Functionalising silk hydrogels with hetero- and homotypic nanoparticles.

Despite many reports detailing silk hydrogels, the development of composite silk hydrogels with homotypic and heterotypic silk nanoparticles and their impact on material mechanics and biology have remained largely unexplored. We hypothesise that the inclusion of nanoparticles into silk-based hydrogels enables the formation of homotropic and heterotropic material assemblies. The aim was to explore how well these systems allow tuning of mechanics and cell adhesion to ultimately control the cell-material interface. We utilised nonporous silica nanoparticles as a standard reference and compared them to nanoparticles derived from Bombyx mori silk and Antheraea mylitta (tasar) silk (approximately 100-150 nm in size). Initially, physically cross-linked B. mori silk hydrogels were prepared containing silica, B. mori silk nanoparticles, or tasar silk nanoparticles at concentrations of either 0.05% or 0.5% (w/v). The initial modulus (stiffness) of these nanoparticle-functionalised silk hydrogels was similar. Stress relaxation was substantially faster for nanoparticle-modified silk hydrogels than for unmodified control hydrogels. Increasing the concentrations of B. mori silk and silica nanoparticles slowed stress relaxation, while the opposite trend was observed for hydrogels modified with tasar nanoparticles. Cell attachment was similar for all hydrogels, but proliferation during the initial 24 h was significantly improved with the nanoparticle-modified hydrogels. Overall, this study demonstrates the manufacture and utilisation of homotropic and heterotropic silk hydrogels.

Microfibre-Functionalised Silk Hydrogels.

Silk hydrogels have shown potential for tissue engineering applications, but several gaps and challenges, such as a restricted ability to form hydrogels with tuned mechanics and structural features, still limit their utilisation. Here, Bombyx mori and Antheraea mylitta (Tasar) silk microfibres were embedded within self-assembling B. mori silk hydrogels to modify the bulk hydrogel mechanical properties. This approach is particularly attractive because it creates structured silk hydrogels. First, B. mori and Tasar microfibres were prepared with lengths between 250 and 500 µm. Secondary structure analyses showed high beta-sheet contents of 61% and 63% for B. mori and Tasar microfibres, respectively. Mixing either microfibre type, at either 2% or 10% (w/v) concentrations, into 3% (w/v) silk solutions during the solution-gel transition increased the initial stiffness of the resulting silk hydrogels, with the 10% (w/v) addition giving a greater increase. Microfibre addition also altered hydrogel stress relaxation, with the fastest stress relaxation observed with a rank order of 2% (w/v) > 10% (w/v) > unmodified hydrogels for either fibre type, although B. mori fibres showed a greater effect. The resulting data sets are interesting because they suggest that the presence of microfibres provided potential 'flow points' within these hydrogels. Assessment of the biological responses by monitoring cell attachment onto these two-dimensional hydrogel substrates revealed greater numbers of human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) attached to the hydrogels containing 10% (w/v) B. mori microfibres as well as 2% (w/v) and 10% (w/v) Tasar microfibres at 24 h after seeding. Cytoskeleton staining revealed a more elongated and stretched morphology for the cells growing on hydrogels containing Tasar microfibres. Overall, these findings illustrate that hydrogel stiffness, stress relaxation and the iPSC-MSC responses towards silk hydrogels can be tuned using microfibres.

Correction: Mixing and flow-induced nanoprecipitation for morphology control of silk fibroin self-assembly.

[This corrects the article DOI: 10.1039/D1RA07764C.].

Smart Silk Origami as Eco-sensors for Environmental Pollution.

Origami folding is an easy, cost-effective, and scalable fabrication method for changing a flat material into a complex 3D functional shape. Here, we created semicrystalline silk films doped with iron oxide particles by mold casting and annealing. The flat silk films could be loaded with natural dyes and folded into 3D geometries using origami principles following plasticization. They performed locomotion under a magnetic field, were reusable, and displayed colorimetric stability. The critical parameters for the design of the semi-autonomous silk film, including ease of folding, shape preservation, and locomotion in the presence of a magnetic field, were characterized, and pH detection was achieved by eye and by digital image colorimetry with a response time below 1 min. We demonstrate a practical application─a battery-free origami silk boat─as a colorimetric sensor for waterborne pollutants, which was reusable at least five times. This work introduces silk eco-sensors and merges responsive actuation and origami techniques.

Mixing and flow-induced nanoprecipitation for morphology control of silk fibroin self-assembly.

Tuning silk fibroin nanoparticle morphology using nanoprecipitation for bottom-up manufacture is an unexplored field that has the potential to improve particle performance characteristics. The aim of this work was to use both semi-batch bulk mixing and micro-mixing to modulate silk nanoparticle morphology by controlling the supersaturation and shear rate during nanoprecipitation. At flow rates where the shear rate was below the critical shear rate for silk, increasing the concentration of silk in both bulk and micro-mixing processes resulted in particle populations of increased sphericity, lower size, and lower polydispersity index. At high flow rates, where the critical shear rate was exceeded, the increased supersaturation with increasing concentration was counteracted by increased rates of shear-induced assembly. The morphology could be tuned from rod-like to spherical assemblies by increasing supersaturation of the high-shear micro-mixing process, thereby supporting a role for fast mixing in the production of narrow-polydispersity silk nanoparticles. This work provides new insight into the effects of shear during nanoprecipitation and provides a framework for scalable manufacture of spherical and rod-like silk nanoparticles.

Pharmacokinetics and pharmacodynamics of single dose of inhaled nebulized sodium nitrite in healthy and hemoglobin E/ß-thalassemia subjects.

Inhaled sodium nitrite has been reported to decrease pulmonary artery pressure in hemoglobin E/ß-thalassemia (HbE/ß-thal) patients with pulmonary hypertension. This study investigated the pharmacokinetics and pharmacodynamics of inhaled nebulized sodium nitrite in 10 healthy subjects and 8 HbE/ß-thal patients with high estimated pulmonary artery pressure. Nitrite pharmacokinetics, fraction exhaled nitric oxide (FENO), estimated right ventricular systolic pressure (eRVSP) measured by echocardiography, and platelet activation were determined. Nebulized sodium nitrite at doses used in this study (37.5 and 75 mg for healthy subjects and 15 mg for HbE/ß-thal patients) was well tolerated and did not cause changes in methemoglobin levels and systemic blood pressure. Absorption of inhaled nitrite was rapid with the absolute bioavailability of 18%. In whole blood, nitrite exhibited the dose-independent pharmacokinetics with clearance (CL) of 1.5 l/h/kg, volume of distribution (Vd) of 1.3 l/kg and half-life (t1/2) of 0.6 h. CL and Vd of nitrite was higher in red blood cells (RBC) than whole blood and plasma. HbE/ß-thal patients had lower nitrite CL and longer t1/2 in RBC than healthy subjects. FENO increased immediately after inhalation. Following nitrite inhalation, eRVSP remained unchanged but platelet activation was suppressed as evidenced by inhibition of adenosine diphosphate (ADP)-induced P-selectin expression and increase in phosphorylated vasodilator-stimulated phosphoprotein (P-VASPSer239) in platelets. There were no changes in markers of oxidative and nitrosative stress after inhalation. Our results support further development of inhaled nebulized sodium nitrite for treatment of pulmonary hypertension in ß-thalassemia.

Abnormal red blood cell morphological changes in thalassaemia associated with iron overload and oxidative stress.

AIMS: Iron overload is a major factor contributing to the overall pathology of thalassaemia, which is primarily mediated by ineffective erythropoiesis and shorter mature red blood cell (RBC) survival. Iron accumulation in RBCs generates reactive oxygen species (ROS) that cause cellular damage such as lipid peroxidation and RBC membrane deformation. Abnormal RBCs in patients with thalassaemia are commonly known as microcytic hypochromic anaemia with poikilocytosis. However, iron and ROS accumulation in RBCs as related to RBC morphological changes in patients with thalassaemia has not been reported. METHODS: Twenty-one patients with thalassaemia, including HbH, HbH with Hb Constant Spring and ß-thalassaemia/HbE (splenectomy and non-splenectomy) genotypes, and five normal subjects were recruited. RBC morphology was analysed by light and scanning electron microscopy. Systemic and RBC iron status and oxidative stress were examined. RESULTS: Decreased normocytes were observed in the samples of patients with thalassaemia, with RBC morphological abnormality being related to the type of disease (α-thalassaemia or ß-thalassaemia) and splenic status. Target cells and crenated cells were mainly found in splenectomised patients with ß-thalassaemia/HbE, while target cells and teardrop cells were found in non-splenectomised patients. Patients with thalassaemia had high levels of serum ferritin, red cell ferritin and ROS in RBCs compared with normal subjects (p<0.05). Negative correlations between the amount of normocytes and serum ferritin (rs=-0.518, p=0.011), red cell ferritin (rs=-0.467, p=0.025) or ROS in RBCs (rs=-0.672, p<0.001) were observed. CONCLUSIONS: Iron overload and its consequent intracellular oxidative stress in RBCs were associated with reduce normocytes in patients with thalassaemia.