Periodic Reporting for period 1 - cubiform (Development of mucosal vaccines based in crosslinked-mucin glycoproteins and chitosan-based nanodevices)
Reporting period: 2021-05-01 to 2023-04-30
Mucin is a family of glycoproteins found in mucus that have important functions related to mucosal health. Mucins have a common core structure and show glycans, uncharged or negatively charged, exposed to the external environment that vary on either inner and outer conditions. Once isolated mucins cannot self-crosslink anymore. The candidate discovered a method for mucin crosslinking that result in biomimetic mucus gels (BMG) of potential use for researching and biomedical and veterinary applications.
Chitosan (CS) is a family of positively charged polymers obtained from chitin. Despite chitin is water-insoluble, it can be deacetylated to yield CS that show interesting properties such as non-toxicity. CS can be used for the development of drug delivery systems (DS). Scientific research studies have shown that mucin and CS can interact between them although the specific interaction mechanism between both molecules is unknown yet.
The overall goal of the action was to characterize the effect of a BMG on the immune system of fish and to glean a deep understanding of the interactions between CS and mucin, and to use all the information obtained to develop new methods for mucosal vaccination strategies. Given the importance of the discovery of BMG, this technology is under patent filling stage. Therefore, no results can be shown yet. Regarding the interaction between mucin and CS, it has been analysed by using several techniques such as Langmuir Blodgett films.
Chitosan (CS) is a family of positively charged polymers obtained from chitin. Despite chitin is water-insoluble, it can be deacetylated to yield CS that show interesting properties such as non-toxicity. CS can be used for the development of drug delivery systems (DS). Scientific research studies have shown that mucin and CS can interact between them although the specific interaction mechanism between both molecules is unknown yet.
The overall goal of the action was to characterize the effect of a BMG on the immune system of fish and to glean a deep understanding of the interactions between CS and mucin, and to use all the information obtained to develop new methods for mucosal vaccination strategies. Given the importance of the discovery of BMG, this technology is under patent filling stage. Therefore, no results can be shown yet. Regarding the interaction between mucin and CS, it has been analysed by using several techniques such as Langmuir Blodgett films.
University of Leeds (UoL):
BMG were characterized by analysing the diffusion of fluorescein through them by UV and by cryo-SEM and SEM.
Prawns were bought frozen and the meat prawn was donated to Saint George Crypt, a charity in Leeds that offers meal to homelessness. Chitin was isolated and deacetylated to yield very low degree of acetylation (DA) and high molecular weight (MW) CS. The reacetylation (RAc) of the CS obtained was attempted in the UoL although there was no time for its characterization prior moving to University of Murcia (UM).
Only one squid pen (SP) supplier company was found worldwide. The SP transport through the border was not allowed due to Brexit rules and SP got spoiled. As a contingency strategy, a small amount of chitin leftover obtained from SP in a previous project in which the candidate had participated in was used to produce high MW and low DA CS.
All the research material was sent through the border by a courier. Due to the Brexit restrictions, the material was kept in the border for one month before being delivered.
University of Murcia
RMN analyses revealed that RAc was unsuccessful. Despite applying the protocols reported previously in literature, no appropriate DA was obtained. Not until changing the CS solubilisation conditions, were the CS successfully reacetylated. The prawn CS obtained in UoL was depolymerized to two different degrees prior of being reacetylated. Finally, three different MW CS with four DA each were prepared. A new methodology was developed for the characterization of the CS MW. Not until the end of the period of the project were the CS produced. As a contingency strategy, commercially available CS that show different MW, DA and origins were used. Namely: Low MW and Medium MW CSs from Sigma Aldrich, CS HMC 70/200, 80/200 and 90/200 from Heppe Medical CS, Ultra Low MW from Glentham Life Sciences, CS from Aspergillus niger and mushroom from Chibio Ltd.
The biocompatibility of mucin type III from Sigma Aldrich, CS and BMG were analysed by MTT on SAF-1 and SAB-1 cell lines obtained from Sparus aurata. The reactive oxygen species production, the cell viability and the effects on the mitochondria were analysed by Seahorse technology from Agilent. The presence of cells within BMG after a week of incubation was qualitatively analysed by confocal microscopy.
The authorisation for in vivo experiments using S. aurata specimens was obtained from the authorities. This part of the project will be carried out as soon as possible.
University of Zaragoza
The interaction between mucin and CS by Dynamic Light Scattering allowed to determine the number of charges in mucin. This data was used for performing the Langmuir films (Lf) at mucin to CS ratio of equal number of charges. The mucin to CS interaction were analysed by Lf of the mixtures and analysing the surface pressure and potential. The changes in the surface of the Lf were analysed by Brewster Angle Microscope. Langmuir Blodgett films were prepared on mica and were analysed by Atomic Force Microscopy. Finally, mucin Lf was prepared at a specific surface pressure and then CS were added in the subphase. The position of the barriers to keep the surface pressure was recorded.
The results obtained in this work has been published in Fish and Shellfish Immunology (2022) 130:1-8. Doi: 10.1016/j.fsi.2022.08.030
The results of this project has been communicated in several congresses and conferences, such as 16th International Meeting on Mucins in Health and Disease and 2nd International Symposium Mucosal Health in Aquaculture, among others.
Also this project has been disseminated in several events such as 6th Virtual International Conference on Education, Innovation and ICT, EDUNOVATIC 2021, IV Science and Technology Fair of Cieza, Marie Sklodowska-Curie actions day MSCA 2022 from the University of Murcia, 1st International Forum on Blue Economy and Growth, European Mednight 2022, Science and Technology Week (SeCyt’22), 4th World Symposium on Sustainability Science and Research, among others. Also the webpage www.mariachit.com has been created.
BMG were characterized by analysing the diffusion of fluorescein through them by UV and by cryo-SEM and SEM.
Prawns were bought frozen and the meat prawn was donated to Saint George Crypt, a charity in Leeds that offers meal to homelessness. Chitin was isolated and deacetylated to yield very low degree of acetylation (DA) and high molecular weight (MW) CS. The reacetylation (RAc) of the CS obtained was attempted in the UoL although there was no time for its characterization prior moving to University of Murcia (UM).
Only one squid pen (SP) supplier company was found worldwide. The SP transport through the border was not allowed due to Brexit rules and SP got spoiled. As a contingency strategy, a small amount of chitin leftover obtained from SP in a previous project in which the candidate had participated in was used to produce high MW and low DA CS.
All the research material was sent through the border by a courier. Due to the Brexit restrictions, the material was kept in the border for one month before being delivered.
University of Murcia
RMN analyses revealed that RAc was unsuccessful. Despite applying the protocols reported previously in literature, no appropriate DA was obtained. Not until changing the CS solubilisation conditions, were the CS successfully reacetylated. The prawn CS obtained in UoL was depolymerized to two different degrees prior of being reacetylated. Finally, three different MW CS with four DA each were prepared. A new methodology was developed for the characterization of the CS MW. Not until the end of the period of the project were the CS produced. As a contingency strategy, commercially available CS that show different MW, DA and origins were used. Namely: Low MW and Medium MW CSs from Sigma Aldrich, CS HMC 70/200, 80/200 and 90/200 from Heppe Medical CS, Ultra Low MW from Glentham Life Sciences, CS from Aspergillus niger and mushroom from Chibio Ltd.
The biocompatibility of mucin type III from Sigma Aldrich, CS and BMG were analysed by MTT on SAF-1 and SAB-1 cell lines obtained from Sparus aurata. The reactive oxygen species production, the cell viability and the effects on the mitochondria were analysed by Seahorse technology from Agilent. The presence of cells within BMG after a week of incubation was qualitatively analysed by confocal microscopy.
The authorisation for in vivo experiments using S. aurata specimens was obtained from the authorities. This part of the project will be carried out as soon as possible.
University of Zaragoza
The interaction between mucin and CS by Dynamic Light Scattering allowed to determine the number of charges in mucin. This data was used for performing the Langmuir films (Lf) at mucin to CS ratio of equal number of charges. The mucin to CS interaction were analysed by Lf of the mixtures and analysing the surface pressure and potential. The changes in the surface of the Lf were analysed by Brewster Angle Microscope. Langmuir Blodgett films were prepared on mica and were analysed by Atomic Force Microscopy. Finally, mucin Lf was prepared at a specific surface pressure and then CS were added in the subphase. The position of the barriers to keep the surface pressure was recorded.
The results obtained in this work has been published in Fish and Shellfish Immunology (2022) 130:1-8. Doi: 10.1016/j.fsi.2022.08.030
The results of this project has been communicated in several congresses and conferences, such as 16th International Meeting on Mucins in Health and Disease and 2nd International Symposium Mucosal Health in Aquaculture, among others.
Also this project has been disseminated in several events such as 6th Virtual International Conference on Education, Innovation and ICT, EDUNOVATIC 2021, IV Science and Technology Fair of Cieza, Marie Sklodowska-Curie actions day MSCA 2022 from the University of Murcia, 1st International Forum on Blue Economy and Growth, European Mednight 2022, Science and Technology Week (SeCyt’22), 4th World Symposium on Sustainability Science and Research, among others. Also the webpage www.mariachit.com has been created.
This project will allow to determine the conditions of CS that result in better interaction with mucins. Moreover, the CS obtained in this project are unique in the sense that their MW and DA are not found in the market, which enhances the impact of the results of this project.
The biocompatibility analysis of CS in fish cell lines will result in the determination of the CS characteristics of interest for its application for the development of drug delivery systems in aquaculture. This is of utmost importance since less than 21 % of research works properly describe CS properties, which result in the lack of reproducibility and a chaotic knowledge on CS.
Designing an effective treatment that can be used through mucosal surfaces in fish will reduce the handling processes and consequently reducing the fish mortality rates associated to the current aquaculture processes. It will have a worldwide repercussion since aquaculture is a practice developed worldwide and it constitutes the only protein source of the local population and it has a key role in the socio-economy in some areas.
The biocompatibility analysis of CS in fish cell lines will result in the determination of the CS characteristics of interest for its application for the development of drug delivery systems in aquaculture. This is of utmost importance since less than 21 % of research works properly describe CS properties, which result in the lack of reproducibility and a chaotic knowledge on CS.
Designing an effective treatment that can be used through mucosal surfaces in fish will reduce the handling processes and consequently reducing the fish mortality rates associated to the current aquaculture processes. It will have a worldwide repercussion since aquaculture is a practice developed worldwide and it constitutes the only protein source of the local population and it has a key role in the socio-economy in some areas.