Yi-Mei Zhang

Novel imidazole-functionalized cyclen cationic lipids: synthesis and application as non-viral gene vectors.

A series of novel 1,4,7,10-tetraazacyclododecanes (cyclen)-based cationic lipids bearing histidine imidazole group 10a-10e were synthesized. These amphiphilic molecules have different hydrophobic tails (long chain, cholesterol or α-tocopherol) and various type of linking groups (ether, carbamate or ester). These molecules were used as non-viral gene delivery vectors, and their structure-activity relationships were investigated. As expected, the imidazole group could largely improve the buffering capabilities comparing to cyclen. The liposomes formed from 10 and dioleoylphosphatidyl ethanolamine (DOPE) could bind and condense plasmid DNA into nanoparticles with proper size and zeta-potentials. Comparing with Lipofectamine 2000, the formed lipoplexes gave lower transfected cells proportion, but higher fluorescence intensity, indicating their good intracellular delivering ability. Furthermore, results indicate that transfection efficiency of the cationic lipids is influenced by not only the hydrophobic tails but also the linking group. The cyclen-based cationic lipid with α-tocopherol hydrophobic tail and an ester linkage could give the highest transfection efficiency in the presence of serum.

Biotinylated Cyclen-Contained Cationic Lipids as Non-Viral Gene Delivery Vectors

A series of 1, 4, 7, 10‐tetraazacyclododecane (cyclen)‐based cationic lipids, namely 5a–c bearing a biotin moiety and a variety of end groups (cholesterol, diosgenin, and α‐tocopherol) via biodegradable carbamate bond linkage were prepared and applied as non‐viral gene delivery vectors. The liposomes formed from 5 and dioleoylphosphatidylethanolamine could bind and condense plasmid DNA into nanoparticles with appropriate size and zeta potentials. All biotinylated cyclen cationic lipids showed higher cell viability than commercially available lipofectamine 2000 even at high N/P ratios, while their transfection efficiency was relatively lower. Further, results indicate that among the three lipids, α‐tocopherol‐containing compound 5c has higher DNA‐binding ability, lower cytotoxicity, and higher transfection efficiency. Transfection in two different cell lines revealed that these lipoplexes have higher gene delivery efficiency toward tumor cells.

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.

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.

Structure–activity relationship studies of symmetrical cationic bolasomes as non-viral gene vectors

Compared to traditional cationic lipids, bola-type lipids have received much less attention despite their advantages including the ability to form more stable and regular-shaped liposomes. In this report, a series of novel symmetric cationic bolalipids based on lysine or cyclen headgroups were designed and synthesized. Structure-activity relationships including the effect of the hydrophobic chain length and cationic headgroup on liposome formation, DNA binding, the physical property of bolasomes, and gene transfection were systematically studied. Results reveal that an appropriate hydrophobic chain length is essential to form nano-sized bolasomes with good DNA binding and condensation ability. MTS-based cell viability assays showed low cytotoxicity of these bolasome/DNA complexes. Lys-14-10, which has a 36-atom-length hydrophobic chain, exhibited the best transfection efficiency in the two cell lines. Flow cytometry and confocal laser microscopy assays reveal that the bolaplexes formed from bolalipids with such a chain might induce the highest cellular uptake. For the cationic headgroup, lysine is more suitable than cyclen for such a bola-type vector. Although the TEs of these bolalipids are still lower than commercially used non-bola lipid lipofectamine 2000, this study may give us some clues for the design of novel bolalipids with higher TE and biocompatibility.

Cross-linked polymers with fluorinated bridges for efficient gene delivery

A new strategy for the construction of fluorinated cationic polymers for gene delivery was introduced. The fluorinated polymers were synthesized by crosslinking low molecular weight PEI with diols containing various lengths of perfluoroalkyl chains via epoxide ring-opening polymerization. Such a study presents the first example of polymeric gene vectors with fluorination on the polymer backbone but not on the side chains. These materials showed good DNA condensation and protection ability and could condense DNA into nanoparticles with appropriate sizes and zeta-potentials. The fluorine atoms might strengthen the interaction toward DNA, leading to more stable polyplexes. In vitro transfection results showed that the fluorinated polymers could mediate efficient gene delivery toward both 2D and 3D cell cultures at low weight ratios, and their transfection efficiency was higher than that of PEI 25 kDa and their non-fluorinated counterparts. Several assays including DLS, TEM, luciferase reporter gene transfection and flow cytometry revealed that fluorination improved the serum resistance of these polymeric vectors, and more fluorine atoms might lead to better serum tolerance. These fluorinated materials exhibited very low cytotoxicity at transfection dosage. A cellular uptake study with uptake inhibitors indicated that macro-pinocytosis and microtubule-mediated endocytosis were the major endocytosis pathways for these polyplexes.

Functionalized Asymmetric Bola-Type Amphiphiles for Efficient Gene and Drug Delivery

The studies of bolaamphiphile-based nanoparticles as delivery vectors are still rudimentary and under development. In this study, several asymmetric bolaamphiphiles containing lysine and another moiety with special functions, such as pH-sensitive or cell-targeting property, were designed and synthesized. The potentials of these bolaamphiphile-based nanoparticles as versatile vectors for both nucleic acids and chemical drugs were studied. With the presence of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), these amphiphiles could be prepared into bolasomes, which showed good DNA binding ability and could condense plasmid DNA into nanoparticles with appropriate size and surface potential. Lys-His, which has a pH-sensitive histidine on one head, exhibited higher transfection efficiency than the symmetric counterpart and comparable efficiency to commercially available transfection reagent. Mechanism studies confirmed that the bolaplexes formed from Lys-His might induce the highest cellular uptake and the best endosomal escape ability. On the other hand, these bolaamphiphiles also exhibited good drug loading ability. The self-assembly vesicles could efficiently encapsulate the hydrophobic anti-cancer drug doxorubicin (DOX) in aqueous solution with high drug loading content and encapsulation efficiency. Confocal laser scanning microscopy (CLSM) experiment and cell viability assay exhibited a controlled release of the drug with the assistance of bolasomes. It was shown that such bolaamphiphiles have great potential as nano-vectors for both drug and gene or their co-delivery.

Cationic lipids with a cyclen headgroup: synthesis and structure–activity relationship studies as non-viral gene vectors

The clarification of the structure–activity relationships of non-viral gene delivery vectors is of great importance. A series of novel cyclen-based cationic lipids were synthesized and applied as gene carriers. The structures of these fifteen lipids varied with different linking groups and hydrophobic tails. The liposomes formed by these lipids and helper lipid DOPE could efficiently bind DNA and condense it into nano-sized particles with positive zeta-potentials and protect DNA from enzymatic digestion. Results revealed that the hydroxyl group or aromatic ring in the lipid structure would affect their DNA binding and transfection ability. Lipids with double hydrophobic tails gave much higher transfection efficiencies than those with a single tail, and aromatic rings in the lipid backbone might facilitate the transfection. The transfection efficiency could be further improved by optimizing the hydrophobic tails. The lipid with C14 tails gave a much higher efficiency than its analogs. A cytotoxicity assay showed that the lipids generally gave higher cell viabilities than a commercially available transfection reagent. It was suggested that such cationic lipids may act as promising non-viral gene delivery carriers.