Acylated and unacylated ghrelin binding to membranes and to ghrelin receptor: towards a better understanding of the underlying mechanisms.

The O-octanoylation of human ghrelin is a natural post-translational modification that enhances its binding to model membranes and could potentially play a central role in ghrelin biological activities. Here, we aimed to clarify the mechanisms that drive ghrelin to the membrane and hence to its receptor that mediates most of its endocrinological effects. As the acylation enhances ghrelin lipophilicity and that ghrelin contains many basic residues, we examined the electrostatic attraction and/or hydrophobic interactions with membranes. Using various liposomes and buffer conditions in binding, zeta potential and isothermal titration calorimetry studies, we found that whereas acylated and unacylated ghrelin were both electrostatically attracted towards the membrane, only acylated ghrelin penetrated into the headgroup and the lipid backbone regions of negatively charged membranes. The O-acylation induced a 120-fold increase in ghrelin local concentration in the membrane. However, acylated ghrelin did not deeply penetrate the membrane nor did it perturb its organisation. Conformational studies by circular dichroism and attenuated total reflection Fourier transformed infrared as well as in silico modelling revealed that both forms of ghrelin mainly adopted the same structure in aqueous, micellar and bilayer environments even though acylated ghrelin structure is slightly more α-helical in a lipid bilayer environment. Altogether our results suggest that membrane acts as a "catalyst" in acylated ghrelin binding to the ghrelin receptor and hence could explain why acylated and unacylated ghrelin are both full agonists of this receptor but in the nanomolar and micromolar range, respectively.

Targeting of tumor endothelium by RGD-grafted PLGA-nanoparticles loaded with paclitaxel.

Paclitaxel (PTX)-loaded PEGylated PLGA-based nanoparticles (NP) have been previously described as more effective in vitro and in vivo than taxol. The aim of this study was to test the hypothesis that our PEGylated PLGA-based nanoparticles grafted with the RGD peptide or RGD-peptidomimetic (RGDp) would target the tumor endothelium and would further enhance the anti-tumor efficacy of PTX. The ligands were grafted on the PEG chain of PCL-b-PEG included in the nanoparticles. We observed in vitro that RGD-grafted nanoparticles were more associated to human umbilical vein endothelial cells (HUVEC) by binding to alpha(v)beta(3) integrin than non-targeted nanoparticles. Doxorubicin was also used to confirm the findings observed for PTX. In vivo, we demonstrated the targeting of RGD and RGDp-grafted nanoparticles to tumor vessels as well as the effective retardation of TLT tumor growth and prolonged survival times of mice treated by PTX-loaded RGD-nanoparticles when compared to non-targeted nanoparticles. Hence, the targeting of anti-cancer drug to tumor endothelium by RGD-labeled NP is a promising approach.

Novel self-assembling PEG-p-(CL-co-TMC) polymeric micelles as safe and effective delivery system for paclitaxel.

Paclitaxel (PTX) is an effective anti-cancer drug currently used to treat a wide variety of cancers. Unfortunately, nonaqueous vehicle containing Cremophor EL is associated with serious clinical side effects. This work aimed to evaluate the ability of polymeric micelles to (i) solubilize PTX without Cremophor EL and to be used as a (ii) safe and (iii) effective delivery system for PTX. Hence, we developed novel self-assembling poly(ethyleneglycol)(750)-block-poly(epsilon-caprolactone-co-trimethylenecarbonate) (PEG-p-(CL-co-TMC)) polymeric micelles which form micelles spontaneously in aqueous solution. The solubility of PTX increased up to three orders of magnitude. The PTX-loaded micelles showed a slow release of PTX with no burst effect. The HeLa cells viability assessed by the MTT test was lower for PTX-loaded micelles than for Taxol (IC(50) 10.6 vs. 17.6 microg/ml). When solubilized in micelles, PTX induced apoptosis comparable with Taxol. The maximum tolerated doses (MTD) of PTX-loaded micelles and Taxol in mice were 80 mg/kg and 13.5mg/kg, respectively, after intraperitoneal administration; and 45 mg/kg and 13.5mg/kg, respectively, after intravenous administration. Similar anti-tumor efficacy of PTX-loaded micelles and Taxol was observed at the dose of 13.5mg/kg on TLT-tumor-bearing mice, while the body weight loss was only observed in Taxol group. However, as higher dose was tolerated (80 mg/kg - IP), a higher growth delay was induced with PTX-loaded micelles. These results demonstrated that PTX-loaded self-assembling micelles present a similar anti-tumor efficacy as Taxol, but significantly reduced the toxicity allowing the increase in the dose for better therapeutic response.

Effect of tape stripping and adjuvants on immune response after intradermal DNA electroporation.

PURPOSE: DNA vaccines require both efficient delivery methods and appropriate adjuvants. Based on their mechanisms of action, we hypothesised that some adjuvants could enhance vaccine immunogenicity or direct the response towards Th1 profile after intradermal DNA electroporation. METHODS: After intradermal electroporation of plasmid DNA encoding luciferase, mice received hyaluronidase, imiquimod, monophosphoryl lipid A or were tape stripped in order to modulate the immune response against the encoded protein. We measured total immunoglobulin G, IgG1, IgG2a titres and the cytokines produced by splenocyte cultures to assess both humoral and cellular response. The effect of tape stripping on the response against intradermally delivered ovalbumin protein was also assessed. RESULTS: Neither hyaluronidase nor imiquimod improved the immune response against the encoded luciferase. Monophosphoryl lipid A did not modify the cytokines production but increased the anti-luciferase IgG2a titres. Tape stripping significantly increased anti-luciferase IgG2a and IFN-gamma responses. It also enhanced the humoral response after intradermal injection of the ovalbumin protein. CONCLUSIONS: Tape stripping is able to increase the Th1 immune response against both DNA and protein vaccines. Therefore, tape stripping appears to have interesting adjuvant effect on intradermal vaccination.

Paclitaxel-loaded PEGylated PLGA-based nanoparticles: in vitro and in vivo evaluation.

The purpose of this study was to develop Cremophor EL-free nanoparticles loaded with Paclitaxel (PTX), intended to be intravenously administered, able to improve the therapeutic index of the drug and devoid of the adverse effects of Cremophor EL. PTX-loaded PEGylated PLGA-based were prepared by simple emulsion and nanoprecipitation. The incorporation efficiency of PTX was higher with the nanoprecipitation technique. The release behavior of PTX exhibited a biphasic pattern characterized by an initial burst release followed by a slower and continuous release. The in vitro anti-tumoral activity was assessed using the Human Cervix Carcinoma cells (HeLa) by the MTT test and was compared to the commercial formulation Taxol and to Cremophor EL. When exposed to 25 microg/ml of PTX, the cell viability was lower for PTX-loaded nanoparticles than for Taxol (IC(50) 5.5 vs 15.5 microg/ml). Flow cytometry studies showed that the cellular uptake of PTX-loaded nanoparticles was concentration and time dependent. Exposure of HeLa cells to Taxol and PTX-loaded nanoparticles induced the same percentage of apoptotic cells. PTX-loaded nanoparticles showed greater tumor growth inhibition effect in vivo on TLT tumor, compared with Taxol. Therefore, PTX-loaded nanoparticles may be considered as an effective anticancer drug delivery system for cancer chemotherapy.