Drug loading and release studies for milled silk particles of different sizes

Milled silk particles with volume median particle size (d(0.5)) of 7 μm and 281 nm as well as silk snippets were used for loading of model drugs Orange G, Azophloxine, Rhodamine B, and Crystal Violet. Loading and release of these chemicals depended on the size of silk particles, pH, and the structure and properties of model drugs. Both types of silk particles reached equilibrium loading in less than 10 min due to high surface area whereas silk fibres needed more than 2-3 days to reach equilibrium, depending on the drug type. The uptake rate in fibres could be improved by increasing temperature. Both fibres and particles could slowly release the drugs over many days at 37 °C without a significant initial burst. As particle size decreased, the amount of model drug release also decreased. The release of drugs by the silk fibres was quicker than the silk particles.

Self-assembly of monolayered lipid membranes for surface-coating of a nanoconfined Bombyx mori silk fibroin film

Regenerated Bombyx mori (B. mori) silk fibroin is a type of widely used biomaterial. The β-sheet structure of it after methanol treatment provides water-insolubility and mechanical stability while on the other side leads to a hydrophobic surface which is less preferred by biological systems. In this work we prepare a novel type of nanoconfined silk fibroin film with a thickness below 100 nm. The film has a flat while hydrophobic surface because of its β-sheet structure due to the z-direction confinement during formation. Different types of lipid monolayers, DOPC, DPPC and MO, are assembled on the silk film surface. The lipid coating, especially the DPPC membrane, provides a much smoother and more hydrophilic surface due to the gel phase tails of the lipids, in comparison with the DOPC and MO ones which are in a liquid phase and have a much stronger interfacial association between silk film surface and lipid tails. Such a lipid coating preserves the biocompatibility and cellular affinity of the silk film which promises potential applications as surface coatings for materials for biological use.

Altered expression of two zinc transporters, SLC30A5 and SLC30A6, underlies a mammary gland disorder of reduced zinc secretion into milk

Two cases of zinc deficiency in breastfed neonates were investigated where zinc levels in the mothers' milk were reduced by more than 75 % compared to normal. The objective of this study was to find the molecular basis of the maternal zinc deficiency condition. Significant reductions in mRNA expression and protein levels of the zinc transporters SLC30A5 and SLC30A6 were found in maternal tissue, suggesting a causal link to the zinc-deficient milk. Novel splice variants of the SLC30A6 transcript were detected. No modifications were found in coding regions, or in transcription binding sites of promoter regions or in 5' and 3' untranslated regions of both transporters in lymphoblasts and fibroblasts isolated from both mothers. Altered DNA methylation in SLC30A5 at two CpG sites was detected and may account for the reduced levels of SLC30A5 mRNA and protein in lymphoblasts. Reduced SLC30A6 mRNA and protein levels in lymphoblasts may be secondary to reduced SLC30A5 expression, as they function as a heterodimer in zinc transport. In conclusion, two cases of zinc deficiency are linked to low levels of the SLC30A5 and SLC30A6 zinc transporters. These two zinc transporters have not been previously associated with zinc deficiency in milk.

A colostrum trypsin inhibitor gene expressed in the Cape fur seal mammary gland during lactation

The colostrum trypsin inhibitor (CTI) gene and transcript were cloned from the Cape fur seal mammary gland and CTI identified by in silico analysis of the Pacific walrus and polar bear genomes (Order Carnivora), and in marine and terrestrial mammals of the Orders Cetartiodactyla (yak, whales, camel) and Perissodactyla (white rhinoceros). Unexpectedly, Weddell seal CTI was predicted to be a pseudogene. Cape fur seal CTI was expressed in the mammary gland of a pregnant multiparous seal, but not in a seal in its first pregnancy. While bovine CTI is expressed for 24-48h postpartum (pp) and secreted in colostrum only, Cape fur seal CTI was detected for at least 2-3months pp while the mother was suckling its young on-shore. Furthermore, CTI was expressed in the mammary gland of only one of the lactating seals that was foraging at-sea. The expression of β-casein (CSN2) and β-lactoglobulin II (LGB2), but not CTI in the second lactating seal foraging at-sea suggested that CTI may be intermittently expressed during lactation. Cape fur seal and walrus CTI encode putative small, secreted, N-glycosylated proteins with a single Kunitz/bovine pancreatic trypsin inhibitor (BPTI) domain indicative of serine protease inhibition. Mature Cape fur seal CTI shares 92% sequence identity with Pacific walrus CTI, but only 35% identity with BPTI. Structural homology modelling of Cape fur seal CTI and Pacific walrus trypsin based on the model of the second Kunitz domain of human tissue factor pathway inhibitor (TFPI) and porcine trypsin (Protein Data Bank: 1TFX) confirmed that CTI inhibits trypsin in a canonical fashion. Therefore, pinniped CTI may be critical for preventing the proteolytic degradation of immunoglobulins that are passively transferred from mother to young via colostrum and milk.

Biodegradable Eri silk nanoparticles as a delivery vehicle for bovine lactoferrin against MDA-MB-231 and MCF-7 breast cancer cells

This study used the Eri silk nanoparticles (NPs) for delivering apo-bovine lactoferrin (Apo-bLf) (~2% iron saturated) and Fe-bLf (100% iron saturated) in MDA-MB-231 and MCF-7 breast cancer cell lines. Apo-bLf and Fe-bLf-loaded Eri silk NPs with sizes between 200 and 300 nm (±10 nm) showed a significant internalization within 4 hours in MDA-MB-231 cells when compared to MCF-7 cells. The ex vivo loop assay with chitosan-coated Fe-bLf-loaded silk NPs was able to substantiate its future use in oral administration and showed the maximum absorption within 24 hours by ileum. Both Apo-bLf and Fe-bLf induced increase in expression of low-density lipoprotein receptor-related protein 1 and lactoferrin receptor in epidermal growth factor (EGFR)-positive MDA-MB-231 cells, while transferrin receptor (TfR) and TfR2 in MCF-7 cells facilitated the receptor-mediated endocytosis of NPs. Controlled and sustained release of both bLf from silk NPs was shown to induce more cancer-specific cytotoxicity in MDA-MB-231 and MCF-7 cells compared to normal MCF-10A cells. Due to higher degree of internalization, the extent of cytotoxicity and apoptosis was significantly higher in MDA-MB-231 (EGFR+) cells when compared to MCF-7 (EGFR-) cells. The expression of a prominent anticancer target, survivin, was found to be downregulated at both gene and protein levels. Taken together, all the observations suggest the potential use of Eri silk NPs as a delivery vehicle for an anti-cancer milk protein, and indicate bLf for the treatment of breast cancer.

The relationship between milk, mammary adipocytes and ECM in regulating murine mammary gland involution

 This thesis investigated the role of milk, extracellular matrix and mammary adipocytes in regulating mammary gland function during involution in mice and explored the use of an in vitro culture model, the mammosphere model system to study the same.

'What if you live on top of a bakery and you like cakes?'- Drug use and harm trajectories before, during and after the emergence of Silk Road

BACKGROUND: Cryptomarkets are digital platforms that use anonymising software (e.g. Tor) and cryptocurrencies (e.g. Bitcoin) to facilitate peer-to-peer (P2P) trade of goods and services. Their emergence has facilitated access to a wide range of high-quality psychoactive substances, according to surveys of users. In this paper, we ask the question 'How does changing access to drugs through cryptomarkets affect the drug use and harm trajectories of their users?'

METHODS: We conducted a digital ethnography spanning 2012-2014, a period that included the seizure of the original Silk Road marketplace and forum by law enforcement. Using encrypted online chat, we interviewed 17 people who reported using Silk Road to purchase illicit drugs. The interviews were in-depth and unstructured, and also involved the use of life history timelines to trace trajectories. Transcripts were analysed thematically using NVivo.

RESULTS: For some, Silk Road facilitated initiation into drug use or a return to drug use after cessation. Typically, participants reported experiencing a glut of drug consumption in their first months using Silk Road, described by one participant as akin to 'kids in a candy store'. There was evidence that very high availability reduced the need for drug hoarding which helped some respondents to moderate use and feel more in control of purchases made online. Cryptomarket access also appeared to affect solitary and social drug users differently. Most participants described using other cryptomarkets after the closure of Silk Road, albeit with less confidence.

CONCLUSION: In the context of high levels of drug access, supply and diversity occurring within a community regulated environment online, the impacts of cryptomarkets upon drug use trajectories are complex, often posing new challenges for self-control, yet not always leading to harmful outcomes. A major policy challenge is how to provide support for harm reduction in these highly volatile settings.

Silk fibroin as a water-soluble bio-resist and its thermal properties

Thermal diffusivity of silk fibroin films, α = (1.6 ± 0.24) × 10-7 m2 s-1, was measured by a direct contact method. It was shown to be reduced down to ∼1 × 10-7 m2 s-1 in the crystallized phase, consistent with the multi-domain composition of β-sheet assemblies. Crystalline silk with β-sheets was made by dipping into alcohol and was used as a positive electron beam lithography (EBL) resist. It is shown by direct IR imaging of the 1619 cm-1 amide-I CO spectral signature and 3290 cm-1 amide-A N-H stretching band that an e-beam is responsible for unzipping β-sheets, which subsequently results in exposed areas returning to a water soluble state. This makes it possible to develop a water-based biocompatible silk resist and use it in lithography applications. The general principles of protein crystallization, traceable to spectral changes in IR amide bands of silk, can be used as a guide for the creation of new protein EBL resists and to quantify the electron dose required for solubility. Foam formation and laser treatments of silk can provide new approaches in surface functionalization and fabrication of 3D bio-scaffolds.

Interactions between fibroin and sericin proteins from antheraea pernyi and bombyx mori silk fibers

Silkworm silk fibers are core-shell composites of fibroin and sericin proteins. Studying the interactions between fibroin and sericin is essential for understanding the properties of these composites. It isobserved that compared to the domestic silk cocoon Bombyx mori (B. mori), the adhesion between fibroin and sericin from the wild silk cocoon, Antheraea pernyi (A. pernyi), is significantly stronger with a higherdegree of heterogeneity. The adsorption of A. pernyi sericin on its fibroin is almost twice the value for B.mori sericin on fibroin, both showing a monolayer Langmuir adsorption. 1H NMR and FTIR studiesdemonstrate on a molecular level the stronger interactions and the more intensive complex formation between A. pernyi fibroin and sericin, facilitated by the hydrogen bonding between glycine and serine.The findings of this study may help the design of composites with superior interfacial adhesion between different components.

Removal of copper ions from aqueous solution by adsorption onto novel polyelectrolyte film-coated nanofibrous silk fibroin non-wovens

In this approach, polyelectrolyte film-coated nanofibrous silk fibroin (SF) nonwovens were prepared from the alternate deposition of positively charged polyethylenimine (PEI) and negatively charged SF using electrostatic layer-by-layer (LBL) self-assembled technology. The composite membranes were characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectrometer. The SF-PEI multilayer-assembled nanofibers (less than five layers) were fine and uniform with the fiber diameter from 400 nm to 600 nm, and had very large surface area and high porosity (more than 70%). The amino groups of PEI were proved to be deposited onto SF nonwovens, which granted the coated nonwovens with potential applicability for copper ions adsorption. The PEI films coated SF substrate showed much higher copper ions adsorption capacity than that of ethanol treated SF nanofibers. Adding the number of PEI coated could enhance the Cu²⁺ adsorption capacity significantly. The maximum milligrams per gram of copper ions adsorbed reached 59.7 mg/g when the SF substrate was coated with 5 bilayers of SF-PEI. However, the copper ions adsorption capacity had no obvious change as the number of PEI continued to increase. These results suggest potential for PEL film-coated nanofibrous nonwovens as a new adsorbent for metal ions.

Functionality of nano and 3D-microhierarchical TiO₂ particles as coagulants for sericin extraction from the silk degumming wastewater

Amine-functionalised TiO2 particles were employed as coagulants to remove sericin from the silk degumming wastewater. Two types of TiO2 particles including 3D-microhierarchical TiO2 (Micron-size) and TiO2 nanoparticles (Degussa P-25) were used in this study. The surfaces of both types of TiO2 particles were functionalised using 3-aminopropyl trimethoxysilane (APTMS). The impacts of TiO2 type, pH, TiO2 concentration, and settling time on sericin removal efficiency were investigated. The efficiency of TiO2 particles in sericin removal was evaluated by measuring the residual turbidity and UV-vis spectra of the solutions before and after the treatment. Moreover, the COD, SDS-PAGE and protein assay tests were conducted to further analyse the treated solutions. The results demonstrated that the sericin removal efficiency of around 67% and turbidity reduction of 95% were achieved at the optimum conditions of 0.04 g TiO2, pH = 5, and 60 min settling time. Nano and micron-size TiO2 particles showed similar efficiency for sericin removal, but micron-size particles outweighed due to their higher efficiency in inducing greater turbidity reduction after 60 min. The obtained sericin/TiO2 composites were stable after several cycles of wash and could be useful in different fields including cosmetic, and textile finishing, among others.

Sunlight-induced coloration of silk

Silk fabrics were colored by gold nanoparticles (NPs) that were in situ synthesized through the induction of sunlight. Owing to the localized surface plasmon resonance (LSPR) of gold NPs, the treated silk fabrics presented vivid colors. The photo-induced synthesis of gold NPs was also realized on wet silk through adsorbing gold ions out of solution, which provides a water-saving coloration method for textiles. Besides, the patterning of silk was feasible using this simple sunlight-induced coloration approach. The key factors of coloration including gold ion concentration, pH value, and irradiation time were investigated. Moreover, it was demonstrated that either ultraviolet (UV) light or visible light could induce the generation of gold NPs on silk fabrics. The silk fabrics with gold NPs exhibited high light resistance including great UV-blocking property and excellent fastness to sunlight.