Jacqueline Marchand-Brynaert

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.

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.

A Plant Plasma Membrane ATP Binding Cassette–Type Transporter Is Involved in Antifungal Terpenoid Secretion

ATP binding cassette (ABC) transporters, which are found in all species, are known mainly for their ability to confer drug resistance. To date, most of the ABC transporters characterized in plants have been localized in the vacuolar membrane and are considered to be involved in the intracellular sequestration of cytotoxins. Working on the assumption that certain ABC transporters might be involved in defense metabolite secretion and their expression might be regulated by the concentration of these metabolites, we treated a Nicotiana plumbaginifolia cell culture with sclareolide, a close analog of sclareol, an antifungal diterpene produced at the leaf surface of Nicotiana spp; this resulted in the appearance of a 160-kD plasma membrane protein, which was partially sequenced. The corresponding cDNA (NpABC1) was cloned and shown to encode an ABC transporter. In vitro and in situ immunodetection showed NpABC1 to be localized in the plasma membrane. Under normal conditions, expression was found in the leaf epidermis. In cell culture and in leaf tissues, NpABC1 expression was strongly enhanced by sclareolide and sclareol. In parallel with NpABC1 induction, cells acquired the ability to excrete a labeled synthetic sclareolide derivative. These data suggest that NpABC1 is involved in the secretion of a secondary metabolite that plays a role in plant defense.

Targeting nanoparticles to M cells with non-peptidic ligands for oral vaccination

The presence of RGD on nanoparticles allows the targeting of beta1 integrins at the apical surface of human M cells and the enhancement of an immune response after oral immunization. To check the hypothesis that non-peptidic ligands targeting intestinal M cells or APCs would be more efficient for oral immunization than RGD, novel non-peptidic and peptidic analogs (RGD peptidomimitic (RGDp), LDV derivative (LDVd) and LDV peptidomimetic (LDVp)) as well as mannose were grafted on the PEG chain of PCL-PEG and incorporated in PLGA-based nanoparticles. RGD and RGDp significantly increased the transport of nanoparticles across an in vitro model of human M cells as compared to enterocytes. RGD, LDVp, LDVd and mannose enhanced nanoparticle uptake by macrophages in vitro. The intraduodenal immunization with RGDp-, LDVd- or mannose-labeled nanoparticles elicited a higher production of IgG antibodies than the intramuscular injection of free ovalbumin or intraduodenal administration of either non-targeted or RGD-nanoparticles. Targeted formulations were also able to induce a cellular immune response. In conclusion, the in vitro transport of nanoparticles, uptake by macrophages and the immune response were positively influenced by the presence of ligands at the surface of nanoparticles. These targeted-nanoparticles could thus represent a promising delivery system for oral immunization.

Targeted nanoparticles with novel non-peptidic ligands for oral delivery☆

Orally administered targeted nanoparticles have a large number of potential biomedical applications and display several putative advantages for oral drug delivery, such as the protection of fragile drugs or modification of drug pharmacokinetics. These advantages notwithstanding, oral drug delivery by nanoparticles remains challenging. The optimization of particle size and surface properties and targeting by ligand grafting have been shown to enhance nanoparticle transport across the intestinal epithelium. Here, different grafting strategies for non-peptidic ligands, e.g., peptidomimetics, lectin mimetics, sugars and vitamins, that are stable in the gastrointestinal tract are discussed. We demonstrate that the grafting of these non-peptidic ligands allows nanoparticles to be targeted to M cells, enterocytes, immune cells or L cells. We show that these grafted nanoparticles could be promising vehicles for oral vaccination by targeting M cells or for the delivery of therapeutic proteins. We suggest that targeting L cells could be useful for the treatment of type 2 diabetes or obesity.

Stability of meropenem and doripenem solutions for administration by continuous infusion

the same pI—despite the negative diffusion test for ESBL detection—we carried out a search for bla SHV using PCR (see Table S2); this, however, was negative for both strains. Another possible explanation for the aztreonam resistance observed in our strains might be overproduction of their chromosomal class A OXY b-lactamase. 5 The promoter region of the OXY b-lactamase was amplified by PCR (see Table S2), and the sequences revealed a mutation in the 210 consensus region of the promoter in both strains; this consisted of the transition (G!A) of the fifth base, described as the most frequent among in vitro mutants and clinical isolates of aztreonam-resistant K. oxytoca. 5 The bla OXY gene was also amplified by PCR (see Table S2), and the sequence analysis indicated that both strains carried a bla OXY-2-type very similar to bla OXY-2-8 (GenBank accession no. AY055205) with only two substitutions, a serine to glycine at position 23 and an aspartic acid to alanine at position 38, considering position 1 as the starting methionine.

Fibronectin adsorption or/and covalent grafting on chemically modified PEEK film surfaces.

Poly(ether ether ketone) (PEEK) films were chemically modified, by surface wet chemistry, into PEEK-OH, PEEK-NH2, and PEEK-NCO. Fibronectin (FN) adsorption, in the presence or absence of two non-ionic surfactants, was compared onto PEEK, PEEK-OH, and PEEK-NH2 on which the protein can only be adsorbed, and onto PEEK-NCO on which FN could be covalently grafted. The amounts of FN present on the various supports were assayed by ELISA and LSC (with 125I-labeled FN). The remarkable effect of Pluronic F68 in preventing non-specific protein adhesion on the less hydrophilic surfaces was pointed out. Accordingly, a procedure could be proposed that allows minimal FN adhesion vs FN fixation on PEEK-NCO. The resulting PEEK-FN film, which immobilized 120-150 ng FN cm(-2), constitutes a new substratum for cell cultivation.

Biological evaluation of RGD peptidomimetics, designed for the covalent derivatization of cell culture substrata, as potential promotors of cellular adhesion

Our aim was to replace the proteins and peptides, generally used for the biocompatibilization of polymer substrata, with synthetic molecules mimicking the RGD (Arg-Gly-Asp) active sequence. Based on the (L)-tyrosine template, RGD peptidomimetics were constructed; one molecule 3 was equipped with an anchorage arm that allowed its covalent grafting on a culture substratum made from poly(ethylene terephthalate) (PET) microporous membrane. The amount of fixed molecules was readily determined by XPS, using a fluorine tag incorporated in the peptidomimetic structure. The binding of peptidomimetics 1-3 to the vitronectin (VN) and fibronectin (FN) receptors could not be revealed in a test of inhibition of MSC 80 cells adhesion, by the synthetic compounds in solution placed in competition with the adhesive proteins (VN and FN) coating polystyrene plates. However, the cell-attachment activity of peptidomimetic 3 was shown by culturing CaCo2 cells, in the absence of serum, on the PET substratum grafted with 3. The performance of this support was similar to that of PET grafted with the reference peptide RGDS (Arg-Gly-Asp-Ser), and only reduced by half comparatively to the PET grafted with FN.

Self-Assembling Doxorubicin−Tocopherol Succinate Prodrug as a New Drug Delivery System: Synthesis, Characterization, and in Vitro and in Vivo Anticancer Activity

Self-assembled prodrugs forming nanoaggregates are a promising approach to enhance the antitumor efficacy and to reduce the toxicity of anticancer drugs. To achieve this goal, doxorubicin was chemically conjugated to d-α-tocopherol succinate through an amide bond to form N-doxorubicin-α-d-tocopherol succinate (N-DOX-TOS). The prodrug self-assembled in water into 250 nm nanostructures when stabilized with d-α-tocopherol poly(ethylene glycol) 2000 succinate. Cryo-TEM analysis revealed the formation of nanoparticles with a highly ordered lamellar inner structure. NMR spectra of the N-DOX-TOS nanoparticles indicated that N-DOX-TOS is located in the core of the nanoparticles while PEG chains and part of the tocopherol are in the corona. High drug loading (34% w/w) and low in vitro drug release were achieved. In vitro biological assessment showed significant anticancer activity and temperature-dependent cellular uptake of N-DOX-TOS nanoparticles. In vivo, these nanoparticles showed a greater antitumor efficacy than free DOX. N-DOX-TOS nanoparticles might have the potential to improve DOX-based chemotherapy.

Adhesion and growth of CaCo2 cells on surface-modified PEEK substrata.

A series of surface-functionalized poly(ether ether ketone) (PEEK) films has been prepared by selective wet-chemistry; they are hydroxylated polymer (PEEK-OH) obtained by reduction, aminated polymer (PEEK-[]-NH2) prepared by coupling a diisocyanate reagent to PEEKOH (PEEK-[]-NCO) followed by hydrolysis, and carboxylated and aminocarboxylated polymers (PEEK-[]-GABA and PEEK-Lysine) resulting from the coupling of aminoacids to PEEK-[]-NCO. The aminated and carboxylated substrata promoted the adhesion and growth of CaCo2 cells in the presence of serum. Fibronectin (FN), an extra-cellular matrix protein, has been covalently fixed and/or adsorbed on various PEEK substrata, in the presence or not of a polymeric surfactant (Pluronic F68). The performances of the FN-grafted substrata (PEEK-[]-FN(1) and PEEK-[]-FN(2)) were significantly higher than those of reference substrata simply coated with FN (PEEK-OH(+FN)(1) and (2), PEEK-[]-NH2(+FN)(1) and (2)), considering the adhesion and spreading of CaCo2 cells in the absence of serum. Moreover, the stability of the adherent cells on the FN-adsorbed substrata dramatically depended on the experimental conditions applied during the PEEK coating with FN.