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.

Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophosphoethanolamine helper lipid.

BACKGROUND: One of the main drawbacks of synthetic, non-viral gene vectors is their relatively low in vivo efficiency when compared with viral vectors. The present paper describes the use of a partially fluorinated glycerophosphoethanolamine (F-PE), a close analog of DOPE, which, as a helper lipid with the cationic lipopolyamine pcTG90, increases its in vitro and in vivo gene transfer capability to a larger extent than DOPE. METHODS: To evaluate the contribution of F-PE to lipoplex-mediated gene transfer, the effect of including F-PE in lipoplexes formulated with the lipopolyamine pcTG90 for various pcTG90/DOPE/F-PE molar ratios [1:(1-x): x; 1:(2-y):y] was examined. For the in vitro analyses on human lung carcinoma epithelial A549 cells, the lipoplexes were formulated with the luciferase reporter plasmid pTG11033 using various N/P ratios (from 10 to 0.8, N = number of pcTG90 amines, P = number of DNA phosphates). The in vivo analyses were performed (1) with the luciferase reporter plasmid pCMV-Luc, which gives higher luciferase expression in the lung than pcTG11033; (2) with pcTG90/co-lipid(s) (1:2) lipoplexes which yield higher expression than the (1:1) formulations; and (3) by intravenous (iv) injection into the tail vein of mice. RESULTS: The efficiency of the F-PE lipoplexes to transfect in vitro A549 cells was significantly higher (5-90-fold) than that of DOPE lipoplexes, when formulated in HEPES. However, when formulated in 5% glucose, both co-lipids display a comparable transfection helper potential. Most remarkably, an up to eight-fold increase of luciferase expression could be measured in the lung after iv injection of pcTG90/F-PE (1:2) N/P 5 lipoplexes as compared with the pcTG90/DOPE lipoplexes. It led also to higher luciferase expression than PEI(ExGen500)/pCMV-Luc N/P 10 polyplexes. Besides expression in lung, low levels of luciferase expression were also observed in heart, spleen and liver. CONCLUSION: The present work, showing a higher in vitro and in vivo transfection potential for lipoplexes formulated with a partially fluorinated co-lipid as compared with its analogous DOPE lipoplexes or PEI polyplexes, indicates that 'fluorinated' lipoplexes are attractive candidates for in vivo applications.

Lipopolycationic telomers for gene transfer: synthesis and evaluation of their in vitro transfection efficiency.

We report on the synthesis of a series of lipopolyamine telomers I-14,n, I-18,n, and II-18,n and on their in vitro gene-transfer capability. Their structure consists of a polyamine polar moiety, including n primary amine functions (from 1 to 70), connected to a hydrophobic moiety, including two hydrocarbon C14 or C18 chains, through a mercaptopropanoyl or mercaptoglyceryl unit and an amide or ether function. They were obtained by telomerization of N-[2-[(BOC)aminoethyl]]acrylamide with N,N-ditetradecyl- and N,N-dioctadecylpropanamide-3-thiol and rac-1,2-dioctadecyloxypropane-3-thiol, respectively, then BOC deprotection. For N/P ratios (N = number of telomer amine equivalents; P = number of DNA phosphates) from 0.8 to 10, these lipopolyamines condensed DNA, with or without the use of DOPE, forming lipopolyplexes or teloplexes of mean sizes less than 200 nm. Some trends, structure-activity and structure-toxicity relationships, were established to achieve both highest in vitro transfection levels and cell viability. Thus, DNA formulations based on telomers I-14,20 and I-18,20 and for N/P ratios lower than 5 led to the most efficient teloplex formulations for plasmid delivery to lung epithelial A549 cells.

Solvoplex: a new type of synthetic vector for intrapulmonary gene delivery.

A novel type of synthetic vector, termed solvoplex, is described that can greatly enhance gene expression in lung after intrapulmonary delivery. Solvoplexes consist of plasmid DNA and organic solvents. Several organic solvents were analyzed, and luciferase reporter gene expression was observed after intrapulmonary delivery of solvoplexes containing DPSO (di-n-propylsulfoxide), TMU (tetramethylurea), or BMSO (butylmethylsulfoxide). Expression levels correlated with the amount of solvent used at constant DNA amounts. Highest expression was obtained in the lung after intratracheal injection with 15% DPSO resulting in an increase up to 440-fold compared with DNA alone. DPSO-solvoplexes (15%) gave higher reporter gene expression than polyplexes (ExGen 500) or lipoplexes (DOTAP-cholesterol or DOTAP-DOPE). Solvoplex-mediated gene expression did not depend on the delivery mode, and was observed in both mice and rats. Readministration of DPSO-solvoplexes was possible. A second injection after 4 weeks resulted in expression levels similar to the first administration. Histological analyses using lacZ and GFP reporter genes demonstrated gene expression in the lung airway epithelium after intratracheal and microspray delivery. When luciferase expression levels in lung homogenates were compared with adenovirus vectors, DPSO-solvoplexes were 4- or 100-fold less efficient, depending on the promoter used in the viral vector. A quantitative histological comparison between solvoplexes and adenovirus vectors in the best expressing regions revealed that solvoplexes yielded about 2% LacZ-positive cells in the lung airway epithelium, and adenovirus vectors about 20%. Using the microsprayer system, we demonstrated that DNA remained intact in solvoplexes on spraying and that reporter gene expression was observed in mice after intrapulmonary delivery of a solvoplex spray. DNA in DPSO-solvoplexes remained stable and functional after prolonged storage at room temperature.

Synthesis of polyallylamine derivatives and their use as gene transfer vectors in vitro.

Cationic polymers possessing primary amine groups are inefficient in transferring nucleic acids into eukaryotic cells. With appropriate chemical modification, namely glycolylation of the amine groups of polylysine and polyallylamine, the actual number of free amino groups was decreased, hydrophilic residues were introduced, and the cytotoxicity of both polymers decreased significantly. Furthermore, in the case of polyallylamine, its ability to mediate gene transfer into cells increased by several orders of magnitude. Transfection efficiency was found to be dependent on the substitution level of amino groups and reached highest levels in the presence of lysosomotropic and/or fusogenic agents. At optimal conditions, glycolylated PAM was shown to be as efficient as the linear polyethylenimine of 22 kDa.

Effect of liposome-encapsulated clodronate pretreatment on synthetic vector-mediated gene expression in mice.

One of the main limitations for the use of synthetic vectors in gene therapy is their relatively low in vivo efficiency when compared with viral vectors. Here, we describe a pretreatment protocol with liposome-encapsulated clodronate in mice by which gene expression levels of a luciferase reporter gene could be increased up to nine-fold in the lung, after intravenous (i.v.) injection of glycerolipoplexes. Optimal results were obtained if mice were pretreated with liposome-encapsulated clodronate 1 day before injection of lipoplexes. The enhancement effect could be observed for lipoplexes prepared with different multivalent cationic glycerolipids. Most remarkably, polyplexes behaved in the opposite way. Liposome-encapsulated clodronate pretreatment strongly reduced reporter gene expression after i.v. injection of polyethylenimine-polyplexes (ExGen500).

Gene transfer with lipospermines and polyethylenimines.

It is an obvious and basic principle that to be efficient, gene therapy requires effective gene transfer followed by adequate gene expression. However, getting DNA, a pro-drug, into the cell and into the nucleus, remains a crucially limiting factor. Even recombinant viral methods still show poor performances in clinical situations and non-viral methods are considered classically to be of yet lower efficiency. Here, we consider the mode of action, the nature of the complexes formed with DNA and the transfection potentials of two categories of inert, cationic vectors, the lipospermines and polyethylenimine. Both are among the best vectors currently available for in vitro work. Moreover, polyethylenimine is proving to be a versatile and effective carrier for different in vivo situations, especially for delivering genes into the mammalian brain.

In vitro gene delivery to hepatocytes with galactosylated polyethylenimine.

A hepatocyte-directed vector has been developed; it includes several key features thought to favor in vivo gene delivery to the liver: electrostatically neutral particles which avoid nonspecific binding to other cells, to the extracellular matrix, and to complement proteins; asialoglycoprotein receptor-mediated endocytosis which may address the complexes to the perinuclear region; and polyethylenimine (PEI)-mediated endosome buffering and swelling as an escape mechanism to the cytoplasm. This system is based on a 5% galactose-bearing polyethylenimine (PEI-gal) polymer which is condensed with plasmid DNA to neutrality. Murine (BNL CL.2) and human (HepG2) hepatocyte-derived cell lines were transfected 10(4)-10(5)-fold more efficiently than murine fibroblasts (3T3), whether transfection was assessed globally (luciferase expression from the cell extract) or following histochemical staining (beta-galactosidase). Under these conditions, over 50% of the hepatocytes were selectively transfected in the presence of 10% serum. Transfection was suppressed by removal of the targeting galactose residues, by their replacement with glucose, or by the addition of excess asialofetuin. Thus, results from comparative and competitive experiments indicate the asialoglycoprotein receptor is involved in transfection of hepatocytes with neutral PEI-gal/DNA complexes.

Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold.

Reproducible and optimized complex formation between polyanionic DNA and a polycationic vector is a key aspect of nonviral gene transfer systems. To this end, several factors relevant to in vivo delivery have been tested repeatedly on several cell types. Gene transfer with a lipopolyamine (transfectam) in the presence of serum was increased over 10-fold by sequential addition of the lipid to DNA. Paradoxically, high complex concentrations (> 200 micrograms DNA/ml) led to large enhancements too, which points to the fact that formation of productive complexes is a slow process. Each parameter, more than compensates for the decreased efficiency generally observed with nonviral vectors in serum. Transfectam and PEI (polyethylenimine)-mediated transfection also improved after mild centrifugation of the complexes on to the cells. These individual factors were shown to be essentially multiplicative, leading altogether to approximately a 1000-fold transfection increase with a luciferase reporter gene. Finally, 25 cell lines and primary cells (including fibroblasts, hepatocytes and endothelial cells) were successfully transfected over a five orders-of-magnitude efficiency range, From this large set of data, a general relation between the overall transfection level (as measured by luciferase reporter gene expression) and the fraction of transfected cells (histochemically stained for beta-galactosidase) could be inferred. Finally, transfectam and PEI displayed similar trends over this large range of efficiencies, which reinforces the hypothesis of a common transfection mechanism where the key endosome-releasing stop occurs through a "proton sponge' effect.

A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.

Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se--i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its gene-delivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.

Efficient gene delivery with neutral complexes of lipospermine and thiol-reactive phospholipids.

The presence of thiol-reactive phospholipid derivatives, such as N-4-(p-maleimidophenyl)butyryl) dipalmitoylphosphatidylethanolamine (MPB-DPPE), in electrically neutral lipospermine/DNA particles results in more than a 100-fold increased transfection efficiency of human hepatoma HepG2 cells and murine 3T3 fibroblasts. These effects could be ascribed to the presence of thiol-reactive functions, such as maleimide, bromoacetamide and dithiopyridyl linkage, on the transfecting particles. We propose that such particles react with thiol groups present at the surface of the cells, leading to their covalent anchoring, a process that is probably followed by an endocytosis of the complex.