ABSTRACT
Immunoliposomes generated by coupling of antibodies to the liposomal surface allow for an active tissue targeting, e.g., through binding to tumor cell-specific receptors. Instead of whole antibodies, single-chain Fv fragments (scFv), which represent the smallest part of an antibody containing the entire antigen-binding site, find increasing usage as targeting moiety. Here we provide protocols for the preparation of type II scFv immunoliposomes by the conventional coupling method as well as the post-insertion method. Furthermore protocols to analyze binding of these immunoliposomes to antigen-expressing cells as well as internalization through receptor-mediated endocytosis are included.
Subject(s)
Drug Delivery Systems/methods , Liposomes/immunology , Nanoparticles/chemistry , Neoplasms/metabolism , Receptors, Cell Surface/metabolism , Single-Chain Antibodies/metabolism , Amino Acid Sequence , Base Sequence , Cell Line, Tumor , Endocytosis , Flow Cytometry , Humans , Liposomes/isolation & purification , Micelles , Microscopy, Fluorescence , Molecular Sequence Data , Nanomedicine/methods , Organ Specificity , Phosphatidylethanolamines/chemistry , Plasmids/chemistry , Plasmids/genetics , Polyethylene Glycols/chemistryABSTRACT
Affibody molecules are small and stable antigen-binding molecules derived from the B domain of protein A. We applied a bivalent, high-affinity epidermal growth factor receptor (EGFR)-specific affibody molecule for the generation of targeted PEGylated liposomes. These sterically stabilized affibody liposomes (SAL) were produced by chemical coupling of the cysteine-modified affibody molecule to maleimide-PEG(2000)-DSPE and subsequent insertion into PEGylated liposomes. These SAL showed strong and selective binding to EGFR-expressing tumor cell lines. Binding was dependent on the amount of inserted affibody molecule-lipid conjugates and could be blocked by soluble EGF. Approximately 30% of binding activity was still retained after 6 days of incubation in human plasma at 37 degrees C. Binding of SAL to cells led to efficient internalization of the liposomes. Using mitoxantrone-loaded liposomes, we observed for SAL, compared to untargeted liposomes, an enhanced cytotoxicity toward EGFR-expressing cells. In summary, we show that SAL can be easily prepared from affibody molecules and thus may be suitable for the development of carrier systems for targeted delivery of drugs.
Subject(s)
Antibodies/metabolism , ErbB Receptors/immunology , ErbB Receptors/metabolism , Gene Expression Regulation, Neoplastic , Liposomes/chemistry , Neoplasms/pathology , Recombinant Fusion Proteins/metabolism , Antibodies/chemistry , Antibodies/immunology , Biological Transport , Cell Line, Tumor , Cysteine/chemistry , Humans , Inhibitory Concentration 50 , Liposomes/metabolism , Maleimides/chemistry , Mitoxantrone/chemistry , Mitoxantrone/pharmacology , Nanostructures/chemistry , Phosphatidylethanolamines/chemistry , Polyethylene Glycols/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , Substrate SpecificityABSTRACT
Medical devices manufactured for implantation into humans must be free of any contamination with viable bacteria. However, remnants of dead bacteria and bacterial components alone may induce an inflammatory immune response. Pyrogen tests for such inflammatory contaminations are generally performed either by determining the content of lipopolysaccharide in rinsing solutions of batch samples by limulus amoebocyte lysate assay, by injecting the rinsing solutions into rabbits or by implanting batch samples into rabbits and measuring change of body temperature. In this study, we show that the in vitro pyrogen test (IPT), which measures the release of the inflammatory cytokine IL-1beta in fresh or cryopreserved human whole blood, can be used to assess the pyrogenic contamination of implantable medical devices. This test was used to check neurosurgical implants, namely aneurysm clips, as a proof of principle. Owing to the direct contact of the test material with the blood cells, this test does not require rinsing procedures, which have variable efficacy. The use of human blood ensures the detection of all substances that are pyrogenic for humans and reflects their relative potency. The safety of the products as delivered could be confirmed. The effects of sterilization and depyrogenization procedures on intentional pyrogenic contaminations of samples could be followed. This new application of the already internationally validated method promises to replace further rabbit pyrogen tests. It generates extremely sensitive results with an extended range of detectable pyrogenic contaminants.
