| Popis: |
The utilization of macromolecules in therapy of cancer and other diseases is becoming increasingly relevant. Recent advances in molecular biology and biotechnology have made it possible to improve targeting and design of cytotoxic agents, DNA complexes, and other macromolecules for clinical applications. To achieve the expected biological effect of these macromolecules, in many cases, internalization to the cell cytosol is crucial. At an intracellular level, the most fundamental obstruction for cytosolic release of the therapeutic molecule is the membrane-barrier of the endocytic vesicles. Photochemical internalization (PCI) is a novel technology for release of endocytosed macromolecules into the cytosol. The technology is based on the use of photosensitizers located in endocytic vesicles that upon activation by light induces a release of macromolecules from their compartmentalization in endocytic vesicles. PCI has been shown to potentiate the biological activity of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), gene-encoding plasmids, adenovirus, oligonucleotides, and the chemotherapeutic bleomycin. PCI has also been shown to enhance the treatment effect of targeted therapeutic macromolecules. The present protocol describes PCI of an epidermal growth factor receptor (EGFR)-targeted protein toxin (Cetuximab-saporin) linked via streptavidin-biotin for screening of targeted toxins as well as PCI of nonviral polyplex-based gene therapy. Although describing in detail PCI of targeted protein toxins and DNA polyplexes, the methodology presented in these protocols are also applicable for PCI of other gene therapy vectors (e.g., viral vectors), peptide nucleic acids (PNA), small interfering RNA (siRNA), polymers, nanoparticles, and some chemotherapeutic agents. |
