4T1 cell membrane fragment reunited PAMAM polymer units disguised as tumor cell clusters for tumor homotypic targeting and anti-metastasis treatment

Cell membrane-based nanoparticles have garnered increasing attention owing to their inherent biomimetic properties, such as homotypic targeting, prolong circulation, and immune escaping mechanisms.

Two Distinct Modes of Exocytotic Fusion Pore Expansion in Large Astrocytic Vesicles

Formation of the fusion pore is a central question for regulated exocytosis by which secretory cells release neurotransmitters or hormones. Here, by dynamically monitoring exocytosis of large vesicles (2–7 μm) in astrocytes with two-photon microscopy imaging, we found that the exocytotic fusion pore was generated from the SNARE-dependent fusion at a ring shape of the docked plasma-vesicular membrane and the movement of a fusion-produced membrane fragment. We observed two modes of fragment movements, 1) a shift fragment that shifted to expand the fusion pore and 2) a fall-in fragment that fell into the collapsed vesicle to expand the fusion pore. Shift and fall-in modes are associated with full and partial collapses of large vesicles, respectively. The astrocytic marker, sulforhodamine 101, stained the fusion-produced membrane fragment more brightly than FM 1-43. Sulforhodamine 101 imaging showed that double fusion pores could simultaneously occur in a single vesicle (16% of large vesicles) to accelerate discharge of vesicular contents. Electron microscopy of large astrocytic vesicles showed shift and fall-in membrane fragments. Two modes of fusion pore formation demonstrate a novel mechanism underlying fusion pore expansion and provide a new explanation for full and partial collapses of large secretory vesicles.

A New Mechanism of Platelet Activation, Oxidation and Death Induced by ADAMTS-18.

Anti-platelet GPIII49–66 Ab obtained from HIV-1-ITP patients (or raised in rabbits) induces platelet oxidation, fragmentation and death by activating platelet 12-lipoxygenase (generating 12(S)-HETE) and NADPH-oxidase with exposure of membrane fragment phosphatidyl serine and thrombin-generating capacity (Cell106:551, 2001; JCI113: 973, 2004). Our recent studies reveal that activation of oxidative platelet death requires classic Ca++ flux (fura-2, AM) (completely inhibited by 100uM EGTA or 10uM BAPTA), and is associated with mild GPIIIa activation (PAC Ab binding) and prominent P-selectin release (n=4). However, robust activation of oxidative fragmentation/death is induced in the presence of 1uM PGE1, 10uM dibutyrl cyclic AMP (n=6) and occurs in Gαq KO mouse platelets (all conditions which inhibit ADP, collagen or thrombin-induced platelet activation). We have also observed that platelet oxidation/fragmentation can be induced independently of anti-GPIIIa49–66 by 10mM A23187, a Ca++ ionophore or 0.4uM PMA, a PKC activator. Both A23187 and PMA induce oxidation of platelets loaded with the oxidative fluorochrome, DCFH. Their reactivity is inhibited by the oxidation scavengers catalase (H2O2) and DPI (inhibitor of NADPH oxidase) and is absent in NADPH oxidase gp91phox(−/−) KO as well as 12-LO(−/−) KO mouse platelets (n=4). Thus, anti-GPIIIa49–66 could be inducing the intracellular effects of ionophore and PMA. We next looked for a possible physiologic mechanism. Platelet GPIIIa49–66 was panned with a phage-peptide display library. Twenty 7-mer peptide clones were found which reacted with GPIIIa49–66. One of these peptides (VHCVQLY) had 70% homology with ADAMTS-18, a disintegrin and metalloprotease with thrombospondin (TSP)-like motifs, constitutively secreted by endothelial cells. An 18-mer peptide of ADAMTS-18 was therefore synthesized from the C-terminal TSP motif and conjugated to biotin, Bio-VQTRSVHCVQQGRPSSSC-OH. The peptide alone had no effect on platelet oxidation/fragmentation. However, an anti-biotin Ab employed to cluster the peptide did induce oxidation/fragmentation (n=6). Recombinant ADAMTS 18 was then made with the expression vector pBudCE4.1 in 293T cells. It induced platelet 12(S)-HETE and oxidation/fragmentation in an identical kinetic fashion as anti-GPIIIa49–66 Ab. Both expressed rADAMTS-18 and HUVEC conditioned media ADAMTS-18 could be activated by thrombin (0.5 u/ml and then neutralized with hirudin) with optimum effect at 1 hr (n=4). HUVEC ADAMTS-18 was inactive in the absence of thrombin. ADAMTS-18 induced oxidation/fragmentation could be inhibited ~50% by an scFV Ab raised against the ADAMTS-18 (18-mer) peptide as well as GPIIIa49–66 peptide, as well as RGDS (GPIIIa ligand binding site) (n=7). Both peptides GPIII49–66 and RGDS were synergistic (~75% inhibited) when combined at optimum individualized concentration, suggesting 2 binding sites on platelet GPIIIa. Thus a mechanism is proposed for platelet thrombus clearance, induced by platelet membrane oxidative fragmentation leading to thrombin generation and activation of constitutively secreted endothelial cell ADAMTS-18.