Regular ArticleAggregation behaviour of triphenylphosphonium bolaamphiphiles
Introduction
Bolaamphiphiles are a class of surfactants featuring one or more hydrophobic chains that connect two identical or different hydrophilic headgroups. Since the late 1970s, a great effort has been spent in the design and synthesis of new bolaamphiphiles with different chemical structure, and in the characterization of their self-assembly [1]. Because of the covalent connection of the two headgroups, in the self-assembling process this kind of molecules can behave cooperatively to a larger extent with respect to conventional surfactants, and various morphologies such as monolayers, micelles, vesicles, nanofibers, nanoribbons, and nanotubes can be formed [2], [3], [4], [5]. Systematic investigations have shown that the aggregate morphology depends on the chemical structure of the bolalipid; in particular, the length of the hydrophobic chain, its modification by introduction of hetero-atoms, triple bonds, or aromatic rings, and the chemical nature of the headgroup strongly influence the aggregation features. Compared with bilayer membranes formed by conventional amphiphiles, it is known that the monolayer membrane formed by spanning bolaamphiphiles is more tightly packed and scarcely permeable [6]. This interesting property has led to exploit bolaamphiphiles in the investigations on membrane models [7], [8] and as components in vesicular systems for several technological applications [9]. In particular, bolaamphiphiles have been investigated as stabilizing agents in liposomes and other lipid-based drug delivery systems [10]; in fact, the inclusion of bolaamphiphiles in liposomal formulations may stabilize liposomal membranes, preventing the formation of both hydrophilic and hydrophobic pores, thus allowing the construction of more robust carriers [11]. In addition to the improvement of bilayer mechanical properties, some structural motifs (e.g. folate [12], lactose, mannose [13], phenylalanine [14], vernonia oil [15]) on bolaamphiphile can give peculiar properties to the liposome. It is the case of Dequalinium (DQA, Chart 1), a cationic bolaamphiphile [16] that spontaneously self-associates into mechanically very stable monolayer vesicles, which have been shown to easily cross the mitochondrial membranes and deliver genetic material only to mitochondria [17]. This ability depends on the “soft” cationic nature of DQA headgroups, characterized by an extended delocalization of the positive charge on the quinolinium moieties. The delocalization of the positive charge, together with the hydrophobicity of the methylenic chain, allows the crossing of the two mitochondrial membranes (the second of which is densely packed) and promotes the accumulation of DQA into mitochondria, driven by the strong negative potential of the inner mitochondrial membrane. Both liposomes composed only of DQA (DQAsome) and mixed liposomes, formulated with DQA and natural or synthetic phospholipids (mitosomes), were reported [18].
Another class of bolaamphiphiles, well known for their ability to enter mitochondria, is represented by triphenylphosphonium (TPP) bolaamphiphiles [19]. These peculiar compounds are able to cross the mitochondrial membranes as they display the same feature of DQA, i.e. a cationic charge highly delocalized on the entire headgroup. Some reported investigations highlighted that TPP bolaamphiphile with hydrophobic chain of five and ten methylene units cause a depolarization of the mitochondrial membrane and have an inhibitory activity on the mitochondria functions, leading to a permanent damage of the organelle [20]. Unfortunately, only few studies have been performed on the interaction of TPP bolaamphiphiles with mitochondria and none of these investigations takes into account the relationship between the biological activity and the aggregation features of the TPP bolaamphiphile. A recent investigation reports that TPP bolaamphiphiles toxicity depends more on the ability of the amphiphile in disrupting the membrane rather than on the depolarization effect [21]. Therefore, it is clear that the colloidal properties of the TPP bolaamphiphiles play a central role also in their mitochondrial toxicity. For this reason, we decided to investigate the colloidal behaviour and the physico-chemical features of a class of dicationic TPP bolaamphiphiles featuring chains of different length (12, 16, 20 and 30 methylene units, 1–4, Chart 1) with the future perspective of using these TPP bolaamphipiles as minor component-targeting agent in mitochondriotropic mixed liposome formulations. To the best of our knowledge, all these symmetric TPP bolaamphiphiles have never been characterized in terms of their aggregation properties, such Krafft point, critical aggregative concentration (cac), spontaneous aggregate dimensions and morphology, all features that should be taken into account for the rational design of a new mitochondriotropic drug delivery system.
In this work, we report the investigation of the colloidal properties of these molecules (see SI for the synthesis and characterization of compounds 1–4) with the aim of elucidating the correlation between chain length, morphology of the spontaneous aggregate and conformation of the monomer inside the aggregate. In fact, with respect to conventional surfactants, the physical properties and the organization of the membrane of bolaamphiphiles are controlled also by an additional feature that is the conformation of the monomer inside the aggregate, which can vary from an extended to a U-shape because of a complex texture of hydrophobic and hydrophilic interactions.
The interest for these bolaamphiphiles arises not only from their potential implications in mitochondrial delivery, but also from the wide polymorphism of their molecular aggregates that renders their investigation very intriguing for the development of nanomaterials.
Section snippets
Conductivity measurements
Aggregation properties in water such as Krafft point and cac of 1–4 were determined by conductivity measurements [22], [23], [24]. All the compounds display Krafft points below 277 K since, after one night at this temperature, solutions of these surfactants above their cac were still clear.
For the determination of the cac, since we did not know the nature of the aggregates formed by 1–4 (kinetically inert or equilibrium systems), all the stock solutions of the surfactants have been prepared in
General discussion
The physico-chemical features of dicationic TPP bolaamphiphiles 1–4, in particular colloidal and thermodynamic behaviour, morphology and monomer conformational arrangements inside spontaneous aggregates, were investigated with many experimental techniques, in some cases complementary, that allowed obtaining a quite complete and convincing picture of the aggregate structure. Bolaamphiphiles 1–4, once dispersed in aqueous solution, are able to spontaneously self-assemble into vesicles,
Conclusions
In this work the single chain triphenylphosphonium bolaamphiphiles 1–4, featuring chains of 12, 16, 20 and 30 methylene units, were synthesized and their self aggregation features investigated by conductivity, dialysis, TEM, Raman spectroscopy, dynamic and dielectrophoretic laser light scattering measurements. These amphiphiles are able to form vesicle stable to dilution, especially 3 and 4, displaying a spacer of 20 and 30 methylene units respectively. This feature makes 3 and 4, in
Instrumentation
NMR spectra were run on a Bruker AC 300 P spectrometer, operating at 300.13 and 75.47 MHz for 1H and 13C, respectively, equipped with a sample tube thermostating apparatus. Signals were referenced with respect to residual proton signal of deuterated solvent, δ in parts per million, J in hertz.
Kolbe reaction was performed in a 250 mL cell equipped with Pt electrodes (2.5 cm × 5.0 cm), at a density current of 60 mA/cm2. After Kolbe reaction, the Pt-electrodes were cleaned with a RCA solution (H2
Supporting information
Synthesis and characterization of bolaamphiphiles 1–4; Conductance plots for cac and α determination; Dynamic Light Scattering measurements of bolaamphiphiles dissolved in ethanol; DLS and conductivity experiments of 1 and 3 aggregates prepared by procedure (b); Raman spectrum of bolaamphiphile 3 (dried sample). “This material is available free of charge via the Internet at https://www.sciencedirect.com.”
Author contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Funding sources
This work was supported by the MIUR Basic Research Investigation Fund FIRB2012 (Grant n° RBFR12BUMH), by the IIT@Sapienza Centre for Life Nanoscience and by "Ministero della Salute" with the "Progetto Ordinario di Ricerca Finalizzata" (Grant n° RF-2013-02355682).
Notes
The authors declare no competing financial interest.
Acknowledgment
We thank Dr. Alessandro Latini (Sapienza University of Rome) for sharing with us his experience about electrolysis; we also thank Roberto Moscardelli (CNR-IMC) and Enrico Rossi (CNR-IMC) for technical support.
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2019, Journal of Molecular LiquidsCitation Excerpt :On the other hand, an increase in counterion binding in the case of high homologue could indicate at sphere-to-rod morphological transition. Taking into account that charge characteristics of aggregates including β values and zeta potential are affected by different factors [62,63], e.g. aggregation numbers, an additional study of IA-n(OH) series was performed using approach based on the quenching of pyrene fluorescence. As an example, primary spectral data for two IA-18(OH) concentrations are depicted in fig. S2,the fitting of which in terms of Eq. (1) allows to obtain aggregation numbers for IA-n(OH) amphiphiles listed in Table 4.
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These authors contributed equally.