A mucopolissacaridose tipo I (MPS I) é uma doença autossômica recessiva causada pela deficiência da enzima α-L-iduronidade (IDUA). Essa deficiência resulta no acúmulo de glicosaminoglicanos levando a diversas manifestações clínicas. A microencapsulação de células recombinantes que superexpressam IDUA tem sido considerada uma estratégia promissora para o tratamento de MPS I. Neste contexto, o presente estudo teve por objetivo a otimização da encapsulação de células BHK (Baby Hamster Kidney) superexpressando IDUA em microcápsulas de alginato revestidas com poli-L-lisina (PLL) utilizando-se um extrusor eletrostático. Em uma primeira etapa, um estudo de otimização das microcápsulas de alginato (MC-A) foi realizado por meio de um desenho experimental do tipo Box-Behnken (software Mini-Tab®) que permitiu avaliar simultaneamente a influência da voltagem (kV), fluxo alginato/células (mL/h) e concentração de alginato (%) sobre o tamanho das microcápsulas e a atividade de IDUA. Após, as microcápsulas foram revestidas sequencialmente com PLL e alginato (MC-APA) com o objetivo de aumentar a sua estabilidade. Nas condições experimentais empregadas, MC-A e MC-APA apresentaram-se monodispersas (span < 1,22) com um diâmetro médio inferior a 350 μm, determinado por difração a laser. O revestimento alterou a morfologia das microcápsulas (microscopia eletrônica de varredura) e a sua resistência mecânica (analisador de textura), sendo observado um aumento de cerca de 6 vezes na força necessária para compressão das mesmas. O revestimento final pelo alginato (MC-APA) parece ter sido parcial de acordo com as análises de infravermelho por transformada de Fourier com refletância atenuada. Em uma última etapa, a atividade enzimática foi avaliada em modelo murino MPS I após implante subcutâneo de MC-APA. Foi observado um aumento significativo da atividade de IDUA na pele, após 30 dias de tratamento. Nas análises histológicas foi observado um infiltrado inflamatório no local da aplicação que não impediu a liberação da enzima nas condições avaliadas. No seu conjunto, esse estudo demonstra a potencialidade das MC-APA para a liberação local de IDUA. / Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disorder caused by the deficiency of α-L-iduronidase (IDUA). This deficiency results in the accumulation of glycosaminoglycans leading to various clinical manifestations. The microencapsulation of recombinant cells overexpressing IDUA has been considered as a promising strategy for the treatment of MPS I. In this context, the present study aimed to optimize the encapsulation of BHK cells overexpressing IDUA in poly-L-lysine (PLL) coated alginate microcapsules using an electrostatic extruder. In a first step, a Box-Behnken experimental design (Mini-Tab® software) was carried out for the optimization of the alginate microcapsules (MC-A), which allowed to evaluate simultaneously the influence of voltage (kV), alginate/cell flow (mL/h) and alginate concentration (%) on the size of the microcapsules and IDUA activity. Thereafter, the microcapsules were sequentially coated with PLL and alginate (MC-APA) in order to increase their stability. In the experimental conditions used, MC-A and MC-APA were monodisperse (span <1.22) with an average diameter of less than 350 μm, determined by laser diffraction. The coating modified microcapsules morphology (scanning electron microscopy) and their mechanical resistance (texture analyzer), being observed a six-fold increase in the required force for their compression. The final alginate coating (MC-APA) appears to have only partially coated the microcapsules, according to the attenuated total reflectance Fourier transform infrared spectroscopy analyses. In a final step, the enzymatic activity was evaluated in a MPS I murine model after subcutaneous implantation of MC-APA. A significant increase in IDUA activity was observed in the skin at 30 days after treatment. Histological analszes revealed an inflammatory infiltrate at the application site, which did not prevent the release of the enzyme under the evaluated conditions. Overall, this study demonstrates the potentiality of MC-APA for the local release of IDUA.

A mucopolissacaridose tipo I (MPS I) é uma doença autossômica recessiva causada pela deficiência da enzima α-L-iduronidade (IDUA). Essa deficiência resulta no acúmulo de glicosaminoglicanos levando a diversas manifestações clínicas. A microencapsulação de células recombinantes que superexpressam IDUA tem sido considerada uma estratégia promissora para o tratamento de MPS I. Neste contexto, o presente estudo teve por objetivo a otimização da encapsulação de células BHK (Baby Hamster Kidney) superexpressando IDUA em microcápsulas de alginato revestidas com poli-L-lisina (PLL) utilizando-se um extrusor eletrostático. Em uma primeira etapa, um estudo de otimização das microcápsulas de alginato (MC-A) foi realizado por meio de um desenho experimental do tipo Box-Behnken (software Mini-Tab®) que permitiu avaliar simultaneamente a influência da voltagem (kV), fluxo alginato/células (mL/h) e concentração de alginato (%) sobre o tamanho das microcápsulas e a atividade de IDUA. Após, as microcápsulas foram revestidas sequencialmente com PLL e alginato (MC-APA) com o objetivo de aumentar a sua estabilidade. Nas condições experimentais empregadas, MC-A e MC-APA apresentaram-se monodispersas (span < 1,22) com um diâmetro médio inferior a 350 μm, determinado por difração a laser. O revestimento alterou a morfologia das microcápsulas (microscopia eletrônica de varredura) e a sua resistência mecânica (analisador de textura), sendo observado um aumento de cerca de 6 vezes na força necessária para compressão das mesmas. O revestimento final pelo alginato (MC-APA) parece ter sido parcial de acordo com as análises de infravermelho por transformada de Fourier com refletância atenuada. Em uma última etapa, a atividade enzimática foi avaliada em modelo murino MPS I após implante subcutâneo de MC-APA. Foi observado um aumento significativo da atividade de IDUA na pele, após 30 dias de tratamento. Nas análises histológicas foi observado um infiltrado inflamatório no local da aplicação que não impediu a liberação da enzima nas condições avaliadas. No seu conjunto, esse estudo demonstra a potencialidade das MC-APA para a liberação local de IDUA. / Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disorder caused by the deficiency of α-L-iduronidase (IDUA). This deficiency results in the accumulation of glycosaminoglycans leading to various clinical manifestations. The microencapsulation of recombinant cells overexpressing IDUA has been considered as a promising strategy for the treatment of MPS I. In this context, the present study aimed to optimize the encapsulation of BHK cells overexpressing IDUA in poly-L-lysine (PLL) coated alginate microcapsules using an electrostatic extruder. In a first step, a Box-Behnken experimental design (Mini-Tab® software) was carried out for the optimization of the alginate microcapsules (MC-A), which allowed to evaluate simultaneously the influence of voltage (kV), alginate/cell flow (mL/h) and alginate concentration (%) on the size of the microcapsules and IDUA activity. Thereafter, the microcapsules were sequentially coated with PLL and alginate (MC-APA) in order to increase their stability. In the experimental conditions used, MC-A and MC-APA were monodisperse (span <1.22) with an average diameter of less than 350 μm, determined by laser diffraction. The coating modified microcapsules morphology (scanning electron microscopy) and their mechanical resistance (texture analyzer), being observed a six-fold increase in the required force for their compression. The final alginate coating (MC-APA) appears to have only partially coated the microcapsules, according to the attenuated total reflectance Fourier transform infrared spectroscopy analyses. In a final step, the enzymatic activity was evaluated in a MPS I murine model after subcutaneous implantation of MC-APA. A significant increase in IDUA activity was observed in the skin at 30 days after treatment. Histological analszes revealed an inflammatory infiltrate at the application site, which did not prevent the release of the enzyme under the evaluated conditions. Overall, this study demonstrates the potentiality of MC-APA for the local release of IDUA.

A mucopolissacaridose tipo I (MPS I) é uma doença autossômica recessiva causada pela deficiência da enzima α-L-iduronidade (IDUA). Essa deficiência resulta no acúmulo de glicosaminoglicanos levando a diversas manifestações clínicas. A microencapsulação de células recombinantes que superexpressam IDUA tem sido considerada uma estratégia promissora para o tratamento de MPS I. Neste contexto, o presente estudo teve por objetivo a otimização da encapsulação de células BHK (Baby Hamster Kidney) superexpressando IDUA em microcápsulas de alginato revestidas com poli-L-lisina (PLL) utilizando-se um extrusor eletrostático. Em uma primeira etapa, um estudo de otimização das microcápsulas de alginato (MC-A) foi realizado por meio de um desenho experimental do tipo Box-Behnken (software Mini-Tab®) que permitiu avaliar simultaneamente a influência da voltagem (kV), fluxo alginato/células (mL/h) e concentração de alginato (%) sobre o tamanho das microcápsulas e a atividade de IDUA. Após, as microcápsulas foram revestidas sequencialmente com PLL e alginato (MC-APA) com o objetivo de aumentar a sua estabilidade. Nas condições experimentais empregadas, MC-A e MC-APA apresentaram-se monodispersas (span < 1,22) com um diâmetro médio inferior a 350 μm, determinado por difração a laser. O revestimento alterou a morfologia das microcápsulas (microscopia eletrônica de varredura) e a sua resistência mecânica (analisador de textura), sendo observado um aumento de cerca de 6 vezes na força necessária para compressão das mesmas. O revestimento final pelo alginato (MC-APA) parece ter sido parcial de acordo com as análises de infravermelho por transformada de Fourier com refletância atenuada. Em uma última etapa, a atividade enzimática foi avaliada em modelo murino MPS I após implante subcutâneo de MC-APA. Foi observado um aumento significativo da atividade de IDUA na pele, após 30 dias de tratamento. Nas análises histológicas foi observado um infiltrado inflamatório no local da aplicação que não impediu a liberação da enzima nas condições avaliadas. No seu conjunto, esse estudo demonstra a potencialidade das MC-APA para a liberação local de IDUA. / Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disorder caused by the deficiency of α-L-iduronidase (IDUA). This deficiency results in the accumulation of glycosaminoglycans leading to various clinical manifestations. The microencapsulation of recombinant cells overexpressing IDUA has been considered as a promising strategy for the treatment of MPS I. In this context, the present study aimed to optimize the encapsulation of BHK cells overexpressing IDUA in poly-L-lysine (PLL) coated alginate microcapsules using an electrostatic extruder. In a first step, a Box-Behnken experimental design (Mini-Tab® software) was carried out for the optimization of the alginate microcapsules (MC-A), which allowed to evaluate simultaneously the influence of voltage (kV), alginate/cell flow (mL/h) and alginate concentration (%) on the size of the microcapsules and IDUA activity. Thereafter, the microcapsules were sequentially coated with PLL and alginate (MC-APA) in order to increase their stability. In the experimental conditions used, MC-A and MC-APA were monodisperse (span <1.22) with an average diameter of less than 350 μm, determined by laser diffraction. The coating modified microcapsules morphology (scanning electron microscopy) and their mechanical resistance (texture analyzer), being observed a six-fold increase in the required force for their compression. The final alginate coating (MC-APA) appears to have only partially coated the microcapsules, according to the attenuated total reflectance Fourier transform infrared spectroscopy analyses. In a final step, the enzymatic activity was evaluated in a MPS I murine model after subcutaneous implantation of MC-APA. A significant increase in IDUA activity was observed in the skin at 30 days after treatment. Histological analszes revealed an inflammatory infiltrate at the application site, which did not prevent the release of the enzyme under the evaluated conditions. Overall, this study demonstrates the potentiality of MC-APA for the local release of IDUA.

Le papier bioactif est obtenu par la modification de substrat du papier avec des biomolécules et des réactifs. Ce type de papier est utilisé dans le développement de nouveaux biocapteurs qui sont portables, jetables et économiques visant à capturer, détecter et dans certains cas, désactiver les agents pathogènes. Généralement les papiers bioactifs sont fabriqués par l’incorporation de biomolécules telles que les enzymes et les anticorps sur la surface du papier. L’immobilisation de ces biomolécules sur les surfaces solides est largement utilisée pour différentes applications de diagnostic comme dans immunocapteurs et immunoessais mais en raison de la nature sensible des enzymes, leur intégration au papier à grande échelle a rencontré plusieurs difficultés surtout dans les conditions industrielles. Pendant ce temps, les microcapsules sont une plate-forme intéressante pour l’immobilisation des enzymes et aussi assez efficace pour permettre à la fonctionnalisation du papier à grande échelle car le papier peut être facilement recouvert avec une couche de telles microcapsules. Dans cette étude, nous avons développé une plate-forme générique utilisant des microcapsules à base d’alginate qui peuvent être appliquées aux procédés usuels de production de papier bioactif et antibactérien avec la capacité de capturer des pathogènes à sa surface et de les désactiver grâce à la production d’un réactif anti-pathogène. La conception de cette plate-forme antibactérienne est basée sur la production constante de peroxyde d’hydrogène en tant qu’agent antibactérien à l’intérieur des microcapsules d’alginate. Cette production de peroxyde d’hydrogène est obtenue par oxydation du glucose catalysée par la glucose oxydase encapsulée à l’intérieur des billes d’alginate. Les différentes étapes de cette étude comprennent le piégeage de la glucose oxydase à l’intérieur des microcapsules d’alginate, l’activation et le renforcement de la surface des microcapsules par ajout d’une couche supplémentaire de chitosan, la vérification de la possibilité d’immobilisation des anticorps (immunoglobulines G humaine comme une modèle d’anticorps) sur la surface des microcapsules et enfin, l’évaluation des propriétés antibactériennes de cette plate-forme vis-à-vis l’Escherichia coli K-12 (E. coli K-12) en tant qu’un représentant des agents pathogènes. Après avoir effectué chaque étape, certaines mesures et observations ont été faites en utilisant diverses méthodes et techniques analytiques telles que la méthode de Bradford pour dosage des protéines, l’électroanalyse d’oxygène, la microscopie optique et confocale à balayage laser (CLSM), la spectrométrie de masse avec désorption laser assistée par matrice- temps de vol (MALDI-TOF-MS), etc. Les essais appropriés ont été effectués pour valider la réussite de modification des microcapsules et pour confirmer à ce fait que la glucose oxydase est toujours active après chaque étape de modification. L’activité enzymatique spécifique de la glucose oxydase après l’encapsulation a été évaluée à 120±30 U/g. Aussi, des efforts ont été faits pour immobiliser la glucose oxydase sur des nanoparticules d’or avec deux tailles différentes de diamètre (10,9 nm et 50 nm) afin d’améliorer l’activité enzymatique et augmenter l’efficacité d’encapsulation. Les résultats obtenus lors de cette étude démontrent les modifications réussies sur les microcapsules d’alginate et aussi une réponse favorable de cette plate-forme antibactérienne concernant la désactivation de E. coli K-12. La concentration efficace de l’activité enzymatique afin de désactivation de cet agent pathogénique modèle a été déterminée à 1.3×10-2 U/ml pour une concentration de 6.7×108 cellules/ml de bactéries. D’autres études sont nécessaires pour évaluer l’efficacité de l’anticorps immobilisé dans la désactivation des agents pathogènes et également intégrer la plate-forme sur le papier et valider l’efficacité du système une fois qu’il est déposé sur papier. / Bioactive paper is obtained through the modification of paper substrate with biomolecules and reagents. It is used in the development of novel biosensors that are portable, disposable and inexpensive, aimed at capturing, detecting and in some cases deactivating pathogens. Generally bioactive papers are made by incorporating biomolecules such as enzymes and/or antibodies on to paper. The immobilization of such biomolecules on solid surfaces is widely used for different diagnostic applications such as in immunosensors and immunoassays but due to the sensitive nature of enzymes, their large scale incorporation into paper has faced several difficulties especially under industrial papermaking conditions. The functionalization of paper at large scale is possible because paper can be easily coated with a layer of microcapsules, which have proven to be an efficient immobilization platform for enzymes and to allow. In this study, we developed a generic alginate-based platform incorporating microcapsules that can be applied to current paper production processes to prepare antibacterial bioactive paper with the ability to capture pathogens on its surface and to deactivate them by producing an anti-pathogenic agent. The design of the antibacterial platform is based on constant production of hydrogen peroxide as the antibacterial agent inside the alginate microcapsules. Hydrogen peroxide production is achieved through oxidation of glucose, catalyzed by the enzyme glucose oxidase encapsulated inside the alginate beads. The different steps of development included the entrapment of glucose oxidase inside alginate microcapsules, the reinforcement and surface activation of microcapsules by adding an additional layer of chitosan, investigating the possibility of immobilization of antibodies (human immunoglobulin G as a model antibody) on the surface of microcapsules and, finally, verifying the antibacterial properties of the system against Escherichia coli K-12 (E. coli K-12) as a representative pathogen. During development, certain measurements and observations were made using various analytical methods and techniques such as Bradford protein assay, oxygen electroanalysis, optical and confocal laser canning microscopy (CLSM), matrix assisted laser desorption/ionization- time of flight mass spectrometry (MALDI-TOF-MS), etc. Appropriate tests were performed to validate the successful modification of microcapsules and to ensure that glucose oxidase is still active after each modification. It was found that the encapsulated glucose oxidase maintained the specific enzymatic activity of 120±30 U/g. Subsequent efforts were made to immobilize glucose oxidase on gold NPs of two different diameters (10.9 nm and 50 nm) to enhance the enzymatic activity and increase the encapsulation efficiency. The results obtained during this study demonstrate successful modifications on alginate microcapsules and also a successful response of such antibacterial platform regarding deactivation of the pathogen representative, E. coli K-12. The threshold for the enzymatic activity was found to be 1.3×10-2 U/ml for E. coli K-12 growth inhibition of 6.7×108 cells/ml. Further studies are needed to assess the efficiency of immobilized antibody in the capture of pathogens and also to incorporate the platform onto paper and to validate the efficiency of the system once it is coated on paper.