Self-assembly of natural and synthetic drug amphiphiles into discrete supramolecular nanostructures

Abstract

Molecular assembly provides an effective approach to construct discrete supramolecular nanostructures of various sizes and shapes in a simple manner. One important technological application of the resulting nanostructures is their potential use as anticancer drug carriers to facilitate targeted delivery to tumour sites and consequently to improve clinical outcomes. In this carrier-assisted delivery strategy, anticancer drugs have been almost exclusively considered as the cargo to be carried and delivered, and their potential as molecular building blocks has been largely ignored. In this discussion, we report the use of anticancer drugs as molecular building units to create discrete supramolecular nanostructures that contain a high and quantitative drug loading and also have the potential for self-delivery. We first show the direct assembly of two amphiphilic drug molecules (methotrexate and folic acid) into discrete nanostructures. Our results reveal that folic acid exhibits rich self-assembly behaviour via Hoogsteen hydrogen bonding under various solvent conditions, whereas methotrexate is unable to assemble into any well-defined nanostructures under the same conditions, despite its similar chemical structure. Considering the low water solubility of most anticancer drugs, hydrophilic segments must be conjugated to the drug in order to bestow the necessary amphiphilicity. We have demonstrated this for camptothecin through the attachment of beta-sheet-forming peptides with overall hydrophilicity. We found that the intermolecular interactions among camptothecin segments and those among beta-sheet peptides act together to define the formation of stable one-dimensional nanostructures in dilute solutions, giving rise to nanotubes or nanofibers depending upon the processing conditions used. These results lead us to believe that self-assembly of drugs into discrete nanostructures not only offers an innovative way to craft self-delivering anticancer drugs, but also extends the paradigm of using molecular assembly as a toolbox to achieve functional nanostructures, to a new area which is specifically focused on the direct assembly of functional molecules (e.g. drugs, or imaging agents) into nanostructures of their own.

Figure 1

TEM images of self-assembled filamentous nanostructures and micron-sized platelets formed by 1 wt% folic acid in mixtures of methanol and water. The samples were prepared through a stepwise mixing method through which folic acid was first dissolved in methanol with subsequent addition of water to reach the desired mixing ratio. Filamentous nanostructures were observed as the dominant morphology in solution samples containing 100% (A), 80% (B), and 70% (C) methanol, with a diameter of 4.2±0.5 nm, 3.5±0.5 nm and 3.9±0.6 nm respectively. Lozenge-shaped platelets of micron size were dominant in solutions containing 50% (D), 25% (E) and 0% (F) methanol.

Figure 2

Circular dichroism spectra of 1wt% folic acid solutions in methanol-water mixtures of varying compositions, prepared by two different methods: step-wise addition of water to a methanolic solution of folic acid (A) and dissolution of folic acid in pre-mixed methanol-water mixtures (B).

Figure 3

TEM images of micron-sized, lozenge-shaped platelets at mixed solvents containing 80% (A), 70% (B), 50% (C) and 25% (D) methanol. The samples were prepared by directly dissolving folic acid into the mixed solvents with predetermined ratio to reach a final concentration of 1%.

Figure 4

Proposed self-assembly pathways of folic acid into nanofibers and micro-lozenges. The pterin ring of folic acid (I) interacts with two adjacent folic acid molecules through Hoogsteen H-bond to form disklike tetramers (II). These tetramers stack up in a rotated manner (III) to form a nanofiber (IV) in methanol. These stacked columns can further associate to give lozenge-shaped platelets in water (V). Addition of water into nanofiber-containing methanol solution leads to a morphological transition into platelets.

Figure 5

TEM images of folic acid formed at three different buffers (A–C) and their corresponding CD spectra (D). (A) Sodium acetate buffer (pH 5), (B) 1×DPBS (~pH 7.4), and (C) borate buffer (~pH 9.5).

Figure 6

Representative TEM micrographs of nanotubes formed by qCPT-Tau (A) and qCPT-Sup35 (B) in water at 100 μM. TEM samples were negatively stained with 2% uranyl acetate. (C) Circular dichroism spectra of qCPT-Tau in water (1 μM) and DMSO (500 nM), and qCPT-Sup35 (50 μM) in water.

Figure 7

TEM micrographs of the assembled structures formed by qCPT-Sup35 via different preparative pathways. Nanotubes of qCPT-Sup35 were observed after dissolution in water at 100 μM after peptide purification (A). Only very few short nanofibers were observed upon reconstitution of qCPT-Sup35 in water at 100 μM after initial lyophilization from HFIP (B). Core-shell nanofibers were observed as the dominant structures when qCPT-Sup35 was reconstituted at 50 μM in 50% aqueous MeCN after initial lyophilization from HFIP (C). All TEM samples were negatively stained by 2% uranyl acetate. (D) CD spectra of qCPT-Sup35 in H₂O (solid line, 50μM), where nanotubes are the dominant self-assembly morphology, and qCPT-Sup35 in 50% aqueous MeCN after lyophilization from HFIP (dashed line, 50 μM). The latter process of solution preparation favors the formation of single filaments rather than nanotubes.

Scheme 1

Chemical structures of natural and synthetic drug amphiphiles used in this study.(A) Both methotrexate and folic acid contain a glutamic acid residue (marked in blue) and can be regarded as amphiphilic molecules when deprotonated at a higher pH. (B) The creation of camptothecin (CPT) drug amphiphiles by conjugating four CPT molecules to one β-sheet forming peptide via a biodegradable linker. Two β-sheet forming sequences (VQIVYK and NNQQNY) were used to create two drug amphiphiles: qCPT-buSS-Tau and qCPT-buSS-Sup35. The linker used to bridge the drug and the peptide is responsive to glutathione, a reducing agent within cells.