Characterization of the complexation phenomenon and biological activity in vitro of polyplexes based on Tetronic T901 and DNA
Graphical abstract
Introduction
In the last two decades, gene therapy has created a great expectation as a potential alternative to conventional drug medical treatments in order to treat inherited genetic diseases but also others related to cell gene malfunction, such as cancer [1], [2] and infection, as HIV [3], [4]. Different therapeutic approaches can be applied depending on the pathology and the stage of evolution of the disease as, for example, (i) the substitution of a mutated gene or the addition of a missing one by a properly working copy (the so-called gene correction therapy); (ii) the inactivation of the mutated gene causing the malfunction; or (iii) the introduction of a new exogenous gene able to encode therapeutic products for secretion inside the body to fight against the disease, to favorably modify its course, and/or to provide protection to healthy cells from complementary therapies [5]. To this end, different kinds of genetic materials can be used as, for example, double-strand DNA, DNA plasmids, single strand oligonucleotides, silencing interfering RNAs, siRNAs, or microRNAs, miRNAs, amongst others [6].
However, genetic materials and, in particular, naked DNA cannot freely cross cell membranes due to its hydrophilicity and negative net electric charge. Also, it is rapidly degraded by nucleases. Thus, to provide protection and subsequently ensure a safe and sustainable delivery to the target cells/tissues as well as to enhance the DNA therapeutic activity different vectors can be used. The transfection of genetic material by these vectors must guarantee (i) DNA compaction, (ii) DNA protection against enzymatic attacks in the vicinity of the cell membrane, and (iii) DNA delivery across the latter with efficiency and specificity. The most efficient transfection vectors known nowadays are those based on viruses. Viral vectors use specific virus (typically adenovirus and retrovirus, but also nowadays lentivirus) whose genes were previously extracted and replaced by a therapeutic gene. Their main advantage is their biological nature and mechanism of action: viruses introduce their genetic material into host cells using these to create more virus copies and expand the infection. Viral vectors have demonstrated a good in vivo efficiency [7], [8], [9], [10] but display several important drawbacks such as immunogenicity, the insert size and their potential oncogenicity [11], [12], [13]. In contrast, non-viral vectors have been found to be an interesting potential alternative to viral systems in gene therapy, especially considering the diversity of available molecules (lipids, polymers, surfactants), the possibility of chemically modifying/functionalizing these types of carriers, and the achievement of synergistic effects by associating two or more excipients/cargoes into these carriers. Moreover, these non-viral vectors are stable upon storage and are easy to produce, and can be administered repeatedly with low toxicity and immunogenicity, which make them safer candidates for efficient DNA transfection. Nonetheless, their main drawback is the lower delivery efficiency of the genetic material compared to viral vectors because of the complex barriers they must overcome to reach the nuclei of target cells [6]. Numerous non-viral carriers have been tested, but a particular attention has been paid to lipids and polymers, especially the cationic ones. Cationic lipid-based carriers have exhibited promising results in in vitro assays, but appear to be poorly efficient in vivo [6], [14], [15]. On the other hand, DNA/polycationic polymer complexes, the so-called polyplexes, have been proven to be more stable than lipidic formulations, achieving larger extents of DNA compaction and better cell transfection efficiencies [16], [17], [18]. Most commonly studied cationic polymers as gene carriers (vectors) include chitosan, polyethylenimine, poly(l-lysine), poly(β-aminoester)s and poly(amidoamine) dendrimers, amongst others [19], [20], [21], [22], [23], [24]. Nevertheless, some problems such as not excessively high transfection efficiencies and toxic/immunogenic concerns, especially related to the excess of cationic charge of the polymers, are still present both in vitro and in vivo [25], [26], [27].
For these reasons, polymers displaying few or no electrical charges have recently raised an important interest to solve such issues. Among these, amphiphilic triblock copolymers composed of ethylene oxide (EO) and propylene oxide (PO) blocks have shown to be very efficient for transfecting DNA in vivo [28], [29], [30], [31], [32]. EOnPOmEOn triblock copolymers, where n y m denote the EO and PO block lengths respectively, can be classified in two families according to the structure of the main chain: the linear Pluronics™ (also known as Synperonics™ or Polaxamers), and the X-shaped poloxamines (also known as Tetronics™). Pluronics™ are lineal and symmetrical non-ionic triblock copolymers displaying a direct EOmPOnEOm or reverse POnEOmPOn structure [33], [34]. These block copolymers have been shown to get relatively success in DNA transfection when used as adjuvants upon injection of naked DNA, or as stabilizing agents of gene vectors to help them in overcoming intracellular barriers. It seems that the enhanced transfection efficiency in the presence of these amphiphilic copolymers might be related to an improved diffusion of DNA [35]; the prevention of vector aggregation [36]; the establishment of Pluronic™ interactions with biological membranes which allows the formation of transient pores in the latter [37], then increasing cellular internalization through endocytosis independent membrane fusion [38]; and/or the increase of transcriptional activity [39]. However, Pluronic™ block copolymers are not able to condensate DNA to form polyplex vectors in order to provide enhanced protection to DNA from nucleases, which gives rise to uneven therapeutical outcomes.
Conversely, Tetronic™ copolymers are four-arm star copolymers in which each arm is formed by an EOm-POn arm attached to a central diamine. The presence of this chemical moiety confers some cationic electrical charge and pH-responsiveness to this class of copolymers since the pKa of the two tertiary amines are pKa1 = 7.5–8.1 and pKa2 = 4.0–5.6 [40], which should help in the compaction of the genetic material. For example, it has been shown that Tetronic T304 is able to partially condensate DNA in the form of some interconnected polyplexes by free DNA chains (somehow resembling a pearl necklace structure), providing a good transfection efficiency of the genetic material into muscular tissue [41], [42]. Nevertheless, complete condensation and full protection of DNA is precluded, which has been explained in terms of the low molecular weight of the copolymer. Conversely, further studies have shown that the complexation capability of Tetronic™ copolymers to DNA decreases as their molecular weight increases [28]. Moreover, as occurs for Pluronics™, Tetronics™ also enhance the transfection efficiency of polyplexes formed by other cationic copolymers in a dose-dependent manner when administered in combination with them [43]. Hence, the mechanisms by which polyplex formation by Tetronic™ copolymers take place and improve polyplex gene delivery in vitro and in vivo are still far to be completely understood, so that a more in-depth research on the factors influencing Tetronic™/DNA complex formation is still highly desired.
Hence, in the present paper we focused on performing a deep physico-chemical analysis about the complexation process between the block copolymer Tetronic™ T901 and DNA under different solution conditions through changes in the solution pH (4.0 and 7.4) and temperature (25 and 37 °C). In particular, we select copolymer T901 since it displays a larger hydrophobicity (due to the large fraction of PO blocks in their composition) and molecular weight than previously analysed Tetronic™ block copolymers (usually T304) with the aim of obtaining more compacted polyplex structures favored not only by electrostatic interactions between the diamine core of the block copolymer and DNA but also by means of hydrophobic/dipolar interactions. We noted that this larger hydrophobicity favors a larger tendency to form self-assembled structures at smaller polymer concentrations than previously used with other Pluronic™/Tetronic™-type copolymers in order to achieve at least a partial DNA condensation and more sustained release profiles. Different population distributions of complexes were observed by dynamic light scattering and electron microscopy, whereas the presence of DNA in their composition, which remains almost structurally unaltered, was largely confirmed by fluorescence spectroscopy and microscopy. Moreover, we assessed the capability of the formed complexes to transfect cells in vitro by using a plasmid DNA encoding the green fluorescent protein (GFP), observing a fair dose-dependent transfection without significant toxicity to cells.
Section snippets
Materials
Tetronic™ T901 (a gift from BASF) was used as received. T901 possesses a molecular weight of 4700 Da with a composition of 10.7% and 72.9% of EO and PO, respectively, and a critical micelle concentration of ca. 2.1 mM in 10 mM HCl at 25 °C [44] and 0.64 mM at pH 5 [45], respectively. DNA from salmon tests having 2000 bp was from Sigma Aldrich, and plasmid DNA encoding green fluorescent protein (GFP) with CMV promoter (pCMV-GFP, 3.5 kbp, 1 mg/mL suspended in water) was from Plasmid Factory
Results and discussion
As commented previously, poloxamines are branched amphiphilic block copolymers consisting of EO and PO blocks linked to an aliphatic diamine core. The presence of this central ethylenediamine moiety renders the molecule positively charged: pKa values for poloxamines are usually in the range of 4.0–5.6 and 7.6–8.1 for pKa1 and pKa2, respectively, so that the diprotonated form is predominant at pH values below 4.0, while the monoprotonated form is predominant in the pH range between the pKa1 and
Conclusions
The present work has demonstrated that block copolymer T901 may partially condense DNA and reasonably transfect the genetic material to cells in a safe way by forming negatively charged polymer-DNA complexes, as also observed for several cationic polyelectrolyte-DNA polyplexes at low [N]/[P] ratios [25], [47]. The relative hydrophobic character of T901 if compared to other Pluronic™ and Tetronic™ copolymers has allowed the use of lower polymer concentrations to achieve partial DNA compaction
Acknowledgements
This work was supported by AIE (funding through Project MAT2016-80266-R and ERDF funds) and Xunta de Galicia (GPC2015-007). A.C. also thanks Xunta de Galicia for her postdoctoral fellowship. E.V.A. and A.P. are also grateful to the Spanish Ministerio de Economia y Competitividad for their FPU fellowship. M.A.-M. thanks funding from CONACyT (Mexico) through research projects INFR-2015-251863 and PDCPN-2015-89. Authors also thank Instituto de Ortopedia and Tejidos Musculo-Esqueléticos for the
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These authors contribute equally to this work.