Introduction

Vesicle aggregation has attracted much interest in the past few decades because of its applications in the modelling of biomembranes [1–3] and in drug and gene delivery [4, 5]. It is known that the change of some experimental conditions can induce the aggregation of vesicles, for example, surfactant composition [6, 7], external additives [8–13], solution pH values [11, 14, 15], temperature [11, 16, 17], etc. In these cases, the reduction, electrostatic interaction or ionic complex formation was considered to be the main mechanism of vesicle aggregation. Besides, Huang et al. assumed inter-vesicular hydrophobic interaction among the exposed hydrophobic part of the surfactant head groups as the driving force for vesicle aggregation in the catanionic surfactant system of sodium n-dodecylsulfate/n-dodecyltributylammonium bromide [16, 17]. In this system, the extended butyl groups on the surface of the vesicles provide such a hydrophobic force as to catch other butyls on another vesicle and bring them together. They further confirmed that the shorter groups such as methyls, ethyls or propyls carried by cationic surfactants cannot achieve this [16]. Another more interesting system utilising hydrophobic interaction to link vesicles was reported by Menger and Peresypkin [18]. In their system, the vesicle was constructed by asymmetric gemini surfactants that consisted of phosphate and quaternary ammonium head groups, a two-carbon spacer, and two hydrophobic tails of different lengths (abbreviated as Cm-Cn, where m and n are the number of carbon atoms on each tail, respectively). They found that some vesicles were cohesive, which was conceptualised as having a “pearls on a string” morphology. This phenomenon was interpreted as owing to the protrusion of short chains from the vesicle surfaces; it thereby created the opportunity to link neighbouring vesicles through the hydrophobic interactions between the extended short chains. This approach might be considerably more effective when associating the neighbouring vesicles, by which those strings of vesicles may form a complex three-dimensional network, thus making the solution very viscous.

Here, we report a new system of vesicle aggregation, based upon the hydrophobic effect, in which the vesicles were formed by mixed anionic gemini surfactant, O,O′-bis(sodium 2-alkylcarboxylate)-p-dibenzenediol (referred to as Cmϕ2Cm, Scheme 1), and cationic single-chain surfactant, alkyltrimethylammonium bromides CnNBr. Here, Cmϕ2Cm was a family of the gemini surfactants and its molecule carried a long, rigid spacer between the two head groups. This molecular structure made its two tails difficult to be brought into close mutual proximity and yielded a pseudo volume between them. Thus, Cmϕ2Cm had a columnar-like molecular geometry and favoured the formation of aggregates with low surface curvature [19]. We have obtained threadlike micelles by using Cmϕ2Cm [20] and hoped that they would form vesicles under certain conditions. Besides, the long, rigid spacer of Cmϕ2Cm made its two tails possibly extend in opposite directions, i.e., in a trans-configuration. This could form novel network aggregates through the interactions between the extending alkyl tails [21]. As mentioned, Menger and Peresypkin have linked the vesicles formed by Cm-Cn with the interactions between their extending short tails [18]. Thus, the Cmϕ2Cm was also expected to create a new case of vesicle aggregation based on its trans-configuration.

Scheme 1
scheme 1

Chemical structure of Cmϕ2Cm

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Experimental

Materials

The carboxylate gemini surfactants, C12ϕ2C12 and C10ϕ2C10, were synthesised in our laboratory [21]. Octyl-, decyl- and dodecyl-trimethylammonium bromides (denoted as C8NBr, C10NBr and C12NBr, respectively) were purchased from Acros at a purity of 98 %. 1,6-Diphenyl-1,3,5-hexatriene (DPH) was sourced from Aldrich and used as a hydrophobic probe. All solutions were prepared with Milli-Q water with a resistivity of 18.2 MΩ⋅cm.

Methods

Turbidity

Turbidity of the solutions was measured with a UV/vis spectrophotometer (Hitach U-3010, Japan) equipped with a thermostat cell holder and a magnetic stirring device, using quartz cells of 1-cm path length. Transmittance at 500 nm, where the surfactants had no absorption, was recorded to characterise the turbidity.

Dynamic light scattering

Dynamic light scattering of micellar solutions was measured with a Brookhaven Instrument which comprised a BI-200SM goniometer, a BI-9000AT digital correlator (522 channels) and a photomultiplier detector. A green laser with an output power of 200 mW and a wavelength of 532 nm was used as the light source. The measurement temperature was controlled by a thermostatic circulator (Poly-science, USA) with an accuracy ±0.01 °C. All solutions were filtered through 0.22-μm Millipore filters into cylindrical light-scattering cells (external diameter, 25 mm). The experimental data were analysed by the CONTIN program to get the intensity-fraction distributions around each characteristic aggregate size.

Freeze-fractured TEM

The solution for the test was put into a 25 ± 0.1 °C water bath for at least 1 h. Then, a small amount of solution was coated onto a 0.1-mm-thick gold disc. The sample was rapidly frozen by plunging it into liquid nitrogen cooled liquid ethane. After a few seconds, the sample was transferred onto a specimen holder immersed in liquid nitrogen and then put into the freeze-etching apparatus (Bal-tec, BALZERS BAF-400D, Liechtenstein). The fracturing and replication process was performed at a temperature of −165 °C and at a pressure of 2 × 10−6 mbar. The sample was replicated with 10 nm carbon at an angle of 90° and then 2 nm Pt at an angle of 45°. After that, acetone/dilute sodium hydroxide solution was used to remove the soluble component to obtain the replica which was then examined with a Philips TECHAI 20 transmission electron microscope.

Results and discussion

Vesicle formation with assistance of CnNBr

Figure 1 shows the solution turbidity expressed as transmittance (T %) at 500 nm as a function of the mole ratio of CnNB to Cmϕ2Cm (β), in which the critical point inducing a rapid fall in T % was denoted as βv (Table 1). The appearance (in photographs) of some typical C12ϕ2C12/CnNB solutions is shown in Fig. 2, in which the two aqueous phases (ASTP) can be seen in some examples. All of these indicated vesicle formation, which was further confirmed by the FF-TEM images (Fig. 3).

Fig. 1
figure 1

Turbidity as a function of β for C12ϕ2C12(10 mmol L−1)/CnNBr (a) and C10ϕ2C10 (10 mmol L−1)/CnNBr (b) systems at 25 °C, in which the onset for an abrupt decrease in transmittance is denoted as the critical βv to indicate vesicle formation

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Table 1 Values of βv
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Fig. 2
figure 2

a Appearance of C12ϕ2C12 (10 mmol L−1)/CnNB solutions at β = 1.6 and 25 °C: from left to right, n = 8, 10 and 12. b Two aqueous phases (ASTP) of C12ϕ2C12 (10 mmol L−1)/C8NBr as an example: from left to right, β = 1.25, 1.30, 1.325 and 1.35

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Fig. 3
figure 3

FF-TEM images of a C12ϕ2C12 (10 mmol L−1)/C12NBr, b C12ϕ2C12 (10 mmol L−1)/C8NBr and c C10ϕ2C10 (10 mmol L−1)/C8NBr at β = 1.6

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The mechanism of vesicle formation can be interpreted as an effective gemini molecular geometry improved by the addition of CnNB. Israelachvili et al. suggested the dimensionless packing parameter P as an index to predict the shape and size of the aggregates [22]. This parameter reflects the effective molecular geometry of a given surfactant molecule and is defined as P = v/(al) where v is the hydrocarbon chain volume, a is the optimum head group area per molecule and l is the hydrocarbon chain length. Vesicles are favoured when 0.5 < P < 1 [22].

For the present systems, it was supposed that all the added CnNBr associated with C12ϕ2C12 or C10ϕ2C10 to form vesicles under strong electrostatic attraction. In the mixed vesicles, two moieties coexisted, i.e., the ion pairs consisted of two CnNBr with one gemini and residual single C12ϕ2C12 or C10ϕ2C10. Thus, the effective packing parameter Peff can be estimated by (1) [23]:

$$ {P}_{\mathrm{eff}}={\left(v/{a}_0l\right)}_{\mathrm{eff}}={x}_1{P}_{\mathrm{ion}\ \mathrm{pairs}}+{x}_2{P}_{\mathrm{gemini}} $$
(1)

where x1 and x2 are the mole fractions of the ion pairs and the single gemini, respectively, in the aggregate.

Figure 4 shows the linear variation of Peff with β for all systems analysed here. The horizontal dashed line indicated the theoretical boundary above which vesicles could have been formed. The crossovers between the Peff plots and the horizontal dashed line indicated the critical β values required to form vesicles for each system. These values listed in parentheses in Table 1 were consistent with those experimental points (βV). This mechanism dominated the improvement of the effective molecular geometry was the main reason for vesicle formation.

Fig. 4
figure 4

Effective packing parameter Peff as a function of β for a C12ϕ2C12/CnNBr and b C10ϕ2C10/CnNBr systems

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Vesicle aggregation at high temperatures

Figure 5 shows the solution turbidity (expressed as transmittance at 500 nm) as a function of temperature for the C12ϕ2C12/CnNBr and C10ϕ2C10/CnNBr systems at β = 1.6. As the temperature increased, the solutions became cloudy at a critical temperature (Tc) which was indicated by an arrow for each system. The dynamic light scattering (DLS) measurements showed a rapid increase in aggregate size when the temperature approached Tc (Fig. 6). All these indicated that the vesicles associated at high temperatures.

Fig. 5
figure 5

Turbidity (expressed as transmittance at 500 nm) curves for the mixed systems at β = 1.6 under heating, in which arrows show the critical temperature to clouding (Tc)

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Fig. 6
figure 6

Variation of average hydrodynamic radius (Rh,ave) with temperature for C12ϕ2C12/CnNBr (left) and C10ϕ2C10/CnNBr (right) systems at β = 1.6

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Mechanism of vesicle aggregation

To understand the mechanism of vesicle aggregation, one detail was important: what was the attractive force to pull neighbouring vesicles together in the present case? According to the characteristics of the Cmϕ2Cm molecular structure, it was believed that this responsibility should be borne by the alkyl tails themselves. As expounded in “Introduction” section, Cmϕ2Cm has a long and rigid spacer in its molecular structure, which made its two alkyl tails able to produce both cis- and trans-configurations, i.e., toward the same or the opposite side. When Cmϕ2Cm formed the vesicles together with CnNBr, it should prefer the cis-configuration so as to make the tails penetrate the inner of the double layer of vesicles. However, this cannot guarantee that all of the Cmϕ2Cm molecules always kept one kind of configuration. When temperature was raised, the action of the alkyl tails was enhanced. A few molecules probably adopted trans-configurations and stretched one tail toward the solution. When the vesicles approached each other in solution, those projecting tails associated hydrophobically, thereby non-covalently attaching one vesicle to another (Fig. 7). This was perhaps the mechanism underpinning the present vesicle aggregation.

Fig. 7
figure 7

Schematic representation of bridging between neighbouring vesicles by the extension of the alkyl tails of Cmϕ2Cm

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A similar effect was suggested by Menger and Peresypkin [18], in which they studied the vesicles generated by a set of zwitterionic geminis with two alkyl tails of different lengths (these geminis had general structure: ROPO2-OCH2CH2N+(CH3)2R′, where R and R′ were hydrocarbon chains and were referred to as Cm-Cn). They obtained strings of vesicles for those zwitterionic geminis with highly asymmetric alkyl tails such as C14-C8, C18-C7, C22-C6, etc., using the short chains extending from the vesicles to associate with the projecting chains of neighbouring vesicles. In this system, the two alkyl tails of Cmϕ2Cm had the same length, and thereby, it may have been relatively difficult to extend one tail compared with the cases described by Menger and Peresypkin. This may have been why the present vesicle aggregation occurred at higher temperatures.

It was of significance to compare the present cases with those generated by the homologue of C12ϕC12, O,O′-bis(sodium 2-dodecylcarboxylate)-p-benzenediol (generally referred to as C12ϕC12, Scheme 2). Compared with C12ϕ2C12, C12ϕC12 has a shorter spacer but can also form vesicles together with CnNBr, which resulted in two aqueous phases (ASTP) [24, 25]. Figure 8 shows the variation of average hydrodynamic radius (Rh,ave) of the vesicles formed by C12ϕC12/C8NBr at β = 1.6 with temperature. It was found that Rh,ave remained practically constant over a wide range of temperatures, indicating no vesicle aggregation in the case of C12ϕC12/C8NBr. This result indicated that responsibility for extending the tail out to the vesicle was indeed borne by Cmϕ2Cm in the present cases, which agreed with the special property of these geminis having both cis- or trans-configuration as mentioned.

Scheme 2
scheme 2

Chemical structure of C12ϕC12

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Fig. 8
figure 8

Variation of average hydrodynamic radius (Rh,ave) of the vesicles with temperature for a C12ϕC12/C8NBr system at β = 1.6

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Figure 9 shows the influence of the hydrophobic interaction between the alkyl tails on Tc. As seen in Fig. 9, the stronger the hydrophobic interaction (which was a result of the longer alkyl tails), the lower the value of Tc. This clearly indicated that a hydrophobic interaction existed between the alkyl tails and strongly influenced vesicle aggregation. If the mechanism of vesicle aggregation was indeed due to the trans-configuration of some C12ϕ2C12 or C10ϕ2C10, the result shown in Fig. 9 supported this mechanism because the strong hydrophobic interaction hindered the extension of the alkyl tail of Cmϕ2Cm.

Fig. 9
figure 9

Tc-dependence on the alkyl tail length of CnNBr

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Effect of vesicle surface charge

Figure 10 shows the effect of the mole ratio of CnNBr to gemini surfactants (β) on Tc, in which Tc decreased with increasing β. The increase in β meant that more CnNBr molecules entered the mixed vesicles and this neutralised the charge thereof and reduces the repulsion between them. As a result, the aggregation of vesicles was promoted and Tc decreased. The addition of NaBr also yielded a similar effect (Fig. 11) because the salt screened the surface charge on the vesicles. The two approaches can provide methods of adjusting the intensity of vesicle aggregation, which may be useful in future applications.

Fig. 10
figure 10

Effect of β on Tc for C12ϕ2C12/CnNBr (left) and C10ϕ2C10/CnNBr (right) systems

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Fig. 11
figure 11

Effect of NaBr concentration on Tc for C12ϕ2C12/CnNBr (left) and C10ϕ2C10/CnNBr (right) systems at β = 1.6 (Arrows indicate precipitation)

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Conclusion

The carboxylate gemini surfactant Cmϕ2Cm exhibited novel self-assembly behaviour, which benefitted from its special molecular structure. Cmϕ2Cm carried a long, rigid spacer that made its two alkyl tails difficult to bring into close proximity. This had two effects: firstly, Cmϕ2Cm had a columnar-like molecular geometry, which was the origin of the formation of the vesicles together with CnNBr, and secondly, the Cmϕ2Cm molecule could rotate its two alkyl tails freely, i.e., adopting either cis- or trans-configurations, which led to the present vesicle aggregation and may also hold a function which produced this novel self-assembly behaviour. All of these aspects indicated that the gemini surfactants, with their long, rigid spacers, were good candidates for novel self-assemblies and further applications.