Introduction

Over the past several decades, two types of materials have received significant attention in the field of materials science, namely, nanosheets and supramolecular polymers. The former are ultrathin anisotropic materials. A representative example is graphene, which was the basis of a Nobel Prize award in 2010 and exhibits remarkable electrical properties, although various nanosheet-based materials have been developed to date [1,2,3,4,5]. The latter consist of two-dimensional molecular assemblies [6,7,8,9,10] that form by precisely controlled self-assembly from simple mixtures of monomers in solution. More recently, supramolecular nanosheets that combine these two concepts (Fig. 1) have emerged in conjunction with progress in the field of nanotechnology [11,12,13,14,15,16,17,18,19,20,21,22,23,24,25], and various interesting functions have been reported. As an example, the two-dimensional (2D) self-assembly of DNA (so-called DNA origami) has become the basis for an important field of study by providing platforms capable of constructing a variety of complex planar nanostructures [19]. Although some supramolecular nanosheets consisting of proteins and phospholipid membranes have been developed for applications as biomaterials, specific principles for the design of such materials have not yet been established, and significant challenges remain in terms of stability and functionality. The present review summarizes the design and application of recently reported tubulin heterodimer (THD) protein-based and phospholipid membrane-based supramolecular nanosheets.

Fig. 1
figure 1

Diagram showing the integration of nanosheets and supramolecular polymers to produce supramolecular nanosheets

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Results and Discussion

Tubulin-based nanosheets

THD is a protein that serves as a component of microtubules (MTs) found in intracellular cytoskeletons [26,27,28,29,30,31,32,33] (Fig. 2a, f). This protein is composed of α-tubulin (shown in green) and β-tubulin (shown in cream), both of which bind to guanosine triphosphate (GTP). GTP attached to the α-tubulin units can neither undergo hydrolysis into guanosine diphosphate (GDP) nor be replaced with other nucleoside phosphates. In contrast, GTP attached to the β-tubulin units can be hydrolyzed to GDP, which, in turn, can be replaced with another substituent. For example, replacement with GTP* (guanylyl 5ʹ-α,β-methylenediphosphonate), a nonhydrolyzable GTP analog, affords THDGTP*. Note that in this figure, only the nucleoside phosphates attached to β-tubulin units are included as subscripts for the sake of convenience. Both THDGTP and THDGTP* have been reported to self-assemble into MTGTP and MTGTP*, respectively, upon heating to 37 °C [26, 27]. Although MTGTP depolymerizes into THDGDP (Fig. 2b, g) in conjunction with the hydrolysis of bound GTP to GDP, MTGTP* does not depolymerize into THDGDP* because of the nonhydrolyzable nature of GTP*. Interestingly, it has been demonstrated that the coassembly of THDGTP and THDGTP* at a ratio of 1 to 5 (mol/mol) results in the formation of 2D nanosheets (NSGTP/GTP*) rather than MTs, as confirmed by transmission electron microscopy (TEM) (Fig. 2c, h). NSGTP/GTP* has a leaf-like 2D structure and a thickness of approximately 5 nm, corresponding to the thickness of the THD units, as confirmed by atomic force microscopy [21]. Both MTGTP and MTGTP*, which are prepared from THDGTP and THDGTP*, respectively, are formed by the edge-closing of NSGTP and NSGTP*, which act as [28], and the long axis of THDGTP is known to be shorter than that of THDGTP* [29]. This mismatch in the size of the long axis is believed to lead to the formation of the unfolded NSGTP/GTP* structure rather than the folded MTGTP/GTP* structure. Interestingly, NSGTP/GTP* can transform into GTP-responsive spherical nanocapsules (NCGTP/GTP*; Fig. 2e, i) in response to interactions with a photoreactive molecular glue (GlueCO2–; Fig. 2d) containing multiple guanidinium ion (Gu+) and benzophenone (BP) groups. GlueCO2– molecules can strongly attach to proteins under physiological conditions through the formation of multivalent salt bridges between Gu+ ions and oxyanionic functional groups followed by photocrosslinking via BP units [21]. Molecular dynamics simulations have suggested that the adhesion of GlueCO2– transforms NSGTP/GTP* into a planar structure, resulting in the stacking and assembly of NSGTP/GTP* to construct spherical NCGTP/GTP*.

Fig. 2
figure 2

Diagram summarizing the multistep procedure for the synthesis of NCGTP/GTP* from MTGTP. In this process, (a) MTGTP is depolymerized into (b) THDGDP, which is subsequently incubated with a mixture of GTP* (83 mol%) and GTP (17 mol%) to form (c) NSGTP/GTP*. Upon treatment with (d) the photoreactive molecular glue GlueCO2–, the NSGTP/GTP* nanosheets are transformed into spherical (e) NCGTP/GTP*. TEM images of (f) MTGTP, (g) THDGDP, (h) NSGTP/GTP*, and (i) NCGTP/GTP*

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GTP-responsiveness of tubulin vesicles

GTP is an intracellular molecule involved in many essential biological processes [30,31,32,33,34,35,36,37], such as cell division [33], nucleotide synthesis [34] and cell signaling [35]. During cell division, THD uses GTP as an energy source for conformational changes that induce the polymerization or depolymerization of MTs [30,31,32]. GTP is also a component for the self-replication of RNA viruses [38,39,40,41], such as coronaviruses. Notably, GTP is abundant in certain diseased cells (at concentrations of 1.5–4.5 mM), such as rapidly proliferating cancer cells [42] and cells infected with RNA viruses [43]. In contrast, the concentration of GTP, unlike that of adenosine triphosphate, is negligibly low in normal cells (<0.3 mM) [44]. Therefore, GTP-responsive nanocarriers could have the potential to efficiently cure cancer and to treat diseases linked to RNA viruses, including coronavirus disease 2019 (COVID-19) [41]. Interestingly, NCGTP/GTP* collapses upon exposure to GTP at concentrations higher than 0.5 mM (Fig. 3c, d), releasing encapsulated cargo in the process. This behavior suggests that NCGTP/GTP* could be used as a GTP-responsive carrier. In the presence of GTP, the GTP* in NCGTP/GTP* is likely to be replaced with GTP to afford NCGTP, which can then collapse in association with the hydrolysis of GTP in a manner analogous to the depolymerization of MTGTP (Fig. 3a). Because NCGTP/GTP* does not collapse in the presence of less than 0.2 mM GTP (Fig. 3b, d) and normal cells contain less than this level, GTP-responsive NCGTP/GTP* could be used to deliver encapsulated drug molecules solely to high-GTP regions such as cancer cells and virus-infected cells. Indeed, it has been shown that the anticancer drug doxorubicin (DOX) can be encapsulated in NCGTP/GTP* and administered to cancerous Hep3B cells, killing these cells more effectively than DOX without encapsulation.

Fig. 3
figure 3

a Diagram of the GTP-responsive collapse of NCGTP/GTP*. TEM images of NCGTP/GTP* upon mixing with GTP at concentrations of (b) 0.2 mM and (c) 0.5 mM. d Size distribution of NCGTP/GTP* following titration with GTP (0–1.0 mM) as determined by DLS

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Kinetically stable phospholipid nanosheets using cholic acid-based surfactants

Phospholipid assemblies such as liposomes and micelles have long been regarded as versatile biomaterials owing to their similarity to cell membranes and their high degree of biocompatibility [45, 46]. Among these, phospholipid nanosheets (sometimes referred to as bicelles) have recently attracted particular attention for the design of a variety of biomaterials. For example, phospholipid nanosheets have been found to orient themselves in relation to the strong magnetic fields applied during nuclear magnetic resonance (NMR) analyses, and membrane proteins embedded in these materials can provide angle-dependent NMR data that can allow structural elucidation [47]. Furthermore, it has recently been reported that phospholipid nanosheets are promising carriers for drug delivery. One advantage of these nanosheets is the extended circulation times associated with disk-shaped carriers [48] together with the stronger adhesion to microvascular networks [49, 50] compared with spherical carriers. In addition, López et al. reported that the characteristic 2D shape of phospholipid nanosheets is advantageous with regard to transdermal drug delivery applications, in which the nanocarriers need to pass through narrow gaps (6–10 nm) between skin cells [51]. The formation of phospholipid nanosheets in solution can be quantitatively evaluated by 31P NMR because 31P atoms in phospholipid nanosheets exhibit shifted NMR peaks that differ from those of phospholipid micelles and vesicles as a consequence of the chemical shift anisotropy of oriented 31P atoms. Unfortunately, nanosheets composed of mixtures of surfactants and phospholipids typically produce unstable self-assemblies that readily transition to other morphologies in response to changes in the surrounding environment. For example, phospholipid nanosheets composed of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and cholic acid-based surfactants, which have been frequently studied, are known to undergo a structural transition when in dilute suspensions. This phenomenon is attributed to the phase transition temperature of DMPC (Tm = 24 °C), which is lower than room temperature, causing DMPC membranes to form a highly fluid liquid crystalline phase [52]. In addition, the critical micelle concentration (CMC) values of cholic acid-based surfactants are generally quite high. Hence, these surfactants easily dissociate from the edges of the phospholipid nanosheets to induce fusion of the nanosheets (Fig. 4a) [53].

Fig. 4
figure 4

Diagrams of the structures of (a) a conventional nanosheet and (b) a kinetically stable nanosheet. Legend: Tm = bilayer melting point, CMC = critical micelle concentration. c Molecular structures of the phospholipid DPPC and of Chol-TEG-C5. d Photographic images and (e) 31P NMR spectra of aqueous dispersions of DPPC/Chol-TEG-C5 nanosheet assemblies ([DPPC]/[Chol-TEG-C5] = 5:1, mol/mol) at concentrations of 2.0, 1.0 and 0.2 wt% and 25 °C. f Large-area and (g) zoomed-in TEM images of Chol-TEG-C5 nanosheets deposited on a hydrophilized carbon grid from a 0.04 wt% aqueous dispersion and stained with uranyl acetate. Merged bright-field and fluorescence images of Hep3B cells incubated with 0.1 wt% aqueous dispersions of DPPC/Chol-TEG-C5/DOX assemblies ([DPPC]/[Chol-TEG-C5] = 5:1 mol/mol, concentration of DOX: 24 μM) at (h) 25 °C (the nanosheet state) and (i) 37 °C (the micelle state). These images were acquired during trials involving the in vitro thermoresponsive delivery of DOX to Hep3B cells by DPPC/Chol-TEG-C5 acting as a carrier. Hep3B cells were incubated with 0.1 wt% aqueous dispersions of DPPC/Chol-TEG-C5/DOX assemblies ([DPPC]/[Chol-TEG-C5] = 5:1 mol/mol, concentration of DOX: 24 μM) at various temperatures. Viability of Hep3B cells incubated with DOX (24 μM) at (j) 25 °C and (k) 37 °C in the presence (blue) or absence (gray) of DPPC/Chol-TEG-C5 assemblies. Reprinted from [22] with permission from Wiley

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Recently, phospholipid nanosheets that overcome the above problems based on the use of optimal phospholipids and surfactants have been reported [22]. One such system comprises nanosheets made of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC, Fig. 4b) and a cholic acid-based surfactant attached to pentyl chains via a triethylene glycol spacer (Chol-TEG-C5, Fig. 4c). This material maintains a nanosheet structure even under dilute conditions [53]. Moreover, because the Tm of DPPC is 41 °C, which is above room temperature, DPPC membranes form a less fluid gel phase under ambient conditions [54] that tends to provide kinetically stable assemblies. Finally, Chol-TEG-C5 has a low CMC ( < 0.5 mM) as a result of the introduction of hydrophobic end-cap groups, such that dissociation from the edges of the DPPC membranes is suppressed [55]. Consequently, a mixture of DPPC and Chol-TEG-C5 ([DPPC]/[Chol-TEG-C5] = 1/5, 5.0 wt% total concentration) was found to generate a clear dispersion without large aggregates even after dilution to 0.2 wt% (Fig. 4d). The 31P NMR spectra acquired from such dispersions exhibited a negative peak shift derived from the orientation of the DPPC/Chol-TEG-C5 nanosheets over the concentration range of 0.2–5.0 wt% (Fig. 4e). Furthermore, TEM images confirmed the formation of nanosheets with diameters ranging from 200 nm to 2 µm (Fig. 4f, g).

Because the Tm of DPPC (41 °C) is close to body temperature (37 °C), DPPC/Chol-TEG-C5 nanosheets could potentially be used as a thermoresponsive material capable of undergoing a structural transition from nanosheets to micelles upon heating in the body to facilitate cellular uptake. Indeed, during trials in which DPPC/Chol-TEG-C5 nanosheets containing the anticancer drug DOX ([DPPC]/[Chol-TEG-C5] = 5:1 mol/mol, concentration of DOX: 24 μM) were incubated with Hep3B cancer cells at 25 °C, minimal cell death was observed (Fig. 4h, j). Conversely, many dead Hep3B cells containing DOX were observed after incubation at 37 °C (Fig. 4i, k). This temperature-responsive behavior of DPPC/Chol-TEG-C5 nanosheets could permit the design of transdermal drug delivery systems to be administered near room temperature and activated by body temperature in skin tissue [56].

Phospholipid nanosheets for blood administration

Phospholipid nanosheets have also been utilized for blood administration based on designing surfactants with higher biocompatibility [23]. In prior work, a cholic acid derivative attached to the metal-binding peptide amino acid sequence PHGGGPHGGG (Chol-MBP; Fig. 5a) was developed as a surfactant and mixed with DPPC ([DPPC]/[Chol-MBP] = 5/1, 5.0 wt% in total) to produce DPPC/Chol-MBP nanosheets. PHGGG is a naturally occurring Cu2+ binding motif because of the metal-coordinating ability of the imidazole group of the histidine residues [57]. Because each Chol-MBP can bind to two Cu2+ ions, these moieties can cross-link at the edges of DPPC/Chol-MBP membranes to inhibit dissociation (Fig. 5c), thus improving the stability of the phospholipid nanosheets. Indeed, when a DPPC/Chol-MBP nanosheet dispersion was diluted to 0.2 wt%, spherical aggregates less than 100 nm in diameter were observed by TEM (Fig. 5c). In contrast, after the addition of 2.0 equiv. of Cu2+ ions to afford DPPC/Chol-MBP/Cu nanosheets, the nanosheet structure was maintained after dilution (Fig. 5d). These results indicate that the Cu2+-mediated crosslinking of Chol-MBP significantly improved the stability of these nanosheets in response to dilution.

Fig. 5
figure 5

a Molecular structure of Chol-MBP. b Diagram showing the preparation of DPPC/Chol-MBP nanosheets and subsequent Cu2+-mediated metal coordination of Chol-MBP to afford crosslinked DPPC/Chol-MBP/Cu nanosheets. After crosslinking, the disk structure of the nanosheets is stable when diluted and in the presence of serum protein. TEM images of mixtures of DPPC and Chol-MBP (total concentration = 0.20 wt%) (c) without and (d) with 2.0 equiv. Cu2+. Scale bars: 100 nm. e Diagram showing the in vivo delivery of FLDPPC and FLDPPC/Chol-MBP/Cu nanosheets. IV-CLSM images (λext = 640 nm) of blood vessels in a mouse earlobe 2 min after the injection of (f) FLDPPC vesicles (total concentration: 5.0 wt% before injection) and (g) FLDPPC/Chol-MBP/Cu nanosheets (total concentration: 5.0 wt% before injection). Scale bars: 100 μm. Fluorescence intensities over time from trials using (h) FLDPPC vesicles and (i) FLDPPC/Chol-MBP/Cu nanosheets. These fluorescence data were acquired from a vein (green) and tissue (gray) as a control. Reprinted from [23] with permission from the Royal Society of Chemistry

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These structurally stable DPPC/Chol-MBP/Cu nanosheets were used to investigate the effect of the morphology of phospholipid aggregates on the blood circulation profile. For this purpose, fluorescent Cy5-labeled DPPC vesicles and DPPC/Chol-MBP/Cu nanosheets (FLDPPC vesicles and FLDPPC/Chol-MBP/Cu nanosheets, respectively, total concentration: 5.0 wt% before injection) were prepared, passed through a membrane with 200 nm pores, and then administered to mice via the tail vein. Confocal laser scanning microscopy observations of the earlobes of the mice (IV-CLSM) were performed to evaluate blood circulation (Fig. 5e). The IV-CLSM data confirmed that the FLDPPC vesicles tended to aggregate and clog blood vessels (Fig. 5f). Fluorescence data acquired over time indicated minimal transfer of these vesicles from the artery to the vein (Fig. 5h). In sharp contrast, FLDPPC/Chol-MBP/Cu nanosheets were homogeneously dispersed in the blood vessels (Fig. 5g), and the time course of fluorescence monitoring revealed prolonged circulation of this material (Fig. 5i). Hence, it was demonstrated that phospholipid nanosheets exhibited longer circulation in blood than spherical vesicles. Therefore, it is likely that stabilized phospholipid nanosheets could be a suitable carrier for delivery to the whole body by blood administration.

Conclusion and future perspectives

There has recently been significant progress in the study of nanosheets and supramolecular polymers, leading to the development of supramolecular nanosheets as a new trend integrating these two concepts. Although supramolecular nanosheets have had limited applications due to their structural instability, recent advances in nanotechnology have made it possible to create several more stable versions of these nanosheets. The author previously developed supramolecular nanosheets composed of tubulin protein and phospholipid membranes that exhibited unique functions. These prior studies clearly demonstrated that supramolecular nanosheets are promising next-generation materials that can be designed to produce various structural motifs.