This could be related to steric hindrance of the terminal amines with the hyperbranched dendrimer structure, or to pH-dependent ionization of the dendrimer amino groups. A further possibility is that the heparin used in the present study Anti-infection Compound Library in vivo was a polydisperse mixture of heparin chains, whereas the assumption in calculating the +/−charge ratio was based on a single molecular weight. To confirm that an electrostatic-type interaction was the driving force for dendriplex formation, MB spectroscopy was employed. MB is a cationic metachromatic dye with an affinity

for polyanions such as heparin [10]. Unbound MB has a λmax of 664 nm whereas MB bound to heparin (MB–heparin) has a λmax of 568 nm ( Fig. 3A). Dendrimer addition to MB–heparin caused a shift in λmax from 568 to 664 nm. MB and MB–dendrimer exhibit the same λmax of 664 nm, which

excludes any interaction between MB and the dendrimer. MB spectroscopy (A664/A568 ratio) was used to identify the ratio at which maximum dendrimer–heparin association occurred. First, the optimum heparin concentration required to produce a minimum A664/568 ratio was found experimentally, i.e. all MB selleck molecules (10 μM) were bound to heparin with no excess heparin (0.725 μM) in the solution; excess free heparin in the medium would give an inaccurate dendrimer/heparin association ratio. The MB–heparin mixture was titrated with dendrimer (0.16–10 μM). A maximum A664/A568 ratio was obtained at a 1:1 mass ratio (2:1+/−charge ratio or molar ratio) ( Fig. 3B). This result agrees with the zeta potential measurement study, which showed a negative zeta potential (−47 mV) at this molar ratio, which then became positive (+52 mV) when the ratio was increased; this PAK6 would indicate the presence of excess dendrimer on the complex surface at higher molar ratios. Antithrombin III (AT-III) is a natural inhibitor of thrombin, factor Xa and other coagulation

proteases in plasma. The rate of inhibition by AT-III is slow, but the rate can be increased several thousand times by the presence of heparin. Thus the antifactor Xa assay is a useful test to evaluate the anticoagulant activity of heparin. A commercial antifactor Xa assay kit was used to estimate the residual anticoagulant activity of heparin. Since both factor Xa and AT-III are present in excess in the assay kit, the rate of factor Xa inhibition is directly proportional to the heparin concentration. The residual activity of factor Xa, as measured by the absorbance of its chromogenic substrate at 405 nm, is inversely proportional to the anticoagulant activity of heparin in plasma. The antifactor Xa assay was employed to study the effect of complexation on the in vitro anticoagulant activity of heparin.