Effect of Degumming in the Characteristics of Silk Fibroin Nanoparticles

Several studies have stated that the process used for sericin removal, or degumming, from silk cocoons has a strong impact on the silk fibroin integrity and consequently in their mechanical or biochemical properties after processing it into several biomaterials (e.g., fibers, films or scaffolds) but still, there is a lack of information of the impact on the features of silk nanoparticles. In this work, silk cocoons were degummed following four standard methods: autoclaving, short alkaline (Na2CO3) boiling, long alkaline (Na2CO3) boiling, and ultrasounds. The resultant silk fibroin fibers were dissolved in the ionic liquid 1-ethyl-3-methylimidazolium acetate and used for nanoparticle synthesis by rapid desolvation in polar organic solvents. The relative efficiencies of the degumming processes and the integrity of the resulting fibroin fibers obtained were analyzed by mass loss, optical microscopy, thermogravimetric analysis, infrared spectroscopy, and SDS-PAGE. Particle sizes and morphology were analyzed by Dynamic Light Scattering and Field Emission Scanning Electronic Microscopy. The results showed that the different treatments had a remarkable impact on the integrity of the silk fibroin chains, as confirmed by gel electrophoresis, which can be correlated with particle mean size and size distribution changes. The smallest nanoparticles (156 ± 3 nm) and the most negative Z potential (−30.2 ± 1.8 mV) were obtained with the combination of long treatment (2 h) of boiling in alkaline solution (Na2CO3 0.02 eq/L). The study confirms that parameters of the process, such as the composition of the solution and time of the degumming step, must be controlled in order to reach an optimum reproducibility of the nanoparticle production.This work has been partially supported (80%) by the European Commission ERDF/FEDER Operational Programme 'Murcia' CCI N° 2007ES161PO001 (Project No. 14-20/20), and the Spanish MINECO (Ref. CTQ2017-87708-R) and the programme of support to the research of the Seneca Foundation of Science and Technology of Murcia, Spain (Ref. 20977/PI/18). A.A.L.-P.’s research contract was partially supported (80%) by the ERDF/FEDER Operational Programme 'Murcia' CCI N° 2007ES161PO001 (Project No. 14-20/20),. M.G. Montalbán’s research contract is funded by the Spanish MINECO (Juan de la Cierva-Formación contract, Ref. FJCI-2016-28081). S.D.A.-C.’s research contract is funded by the program INIA-CCAA (DOC INIA 2015), announced by the National Institute for Agricultural and Food Research and Technology (INIA) and supported by the Spanish State Research Agency (AEI) under the Spanish Ministry of Economy, Industry and Competitiveness

Revealing the Influence of the Degumming Process in the Properties of Silk Fibroin Nanoparticles

Several studies have stated that the process used for sericin removal, or degumming, from silk cocoons has a strong impact in the silk fibroin integrity and consequently in their mechanical or biochemical properties after processing it into several biomaterials (e.g. fibers, films or scaffolds) but still, there is a lack of information of the impact on the features of silk nanoparticles. In this work, silk cocoons were degummed following four standard methods: autoclaving, short alkaline (Na2CO3) boiling, long alkaline (Na2CO3) boiling and ultrasounds. The resultant silk fibroin fibers were dissolved in the ionic liquid 1-ethyl-3-methylimidazolium acetate and used for nanoparticle synthesis by rapid desolvation in polar organic solvents. The relative efficiencies of the degumming processes and the integrity of the resulting fibroin fibers obtained were analyzed by mass loss, optical microscopy, thermogravimetric analysis, infrared spectroscopy and SDS-PAGE. Particle sizes and morphology were analyzed by Dynamic Light Scattering and Field Emission Scanning Electronic Microscopy. The results showed that the different treatments had a remarkable impact on the integrity of the silk fibroin chains, as confirmed by gel electrophoresis, which can be correlated with particle mean size and size distribution changes. The smallest nanoparticles (156 ± 3 nm) and the most negative Z potential (−30.2 ± 1.8 mV) were obtained with the combination of long treatment (2 h) of boiling in alkaline solution (Na2CO3 0.02 eq/L). The study confirms that parameters of the process, such as composition of the solution and time of the degumming step, must be controlled in order to reach an optimum reproducibility of the nanoparticle production.This work has been partially supported (80%) from the European Commission ERDF/FEDER Operational Programme 'Murcia' CCI N° 2007ES161PO001 (Project No. 14-20/20), and the Spanish MINECO (Ref. CTQ2017-87708-R) and the programme of support to the research of the Seneca Foundation of Science and Technology of Murcia, Spain (Ref. 20977/PI/18).A.A. Lozano-Pérez’s research contract was partially supported (80%) by the ERDF/FEDER Operational Programme 'Murcia' CCI N° 2007ES161PO001 (Project No. 14-20/20),. M.G. Montalbán’s research contract is funded by the Spanish MINECO (Juan de la Cierva-Formación contract, Ref. FJCI-2016-28081). S.D. Aznar-Cervantes’s research contract is funded by the program INIA-CCAA (DOC INIA 2015), announced by the National Institute for Agricultural and Food Research and Technology (INIA) and supported by The Spanish State Research Agency (AEI) under the Spanish Ministry of Economy, Industry and Competitiveness

Impact of Covalent Functionalization on the Aqueous Processability, Catalytic Activity, and Biocompatibility of Chemically Exfoliated MoS2 Nanosheets

Chemically exfoliated MoS2 (ce-MoS2) has emerged in recent years as an attractive two-dimensional material for use in relevant technological applications, but fully exploiting its potential and versatility will most probably require the deployment of appropriate chemical modification strategies. Here, we demonstrate that extensive covalent functionalization of ce-MoS2 nanosheets with acetic acid groups (∼0.4 groups grafted per MoS2 unit) based on the organoiodide chemistry brings a number of benefits in terms of their processability and functionality. Specifically, the acetic acid-functionalized nanosheets were furnished with long-term (>6 months) colloidal stability in aqueous medium at relatively high concentrations, exhibited a markedly improved temporal retention of catalytic activity toward the reduction of nitroarenes, and could be more effectively coupled with silver nanoparticles to form hybrid nanostructures. Furthermore, in vitro cell proliferation tests carried out with murine fibroblasts suggested that the chemical derivatization had a positive effect on the biocompatibility of ce-MoS2. A hydrothermal annealing procedure was also implemented to promote the structural conversion of the functionalized nanosheets from the 1T phase that was induced during the chemical exfoliation step to the original 2H phase of the starting bulk material, while retaining at the same time the aqueous colloidal stability afforded by the presence of the acetic acid groups. Overall, by highlighting the benefits of this type of chemical derivatization, the present work should contribute to strengthen the position of ce-MoS2 as a two-dimensional material of significant practical utility.Financial support from the Spanish Ministerio de Economía y Competitividad (MINECO) and the European Regional Development Fund (FEDER) through project MAT2015-69844-R is gratefully acknowledged, as well as partial support of the ERDF/FEDER. Operative Program of the Región de Murcia 2007–2013. We also acknowledge partial funding by Plan de Ciencia, Tecnología e Innovación 2013-2017 del Principado de Asturias and FEDER through grant GRUPIN14-056.Peer reviewe

Unexpected high toughness of Samia cynthia ricini silk gut

Silk gut fibers were produced from the silkworm Samia cynthia ricini silk glands by the usual procedure of immersion in a mildly acidic solution and subsequent stretching. The morphology of the silk guts was assessed by scanning electron microscopy, and their microstructure was assessed by infrared spectroscopy and X-ray diffraction. It was found that both naturally spun and Samia silk guts share a common semicrystalline microstructure. The mechanical characterization of the silk guts revealed that these fibers show an elastomeric behavior when tested in water, and exhibit a genuine ground state to which the fiber may revert independently of its previous loading history. In spite of its large cross-sectional area compared with naturally spun silk fibers, Samia silk guts show values of work to fracture up to 160 MJ m, much larger than those of most of their natural counterparts, and establish a new record value for this parameter in silk guts

Mechanical behaviour and formation process of silkworm silk gut

High performance silk fibers were produced directly from the silk glands of silkworms ("Bombyx mori") following an alternative route to natural spinning. This route is based on a traditional procedure that consists of soaking the silk glands in a vinegar solution and stretching them by hand leading to the so called silkworm guts. Here we present, to the authors’ best knowledge, the first comprehensive study on the formation, properties and microstructure of silkworm gut fibers. Comparison of the tensile properties and microstructural organization of the silkworm guts with those of naturally spun fibers allows gain of a deeper insight into the mechanisms that lead to the formation of the fiber, as well as the relationship between the microstructure and properties of these materials. In this regard, it is proved that an acidic environment and subsequent application of tensile stress in the range of 1000 kPa are sufficient conditions for the formation of a silk fiber

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

El gusano de seda, bombyx mori (Linneo, 1758)(Lepidoptera : Bombycidae) como plataforma de producción de proteínas naturales y recombinantes : aplicaciones en biotecnología e ingeniería de tejidos

La presente Tesis se propone desarrollar algunas aplicaciones recientes no textiles del gusano de seda (Bombyx mori). La primera es el uso de las larvas del insecto como biorreactor para la producción de proteína recombinante, mediante la inoculación con un vector de expresión baculovirus. Se realizó un ciclo completo de producción de una proteína modelo, la dihidrofolato reductasa. Se desarrolló la cría del insecto en una dieta artificial, se preparó la construcción viral adecuada, se inoculó en larvas y se extrajo y purificó la proteína expresada. La segunda aplicación consiste en la elaboración de la seda como biomaterial para crecimiento y diferenciación celular en la reconstrucción de tejidos. Se optimizó la fabricación de mallas de micro y nanofibras mediante electrohilatura, funcionalizándolas posteriormente con polipirrol para hacerlas electroconductoras. Se evaluó la biocompatibilidad de las mismas con cultivos de fibroblastos y células madre mesenquimales de médula ósea. Palabras clave: Gusano de seda, fibroína, sericicultura, biomateriales, polipirrol, electrohilado. Summary: The present Thesis aims to develop some recent non-textile applications of silkworm (Bombyx mori). The first is the use of the insect larvae as a bioreactor for the production of recombinant protein, through the inoculation with a baculovirus expression vector. A complete cycle of production was made with the model enzyme dihydrofolate reductase. The rearing of the insect in an artificial diet was developed. After preparing a suitable construct, it was inoculated in larvae and the expressed protein was extracted from the insects and purified. The second application consists of the elaboration of silk as biomaterial for the fabrication of scaffolds for growth and cellular differentiation in tissue regeneration. The fabrication of micro- and nanofiber mats by electrospinning was optimized. The mats were also functionalized with polypyrrole for making them electroconductive. Cell culture methods were carried out, using human fibroblasts and human mesenchymal stem cells, for testing biocompatibility. Key words: Silkworm, fibroin, sericulture, biomaterials, polypyrrole, electrospinning