Cells as factories for humanized encapsulation.

Biocompatibility is of paramount importance for drug delivery, tumor labeling, and in vivo application of nanoscale bioprobes. Until now, biocompatible surface processing has typically relied on PEGylation and other surface coatings, which, however, cannot minimize clearance by macrophages or the renal system but may also increase the risk of chemical side effects. Cell membranes provide a generic and far more natural approach to the challenges of encapsulation and delivery in vivo. Here we harness for the first time living cells as "factories" to manufacture cell membrane capsules for encapsulation and delivery of drugs, nanoparticles, and other biolabels. Furthermore, we demonstrate that the built-in protein channels of the new capsules can be utilized for controlled release of encapsulated reagents.

In vitro and in vivo performance of biocompatible negatively-charged salbutamol-loaded nanoparticles.

The development and performance of a novel nanoparticle-based formulation for pulmonary delivery has been characterized chronologically through the particle preparation process, in vitro testing of drug release, biocompatibility, degradation, drug transport in cell culture, and in vivo bronchoprotection studies in anaesthetised guinea pigs. This study demonstrates excellent agreement of the in vitro and in vivo experiments undertaken to prove the feasibility of the design, thereby serving as an example highlighting the importance of in vitro test methods that predict in vivo performance. Nanoparticles were prepared from the newly designed negatively-charged polymer poly(vinyl sulfonate-co-vinyl alcohol)-g-poly(d,l-lactic-co-glycolic acid) loaded with salbutamol free base. Average particle sizes of blank and drug-loaded nanoparticles prepared at the various stages of the investigations were between 91 and 204nm; average zeta potential values were between -50.1 and -25.6mV. Blank nanoparticles showed no significant toxicity, and no inflammatory activity was detected in Calu-3 cells. Sustained release of salbutamol from the nanoparticles was observed for 2.5h in vitro, and a prolonged effect was observed for 120min in vivo. These results demonstrate good agreement between in vitro and in vivo tests and also present a promising foundation for future advancement in nanomedicine strategies for pulmonary drug delivery.

One-pot synthesis of pegylated ultrasmall iron-oxide nanoparticles and their in vivo evaluation as magnetic resonance imaging contrast agents.

A well-defined copolymer poly(oligo(ethylene glycol) methacrylate-co-methacrylic acid) P(OEGMA-co-MAA) was studied as a novel water-soluble biocompatible coating for superparamagnetic iron oxide nanoparticles. This copolymer was prepared via a two-step procedure: a well-defined precursor poly(oligo(ethylene glycol) methacrylate-co-tert-butyl methacrylate), P(OEGMA-co-tBMA) (M(n) = 17300 g mol(-1); M(w)/M(n) = 1.22), was first synthesized by atom-transfer radical polymerization in the presence of the catalyst system copper(I) chloride/2,2'-bipyridyl and subsequently selectively hydrolyzed in acidic conditions. The resulting P(OEGMA-co-MAA) was directly utilized as a polymeric stabilizer in the nanoparticle synthesis. Four batches of ultrasmall PEGylated magnetite nanoparticles (i.e., with an average diameter below 30 nm) were prepared via aqueous coprecipitation of iron salts in the presence of variable amounts of P(OEGMA-co-MAA). The diameter of the nanoparticles could be easily tuned in the range 10-25 nm by varying the initial copolymer concentration. Moreover, the formed PEGylated ferrofluids exhibited a long-term colloidal stability in physiological buffer and could therefore be studied in vivo by magnetic resonance (MR) imaging. Intravenous injection into rats showed no detectable signal in the liver within the first 2 h. Maximum liver accumulation was found after 6 h, suggesting a prolongated circulation of the nanoparticles in the bloodstream as compared to conventional MR imaging contrast agents.

Structural aspects of histone H1-DNA complexes and their relation to transfection efficiency.

During transfection, polycation-DNA complexes are normally diluted by the transfection medium, which often contains salt in the physiological concentration range and serum. It is not exactly known to what extent this dilution step influences the properties of the complexes, which in turn influence the transfection efficiency. In order to gain more insight into the size-structure-transfection activity relationship, we prepared histone H1-DNA complexes in NaCl solutions at various concentrations known to determine the size and structure of the resulting complexes. We characterized the complexes by physicochemical methods. Fluorescence correlation spectroscopy enabled relative measurements of complex sizes even under physiological conditions. The different appearances of the complexes were correlated with their transfection efficiency. When transfection was performed by dilution of the complexes in cell-cultivation media, the initial structure of H1-DNA complexes preformed under distinct salt conditions had no significant influence on the transfection efficiency. The dilution of the preformed complexes with cell-cultivation medium resulted in re-formation and aggregation of the complexes. The addition of the complexes to the cells without cell-cultivation medium, however, showed a direct correlation between the size of the complexes and the transfection efficiency (correlation coefficient 0.91). Small complexes did not contribute to the transfection.

Polyelectrolyte nanoparticles mediate vascular gene delivery.

PURPOSE: The purpose is to develop a non-viral gene delivery system that meets the requirements of colloidal stability of DNA complexes expressed in terms of no particle aggregation under physiologic conditions. The system should be used to transfect cardiovascular tissues. METHODS: We used a strategy based on the formation of polyelectrolyte nanoparticles by deposition of alternatively charged polyelectrolytes onto a DNA core. Polyelectrolytes were transfer RNA as well as the synthetic polyanion, polyvinyl sulfate (PVS), and the polycation polyethylenimine (PEI). The PEI/DNA complex formed the DNA core. RESULTS: We observed that the DNA is condensed by polycations and further packaged by association with a polyanion. These nanoparticles exhibited negative surface charge and low aggregation tendency. In vivo rat carotid artery experiments revealed high transfection efficiency, not only with the reporter gene but also with the gene encoding human urokinase plasminogen activator (Hu-uPA). Hu-uPA is one of the proteins involved in the recovery of the blood vessels after balloon catheter injury and therefore clinically relevant. CONCLUSIONS: A strategy for in vivo gene transfer is proposed that uses the incorporation of polyanions as RNA or PVS into PEI/DNA complexes in order to overcome colloidal instability and to generate a negative surface charge. The particles proved to be transfectionally active in vascular gene transfer.

Integrin specificity of the cyclic Arg-Gly-Asp motif and its role in integrin-targeted gene transfer.

Targeted gene transfer, addressing the alphavbeta3 integrin by coupling the appropriate ligand, cRGD (S(2)-bridged cyclic Arg-Gly-Asp containing peptide) motif, on to a DNA condensing sequence was described as early as 1995 by Hart, Harbottle, Cooper, Miller, Williamson and Coutelle [(1995) Gene Ther. 2, 552-554]. Their work was followed by a series of publications, introducing the cRGD motif in polycationic DNA carriers, such as peptides, proteins and liposomes. Polyethylenimine and even adenoviruses were additionally ligated using the cRGD motif. 'Integrin specificity' has been determined from the significantly improved transfection efficiency compared with the DNA carriers with control ligands, mainly the cRGE (S(2)-bridged cyclic-Arg-Gly-Glu-containing peptide) motif. However, by observing the physicochemical appearance of the resulting complexes and their controls such as the poly(L-lysine)-DNA complexes carrying the cRGD and the cRGE motifs, we doubted the integrin-mediated specificity of the increased transfection efficiency. To clarify this contradiction, we investigated the suitability of the cRGD motif for targeted gene transfer. We proved the specificity of the RGD motif and its controls using computational docking procedures and molecular modelling methods. Since we were confident of the motifs used, we improved our transfection method. Since aggregation of the RGD-ligated poly(L-lysine)-DNA complexes under physiological conditions caused an enormous amount of unspecific cell uptake and transfection, a method had to be designed to exclude aggregation processes of the motif-polycation-DNA complexes. Small complex sizes are necessary for receptor-specific uptake. The complexes were therefore recharged using poly(vinyl sulphate). Inhibited aggregation of the targeted DNA carriers under physiological conditions is a necessary prerequisite for successful in vivo gene transfer.

Ultrastructural analysis of DNA complexes during transfection and intracellular transport.

We present a simple method based on transmission electron microscopy that allows investigation of the early steps of polyplex-mediated transfection without the use of labeled DNA. The ultrastructural analysis showed internalization of 0.2-1-micro m aggregates composed of 30-50-nm subunits. In addition, new details of the internalization process were revealed, suggesting an unspecific cell entry mechanism of large DNA aggregates.