Passive targeting of large nanoparticles by the enhanced permeability and retention (EPR) effect is a crucial concept for solid tumor targeting in cancer nanomedicine. There is, however, a trade-off between the long-term blood circulation of nanoparticles and their nonspecific background tissue uptake. To define this size-dependent EPR effect, we designed near-infrared fluorophoreconjugated polyethylene glycols (PEG-ZW800s; 1-60 kDa) and evaluated their biodistribution, pharmacokinetics, and renal clearance in tumor-bearing mice. The targeting efficiency of sizevariant PEG-ZW800s was investigated in terms of tumor-to-background ratio (TBR). Interestingly, smaller sized PEGs (≤20 kDa, 12 nm) exhibited significant tumor targeting with minimum to no nonspecific uptakes, while larger sized PEGs (>20 kDa, 13 nm) accumulated highly in major organs, including the lungs, liver, and pancreas. Among those tested, 20 kDa PEG-ZW800 exhibited the highest TBR, while excreting unbound molecules to the urinary bladder. This result lays a foundation for engineering tumor-targeted nanoparticles and therapeutics based on the sizedependent EPR effect.

No abstract

The prophenoloxidase (proPO) activation pathway, like the vertebrate complement system, consists of a protease cascade and functions as a non-self-recognition system in these animals. Determining the molecular mechanism by which pattern recognition molecules differentiate non-self from self and transduce signals that stimulate defense responses is a key for understanding the ways in which innate immune systems are regulated. However, the proPO system is poorly defined at the molecular level. The proPO-activating system of the insect Holotrichia diomphalia comprises several components, some of which have been cloned and characterized, such as the novel 27-kDa proPO-activating factor-III (PPAF-III) from the plasma of H. diomphalia larvae and two prophenoloxidases. The PPAF-III gene encodes an easter-type serine protease zymogen consisting of 351 amino acid residues with a mass of 40 kDa. The purified 27-kDa PPAF-III specifically cleaved a 55-kDa proPPAF-II to generate a 45-kDa PPAF-II with or without Ca 2؉ present. Furthermore, two Holotrichia prophenoloxidases (proPO-I and -II) have been characterized, and their structural changes during activation were examined by in vitro reconstitution experiments. When the proPOs were incubated with PPAF-I, the 79-kDa proPOs were converted to 76-kDa proPOs, which did not exhibit any phenoloxidase (PO) activity. However, when the proPOs were incubated simultaneously with PPAF-I, proPPAF-II, and PPAF-III in the presence of Ca 2؉ , a 60-kDa protein (PO-1) with PO activity was detected in addition to the 76-kDa proPO-II protein. These results indicate that the conversion of Holotrichia proPOs to enzymatically active phenoloxidase is accomplished by PPAF-I, PAF-II, and PPAF-III through a twostep limited proteolysis in the presence of Ca 2؉ .The prophenoloxidase (proPO) 1 -activating system in invertebrates plays an important role in defense against pathogens and parasites and during cuticular sclerotization. The activation of the proPO system is triggered by elicitors derived from microbial cell walls, such as lipopolysaccharide (LPS), peptidoglycan, and ␤-1,3-glucan (1-3). Several pattern recognition molecules involved in the proPO system, such as peptidoglycan-binding proteins (4), proteins that bind both LPS and ␤-1,3-glucan (5, 6), and ␤-1,3-glucan-binding proteins (7, 8), have been found in various invertebrates. However, the key question is how these pattern recognition molecules can induce activation of the proPO system in response to microbial infection. One hypothesis is that the pattern recognition molecules make a complex with the proPO-activating enzyme(s) and microbial cell wall components, and then activated proPO activating enzyme(s) will convert proPO to active phenoloxidase (PO) by limited proteolysis (1-3).Recently, we characterized two new proPO-activating factors (PPAF-I and PPAF-II) from the coleopteran Holotrichia diomphalia larvae (9, 10). The overall structure of the 37-kDa proPPAF-I is highly similar to that of Drosophila easter, a serine protease that is ...

No abstract

Previously, we reported the molecular cloning of cDNA for the prophenoloxidase activating factor‐I (PPAF‐I) that encoded a member of the serine proteinase group with a disulfide‐knotted motif at the N‐terminus and a trypsin‐like catalytic domain at the C‐terminus [Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K.I., Natori, S., Kawabata, S.I., Iwanaga, S. & Lee, B.L. (1998) Eur. J. Biochem. 257, 615–621]. PPAF‐I is directly involved in the activation of pro‐phenoloxidase (pro‐PO) by limited proteolysis and the overall structure is highly similar to that of Drosophila easter serine protease, an essential serine protease zymogen for pattern formation in normal embryonic development. Here, we report purification and molecular cloning of cDNA for another 45‐kDa novel PPAF from the hemocyte lysate of Holotrichia diomphalia larvae. The gene encodes a serine proteinase homologue consisting of 415 amino‐acid residues with a molecular mass of 45 256 Da. The overall structure of the 45‐kDa protein is similar to that of masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster. The 45‐kDa protein contained a trypsin‐like serine proteinase domain at the C‐terminus, except for the substitution of Ser of the active site triad to Gly and had a disulfide‐knotted domain at the N‐terminus. A highly similar 45‐kDa serine proteinase homologue was also cloned from the larval cDNA library of another coleopteran, Tenebrio molitor. By in vitro reconstitution experiments, we found that the purified 45‐kDa serine proteinase homologue, the purified active PPAF‐I and the purified pro‐PO were necessary for expressing phenoloxidase activity in the Holotrichia pro‐PO system. However, incubation of pro‐PO with either PPAF‐I or 45‐kDa protein, no phenoloxidase activity was observed. Interestingly, when the 45‐kDa protein was incubated with PPAF‐I and pro‐PO in the absence, but not in the presence of Ca2+, the 45‐kDa protein was cleaved to a 35‐kDa protein. RNA blot hybridization revealed that expression of the 45‐kDa protein was increased in the Holotrichia hemolymph after Escherichia coli challenge.