The in vitro sub-cellular localization and in vivo efficacy of novel chitosan/GMO nanostructures containing paclitaxel.

PURPOSE: To determine the in vitro sub-cellular localization and in vivo efficacy of chitosan/GMO nanostructures containing paclitaxel (PTX) compared to a conventional PTX treatment (Taxol). METHODS: The sub-cellular localization of coumarin-6 labeled chitosan/GMO nanostructures was determined by confocal microscopy in MDA-MB-231 cells. The antitumor efficacy was evaluated in two separate studies using FOX-Chase (CB17) SCID Female-Mice MDA-MB-231 xenograph model. Treatments consisted of intravenous Taxol or chitosan/GMO nanostructures with or without PTX, local intra-tumor bolus of Taxol or chitosan/GMO nanostructures with or without PTX. The tumor diameter and animal weight was monitored at various intervals. Histopathological changes were evaluated in end-point tumors. RESULTS: The tumor diameter increased at a constant rate for all the groups between days 7-14. After a single intratumoral bolus dose of chitosan/GMO containing PTX showed significant reduction in tumor diameter on day 15 when compared to control, placebo and intravenous PTX administration. The tumor diameter reached a maximal decrease (4-fold) by day 18, and the difference was reduced to approximately 2-fold by day 21. Qualitatively similar results were observed in a separate study containing PTX when administered intravenously. CONCLUSION: Chitosan/GMO nanostructures containing PTX are safe and effective administered locally or intravenously. Partially supported by DOD Award BC045664.

A novel nanoparticle formulation for sustained paclitaxel delivery.

PURPOSE: To develop a novel nanoparticle drug delivery system consisting of chitosan and glyceryl monooleate (GMO) for the delivery of a wide variety of therapeutics including paclitaxel. METHODS: Chitosan/GMO nanoparticles were prepared by multiple emulsion (o/w/o) solvent evaporation methods. Particle size and surface charge were determined. The morphological characteristics and cellular adhesion were evaluated with surface or transmission electron microscopy methods. The drug loading, encapsulation efficiency, in vitro release and cellular uptake were determined using HPLC methods. The safety and efficacy were evaluated by MTT cytotoxicity assay in human breast cancer cells (MDA-MB-231). RESULTS: These studies provide conceptual proof that chitosan/GMO can form polycationic nano-sized particles (400 to 700 nm). The formulation demonstrates high yields (98 to 100%) and similar entrapment efficiencies. The lyophilized powder can be stored and easily be resuspended in an aqueous matrix. The nanoparticles have a hydrophobic inner-core with a hydrophilic coating that exhibits a significant positive charge and sustained release characteristics. This novel nanoparticle formulation shows evidence of mucoadhesive properties; a fourfold increased cellular uptake and a 1000-fold reduction in the IC(50) of PTX. CONCLUSION: These advantages allow lower doses of PTX to achieve a therapeutic effect, thus presumably minimizing the adverse side effects.

Release of TNF-alpha and IL-1beta from porcine brain endothelium corresponds to the pyrogenic potential of three marketed formulations of amphotericin.

BACKGROUND: Formulations of amphotericin include a deoxycholate suspension (d-Amph), an amphotericin-B lipid complex (Ablc), and a liposomal product (L-Amph). Fever is most frequent with d-Amph, intermediate with Ablc, and lowest with L-Amph. OBJECTIVE: To determine if the release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1) from brain endothelium corresponds to the incidence of amphotericin fever. RESULTS: Release of TNF-alpha and IL-1beta after L-Amph treatment was similar to negative controls while after d-Amph treatment release was similar to lipopolysaccharide. Ablc treatment produced intermediate pyrogen release.NF-kappaB expression, a transcriptional regulator for TNF-alpha and IL-1beta genes, corresponded to this secretion pattern. TNF-alpha release was elevated 2 hours (p = 0.0021) after treatment while significant elevations in IL-1beta required 6 hours (p = 0.0009). CONCLUSION: Results from this in vitro study suggest that amphotericin fever may be directly mediated by brain endothelium. These experiments also suggest that amphotericin fever is initially mediated by TNF-alpha.

Brain microvessel endothelial cell responses to tumor necrosis factor-alpha involve a nuclear factor kappa B (NF-kappaB) signal transduction pathway.

The involvement of nuclear factor kappa B (NF-kappaB) in TNF-induced increases in cerebral microvascular permeability was evaluated both in vitro, using primary cultured bovine brain microvessel endothelial cells (BBMEC), and in vivo, using the rat cranial window model. In primary cultured BBMEC, TNF exposure resulted in an increased appearance of the Rel A subunit of NF-kappaB in immunoblots of cell lysates. Increases in the Rel A subunit of NF-kappaB were observed as early as 30-min after administration of TNF. The increased permeability and the secretion of prostaglandin E2 in response to TNF exposure in BBMEC monolayers were significantly reduced by several different NF-kappaB inhibitors, including PDTC, CAPE, BAY 11-7085, and lactacystin. Similar results were also obtained in the rat cranial window model where treatment with the COX-2 inhibitor, NS-398 (0.1 microM), or the NF-kappaB inhibitor, PDTC (10 microM), significantly reduced the permeability increases produced by TNF. These studies suggest that the increases in BBB permeability following TNF exposure are attributable to activation of an NF-kappaB-mediated signaling pathway in the cerebral microvasculature.

Tumor necrosis factor-alpha induces cyclooxygenase-2 expression and prostaglandin release in brain microvessel endothelial cells.

Primary cultured bovine brain microvessel endothelial cells (BBMECs), were used as an in vitro model of the blood-brain barrier to examine the involvement of eicosanoids in the permeability and cytoskeletal structural changes observed following exposure to tumor necrosis factor-alpha (TNF-alpha). Compared with control monolayers, BBMECs exposed to TNF-alpha formed actin filament tangles and extracellular gaps with a resultant increase in permeability. Both the permeability and cytoskeletal changes observed with TNF-alpha were significantly reduced following pretreatment with NS-398 or indomethacin, inhibitors of cyclooxygenase (COX). Western blot analysis showed that TNF-alpha had no apparent effect on the expression of COX-1, but did induce the expression of COX-2 in the BBMECs. The induction of COX-2 expression occurred within the same time frame (2-4 h following TNF-alpha exposure) as the permeability increases observed with the cytokine. Consistent with the increased expression of COX-2, BBMEC monolayers exposed to TNF-alpha had significantly greater secretion and release of prostaglandin E(2) (PGE(2)) and prostaglandin F(2alpha) (PGF(2alpha)). Furthermore, BBMEC monolayers treated with PGE(2) or PGF(2alpha) showed significant increases in permeability and cytoskeletal structural changes when compared with control monolayers. Together, these results suggest that the TNF-alpha-induced permeability and cytoskeletal structural effects are due, in part, to an induction of the COX-2 system and increased release of prostaglandins in the cerebral microvasculature.