Cationic liposomes and polymers are both important candidates for use as non-viral gene vectors. However, both of them have special shortcomings and application limits. This work is devoted to the combination of advantages of liposomes and polymers. The ring-opening polymerization strategy was used for the preparation of amphiphilic polymers from cyclen-based cationic small lipids. The non-hydrophobic polymer and the corresponding lipids were also prepared for performing structure–activity relationship studies. Gel electrophoresis results reveal that both the lipopolymers and liposomes could effectively condense DNA into nanoparticles and protect DNA from degradation. Compared to polymers, the DNA binding ability of liposomes is more affected by hydrophobic tails. Under the same dosage, the synthetic polymers have stronger DNA binding ability than the liposomes. In vitro transfection experiments show that the polymers could give better transfection efficiency, which was much higher than those of the corresponding liposomes and non-hydrophobic polymer. The oleyl moiety is suitable for lipidic vectors, but things were different for polymers. Under optimized conditions, up to 14.2 times higher transfection efficiency than that for 25 kDa bPEI could be obtained. More importantly, the lipopolymers showed much better serum tolerance, which was further confirmed by protein adsorption, gel electrophoresis, flow cytometry, and CLSM assays. The results indicate that ring-opening polymerization is a promising strategy for the enhancement of the gene delivery efficiency and biocompatibility of cationic lipids.

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.

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.

Carbon dot (CD)-based multifunctional delivery systems have shown great potential in both drug/gene delivery and bio-imaging. In this work, we present a strategy to simply construct amphiphilic CDs (ACDs) by conjugating hydrophobic alkyl epoxide to the surface amino groups of PEI 600-derived CDs. ACDs could well dissolve in water or organic solvents and emit bright fluorescence both in solutions and cells. 1HNMR also suggested that ACDs may form micelle-like structures in water, and their CMC could be determined. Enhanced green fluorescent protein (EGFP) expression and flow cytometry experiments showed that ACDs have higher transfection efficiency than Lipofectamine 2000 in A549 cells. Besides DNA, ACDs could also effectively transfect Sur siRNA toward A549 cells and cause early cell apoptosis. The 3D multicellular spheroids further confirmed their high potential for delivering therapeutic genes into the tumor tissue. On the other hand, ACDs also exhibited good drug loading ability. CLSM experiment results showed that DOX could be effectively internalized by the cell and slowly released from the drug/ACD complex. These results suggest that ACDs may not only serve as versatile delivery vectors with potential for applications in clinical cancer treatment, but also offer an inspiration for the discovery of CD-based gene/drug delivery systems.

With the aim to develop a novel multifunctional gene delivery system that may overcome the common barriers of gene transfection, two amphiphilic molecules with tetraphenyl ethylene (TPE) as a hydrophobic moiety and linear PEI-galactose as a hydrophilic part were elaborately designed and synthesized. The molecules could self-assemble to form micelles and exhibited aggregation-induced emission (AIE) property, which may be used for cell imaging. Several assays revealed that the disulfide bond in TSPG may facilitate vector decomposition and DNA release, resulting in higher transfection efficiency and lower cytotoxicity than TCPG and PEI 25 kDa. These vectors showed excellent serum tolerance, and even higher efficiency could be obtained in transfection with the presence of serum. Moreover, the transfection in different cell lines showed that TSPG complex could induce higher gene expression in asialoglycoprotein receptor-rich HepG2 cells than in HeLa cells, which lack such receptors. Galactose competition assay also demonstrated that the galactose moiety could afford the vector with targeting ability toward hepatoma cells. Cellular uptake inhibition assay indicated that the DNA complex entered the cell mainly via caveolae-mediated endocytosis. Results showed that TSPG may serve as a promising reagent for simultaneous gene transfection and imaging with good biocompatibility and cell targeting ability.

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.

•Effect of the rigidity of lipid's linkage on their gene delivery ability was investigated.•Rigid aromatic linkage facilitates tight liposome formation and DNA condensation.•Lipids with rigid linkage have higher gene transfection efficiency and serum tolerance.•Transfection efficiency may be further improved according to mechanism study results.Although numerous cationic lipids have been developed as non-viral gene vectors, the structure-activity relationship (SAR) of these materials remains unclear and needs further investigation. In this work, a series of lysine-derived cationic lipids containing linkages with different rigidity were designed and synthesized. SAR studies showed that lipids with rigid aromatic linkage could promote the formation of tight liposomes and enhance DNA condensation, which is essential for the gene delivery process. These lipids could give much higher transfection efficiency than those containing more flexible aliphatic linkage in various cell lines. Moreover, the rigid aromatic linkage also affords the material higher serum tolerance ability. Flow cytometry assay revealed that the target lipids have good cellular uptake, while confocal microscopy observation showed weaker endosome escape than Lipofectamine 2000. To solve such problem and further increase the transfection efficiency, some lysosomotropic reagents were used to improve the endosome escape of lipoplex. As expected, higher transfection efficiency than Lipofectamine 2000 could be obtained via this strategy. Cytotoxicity assay showed that these lipids have lower toxicity in various cell lines than Lipofectamine 2000, suggesting their potential for further application. This work demonstrates that a rigid aromatic linkage might distinctly improve the gene transfection abilities of cationic lipids and affords information to construct safe and efficient gene vector towards practical application.Download high-res image (326KB)Download full-size image

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.

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.Keywords: aromatic backbone; cationic oligomer; nonviral gene vector; serum tolerance; structure−activity relationship

Synthetic polycations show great potential for the construction of ideal non-viral gene delivery systems. Several cationic polymers were synthesized by the epoxide ring-opening polymerization between diepoxide and various polyamines. Disulfide bonds were introduced to afford the polymers bio-reducibility, while the oxygen-rich structure might enhance the serum tolerance and biocompatibility. The polycations have much lower molecular weights than PEI 25 kDa, but still could well bind and condense DNA into nano-sized particles. DNA could be released from the polyplexes by addition of reductive DTT. Compared to PEI, the polycations have less cytotoxicity possibly due to their lower molecular weights and oxygen-rich structure. More significantly, these materials exhibit excellent serum tolerance than PEI, and up to 6 times higher transfection efficiency than PEI could be obtained in the presence of serum. The transfection mediated by TETA-SS was seldom affected even at a high concentration of serum. Much lower protein adsorption of polycations than PEI was proved by bovine serum albumin adsorption experiments. Flow cytometry also demonstrates their good serum resistance ability.

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.

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 series of novel cationic lipids based on 1,4,7,10-tetrazacyclododecane (cyclen) with the imidazole group as the pH-sensitive moiety and various aliphatic long chains were designed and synthesized. Cationic liposomes were prepared by mixing the lipids and the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in an appropriate molar ratio. The liposomes showed good stability and could condense plasmid DNA into nanosized particles (∼100 to ∼250 nm) with a positive zeta-potential (+10–25 mV). CCK-8-based cell viability assays showed a relatively lower cytotoxicity of the lipoplexes compared to commercially available lipofectamine 2000. Both enhanced green fluorescent protein and luciferase assays were carried out to investigate the in vitro transfection efficiency (TE) of the lipoplexes. Results showed that both the structures of the hydrophobic chain and the linking bond significantly affected the TE, and the linoleyl-containing lipoplex gave the best TE, which is comparable to lipofectamine 2000. The imidazole group was demonstrated to play an important role in the transfection, and the imidazole-absent analog gave dramatically lower TE. Furthermore, it was also found that Ca2+ could largely enhance the TE of these lipids, and the optimized TE was about 5 times higher than lipofectamine 2000. Flow cytometry demonstrates that the enhancement of TE by Ca2+ was caused by the improvement of cellular uptake. These results suggest that the cyclen-imidazole containing lipids might be promising non-viral gene delivery vectors.

Cationic lipids are the most widely used non-viral gene vectors, and it is important to develop novel lipids that have high transfection efficiency (TE) and good biocompatibility. A series of macrocyclic polyamine (cyclen and TACN)-based cationic lipids bearing different hydrophobic tails were synthesized with the ring-opening reaction. Several assays were used to study their interactions with plasmid DNA, and these lipoplexes could efficiently condense DNA into nanoparticles with proper sizes and zeta potentials. CCK-8-based cell viability assays showed relatively lower cytotoxicity of the lipoplexes compared with the commercially available Lipofectamine 2000. With transfection in vitro, lipid 6a had comparable or better TE than Lipofectamine 2000 in both 7402 and A549 cells. Interestingly, the TE of 6a was significantly increased in the presence of serum. The results not only demonstrate that lipid 6a can be a promising non-viral gene vector, they also provide clues for developing cationic gene vectors for future in vivo applications.

•A series of polymeric vectors were synthesized via Michael addition from LMW PEI.•Enhanced transfection efficiency and reduced toxicity than 25 KDa PEI were obtained.•These non-viral vectors exhibit excellent serum tolerance in gene transfection.A series of polycationic gene delivery vectors were synthesized via Michael addition from low molecular weight PEI and linking compounds with various heteroatom compositions. Agarose gel electrophoresis results reveal that these polymers can well condense plasmid DNA and can protect DNA from degradation by nuclease. The formed polyplexes, which are stable toward serum, have uniform spherical nanoparticles with appropriate sizes around 200–350 nm and zeta-potentials about +40 mV. In vitro experiments show that these polymers have lower cytotoxicity and higher transfection efficiency than 25 KDa PEI. Furthermore, the title materials exhibit excellent serum tolerance. With the present of 10% serum, up to 19 times higher transfection efficiency than PEI was obtained, and no obvious decrease of TE was observed even the serum concentration was raised to >40%. Flow cytometry and confocal microscopy studies also demonstrate the good serum tolerance of the materials.

•Novel bioreducible disulfide bridged polymers based on G1 dendrimer were prepared.•These polymers gave 15 times higher transfection efficiency than PEI/DNA complex.•Polymer prepared from ring-opening reaction may have better serum tolerance.•The disulfide linkage plays important role in the transfection process.A series of cationic polymers based on low generation (G1) peptide dendrimer were synthesized with disulfide-containing linkages. The DNA binding abilities of the target polymers were studied by gel electrophoresis and fluorescence quenching assay. The bioreducible property of the disulfide-containing polymers P2 and P3 was also investigated in the presence of dithiothreitol (DTT). Results from dynamic light scattering (DLS) and transmission electron microscopy (TEM) assays reveal that these materials may condense DNA into nanoparticles with proper sizes and zeta-potentials. In vitro cell experiments show that compared to branched 25 KDa PEI, P2 and P3 may exhibit much higher gene transfection efficiency and lower cytotoxicity in both HEK293 and U-2OS cells. Additionally, polymer prepared from Michael addition gives better gene transfection ability, while polymer prepared from ring-opening reaction has better serum tolerance. Results indicate that these polymers might be promising non-viral gene vectors for their easy preparation, very low cytotoxicity, and good transfection efficiency.

•Novel low molecular weight PEI-appended biodegradable polymers were prepared.•These polymers have much lower cytotoxicity than 25 kDa PEI in several cell lines.•The polyplex gave 54 times higher transfection efficiency than PEI/DNA complex.Routine clinical implementation of human gene therapy requires safe and efficient gene delivery methods. Linear biodegradable polyesters with carbon–carbon double bonds are prepared from unsaturated diacids and diols. Subsequent appending of low molecular weight PEI by Michael addition gives target cationic polymers efficiently. Agarose gel retardation and fluorescence quenching assays show that these materials have good DNA binding ability and can completely retard plasmid DNA at weight ratio of 0.8. The formed polyplexes have appropriate sizes around 275 nm and zeta-potential values about +20–35 mV. The cytotoxicities of these polymers assayed by MTT are much lower than that of 25 kDa PEI. In vitro transfection toward 7402, HEK293 and U-2OS cells show that polymer P1 may give dramatically higher transfection efficiency (TE) than 25 kDa PEI, especially in U-2OS cells, suggesting that such polymer might be promising non-viral gene vectors.Novel low molecular weight PEI-appended biodegradable polymers were prepared and applied as non-viral gene vectors. Compared to 25 kDa PEI, these polymers have much higher transfection efficiency and lower cytotoxicity.

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.

A series of novel 1,4,7,10-tetraazacyclododecane (cyclen)-based cationic lipids with asymmetric double hydrophobic tails (cholesteryl and long aliphatic chains) were designed and synthesized. Lysine was chosen as a linking moiety in the molecular backbone. The liposomes formed from 8 and dioleoylphosphatidylethanolamine (DOPE) could bind and condense plasmid DNA into nanoparticles under a low N/P ratio. These nano-scaled lipoplexes have low cytotoxicity, and might efficiently transfect A549 cells. In vitro transfection results revealed that all cationic lipids showed a comparable or better transfection efficiency (TE) than commercially available Lipofectamine 2000. The length and saturation degree of the aliphatic chain would affect their gene transfection performance, and the linoleic acid-containing 8e could give the best TE.

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.

A series of linear cationic polymers were synthesized by the ring-opening polymerization between diglycidyl ethers and 1-Cbz-1,4,7-triazacyclononane (TACN). Besides the good pH buffering capacity in endosome pH range caused by TACN, these polymers have evenly distributed hydroxyl groups, which may benefit not only the water solubility but also their biocompatibility and serum tolerance. The polymers could condense DNA into nanoparticles with appropriate sizes and zeta-potentials. Cytotoxicity assays reveal that most of the polyplexes formed from title polymers have lower cytotoxicity than those derived from PEI. In vitro transfection assays show that some of these materials have higher transfection efficiency than bPEI, especially in tumor cells with the presence of serum. Flow cytometry and confocal microscopy were applied to further confirm their good serum tolerance. The structure–activity relationship of such type of polymeric vectors was also discussed. Results suggest that the ring-opening polymerization may be an effective synthetic approach toward gene delivery materials with high biological activity.

An efficient oxidative coupling of estrogen derivatives was developed. Several 2-substitutent-17-deoxyestrones were applied to the aerobic oxidative coupling reactions catalyzed by CuCl2/TMEDA. The products were obtained as diastereoisomers with moderate to good yields. As chiral ligands, three couples of diastereoisomers were applied to the asymmetric HDA reaction to investigate their chemical properties.

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.

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.

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.

A series of novel 1,4,7,10-tetraazacyclododecane (cyclen)-based cationic lipids with asymmetric double hydrophobic tails (cholesteryl and long aliphatic chains) were designed and synthesized. Lysine was chosen as a linking moiety in the molecular backbone. The liposomes formed from 8 and dioleoylphosphatidylethanolamine (DOPE) could bind and condense plasmid DNA into nanoparticles under a low N/P ratio. These nano-scaled lipoplexes have low cytotoxicity, and might efficiently transfect A549 cells. In vitro transfection results revealed that all cationic lipids showed a comparable or better transfection efficiency (TE) than commercially available Lipofectamine 2000. The length and saturation degree of the aliphatic chain would affect their gene transfection performance, and the linoleic acid-containing 8e could give the best TE.

A series of novel cationic lipids based on 1,4,7,10-tetrazacyclododecane (cyclen) with the imidazole group as the pH-sensitive moiety and various aliphatic long chains were designed and synthesized. Cationic liposomes were prepared by mixing the lipids and the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in an appropriate molar ratio. The liposomes showed good stability and could condense plasmid DNA into nanosized particles (∼100 to ∼250 nm) with a positive zeta-potential (+10–25 mV). CCK-8-based cell viability assays showed a relatively lower cytotoxicity of the lipoplexes compared to commercially available lipofectamine 2000. Both enhanced green fluorescent protein and luciferase assays were carried out to investigate the in vitro transfection efficiency (TE) of the lipoplexes. Results showed that both the structures of the hydrophobic chain and the linking bond significantly affected the TE, and the linoleyl-containing lipoplex gave the best TE, which is comparable to lipofectamine 2000. The imidazole group was demonstrated to play an important role in the transfection, and the imidazole-absent analog gave dramatically lower TE. Furthermore, it was also found that Ca2+ could largely enhance the TE of these lipids, and the optimized TE was about 5 times higher than lipofectamine 2000. Flow cytometry demonstrates that the enhancement of TE by Ca2+ was caused by the improvement of cellular uptake. These results suggest that the cyclen-imidazole containing lipids might be promising non-viral gene delivery vectors.

Cationic liposomes and polymers are both important candidates for use as non-viral gene vectors. However, both of them have special shortcomings and application limits. This work is devoted to the combination of advantages of liposomes and polymers. The ring-opening polymerization strategy was used for the preparation of amphiphilic polymers from cyclen-based cationic small lipids. The non-hydrophobic polymer and the corresponding lipids were also prepared for performing structure–activity relationship studies. Gel electrophoresis results reveal that both the lipopolymers and liposomes could effectively condense DNA into nanoparticles and protect DNA from degradation. Compared to polymers, the DNA binding ability of liposomes is more affected by hydrophobic tails. Under the same dosage, the synthetic polymers have stronger DNA binding ability than the liposomes. In vitro transfection experiments show that the polymers could give better transfection efficiency, which was much higher than those of the corresponding liposomes and non-hydrophobic polymer. The oleyl moiety is suitable for lipidic vectors, but things were different for polymers. Under optimized conditions, up to 14.2 times higher transfection efficiency than that for 25 kDa bPEI could be obtained. More importantly, the lipopolymers showed much better serum tolerance, which was further confirmed by protein adsorption, gel electrophoresis, flow cytometry, and CLSM assays. The results indicate that ring-opening polymerization is a promising strategy for the enhancement of the gene delivery efficiency and biocompatibility of cationic lipids.

Synthetic polycations show great potential for the construction of ideal non-viral gene delivery systems. Several cationic polymers were synthesized by the epoxide ring-opening polymerization between diepoxide and various polyamines. Disulfide bonds were introduced to afford the polymers bio-reducibility, while the oxygen-rich structure might enhance the serum tolerance and biocompatibility. The polycations have much lower molecular weights than PEI 25 kDa, but still could well bind and condense DNA into nano-sized particles. DNA could be released from the polyplexes by addition of reductive DTT. Compared to PEI, the polycations have less cytotoxicity possibly due to their lower molecular weights and oxygen-rich structure. More significantly, these materials exhibit excellent serum tolerance than PEI, and up to 6 times higher transfection efficiency than PEI could be obtained in the presence of serum. The transfection mediated by TETA-SS was seldom affected even at a high concentration of serum. Much lower protein adsorption of polycations than PEI was proved by bovine serum albumin adsorption experiments. Flow cytometry also demonstrates their good serum resistance ability.