Elsevier

Biomaterials

Volume 33, Issue 32, November 2012, Pages 8160-8166
Biomaterials

A biomimetic lipid library for gene delivery through thiol-yne click chemistry

https://doi.org/10.1016/j.biomaterials.2012.07.044Get rights and content

Abstract

The delivery of nucleic acids such as plasmid DNA and siRNA into cells is a cornerstone of biological research and is of fundamental importance for medical therapeutics. Although most gene delivery therapeutics in clinical trials are based on viral vectors, safety issues remain a major concern. Non-viral vectors, such as cationic lipids and polymers, offer safer alternatives but their gene delivery efficiencies are usually not high enough for clinical applications. Thus, there is a high demand for more efficient and safe non-viral vectors. Here, we present a facile two-step method based on thiol-yne click chemistry for parallel synthesis of libraries of new biomimetic cationic thioether lipids. A library of novel lipids was synthesized using the developed method and more than 10% of the lipids showed highly efficient transfection in different cell types, surpassing the efficiency of several popular commercial transfection reagents. One of the new lipids showed highly efficient siRNA delivery to multiple cell types and could successfully deliver DNA plasmid to difficult-to-transfect mouse embryonic stem cells (mESC). Analysis of structure–activity relationship revealed that the length of the hydrophobic alkyl groups was a key parameter for efficient cell transfection and was more important for transfection efficiency than the nature of cationic head groups. The correlation of the size and surface charge of liposomes with transfection efficiency is described.

Introduction

The delivery of nucleic acids such as plasmid DNA and siRNA into cells is a cornerstone of biological research and is of fundamental importance for medical therapeutics. Indeed, since the first FDA-approved gene therapy experiment in 1990 [1], over 1700 clinical trials have been conducted for gene delivery [2]. Although most gene delivery therapeutics in clinical trials are based on viral vectors, safety issues remain a major concern [3]. Non-viral vectors, including cationic lipids [4], [5], polymers [6], [7], dendrimers [8], cationic proteins [9] and inorganic nanoparticles [10], [11], offer safer alternatives but their gene delivery efficiencies are usually not high enough for clinical applications. Although lipid-based vectors have only 4.8% of all gene therapy clinical trials [12], they are already the most commonly used systems for in vitro delivery of nucleic acids into cells [4], [13], [14], [15]. However, most of the lipid-based delivery systems are synthesized using a multi-step synthesis route, requiring protecting groups and excessive purifications [15], thus limiting the possibility for successful and fast structural optimizations. Recently Anderson et al. reported two important combinatorial approaches to synthesize libraries of alkyl amines for siRNA delivery using aza-michael addition [16] and epoxide-amine [17] reactions. However, there are still no convenient combinatorial methods that could lead to lipid-like molecules structurally similar to natural phospholipids, the main lipid components of the cell membrane.

Here we report a facile modular and scalable approach employing thiol-yne “click” chemistry [18], [19], [20], [21] for the parallel synthesis of a library of cationic thioether lipids with two hydrophobic tails of variable lengths and possessing a linker group structurally mimicking the glycerol core of the phospholipids. We used the method to synthesize more than 100 novel lipids and found that more than 10% of all lipids showed highly efficient transfection in different cell types. Both siRNA delivery and transfection of difficult-to-transfect cell lines, such as mESC, was analyzed. Analysis of structure–activity relationship and the correlation of the size and surface charge of liposomes with transfection efficiency are described.

Section snippets

Chemicals, plasmids and siRNAs

1-Hexanethiol (95%), 1-heptanethiol (98%), 1-octanethiol (≥98.5%), 1-nonanethiol (95%), 1-decanethiol (96%), 1-undecanethiol (98%), 1-dodecanethiol (≥98%), 1-hexadecanethiol (≥95%), 2,2-dimethoxy-2-phenylacetophenone (99%), 4-pentynoic acid (95%), 5-hexynoic acid (97%), 4-(2-aminoethyl)morpholine (99%), 1-(2-aminoethyl)pyrrolidine (98%), N′,N′-diethylethane-1,2-diamine (99%), N′,N′-diethylpropane-1,3-diamine (≥99%), N′,N′-dimethylpropane-1,3-diamine (≥98%) and

Synthesis of thiol-yne lipids

In order to produce a library of cationic lipids parallel synthesis was performed using 8 different alkyl thiols (alkyl chain length from C6 to C16), 2 alkynyl carboxylic acid linkers bearing a terminal triple bond, and 7 different cationic amines (Fig. 1). The synthesis of lipids is based on two consecutive modular steps. First, the thiol-yne click reaction of an alkyl thiol with an alkynyl carboxylic acid gives a lipid molecule with two hydrophobic tails and one carboxylic head group. In the

Conclusion

We envision that the developed modular method for the parallel synthesis of cationic biomimetic thioether lipids will greatly help the intelligent design of new efficient gene delivery systems. The simple and rapid synthesis scheme enables efficient parallel synthesis of hundreds of cationic lipids that can be used for in vitro or in vivo delivery of both DNA plasmids and siRNAs. The analysis of structure–activity relationship revealed a particular importance of the length of the hydrophobic

Conflict of interest

The authors declare no conflict of interest.

Acknowledgment

The authors would like to thank Dr. Liebel (IAI) and his group for their assistance with the Olympus IX81 automated fluorescent imaging microscope, Dr. Welle (IBG) for his help on Vilber Lourmat BLX-254 crosslinker, Dr. Brenner-Weiß (IFG) and his group, especially Michael Nusser and Boris Kühl, for their help on API 4000 Quadruple mass spectrometer and 4800 MALDI-TOF/TOF mass spectrometer, and Sven Stahl (INT) for his help with NMR Bruker AMX 500 spectrometer. P.A.L. would like to thank the

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