Research paper
Understanding the mechanism of protamine in solid lipid nanoparticle-based lipofection: The importance of the entry pathway

https://doi.org/10.1016/j.ejpb.2011.06.005Get rights and content

Abstract

The aim of our study was to evaluate the effect of protamine on the transfection capacity of solid lipid nanoparticles (SLNs) by correlating it to the internalization mechanisms and intracellular trafficking of the vectors. Vectors were prepared with SLN, DNA, and protamine. ARPE-19 and HEK-293 cells were used for the evaluation of the formulations. Protamine induced a 6-fold increase in the transfection of SLNs in retinal cells due to the presence of nuclear localization signals (NLS), its protection capacity, and a shift in the internalization mechanism from caveolae/raft-mediated to clathrin-mediated endocytosis. However, protamine produced an almost complete inhibition of transfection in HEK-293 cells. In spite of the high DNA condensation capacity of protamine and its content in NLS, this does not always lead to an improvement in cell transfection since it may impair some of the limiting steps of the transfection processes.

Graphical abstract

Protamine induced an increase in the transfection of SLNs in retinal cells but a decrease in HEK-293 cells, which is related to the change in the internalization mechanism.

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Introduction

Gene therapy, which involves systems for the targeted delivery of DNA into cells, has a great potential for treating a large number of diseases [1], [2], [3], [4]. Non-viral vectors are safer, cheaper, and more reproducible than viral vectors and have no limitation in the size of DNA they can transport, but their transfection efficacy is still low [5]. Therefore, the enhancement of non-viral vectors is a priority in the field of gene therapy, in order to manage effective and safe vectors.

Non-viral vectors based on cationic lipids are being extensively studied [6], [7], [8], [9], and among lipidic systems, solid lipid nanoparticles (SLNs) have become a promising strategy as gene delivery systems. In previous studies, we showed the capacity of SLNs for transfection “in vitro” [10], [5] and “in vivo” [11], and from the point of view of application, SLNs have good stability and are subject to be lyophilized [12], which facilities the industrial production.

Transfection efficacy is conditioned by the entry and posterior intracellular trafficking of gene delivery vectors. These processes are cell line dependent, but there are steps common to all cells [13]: binding to cell surface by electrostatic interactions between the positively charged systems and the negative charges of the cell membrane, entry into the cell by following a particular pathway [14], [15], release of DNA into the cytoplasm and crossing the nuclear envelope to reach the cellular machinery for protein synthesis. The entry into the nucleus is, in general, quite difficult, as the nuclear membrane is a selective barrier to molecules longer than 40 kDa, and plasmids surpass that size. There are two mechanisms those molecules can use to overcome that barrier: the disruption of the nuclear membrane during mitosis, which is conditioned by the division rate of the targeted cells, or the import through the nuclear pore complex (NPC). This latter mechanism requires nuclear localization signals (NLS), which can be used to improve transfection by non-viral vectors [16].

NLS peptides have sequences rich in basic amino acids (lysine or arginine) that promote active nuclear transport through the NPC, mediated by importin α–importin β interactions [17]. Peptides with arginine residues in their sequences have shown translocation activity, and those containing six or eight consecutive arginine residues exhibited the maximum internalization and accumulation in the nucleus [18], [19]. Among these peptides, protamine sulfate is an USP (United States Pharmacopeia) compound isolated from the sperm of mature fish. It condenses DNA and presents sequences of six consecutive arginine residues [20], which make this peptide able to translocate molecules such as DNA from the cytoplasm to the nucleus of living cells. This feature would solve the passage of DNA from the cytoplasm into the nucleus. Moreover, protamine improves intra-nuclear transcription [21].

The present study considers the effect of protamine on the transfection capacity of SLNs composed by the core lipid Precirol® ATO 5, and the surfactants DOTAP and Tween 80 as surface components. Protamine–DNA–SLN complexes were characterized in terms of size, superficial charge, and DNA protection capacity. The mechanism of internalization and the intracellular trafficking of the vectors were also studied into cell lines with different division rates and related to the transfection capacity of the vectors.

Section snippets

Materials

Precirol® ATO 5 was provided by Gattefossé (Madrid, Spain). 1,2-Dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) was acquired from Avanti Polar Lipids, Inc. Deoxyribonuclease I (DNase I), lauryl sulfate sodium (SDS), Nile Red, and protamine sulfate were purchased from Sigma–Aldrich (Madrid, Spain). Tween 80 was provided by Vencaser (Bilbao, Spain), and dichloromethane by Panreac (Barcelona, Spain).

The plasmid pCMS-EGFP, which encodes the enhanced green fluorescent protein (EGFP), was

Binding of protamine to DNA

The gel in Fig. 1A illustrates that the capacity of protamine to condensate DNA depends on the protamine to DNA ratio. In lanes 1–3 (protamine to DNA ratios from 0.25:1 to 1:1), the intensity of the bands indicates that most DNA was free. However, in lines 4 and 5 (protamine to DNA ratios 2:1 and 5:1, respectively), no band is present in the gel, indicating that all DNA was completely bound to protamine.

Particle size and zeta potential of the formulations

When protamine–DNA (ratio 2:1) complexes were bound to SLNs, protamine did not induce any

Discussion

The incorporation of NLS into non-viral vectors has been studied by several authors, and it has shown improvement in transfection due to an effect on the nuclear membrane [25], [26], [27]. Protamine is a peptide widely used to improve lipofection due to its content in NLS. The enhancement of transfection efficacy with protamine is attributed to nuclear localization and stabilization against DNase degradation [28]. However, there is very little information describing its role in lipofection [29]

Conclusions

In summary, protamine induced a 6-fold increase in the transfection capacity of SLNs in retinal cells due to a shift in the internalization mechanism from caveolae/raft-mediated to clathrin-mediated endocytosis, which promotes the release of the protamine–DNA complexes from the SLNs; afterward the transport of the complexes into the nucleus is favoured by the NLS of the protamine. On the contrary, protamine diminished transfection in HEK-293 cells, due to the decrease in the cell uptake of the

Acknowledgements

We would like to thank the University of the Basque Country (UPV-EHU) for research grants awarded to Diego Delgado. The authors also acknowledge the General Service of Analytical Microscopy and High Resolution in Biomedicine of the University of the Basque Country (UPV-EHU) for technical advice on confocal microscopy.

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