Fluorescent molecular imaging of metastatic lymph node using near-infrared emitting low molecular weight heparin modified nanoliposome based on enzyme-substrate interaction.

Tumor metastatic lymph node mapping has been widely used to predict the metastatic spread of primary tumor and guide the lymph node dissection in clinical practice. In this research, a new near-infrared (NIR)-emitting low molecular weight heparin (LMWHEP)-modified Cy7-loaded nanoliposome (LMWHEP-NLips/Cy7) was developed and had the particle size of about 80 nm and the fluorescence intensity of about 2300, which is optimal for metastatic lymph node uptake and imaging. The NIR-emitting nanoliposomes were designed by LMWHEP coating on the surface of Cy7-loaded nanoliposome (NLips/Cy7) according to electrostatic attraction. The LMWHEP-NLips/Cy7 with negligible cytotoxicity for Hela and RAW264.7 cells and was found to be fluorescent stability compared with the Cy7-free dye at room temperature. The BALB/c nude mice bearing tumor lymphatic metastasis was established at eighth week post-injection by subcutaneously injecting Hela cells suspension. Heparanase (HPA) expression concentrations quantitatively measured by ELISA kit respectively were 237.42U/mL, 214.82U/mL and 128.45U/mL in the extracellular Hela cells, metastatic popliteal and iliac lymph node. LMWHEP-NLips/Cy7 successfully increased fluorescence signal in the metastatic lymph node compared with normal lymph node and achieve in vivo and ex vivo high fluorescence signal within 10 min and retention time up to 4 h post-injection. Maximum mean fluorescence intensity of the LMWHEP-NLips/Cy7 group was significantly more than NLips/Cy7 group (increase 2-fold in the metastatic popliteal lymph node and 4.8-fold in the metastatic iliac lymph node, p < 0.05). The experimental results demonstrating LMWHEP-NLips/Cy7 have the potential utility as specific, biosafe and stable near-infrared imaging contrast agents for HPA-expression tumor metastatic lymph node mapping. Copyright © 2016 John Wiley & Sons, Ltd.

Preserving the original heparin structure of a novel low molecular weight heparin by gamma-irradiation.

Several preparations of low molecular weight heparins (LMWHs) obtained by physical depolymerisation (irradiation with gamma-rays) of pig mucosal heparin have been characterised by mono- and two-dimensional NMR spectroscopy. Integration of typical 1H- and 13C-NMR signals provided a useful quantification of their sulfation pattern. The availability of the corresponding parent heparins showed that the original structure (including that of the active site for antithrombin, as also confirmed by affinity chromatography) had not been significantly modified by the depolymerisation procedure. This process involves only a slight decrease of undersulfated sequences. A peculiarity of gamma-LMWH is the absence of the 'linkage region', commonly present in unmodified heparins and most LMWHs.

Pharmacologic profile of a low-molecular-weight heparin depolymerized by gamma-irradiation.

Low molecular weight heparins (LMWHs) are considered to be the agent of choice for the prophylaxis of DVT in medical and surgical patients. Conventionally, these agents have been produced by fractionation of or by chemical or enzymatic depolymerization of native heparin. The fractionated heparin retains many of its biological properties such as AT III affinity and sulfate content gamma-irradiation (60Co) has been used to depolymerize GAGs (De Ambrosi et al. In: biomedical and Biotechnological Advances in Industrial Polysaccharides, pp. 45-53). This procedures has now been used for the preparation of LMWH derivatives of varying molecular weight. The current studies examine the biochemical and pharmacologic profile of one such gamma-irradiated depolymerized heparin. In standard clotting and amidolytic antiprotease assays (PT, APTT, AXa, Alla), gamma-irradiated depolymerized heparin produced equal or stronger activity when compared to a LMWH produced by nitrous acid depolymerization and retained the ability to active AT III and HCHII. Initial results indicate that LMWHs produced by gamma-irradiation exhibit comparable antithrombotic actions to those produced by chemical depolymerization when measured in animal models of thrombosis. gamma-Irradiation may be a useful method for the production of LMWHs.

Chemical composition, particle size range, and biological activity of some low molecular weight heparin derivatives.

Several low molecular weight (LMW) heparin sodium derivatives from different sources, as well as some related regular heparin sodium preparations, were examined for chemical composition by high field (300 MHz) 1H NMR spectroscopy, for particle size range by quasi-elastic light scattering (QELS) methods, and for anti-coagulation potency and anti-factor Xa activity by the standard U.S. Pharmacopeial assays described for regular heparin. The NMR spectra provided insight into possible modes of depolymerization used to generate the LMW heparins, as well as into the presence of dermatan sulfate or other chemical contaminants. The QELS analysis permitted the heparin preparations to be characterized and compared by virtue of their distinctive particle size distributions.