An Investigation into the Acidity-Induced Insulin Agglomeration: Implications for Drug Delivery and Translation.

Insulin undergoes agglomeration with (subtle) changes in its biochemical environment, including acidity, application of heat, ionic imbalance, and exposure to hydrophobic surfaces. The therapeutic impact of such unwarranted insulin agglomeration is unclear and needs further evaluation. A systematic investigation was conducted on recombinant human insulin-with or without labeling with fluorescein isothiocyanate-while preparing insulin suspensions (0.125, 0.25, and 0.5 mg/mL) at pH 3. The suspensions were incubated (37 °C) and analyzed at different time points (t = 2, 4, 24, 48, and 72 h). Transmission electron microscopy and nanoparticle tracking analysis identified colloidally stable (zeta potential 15 ± 5 mV) spherical agglomerates of unlabeled insulin (100-500 nm). Circular dichroism established the preservation of insulin's secondary structure rich in α-helices despite exposure to an acidic environment (pH 3) for 72 h. Furthermore, fluorescence lifetime imaging microscopy illustrated an acidic core inside these spherical agglomerates, while the acidity gradually lessened toward the periphery. Some of these smaller agglomerates fused to form larger chunks with discrete zones of acidity. The data indicated a primary nucleation-driven mechanism of acid-induced insulin agglomeration under physiologically relevant conditions.

A novel antiviral formulation containing caprylic acid inhibits SARS-CoV-2 infection of a human bronchial epithelial cell model.

A novel proprietary formulation, ViruSAL, has previously been demonstrated to inhibit diverse enveloped viral infections in vitro and in vivo. We evaluated the ability of ViruSAL to inhibit SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) infectivity, using physiologically relevant models of the human bronchial epithelium, to model early infection of the upper respiratory tract. ViruSAL potently inhibited SARS-CoV-2 infection of human bronchial epithelial cells cultured as an air-liquid interface (ALI) model, in a concentration- and time-dependent manner. Viral infection was completely inhibited when ViruSAL was added to bronchial airway models prior to infection. Importantly, ViruSAL also inhibited viral infection when added to ALI models post-infection. No evidence of cellular toxicity was detected in ViruSAL-treated cells at concentrations that completely abrogated viral infectivity. Moreover, intranasal instillation of ViruSAL to a rat model did not result in any toxicity or pathological changes. Together these findings highlight the potential for ViruSAL as a novel and potent antiviral for use within clinical and prophylactic settings.

Case Report: Hypergranular Platelets in Vaccine-Induced Thrombotic Thrombocytopenia After ChAdOx1 nCov-19 Vaccination.

BACKGROUND: Vaccine-induced thrombotic thrombocytopenia (VITT) post SARS-CoV-2 vaccination is characterized by thrombocytopenia and severe thrombosis. Platelet function during patient recovery in the medium-/long-term has not been investigated fully. Here, we undertook a 3-month study, assessing the recovery of a VITT patient and assessing platelet morphology, granule content and dense-granule release at two distinct time points during recovery. CASE PRESENTATION: A 61 year-old female was admitted to hospital 15 days post ChAdOx1 nCov-19 vaccination. Hematological parameters and peripheral blood smears were monitored over 3 months. Platelet morphology and granule populations were assessed using transmission electron microscopy (TEM) at two distinct time points during recovery, as was agonist-induced platelet dense-granule release. Upon admission, the patient had reduced platelet counts, increased D-dimer and high anti-PF4 antibodies with multiple sites of cerebral venous sinus thrombosis (CVST). Peripheral blood smears revealed the presence of large, hypergranular platelets. Following treatment, hematological parameters returned to normal ranges over the study period. Anti-PF4 antibodies remained persistently high up to 90 days post-admission. Two days after admission, VITT platelets contained more granules per-platelet when compared to day 72 and healthy platelets. Additionally, maximal ATP release (marker of dense-granule release) was increased on day 2 compared to day 72 and healthy control platelets. CONCLUSION: This study highlights a previously unreported observation of platelet hypergranularity in VITT which may contribute to the thrombotic risk associated with VITT. Optimal approaches to monitoring recovery from VITT over time remains to be determined but our findings may help inform therapeutic decisions relating to anticoagulation treatment in this novel pathology.

One particle, two targets: A combined action of functionalised gold nanoparticles, against Pseudomonas fluorescens biofilms.

Attempts to deal with the problem of detrimental biofilms using nanoparticle technologies have generally focussed on exploiting biocidal approaches. However, it is now recognised that biofilm matrix-components may be targets for the disruption or dispersion of biofilms. Here, we show that the functionalization of gold nanoparticles with the enzyme, proteinase-K (PK) led to both biocidal and matrix disruption effects within Pseudomonas fluorescens biofilms and released cells. This study highlights the potential mechanisms underpinning the properties of Proteinase-K functionalized gold nanoparticles. With the emergence of biocide-resistant biofilm-forming organisms, novel nanoparticle strategies may provide the ideal solution for disrupting and inactivating biofilm cells, thereby minimising the use of biocides or antibiotics.

Immunogold labeling reveals subcellular localisation of silica nanoparticles in a human blood-brain barrier model.

Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles.

A safe-by-design approach to the development of gold nanoboxes as carriers for internalization into cancer cells.

Gold nanomaterials are currently raising a significant interest for human welfare in the field of clinical diagnosis, therapeutics for chronic pathologies, as well as of many other biomedical applications. In particular, gold nanomaterials are becoming a promising technology for developing novel approaches and treatments against widespread societal diseases such as cancer. In this study, we investigated the potential of proprietary gold nanoboxes (AuNBs) as carriers for their perspective translation into multifunctional, pre-clinical nano-enabled systems for personalized medicine approaches against lung cancer. A safe-by-design, tiered approach, with systematic tests conducted in the early phases on uncoated AuNBs and more focused testing on the coated, drug-loaded nanomaterial toward the end, was adopted. Our results showed that uncoated AuNBs could effectively penetrate into human lung adenocarcinoma (A549) cells when in simple (mono-cultures) or complex (co- and three-dimensional-cultures) in vitro microenvironments mimicking the alveolar region of human lungs. Uncoated AuNBs were biologically inert in A549 cells and demonstrated signs of biodegradability. Concurrently, preliminary data revealed that coated, drug-loaded AuNBs could efficiently deliver a chemotherapeutic agent to A549 cells, corroborating the hypothesis that AuNBs could be used in the future for the development of personalized nano-enabled systems for lung cancer treatment.