Multifunctional Neomycin-Triazine-Based Cationic Lipids for Gene Delivery with Antibacterial Properties

Abstract

Cationic lipids (CLs) have gained significant attention among nonviral gene delivery vectors due to their ease of synthesis and functionalization with multivalent moieties. In particular, there is an increasing request for multifunctional CLs having gene delivery capacity and antibacterial activity. Herein, we describe the design and synthesis of a novel class of aminoglycoside (AG)-based multifunctional vectors with high transfection efficiency and noticeable antibacterial properties. Specifically, cationic amphiphiles were built on a triazine scaffold, allowing for an easy derivatization with up to three potentially different substituents, such as neomycin (Neo) that serves as the polar head and one or two lipophilic tails, namely stearyl (ST) and oleyl (OL) alkyl chains and cholesteryl (Chol) tail. With the aim to shed more light on the effect of different types and numbers of lipophilic moieties on the ability of CLs to condense and transfect cells, the performance of Neo-triazine-based derivatives as gene delivery vectors was evaluated and compared. The ability of Neo-triazine-based derivatives to act as antimicrobial agents was evaluated as well. Neo-triazine-based CLs invariably exhibited excellent DNA condensation ability, even at a low charge ratio (CR, +/-). Besides, each derivative showed very good transfection performance at its optimal CR on two different cell lines, along with negligible cytotoxicity. CLs bearing symmetric two-tailed OL proved to be the most effective in transfection. Interestingly, Neo-triazine-based derivatives, used as either free lipids or lipoplexes, exhibited strong antibacterial activity against Gram-negative bacteria, especially in the case of CLs bearing one or two aliphatic chains. Altogether, these results highlight the potential of Neo-triazine-based derivatives as effective multifunctional nonviral gene delivery vectors.

Scheme 1. Synthesis of Monotailed 5a–c, Symmetric Two-Tailed 6aa–cc, and Asymmetric Two-Tailed 6ab–bc Triazine-PEG₂-N₃

Scheme 2. Synthesis of Monotailed Neo–Triazine 8a–c and Two-Tailed Neo–Triazine 9aa–cc and 9ac–bc

Figure 1

Structures of monotailed Neo–triazine conjugates 8a–c (1_ST, 1_OL, 1_Chol), symmetric two-tailed Neo–triazine conjugates 9aa–cc (2_ST, 2_OL, 2_Chol), and asymmetric two-tailed Neo–triazine conjugates 9ab, 9ac, and 9bc (1_ST-1_OL, 1_ST-1_Chol, 1_OL-1_Chol).

Figure 2

Comparative DNA complexation ability: (A) monotailed Neo–triazine conjugates 1_ST, 1_OL, and 1_Chol; (B) symmetric two-tailed Neo–triazine conjugates 2_ST, 2_OL, and 2_Chol; (C) asymmetric two-tailed Neo–triazine conjugates 1_ST-1_OL, 1_ST-1_Chol, and 1_OL1_Chol. Complexation was evaluated by monitoring the fluorochrome (SYBR Green I) exclusion from lipoplexes as a function of the charge ratio (CR, +/−).

Figure 3

Comparative cytotoxicity and transfection efficiency, evaluated on L929 cells, of complexes prepared by mixing pGL3 with Neo–triazine derivatives at different charge ratios (CR, +/−), 25 kDa bPEI and Lipofectamine 2000 (Lipo). Transfection efficiency is expressed as luminescence signal (RLU) normalized to the total protein content in each cell lysate: (A) monotailed Neo–triazine conjugates 1_ST, 1_OL, and 1_Chol; (B) symmetric two-tailed Neo–triazine conjugates 2_ST, 2_OL, and 2_Chol; (C) asymmetric two-tailed Neo–triazine conjugates 1_ST-1_OL, 1_ST-1_Chol, and 1_OL-1_Chol. Results are expressed as mean ± SD (n ≥ 3) (*, p < 0.05 vs other lipids for any given CR).

Figure 4

Comparative antibacterial activity on E. coli of DNA-free lipids and lipoplexes prepared by mixing pGL3 with Neo–triazine derivatives at different charge ratios (CRs, +/−), corresponding to different [Neo]: (A) monotailed Neo–triazine conjugates 1_ST, 1_OL and 1_Chol; (B) symmetric two-tailed Neo–triazine conjugates 2_ST, 2_OL, and 2_Chol; (C) asymmetric two-tailed Neo–triazine conjugates 1_ST-1_OL, 1_ST-1_Chol, and 1_OL-1_Chol. Results are expressed as mean ± SD (n ≥ 3) (*, p < 0.05 vs each respective lipid counterpart for any given CR).