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Although peptides notoriously have poor intrinsic pharmacokinetic properties, it is well-known that nanostructures with excellent pharmacokinetic properties can be designed. Noticing that peptide inhibitors are generally nonpolar, here, we consolidate the peptide inhibitor targeting intracellular protein-protein interactions (PPIs) as an integral part of biodegradable self-assembled depsipeptide nanostructures (SdPNs). Because the peptide inhibitor has the dual role of PPI inhibition and self-assembly in this design, problems associated with the poor pharmacokinetics of peptides and encapsulation/entrapment processes can be overcome. Optimized SdPNs displayed better tumor targeting and PPI inhibition properties than the comparable small molecule inhibitor in vivo. Kinetics of PPI inhibition for SdPNs were gradual and controllable in contrast to the rapid inhibition kinetics of the small molecule. Because SdPN is modular, any appropriate peptide inhibitor can be incorporated into the platform without concern for the poor pharmacokinetic properties of the peptide., Competing Interests: Declaration of Competing Interest Y.B.L. and H.H.S. have filed a patent for the development of the SdPN platform. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yong-beom Lim has patent pending to Yonsei University. Hyun-seok Hwang has patent pending to Yonsei University., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The quaternary structure of proteins extends the functionality of monomeric proteins. Similarly, self-assembled protein nanostructures (SPrNs) have great potential to improve the functionality and complexity of proteins; however, the difficulty associated with the fabrication of SPrNs is far greater than that associated with the fabrication of self-assembled peptides or polymers and often requires sophisticated computational design. To make the process of SPrN formation simpler and more intuitive, herein, we devise a strategy to adopt an irreversible self-assembled peptide nanostructure (SPeN) process en route to the formation of SPrNs. The strategy employs three sequential steps: first, the formation of SPeNs (an equilibrium process); second, covalent capture of SPeNs (an irreversible process); third, the final assembly of SPrNs via protein-peptide interactions (an equilibrium process). This strategy allowed us to fabricate SPrNs in which the size of the protein was approximately 9 times higher than that of the self-assembling peptide. Furthermore, we demonstrated that the irreversible SPeN could be used as a primary building block for assembly into superstructures. Overall, this strategy is conceptually as simple as SPeN fabrication and is potentially applicable to any soluble protein.

Self-assembling peptides are biomedical materials with unique structures that are formed in response to various environmental conditions. Governed by their physicochemical characteristics, the peptides can form a variety of structures with greater reactivity than conventional non-biological materials. The structural divergence of self-assembling peptides allows for various functional possibilities; when assembled, they can be used as scaffolds for cell and tissue regeneration, and vehicles for drug delivery, conferring controlled release, stability, and targeting, and avoiding side effects of drugs. These peptides can also be used as drugs themselves. In this review, we describe the basic structure and characteristics of self-assembling peptides and the various factors that affect the formation of peptide-based structures. We also summarize the applications of self-assembling peptides in the treatment of various diseases, including cancer. Furthermore, the in-cell self-assembly of peptides, termed reverse self-assembly, is discussed as a novel paradigm for self-assembling peptide-based nanovehicles and nanomedicines.

The interactions between graphene oxide (GO) and various biological entities have been actively investigated in recent years, resulting in numerous potential bioapplications of these nanomaterials. Despite this, the biological interactions between GO and disease-causing protozoan parasites have not been well elucidated and remain relatively unexplored. Here, we investigate the in vitro interactions between GO nanosheets and a particular species of malaria parasites, Plasmodium falciparum (P. falciparum). We hypothesize that GO nanosheets may exhibit antimalarial characteristic via action mechanisms of physical obstruction of P. falciparum parasites as well as nutrient depletion. To ascertain this, we characterize the physical interactions between GO nanosheets, red blood cells (RBCs), and malarial parasites as well as the adsorption of several biomolecules necessary for parasitic survival and growth on GO nanosheets. Subsequent to establishing the origin of this antimalarial behavior of GO nanosheets, their efficiency in inhibiting parasite invasion is evaluated. We observe that GO nanosheets at various tested concentrations significantly inhibit the invasion of malaria parasites into RBCs. Furthermore, GO nanosheets delay parasite progression from the ring to the trophozoite stage. Overall, this study may further shed light on the graphene-parasite interactions and potentially facilitate the development of nanomaterial-based strategies for combating malaria.

The first step in the conventional approach to self-assembled biomaterials is to develop well-defined nanostructures in vitro, which is followed by disruption of the preformed nanostructures at the inside of the cell to achieve bioactivity. Here, we propose an inverse strategy to develop in-cell gain-of-function self-assembled nanostructures. In this approach, the supramolecular building blocks exist in a unimolecular/unordered state in vitro or at the outside of the cell and assemble into well-defined nanostructures after cell internalization. We used block copolypeptides of an oligoarginine and a self-assembling peptide as building blocks and investigated correlations among the nanostructural state, antiprion bioactivity, and cytotoxicity. The optimal bioactivity (i.e., the highest antiprion activity and lowest cytotoxicity) was obtained when the building blocks existed in a unimolecular/unordered state in vitro and during the cell internalization process, exerting minimal cytotoxic damage to cell membranes, and were subsequently converted into high-charge-density vesicles in the low pH endosome/lysosomes in vivo, thus, resulting in the significantly enhanced antiprion activity. In particular, the in-cell self-assembly concept presents a feasible approach to developing therapeutics against protein misfolding diseases. In general, the in-cell self-assembly provides a novel inverse methodology to supramolecular bionanomaterials.

Although self-assembled peptide nanostructures (SPNs) have shown potential as promising biomaterials, there is a potential problem associated with the extremely slow hydrolysis rate of amide bonds. Here, we report the development of cell-penetrating cross-β SPNs with a controllable biodegradation rate. The designed self-assembling β-sheet peptide incorporating a hydrolyzable ester bond (self-assembling depsipeptide; SADP) can be assembled into bilayer β-sandwich one-dimensional (1D) fibers similarly to conventional β-sheet peptides. The rate of hydrolysis can be controlled by the pH, temperature, and structural characteristics of the ester unit. The 1D fiber of the SADP transforms into vesicle-like 3D structures when the hydrophilic cell-penetrating peptide segment is attached to the SADP segment. Efficient cell internalization of the 3D nanostructures was observed, and we verified the intracellular degradation and disassembly of the biodegradable nanostructures. This study illustrates the potential of biodegradable cross-β SPNs and provides a valuable toolkit that can be used with self-assembling peptides.

To overcome the limitations of molecular assemblies, the development of novel supramolecular building blocks and self-assembly modes is essential to create more sophisticated, complex, and controllable aggregates. The self-assembly of peptide-DNA conjugates (PDCs), in which two orthogonal self-assembly modes, that is, β-sheet formation and Watson-Crick base pairing, are covalently combined in one supramolecular system, is reported. Despite extensive research, most self-assembly studies have focused on using only one type of building block, which restricts structural and functional diversity compared to multicomponent systems. Multicomponent systems, however, suffer from poor control of self-assembly behaviors. Covalently conjugated PDC building blocks are shown to assemble into well-defined and controllable nanostructures. This controllability likely results from the decrease in entropy associated with the restriction of the molecular degrees of freedom by the covalent constraints. Using this strategy, the possibility to thermodynamically program nano-assemblies to exert gene regulation activity with low cytotoxicity is demonstrated., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although there has been substantial advancement in the development of nanostructures, the development of self-assembled nanostructures that can selectively recognize multivalent targets has been very difficult. Here we show the proof of concept that topology-controlled peptide nanoassemblies can selectively recognize and detect a multivalent RNA target. We compared the differential behaviors of peptides in a linear or cyclic topology in terms of peptide-gold nanoparticle hybrid nanostructure formation, conformational stabilization, monovalent and multivalent RNA binding in vitro, and multivalent RNA recognition in live cells. When the topology-dependent selectivity amplification of the cyclic peptide hybrids is combined with the noninvasive nature of dark-field microscopy, the cellular localization of the viral Rev response element (RRE) RNA can be monitored in situ. Because intracellular interactions are often mediated by overlapping binding partners with weak affinity, the topology-controlled peptide assemblies can provide a versatile means to convert weak ligands into multivalent ligands with high affinity and selectivity.

Self-assembled peptide nanostructures with actively folded secondary structures have potential to mimic the function of proteins. We here show that α-helix-stabilized self-assembled peptide nanostructures (αSSPNs), whose sizes are comparable to those of proteins, have potential to be developed as protein-protein interaction (PPI) inhibitors along with several unprecedented properties. Using p53-MDM2 PPI as a model system, the molecular recognition and modulation of PPIs by αSSPN grafted with a p53 α-helix (p53 αSSPN) were investigated. The competition assay showed that the p53 αSSPN can inhibit the p53-MDM2 interaction. Interestingly, the p53 αSSPN was far more resistant to degradation by the protease chymotrypsin than the monomeric p53 peptide and had high thermal stability. These results suggest that the αSSPN scaffold holds great potential to be developed as a novel class of PPI inhibitors.

Understanding the dynamic behavior of nanostructural systems is important during the development of controllable and tailor-made nanomaterials. This is particularly true for nanostructures that are intended for biological applications because biomolecules are usually highly dynamic and responsive to external stimuli. In this Article, we investigated the structural and conformational dynamics of self-assembling bioactive β-sheet peptide nanostructures using electron paramagnetic resonance (EPR) spectroscopy. The model peptide nanostructures are characterized by the cross-β spine of β-ribbon fibers and multiple RNA-binding bioactive peptides that constitute the shell of the nanostructures. We found first, that bioactive peptides at the shell of β-ribbon nanostructure have a mobility similar to that of an isolated monomeric peptide. Second, the periphery of the cross-β spine is more immobile than the distal part of surface-displayed bioactive peptides. Third, the rotational dynamics of short and long fibrils are similar; that is, the mobility is largely independent of the extent of aggregation. Fourth, peptides that constitute the shell are affected first by the external environment at the initial stage. The cross-β spine resists its external environment to a certain extent and abruptly disintegrates when the perturbation reaches a certain degree. Our results provide an overall picture of β-sheet peptide nanostructure dynamics, which should be useful in the development of dynamic self-assembled peptide nanostructures.

β-Barrel proteins that take the shape of a ring are common in many types of water-soluble enzymes and water-insoluble transmembrane pore-forming proteins. Since β-barrel proteins perform diverse functions in the cell, it would be a great step towards developing artificial proteins if we can control the polarity of artificial β-barrel proteins at will. Here, we describe a rational approach to construct β-barrel protein mimics from the self-assembly of peptide-based building blocks. With this approach, the direction of the self-assembly process toward the formation of water-soluble β-barrel nanorings or water-insoluble transmembrane β-barrel pores could be controlled by the simple but versatile molecular manipulation of supramolecular building blocks. This study not only delineates the basic driving force that underlies the folding of β-barrel proteins, but also lays the foundation for the facile fabrication of β-barrel protein mimics, which can be developed as nanoreactors, ion- and small-molecule-selective pores, and novel antibiotics., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We demonstrate here that rationally designed block copolypeptides based on poly(L-arginine) and β-sheet peptides can form toroidal nanostructures. In aqueous solution, we found that 1D nanoribbons roll up and connect in an end-to-end fashion under charge-balanced conditions, resulting in the formation of barrel-like toroidal nanostructures. Toroidal diameter was highly uniform (10 nm), indicating that there is a preferred geometrical packing requirement for toroid formation. Our results demonstrate that, when suitably designed, β-sheet nanostructures can be manipulated to form more complex 2D nanostructures. This finding offers new opportunities not only for the fabrication of more sophisticated peptide-based nanobiomaterials, but also for understanding and inhibiting protein misfolding diseases., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Importance of the Field: Among many nanostructural shapes, filamentous shapes can have unique advantages over the others, including longer persistence in the bloodstream. With the advent of nanotechnology in recent years, a variety of shape-controlled nanostructures has been fabricated. As a wide variety of building blocks can self-assemble into filamentous nanostructures, there are many options available for biomaterial developments with filamentous nanostructures. Similarly to conventional spherical micelles, most filamentous nanostructures have hydrophobic cores where hydrophobic guest molecules can be encapsulated, which enable them to be used in drug delivery applications. Moreover, on suitable molecular design and self-assembly process control, filamentous nanostructures can also be made to deliver nucleic acids or even both drugs and nucleic acids simultaneously., Areas Covered in This Review: This review describes the self-assembly process, current developments, and prospects of filamentous nanostructures in drug and gene delivery applications., What the Reader Will Gain: This review should be helpful in gaining insight into the self-assembly process and nanostructural shape control, the advantages of constructing filamentous nanostructures, and the design of more advanced nanobiomaterials., Take Home Message: At present, the development of multifunctional nanostructures is one of the main focuses in nanobiomaterial developments. In this regard, filamentous nanostructures can be good initial targets for tailor-made nanostructure developments because they have more structural variables, as compared with spherical micelles.

Supramolecular nanostructures covered with bioactive functional molecules have been actively explored as promising materials in the field of biotechnology. Recent advances in nano-sized chemistry have made it possible to fabricate various kinds of nanostructures with tailor-made nanostructural properties. This, combined with appropriate bioactive functionalization, has led to the successful utilization of supramolecular nanostructures in diverse biomaterials applications. This tutorial review describes the concept, current developments, and prospects of self-assembled bioactive nanostructures, which are assembled directly from bioactive supramolecular building blocks.

We have demonstrated that polymerizable peptides self-assemble into a unique sheet-like 2D structure in bulk solution that can be covalently fixed to produce 2D molecular objects which were shown to be efficient at delivering cargos into living cells and are nearly nontoxic in contrast to non-polymerized nanostructures.

We have synthesized a peptide-based supramolecular building block consisting of a cyclic Arg-Gly-Asp (cRGD) peptide segment and a beta-sheet-forming peptide segment. The block peptide was shown to self-assemble into a cRGD-coated nanoribbon structure, as revealed by circular dichroism (CD), dynamic light scattering (DLS), and transmission electron microscopy (TEM) studies. We have shown that this cRGD-coated nanoribbon can encapsulate hydrophobic guest molecules and deliver them into cells. Colocalization of the nanoribbon with LysoTracker and the selective intracellular delivery results suggests that the cRGD-coated nanoribbon is likely to be internalized into the cells through integrin receptors.

We explored a method of controlling bacterial motility and agglutination by using self-assembled carbohydrate-coated beta-sheet nanoribbons. To this aim, we synthesized triblock peptides that consist of a carbohydrate, a polyethylene glycol (PEG) spacer, and a beta-sheet-forming peptide. An investigation into the effect of PEG-spacer length on the self-assembly of the triblock peptides showed that the PEG should be of sufficiently length to stabilize the beta-sheet nanoribbon structure. It was found that the stabilization of the nanoribbon led to stronger activity in bacterial motility inhibition and agglutination, thus suggesting that antibacterial activity can be controlled by the stabilization strategy. Furthermore, another level of control over bacterial motility and agglutination was attained by co-assembly of bacteria-specific and -nonspecific supramolecular building blocks. The nanoribbon specifically detected bacteria after the encapsulation of a fluorescent probe. Moreover, the detection sensitivity was enhanced by the formation of bacterial clusters. All these results suggest that the carbohydrate-coated beta-sheet nanoribbons can be developed as promising agents for pathogen capture, inactivation, and detection, and that the activity can be controlled at will.

We demonstrate here the rational design strategy to control the length of 1-dimensional beta-sheet peptide nanoassembly. We synthesized the beta-sheet peptides with attached coils and carbohydrates. We reasoned that the bulkiness of the coils affects the final length of the assembled beta-sheet peptide nanostructures because of the steric crowding effect. The nanostructure from the peptide with a small and linear coil was several micrometers long, whereas the one from the peptide with a high-volume-fraction dendritic coil was only about 150 nm long. For potential biological applications of the peptide nanoassemblies, we investigated the interactions of the carbohydrate-coated nanoassemblies with E. coli cells containing cognate binding proteins. The results showed that both of the nanoassemblies could immobilize and/or aggregate bacterial cells. The degrees of immobilization were similar for both nanoassemblies; however, only the long nanoribbon was shown to induce the formation of bacterial clusters.

Triblock rigid-flexible dendritic block molecules consisting of a rigid aromatic segment as a stem segment, carbohydrate-branched dendrons as a flexible head, and a hydrophobic alkyl chain were synthesized and characterized. The carbohydrate conjugate molecule based on a methyl group as a hydrophobic tail, in the solid state, self-assembles into a 1D nanostructure, whereas the molecule based on a longer hydrophobic tail self-assembles into 2D nanosheets, as confirmed by X-ray scatterings. In aqueous solution, however, both molecules were observed to self-assemble into carbohydrate-coated cylindrical aggregates with a uniform diameter, as confirmed by dynamic light scatterings and transmission electron microscopic (TEM) investigations. Notably, these cylindrical objects reversibly transformed into spherical objects on addition of guest molecules. Investigation of the interactions of the carbohydrate-coated nanostructures with E. coli cells showed that both nano-objects could immobilize bacterial cells, while the degrees of immobilization were significantly dependent on the shape of nanostructure. These results demonstrated that the supramolecular materials that are responsive to external stimuli can provide novel opportunities to control many biological activities.

Materials that display multiple carbohydrate residues have gained much attention due to their potential to inhibit or modulate biological multivalent interactions. These materials can be grouped accordingly to the way they are prepared, as unimolecular or as self-assembled systems. Both systems take advantage of the fact that multivalent interactions have significantly higher binding affinity than the corresponding monovalent interactions. The self-assembled system is a more recent field of research compared to the unimolecular system. In this review, we describe current efforts to realize multivalent carbohydrate interactions from the perspective of synthetic self-assembled systems. We limit the scope to self-assembled systems that are stable, soluble in aqueous solution and morphologically discrete. We grouped them into two separate categories. In the first category carbohydrate ligands self-assemble onto a pre-organized nanostructure, and in the second carbohydrate-conjugated block molecules spontaneously assemble to construct morphologically distinct nanostructures.