Lipid nanoparticles protect from edelfosine toxicity in vivo.

Edelfosine, an alkyl-lysophospholipid antitumor drug with severe side-effects, has previously been encapsulated into lipid nanoparticles (LN) with the purpose of improving their toxicity profile. LN are made of lipids recognized as safe by the Food and Drug Administration (FDA) and, therefore, these systems are generally considered as nontoxic vehicles. However, toxicity studies regarding the use of LN as vehicles for drug administration are limited. In the present study, we investigated the in vivo toxicity of free edelfosine, and the protection conferred by LN. The free drug, non-loaded LN and edelfosine-loaded LN were orally administered to mice. Our results show that the oral administration of the free drug at 4 times higher than the therapeutic dose caused the death of the animals within 72h. Moreover, histopathology revealed gastrointestinal toxicity and an immunosuppressive effect. In contrast, LN showed a protective effect against edelfosine toxicity even at the higher dose and were completely safe. LN are, therefore, a safe vehicle for the administration of edelfosine by the oral route. The nanosystems developed could be further used for the administration of other drugs.

Delay of corneal epithelial wound healing and induction of keratocyte apoptosis by platelet-activating factor.

PURPOSE: To examine the role of the lipid mediator platelet-activating factor (PAF) in epithelial wound healing. METHODS: A 7-mm central de-epithelializing wound was produced in rabbit corneas, and the tissue was incubated with 125 nM carbamyl PAF (cPAF), an analogue of PAF. Rabbit corneal epithelial and stromal cells were also cultured in the presence of cPAF. Cell adhesion, proliferation, and migration assays were conducted. Apoptosis was assayed by TUNEL staining on preparations of corneal tissue sections and in cells in culture. RESULTS: Twenty-four hours after injury, 50% of the wounded area was covered by new epithelium, whereas only 30% was covered in the presence of cPAF. At 48 hours, the epithelium completely closed the wound, but only 45% of the original wound was covered in corneas treated with cPAF. Similar inhibition of epithelial wound closure was found with human corneas incubated with PAF in organ culture. Moreover, addition of several growth factors involved in corneal wound healing, such as epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, could not overcome the inhibitory action of PAF in wound closure. Three PAF antagonists, BN50727, BN50730, and BN50739, abolished the effect of PAF. A significant increase in TUNEL-positive staining occurred in corneal stromal cells (keratocytes), which was inhibited by preincubating the corneas with PAF antagonists. However, no TUNEL-positive staining was found in epithelial cells. TUNEL-staining results in cultured stromal cells (keratocytes) and epithelial cells in first-passage cell culture were similar to those in organ-cultured corneas. In addition, PAF caused 35% to 56% inhibition of adhesion of epithelial cells to proteins of the extracellular matrix: collagen I and IV, fibronectin, and laminin. There were no significant changes in proliferation or migration of epithelial cells induced by the lipid mediator. CONCLUSIONS: The results suggest PAF plays an important role in preventing corneal wound healing by affecting adhesion of epithelial cells and increasing apoptosis in stromal cells. PAF antagonists could be of therapeutic importance during prolonged ocular inflammation, helping to avoid loss of corneal transparency and visual acuity.

Platelet-activating factor (PAF) enhances apoptosis induced by ultraviolet radiation in corneal epithelial cells through cytochrome c-caspase activation.

PURPOSE: To examine the role of platelet-activating factor (PAF) on apoptosis of corneal epithelial cells exposed to radiation. METHODS: Rabbit corneal epithelial (RCE) and human corneal epithelial (HCE) cells were exposed to UVC radiation and then to carbamyl PAF (cPAF) for different increments of time. The PAF antagonist BN50739 was added 30 min before cPAF. The caspase inhibitors Ac-DEVD-CHO and Ac-YVAD-CHO were added 1 h before, and the phospholipase A(2) (PLA(2)) inhibitor MAFP was added 3 h before UVC irradiation. FITC-dUTP TUNEL and DAPI staining were performed to assess the percentage of apoptotic cells. DNA ladder analysis was used to investigate apoptosis induced by different intensities of UVC (50-600 J/m(2)) with or without cPAF. Caspase activation and release of cytochrome c from mitochondria to cytosol were determined by Western blot. RESULTS: While only 2.7% of RCE cells were DAPI positive in controls incubated for 12 h, 44% of cells were stained positive 4 h after irradiation; these values increased to 63% in the presence of cPAF. Cells incubated with cPAF alone were similar to controls. TUNEL staining and DNA laddering showed also increased in apoptosis after PAF treatment of UV-irradiated cells and BN50739 blocked the effect of cPAF. cPAF increased caspase-3 activation induced by UV irradiation in HCE cells. Cytochrome c release from mitochondria to cytosol was observed 30 min after irradiation. cPAF almost doubled the release of cytochrome c at 30 min and 1 h. Here, too, BN50739 blocked the PAF effect. No release of cytochrome c by PAF was seen in non-irradiated cells, even at higher concentrations. MAFP caused a decrease in cytochrome c release from UV-treated cells, and caused an even greater inhibition of cytochrome c release in cells stimulated with PAF. CONCLUSIONS: PAF increases RCE and HCE apoptosis caused by UV irradiation by stimulating PLA(2), producing an early release of cytocrome c from mitochondria and activating caspase-3 by a receptor-mediated mechanism. This accelerating effect of PAF on the apoptotic cascade only occurred when corneal epithelial cells had been previously damaged by UV radiation.

Effects of ether lipid 1-O-octadecyl-2-methoxy-rac-glycero- 3-phosphocholine and its analogs PAF and CPAF on the release of nitric oxide in primary cultures of rat astrocytes.

Ether lipid 1-O-octadecyl-2-O-methoxy-rac-glicero-3-phosphocholine (ET-18-OCH3) is an immunomodulator with antineoplastic activity. Its analog compounds PAF and CPAF share some of its biological effects. In our experiments, even very small amounts of ET-18-OCH3 released a remarkable quantity of nitric oxide (NO) from rat astrocytes cultured in vitro. The NO biosynthesis was inhibited by pretreatment with the antagonist BN 50730. The effect of ET-18-OCH3 was greater than that of the LPS inducer. PAF did not produce NO, even at high doses, while the nonmetabolizable CPAF only induced a significant release of NO from 12 micrograms/ml onwards. These results demonstrate that ET-18-OCH3 is functionally active also in astrocyte cultures. Stimulation of NO biosynthesis is of a great value on account of its the known effect as a neurotransmitter, potentiator of immune defences and possible modulator of cerebral circulation.

Lysophosphatidylcholine attenuates the cytotoxic effects of the antineoplastic phospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3- phosphocholine.

A colony-stimulating factor 1-dependent cell line was used to determine the relationship between the inhibition of phospholipid synthesis and the cytotoxic activity of the antineoplastic ether lipid, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). ET-18-OCH3 inhibited choline incorporation into phosphatidylcholine as well as total phospholipid synthesis. Exposure to ET-18-OCH3 at the G1/S boundary led to the accumulation of cells in G2, whereas the addition of ET-18-OCH3 in the G1 phase of the cell cycle prevented entry into the S phase. In both cases, ET-18-OCH3 treatment triggered DNA fragmentation and morphological changes associated with apoptosis within 10 h. The addition of lysophosphatidylcholine provided an exogenous source of cellular phospholipid and prevented ET-18-OCH3-dependent accumulation of cells in G2 and apoptosis. However, lysophosphatidylcholine did not overcome the ET-18-OCH3-dependent G1 block, although the growth-arrested cells remained viable. These data indicate that restoring phosphatidylcholine synthesis by supplementation with lysophosphatidylcholine overrides the cytotoxic but not the cytostatic activity of ET-18-OCH3.