Quantification of the Intracellular pH Environment of Oligonucleotides Delivered with Novel Poly(glycoamidoamine)s

摘要

Genetic-based therapies have become an area of intense research focus because of their wide application to combat many diseases. However, one of the major obstacles to applying this treatment strategy is the physical delivery of the therapeutic DNA to the ailing cell. Viruses have traditionally been used as gene delivery vectors, but a variety of problems have arisen such as severe immune responses, insertional mutagenesis, and other cytotoxic effects1. This has prompted the development of polymeric vectors to replace viral vectors. In comparison to a common polycation vector, PEI, a set of poly(glycoamidoamine)s have been previously synthesized and characterized2. These glycopolymers have been found to efficiently transfect a variety of mammalian cell lines with much lower toxicity than PEI, however, the intracellular path of these polymeric delivery vehicles remains elusive. Although it is accepted that the polymer/DNA complexes (polyplexes) are taken up by endocytosis, the mechanism of escape from the acidic vesicles in the endocytic pathway is still being investigated3. In this study, a flow cytometry assay has been used to investigate this delivery route4. By probing the intracellular pH environment of the delivered DNA, it may be determined if the DNA is being retained in acidic vesicles such as endosomes or lysosomes, or if the DNA is being released into the cytoplasm of the cell. In addition, this assay will help determine how the polymer structure affects the cellular delivery mechanism.