A series of cationic sterol lipids with gene transfer and bactericidal activity

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Abstract

A family of cationic lipids was synthesized via direct amide coupling of spermine to the C-24 position of cholic acid analogs. Four monosubstituted spermines and a bis-substituted spermine were evaluated as plasmid transfection reagents, as bacteriostatic agents, and as bactericidal agents. The incorporation of a double bond in the sterol moiety enhanced transfection efficiency significantly and produced two compounds with little cytotoxicity and transfection potency comparable to Lipofectamine2000. Inclusion of the double bond had no effect on the general trend of increasing bactericidal activity with increasing sterol hydrophobicity. Co-formulation of the most hydrophilic of the compounds with its bis-substituted analogue led to enhancement in transfection activity. The bis-substituted compound, when tested alone, emerged as the most bacteriostatic compound in the family with minimum inhibitory concentrations (MIC) of 4 μM against Bacillus subtilis and 16 μM against Escherichia coli and therapeutic indexes (minimum hemolytic concentration/minimum inhibitory concentration) of 61 and 15, respectively. Cationic lipids can be optimized for both gene delivery and antibacterial applications by similar modifications.

Graphical abstract

A family of cationic lipids was synthesized and evaluated as plasmid transfection reagents and antibacterials. Double bond incorporation in the sterol moiety significantly enhanced transfection efficiency but not bactericidal activity.

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Introduction

Cationic lipids were first reported over 20 years ago to facilitate liposomal gene delivery1 and many are commercially available.2 Lipofection efficiency remains low compared to viral-based gene delivery. Less than 8% of clinical gene therapy trials have used cationic lipid vectors.3 In reviewing common cationic lipid classes, such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)1, 4 or 3β[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol)5 (Fig. 1), it is notable that similar structures identified as cationic lipid amphiphiles, synthetic ionophores, and cationic steroid antibiotics, are cited throughout anti-microbial literature. Squalamine, isolated from the stomach tissue of the dogfish shark Squalus acanthias, was the first of these steroid derivative amphiphiles proven to have significant antibiotic activity against a broad-spectrum of organisms.6 A number of synthetic analogues mimicking the functionality of squalamine in simplified forms also have bactericidal activity.7, 8, 9, 10

The potential for dual functionality8, 11 has led us to design a family of cationic lipids with varying structures to investigate their membrane activity in two different contexts of gene transfer and antimicrobial activity. We chose the naturally occurring spermine, spermidine, and putrescine, known for their abilities to aid in DNA condensation as well as cell proliferation and differentiation.12, 13, 14 Further, free spermine and putrescine have been shown to associate in the minor groove of B-DNA and the major groove of A-DNA.15

Inspired by the success of DC-Chol which pioneered the use of cholesterol in place of aliphatic chains,5 we elected to use cholesterol-derivatives as our hydrophobic tails. The added rigidity of the structure provided by cholesterol favors a large cross-sectional area on the hydrophobic tail.

Consideration of the linkage strategy led us to select a bile acid subset of cholesterol derivatives that could be linked to polyamines by amide coupling. These bile acid derivatives characteristically display a carboxylic acid moiety on their alkyl chain that reacts with amines through a carbodiimide-assisted mechanism to form amides.16 Conjugating the sterols at this position as opposed to on the ring structure, as seen in DC-Chol and many others in the literature, further increases the cross-sectional area of the hydrophobic tail. The susceptibility of amides to hydrolysis in acidic environments facilitates the eventual degradation of the compounds in physiological environments such as the late endosome.

In this work, we present a synthesis strategy and promising results for both in vitro transfection and antibacterial applications for a unique family of cationic lipids. Structural variations across the family provide an opportunity to consider the relationships between these features and their functional impacts.

Our approach began with a focus on design for transfection activity. Generally, correlations between lipid structure, hydration and in vivo transfection activity have shown that maximizing the imbalance between the large cross sectional area occupied by the hydrophobic tail and the small cross sectional area occupied by the cationic head promotes transfection activity, potentially due to improvements in membrane fusion.17

Section snippets

Chemistry

In an effort to span a range of hydrophobicities on the sterol tail group, compounds 14 were synthesized from a variety of bile acid derivatives (Fig. 2). Compounds 2 and 3 incorporate double bonds in the base sterol structure. The octanol–water partition coefficients (log D) calculated for each of the compounds provide a quantitative comparison of the hydrophobicities across the family in Table 1.

Molecular weights and 1H NMR were determined for each compound.

Lipofection: efficiency and toxicity

All transfection results were

Discussion

Cationic lipids have received attention as both gene delivery vehicles and as antibacterials. In this work, we set out to synthesize a family of cationic lipids bearing structural features potentially beneficial for both applications and evaluate the effects of varying these features on both gene delivery and antibacterial function. The synthesis of our compound family employed a facile amide coupling that produced a clean product with few significant impurities. A dicyclohexylurea was

Conclusions

We employed a single-step amide coupling to synthesize a family of cationic lipids with varying functionality that allowed us to study performance in both gene delivery and antibacterial applications. We found that inclusion of a single double bond in the hydrophobic tail sterol structure significantly enhanced transfection efficiency without exhibiting significant cytotoxicity over naked plasmid delivery.

A positive effect when including the double bond was not observed in our antibacterial

Chemistry

The carboxylic acid starting materials for the amide couplings to make compound 13 were purchased from Steraloids Inc. (Newport, RI). All other reagents were obtained from Sigma–Aldrich (St. Louis, MO) and used as received. Solvents were of HLPC grade.

Compounds 14 were synthesized in a similar manner, starting with the carboxylic acid sterol derivatives. The carboxylic acid was charged to a clean, dry three-neck round bottom flask equipped with magnetic stirring. Dicyclohexylcarbodiimide was

Acknowledgement

This work was supported by NIH grant HL-066565.

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