ARTICLE | doi:10.20944/preprints201805.0419.v1
Online: 29 May 2018 (08:54:56 CEST)
A heterobifunctional reactive oxygen species (ROS)-responsive linker for directed drug assembly onto and delivery from a quantum dot (QD) nanoparticle carrier was synthesized and coupled to doxorubicin using EDC/sulfo-NHS coupling. The doxorubicin conjugate was characterized using 1H NMR and LC-MS and subsequently reacted under conditions of ROS formation (Cu2+/H2O2) resulting in successful and rapid thioacetal oxidative cleavage which was monitored using 1H NMR. The deprotected amine linker is amenable to peptide or protein conjugation prior to QD assembly or to direct conjugation to cognate reactive groups on ligands that cap the QD surface.
SHORT NOTE | doi:10.20944/preprints201801.0087.v1
Subject: Chemistry, Medicinal Chemistry Keywords: redox-sensitive; disulfide linker; gemini amphiphiles; gene therapy
Online: 10 January 2018 (09:12:03 CET)
The absence of highly effective delivery systems is a major challenge for gene therapy. Our work was aimed at the development of novel cationic liposomes possessing high transfection efficiency. For this purpose, a novel disulfide polycationic amphiphile 2S4 was synthesized. Cationic liposomes based on 2S4 and a helper lipid DOPE were formed by the thin film hydration method and exhibited effective pDNA delivery into the HEK293 cells, with a maximal transfection activity superior to that of the commercial agent Lipofectamine® 2000. Our results suggest that the polycationic amphiphile 2S4 is a promising candidate for in vitro nucleic acid delivery.
CONCEPT PAPER | doi:10.20944/preprints202003.0401.v1
Subject: Life Sciences, Biophysics Keywords: SNAP25; linker; protein lipid interaction; acceptor complex; exocytosis; fusion
Online: 27 March 2020 (02:56:59 CET)
A recent paper demonstrates the importance of the linker region joining the two SNARE motifs of the neuronal t-SNARE SNAP25 for maintaining rates of secretion with roles for distinct segments in speeding fusion pore expansion (Shaaban et al., 2019, Elife. 8). Remarkably, lipid perturbing agents rescue a palmitoylation-deficient phenotype that includes slow fusion pore expansion, suggesting that protein-protein interactions have a role not only in bringing together the granule or vesicle membrane with the plasma membrane but also in orchestrating protein-lipid interactions leading to the fusion reaction. Furthermore, biochemical investigations demonstrate the importance of the C-terminal domain of the linker in the formation of the plasma membrane t-SNARE acceptor complex for synaptobrevin2 (Jiang, et al., 2019, FASEB J. 33:7985-7994;Shaaban et al., 2019, Elife. 8). This insight, together with biophysical and optical studies from other laboratories (Wang, et al., 2008, Molecular Biology of the Cell. 19:3944-3955; Zhao, et al., 2013, Proc Natl Acad Sci U S A. 110:14249-14254) suggests that the plasma membrane SNARE acceptor complex between SNAP25 and syntaxin and the resulting trans SNARE complex with the v-SNARE synaptobrevin form just milliseconds before fusion.
ARTICLE | doi:10.20944/preprints202007.0180.v1
Subject: Medicine & Pharmacology, Other Keywords: Cationic cyclodextrin; cyclodextrin polymer; epichlorohydrin cross-linker; nasal delivery; cytotoxicity; cell uptake
Online: 9 July 2020 (08:05:35 CEST)
Cyclodextrin polymers have high applicability in pharmaceutical formulations due to better biocompatibility, solubility enhancement, loading capacity and controlled drug release than parent the cyclodextrins. The cytotoxicity and cell uptake of new cationic beta-cyclodextrin monomers and polymers were evaluated as suitable material for nasal formulations and their protective effects on cells exposed to hydrogen peroxide were studied. PC12 and CACO-2 cells were selected as the neuronal and epithelial type cells, respectively, to mimic the structure of respiratory and olfactory epithelia of the nasal cavity. All cationic beta-cyclodextrin polymers tested showed dose- and time-dependent toxicity; nevertheless, at 5 µM concentration and 60 min of exposure, the quaternary-ammonium-beta-cyclodextrin soluble polymer could be recognized as non-toxic. Based on these results, fluorescently labelled quaternary-ammonium-beta-cyclodextrin monomer and polymer were selected for uptake studies in CACO-2 cells. The monomeric and polymeric beta-cyclodextrins were internalized in the cytoplasm of CACO-2 cells; the cationic monomer showed higher permeability than the hydroxypropyl-beta-cyclodextrin, employed as comparison. Therefore, these cationic beta-cyclodextrins showed potential as excipients able to improve the nasal absorption of drugs. Furthermore, amino-beta-cyclodextrin and beta-cyclodextrin soluble polymers were able to reduce oxidative damage in PC12 and CACO-2 cells and thus could be studied as bioactive carriers or potential drugs for cells protection against oxidative stress.
ARTICLE | doi:10.20944/preprints201801.0245.v1
Subject: Life Sciences, Biochemistry Keywords: sulfoxidation; epoxidation; two-component monooxygenase; flavoprotein; enantioselective biotransformation; fusion protein; protein linker; soil microorganism
Online: 25 January 2018 (17:14:33 CET)
VpStyA1 and VpStyA2B of Variovorax paradoxus EPS is annotated and characterized as the first representative of an E2-type styrene monooxygenase of proteobacteria. It comprises a single epoxidase (VpStyA1) and a fusion protein (VpStyA2B) which serves mainly as NADH:FAD-oxidoreductase. VpStyA2B had a Km of 33.6 ± 4.0 µM for FAD and a kcat of 22.3 ± 1.1 s-1. VpStyA2B and VpStyA1 showed monooxygenase activity on styrene of 0.14 U mg-1 and 0.46 U mg-1 as well as on benzyl methyl sulfide of 1.62 U mg-1 and of 3.11 U mg-1. A putative fusion region at position 408 (AREAV) was mutated to provide insights on VpStyA2B-function. The best mutant (408-AAAAA) obtained showed a 6.6-times higher affinity for FAD while keeping the NADH-affinity and -oxidation activity. Corresponding epoxidase activity increased (1.6-times). But, other mutants showed still NADH:FAD-oxidoreductase activity, but lost mostly their epoxidase activity indicating effects on the monooxygenase-part as well. Thus, this monooxygenase system represents an interesting candidate for biocatalyst development.
ARTICLE | doi:10.20944/preprints202204.0072.v1
Subject: Life Sciences, Immunology Keywords: SARS-CoV-2; Variants; COVID-19 vaccine; Chimeric adenovirus-vectored vaccine; GS linker; Neutralizing activity; Th1 immune responses
Online: 8 April 2022 (05:03:04 CEST)
Several COVID-19 platforms have been licensed across the world thus far, but vaccine platforms research that can lead to effective antigen delivery is still ongoing. Here, we constructed AdCLD-CoV19 that could modulate humoral immunity by harboring SARS-CoV-2 antigens onto a chimeric adenovirus 5/35 platform that was effective in cellular immunity. By replacing the S1/S2 furin cleavage sequence of the SARS-CoV-2 Spike (S) protein mounted on AdCLD-CoV19 with the linker sequence, high antigen expression was confirmed in various cell lines. The high levels of antigen expression contributed to antigen-specific antibody activity in mice and non-human primates (NHPs) with single vaccination of AdCLD-CoV19. Furthermore, the adenovirus-induced Th1 immune response was specifically raised for the S protein, and these immune responses protected the NHP against live viruses. While AdCLD-CoV19 maintained neutralizing antibody activity against various SARS-CoV-2 variants, it was reduced to single vaccination for β and ο variants, and the reduced neutralizing antibody activity was restored with booster shots. Hence, AdCLD-CoV19 can prevent SARS-CoV-2 with single vaccination, and the new vaccine administration strategy that responds to various variants can maintain the efficacy of the vaccine.
ARTICLE | doi:10.20944/preprints201911.0023.v1
Subject: Life Sciences, Biotechnology Keywords: monoclonal antibodies; Mabs; fusion; false positives; hapten immunoassays; competitive immunoassays; ELISA; antibody validation; antibody quality; microarray; hybridoma technology; linker recognition; high-throughput screening; HTS; heterology concept
Online: 3 November 2019 (17:00:59 CET)
The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence a risky venture. We think that it is crucial to improve the screening process to eliminate most of the immanent deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and simultaneous performance of competition experiments. The latter can directly be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones and blank supernatant has been designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the IgG concentration, which is unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration is not possible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media had been used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system, we conclude that this approach should be preferable to most other protocols leading to many of false positives, causing expensive and lengthy confirmation steps to weed out the poor clones.