Comparison Of Hemolytic Activities Research Articles

Drug-delivering-drug platform-mediated potent protein therapeutics via a non-endo-lysosomal route

Protein therapeutics is playing an increasingly critical role in treatment of human diseases. However, current vectors are captured by the digestive endo-lysosomal system, which results in an extremely low fraction (<2%) of protein being released in the cytoplasm. This paper reports a drug-delivering-drug platform (HA-PNPplex, 200 nm) for potent intracellular delivery of protein and combined treatment of cancer.Methods: The platform was prepared by loading functional protein on pure drug nanoparticles (PNPs) followed by hyaluronic acid coating and was characterized by dynamic light scattering, transmission electron microscopy, and gel electrophoresis. In vitro, cellular uptake, trafficking, and cytotoxicity were evaluated by flow cytometry and confocal laser microscopy. Protein expression was assayed by western blot. In vivo, blood circulation and biodistribution were studied using a fluorescence imaging system, antitumor efficacy was assessed in a caspase 3-deficient tumor model, and biocompatibility was determined by comparison of hemolytic activity and proinflammatory cytokines and tissue histology.Results: HA-PNPplex delivered the functional protein, caspase 3, to cells via bypassing endo-lysosomes and raised the caspase-3 level 6.5-fold in caspase 3-deficient cells. Promoted tumor accumulation (1.5-fold) and penetration were exhibited, demonstrating a high tumor-targeting ability of HA-PNPplex. HA-PNPplex rendered a 7-fold increase in caspase 3 in tumor and allowed for a 100% tumor growth inhibition and >60% apoptosis, implying significant antitumor activities.Conclusions: This platform gains cellular entry without entrapment in the endo-lysosomes and enables efficient intracellular protein delivery and resultant profound cancer treatment. This platform, with extremely high drug-loading, is a valuable platform for combined cancer therapy with small-molecule drugs and proteins. More importantly, this work offers a robust and safe approach for protein therapeutics and intracellular delivery of other functional peptides, as well as gene-based therapy.

Comparison of hemolytic activity in eight species of polychaetes

Hemolytic capacity was investigated in homogenates of eight species of polychaetes, belonging to five different orders.Nainereis laevigata, Orbinia cuvieri, Cirriformia tentaculata, Notomastus latericeus, Arenicola claparedii andPetaloproctus terricola were collected during February 1990 from the coastal lagoon of Acquatina (Puglia, Italy).Nereis sp. (from the Yellow Sea, Japan) andMarphysa sanguinea (from Venice lagoon, Italy) were both purchased from a retail shop in Lecce (Italy). Homogenates from only three species [Nainereis laevigata andO. cuvieri (family Orbinidae, order Orbinia) andP. terricola (family Maldanidae, order Capitellida)] induced hemolysis in sheep red blood cells. Important differences in hemolytic titers, Ca- or Mg-dependence and in timecourse responses were noted between the three hemolytic species. On the other hand, thermolability and proteinase-inhibitor resistance (with the exception ofO. cuvieri) were features common to all three. Electrophoretic analysis revealed that hemolysis was mediated by apparently only one protein inN. laevigata andP. terricola, with no correlation between them or with the proteins responsible for hemolysis in the oligochaeteEisenia fetida andrei (Lumbricidae). No lytic protein was localized in hemolyticO. cuvieri homogenate, which was also the only homogenate to be partially inhibited by benzamidine, but not by phenylmethylsulfonyl fluoride or soybean trypsin inhibitor. These results underline the extreme variety existing among the species constituting the polychaete group.

Hemolytic activity in enterotoxigenic and non-enterotoxigenic strains of Escherichia coli.

We screened 223 strains of Escherichia coli belonging to serotypes previously associated with the production of enterotoxin for hemolytic activity, using horse erythrocytes in liquid and in agar media. Thirty-eight were hemolytic. They belonged to nine different serotypes; most (65.8%) belonged to one serotype, O6: H-. Additionally, all 38 strains were specifically assayed for a filterable, heat-labile hemolytic activity previously associated with a hemolysin plasmid. A comparison of hemolytic activity and enterotoxicity showed that none of 32 strains hemolytic in both media was enterotoxigenic; 28 of the 32 expressed heat-labile hemolytic activity. Four of the six strains hemolytic in only one of the media were enterotoxigenic; none of these six expressed heat-labile hemolytic activity. Of 223 strains, 176 that were of human origin and isolated in the United States were further assayed for three traditionally plasmid-mediated characteristics: heat-labile enterotoxin, heat-stable enterotoxin, and colonization factors. The interrelationships of these characteristics, including hemolytic activity, may reflect varying degrees of plasmid compatibility.

Interaction of staphylococcal alpha-toxin with artificial and natural membranes.

Comparison of hemolytic activity and chromate-releasing activity of partially purified preparations of staphylococcal alpha-toxin indicated the presence of a lytic factor other than alpha-toxin. This lytic release factor (RF) was isolated from the preparations and was shown to be active against both lipid spherules and erythrocytes. Heat-purified alpha-toxin (HP alpha-toxin) disrupted spherules, with the formation of fragments which always showed the presence of ring structures similar in dimensions (ca. 90 A) to pure alpha 12S-toxin. The interaction of HP alpha-toxin with spherules was accompanied by loss of hemolytic activity and adsorption of toxic protein. The alpha 12S-toxin, although only weakly hemolytic, was shown to be lytic for spherules. An alpha 12S-free toxin rapidly disrupted spherules, with formation of fragments with attached rings similar in dimensions to the alpha 12S molecule. Lipid monolayer experiments showed that HP alpha-toxin could penetrate lipid monolayers by virtue of a hydrophobic interaction. Effects of HP alpha-toxin on rabbit and human erythrocyte ghosts were similar to its effects on spherules, in that rings appeared on membrane fragments. Toxin-lysed rabbit erythrocytes showed similar rings on the resulting membrane fragments. However, rings were not seen on toxin-treated rabbit erythrocytes in the prelytic lag phase; this result and the fact that human erythrocytes are largely insensitive to alpha-toxin were interpreted as evidence against a lytic mechanism involving ring formation as the primary event. Rings were interpreted as toxin polymers similar to alpha 12S molecules, formed from specifically orientated active toxin molecules at the surface of lipid structures. Possible mechanisms for toxin lysis of spherules and erythrocytes are discussed.