Alkyl chain moieties of polyamidoamine dendron-bearing lipids influence their function as a nonviral gene vector

We recently developed a novel family of cationic lipids consisting of a polyamidoamine (PAMAM) dendron and two dodecyl chains. Their transfection activity increases with increasing generation of the dendron moiety [Takahashi et al. (2003) Bioconjugate Chem. 14, 764-773]. In the present study, to elucidate the effect of hydrophobic tail moieties of the dendron-bearing lipids, two kinds of PAMAM G3 dendron-bearing lipids were synthesized with different alkyl lengths, DL-G3-2C18 and DL-G3-2C12. Their functions as gene vectors were compared. Irrespective of their different alkyl chain lengths, these dendron-bearing lipids formed complexes with plasmid DNA with similar efficiency. However, their complex sizes differed markedly: DL-G3-2C18 lipoplexes exhibited much smaller diameters than DL-G3-2C12 lipoplexes. Interaction of the lipoplexes with heparin revealed that the DL-G3-2C18 lipoplexes required more heparin than DL-G3-2C12 lipoplexes to cause dissociation of plasmid DNA from the lipoplexes. Although the DL-G3-2C12 lipoplexes and DL-G3-2C18 lipoplexes transfected CV1 cells with similar efficiency in the absence of serum, only the latter retained high transfection activity in the presence of serum. These results indicate that hydrophobic interaction of alkyl chain moieties plays an important role in the increment of stability and the serum-resistant transfection activity for dendron-bearing lipid lipoplexes.     

Polyamidoamine dendron-bearing lipids as a nonviral vector: influence of dendron generation

Recently, we demonstrated that octadecyl chains are important as alkyl chain moieties of polyamidoamine (PAMAM) dendron-bearing lipids for their serum-resistant transfection activity [Bioconjugate Chem.2007, 18, 1349-1354]. Toward production of highly potent vectors, we examined the influence of the generation of dendron moiety on transfection activity of PAMAM dendron-bearing lipids having two octadecyl chains. We synthesized dendron-bearing lipids with PAMAM G1, G2, and G3 dendrons, designated respectively as DL-G1-2C(18), DL-G2-2C(18), and DL-G3-2C(18). The DL-G2-2C(18) and DL-G3-2C(18) interacted with plasmid DNA effectively and formed stable lipoplexes with small sizes and spherical shape. However, DL-G1-2C(18) interacted with plasmid DNA less effectively and formed tubular-shaped lipoplexes with lower stability and larger size. Cells took up DL-G2-2C(18) and DL-G3-2C(18) lipoplexes efficiently, but cellular uptake of the DL-G1-2C(18) lipoplexes was less efficient. Nevertheless, DL-G1-2C(18) lipoplexes achieved 100-10?000 times higher levels of transgene expression, which was evaluated using luciferase gene as a reporter gene. Confocal scanning laser microscopic analysis of intracellular behaviors of the lipoplexes revealed that DL-G1-2C(18) lipoplexes generated free plasmid DNA molecules in the cytosol more effectively than other lipoplexes did. Moderate binding ability of DL-G1-2C(18) might be responsible for generation of lipoplexes which deliver plasmid DNA into cells, liberate it in the cytoplasm, and induce efficient transgene expression.     

Synthesis of novel cationic lipids having polyamidoamine dendrons and their transfection activity

We designed a novel type of cationic lipid, lipids with a cationic polar group in the polyamidoamine dendron, because these dendron-bearing lipids are expected to form complexes with plasmid DNA and achieve efficient transfection of cells by synergy of endosome buffering and membrane fusion with the endosome, both of which are useful for the promotion of the transfer of plasmid DNA from endosome to cytosol. Four kinds of lipids with polyamidoamine dendrons of first to fourth generations, DL-G1, DL-G2, DL-G3, and DL-G4, were synthesized. The lipid with a dendron of a higher generation exhibited greater ability to form lipoplexes with plasmid DNA, as estimated by agarose gel electrophoresis. While the DL-G1 lipoplex did not transfect CV1 cells, the lipoplexes containing the DL-G2, DL-G3, or DL-G4 could induce transfection of the cells, and their activity was elevated with increasing generation of the dendron. Addition of dioleoylphosphatidylethanolamine (DOPE), which is known to increase fusion ability of a lipid membrane, into the lipoplexes greatly enhanced their transfection activity. In addition, the comparison with DC-Chol-containing lipoplex, which is widely used as a nonviral vector, showed that the DL-G3-DOPE lipoplex exhibits more efficient transfections. These findings imply that these dendron-bearing lipids may form the basis for a novel family of cationic lipids for efficient gene delivery.     

Preparation of efficient gene carriers using a polyamidoamine dendron-bearing lipid: improvement of serum resistance

In a previous study, we developed a novel cationic lipid consisting of polyamidoamine dendron of third generation and two dodecyl chains, designated as DL-G3, which in combination with a fusogenic lipid dioleoylphosphatidylethanolamine (DOPE) achieves efficient transfection of CV1 cells by synergetic action of the proton sponge effect and membrane fusion. This study examines the effect of serum on the transfection activity of the DL-G3-DOPE-plasmid DNA lipoplexes. The transfection activity of a lipoplex with a composition optimized in the absence of serum decreased markedly in the presence of serum. However, the lipoplexes that induce efficient transfection in the presence of serum were obtainable by controlling the charge ratio of the primary amine of the DL-G3 to the phosphate group (N/P ratio) and DOPE content. The complex, which exhibited the highest transfection activity in the presence of serum, has a lower N/P ratio and higher DOPE content than that optimized in the absence of serum. Whereas disintegration of these complexes was induced by addition of heparin, which is a polysaccharide with negatively charged groups, the complex that retained transfection activity in the presence of serum required more negative charges of heparin for complex disintegration. That result implies its higher stability against negatively charged serum proteins. Comparison of the serum-resistant complex with some commercially available transfection reagents, such as Lipofectamine and SuperFect, indicates that the DL-G3 complex achieved more efficient transfection of these cells in the presence of serum.     

Highly fluorinated lipospermines for gene transfer: synthesis and evaluation of their in vitro transfection efficiency

Fluorinated double-chain lipospermines (one or both of these chains being ended by a highly fluorinated tail of various length) which are close analogues of DOGS (Transfectam) were designed as synthetic vectors for gene delivery. For N/P ratios (N = number of amine functions of the lipid; P = number of DNA phosphates) from 0.8 to 10, these lipospermines condensed DNA, with or without the use of DOPE, to form fluorinated lipoplexes. The efficiency of the fluorinated lipoplexes to transfect lung epithelial A549 cells was significantly higher than that of the DOGS lipoplexes. No specific cell toxicity was evidenced for the fluorinated lipoplexes as compared to that of the DOGS ones. The palette of structural elements explored allowed to determine those required for efficient transfection, highlighting the importance of highly fluorinated chains, the unique properties of unsaturated double-chain lipids and of the use of DOPE as helper lipid on transfection.     

Transfection with fluorinated lipoplexes based on fluorinated analogues of DOTMA,DMRIE and DPPES

Fluorinated double-chain (poly)cationic lipids (one or both of these chains being ended by a highly fluorinated tail) which are close analogues of DOTMA, DMRIE or DPPES were designed as synthetic vectors for gene delivery. For N/P ratios (N=number of amine functions of the lipid; P=number of DNA phosphates) from 0.8 to 5, these fluorinated cationic lipids condensed DNA, with or without the use of DOPE, to form fluorinated lipoplexes. No specific cell toxicity was evidenced for these new fluorinated lipoplexes. The efficiency of some of the fluorinated lipoplexes to transfect lung epithelial A549 cells was comparable to that of the first generation of fluorinated lipoplexes made from fluorinated analogues of DOGS (Transfectam) [Bioconjug. Chem. 12 (2001) 114]. These results, combined with the higher in vivo transfection potential found for fluorinated lipoplexes than for conventional lipoplexes or PEI polyplexes [J. Gene Med. 3 (2001) 109], confirm that fluorinated lipoplexes are very promising gene transfer systems.     

‘Click’ synthesized sterol-based cationic lipids as gene carriers,and the effect of skeletons and headgroups on gene delivery

In this work, we have successfully prepared a series of new sterol-based cationic lipids (1–4) via an efficient ‘Click’ chemistry approach. The pDNA binding affinity of these lipids was examined by EB displacement and agarose-gel retardant assay. The average particle sizes and surface charges of the sterol-based cationic lipids/pDNA lipoplexes were analyzed by dynamic laser light scattering instrument (DLS), and the morphologies of the lipoplexes were observed by atomic force microscopy (AFM). The cytotoxicity of the lipids were examined by MTT and LDH assay, and the gene transfection efficiencies of these lipid carriers were investigated by luciferase gene transfection assay in various cell lines. In addition, the intracellular uptake and trafficking/localization behavior of the Cy3-DNA loaded lipoplexes were preliminarily studied by fluorescence microscopy. The results demonstrated that the pDNA loading capacity, lipoplex particle size, zeta potential and morphology of the sterol lipids/pDNA lipoplexes depended largely on the molecular structure factors including sterol-skeletons and headgroups. Furthermore, the sterol-based lipids showed quite different cytotoxicity and gene transfection efficacy in A549 and HeLa cells. Interestingly, it was found that the cholesterol-bearing lipids 1 and 2 showed 7–10⁴ times higher transfection capability than their lithocholate-bearing counterparts 3 and 4 in A549 and HeLa cell lines, suggested that the gene transfection capacity strongly relied on the structure of sterol skeletons. Moreover, the study on the structure–activity relationships of these sterol-based cationic lipid gene carriers provided a possible approach for developing low cytotoxic and high efficient lipid gene carriers by selecting suitable sterol hydrophobes and cationic headgroups.     

trans-2-Aminocyclohexanol-based amphiphiles as highly efficient helper lipids for gene delivery by lipoplexes

Lipidic amphiphiles equipped with the trans-2-aminocyclohexanol (TACH) moiety are promising pH-sensitive conformational switches (“flipids”) that can trigger a lipid bilayer perturbation in response to increased acidity. Because pH-sensitivity was shown to improve the efficiency of several gene delivery systems, we expected that such flipids could significantly enhance the gene transfection by lipoplexes. Thus a series of novel lipids with various TACH-based head groups and hydrocarbon tails were designed, prepared and incorporated into lipoplexes that contain the cationic lipid 1,2-dioleoyl-3-trimethylammonio-propane (DOTAP) and plasmid DNA encoding a luciferase gene. B16F1 and HeLa cells were transfected with such lipoplexes in both serum-free and serum-containing media. The lipoplexes consisting of TACH-lipids exhibited up to two orders of magnitude better transfection efficiency and yet similar toxicity compared to the ones with the conventional helper lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol. Thus, the TACH-lipids can be used as novel helper lipids for efficient gene transfection with low cytotoxicity.     

In vitro cationic lipid-mediated gene delivery with fluorinated glycerophosphoethanolamine helper lipids.

There is a need for the development of nonviral gene transfer systems with improved and original properties. "Fluorinated" lipoplexes are such candidates, as supported by the remarkably higher in vitro and in vivo transfection potency found for such fluorinated lipoplexes as compared with conventional ones or even with PEI-based polyplexes (Boussif, O., Gaucheron, J., Boulanger, C., Santaella, C., Kolbe, H. V. J., Vierling, P. (2001) Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophosphoethanolamine helper lipid. J. Gene Med. 3, 109-114). Here, we describe the synthesis of fluorinated glycerophosphoethanolamines (F-PEs), close analogues of dioleoylphosphatidylethanolamine (DOPE), and report on their lipid helper properties vs that of DOPE, as in vitro gene transfer components of fluorinated lipoplexes based on pcTG90, DOGS (Transfectam), or DOTAP. To evaluate the contribution of the F-PEs to in vitro lipoplex-mediated gene transfer, we examined the effect of including the F-PEs in lipoplexes formulated with these cationic lipids (CL) for various CL:DOPE:F-PE molar ratios [1:(1 - x):x with x = 0, 0.5 and 1; 1:(2 - y):y with y = 0, 1, 1.5, and 2], and various N/P ratios (from 10 to 0.8, N = number of CL amines, P = number of DNA phosphates). Irrespective of the F-PE chemical structure, of the colipid F-PE:DOPE composition, and of the N/P ratio, comparable transfection levels to those of their respective control DOPE lipoplexes were most frequently obtained when using one of the F-PEs as colipid of DOGS, pcTG90, or DOTAP in place of part of or of all DOPE. However, a large proportion of DOGS-based lipoplexes were found to display a higher transfection efficiency when formulated with the F-PEs rather than with DOPE alone while the opposite tendency was evidenced for the DOTAP-based lipoplexes. The present work indicates that "fluorinated" lipoplexes formulated with fluorinated helper lipids and conventional cationic lipids are very attractive candidates for gene delivery. It confirms further that lipophobicity and restricted miscibility of the lipoplex lipids with the endogenous lipids does not preclude efficient gene transfer and expression. Their transfection potency is rather attributable to their unique lipophobic and hydrophobic character (resulting from the formulation of DNA with fluorinated lipids), thus preventing to some extent DNA from interactions with lipophilic and hydrophilic biocompounds, and from degradation.     

Low-pH-sensitive poly(ethylene glycol) (PEG)-stabilized plasmid nanolipoparticles: effects of PEG chain length, lipid composition and assembly conditions on gene delivery

BACKGROUND: We have studied the effects of the poly(ethylene glycol) (PEG) chain length and acyl chain composition on the pH-sensitivity of acid-labile PEG-diorthoester (POD) lipids. The optimal conditions are described for preparation of DNA plasmid encapsulated POD nanolipoparticles (NLPs) which mediate high gene delivery activity in vitro with moderate cytotoxicity. METHODS AND RESULTS: A series of POD lipids with various PEG chain lengths (750, 2000, and 5000 Da) or acyl chains (distearoyl 18:0 or dioleoyl 18:1) were incorporated into DNA containing NLPs or model liposomes as a stealth and bioresponsive component. We investigated the collapse kinetics of the POD-stabilized liposomes when the PEG chain length was changed. We optimized a detergent dialysis method to encapsulate plasmid DNA into NLPs prepared from a mixture of the various POD lipids, cationic lipid and phosphatidylethanolamine lipid. A critical concentration (28 mM) of n-octyl-beta-D-glucopyranoside (OG) enabled high encapsulation of DNA plasmid into 100 nm particles with a neutral surface charge. The POD NLPs are stable at pH 8.5 but rapidly collapse (approximately 10 min) into aggregates at pH 5.0. In the detergent solution there is a metastable DNA-lipid intermediate that evolves into a stable NLP if the detergent is removed shortly after adding DNA to the lipid-detergent mixture. The rank order of transfection activity from NLPs containing PEG-lipid was POD 750 > POD 5000 = POD 2000 > non-pH-sensitive PEG-lipid. The particle size stability was in the reverse order. Binding of the NLPs to cells reached a maximum level by 12 hours. The POD NLPs had slightly less transfection activity but considerably lower cytotoxicity than the PEI-DNA polyplex. CONCLUSIONS: Of the PEG-orthoester lipids tested, POD 2000 is the better choice for the preparation of sterically stabilized NLPs with a small particle diameter, good stability, low cytotoxicity, and satisfactory transfection activity.     

Transfection efficiency boost by designer multicomponent lipoplexes

Cationic liposome-DNA complexes (lipoplexes) have emerged as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. We investigated the correlation between structural evolution of multicomponent lipoplexes when interacting with cellular lipids, the extent of DNA release and the efficiency in transfecting mouse fibroblast (NIH 3T3), ovarian (CHO) and tumoral myofibroblast-like (A17) cell lines. We show, for the first time, that the transfection pattern increases monotonically with the number of lipid components and further demonstrate by means of synchrotron small angle X- ray scattering (SAXS) that structural changes of lipoplexes induced by cellular lipids correlate with the transfection efficiency. Specifically, inefficient lipoplexes either fused too rapidly upon interaction with anionic lipids or, alternatively, are found to be extremely resistant to solubilization. The most efficient lipoplex formulations exhibited an intermediate behaviour. The extent of DNA unbinding (measured by electrophoresis on agarose gel) correlates with structural evolution of the lipoplexes but DNA-release does not scale with the extent of transfection. The general meaning of our results is of broad interest in the field of non-viral gene delivery: rational adjusting of lipoplex composition to generate the proper interaction between lipoplexes and cellular lipids may be the most appropriate strategy in optimizing synthetic lipid transfection agents.     

Transfection efficiency boost by designer multicomponent lipoplexes

Cationic liposome-DNA complexes (lipoplexes) have emerged as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. We investigated the correlation between structural evolution of multicomponent lipoplexes when interacting with cellular lipids, the extent of DNA release and the efficiency in transfecting mouse fibroblast (NIH 3T3), ovarian (CHO) and tumoral myofibroblast-like (A17) cell lines. We show, for the first time, that the transfection pattern increases monotonically with the number of lipid components and further demonstrate by means of synchrotron small angle X- ray scattering (SAXS) that structural changes of lipoplexes induced by cellular lipids correlate with the transfection efficiency. Specifically, inefficient lipoplexes either fused too rapidly upon interaction with anionic lipids or, alternatively, are found to be extremely resistant to solubilization. The most efficient lipoplex formulations exhibited an intermediate behaviour. The extent of DNA unbinding (measured by electrophoresis on agarose gel) correlates with structural evolution of the lipoplexes but DNA-release does not scale with the extent of transfection. The general meaning of our results is of broad interest in the field of non-viral gene delivery: rational adjusting of lipoplex composition to generate the proper interaction between lipoplexes and cellular lipids may be the most appropriate strategy in optimizing synthetic lipid transfection agents.     

A new triantennary galactose-targeted PEGylated gene carrier, characterization of its complex with DNA, and transfection of hepatoma cells

Nonviral gene vectors remain inefficient in vivo largely because of their rapid clearance from the circulation and also their nonspecific association with the extracellular matrix. To overcome such drawbacks, cationic lipoplexes are now frequently coated with hydrophilic polymers such as PEGs to reduce nonspecific interactions, and ligands are also linked to their surface to obtain cell-specific gene transfer. In view of the development of vectors for systemic gene delivery, we have designed and studied lipoplexes that carry a triantennary galactosyl ligand attached to the distal end of a (PEG)45-conjugated lipid. We incorporated this targeted PEGylated lipid into lipoplexes using two strategies of formulation, i.e., using either preformed micelles or liposomes. We demonstrated that the incorporation of PEG chains stabilized lipoplexes and masked, but only partially, the positive charges exposed on the surface of the particles. We have also shown that incorporation into lipoplexes of a lipidated PEG chain, bearing a ligand at its distal end, yielded particles that exhibited an accessible ligand throughout the whole range of cationic lipid to DNA ratios. We obtained a targeted transfection in HepG2 cells with one of the formulations. Our results strengthen the validity of using a ligand carried by a long PEG spacer arm for targeted gene transfer.     

Transfection with fluorinated lipoplexes based on fluorinated analogues of DOTMA, DMRIE and DPPES

Fluorinated double-chain (poly)cationic lipids (one or both of these chains being ended by a highly fluorinated tail) which are close analogues of DOTMA, DMRIE or DPPES were designed as synthetic vectors for gene delivery. For N/P ratios (N=number of amine functions of the lipid; P=number of DNA phosphates) from 0.8 to 5, these fluorinated cationic lipids condensed DNA, with or without the use of DOPE, to form fluorinated lipoplexes. No specific cell toxicity was evidenced for these new fluorinated lipoplexes. The efficiency of some of the fluorinated lipoplexes to transfect lung epithelial A549 cells was comparable to that of the first generation of fluorinated lipoplexes made from fluorinated analogues of DOGS (Transfectam) [Bioconjug. Chem. 12 (2001) 114]. These results, combined with the higher in vivo transfection potential found for fluorinated lipoplexes than for conventional lipoplexes or PEI polyplexes [J. Gene Med. 3 (2001) 109], confirm that fluorinated lipoplexes are very promising gene transfer systems.     

Lipid composition of cationic nanoliposomes implicate on transfection efficiency

Abstract

Cationic liposome (CL)-DNA complexes (lipoplexes) have appeared as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. However, using CLs to deliver therapeutic nucleic acids and drugs to target organs have some problems, including low transfection efficiency. The aim of this study was developing novel CLs containing four neutral lipids; cholesterol, 1,2-dioleoyl phosphatidylethanolamine, distearoylphosphatidylcholine and dipalmitoylphosphatidylcholine as a helper lipid and dimethyl dioctadecyl ammonium bromide as a cationic lipid to increase transfection efficiency. We have investigated the correlation between number of lipid composition and transfection efficiency. The morphology, size and zeta potential of liposomes and lipoplexes were measured and lipoplexes formation was monitored by gel retardation assay. Transfection efficiency was assessed using firefly luciferase reporter assay. It was found that transfection efficiency markedly depended on liposome to plasmid DNA (pDNA) weight ratio, lipid composition and efficiency of pDNA entrapment. High transfection efficiency of plasmid by four component lipoplexes was achieved. Moreover, lipoplexes showed lower transfection efficiency and less cytotoxicity compared to Lipofectamine?. These results suggest that lipid composition of nanoliposomes is an important factor in control of their physical properties and also yield of transfection.     

Biophysical and lipofection studies of DOTAP analogs

In order to investigate the relationship between lipid structure and liposome-mediated gene transfer, we have studied biophysical parameters and transfection properties of monocationic DOTAP analogs, systematically modified in their non-polar hydrocarbon chains. Stability, size and (by means of anisotropy profiles) membrane fluidity of liposomes and lipoplexes were determined, and lipofection efficiency was tested in a luciferase reporter gene assay. DOTAP analogs were used as single components or combined with a helper lipid, either DOPE or cholesterol. Stability of liposomes was a precondition for formation of temporarily stable lipoplexes. Addition of DOPE or cholesterol improved liposome and lipoplex stability. Transfection efficiencies of lipoplexes based on pure DOTAP analogs could be correlated with stability data and membrane fluidity at transfection temperature. Inclusion of DOPE led to rather uniform transfection and anisotropy profiles, corresponding to lipoplex stability. Cholesterol-containing lipoplexes were generally stable, showing high transfection efficiency at low relative fluidity. Our results demonstrate that the efficiency of gene transfer mediated by monocationic lipids is greatly influenced by lipoplex biophysics due to lipid composition. The measurement of fluorescence anisotropy is an appropriate method to characterize membrane fluidity within a defined system of liposomes or lipoplexes and may be helpful to elucidate structure-activity relationships.     

Efficient gene transfection by bisguanylated diacetylene lipid formulations

We have previously shown that cationic cholesterol derivatives bearing guanidinium groups were efficient vectors for gene transfer. To further evaluate the potentiality of this novel class of cationic lipids, we undertook to study the transfection efficiency of guanidinium-based lipids with other hydrophobic moieties. Specifically, we synthesized a reagent where two guanidinium groups are linked to a diacetylene lipid which may provide the lipoplexes with favorable structural features. We report here that the cationic lipid bisguanidinium-diacetylene (BGDA) is highly efficient for in vitro gene transfection when formulated with dioleoylphosphatidyl ethanolamine (DOPE). We also show that liposomes composed of BGDA, DOPE, and a neutral diacetylene colipid, hydroxyethylenediacetylene (HEDA), are efficient for transfection. Thus, diacetylene-based lipids provide a novel scaffold for gene transfection and will be particularly useful for gaining new insights into the structure-activity relationships of the lipid/DNA complexes as they offer a means to study the effects of polymerizable domains.     

Transfection activity of polyamidoamine dendrimers having hydrophobic amino acid residues in the periphery

We designed poly(amidoamine) dendrimers with phenylalanine or leucine residues at their chain ends. Thereby, we achieved efficient gene transfection of cells through synergy of the proton sponge effect, which is induced by the internal tertiary amines of the dendrimer, and hydrophobic interaction by the hydrophobic amino acid residues in the dendrimer periphery. Dendrimers having 16, 29, 46, and 64 terminal phenylalanine residues were prepared by the reaction of the amine-terminated poly(amidoamine) G4 dendrimer and L-phenylalanine using condensing reagent 1,3-dicyclohexylcarbodiimide. Transfection activity of these phenylalanine-modified dendrimers for CV1 cells, an African green monkey kidney cell line, increased concomitant with the increasing number of the terminal phenylalanine residues, except for the dendrimer with 64 phenylalanine residues, which showed poor water solubility and hardly formed a complex with DNA at neutral pH. However, under weakly acidic conditions, the dendrimer with 64 phenylalanine residues formed a complex with DNA, thereby achieving highly efficient transfection. In contrast, the attachment of L-leucine residues was unable to improve the transfection activity of the parent dendrimer, probably because of the relatively lower hydrophobicity of this amino acid. The phenylalanine-modified dendrimer exhibited a higher transfection activity and a lower cytotoxicity than some widely used transfection reagents. For that reason, the phenylalanine-modified dendrimers are considered to be promising gene carriers.     

In vitro lipofection with novel series of symmetric 1,3-dialkoylamidopropane-based cationic surfactants containing single primary and tertiary amine polar head groups

A novel series of symmetric double-chained primary and tertiary 1,3-dialkoylamido monovalent cationic lipids were synthesized and evaluated for their transfection activities. In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), only the primary and tertiary dioleoyl derivatives 1,3lmp5 and 1,3lmt5, respectively elicited transfection activity. This is a striking difference between symmetrical 1,2-diacyl glycerol-based monovalent cationic lipids that always found both dioleoyl and dimyristoyl analogues being efficient transfection reagents. In the presence of helper lipid, all cationic derivatives induced marker gene expression, except the dilauroyl analogues 1,3lmp1 and 1,3lmt1 that elicited no transfection activity. Combining electrophoretic mobility data of the lipoplexes at different charge ratios with transfection activity suggested two requirements for high transfection activity with monovalent double-chained cationic lipids, that is, binding/association of the lipid to the plasmid DNA and membrane fusion properties of the lipid layers surrounding the DNA.     

Development of poly(amino ester glycol urethane)/siRNA polyplexes for gene silencing

Nonviral vectors, with their low immunogenicity and lack of pathogenicity, offer significant promise for siRNA therapy with fewer safety concerns. Nonviral vectors were also preferred in most transient siRNA delivery due to their ease of preparation. Previously, we incorporated tertiary amines and polyethylene glycol (PEG) into poly(ester urethane) to synthesize a soluble poly(amino ester glycol urethane), PaE(G)U, as a novel DNA transfection reagent for transgene delivery. The aim of this study was to develop PaE(G)U/siRNA polyplexes for gene silencing. We characterized the properties of PaE(G)U/siRNA polyplexes and compared them with those of PaE(G)U/DNA polyplexes. Using the Alexa Fluor 488-labeled, nonsilencing control siRNA as the reporter, we visualized cellular uptake of PaE(G)U/siRNA polyplexes and optimized the mass ratio of PaE(G)U/siRNA for delivery at 80/1. At this ratio, the average diameter of polyplexes was 540 nm, which was significantly larger than the average diameter of PaE(G)U/DNA polyplexes at 155 nm for efficient DNA delivery. Using the optimized PaE(G)U/siRNA polyplexes, transient silencing of constitutive luciferase expression (up to 92%) was achieved in our recombinant human HT-1080 fibroblast model via anti-luciferase siRNA delivery. In conclusion, PaE(G)U/siRNA polyplexes were developed and optimized for cellular uptake to allow efficient gene silencing. Engineering of soluble biodegradable polymers to incorporate amino, ester, PEG, and urethane units in the backbone constitutes a useful approach for the future design of siRNA carriers.