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Exploração dos recursos genéticos dos Açores para o desenvolvimento de uma enzima com aplicações na saúde: desenvolvimento de uma abordagem direcionada baseada em nanotecnologia (Enzyme4Thrombus)
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Abstract(s)
As doenças cardiovasculares, como a trombose, são a primeira causa de morte a nível mundial, sendo que a trombose resulta da ativação descontrolada da cascata fibrinolítica.
Os agentes mais eficazes para o tratamento de doenças trombóticas são as enzimas fibrinolíticas. Neste âmbito, a enzima AprE127, produzida por um Bacillus subtilis isolado de amostras de solo da ilha de São Miguel, demonstrou ter atividade fibrinolítica específica, sem atuar como ativador do plasminogénio, possibilitando uma dissolução rápida e completa de trombos sanguíneos.
Assim, o presente estudo teve como objetivo obter a enzima fibrinolítica AprE127 por expressão heteróloga, para possibilitar a sua produção em larga escala, e desenvolver uma nanoformulação baseada em lipossomas, para melhorar a entrega da enzima no organismo, reduzindo a possibilidade de reações imunológicas e de efeitos secundários.
Para tal, desenvolveram-se, por tecnologia de DNA recombinante, duas versões da AprE127: a proteína AprE127 madura e a AprE127 sob a forma de pró-proteína. A expressão de ambas as versões da AprE127 foi testada em múltiplos vetores de expressão citoplasmática em E. coli, e em quatro linhagens celulares distintas (BL21, C41, Rosetta II e MC1061).
Os resultados mostram que a proteína AprE127 madura foi expressa sob a forma insolúvel, em corpos de inclusão, mesmo no clone em que foi expressa em fusão com a enzima glutationa S-transferase (no vetor pGEX-4T-1), que não promoveu a solubilidade da AprE127. Apesar da proteína ter sido produzida em corpos inclusão, foram também obtidos bons rendimentos de produção médios de expressão (4 mg de proteína por litro de cultura) nos vetores pET23a(+) e pHTP1 (com otimização de codões para E. coli no caso do último vetor).
De forma a obter a configuração funcional da proteína, esta foi desnaturada e renaturada a diferentes pHs, ambientes redox e força iónica, e com diferentes cofatores. Após os ensaios de desnaturação e renaturação da proteína madura, verificou-se que esta não adquiriu a conformação tridimensional correta, que lhe conferisse o nível de atividade proteolítica da proteína AprE127 nativa.
Por outro lado, a proteína AprE127 foi obtida na forma solúvel, quando expressa sob a forma de pró-proteína, em fusão com as proteínas MBP ou SUMO. Não obstante, a expressão da pró-proteína solúvel apresentou um rendimento de produção médio mais baixo (1 mg de proteína por litro de cultura), e agravado pela necessidade de clivagem da proteína de fusão a jusante.
A expressão heteróloga de ambas as versões da proteína AprE12 foi confirmada por Western-Blot e por espectrometria de massa.
A proteína AprE127 madura foi incorporada na formulação patenteada de lipossomas DODAB/MO 1:2 (mol/mol), aproveitando as caraterísticas estruturais singulares do lípido monooleina, que potenciam a solubilização de proteínas. Estes lipossomas apresentaram características físico-químicas favoráveis à sua aplicação na entrega de fármacos, tendo tamanhos abaixo dos 200 nm e carga superficial positiva (entre os 40 e 60 mV).
Estudou-se ainda a citotoxicidade dos lipossomas contendo a proteína recombinante madura, que demonstraram citotoxicidade dependente de dose.
Desenvolveram-se adicionalmente lipossomas miméticos de exossomas, inovadores por mimetizarem a composição lipídica destas vesículas naturais, e que retêm algumas das suas caraterísticas desejáveis para aplicação na entrega controlada de biomoléculas, permitindo elevadas eficiências de encapsulamento e produção em larga escala, que o uso dos próprios exossomas não possibilita. Estes lipossomas apresentaram características físico-químicas favoráveis à sua aplicação por injeção intravenosa (tamanhos inferiores a 150 nm e carga superficial neutra), tendo também demostrado ser estáveis em condições fisiológicas, uma vez que não se verificaram alterações substanciais no tamanho, PDI e carga superficial após incubação em plasma humano.
Este trabalho representou um primeiro passo para o desenvolvimento de uma tecnologia inovadora para o tratamento da trombose, recorrendo a um recurso natural dos Açores, i.e. a proteína AprE127, cujo potencial se encontrava ainda largamente inexplorado devido à sua baixa biodisponibilidade. Neste contexto, o recurso aos lipossomas nanométricos para encapsular a AprE127, permite potenciar a sua aplicação para a degradação de trombos sanguíneos.
ABSTRACT: Cardiovascular diseases, such as thrombosis, are the main cause of death worldwide, with thrombosis resulting from the uncontrolled activation of the fibrinolytic cascade. Fibrinolytic enzymes are the most effective agents for the treatment of thrombotic diseases. In this regard, the AprE127 enzyme, produced by a Bacillus subtilis isolated form soil samples from the island of São Miguel, has shown to have specific fibrinolytic activity, without plasminogen activation, allowing for the rapid and complete dissolution of blood thrombi. Hence, the goal of the present study was to obtain the AprE127 enzyme by heterologous expression, to allow its large scale production, and develop a nanoformulation, based in liposomes, to improve this enzyme’s delivery in the organism, by reducing the possibility of immunologic reactions and side effects. To do so, two versions of the AprE127 protein, the mature AprE127 and the pro-protein AprE127, were developed by recombinant DNA technology. The expression of both variants was tested in multiple cytoplasmic expression vectors in E. coli, and in four different cell lines (BL21, C41, Rosetta II and MC1061). The results have shown that the mature AprE127 protein was expressed in an insoluble form, in inclusion bodies, even in the clone where the AprE127 protein was expressed in fusion with the glutathione S-transferase enzyme (in the pGEX-4T-1 vector). In spite of being produced in inclusion bodies, good yields of expression of the protein were achieved (4 mg of protein per liter of cell culture) with the pET23a(+) and pHTP1 vectors (with codon optimization for E. coli expression, in the case of the latter vector). In order to obtain its functional conformation, the protein was denaturated and renaturated in different pHs, redox environments and ionic strength, and with different cofactors. After the refolding assays, it was verified that the protein was unable to obtain the correct tridimensional conformation, to allow proteolytic activity comparable to that of the native protein. On the other hand, the AprE127 protein was obtained in a soluble form when expressed as a pro-protein, in fusion with the MBP and SUMO proteins. Regardless, the yield of expression of the soluble pro-protein was lower (1 mg of protein per liter of cell culture), and aggravated by the need to cleave the fusion protein downstream. The heterologous expression of both forms of the AprE127 protein was confirmed by Western-Blot and mass spectroscopy. The mature AprE127 protein was incorporated in the patented liposome formulation DODAB/MO 1:2 (mol/mol), to take advantage of the structural characteristics of the monoolein lipid, that enhances protein solubilization. These liposomes showed favorable physicho-chemical characteristics for application in drug delivery, with sizes below 200 nm and positive superficial charge (ranging between 40 and 60 mV). The cytotoxicity of the liposomes, incorporating the mature recombinant protein, was studied and verified to be dosage-dependent. Innovative exosome-like liposomes were also developed by mimicking the lipid composition of the exosomes, retaining some of their advantageous characteristics for controlled drug delivery, whilst allowing high encapsulation efficiencies and large scale production, that naturally occurring exosomes themselves do not. These liposomes had physico-chemical characteristics that are favorable to their application by intravenous injection (sizes below 150 nm and neutral superficial charge), and also demonstrated to be stable in physiological conditions, as there were no substantial changes in their size, PDI and superficial charge after incubation in human plasma. This work represented a first step in the development of an innovative technology for the treatment of thrombosis, employing a natural resource of the Azores, i.e. the protein AprE127, whose potential was still largely unexplored due to it low bioavailability. In this context, its encapsulation in nanometric liposomes, can largely enhance its application for blood clot degradation.
ABSTRACT: Cardiovascular diseases, such as thrombosis, are the main cause of death worldwide, with thrombosis resulting from the uncontrolled activation of the fibrinolytic cascade. Fibrinolytic enzymes are the most effective agents for the treatment of thrombotic diseases. In this regard, the AprE127 enzyme, produced by a Bacillus subtilis isolated form soil samples from the island of São Miguel, has shown to have specific fibrinolytic activity, without plasminogen activation, allowing for the rapid and complete dissolution of blood thrombi. Hence, the goal of the present study was to obtain the AprE127 enzyme by heterologous expression, to allow its large scale production, and develop a nanoformulation, based in liposomes, to improve this enzyme’s delivery in the organism, by reducing the possibility of immunologic reactions and side effects. To do so, two versions of the AprE127 protein, the mature AprE127 and the pro-protein AprE127, were developed by recombinant DNA technology. The expression of both variants was tested in multiple cytoplasmic expression vectors in E. coli, and in four different cell lines (BL21, C41, Rosetta II and MC1061). The results have shown that the mature AprE127 protein was expressed in an insoluble form, in inclusion bodies, even in the clone where the AprE127 protein was expressed in fusion with the glutathione S-transferase enzyme (in the pGEX-4T-1 vector). In spite of being produced in inclusion bodies, good yields of expression of the protein were achieved (4 mg of protein per liter of cell culture) with the pET23a(+) and pHTP1 vectors (with codon optimization for E. coli expression, in the case of the latter vector). In order to obtain its functional conformation, the protein was denaturated and renaturated in different pHs, redox environments and ionic strength, and with different cofactors. After the refolding assays, it was verified that the protein was unable to obtain the correct tridimensional conformation, to allow proteolytic activity comparable to that of the native protein. On the other hand, the AprE127 protein was obtained in a soluble form when expressed as a pro-protein, in fusion with the MBP and SUMO proteins. Regardless, the yield of expression of the soluble pro-protein was lower (1 mg of protein per liter of cell culture), and aggravated by the need to cleave the fusion protein downstream. The heterologous expression of both forms of the AprE127 protein was confirmed by Western-Blot and mass spectroscopy. The mature AprE127 protein was incorporated in the patented liposome formulation DODAB/MO 1:2 (mol/mol), to take advantage of the structural characteristics of the monoolein lipid, that enhances protein solubilization. These liposomes showed favorable physicho-chemical characteristics for application in drug delivery, with sizes below 200 nm and positive superficial charge (ranging between 40 and 60 mV). The cytotoxicity of the liposomes, incorporating the mature recombinant protein, was studied and verified to be dosage-dependent. Innovative exosome-like liposomes were also developed by mimicking the lipid composition of the exosomes, retaining some of their advantageous characteristics for controlled drug delivery, whilst allowing high encapsulation efficiencies and large scale production, that naturally occurring exosomes themselves do not. These liposomes had physico-chemical characteristics that are favorable to their application by intravenous injection (sizes below 150 nm and neutral superficial charge), and also demonstrated to be stable in physiological conditions, as there were no substantial changes in their size, PDI and superficial charge after incubation in human plasma. This work represented a first step in the development of an innovative technology for the treatment of thrombosis, employing a natural resource of the Azores, i.e. the protein AprE127, whose potential was still largely unexplored due to it low bioavailability. In this context, its encapsulation in nanometric liposomes, can largely enhance its application for blood clot degradation.
Description
Tese de Doutoramento, Biologia, 20 de julho de 2023, Universidade dos Açores.
Keywords
Azores Doenças Cardiovasculares Trombose Enzimas Fibrinolíticas Trombos Sanguíneos
Pedagogical Context
Citation
Lopes, Ivo Edgar Araújo. (2022). "Exploração dos recursos genéticos dos Açores para o desenvolvimento de uma enzima com aplicações na saúde: desenvolvimento de uma abordagem direcionada baseada em nanotecnologia (Enzyme4Thrombus)". 181 p. (Tese de Doutoramento em Biologia). Ponta Delgada: Universidade dos Açores, 2022. Disponível em http://hdl.handle.net/10400.3/7111