title: Endoplasmic reticulum localization of poly(ω-aminohexyl methacrylamide)s conjugated with (L-)-arginines in plasmid DNA delivery.
abstract: In this work, we first synthesized a ω-amino group Boc-protected poly(ω-aminohexyl methacrylamide) PAHMAA-Boc precursor via controlled reversible addition-fragmentation chain transfer (RAFT) polymerization of the Boc-AHMAA monomer in THF solution, and poly(ω-aminohexyl methacrylamide) (PAHMAA) was then prepared via Boc deprotection, and was further conjugated with (L-)-arginines at the ω-amino group sites to give a series of new (L-)-arginine conjugated PAHMAA-R7, PAHMAA-R16 and PAHMAA-R22. Employing these PAHMAA and PAHMAA-Rs as functional gene vectors, their plasmid DNA binding affinities were examined by agarose gel retardant assay. By means of dynamic light scattering (DLS), mean particle sizes and zeta potentials of the polyplexes were analyzed. Moreover, cytotoxicities of the PAHMAA and PAHMAA-Rs were evaluated by MTT and lactate dehydrogenase (LDH) release assays with COS-7 cells, and luciferase and EGFP gene transfection efficacies by these vectors were assayed in COS-7 and HeLa cells. Furthermore, intracellular uptake of the vector/Cy3-labeled pDNA polyplexes was studied with a flow cytometer (FACS), and the most efficient PAHMAA-R16 vector was employed to investigate the endocytic gateways with various inhibitors. In addition, colocalization of the Cy3-labeled pDNA and Oregon Green labeled PAHMAA-R16 vector in the intracellular organelles of COS-7 cells was visualized on a fluorescence microscopy. As a result, it was revealed that the PAHMAA-R vectors showed lower cytotoxicities and transfection efficacies significantly higher than those of the PAHMAA, strongly depending on their percentage of arginine conjugation, and that the results of endocytic inhabitation and fluorescence colocalization in endoplasmic reticulum may suggest a caveolae-mediated efficient intracellular trafficking route for the synthesized PAHMAA-R vectors.
authors.affiliation: Laboratory for Polymer Materials, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.forename: Ting
authors.initials: T
authors.lastname: Luo
authors.forename: Ruilong
authors.initials: R
authors.lastname: Sheng
authors.forename: Jingjing
authors.initials: J
authors.lastname: Sun
authors.forename: Zhao
authors.initials: Z
authors.lastname: Wang
authors.forename: Amin
authors.initials: A
authors.lastname: Cao
mesh_terms: D000178:Acrylamides / Q000138:chemical synthesis*; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001672:Biocompatible Materials / Q000138:chemical synthesis*; D019556:COS Cells; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry / Q000235:genetics; D016503:Drug Delivery Systems / Q000379:methods*; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010316:Particle Size; D010957:Plasmids / Q000737:chemistry; D011108:Polymers / Q000138:chemical synthesis*; D014162:Transfection
chemical_list: D000178:Acrylamides; D001672:Biocompatible Materials; D011108:Polymers; D004247:DNA; D001120:Arginine; D007770:L-Lactate Dehydrogenase; D008156:Luciferases; C045985:methacrylamide
keywords: Arginine; Endocytic pathway; Fluorescence colocalization; Gene delivery; Vector