Self-assembly of folic acid/melamine complexes with hierarchy levels: from membranes to porous spherulites and networks

Abstract

We report a hierarchical self-assembly shown by the complexation of folic acid and melamine in water. With the increase in folic acid (FA) concentration, 2D membranes, 0D spherulites and 3D networks with porous structures are constructed. H-bonds and π–π stacking interaction are proven to be the basic driving forces for the formation of membrane and plates with high inter-affinity, which further self-organize into anisotropic spherulites that show strong birefringence. When the concentration of FA increases further, cross-linked networks with high viscoelasticity are generated.

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Introduction

Self-assembled soft materials with various hierarchy levels, including 0D vesicles/spheres, 1D fibers/tubes, 2D membranes and 3D networks, have attracted considerable attentions due to their amazing features and wide applications.¹ In order to fabricate soft materials used in medical and life sciences, people often pay attention to nature which provides materials superior and more complicated than artificial systems.² Polypeptides, DNA, polysaccharide/cyclodextrins, vitamins and other biotins have been investigated as building blocks to build functional materials in the past decades.³ In contrast to the rationally designed molecules, it is rather difficult to obtain ordered aggregates from natural products due to their multiple functional groups and complex molecular structures. In spite of the obstacles, a variety of self-assembled aggregates have been prepared using natural assemblers, i.e., membranes from iron(III)/tannins⁴ and well-defined nanoparticles from catechols.⁵ However, the fabrication of hierarchical self-assemblies of multi-dimensional materials from natural products still remains challenging.^3–5

Membranes play a significant role in maintaining the cellular shape and biological components in biological tissues. Membrane-based materials have potential uses in sensing and catalysis, as well as biological applications.⁶ Membranous aggregates such as films, vesicles, ribbons also show the possibility of topological transformation (hierarchical self-assembly) into other dimensional aggregates such as tubes and 3D networks.⁷ Spherulite is a kind of polycrystalline aggregates with a spherical shape.⁸ Similar to the membrane (such as liposome) self-assembled in cells, spherulites as polycrystals, are found not only in volcanic rocks, silicate minerals, metals and simple organic molecules, but also widely in living organisms like brain, muscle, nerve, blood and bile.⁹ Thus, self-organized spherulites from natural assemblers are of great importance in biological/medical areas. Generally, spherulites are produced by the process of polymer crystallization; the cooling down of the melts of some small organic molecules gives rise to spherulites as well. Spherulites can be categorized optically using polarized light and exhibit characteristic properties (Maltese cross pattern). These features of spherulite endow it with amazing and useful applications, mainly in medical and arts.¹⁰ For example, the nerve fibrils of people who have Alzheimer's disease or Parkinson's disease would accumulate into radial spherulites that can be detected conveniently by crossed polarizers.¹¹

Folic acid (FA), as a vitamin, is a natural product which has been extensively studied due to its anti-tumor effect and G-quartet formation property.¹² FA and its derivatives have been proven to be the effective building blocks in fabricating supramolecular gels, liquid crystals or other aggregates.^12,13 However, due to poor solubility, materials self-assembled by native FA in pure water have rarely been probed although native FA can form gels in DMSO–water mixtures as elucidated by Nandi^13a and our group.^13b The pterin ring moiety of FA provides A–D–A–D (A: acceptor; D: donor) H-bonding sites, while the carboxylic groups of glutamic acid moieties provide protons and H-bonding sites.¹⁴ Therefore, FA can probably form complementary H-bonds with some molecules, such as melamine. Melamine (Mm) which contains 9 H-bonding sites in D–A–D arrays, has a slight solubility in water and acts as an effective co-assembler in assisting the self-assemblies of organic acids or pterin analogues like riboflavin in aqueous media.¹⁵ For example, Liu and coworkers utilized Mm to tailor the size and self-assembly routes of nanotubes from a bola-type glutamic acid derivative;^14a Nandi and coworkers contributed mostly to the Mm-participated supramolecular gels with controllable fluorescent emissions.¹⁵ Herein, we report a hierarchical self-assembly of FA/Mm complexes in water, which give rise to membranes, spherulites and networks with the increase in FA concentrations (Scheme 1). Spherulites and networks exhibit porous structures that consist of micro-plates and rods. Such plates and rods are derived from the growth and folding of initial membranes, predicted by the topological revolutions. H-bonds and π–π stacking are the primary driving forces in aggregate formation, whereas the radical growth of plates crystallized from super-cooling enables the formation of the outstanding spherulites.

Scheme 1 Schematic representation of the hierarchical self-assembly of FA/Mm complexes.

Results and discussion

FA is almost insoluble (0.0016 mg ml⁻¹) in water even at a high temperature. However, the mixture of FA and Mm has a relatively high solubility (up to 4.0 mg ml⁻¹) at a high temperature (ca. 70 °C). Except at a low concentration range, FA can dissolve well in water at just 70 °C when ca. 3 equiv. (R = 3) or more Mm is added, indicating that FA provides multiple sites to interact with Mm. By cooling down the aqueous solutions of FA/Mm, aggregates with different macroscopic appearances were obtained. As shown in Fig. 1, transparent solutions, precipitates and viscous solutions were obtained with the increase in FA concentration.

Fig. 1 Phase diagram of the two-component system. Insets: digital images of solution, precipitate and gel-like viscous solution. Dashed line: speculated boundary.

Those precipitates and viscous solutions were first examined by optical microscopy. It was found that all the precipitates were actually composed of spherical microparticles with multi-dispersity (Fig. 2 and S1†). Under crossed polarizer, these spherical particles exhibited strong anisotropic birefringence. The well-defined Maltese cross patterns with blue and yellow colors indicate the formation of spherulites.^8–10 In the viscous solution phase, we also detected the presence of spherulites, which were embedded in small size aggregates with barely any birefringence (Fig. S1a and S1b′†). If more than 10 equiv. Mm participates in the self-assembly, crystals of Mm would form, revealing an upper limit in the concentration of Mm. The formation of spherulites undergoes a nucleation-growth process, regulated by multiple factors such as concentration and cooling rate.¹⁰ Under these circumstances, the size of spherulite cannot be controlled precisely.

Fig. 2 Optical microscopy images of precipitates (c_FA = 2 mM, R = 6) under natural and polarized light.

SEM experiments were carried out to probe the micro-morphologies of the self-assembled spherulites and viscous solutions. As displayed in Fig. 3 and S2–S6,† the surfaces of the spherulites are mainly composed of plates, which further self-assemble into porous structures with nanopockets. These particular morphologies show great similarity to graphene-based gels.¹⁶ Notably, amongst the nanopockets, there are microrods exhibiting fibrous networks (red dashed circle in Fig. 3b, S2 and S3†). The co-existence of microrods and microplates implies that there would be two distinct self-assembly routes in the formation process of spherulites. Viscous solution samples display scattered microplates under SEM observations (Fig. S4†). Similarly, the stacking of microplates affords the porous structure of viscous solutions. The common plate-like, porous feature of spherulite and networks strongly suggest the formation of hierarchical self-assembly. The thin-plates might be the basic building units in fabricating the spherical particles and infinite networks.

Fig. 3 SEM images of spherulites (a–d, c: enlarged porous structures, d: enlarged images of red dashed circles) and networks (e–f).

To confirm that hypothesis, TEM experiments which can provide insights into the inner structure of aggregates were performed. As shown in Fig. 4, irregular micron-sized thin membranes are the dominant aggregates in solutions. The small size spherulites have the typical radical, porous structures, and this radical growth from nucleation sites reflects the category-one growing type.¹⁷ The growth units, just as evidenced by SEM images, are membranes and nanorods. Unlike the spherulites, the membrane-encapsulated nanorods in viscous solution sample link together to give networks (Fig. 4e and f). As shown in the red dotted squares, the initial spherulites and initial networks share the same building units. It seems that the nanorods are generated from the folding and overlapping of membranes. These structures collected from the bulk liquids are in good agreement with the SEM results, reflecting the hierarchical self-assembly process of FA/Mm complexes. These observations indicate that the formation and growth of the initial membrane play a dominant role in tuning the topological morphologies.

Fig. 4 TEM images collected from the bulk phases of solution (a), precipitate (b–d) and viscous solution (e and f).

As stated in the last part, at high concentration range, FA would generate the viscous aggregates (inset of Fig. 1). We conducted rheological experiments on the resultant viscous solutions. Frequency sweep indicates that G′ (storage modulus) values of all performed samples are about one order of magnitude higher than that of G′′ (loss modulus) over the entire range of applied frequencies. Moreover, both the G′ and G′′ of samples display the frequency-independent behavior, indicating a solid-like behavior.¹⁸ The viscosity of complex decreases linearly against frequency sweep with a slope of −1. Therefore, the aggregates at high FA concentrations have high viscoelastic properties (a hydrogel-like behavior) (Fig. 5).

Fig. 5 Dynamic oscillatory stress sweep (a, F = 1 Hz), frequency sweep (b, applied stress = 1.0 Pa) and frequency dependency of the complex viscosity (c, applied stress = 1.0 Pa) of a high concentration sample (9 mM with 9 equiv. Mm).

Fig. 6 displays the TGA and DSC curves of samples with different FA concentrations and molar ratios. The first weight loss before ca. 100 °C is attributed to the evaporation of entrapped water. At 250–300 °C, a significant weight loss occurs due to the degradation/cleavage of hydroxyl, amine and carboxyl groups in FA/Mm. In DSC curves, the two endothermic peaks observed were attributed to water evaporation and functional group degradation. The peaks at around 300 °C show a dependence on FA concentrations and R values. With the increase in FA concentration or R value, the endothermic peaks will be greatly elevated (Fig. 6b). For example, a sample of 1.8 mM of FA (R = 3.5) has a peak at 280 °C, whereas it increases to ca. 330 °C when more Mm (R = 16) is involved in the self-assembly. The appearance of this peak at higher temperatures implies better thermal stability. Therefore, by increasing the FA concentration and R value, the thermal stability can be enhanced remarkably, probably due to a more compact structure. Effective proton transfer between Mm and FA might contribute greatly to this phenomenon.

Fig. 6 TGA and DSC curves of different samples. The marker implies R (c_FA).

Compared with the free molecule state in DMSO, UV-vis absorption peaks of FA/Mm complexes in water at 280 and 370 nm all show blue shifts (Fig. 7a). Such blue shifts reflect a typical H-aggregate that contains face-to-face stacking arrays of FA molecules.¹⁹ Hence, the chromophores of FA would be π–π stacked in the self-organized structures. It can also be found that, after self-organization, the absorption bands were barely broadened compared to the disassembled state, suggesting an ordered organization. With the increase in the concentrations of the complexes, the aromatic protons of FA shift to higher field, confirming the π–π stacking interaction (Fig. 7b).

Fig. 7 UV-vis spectra (a), selected ¹H NMR spectra (b), FT-IR spectra (c) and XRD patterns (d) of different samples; in (d) the red hkl values stand for planes of aggregate while black hkl values stand for the planes of crystals.

FT-IR was employed to further confirm the interactions between FA and Mm (Fig. 7c). Sharp peaks at 3469 and 3413 cm⁻¹ are assigned to the –NH₂ vibration stretching band of Mm ring. After complexing with FA, the relative intensities of the two bands of Mm gradually decrease, accompanied by the appearance of the broadened peak at 3350 cm⁻¹. This implies the formation of H-bonds between FA and Mm.²⁰ Moreover, the –NH₂ and –OH stretching peaks of native FA at 3553, 3416 and 3319 cm⁻¹ are absent after aggregation, indicating that the pterin ring of FA forms H-bonds with Mm. The disappearance of C=O stretching vibrations (1693 cm⁻¹) of free carboxylic acid suggests that the proton transfer effect occurs between the entire carboxylic groups and Mm. Proton transfer between the nitrogen in triazine ring and carboxylic acids can also be evidenced by the changes of the C=N vibration in melamine.¹⁴ In addition, the symmetrical stretching vibration (ν_s) peak of carboxylate at 1392 cm⁻¹ also supports the presence of intermolecular proton transfer. The peak of 1510 cm⁻¹ is contributed by amide-II band, suggesting the formation of intermolecular H-bonds between amides of glutamic acid moieties of FA.²¹

XRD patterns could verify the above-mentioned viewpoints, as shown in Fig. 7d. For samples with low concentration and R value, the peaks are totally different from that of pure FA. Three new small peaks at 6.43, 9.08 and 12.88° correspond to the 100, 110 and 111 planes of tetragonal molecular packing, indicating the molecular arrangement of FA backbones (Scheme 1). However, peaks of 17.8° and 26.3° appear in self-assembled samples, ascribed to the π–π stacking between Mm molecules.¹⁴ It should be noted that for samples with high Mm concentrations and R values, peaks that show great similarity to pure Mm start to emerge. Such peaks indicate the formation of Mm crystals, which are in good agreement with morphologies detected by SEM and OM. According to the XRD patterns, the crystals of Mm belong to monoclinic syngony, where the 100 plane (2θ = 13.0°)and the 111 plane in aggregates (12.88°)slightly overlap.

Discussion

As elucidated above, we knew that Mm could interact with glutamic acid and pterin ring moieties of FA by complementary H-bonds and proton transfer. These interactions assist the self-assembly and contribute to a better solubility of FA in water. The outstanding hierarchical self-assembly or concentration-dependent typological revolution is clearly the basis of membrane formation and growth. Moreover, the nucleation and growth play an essential role as well, e.g., in the process of transition from graphene-like thin membranes to thick plates in spherulites or networks, and from plates to radical spherulites with strong birefringence. Apart from the growth of membranes driven by the intermolecular π–π stacking and H-bonds, the formation of the particular rods might be due to the folding and overlap of initial membranes. This hypothesis can be proven to be valid, evidenced by the network-like membrane surface (Fig. S7†) and their effective folds and overlaps (Fig. S8†). These morphologies are similar to the nano- and microrods detected by SEM and TEM (Fig. 3 and 4). According to the AFM results (Fig. 8), the surface of the membrane is rough, which is consistent with TEM observations. Also the cross-section profile indicates that, the thickness of the membrane is ca. 2–3 nm, corresponding to the single molecular length of FA/Mm complexes. Furthermore, in the supernatant of high concentration samples, we also observed the presence of some membrane-like aggregates with thickness of several nanometers (but with more regular shapes, see Fig. S9†), implying a similar self-assembly pathway in samples with different concentrations.

Fig. 8 AFM image (a) of the aggregates from diluted solution (1 mM, 3 equiv. Mm), b: cross-section profile from (a).

At ultralow concentrations, Mm interacts with FA to form H-bonded complexes which further π–π stacked into the very thin membranes. As the solution is highly diluted, the inter-membrane interactions are too weak to further aggregate. But when the concentration increases, membranes are first formed, followed by the growth and folding to generate thicker membranes or plates which also act as nuclei to trigger the radical growth (spherulites). At high concentration range, more nanoplates or thicker membranes would form, which disfavors the radical growth but favors the linear growth to form networks. The networks are infinite aggregates and clearly have a larger aggregation number than that of sperulites. According to the equation:²²

when the concentration increases, networks will form to generate a high aggregation number (N) which corresponds to a lower chemical potential. The low chemical potential means that the networks are thermodynamically stable at high concentrations. On the other hand, when the concentration decreases, aggregation number will decrease to generate a higher chemical potential. The increased mean free energy per molecules will be compensated by the disappearance of the unfavorable rim energy of networks to produce spherical aggregates.

Another amazing feature of the FA/Mm self-assembly is the spherulite formation owing to the fact that plate-like spherulites are rare, not to mention coming from natural small molecules. In contrast to the compact morphologies from the melts, open morphologies often occur in solution-growing spherulites. This theory is confirmed by the porous structure of the resultant FA/Mm spherulites. The super-cooling of super-saturated FA/Mm complex solutions provides a strong driving force for crystallization that is the prerequisite for spherulites. The networks hardly show birefringence, although spheres and networks are assembled from the same building units—microplates/rods. Therefore, the birefringence may be derived from the secondary self-organization or crystallization of microplates/membranes ascribed to their high affinities (electrostatic forces). This is in sharp contrast to the conventional polymeric spherulite formation. Although the details need to be further studied, this finding would broaden the concept and category of spherulites and self-assemblies.

Conclusions

In summary, FA/Mm complexes showed an outstanding hierarchical self-assembly that generates multiple dimensional aggregates at different concentration ranges. H-bonds and π–π stacking allow the formation of membranes, which further grow into plate-constituted spherulites and networks. The super-cooling of saturated FA/Mm solution enables the anisotropic growth (or crystallization) of plates and membranes to form spherulites with strong birefringence. Due to the secondary self-organization of the plates, spherulites and networks exhibit porous structures. Natural assemblers have opened a brand new path for advanced soft materials, and this system that shows multiple topological morphologies would greatly enrich the categories and concepts.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra04585h

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