Cell-mediated transfer of catalase nanoparticles from macrophages to brain endothelial, glial and neuronal cells.

BACKGROUND: Our laboratories forged the concept of macrophage delivery of protein antioxidants to attenuate neuroinflammation and nigrostriatal neurodegeneration in Parkinson's disease. Notably, the delivery of the redox enzyme, catalase, incorporated into a polyion complex micelle ('nanozyme') by bone marrow-derived macrophages protected nigrostriatum against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intoxication. Nonetheless, how macrophage delivery of nanozyme increases the efficacy of catalase remains unknown. METHODS: In this study, we examined the transfer of nanozyme from macrophages to brain microvessel endothelial cells, neurons and astrocytes. RESULTS: Facilitated transport of the nanozyme from macrophages to endothelial, neuronal and glial target cells occurred through endocytosis-independent mechanisms that involved fusion of cellular membranes, macrophage bridging conduits and nanozyme lipid coatings. Nanozyme transfer was operative across an artificial blood-brain barrier and showed efficient reactive oxygen species decomposition. CONCLUSION: This is the first demonstration, to our knowledge, that drug-loaded macrophages discharge particles to contiguous target cells for therapeutic brain enzyme delivery. The data shown are of potential value for the treatment of neurodegenerative disorders and notably, Parkinson's disease.

The modification of siRNA with 3' cholesterol to increase nuclease protection and suppression of native mRNA by select siRNA polyplexes.

Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3' cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC.

Effects of the PPARgamma agonist troglitazone on endothelial cells in vivo and in vitro: differences between human and mouse.

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists and PPARgamma/alpha dual agonists have been or are being developed for clinical use in the treatment of type 2 diabetes mellitus and hyperlipidemias. A common tumor finding in rodent carcinogenicity studies for these agonists is hemangioma/hemangiosarcoma in mice but not in rats. We hypothesized that increased endothelial cell proliferation may be involved in the mechanism of PPAR agonist-induced vascular tumors in mice, and we investigated the effects on endothelial cells utilizing troglitazone, the first clinically used PPARgamma agonist, in vivo and in vitro. Troglitazone (400 and 800 mg/kg/day) induced hemangiosarcomas in mice in a 2-year bioassay. We showed that troglitazone increased endothelial cell proliferation in brown and white adipose tissue and liver in mice at sarcomagenic doses after 4 weeks of treatment. Troglitazone was cytotoxic both to human dermal microvascular endothelial cells (HMEC1) and mouse mammary fat pad microvascular endothelial cells (MFP MVEC) at high concentrations. However, MFP MVEC were more resistant to the cytotoxic effects of troglitazone based on the much lower LC(50) in HMEC1 (17.4 muM) compared to MFP MVEC (92.2 muM). Troglitazone increased the proliferation and survival of MFP MVEC but not HMEC1 in growth factor reduced conditions. Our data demonstrate that troglitazone may induce hemangiosarcomas in mice, at least in part, through enhancement of survival and proliferation of microvascular endothelial cells. Such an effect does not occur with human cells, suggesting that human may react differently to exposure to PPAR agonists compared with mice.

Formulations of biodegradable Nanogel carriers with 5'-triphosphates of nucleoside analogs that display a reduced cytotoxicity and enhanced drug activity.

Therapies including nucleoside analogs are associated with severe toxic side effects and acquirement of drug resistance. We have previously reported the drug delivery in the form of 5'-triphosphates (NTP) encapsulated in cross-linked cationic networks of polyethylenimine (PEI) and PEG/Pluronic polymers (Nanogels). In this study, Nanogels, containing biodegradable PEI that could easily dissociate in reducing cytosolic environment and form products with minimal toxicity, were synthesized and displayed low cytotoxicity. Toxicity of Nanogels was clearly dependent on the total positive charge of carriers and was 5-6 fold lower for carriers loaded with NTP. Though intracellular ATP level was immediately reduced by ca. 50% following the treatment with Nanogels, it was largely restored 24 h later. Effect of Nanogels on various respiratory components of cells was reversible too, and, therefore, resulted in low immediate cell death. Nanogel alone and formulations with AZT-TP demonstrated a much lower mitochondrial toxicity than AZT. As an example of potential antiviral applications of low-toxic Nanogel carriers, a 5'-triphosphorylated Ribavirin-Nanogel formulation was prepared that demonstrated a 30-fold decrease in effective drug concentration (EC(90)) and, totally, a 10-fold increase in selectivity index compared to the drug alone in MDCK cells infected with influenza A virus.