da Silva-Júnior

Figure S1. Effect of N-methyl-pyrrolidone (NMP) in association with mixtures of soy p0hosphatidylcholine (SPC) with The polysorbate 80 (T80) or poloxamer 407 (PO407) on the creaming index of emulsions of medium-chain triglyceride (MCT) in water.

Figure S2. Droplet size distribution (zeta-sizer images) with respective PdI and zeta potential values assessed for the studied formulations: (a) nanoemulsion (NE) and (b) cationic-covered nanoemulsion NE-PEI after 24 hours of preparing stored at 25 °C (c) TsV-loaded NE and d) TsV-loaded NE-PEI after 6 weeks, stored at 4 °C storage.

Table S1. Size, PdI, and Zeta potential measurements for different formulations, at distinct intervals of physical stability of NE formulations against pH and saline content.

Samples Colloidal NE NE pH 5,5 NE pH 7,4 NE pH 8,5 NE-PEI properties 1 month Size (nm) ± SD 130.73 ± 0.38 153.50 ± 0.8 150.65 ± 0.43 155.4 ± 0.85 175,5 ± 3.4

PdI ± SD 0.14 ± 0.02 0.07 ± 0.01 0.1 ± 0.003 0.13 ± 0.003 0.24 ± 0.02 ZP ± SD -17.6 ± 1.20 -16.3 ± 1.30 -15.3 ± 0.25 -14.2 ± 0.65 10.76 ± 0.65 2 month Size (nm) ± SD 130.50 ± 0.38 153.40 ± 0.09 151.6 ± 0.55 157.3 ± 0.5 175,6 ± 3.1

PdI ± SD 0.14 ± 0.02 0.09 ± 0.01 0.09 ± 0.005 0.14 ± 0.002 0.21 ± 0.02 ZP ± SD -17.6 ± 1.30 -15.6 ± 0.35 -14.7 ± 0.32 -13.6 ± 0.35 7.76 ± 0.54 3 month Size (nm) ± SD 129.90 ± 0.40 152.80 ± 0.05 150.6 ± 0.45 155.6 ± 0.4 150,4 ± 4.1

PdI ± SD 0.14 ± 0.02 0.1 ± 0.01 0.08 ± 0.008 0.13 ± 0.004 0.22 ± 0.02 ZP ± SD -16.7 ± 1.50 -16.5 ± 0.29 -15.6 ± 0.34 -13.5 ± 0.35 4.45 ± 0.34 4 month Size (nm) ± SD 130.20 ± 0.74 153.06 ± 0.65 150.8 ± 0.85 153.4 ± 0.8 150.3 ± 3.2 PdI ± SD 0.13 ± 0.02 0.1 ± 0.01 0.11 ± 0.02 0.10 ± 0.009 0.18 ± 0.02

ZP ± SD -16.4 ± 1.20 -10.6 ± 0.25 -12.9 ± 0.36 -9.5 ± 0.35 0.56 ± 0.09

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5.2 Produção de artigo científico

Mini review Adjuvant use and immunization protocols for anti-venom production against scorpion venoms

Arthur Sérgio Avelino de Medeiros 1, Matheus de Freitas Fernandes-Pedrosa 1, Arnóbio Antônio da Silva-Júnior 1.

Abstract The scorpionism treatment comprises symptomatology monitoring and treatment and anti-venom administration in severe cases. Anti-venom production supply local endemic regions according scorpion species, however, continue research have made efforts to optimize the industrial chain, using new adjuvants, classic adjuvants with different protocols and innovations in antigen obtainment. This review displays the several adjuvants that have been investigated to increase serum production efficacy, the mechanism that each act and other innovations in antigen selection for immunization, in order to demonstrate some possibilities to integrate advances in anti- venom field.

1. Introduction Scorpionism is an important medical issue, in which annually around 1.2 million stings are notified worldwide, mainly in tropical and subtropical countries. The lethality of scorpionism can be around 0.27% (more than 3,000 deaths annually), but may vary in each country based on endemic species and envenomation management, with reports of much less lethality in the range of 0.05% 101. Scorpion venoms have noxious effect in humans and also in many animals, including mammals, crustaceans, insects. Toxins that affect mammals generally include peptides that act in Na+, K+ and Ca2+ channels 102. Some scorpion genus are more endemic in each country, and about 30 genus are of medical importance like Centruroides (North and Central America, Colombia), Tityus (South America), Androctonus, Leiurus, Buthus (North Africa and Middle East), Parabuthus (South Africa) and Mesobuthus (India) 103,104. The most severe effects of scorpion venoms include myocardial damage, cardiac arrythmias, pulmonary edema and shock, targeting central and autonomic nervous system and inflammation 51. Considering the importance of scorpion envenomation, appropriate treatment is necessary depending on patient’s symptomatology. The Brazilian universal health system (SUS), for example, indicates serotherapy for moderate symptoms (nausea, vomit and elevated heart pressure) and severe symptoms (seizure, drowsiness, heart failure), with other symptoms treated by management of saline administration and specific anti-inflammatory and analgesic medicines 105. Anti-venom production 60 requires expensive production chain, which include scorpion venom extraction, horse hyper immunization and immunoglobulins purification. To reduce adverse effect of xeno-reactivity against immunoglobulins after serotherapy, purified sera still are subjected to pepsine cleavage to generate Fab fragments to neutralize venom. Despite high cost of production, the low efficiency is an issue since only 1-2.5% of equine antibodies are able to neutralize scorpion venom 106. To increase efficiency of anti- venom production, the immunization uses vaccine adjuvants to increase recognition of antigens. Historically, the Freund’s adjuvants and aluminum have been used for horses immunization, and adjusting number of doses and adjuvant combination are the strategies to increase antigen recognition 107. Meanwhile, adjuvants research brings advances in new forms to increase venom peptides presentation and efficiency to anti-venom production. The world health organization guideline for anti-venom production 46 recommend that advances in the vaccine field concerning new adjuvants should be transferred to anti-venom field, yet commercial protocols using new adjuvants for anti-venom production has not been published. In the past decade, new strategies have been applied to increase anti- venom production or reduce costs. The research for new adjuvants can bring some benefits as less side effects with superior effectiveness 108. New adjuvants include nanotechnology like nanoparticles to increase effectiveness and reduce toxicity 109, nanoemulsions 11, proteins carrier for antigens 110, immune activators 12 and many others as new protocols for antigens obtainment, like recombinant peptides 111. This paper reviews the scientific published data about adjuvants applied for scorpion anti-venom production and antigens used. The effectiveness of each strategy is the focus of this work, in order to analyze which adjuvants can represent new efforts for commercial anti-venom production.

2. Adjuvants for anti-venom immunization

2.1 Use of antibodies linked to immunogen The immunogens recognition is priority when an immunization takes place. A faster or stronger recognition may influence the response of immune system by innate immunity that will induce adaptive immunity. One of the most specific ways to recognize some molecule is through immunoglobulins. Epino-Solis and collaborators developed an immunization protocol 112 that use monoclonal antibody to recognize a marker of dendritic cells of mice, CD11c, and the antibody was attached to Cn2, the most abundant component of Centruroides noxius scorpion venom. The specificity of this type of immunization allowed a rapid production of anti-toxin immunoglobulin (7 days after only one immunization), which proved to raise a neutralizing efficiency of 50% compared to 100% of traditional protocol that requires several immunizations and 90 days duration. This study shows a very promising way of enhance immune recognition, in which the largest portion as possible of antigens will encounter immune cells and promote a response. This technique may be more efficient than using other strategies that recruit immune cells or increase the recognition of antigen, but the disadvantage 61 is the need to produce the monoclonal antibody before immunization, which have to be analyzed carefully about cost-benefit.

2.2 Bentonite venom adsorption Bentonite clays comprise of an octahedral alumina sheet that have nanometers range 113 of length and composition of Al2O3, MgO, CaO, SiO2, K2O, and Fe2O3 . The venom adsorption in bentonite seems to improve the protective sera production due its adsorptive capacity for different organic and inorganic ions 114. Bentonite venom adsorption was used to induce neutralizing sera production against Buthus tamalus 115 indian scorpion venom and and Leiurus quinquestriatus hebraeus 116 scorpion venoms. The produced anti-venom in mice were able to neutralize 1.2 mg of scorpion venom per mL of sera and protect from death guinea pigs, dogs and langurs when compared with non-immunized animals 115. Anti-venom against Leiurus quinquestriatus hebraeus increased the neutralizing capacity more than 5 times. The interesting technique used by Thalley and coworkers 116 to produce anti-venom against Leiurus quinquestriatus scorpion is that they immunize hens and collected the produced antibodies from egg yolks, with a promise to diminish antisera cost compared to horses immunizing and reduce reactivity and toxicity of sera when administering in patients. However, the bentonite efficacy as anti-venom adjuvant has been tested for snake anti-venom production, and was reported that has lower potency than complete Freund adjuvant to generate neutralizing antisera, and its use in some protocols was deprecated 117.

2.3 Aluminum salts immunization The use of aluminum for sera production in immunization programs is benefitted from the activity of this adjuvant with a number of applications 118. The research of aluminum hydroxide as adjuvant has showed its activity against several scorpion venoms. Kharrat et al 119 immunized horses and monkeys with scorpions venoms using aluminum hydroxide as adjuvant, and horse sera was able to neutralize 25 and 8 LD50 of Buthus occitamus tunetanus and Androctonus australis hector G-50 fractions, respectively. All the immunized monkeys showed no signs of envenomation during immunization, and the neutralization was more effective when aluminum gel was mixed with zinc. Alum was compared to MF59 against Androctonus australis hector venom and both adjuvants induced comparative specific IgG titters, and aluminum and MF59 produced higher IgG1 titter than IgG2, indicating a biased stimulation of humoral immunity, but alum was still less effective for mice protection against venom challenge 11. Other researches also demonstrated alum protective activity against Androctonus australis hector venom 32,35,47,69 and Zenouaki et al demonstrated an incredible high efficiency immunization with a synthetic toxin II using alum as adjuvant which could 121 protect 50% of mice from 20 LD50 challenge . Aluminum hydroxide was compared with chitosan nanoparticles against Tityus serrulatus venom demonstrating similar IgG production to both adjuvants 47, also demonstrating alum activity as adjuvant for another scorpion venom species. The efficacy, low cost material and activity as adjuvant against several species of scorpion venom probably explains why this adjuvant is present in immunization protocols until nowadays. 62

2.4 Emulsions formulations as immune adjuvants Emulsions have been used for long time as adjuvants in vaccines, as Freund’s adjuvants and more recently used as delivery systems for more innovative formulations. One of most oldest tested adjuvants is complete Freund’s adjuvant (CFA), developed in 1930 containing water-in-mineral oil emulsion that contained killed mycobacteria (mycobacterium tuberculosis or others) 8. The use of CFA described in literature allowed protection against infection of virulent tubercle bacilli 125 but due its reactogenic effect can offer protection against other antigens when mixing them to CFA, as scorpion antigens 126 127 119 128 129 45,121,130–133. Nouri and coworkers 45 performed a comparative study in rabbits incorporating Androctonus australis hector (Aah) venom in CFA. This adjuvant is quite reactogenic and increased neutrophils, eosinophils, monocytes and lymphocyte count after 24 hours. The produced specific IgG against the venom showed a higher IgG1 titter than IgG2 from day 15 of immunization, indicating a biased humoral response, inducing Th2 lymphocytes activation that induce IL-4, IL-5 and IL-6 cytokines activation and B lymphocytes to produce neutralizing antibodies. Incomplete Freund adjuvant (IFA) is a less reatogenic emulsion without mycobacteria, and IFA along with CFA were also used to immunize sheep against Tityus serrulatus venom, allowing in vivo protection with challenge test to 2 LD50 133. The CFA and IFA causes some side effect due immune response after inoculation. Horses when immunized with CFA combined to snake venoms, for example, present granuloma formation at injection site, that usually ruptures, formatting large infected wounds 46. However, Pratanaphon et al 134 divided the injection of snake venom dose in small volumes of 0.1 – 0.2 mL at multiple sites, in which adverse local reactions were reduced at each site, a protocol that was replicated by its success 46,135,136. Also, dividing the injection volumes may stimulate greater number of immune cells since total surface area are also increased. MF59 is an oil-in-water emulsion within nanoscale containing squalene with important adjuvant activity. The use of MF59 for Aah scorpion venom antigens is reported 11,137, demonstrating capacity to increase IgG titers compared to alum adjuvant and biased Th2 response with more pronounced titer of IgG1 since day 7, also increasing lymphocyte, monocyte and neutrophils count since day 1. The nanoscale of this adjuvant may also influence in adjuvant activity, as demonstrated for nanotechnology feature to enhance antigen presenting to immune cells 14.

2.5 Lipossomes The use of lipossomes as adjuvant to produce protecting IgG against scorpion venoms has been reported 138,139. The liposomes are self-assembly molecules used as delivery systems, composed of spherical vesicles with bilayers of phospholipids. Lipossome can induce immune response and also prevent the antigen from having toxic effect. Tityus serrulatus toxic fraction TstFG50 of scorpion venom were entrapped into liposomes, which allowed in vivo production of neutralizing IgG against venom toxins 139 and reduced mice deaths after exposure to the venom 138. A liposome immunization of ovoalbumin antigen was compared to aluminum adjuvant, which has showed that liposome-OVA immunization did not trigger IgE production, and in comparison, alum- 63

OVA immunization did induce. Also, mixed immunization liposome-OVA and alum- OVA did not induce IgE, which is desirable to produce a sera with reduced allergic reactions140 and thus much more safer sera. Also, liposome can modify some pharmacokinetic properties as a drug delivery system, since it has been described its feature to protect targeted molecules from degradation and depuration and extend drug release, properties which it has to be evaluated if are desirable for each antigen used to sera production 141,142.

2.6 Nanoparticles Nanoparticles are the most developed formulations inside nanotechnology for medical purpose, mainly by the wide number of components that can be used, relativity stability and effectiveness. Polimeric nanoparticles, for example, are widely described due large option numbers of materials used, also being relatively stable in biological fluids and yet biodegradable for some materials 23,47,123, a safety issue that prevent adjuvant accumulation in the body. Polymeric nanoparticles have been studied as vaccine adjuvants capable of promote uptake for dendritic cells and induce IL-β production that contributes for adaptive immune activation. For anti-venom production many were studied. Calcium-alginate nanoparticles encapsulating Androctonus australis hector venom were developed as a pre-clinical study demonstrating adjuvant activity 10,109,137. Some of the most promising results shows that nanoparticle formulation did not presented inflammatory response, while formulations containing venom raise neutrophils count and MPO and EPO enzyme activity. Besides no inflammatory activity, the nanoparticle loaded with irradiated venom also presented higher IgG titter than adjuvant free formulations or nanoparticles loaded with natural venom, which allied with the lower inflammatory response shows promising results for the calcium- alginate nanoparticles. In another moment, it was evaluated for alginate nanoparticles the systemic and local inflammatory response 109, demonstrating no systemic inflammatory activity and transient edema-erythema at the injection site. Alginate nanoparticles without venom also did not induced IL-10, IL-17 and TNF-α, the irradiated venom-loaded nanoparticles induced IL-10 production but lower than non- irradiated venom-loaded nanoparticles. This result demonstrate that the alginate nanoparticle formulation did not have an inflammatory response of its own but can increase the efficacy of the venom immunization, acting possible as a delivery system of the antigens rather than stimulating the immune cells. Cationic chitosan nanoparticles also demonstrated adjuvant activity increasing sera production against Tityus serrulatus scorpion venom 23,47. The technology research of formulation and technique adjustment allowed nanoparticles loaded with crude venom and BSA model proteins ranging from 130 and 209 nm and with positive zeta potential, allowing electrostatic interaction beyond adsorption in polymer structure, with release of proteins from matrix of nanoparticles for more than 6 days 23. Chitosan nanoparticles were also compared as scorpion venom immunizer against the well stablished aluminum hydroxide. Turns out, both chitosan and aluminum hydroxide allowed specific IgG anti-venom production, with chitosan nanoparticles showing very similar activity to alum adjuvant. Beyond nanotechnology delivery systems having intrinsic potential to increase antigen recognition 109, prolonged release of antigens 64 also may change interaction with immune cells since the antigen have prolonged contact and prolonged stimulation. PLA nanoparticles have also been investigated as adjuvant to anti-venom production against Androctonus australis hector (Aah) and Buthus occitanus tunetanus (Bot) scorpion venoms 123. PLA is a biodegradable polymer, as chitosan and calcium- alginate themselves, and is a promising alternative of efficient and safety material for this purpose. The purposed adjuvants showed increase in IgG content after each one of the 5 immunizations and PLA adjuvant activity was identical to PLA/Alhydrogel combined activity against Aah venom, demonstrating nanotechnology value for immune stimulation. In case of immunization of Bot venom, the comparative between PLA and complete Freund adjuvant (CFA), CFA demonstrated faster and higher production of IgG in the first 42 days of immunization protocol. In vivo neutralization assay of mice anti-venom demonstrated the specificity of the anti-venom, with approximately 12 and 14 times increase in LD50 of mice with pre-neutralized of toxic fractions of venoms of Aah and Bot. Also, the formulations demonstrated effective for active protection in animals against venom toxicity 6 months after immunization.

2.7 Proteins The venom toxicity relies in their toxins with variety of effects; thus, the immunization searches produce neutralizing antibodies against all these harmful proteins. To increase the low immunogenicity of some venom peptides, one strategy is the fusion of the targeted toxin with another innocuous protein 110. De Avila et al 143 studied the most inert used protein in research, BSA, fused to the toxic fraction (TstFG50) of Tityus serrulatus scorpion venom. The resultant fused proteins did not show the native toxicity of scorpion proteins and the sera from immunized mice did protected naive mice from challenge of 2LD50 1 week after last immunization and 9 weeks after last immunization. Thus, the selected technique succeeded to present protective and activity and also decrease toxicity of the toxins. Another research demonstrated fusion protein immunogen capable of generate protection against Androctonus australis hector (AaH) scorpion toxins 122. Recombinant proteins were produced from E. coli for AaH I, AaH II and AaH III (three major α-type toxins of Androctonus australis hector), and each of the toxins were fused to maltose binding protein (MBP). The recombinant monomeric proteins were used to immunize mice and the resultant specific IgG were able to bind to native toxins, demonstrated through radioimmune assay, but fused MBP-AaH I+II (two fused toxins) immunized animals sera did not recognized native AaH I and did recognized AaH II toxin, probably by folding error of the process. The produced anti-venom sera of recombinant proteins were able to protect mice challenged with toxic fraction of the venom and increase DL50 3 times. This particular technique, besides increase immunogenicity is promising since eliminate the need of actual venom extraction from scorpions, reducing costs and variables that can induce batches variability in sera production.

2.8 QS saponin 65

The purified saponin (QS) of Quilaja Saponaria Molina have been described as a potent adjuvant. Formulations AS01 (saponin in liposome system) and AS02 (saponin in oil-in-water emulsion system) were developed with QS for clinical trials and induced activation of innate immune system via TLR-4 and specific antigen-specific T cells (AS01) 12. Saponin QS were also studied as adjuvant associated to attenuated Androctonus australis hector scorpion venom and compared to complete Freund adjuvant 45. Compared to the reactogenic CFA, immunizing with saponin demonstrated restoration of normal levels of cell recruitment and enzymatic activity more rapidly, presenting a lower and temporary inflammatory response. However, the specific response was higher to saponin, with higher lymphocyte count 24 hours after immunization. IgG level were also higher to saponin as adjuvant. The IgG balance to its subtypes is biased with higher titter of IgG1 than IgG2 type, indicating Th2 more intense response, which implicated in a more capable anti-venom, with FCA adjuvant neutralizing 2 LD50 and saponin 3 LD50.

3. Alternative antigens for venom immunization 3.1 Immunization with venom fractions, recombinant or synthetic toxins The venoms are complex materials, composed of proteins, peptides, nucleotides, inorganic salts, polysaccharides and amino acids 104. In order to increase anti-venom specificity and homogeneity of produced sera, it has been researched immunization with specific fractions of the venoms that contains some antigens responsible for generate immunostimulation for IgG production of neutralizing antibodies. Toxic and nontoxic components can be separated from native venom from gel filtration chromatography 129,144. The venom toxicity is mainly caused by neurotoxic peptides that can act on ion channels, mainly toxins that block sodium and potassium channels 145,146 and also enzimes like hyaluronidases, proteases, and phospholipases that can contribute for envenomation symptoms 97,104,147,148. Thus, immunization protocols using toxic peptides or toxic fraction of scorpion venoms are intended to generate protective sera against severe effects of envenomation without generating antibodies against non-toxic peptides, which are immunogenic and may contribute to produce less potent sera in some cases 112,121,123,130,143,149–151. The immunization with single peptide often generates partial protection against crude venom. Zenouaki et al 121 immunized mice with Androctonus australis hector toxin II and 2 month after immunization all mice survived 3 LD50. Immunization with Heminecrolysin toxin from Androctonm australis hector scorpion also prevented 4 times from minimal necrotic dose of venom by rabbit immunization, but venom immunization showed more effective to prevent venom effect. Despite some cases shows superior protective effect immunizing with crude venom, the strategies using isolated toxin or venom fraction may be desirable to implement new protocols to enhance process variables or to facilitate immunizing with synthetic and recombinant antigens. Some adverse effects due immunization with toxic peptides can still present some manifestation in the animals, thus immunization with non-toxic peptides were investigated to recognize other toxic peptides with cross-reactivity 119,120,127–129,151, besides, immunization with non-toxic peptides can enable future human active immunization against scorpion venoms. This recognition of toxic peptides is possible 66 due some similarities among produced peptides from the same venom. Chavez- Olortegui et al 128 demonstrated that N- and C- terminal of non-toxic TsNTxP peptide have continuous epitopes responsible for generating polyclonal cross-reactivity antibodies against peptides of Tityus serrulatus. KAaH1 non-toxic peptide of Androctonus australis hector also were used in immunization to protect against toxic fraction of the venom and toxic peptide AahII, neutralizing 5LD50/ml of sera against the toxic fraction 120. To optimize anti-venom cost the immunizing chain is under constant investigation. The several processes using animals, including captive scorpions and extraction is a contributing factor to increase the cost, not only due to maintaining animals but also to the time consuming of the process and the difficulty to scale up the process to a certain level. Synthetic and recombinant antigens are valuable strategies to reduce cost of production and their development were dependent of encountering representative antigens as single peptides or a pool of them. Recombinant proteins are produced inoculating specific genes in bacteria culture and later purification process. Recombinant antigens have been described 111,122,158–160,126,130,152–157 and the protection can reach the whole venom effect 152,154. The immunogenicity and degree of similarity would be crucial to determine the effectiveness of each toxin as antigen, for example, recombinant phospholipase D1 immunization showed the high immunogenicity of this antigen generating protection against incredible 200 times LD100 dose of the venom 111. However, the medium protection of anti-venoms are around 152 against 2-5 LD50 of the venom . The synthetic peptides are also productive alternatives to antigen obtainment, with much more potential to fast production 110,121,128,161–165. This technique is very specific and allow identify specific regions in the peptide and construct the synthetic one only with the epitopes, with discontinuous peptides obtainment 110,161. The identification of epitopes can be done mapping epitopes with anti-peptide antibodies recognition to immobilize overlap peptides, covering amino-acid sequence and identifying the regions that were attached 128. Modifications of specific residues may also be performed, as Zenouaki et al research 121 that substituted cystine residues and eliminate disulfide bridges, obtaining a synthetic toxin without the toxicity of the original peptide, even with the dose of 1000 LD50 of the correspondent non-modified toxin.

3.2 Detoxification of venom antigens The venom innoculation cause noxious effect in the animals in sera production procedure. To reduce toxicity in anti-venom chain or to allow vaccine approach against scorpion envenomation, many strategies have been used to detoxify toxins. As demonstrated with synthetic peptides strategies, some modifications in the toxins structure can decrease toxicity and maintain epitopes intact, which can be much more useful. Detoxifying strategies relies in small modifications in the chemical structure of the toxins. For example, iodination of toxins 166,167, glutaraldehyde conjugation 131,133, gamma radiation 10,109,168, liposomal entrapment 138,139. Chemical alterations can be achieved by both iodination or glutaraldehyde conjugation. In case of iodination the iodine atom is included in toxins molecule and became less toxic or anatoxic in some cases with increasing number of iodine inclusion, causing biological alteration of 67 protein while maintain immunogenic activity, which occur with other protein types as well 167. Another chemical modification method to detoxification is glutaraldehyde conjugation 131,133. Glutaraldehyde conjugation depends also on its concentration and incubation time, with Possani et al 131 demonstrating that a concentration around 26 mM and an incubation time around 120-240 minutes is needed to detoxify 1 mg/mL of toxic fraction II of Centruroides noxius hoffmann venom. This technique demonstrated effective to protect sheep from clinical signs of envenomation as increased heart and respiratory rates compared to crude venom group and also, glutaraldehyde 133 conjugation protected from 16 LD50 doses effect . Venom irradiation is mostly affected by free radicals that interact with toxins, causing chemical and physical- chemical changes in secondary and tertiary structure of proteins. Irradiation could reduce inflammatory response after immunization and also induce higher IgG titers, as showed for attenuated Androctonus australis hector (Aah) venom, which can be caused by the less direct toxic effect on the immune cells that diminish efficiency of immunization 10. The irradiated venom can diminish extent of acute cytokine production and still stimulate IgG production, which is diminish toxicity and maintain effectiveness 109 . The less toxic effect of irradiated venom was demonstrated reducing LD50 and lesions in lungs, heart and liver in mice and demonstrated capable of stimulate anti- venom IgG production, but the irradiated venom showed a difference in IgG1/IgG2 balance, with higher ratio to the non-irradiated venom, but practical effect of this would have to be demonstrated with neutralization assay 168.

3.3 Immunization protocols The protocols used for horse immunization do not use a single adjuvant. Scorpion anti- venom production from Refik Saydam Hygiene Center, in Turkey, uses a protocol of hyperimmunization method of 6 venom inoculations combining CFA, IFA and 107 aluminum hydroxide (Al(OH)3) or aluminum phosphate (AlPO) . Due the high reactogenic and adjuvant efficiency of Freund’s adjuvants, the first two innoculations are generally with CFA and IFA adjuvants, respectively, to optimize the first antigen inoculation and to prevent adverse effects in all inoculation 46,107. Similar results of Freund’s adjuvant activity were observed for researchers of Fundação Ezequiel Dias (Belo Horizonte, MG, Brazil)169 against snake Crotalus durissus terrificus venom with low dose of antigen (37,5 mg per horse). In their study, the Freund’s emulsions combination were compared to other adjuvants immunization protocols, demonstrating that FCA/IFA adjuvants allowed more sustained and protective activity when compared to Al(OH)3 or liposome at low or high antigen dose (50 mg and 870 mg for Al(OH)3 or 5 mg and 20 mg for liposome per horse). The Fundação Ezequiel Dias protocol for scorpion immunization is similar to these first two. The first dose is administered using Complete Freund’s adjuvant, after 21 days of interval two more doses are administered using incomplete Freund’s adjuvants, followed by three more doses of crude venom 133. This protocol is administered in cycles of 6 doses that can be repeated to acquire desired sera titter. Table 1 demonstrate the studied adjuvants for scorpion anti-venom production compared by neutralization immunoprotection assay. The protocol for immunization in commercial anti-venom production (CFA, IFA, alum) is demonstrated for two studies, with one being highly efficient in protect mice from 35 LD50 of Androctonus crassicauda venom 132. Most of the papers used the combination of CFA/IFA adjuvants, a slight difference from commercial protocol, and in one case mice 68 could be protected from the astonish dose > 150 LD50 of toxoid phospholipase D1 from Hemiscorpius lepturus scorpion 111. The Freund’s adjuvant is efficient to induce specific immunoglobulins, as showed in Table 1, with most of immunization using Freund’s adjuvant or combinations protecting 111,121,132 against high doses of scorpion LD50 . However, the reactogenic effect also induce lesions at injection sites as granuloma and infected wounds 46. In fact, with the advance in vaccine field, new adjuvants have showed considerable increase in protection against scorpion venoms, as Alginate and PLA nanoparticles and MF59 nanoemulson, as showed in Table 1. However, some variables in each research may influence the final protection activity, as number of injection, bleeding day, whole venom immunization or venom fraction and isolated toxins, etc. Therefore, more investigation for standardized comparison between several adjuvants may bring answers about modification of used immunization protocols to achieve more safe and effective results. The immunizing inoculations also vary at antigen obtainment. The electrical stimulation of scorpion telson allow obtainment of crude venom, but immunization with macerated scorpion telsons are also described, in which telson weigh are much greater than crud 107,132 venom for immune stimulations and LD50 . Besides the low number of immunization protocols available for public, the number of marketed scorpions antivenoms in the world are in the tenths.

Table 1. Neutralization effect and immunoprotection of anti-venom against scorpion venoms divided by the adjuvants used in the immunizing.

Adjuvant Number Neutralizing / Bleeding Animal for Scorpion Reference of LD50 Immunoprotection day challenge species protected assay assay

Bentonite 5 0.63 mg venom/Kg + 3.1 57 days Mice Leiurus 116 anti-venom/Kg quinquestriatus hebraeus

CFA 20 20 LD50 venom + 5 µL anti- 180 days Mice Androctonm 121 venom australis hector CFA 2 Immune rabbit 120 Days Rabbit Androctonm 45 australis hector CFA > 150 Immune mice 64 days Mice Hemiscorpius 111 lepturus CFA, IFA 2 Immune mice 130 days Mice Androctonm 165 australis hector CFA, IFA 4 4 LD50 venom + 150 µL 42 days Mice Buthus occitanus 126 anti-venom tunetanus 133 CFA, IFA 2 2 LD50 venom + 200 µL 67 Mice Tityus serrulatus anti-venom CFA, IFA 3 LD50 venom + 50 µL anti- 42 days Mice Tityus serrulatus 129 venom CFA, IFA > 2 2 LD50 venom + 100 µL 42 days Mice Tityus serrulatus 160 anti-venom CFA, IFA >3 3 LD50 venom + 100 µL 42 days Mice Tityus serrulatus 152 anti-venom CFA, IFA 2 2 LD50 venom + 150 µL 63 days Mice Tityus serrulatus 161 anti-venom 132 CFA, IFA, Alum 35 35 LD50 venom + 0.5 mL 51 days Mice Androctonus anti-venom9 crassicauda CFA, IFA, Alum >3 3 LD50 venom + 250 µL Mice Centruroides (C.) 153 anti-venom noxius C. limpidus C.suffusus C.tecomanus C. elegans C. sculpturatus MF59 6 Immune rabbit 90 days Rabbit Androctonm 11 australis hector Alum 6 Immune rabbit 90 days Rabbit Androctonm 11 australis hector Alum 2 2 LD50 venom + 150 µL 42 days Mice Androctonm 120 anti-venom australis hector Lipossome 3 Immune mice 87 days Mice Tityus serrulatus 138 139 Lipossome 15 15 LD50 venom + 50 µL 46 days Mice Tityus serrulatus anti-venom Saponin 3 Immune rabbit 120 days Rabbit Androctonm 45 australis hector Calcium alginate >6 Immune rabbit 90 days Rabbit Androctonm 10 nanoparticle australis hector 123 PLA nanoparticle 5.5 12 LD50 venom + 5 times 35 days Mice Androctonm anti-venom volume australis hector Coupled BSA > 16 Immune mice 93 days Mice Tityus serrulatus 143 protein, IFA Fused toxin- 3 3 LD50 venom + 200 µL 75 days Mice Androctonm 143 maltose binding anti-venom australis hector protein

4. Method This paper have been revised what has been reported to date in the literature regarding adjuvant used for stimulation of protective antibodies against scorpion venoms and representative proteins or toxins. The research was made using the follow key words in pubmed, Scopus and Science Direct databases: 70

- Immunization scorpion venom; - Immunization scorpion venom AND nanoparticles OR nanoemulsions OR proteins OR toxins OR lipossomes OR aluminum OR Freund’s adjuvant; - Vaccine scorpion venom; - Vaccine scorpion venom AND nanoparticles OR nanoemulsions OR proteins OR toxins OR lipossomes OR aluminum OR Freund’s adjuvant.

Conclusion The adjuvants use for anti-venom field have demonstrated a considerable increase in options to be used by the pharmaceutical industries, despite few changes in the current protocols for hyper-immunization in anti-venom production. The drug delivery approach using nanotechnology certainly bring several formulation options regarding emulsions, nanoparticles and liposome, that can be benefitted every time that a new material is approved and bring novel features, as biodegradable new polymers. The research field have demonstrated some advantages of nanotechnology for adjuvant field, increasing antigen recognition and with safer profile, in some cases without inflammatory activity of its own, but increasing antigen recognition and adaptive immunity. More standardized studies may demonstrate for each venom type if new adjuvant platform is more effective and safer or its combination with other adjuvants is more indicated. Immunization research can also potentially induce modifications in the entire antivenom production chain, since it has been discovered specific toxins, peptides or venom fractions that can induce immunoglobulin production with cross- reactivity against the whole venom, in some cases obtained from recombinant or synthetic sources, saving the amount of the biological raw material used in comparison with expensive extraction from captive animals.

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6 CONCLUSÕES Carreadores nanoemulsionados foram desenvolvidos nesse trabalho para atuar como sistemas de liberação de antígenos da peçonha do escorpião TsV. A formulação das nanoemulsões foi otimizada através de testes que avaliaram diferentes tensoativos, sendo selecionada a mistura de tensoativos e co-solvente FC:T80:NMP (6:2:15 m/m), respectivamente. A obtenção de nanoemulsões foi possível pela diluição de cristais líquidos lamelares, possibilitando uma transição de fases para nanoemulsões. As interações intermoleculares entre as nanoemulsões, o polímero PEI e o TsV foram demonstradas por modificação no potencial zeta e por alterações espectroscópicas FTIR das nanoemulsões, tanto após adição do polímero catiônico, quanto após adição da peçonha nas nanoemulsões catiônicas e não catiônicas. Estudos de biocompatibilidade em células foram utilizados para demonstrar a segurança das formulações desenvolvidas; as formulações associadas ao TsV apresentaram menor atividade hemolítica do que a solução aquosa da peçonha e apresentaram baixa toxicidade em macrófagos de camundongos, uma das células APC alvo para reconhecimento dos antígenos. Os adjuvantes nanoemulsionados utilizados nas imunizações de camundongos provocaram um aumento no título de imunoglobulinas IgG em comparação com o hidróxido de alumínio, um resultado muito promissor para aumentar a produção de soro antiescorpiônico. Portanto, esta pesquisa mostrou o potencial de nanoemulsões para aumentar a resposta imune adaptativa na produção de imunoglobulinas para a peçonha do escorpião Tityus serrulatus.

7 COMENTÁRIOS, CRÍTICAS E CONCLUSÕES

O projeto inicial de doutorado do aluno Arthur Medeiros tinha o objetivo de desenvolvimento de novo insumo adjuvante com nanoemulsões catiônicas tipo água em óleo para imunização contra a peçonha do escorpião Tityus serrulatus e testá-lo in vivo em camundongos. Neste sentido, o planejamento original e o resultado final foram bem compatíveis, onde pudemos ainda avaliar a biocompatibilidade em células de camundongos RAW 264.7 e em hemácias humanas. O projeto apresentou algumas limitações relacionadas com as propriedades do insumo desenvolvido. Em nosso grupo de pesquisa dispomos de recursos para análise e doseamento de proteínas que foram submetidos ao projeto. Dentre eles cito: (i) eletroforese de proteínas, que poderia demonstrar bandas referentes às proteínas e peptídeos da peçonha e diferenças de migração quando incorporadas às nanoemulsões, mas as bandas não puderam ser observadas utilizando os reveladores comassie blue e nem nitrato de prata devido a baixíssima concentração da peçonha nos insumos (18 µg/mL) e a alta concentração de lipídeos (8% m/m) que também são revelados na eletroforese. Outra dificuldade para análise das nanoemulsões em eletroforese seria a utilização da etapa desnaturante com SDS e aquecimento para caracterizar as proteínas, o que afetaria a estrutura do nano-sistema; (ii) o doseamento de proteínas da peçonha incorporadas nas nanoemulsões também não foi possível com os métodos que tínhamos disponíveis, com o kit para doseamento de proteínas pelo método de Bradford e com o kit para doseamento de proteínas pelo método do ácido bicincronínico, onde ambos também eram afetados por altas concentrações de lipídeos (em torno de 8%) e não tinham sensibilidade para detectar a quantidade de proteínas da peçonha que foi adicionada (em torno de 18 µg/mL). Estas dificuldades, no entanto, apesar de nos privarem de algumas respostas, como eficiência de encapsulação e cinética de liberação, não nos impediram de visualizar os efeitos da encapsulação através das técnicas que tínhamos disponíveis, como mudanças na espectroscopia de infra-vermelho médio após adsorção nas nanoemulsões, efeito da adsorção na modificação da atividade hemolítica da peçonha em suspensões de hemácias humanas e ainda o efeito na potencialização da atividade adjuvante à medida que as formulações eram modificadas em relação à presença de nanoemulsões e carga positiva. O insumo desenvolvido tem alto potencial no campo das imunizações, tendo demonstrado o aumento de eficácia para produção de anticorpos utilizando nanoemulsões catiônicas funcionalizadas com o polímero poli(etilenoimina) e sem o polímero. Os adjuvantes demonstraram atuar através de propriedades dos sistemas de liberação nanoemulsionados, como o tamanho reduzido e uniforme e a carga. A independência tecnológica do projeto também tem de ser ressaltada, como demonstrado experimentalmente, a formulação não partiu de fórmulas já desenvolvidas, mas sim de um estudo amplo de cada componente a ser utilizado e suas concentrações, sendo importante para a universidade como fonte de pesquisas originais, desenvolvimento de novas tecnologias e treinamento de pesquisadores qualificados para a continuação deste tipo de prática científica. 80

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