Total RNA was extracted from cells harvested at 48 h, 7 days, and 28 days of exposure to GO-US, GO-S, and GO-L (Graphene Oxide)

3-6-2020    Treatment protocol

For short-term exposure (48 h), BEAS-2B were seeded in 6 well-plates (2.5x104 cells/mL) and were allowed to attach for 3 h prior to the exposure.  For long-term exposure (up to 28 days), BEAS-2B were seeded in T25 flasks (2.5x104 cells/mL) and allowed to attach for 3 h prior to the exposure.  After cell attachment, cells were exposed to Graphene Oxide (GO).  For long-term experiments, cells were exposed for 7 and 28 days, in triplicate, to 1 μg/mL and 5 μg/mL GO. To this end, the cells were split, counted, reseeded twice a week (2.5x104 cells/mL) and re-exposed to the respective GOs.  While for short term exposure, cells were exposed for 48 h to 8 μg/mL, 40 μg/mL, or 80 μg/mL

Extraction protocol

Total RNA was extracted from cells harvested at 48 h, 7 days, and 28 days of exposure to GO-US, GO-S, and GO-L using the RNeasy Mini Columns (Qiagen) in accordance with manufacturer’s instructions (including the purification step with DNase I). Total RNA concentration was determined spectrophotometrically using NanoDrop (NanoDrop Technologies). Quality control was conducted using the Bioanalyzer 2100 (Agilent Technologies) and all samples had RNA integrity numbers (RIN) above 8. Triplicates of each sample at different time-points and exposures were submitted for sequencing.

Illumina TruSeq Stranded mRNA Library Prep Kit (Illumina, Inc., San Diego, CA, USA) was used to prepare mRNA sequencing libraries.  Briefly, 1000 ng of total RNA was used for mRNA isolation using poly dT-coated beads   https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM4386965

…………………………………………............................................

      (A SUBTLE METHOD TO KILL BRAIN CELLS:)

9-23-2020    Nose-to-Brain Translocation and Cerebral Biodegradation of Thin Graphene Oxide Nanosheets     

  We explore tissue location and in vivo biodegradability of the translocated materials using various techniques.  Mass spectrometry and confocal Raman analyses indicate that trace amounts of GO undergo nose-to-brain translocation in a size-dependent manner.  The smallest GO-sheet size category (us-GO, 10-550 nm) gains the greatest access to the brain in terms of quantity and coverage.    https://www.sciencedirect.com/science/article/pii/S2666386420301879

………………………………………………………….......................

2009  Lonza (hq: Switz, who make the Moderna vax and then ship it for filling/bottling to Spain, from thence across the world) and Bio*One Capital celebrated the groundbreaking of their second manufacturing facility in Singapore at the Tuas Biomedical Park. This was the second large-scale mammalian manufacturing plant in Singapore, and the third one globally that Lonza had built.

Lonza initiated large-scale production capacity and an innovative technology platform for an emerging product class:  Antibody Drug Conjugates, (ADCs) which are utilized primarily in the treatment of cancer

………….

2011 Lonza expanded cytotoxic manufacturing capabilities in Visp, Switzerland to serve the growing oncology API market.  Cytotoxic APIs are commonly used in oncology therapeutics, which represent one of the fastest growing segments of the pharma and biotech industry.

In its largest-ever acquisition to date, Lonza acquires Arch Chemicals, Inc., a global biocides company providing innovative solutions to destroy or to selectively inhibit the growth of harmful microorganisms, to create the world’s leading Microbial Control business.  The combined businesses were well positioned to develop innovative microbial control formulations based on a broad portfolio of registered and approved active ingredients.

……..

2012 Lonza announced successful completion of a new GMP suite for the growing Viral Vaccine and Gene Therapy business in Houston, TX (USA). The flexibly designed clean room supports viral-based GMP manufacturing projects of up to 2,000 liter working volumes, using state-of-the-art disposable manufacturing technologies.

……………………………………………..

2017  With the intended acquisition of Capsugel, Lonza gets closer to its goal of becoming the world’s leading integrated solutions provider in the pharma and consumer healthcare and nutrition markets.  We become a fully integrated development, manufacturing and delivery technology partner.  https://history.lonza.com/history/all_years?q=%2Fhistory%2Fall_years

…………………………………………………………………………

2-27-2019       Among the reported effects of GO in mammalian cells is the delineation of induction of cell death.8  For instance, studies have shown that GO could induce dose-dependent cell death in normal lung fibroblasts (HLF), macrophages (THP-1 and J744A), epithelial (BEAS-2B) cells, lung cancer cells A549, etc.8  However, the data are inconsistent and even contradictory with respect to how physicochemical properties like the lateral dimentional size, surface coating (PVP, PEG, Pluronic), and oxidation states contribute to toxicological effects in mammalian systems.8   Since GO nanosheets have also been reported to induce inflammation and fibrogenic effects in the lung,17 we hypothesized that the oxidation status and surface reactivity of the material play a key role in these adverse outcomes, and that this organ system could be useful to delineate the structure-activity relationships related to deliberate variation of the surface properties.4 …\

Following exposure for 40 h, animals were sacrificed and bronchoalveolar lavage fluid (BALF) obtained to examine the effects of GO on cells and cytokines.  Raman microscopy was used to assess GO uptake in pulmonary macrophages (Figure 5A). 

Assessment of lung cell death by TUNEL staining or immunohistochemistry analysis of the expression of activated caspase-3, showed significantly more cytotoxicity in the lungs of animals exposed to GO and hGO compared to rGO (Figure S4A and S4B),  Pulmonary cytotoxicity was further confirmed by assessment of lactate dehydrogenase (LDH) release in the BALF, which confirmed higher levels in GO and hGO exposed animals than mice aspirating rGO (Figure S5)….  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834379/

…………………………………………………................................