Comparison of Syringes With Intravitreal Anti-VEGF Drugs: Particle Burden and Protein Aggregates in Brolucizumab, Aflibercept and Bevacizumab
Schargus, Marc, Katharina Tatjana Kopp, Constanze Helbig, Andreas Frings, and Gerhard Winter. "Comparison of Syringes With Intravitreal Anti-VEGF Drugs: Particle Burden and Protein Aggregates in Brolucizumab, Aflibercept and Bevacizumab." Translational Vision Science & Technology 10, no. 9 (2021): 21-21.
Purpose: In a benchwork particle counting analytical evaluation, the number and type of particles in intravitreal injection formulations of three different agents against vascular endothelial growth factor were investigated. Methods: Commercially available ready-to-use aflibercept and brolucizumab glass syringes, vials containing bevacizumab (off-label use in ophthalmology), and repackaged ready-to-use plastic syringes containing bevacizumab were tested without filtration. Total visible, subvisible, and nanoparticles numbers and size distributions were quantified using light obscuration, flow imaging, resonant mass measurement (RMM), tunable resistive pulse sensing, and dynamic light scattering. Results: Repackaged bevacizumab showed overall low particle numbers, aflibercept showed high numbers of micrometer sized particles but low nanoparticle numbers, brolucizumab showed low to moderate numbers of micrometer sized particles but high nanoparticle numbers. RMM measurements identified particles in the nanometer range as either proteinaceous or silicon oil; the nature of the other particles was not further evaluated. Conclusions: Repackaged bevacizumab shows no inferior particle quality compared to ready-to-use products. It is relevant to study nanoparticle load of the products as the micrometer-sized particle numbers do not in all cases correlate to nanoparticle counts. Particularly for the high concentration product Beovu (brolucizumab), high nanoparticle numbers were found despite low numbers of micrometer sized particles. Silicone oil droplets did not account for high particle numbers as the measured numbers were low. Translational Relevance: Different side effects are registered in different frequencies with different intravitreal anti-VEGF-drugs and syringes, which are applied by injection by small 30G needles through the sclera directly to the intravitreal cavity. The study of nanoparticles and silicone oil droplets may be able to contribute to narrowing down the causes.
Bioengineered 3D tissue model of intestine epithelium with oxygen gradients to sustain human gut microbiome
The human gut microbiome is crucial to host physiology and health. Therefore, stable in vitro coculture of primary human intestinal cells with a microbiome community is essential for understanding intestinal disease progression and revealing novel therapeutic targets. Here, we present a three-dimensional (3D) scaffold system to regenerate an in vitro human intestinal epithelium that recapitulates many functional characteristics of the in vivo small intestine. The epithelium, derived from human intestinal enteroids, contains mature intestinal epithelial cell types and possesses selectively permeable barrier functions. Importantly, by properly positioning the scaffolds cultured under normal atmospheric conditions, two physiologically relevant oxygen gradients, a proximal-to-distal oxygen gradient along the gastrointestinal (GI) tract and a radial oxygen gradient across the epithelium, were distinguished in the tissues when the lumens were faced up and down in cultures, respectively. Furthermore, the presence of the low oxygen gradients supported the coculture of intestinal epithelial cells along with a complex living commensal gut microbiome (including obligate anaerobes) to simulate temporal microbiome dynamics in the native human gut. This unique silk scaffold platform may enable the exploration of microbiota-related mechanisms of disease pathogenesis and host-pathogen dynamics in infectious diseases including the potential to explore the human microbiome-gut-brain axis and potential novel microbiome-based therapeutics.
Citrus limon L.-Derived Nanovesicles Show an Inhibitory Effect on Cell Growth in p53-Inactivated Colorectal Cancer Cells via the Macropinocytosis Pathway
Edible plant-derived nanovesicles have been explored as effective materials for preventing colorectal cancer (CRC) incidence, dependent on gene status, as a K-Ras-activating mutation via the macropinocytosis pathway. Approximately 70% of CRC harbors the p53 mutation, which is strongly associated with a poor prognosis for CRC. However, it has not been revealed whether p53 inactivation activates the macropinocytosis pathway or not. In this study, we investigated parental cells, wild-type or null for p53 treated with Citrus limon L.-derived nanovesicles, as potential materials for CRC prevention. Using ultracentrifugation, we obtained C. limon L.-derived nanovesicles, the diameters of which were approximately 100 nm, similar to that of the exosomes derived from mammalian cells. C. limon L.-derived nanovesicles showed inhibitory effects on cell growth in not p53-wild, but also in p53-inactivated CRC cells. Furthermore, we revealed that the macropinocytosis pathway is activated by p53 inactivation and C. limon L.-derived nanovesicles were up taken via the macropinocytosis pathway. Notably, although C. limon L.-derived nanovesicles contained citrate, the inhibitory effects of citrate were not dependent on the p53 status. We thus provide a novel mechanism for the growth inhibition of C. limon L.-derived nanovesicles via macropinocytosis and expect to develop a functional food product containing them for preventing p53-inactivation CRC incidence.
Interleukin-1β released from macrophages stimulated with indium tin oxide nanoparticles induces epithelial-mesenchymal transition in A549 cells
Indium tin oxide (ITO) nanoparticles triggered the release of IL-1β from macrophages, followed by the significant induction of epithelial-mesenchymal transition (EMT) in alveolar epithelial cells. Epithelial–mesenchymal transition (EMT) is a crucial process by which epithelial cells lose polarity and acquire migratory mesenchymal properties, eventually leading to tissue fibrosis and cancer. Indium tin oxide (ITO) is one of the most widely manufactured materials with broad applications, such as flat panel displays, touch panels, and solar panels. Whereas cases of indium-related lung disease have been reported worldwide, the effects of ITO on the progression of EMT are completely unknown. In the current study, we explored whether ITO nanoparticles (NPs) induce EMT in human alveolar epithelial cells (A549 cells). We found that although ITO NPs did not directly induce EMT in A549 cells, a conditioned medium (CM) obtained from THP-1-derived macrophages (dTHP-1 cells) stimulated with ITO NPs induced morphological changes, high motility, and EMT progression in A549 cells. After co-culture with ITO NP-treated dTHP-1 cells, A549 cells exhibited morphological and molecular signatures of EMT. Furthermore, we identified that interleukin-1β (IL-1β) produced via the activation of nod-like receptor protein 3 (NLRP3) inflammasome is an ITO NP-mediated EMT inducer based on the results of cytokine array as well as cellular physiological and biochemical analysis. Our results also indicated that the IL-1β-mediated EMT occurs not only in A549 cells, but also in bronchial epithelial cells (BEAS-2B cells) and primary human alveolar epithelial cells (hAEC). In addition, a neutralizing antibody against IL-1 receptor can effectively inhibit the induction of EMT caused by CM from ITO NP-treated dTHP-1 cells. Taken together, these findings suggest that IL-1β is released from macrophages stimulated with ITO NPs and is able to induce EMT progression in A549 cells, thereby potentially triggering the genesis and development of pulmonary fibrosis.