RESUMO
BACKGROUND: Hollow vesicles formed from block copolymers, so-called polymersomes, have been extensively studied in the last decade for their various applications in drug delivery, in diagnostics and as nanoreactors. The immobilization of proteins on the polymersomes' surface can aid in cell targeting, lead to functional biosensors or add an additional reaction space for multistep syntheses. In almost all surface functionalization strategies to date, a chemical pre-conjugation of the polymer with a reactive group or ligand and the functionalization of the protein are required. To avoid chemical pre-conjugation, we investigated the simple and quick functionalization of preformed poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA) polymersomes through the spontaneous insertion of four hydrophobic, non-antibacterial peptide anchors into the membrane to display enhanced green fluorescent protein (eGFP) on the polymersomes' surface. RESULTS: Three of the four hydrophobic peptides, the transmembrane domains of a eukaryotic cytochrome b 5 , of the viral lysis protein L and of the yeast syntaxin VAM3 could be recombinantly expressed as soluble eGFP-fusion proteins and spontaneously inserted into the polymeric membrane. Characterization of the surface functionalization revealed that peptide insertion was linearly dependent on the protein concentration and possible at a broad temperature range of 4-42 °C. Up to 2320 ± 280 eGFP molecules were immobilized on a single polymersome, which is in agreement with the calculated maximum loading capacity. The peptide insertion was stable without disrupting membrane integrity as shown in calcein leakage experiments and the functionalized polymersomes remained stable for at least 6 weeks. CONCLUSION: The surface functionalization of polymersomes with hydrophilic proteins can be mediated by several peptide anchors in a spontaneous process at extremely mild insertion conditions and without the need of pre-conjugating polymers.
Assuntos
Proteínas de Fluorescência Verde/química , Proteínas Imobilizadas/química , Oxazóis/química , Peptídeos/química , Polímeros/química , Clonagem Molecular/métodos , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Interações Hidrofóbicas e Hidrofílicas , Proteínas Imobilizadas/genética , Microscopia Confocal , Microscopia de Fluorescência , Peptídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Propriedades de SuperfícieRESUMO
BACKGROUND: It was our intention to develop cathepsin B-sensitive nanoparticles for tumor-site-directed release. These nanoparticles should be able to release their payload as close to the tumor site with a decrease of off-target effects in mind. Cathepsin B, a lysosomal cysteine protease, is associated with premalignant lesions and invasive stages of cancer. Previous studies have shown cathepsin B in lysosomes and in the extracellular matrix. Therefore, this enzyme qualifies as a trigger for such an approach. METHODS: Poly(dimethylsiloxane)-b-poly(methyloxazoline) (PDMS-PMOXA) nanoparticles loaded with paclitaxel were formed by a thin-film technique and standard coupling reactions were used for surface modifications. Despite the controlled release mechanism, the physical properties of the herein created nanoparticles were described. To characterize potential in vitro model systems, quantitative polymerase chain reaction and common bioanalytical methods were employed. CONCLUSIONS: Stable paclitaxel-loaded nanoparticles with cathepsin B digestible peptide were formed and tested on the ovarian cancer cell line OVCAR-3. These nanoparticles exerted a pharmacological effect on the tumor cells suggesting a release of the payload.