Interaction of novel Megamers (MG) with Caco-2 cells.

PRISCILA SCHILRREFF, JULIETA GROETZNER, EDER L. ROMERO AND MARÍA J. MORILLA

Munster, Alemania

Congreso; NanoBio-Europe 2010 International congress and exhibition on NanoBiotechnology; 2010

Interaction of novel Megamers (MG) with Caco-2 cells Priscila Schilrreff, Julieta Groetzner, Eder L. Romero and María J. Morilla, NANOMEDICINE RESEARCH PROGRAM (NRP). Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Argentina. The poor stability in the harsh environment of the GUT of oligo and/or Polypeptides (P) is responsible for their low bioavailability. Because of this, P must be administrated by parenteral route. However, their short plasma half lives requires of repeated administrations in order to achieve therapeutic levels. In this scenario, it is of relevance to find suitable alternative routes of administration of P, preferably excluding the use of injectables. A successful oral administration of P relies for instance, on the maintenance of their chemical structure along the gastrointestinal transit and on their absorption upon endocytic/transcytotic or alternatively paracellular pathways across the epithelial cells. With the purpose of increasing the oral bioavailability of P, we propose to generate electrostatic complexes between novel core-shell nanoparticles known as megamers (MG) and P. Those complexes should protect the structure of P and increase its paracellular absorption. In the present work we present for the first time the impact of the interaction of MG with cell cultures, measured as the activity of mitochondrial succinate dehydrogenase and stability of enterocytes cell membrane, by the MTT and LDH methods, respectively. Besides, we determined the degree of paracellular transport of fluorescently labeled MG on Caco-2 cells monolayers grown in Transwells. Briefly, the synthesis of the MG (saturated core-shell (tecto) dendrimers), involved the combination of an amine-terminated core reagent (PAMAM dendrimer  generation (G) 5), with an excess of a carboxylic acid terminated dendrimeric shell reagent G 2.5 and subsequent covalent bond formation. The resultant product was purified by size exclusion chromatography (SEC) to remove the excess shell reagent and lyophilized as a white rubbery solid. The final yield achieved was 50 %. Formation of MG was checked by: polyAcrylamide gel electrophoresis (PAGE), MALDI-TOF-MS, SEC, high performance liquid chromatography (HPLC), atomic force microscopy (AFM). Size and Z potential of MG was determined by dynamic light scattering (DLS). In vitro citotoxicity of MG on nude and mucins-covered Caco-2 cells was evaluated by determination of mitochondrial succinate dehydrogenase activity employing a tetrazolium salt (MTT) and as lactate dehydrogenase (LDH) leakage assay. Transepithelial permeability across Caco-2 cell monolayers was determined on a 24-well Transwell filters (pore size 3.0 mm) at non-toxic MG concentrations (previously determined by cytotoxicity assays). Monolayer integrity was assessed by measuring the transepithelial electrical resistance (TEER) and by measuring the permeation of lucifer yellow molecules. The effect of MG on the integrity of Caco-2 cell monolayers was investigated by monitoring the TEER before and after a recovery period of 24 h.                                                                                                                                                                                                                              MG showed to be structurally stable, monodisperse, with a molecular size distribution of 8 ± 1.5 nm determined by DLS and 16.9 ± 4.3 nm (height 1.12 ± 0.2 nm) determined by AFM. Molecular Weight of MG determined by MALDI-TOF-MS was 98000 Da, consistent with data obtained by PAGE. Shell saturation levels of 85-90% were observed, corresponding with 10-11 shell dendrimers surrounding the core as expected from Mansfield-Tomalia-Rakesh´s equation (theoric N máx: 11,7).                                                                                                                                                                                                                                                                                                                                     It is well documented the inherent toxicity of cationic PAMAM on nude Caco-2 cells. However, we found that this new structures, the MG, were non cytotoxic up to 150 mM when incubated with nude and mucin-covered caco-2 cells. Finally, FITC labelled MG were able cross the epithelial barrier when incubated with Caco-2 cells. In conclusion, MG demonstrated to be a promising relatively safe nano-structure capable of crossing the epithelial barrier in an oral administration. Ongoing research will ultimately show their suitability to translocate P -MG electrostatic complexes.