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  • Despite a large amount of uptake for both nanoparticles only

    2022-10-02

    Despite a large amount of uptake for both nanoparticles, only after transport across intestinal epithelia do the nanoparticles really contribute to improving absorption. Though the transport of nanorods across monolayer was a little bit more than nanospheres, the total transport was less than 2% (A1 and B1). Furthermore, the mucus layer in co-culture model of Caco-2 and HT29-MTX also impeded the transport of both nanoparticles. After 4 h of transport experiments, the nanoparticles were removed absolutely, and then the cell monolayers were washed by cold HBSS for three times. The fresh HBSS were added in apical (AP) and basolateral (BL) chambers for exocytosis towards AP and BL sides, respectively. Interestingly, more nanorods were excreted from BL side (Figs. 3A3 and B3) while more nanospheres were exocytosed from AP side (Figs. 3A2 and 3B2), which implied that the exocytosis mechanisms at AP and BL membranes were absolutely different. After 4 h of exocytosis, the monolayers were observed by CLSM, which showed more nanorods (D) stayed in cells compared to nanospheres (C). The similar results were obtained from co-culture monolayer of Caco-2 and HT29-MTX (Fig. S3 in Supporting information). It has been proven that the nanoparticles were difficult to transport through epithelia in a great amount. In fact, most biodegradable nanoparticles enhance oral Canrenone by increasing dispersity or stability of drugs in GI tract, or by improving the apparent dissolution of poorly soluble drugs. The contribution of intact nanoparticles absorption to bioavailability was even negligible []. In contrast to transport, epithelial uptake of nanoparticles is tremendous, which leads to a viewpoint of “easy entry and hard across” for polymeric nanoparticles []. In this paper, this point of view was clarified again and we also demonstrated that most of nanoparticles were exocytosed from cells very fast. In addition, the shape of nanoparticles was found to be a critical factor influencing the exocytosis of nanoparticles. More nanorods stayed in the cells for a long time compared to nanospheres. Hence, the shape of nanoparticles should be intently considered in oral drug delivery because the shape could influence the therapeutic effects, toxicity or oral absorption.
    In 1973, Ricardo Miledi published a lone-author paper entitled ‘Transmitter release induced by injection of calcium ions into nerve terminals’ . This elegant paper rigorously established the Ca hypothesis for neurotransmitter release by demonstrating that Ca is necessary and sufficient for triggering exocytosis in nerve terminals. Two key questions emerged: what is the Ca sensor that mediates this process and what are the immediate consequences of Ca binding to this protein? In other words, how – biophysically – could Ca binding trigger a SV to fuse with the plasma membrane? The molecular part of this story began in 1981, when Matthew . generated monoclonal antibodies against isolated synaptic junctions, which contain both pre- and postsynaptic elements. This effort resulted in the characterization of two antibodies that specifically recognized a 65-kDa SV and large dense core vesicle protein that the authors named p65. At the time, SVs could already be purified in large amounts and the systematic cloning of all constituent proteins was under way. In 1990, Perin . cloned a cDNA encoding p65 and eventually renamed the protein syt1 . The sequence of syt1 revealed a striking clue regarding a potential function: the presence of tandem C2 domains, conserved ∼135-residue motifs found in protein kinase C isoforms that are activated by phosphatidylserine and – notably – Ca. This hinted at the possibility of p65/syt1 acting as a Ca sensor, but does this protein actually bind Ca? In 1992, Reinhard Jahn’s laboratory directly addressed this question when a graduate student, Nils Brose, conducted a groundbreaking experiment: he immunopurified syt1 from brain tissue and used a number of rigorous, independent methods to determine whether it sensed Ca. The use of native protein was crucial, as the original cDNA encoding syt1 harbored a point mutation (G374D) in its second C2 domain (C2B) that abolished function . Brose . observed that syt1 bound multiple Ca ions in the presence of acidic phospholipids. Tantalizingly, the estimated stoichiometry was approximately four Ca ions per molecule of syt1. This was provocative because the Hill coefficient for the Ca dependence of neurotransmitter release from nerve terminals is often observed to be approximately four, a value that represents a lower bound for the number of Ca ions that cooperate to trigger exocytosis.