Integration of [12]aneN3 and Acenaphtho[1,2-b]quinoxaline as non-viral gene vectors with two-photon property for enhanced DNA/siRNA delivery and bioimaging.

Two-photon fluorescent Acenaphtho[1,2-b]quinoxaline (ANQ) and the hydrophilic di-(triazole-[12]aneN3) moieties were combined through an alkyl chain (ANQ-A-M) or a ß-hairpin motif with two aromatic γ-amino acid residues (ANQ-H-M) to explore their capabilities for in vitro and in vivo gene delivery and tracing. ANQ-A-M and ANQ-H-M showed the same maximum absorption at 420 nm, and their fluorescent intensities around 650 nm were varied in different solvents and became poor in the protic solvents. Gel electrophoresis assays indicated that both compounds completely retarded the migration of pDNA at 20 µM in the presence of DOPE. However, the DNA condensation with ANQ-H-M was not reversible, and the particle size of the corresponding complexes were larger indicated from the SEM and DLS measurements. In vitro transfections indicated ANQ-A-M/DOPE achieved Luciferase and GFP expressions were to be 7.9- and 5.7-fold of those by Lipo2000 in A549 cells respectively. However, ANQ-H-M showed very poor transfection efficiency in Luciferase expression. With the help of single/two-photon fluorescence imaging it clearly demonstrated that the successful transfection of ANQ-A-M was attributed to its cellular uptake, apparent lysosomal escape, and reversible release of DNA; and the poor transfection of ANQ-H-M was resulted from the aggregation of the DNA complexes which prevented them from the cellular uptake, and also the strong binding ability which is not easy to release DNA. ANQ-A-M/DOPE also exhibited robust gene silencing (83% knockdown of Luciferase) and GFP expression (2.47-fold higher) efficiency compared with Lipo2000 in A549 and zebrafish, respectively. The work demonstrated that the linkage structure between fluorescent and di(triazole-[12]aneN3) played the important role for their gene delivery performance, and that ANQ-A-M represents a vector with the strong transfection efficiency in vitro and in vivo as well as the efficient real time bioimaging properties, which is potential for the development in biomedical research.

Di[12]aneN3-Functionalized Green Fluorescent Protein Chromophore for GFP Luminescence Simulation and Efficient Gene Transfection In Vitro and In Vivo.

Although a plethora of gene carriers have been developed for potential gene therapy, imageable stimuli-responsive gene vectors with fast access to the nucleus, high biocompatibility, and transfection efficiency are still scarce. Herein, we report the design and synthesis of four dendrite-shaped cationic liposomes, MPA-HBI-R/DOPE (R: n-butyl, 1; n-octyl, 2; n-dodecyl, 3; palmyl, 4), prepared via esterification of 4-alkoxybenzylideneimidazolinone containing aliphatic chains of different lengths (HBI-R), the green fluorescent protein (GFP) chromophore, with a di[12]aneN3 unit. Liposomes were fabricated via the self-assembly of MPA-HBI-R, assisted with 1,2-dioleoyl-sn-glycerol-3-phosphorylethanolamine (DOPE). These liposomes (MPA-HBI-R/DOPE) exhibited efficient DNA condensation, pH-responsive degradation, excellent cellular biocompatibility (up to 150 µM), and high transfection efficiency. Molecular docking experiments were also used to verify the optimal interaction between MPA-HBI-R and DNA, as well as the fluorescence enhancements. In particular, MPA-HBI-2/DOPE delivered DNA into the nucleus in less than an hour, and its luciferase transfection activity was more than 10 times that by Lipo2000, across multiple cell lines. The GFP chromophore conjugation allowed trackable intracellular delivery and release of DNA in real time via fluorescence imaging. Furthermore, efficient red fluorescent protein (RFP) transfection in zebrafish, with an efficiency of more than 6 times that by Lipo2000, was also achieved. The results not only realized, for the first time, the combination of gene delivery and GFP-simulated light emission, allowing fluorescent tracking and highly efficient gene transfection, but also offered valuable insights into the use of biomimetic chromophore for the development of the next-generation nonviral vectors.