Shiraishi Takehiko, Nielsen Peter E
Department of Cellular and Molecular Medicine, Faculty of Health Sciences, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.
Methods Mol Biol. 2011;683:391-7. doi: 10.1007/978-1-60761-919-2_28.
Cell-penetrating peptides (CPPs) have been widely used for a cellular delivery of biologically relevant cargoes including antisense peptide nucleic acids (PNAs). Although chemical conjugation of PNA to a variety of CPPs significantly improves the cellular uptake of the PNAs, bioavailability (antisense activity) is still limited by endocytotic entrapment. We have shown that this low bioavailability can be greatly improved by combining CPP-PNA conjugate administration with a photochemical internalization technique using photosensitizers such as aluminum phthalocyanine (AlPcS(2a)) or tetraphenylporphyrin tetrasulfonic acid (TPPS). Cellular uptake of the PNA conjugates were evaluated by using a sensitive cellular method with HeLa pLuc705 cells based on the splicing correction of luciferase gene by targeting antisense oligonucleotides to a cryptic splice site of the mutated luciferase gene. The cellular efficacy of CPP conjugates were evaluated by measuring luciferase activity as a result of splicing correction and was also confirmed by RT-PCR analysis of luciferase pre-mRNA.
细胞穿透肽（CPPs）已被广泛用于细胞递送包括反义肽核酸（PNA）在内的生物相关货物。尽管将PNA化学偶联到多种CPPs上可显著提高PNA的细胞摄取，但生物利用度（反义活性）仍受内吞截留的限制。我们已经表明，通过将CPP-PNA偶联物给药与使用诸如铝酞菁（AlPcS(2a)）或四苯基卟啉四磺酸（TPPS）等光敏剂的光化学内化技术相结合，这种低生物利用度可得到极大提高。基于将反义寡核苷酸靶向突变型荧光素酶基因的隐蔽剪接位点对荧光素酶基因进行剪接校正，使用HeLa pLuc705细胞的灵敏细胞方法评估PNA偶联物的细胞摄取。通过测量剪接校正后的荧光素酶活性评估CPP偶联物的细胞功效，并且还通过对荧光素酶前体mRNA的RT-PCR分析得到证实。
