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This paper reports a clear elucidation of the pathway for the cellular delivery of layered double hydroxide (LDH) nanoparticles intercalated with anti-restenotic low molecular weight heparin (LMWH). Cellular uptake of LMWH-LDH conjugates into cultured rat vascular smooth muscle cells (SMCs) measured via flow cytometry was more than ten times greater than that of LMWH alone. Confocal and transmission electron microscopy showed LMWH-LDH conjugates taken up by endosomes, then released into the cytoplasm. We propose that LMWH-LDH is taken up via a unique 'modified endocytic' pathway, whereby the conjugate is internalized by SMCs in early endosomes, sorted in late endosomes, and quickly released from late endosomes/lysosomes, avoiding degradation. Treatment of cells with LMWH-LDH conjugates suppressed the activation of ERK1/2 in response to foetal calf serum (FCS) for up to 24h, unlike unconjugated LMWH which had no significant effect at 24h. Improved understanding of the intracellular pathway of LMWH-LDH nanohybrids in SMC will allow for refinement of design for LDH nanomedicine applications., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Surgical procedures to remove atherosclerotic lesions and restore blood flow also injure the artery wall, promoting vascular smooth muscle cell (SMC) phenotypic change, migration, proliferation, matrix production and ultimately, restenosis of the artery. Hence identification of effective anti-restenotic strategies is a high priority in cardiovascular research, and SMCs are a key target for intervention. This paper presents the in vitro study of layered double hydroxides (LDHs) as drug delivery system for an anti-restenotic drug (low molecular weight heparin, LMWH). The cytotoxicity tests showed that LDH itself had very limited toxicity at concentrations below 50 microg/mL over 6-day incubation. LDH nanoparticles loaded with LMWH (LMWH-LDHs) were prepared and tested on rat vascular SMCs. When conjugated to LDH particles, LMWH enhanced its ability to inhibit SMC proliferation and migration, with greater than above 60% reduction compared with the control (growth medium) over 3 or 7-day incubation. Cellular uptake studies showed that compared with LMWH alone, LMWH-LDH hybrids were internalized by SMCs more rapidly, and uptake was sustained over a longer time, possibly revealing the mechanisms underlying the enhanced biological function of LMWH-LDH. The results suggest the potential of LMWH-LDH as an efficient anti-restenotic drug for clinical application., (Copyright 2010 Elsevier Ltd. All rights reserved.)

We investigate the possibility of coating polymer-covered stents with heparin-encapsulating liposomes for improving their haemocompatibility. Thin-film hydration (for multilamellar vesicles, MLV), and the dehydration-rehydration vesicle (DRV) methods are used for preparation of low-molecular weight heparin (LMWH)-encapsulating liposomes with varying lipid compositions. Liposomes are characterized for LMWH encapsulation and retention. For measurement of LMWH, a chromogenic technique is adjusted. For evaluation of heparin release from vesicles in platelet poor plasma (PPP) coagulation time is measured in presence of liposomal samples. Results reveal that LMWH encapsulation in liposomes is higher in DRV, however compositions with high encapsulation are leaky during buffer incubation. Most liposomes release LMWH slowly during plasma incubation (retention after 24 h ranges between 74% and 95%). Concerning the haemocompatibility of polyethylene terephthlate-covered stents after coating with LMWH-encapsulating liposomes, there is a marked increase (higher for DRV-coated stents compared to MLV) in plasma recalcification time compared to the control (plain blood) and reference (non-coated stent), which increases with blood-material contact time. This is probably due to LMWH release, demonstrating that encapsulated LMWH retains its biological functionality. Interestingly, the DRV-coated stents retained a high plasma recalcification time and a large number of liposomes on the stents (as proven by SEM studies) even after extensive washing (high shear conditions), proving that this method may be functional under high flow applying in vivo conditions.

To increase topical delivery of low-molecular-weight heparin (LMWH), cationic, neutral, and anionic flexible liposomes (cFlexosome, nFlexosome, and aFlexosome) were prepared. The effects of surface charge of Flexosome on physicochemical properties and skin penetration of LMWH were also investigated. Among the different formulations of Flexosome, cFlexosome demonstrated three-times higher entrapment efficiency of LMWH, and better physicochemical stability than nFlexosome and aFlexosome. In vitro skin penetration and in vivo localization into the deeper skin layer of LMWH were significantly greater from cFlexosome compared to other formulations. Changes of skin surface charge after LMWH-cFlexosome application were investigated as a function of time. In the process of skin penetration, the Flexosomes act as drug carrier with the associated LMWH. Overall, macromolecular LMWH could be delivered deeply into the skin by topical application of cFlexosome for the treatment of superficial thrombosis, subcutaneous wounds, bruise, and burns.