Yan-Hong Liu

Mitochondria-targeted ratiometric fluorescent probe for real time monitoring of pH in living cells

Pyridinium functioned 7-hydroxy coumarin was presented as the first mitochondria-targeted ratiometric fluorescent probe CP for real time monitoring pH in living cells. Compared with commercially available mitochondrial trackers, CP possesses high specificity to mitochondria in living cells as well as good biocompatibility. Meanwhile, CP displays excellent pH sensitivity and anti-interference capability. Confocal image experiments confirm that CP can monitor mitochondrial pH changes associated with the mitochondrial acidification, cellular apoptosis and stress response efficiently in real time.

Diol glycidyl ether-bridged low molecular weight PEI as potential gene delivery vehicles

A series of cationic polymers (P1-P5) were designed and synthesized using a ring-opening polymerization strategy based on low molecular weight polyethyleneimine (PEI) and using diglycidyl ethers as the bridging moiety. Although these polymers have reduced amino group density relative to 25 kDa PEI, their pH buffering capacity and deoxyribonucleic acid (DNA) binding ability were seldom affected. They were able to condense plasmid DNA (pDNA) well to form nanoparticles of suitable sizes (150-300 nm) and positive zeta potentials (+25-40 mV). Cell Counting Kit-8 (CCK-8) assays revealed that polyplexes formed from these polymers have lower cytotoxicity than those derived from PEI. Luciferase reporter gene delivery experiments indicated that these polymers have much better transfection efficiency than 25 kDa PEI, especially P2 and P5. Unlike PEI, serum has little negative effect on the transfection by these materials, and their transfection efficiencies were seldom reduced even with high concentrations of serum. Under optimized conditions, up to 400 times higher transfection efficiency than with PEI could be achieved. Several assays including gel electrophoresis, dynamic light scattering and transmission electron microscopy also confirmed the good serum tolerance of these polyplexes. The evenly distributed hydroxyl groups formed by the ring-opening polymerization are considered to contribute much to their high serum tolerance, and such polymerization might be a promising strategy for the design of efficient non-viral gene delivery vectors.

A highly sensitive and selective “turn-on” fluorescent probe for hypochlorous acid monitoring

A novel “turn-on” fluorescent probe 3 for monitoring hypochlorous acid was investigated, which showed high selectivity and sensitivity to hypochlorous acid over other ROS and was loaded into cell media to image HClO.

Cyclen-based cationic lipids with double hydrophobic tails for efficient gene delivery

Cationic lipids have been regarded as an important type of non-viral gene vector; to develop novel lipids with high transfection efficiency (TE) and biocompatibility is of great importance. A series of cyclen-based cationic lipids bearing double hydrophobic tails were synthesized herein. To study their structure-activity relationship (SAR), several analogs including the amide-contained double-tailed lipids, lipids containing ester bonds with the reverse direction, and lipids with a single tail were also prepared. Several assays were used to study their interactions with plasmid DNA, and results reveal that these lipids could smoothly condense DNA into nanosized particles. CCK-8-based cell viability assays showed relatively lower cytotoxicity of the lipoplexes compared to commercially available Lipofectamine 2000. In vitro transfection assays exhibited that some of the lipids (5a, 5b and 8b) may give excellent TEs, which were up to 10 times higher than Lipofectamine 2000. SAR of these lipids was studied in detail by investigating the effects of several structural aspects including the chain length and saturation degree, chain numbers, the type of linkage bond, and orientation of ester bonds. The results not only demonstrate that these lipids might be promising non-viral gene vectors, but also afford us clues for further optimization of lipidic gene delivery materials.

Low Molecular Weight Oligomers with Aromatic Backbone as Efficient Nonviral Gene Vectors

A series of oligomers were synthesized via ring-opening polymerization. Although the molecular weights of these oligomers are only ∼2.5 kDa, they could efficiently bind and condense DNA into nanoparticles. These oligomers gave comparable transfection efficiency (TE) to PEI 25 kDa, while their TE could even increase with the presence of serum, and up to 65 times higher TE than PEI was obtained. The excellent serum tolerance was also confirmed by TEM, flow cytometry, and BSA adsorption assay. Moreover, structure-activity relationship studies revealed some interesting factors. First, oligomers containing aromatic rings in the backbone showed better DNA binding ability. These materials could bring more DNA cargo into the cells, leading to much better TE. Second, the isomerism of the disubstituted phenyl group on the oligomer backbone has large effect on the transfection. The ortho-disubstituted ones gave at least 1 order of magnitude higher TE than meta- or para-disubstituted oligomers. Gel electrophoresis involving DNase and heparin indicated that the difficulty to release DNA might contribute to the lower TE of the latter. Such clues may help us to design novel nonviral gene vectors with high efficiency and biocompatibility.

Cationic gemini lipids with cyclen headgroups: interaction with DNA and gene delivery abilities

A series of novel 1,4,7,10-tetraazacyclododecane (cyclen)-based gemini cationic lipids were synthesized, and L-cystine was used as backbone between the two amphiphilic units. The liposomes formed from the lipids and DOPE could efficiently condense plasmid DNA into nanoparticles with suitable size and zeta-potentials, which might be suitable for gene transfection. These lipids were applied as non-viral gene delivery vectors, and their structure–activity relationship was studied. It was found that both the hydrophobic tails and the linking group could largely influence the transfection efficiency, and the oleylamine derived lipid gave the best transfection results, which were close to the commercially available transfection reagent lipofectamine 2000. The gemini structure would favor the gene transfection, and the transfection efficiency of the gemini lipid was much higher than the mono counterpart. Besides, these lipids have very low cytotoxicity, suggesting their good biocompatibility. Results indicate that such gemini lipids might be promising non-viral gene delivery vectors.

Hyaluronic acid-based carbon dots for efficient gene delivery and cell imaging

A strategy that uses hyaluronic acid (HA) as a carbon source and polyethylenimine (PEI) as a passivant to construct carbon dots (HA-Cds) was proposed. The synthetic method is simple and green and no additive was required. These carbon dots could emit strong blue fluorescence under UV light. FT-IR and 1H NMR spectra confirmed that part of characteristic residues of HA and PEI remained in the HA-Cds structure. These materials had much lower cytotoxicity than PEI and high serum tolerance. Up to 50 times higher transfection efficiency than that of PEI was obtained in the presence of 10% serum. BSA protein adsorption, flow cytometry, and confocal microscopy assays also supported their good performance with serum. Furthermore, as multifunctional materials, HA-Cds had good intracellular imaging ability and displayed tunable fluorescence emission under varying excitation wavelengths. An HA competition assay showed that they may have target cell imaging ability in CD44 overexpressed cells. These materials with fluorescence activity also facilitated co-localization experiments by CLSM, which revealed that the DNA cargo could be effectively released into the cytosol, leading to effective gene transfection. These properties make the carbon dots promising candidates for in vivo diagnosis and gene therapy.

BODIPY-Based Two-Photon Fluorescent Probe for Real-Time Monitoring of Lysosomal Viscosity with Fluorescence Lifetime Imaging Microscopy

The viscosity of lysosome is reported to be a key indicator of lysosomal functionality. However, the existing mechanical methods of viscosity measurement can hardly be applied at the cellular or subcellular level. Herein, a BODIPY-based two-photon fluorescent probe was presented for monitoring lysosomal viscosity with high spatial and temporal resolution. By installing two morpholine moieties to the fluorophore as target and rotational groups, the TICT effect between the two morpholine rings and the main fluorophore scaffold endowed the probe with excellent viscosity sensitivity. Moreover, Lyso-B succeeded in showing the impact of dexamethasone on lysosomal viscosity in real time.

Polyethylenimine analogs for improved gene delivery: effect of the type of amino groups

A significant gap currently exists in our understanding of how the detailed chemical characteristics of polycationic gene carriers can improve their delivery performance. The aim of this contribution is to develop a branched polyethylenimine (PEI)-mimetic biodegradable polymer that can increase the transfection efficiency (TE). Michael addition between tris(2-aminoethyl)amine and diacryl amides with special structures was applied to synthesize several degradable polymers with different amine compositions (primary/secondary/tertiary). The 1:1 polymerization could be confirmed by NMR, making the amine composition controllable. Such composition was found to be able to affect the buffering ability of the materials, and the ratio of 1°:2°:3° amine = 1:4:1 is preferable for the pH buffering ability, also for the in vitro gene transfection. Up to 64 times higher TE than PEI was obtained in HeLa cells with the presence of 10% serum. Their excellent serum tolerance was also demonstrated by a bovine serum albumin (BSA) adsorption assay, in which much lower protein adsorption than 25 kDa PEI was observed. Structure-activity relationship studies also revealed that higher proportion of 2° amines may benefit the DNA binding ability, but the balance between the DNA condensation and release is more essential for non-viral vectors.

Cyclen-based double-tailed lipids for DNA delivery: Synthesis and the effect of linking group structures.

The gene transfection efficiency (TE) of cationic lipids is largely influenced by the lipid structure. Six novel 1, 4, 7, 10-tetraazacyclododecane (cyclen)-based cationic lipids L1-L6, which contain double oleyl as hydrophobic tails, were designed and synthesized. The difference between these lipids is their diverse backbone. Liposomes prepared by the lipids and DOPE showed good DNA affinity, and full DNA condensation could be achieved at N/P of 4 to form lipoplexes with proper size and zeta-potentials for gene transfection. Structure-activity relationship of these lipids as non-viral gene delivery vectors was investigated. It was found that minor backbone structural variations, including linking group and the structural symmetry would affect the TE. The diethylenetriamine derived lipid L4 containing amide linking bonds gave the best TE, which was several times higher than commercially available transfection reagent lipofectamine 2000. Besides, these lipids exhibited low cytotoxicity, suggesting their good biocompatibility. Results reveal that such type of cationic lipids might be promising non-viral gene vectors, and also afford us clues for the design of novel vectors with higher TE and biocompatibility.