Soft Matter

Controlling self-assembly within nanospace for peptide nanoparticle fabrication

Weiping Wang ^ab, Zhimou Yang ^c, Siamrut Patanavanich ^d, Bing Xu ^bc and Ying Chau *^ab
aDepartment of Chemical and Biomolecular Engineering, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China. E-mail: ying.chau@ust.hk; Fax: +852-23580054; Tel: +852-23588935
^bGraduate Program of Nano Science and Technology, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China
^cDepartment of Chemistry, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China
^dDepartment of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

Here, for the first time, we present a new approach to produce peptide nanoparticles, which involves controlling the self-assembly process with an enzymatic trigger and confining it within nanoscale reverse micelles.

Research in the area of peptide self-assembly into supramolecular nanostructures has attracted growing interest in recent years due to its promising biomedical and nanobiotechnological applications, especially in tissue engineering, drug delivery and biosensor fabrication.^1–4 Self-assembling peptides provide natural building blocks for the fabrication of well-ordered structures and advanced materials.^4,5 Moreover, short peptide building blocks can be designed to enable a bottom-up construction of smart biomaterials in response to physical or chemical changes, (i.e. pH, temperature, presence of small molecules or enzymes, etc.).⁶ Various morphologies, including nanotubes, nanovesicles, nanofibers, and nanotapes,^7–11 have been self-assembled using oligopeptides. Interestingly, more than one nanostructure can simultaneously be obtained from the same building blocks.⁹

Different nanostructure morphologies are desired to satisfy different applications. For example, nanoparticles are useful for drug delivery, nanotubes for biosensor fabrication and nanofibers for tissue engineering. Using processing parameters to control the morphology of self-assembling peptide nanostructures continues to be a key challenge in the field, and is encouraging very active research. To our knowledge, there are currently only a few examples of the nanostructural transformation of peptide assemblies: Song et al. observed the transition of dipeptide nanotubes into vesicles when the nanotube dispersion was diluted,¹² Yan et al. exploited this phenomenon to fabricate vesicles around 100 nm in diameter from cationic dipeptide nanotubes by dilution,¹³ and Banerjee et al. reported the pH-sensitive nanostructural transformation of a tripeptide from nanotube to nanovesicle.¹⁴ Here, we describe a new strategy to produce self-assembled nanostructures by enzymatic trigger and spatial confinement. This approach uses enzyme-responsive aromatic dipeptides as building blocks, and provides a nanoscale space for self-assembly to take place by reverse microemulsion.

We chose a 9-fluorenylmethoxycarbonyl-modified aromatic dipeptide, Fmoc-Phe-Tyr (FPT), as the self-assembling building blocks (Scheme 1). The molecular structure is similar to Fmoc-Phe-Phe, which was demonstrated to self-assemble into amyloid-like fibrils by the groups of Ulijn¹⁵ and Gazit.¹⁶ The side chain of tyrosine residue is modified with a phosphate group to render the dipeptide enzyme-responsive. This modification also reduces the hydrophobicity of FPT, allowing for higher concentrations of dipeptide in the aqueous phase. When Fmoc-Phe-Tyr-Phosphate (FPTP) was dissolved at 10 mg mL⁻¹ in water at pH 7.4, a clear solution was formed. Upon addition of alkaline phosphatase, FPTP was converted to FPT and a transparent hydrogel was obtained. This enzymatic cleavage was confirmed using mass spectrometry, which, as expected, showed a decrease of molecular weight from 630 to 550. The TEM image (Fig. 2A) of the hydrogel reveals a network of nanofibers with diameters of around 28 nm. This dimension is similar to that of a supramolecular hydrogel formed by Fmoc-tyrosine using an enzymatic trigger.¹⁷ The enzymatic reaction modifies the hydrophobic–hydrophilic balance on the designed peptide blocks, which then assemble and disassemble due to changes in the non-covalent interactions.^18,19

	Scheme 1 The chemical structures and digital photographs of two modified dipeptides illustrating the enzyme-triggered gelation process.

Naturally, FPT self-assembles into nanofibers. We explored whether nanoparticles can be formed from the same building blocks by the spatial restriction within reverse micelles. A reverse microemulsion is formed by dispersing an aqueous solution in a surfactant-containing oil phase. At thermodynamic equilibrium, the mixture produces nano-sized reverse micelles—aqueous droplets stabilized by surfactants in the oil. In recent decades, polymeric nanoparticles, inorganic nanoparticles and inorganic–organic nanocomposites have been successfully synthesized using reverse microemulsion.^20–22 In essence, these droplets act as nano-reactors or templates for the resulting nanostructures.²³ However, the technique has not been explored for controlling the supramolecular assembly of biomolecules including peptides.

We started our process by dissolving FPTP in water at 5 mg mL⁻¹. After adding alkaline phosphatase, the aqueous mixture was immediately added to a heptane phase containing 200 mg mL⁻¹ of an anionic surfactant, bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT). Brief vortexing gave a transparent single phase that corresponded to a thermodynamically stable water-in-oil (W/O) microemulsion. The molar ratio of water to AOT in the microemulsion system was 15. At this ratio, the expected diameter of the aqueous core of these nearly spherical micelles is about 6.27 nm from the equation proposed by Moulik et al.²⁴ The reverse micelles were characterized by dynamic light scattering (DLS), and an average size of 10.3 nm was found with a polydispersity of 0.075 (see Fig. S1). The size is close to that expected by considering the length of the hydrocarbon chain of AOT (1.2 nm).²⁵

Incubation of the microemulsion at 37 °C enabled the enzymatic cleavage of FPTP to FPT, which then self-assembled within reverse micelles by hydrogen bonding and – interaction.¹⁷ We believe that the supramolecular formation process of peptide molecules inside reverse micelles is not interfered with by the self-assembly of surfactant molecules. As shown by Heeres et al., self-assemblies of low molecular weight hydrogelators and of surfactants are orthogonal processes, due to the partial incompatibility of the non-covalent interactions between the two systems.²⁶ In our system, there is no electrostatic attraction between FPTP and AOT molecules, as both carry a negative charge; their self-assembly is driven by different forces.

The reverse microemulsion system was then laid aside for 3.5 h to allow the self-assembly process to continue at room temperature, resulting in the formation of dipeptide nanoparticles in the reverse micelles in the heptane phase. The diameter of these micelles was around 10.1 nm from DLS measurement (see Fig. S1), which was similar to the earlier measurement. This result indicates that peptide self-assembly is confined within the reverse micelles. To extract the peptide nanoparticles into the aqueous phase, a 1 : 1 methanol–water mixture at a two-fold volume to heptane was added. After extensive dialysis of the aqueous layer to remove AOT, a peptide nanoparticle solution was obtained. The fabrication procedure of the peptide nanoparticles is presented in Scheme 2.

	Scheme 2 Schematic representation of the peptide nanoparticle fabrication process.

An enzymatic trigger is equivalent to an on–off switch in the self-assembly process. To obtain the desired morphology, it is critical to delay self-assembly until the building blocks are localized within the templates (i.e. reverse micelles in the current process). Phosphatase and FPTP were mixed immediately before forming the microemulsion in order to distribute the reacting components evenly. The time from mixing to forming microemulsion was very short, less than 20 s. We confirmed by HPLC that the extent of enzymatic reaction within this period was insignificant, about 2.3%. This means that very few FPTP molecules were converted to FPT before being dispersed into the aqueous droplets. Therefore, the self-assembly process that accompanied the enzymatic reaction happened after the components were confined within the reverse micelles.

The molecular assemblies of FPT in hydrogel and in nanoparticles were examined using circular dichroism (CD) and fluorescence spectroscopy. As shown in Fig. 1A, the peak at 194 nm and the trough at 205 nm (both from the –* transition) indicate an -helical arrangement of the dipeptide backbone. Another peak at 230 nm (the n–* transition) further demonstrates the helical arrangement.^13,17 The dipeptide orientation induces the helical arrangement of the fluorenyl groups, which is demonstrated by induced CD in the near-UV region (Cotton effects at about 260–310 nm which correspond to the –* transitions of the fluorenyl groups).²⁷ In Fig. 1B, the peak at 228 nm indicates a helical arrangement of dipeptide molecules.¹⁷ However, the lack of a peak at 194 nm, which is present in Fig. 1A, suggests a change of the helical form in which the dipeptide molecules assemble, although it does not provide proof of any particular helical arrangement. Moreover, there are Cotton effects in the CD spectra of FPT nanoparticles in the near-UV region, corresponding to the – stacking of fluorenyl groups (Fig. 1B).²⁸ The weaker signal from FPT nanoparticles indicates a less ordered arrangement due to the restriction of the small size and spherical morphology. The peak of fluorescence emission at 322 nm and the shoulder at 375 nm of FPT hydrogel (Fig. 1C) suggest that the two fluorenes overlap in an antiparallel mode, and a small amount of parallel – stacking is present. The presence of a broad peak centered around 446 nm indicates multiple stacking of fluorenyl groups.²⁷ As shown in Fig. 1D, although a red-shift from 310 nm in the FPTP solution to around 315 nm for the FPT nanoparticles is not significant, a small broad peak centered at 418 nm supports the stacking of fluorenyl moieties within FPT nanoparticles, albeit less efficiently than in the FPT hydrogel.

	Fig. 1 CD spectra of FPT hydrogel (A) and the nanoparticle solution (B). Fluorescence spectra of FPT hydrogel (C), FPT nanoparticle and FPTP solution (D).

	Fig. 2 TEM images of FPT hydrogel (10 mg mL−1) composed of nanofibers (A); dilute FPT solution (10 g mL−1) forming dendritic structures (B); FPT nanoparticle solution immediately after fabrication (C), after 2 months (D) and after 9 months (E); and the other batch of nanoparticle solution under higher magnification (F).

Fig. 2C shows the TEM image of nanoparticles obtained after dialysis. These nanoparticles have a size around 6.5 nm, and are not spherical—this distortion may be a drying artifact due to the property of soft matter. A simple molecular model for the possible peptide arrangement within a reverse micelle and the subsequent formation of a nanoparticle are presented in Fig. 3. Hydroxyl groups and carboxyl groups from tyrosine residues of dipeptide molecules are in close proximity to the hydrophilic heads of surfactants, forming hydrogen bonds. The aromatic side chains of dipeptide molecules overlap with each other and form helical – stacks, resulting in a 3D spherical structure. The fluorenyl groups also stack with each other, giving rise to a helical arrangement; some are stacked in a parallel mode and others in an antiparallel mode. From the model, the – stacking needs to be twisted to fit the 3D spherical morphology. After removal of surfactants, the peptide nanoparticle remains in the same molecular arrangement, except that the hydrogen bonds on the surface of peptide nanoparticles are formed by FPT molecules and water instead of surfactant molecules.

	Fig. 3 A possible molecular model of the cross-sectional structure of a peptide nanoparticle. (A) Peptide nanoparticle formed in a reverse micelle. (B) Peptide nanoparticle in the aqueous phase. Size representation: AOT, 1.2 nm; FPT, 1.0 nm; peptide nanoparticle: 7.0 nm. Water molecules are omitted.

When the 10 mg mL⁻¹ FPT hydrogel was diluted to low concentration, close to that of the FPT nanoparticle aqueous solution, dendritic structures of micron-scale length were seen in the TEM images (Fig. 2B). The different structures obtained by the reverse microemulsion self-assembly method and direct dilution illustrate the nanostructural controlling ability of the reverse-micelle method. Similar peptide nanoparticles were found in other batches (see Fig. S2), demonstrating the reproducibility of our method. As shown in Fig. 2D, nanoparticles could still be observed by TEM two months after fabrication, although there was significant aggregation. After four months, however, nanotubes of around 38 nm were found in the solution, and these became the majority after nine months (Fig. 2E). These structures are different from the nanofibers that make up the FPT hydrogel (Fig. 2A). Interestingly, some nanotubes with 7.0 nm nanoparticles attached were found under higher magnification in another particle solution (Fig. 2F).

We propose a schematic illustration for a possible nanostructural transformation from peptide nanoparticles to a peptide nanotube (see Fig. S3). We speculate that nanoparticles are first bound together by hydrogen bonds to form a one-shell column, and then the interior dipeptide molecules rearrange, increasing the entropy by forming a nanotube from a confined spherical morphology, and decreasing the enthalpy by forming more efficient – stacks and hydrogen bonds. The transformation indicates that the peptide nanoparticles are in a metastable state, consistent with the less ordered stacking indicated by CD and fluorescence spectra.

We have shown for the first time a novel reverse microemulsion self-assembly method for the fabrication of peptide nanoparticles. The self-assembly process is controlled with an enzymatic trigger and confined within a nanospace. These new particles are potentially useful for applications in drug delivery. The approach that we have demonstrated could be applied to other stimuli-responsive self-assembly blocks, as well as providing a means to study the self-assembly process.

Footnote

Electronic supplementary information (ESI) available: Experimental details of nanoparticle fabrication, DLS results of reverse micelles, TEM images of other FPT nanoparticle solutions, and schematic illustration of nanostructural transformation. See DOI: 10.1039/b801890a