RESUMEN
The major bottleneck in gene therapy remains the issue of delivery. In this work, various modified poly(propylene imine) (PPI) dendrimers are introduced as gene transfection agents. Commercially available PPI-dendrimers have been modified (i) at the exterior primary amines with acetyl groups or glycol gallate (PEG-like) groups, and (ii) at the interior tertiary amines with methyl iodide (MeI) or MeCl to produce multiple quaternized cationic sites in the core of the dendrimer. The prepared materials have been tested with respect to their binding capabilities to DNA, their toxicity in cell cultures, their in vitro transfection efficiency and their in vivo delivery possibilities. In all cases, a 33-mer oligonucleotide (DNAzyme) was used. Polyacrylamide gel electrophoresis (PAGE) studies have demonstrated strong but reversible binding, where the quarternized and higher generation dendrimer species have shown more potent binding. Typically, for the modified fourth PPI-dendrimers, binding is observed at a concentration of about 4 microM DNA and a dendrimer-DNA charge ratio of around 2:1-1:1. All the tested PPI-dendrimers display a low cellular toxicity, especially when higher serum contents are used in the culture medium. For example, most of the prepared fourth generation PPI-dendrimers are not or hardly toxic up to at least 20 microM in 20% serum. An in vitro characterization has revealed a high dendrimer-mediated intracellular uptake of the DNAzyme: all the tested fourth generation PPI-dendrimers display transfection efficiencies close to or exceeding 80%, even when the concentration of serum in the medium is increased from 10 to 40%. Finally, the potential of using modified PPI-dendrimers for in vivo gene therapy experiments is demonstrated. Injecting a G4-PEG(MeI)-ssDNA complex intravenously into Nude mice has resulted in a high nuclear uptake as confirmed by co-localization studies.
Asunto(s)
ADN Catalítico/administración & dosificación , ADN Catalítico/genética , Portadores de Fármacos/química , Polipropilenos/química , Transfección/métodos , Animales , Catálisis , Línea Celular Tumoral , Estabilidad de Medicamentos , Electroforesis en Gel de Poliacrilamida , Humanos , Espectroscopía de Resonancia Magnética , Masculino , RatonesRESUMEN
The objective of this study was to develop and characterize a biodegradable drug-loaded nerve guide for peripheral nerve regeneration. Sabeluzole, a nerve growth agent, was selected as model compound. Four biodegradable polymers were selected for this study: a copolymer of polylactic acid and polycaprolactone (PCL); a copolymer of polyglycolic acid and polycaprolactone PCL; a copolymer of PCL/polydioxanone (PDO) and PDO. Placebo and drug loaded nerve guides were obtained by melt compression and melt extrusion. It was observed that melt compression and melt extrusion are feasible techniques to prepare the nerve guides. Based on the physicochemical characterization, all samples show absence of crystalline sabeluzole, indicating the formation of an amorphous dispersion. The in vitro release measurements show that the release of sabeluzole is complete, reproducible and can be controlled by the proper selection of the polymer. The release mechanism for all samples follows Fickian release behaviour.
Asunto(s)
Implantes Absorbibles , Líquidos Corporales/química , Implantes de Medicamentos/química , Ácido Láctico/química , Piperidinas/administración & dosificación , Piperidinas/química , Poliésteres/química , Polímeros/química , Tiazoles/administración & dosificación , Tiazoles/química , Animales , Materiales Biocompatibles , Difusión , Estabilidad de Medicamentos , Análisis de Falla de Equipo , Humanos , Ensayo de Materiales , Factores de Crecimiento Nervioso/administración & dosificación , Factores de Crecimiento Nervioso/química , Regeneración Nerviosa/efectos de los fármacos , Traumatismos de los Nervios Periféricos , Nervios Periféricos/efectos de los fármacos , Nervios Periféricos/cirugía , Diseño de Prótesis , TemperaturaRESUMEN
A well-known complication in corneal repair surgery is (recurrent) rejection of donor corneal tissue. particularly in patients suffering from an auto-immune disease such as rheumatoid arthritis. Down-regulation of their immune system, by means of drugs, is necessary in order to perform an allograft implantation afterwards. The patient may need a temporary prosthetic cornea while the immune system is inactivated. Recently, NeuroPatch, a mesh-type polyurethane, was used for this purpose. The material exhibits excellent biocompatibility and allows ingrowth of stromal fibroblasts which deposit matrix material into the pores. A serious drawback of NeuroPatch is its non-transparency, which impairs vision. In this work we attempted to develop an improved biomaterial that combines the advantages of NeuroPatch with optical transparency. Based on previous findings that copolymers of hexaethyleneglycolmethacrylate (HEGMA) and butylmethacrylate (BMA), are transparent and well accepted by human corneal epithelial cells, we studied these materials further in detail. (Bruining et al., Bio-Macromolecules 1 (2000) 418) Copolymerizations were studied by means of 1H NMR. The influence of the HEGMA content on hydrophilicity, flexibility and resistance to protein adsorption was studied. The results indicate that materials with a HEGMA content of approximately 20 mol% are potentially useful in corneal repair surgery. These biomaterials meet most of the stringent physical and biological requirements.
