Honggang Cui

Self-Assembly of Peptide Amphiphiles: From Molecules to Nanostructures to Biomaterials

Peptide amphiphiles are a class of molecules that combine the structural features of amphiphilic surfactants with the functions of bioactive peptides and are known to assemble into a variety of nanostructures. A specific type of peptide amphiphiles are known to self‐assemble into one‐dimensional nanostructures under physiological conditions, predominantly nanofibers with a cylindrical geometry. The resultant nanostructures could be highly bioactive and are of great interest in many biomedical applications, including tissue engineering, regenerative medicine, and drug delivery. In this context, we highlight our strategies for using molecular self‐assembly as a toolbox to produce peptide amphiphile nanostructures and materials and efforts to translate this technology into applications as therapeutics. We also review our recent progress in using these materials for treating spinal cord injury, inducing angiogenesis, and for hard tissue regeneration and replacement.

Supramolecular nanostructures formed by anticancer drug assembly.

We report here a supramolecular strategy to directly assemble the small molecular hydrophobic anticancer drug camptothecin (CPT) into discrete, stable, well-defined nanostructures with a high and quantitative drug loading. Depending on the number of CPTs in the molecular design, the resulting nanostructures can be either nanofibers or nanotubes, and have a fixed CPT loading content ranging from 23% to 38%. We found that formation of nanostructures provides protection for both the CPT drug and the biodegradable linker from the external environment and thus offers a mechanism for controlled release of CPT. Under tumor-relevant conditions, these drug nanostructures can release the bioactive form of CPT and show in vitro efficacy against a number of cancer cell lines. This strategy can be extended to construct nanostructures of other types of anticancer drugs and thus presents new opportunities for the development of self-delivering drugs for cancer therapeutics.

Tuning Supramolecular Rigidity of Peptide Fibers through Molecular Structure

We synthesized a series of peptide amphiphiles (PAs) with systematically modified amino acid sequences to control the mechanical properties of the nanofiber gels they form by self-assembly. By manipulating the number and position of valines and alanines in the peptide sequence, we found that valines increase the stiffness of the gel, while additional alanines decrease the mechanical properties. Vitreous ice cryo-transmission electron microscopy shows that all PA molecules investigated here form nanofibers 8-10 nm in diameter and several micrometers in length. We found through Fourier transform IR experiments a strong correlation between gel stiffness and hydrogen bond alignment along the long axis of the fiber. Molecules that form supramolecular structures with the highest mechanical stiffness were found by circular dichroism to self-assemble into beta-sheets with the least amount of twisting and disorder, a result that is consistent with IR experiments. Molecular control of mechanical stiffness in three-dimensional artificial peptide amphiphile matrices offers a chemical strategy to control biological phenomena such as stem cell differentiation and cell morphology.

Light‐Triggered Bioactivity in Three Dimensions

Hydrogelators that undergo stimuli-responsive sol–gel transitions have attracted attention as biomaterials because of their potential applications, for example as sophisticated cell culture substrates, drug carriers, microvalves, and microactuators. 2] Among the various stimuli possible, light is unique because it allows both spatial and temporal control of a specific reaction without requiring physical contact. Many photochemical sol–gel transitions have been reported, mostly in polymers but also in low-molecular-weight peptides. 3] The importance of peptide systems is their potential biocompatibility and the opportunity for researchers to molecularly design bioactive functions. The advent of two-dimensional self-assembling systems and patterning technologies has generated some examples of stimulus-driven bioactivity on surfaces. Using a peptide amphiphile (PA) containing the fibronectin epitope Arg-Gly-Asp-Ser (RGDS) for cell adhesion, we demonstrate here light-triggered enhancement of bioactivity in a three-dimensional system. PAs are known as highly versatile molecules that selfassemble into nanostructures such as spherical micelles, fibers, and helices through hydrogen-bond formation and hydrophobic collapse. 5, 8–10] Our laboratory first reported on PAs that form high-aspect-ratio nanofibers and therefore gels at very low concentrations. These specific PAs self-assemble into nanofibers as a result of their b-sheet peptide domains, and their self-assembly can be triggered by charge screening through changes in pH or the addition of salts. Molecular changes in the b-sheet domains can disrupt nanofiber formation. For example, Hartgerink and co-workers reported the formation of spherical micelles and not fibers as a result of the N-methylation on the amide nitrogen closest to the alkyl tail of a PA. Our laboratory also reported recently the formation of quadruple helices in PAs containing a photolabile 2-nitrobenzyl group in the b-sheet domain. These quadruple helices in turn dissociate into single nonhelical fibrils in response to light. In this work, we have synthesized PA molecule 1 containing both the photocleavable 2-nitrobenzyl group as well as the bioactive epitope Arg-Gly-Asp-Ser (RGDS) (Scheme 1). Based on the previous work, photoirradiation

Quadruple Helix Formation of a Photoresponsive Peptide Amphiphile and Its Light-Triggered Dissociation into Single Fibers

Using a peptide amphiphile having a bulky photolabile 2-nitrobenzyl group between the alkyl chain and the peptide segment, we demonstrated quadruple helical fiber formation and its dissociation into single fibrils in response to light. Putting the bulky group close to the core of a fibril is thought to induce a distortion of the alignment of molecules, which can in turn lead to quadruple helices. Photoirradiation to cleave the bulky group transforms the helices into single fibrils.

The Role of Micelle Size in Tumor Accumulation, Penetration, and Treatment

The specific sizes that determine optimal nanoparticle tumor accumulation, penetration, and treatment remain inconclusive because many studies compared nanoparticles with multiple physicochemical variables (e.g., chemical structures, shapes, and other physical properties) in addition to the size. In this study, we synthesized amphiphilic block copolymers of 7-ethyl-10-hydroxylcamptothecin (SN38) prodrug and fabricated micelles with sizes ranging from 20 to 300 nm from a single copolymer. The as-prepared micelles had exactly the same chemical structures and similar physical properties except for size, which provided an ideal platform for a systematic investigation of the size effects in cancer drug delivery. We found that the micelles blood circulation time and tumor accumulation increased with the increase in their diameters, with optimal diameter range of 100 to 160 nm. However, the much higher tumor accumulation of the large micelles (100 nm) did not result in significantly improved therapeutic efficacy, because the large micelles had poorer tumor penetration than the small ones (30 nm). An optimal size that balances drug accumulation and penetration in tumors is critical for improving the therapeutic efficacy of nanoparticulate drugs.

Kinetic quantification of protein polymer nanoparticles using non-invasive imaging

Protein polymers are repetitive amino acid sequences that can assemble monodisperse nanoparticles with potential applications as cancer nanomedicines. Of the currently available molecular imaging methods, positron emission tomography (PET) is the most sensitive and quantitative; therefore, this work explores microPET imaging to track protein polymer nanoparticles over several days. To achieve reliable imaging, the polypeptides were modified by site-specific conjugation using a heterobifunctional sarcophagine chelator, AmBaSar, which was subsequently complexed with (64)Cu. AmBaSar/(64)Cu was selected because it can label particles in vivo over periods of days, which is consistent with the timescales required to follow long-circulating nanotherapeutics. Using an orthotopic model of breast cancer, we observed four elastin-like polypeptides (ELPs)-based protein polymers of varying molecular weight, amino acid sequence, and nanostructure. To analyze this data, we developed a six-compartment image-driven pharmacokinetic model capable of describing their distribution within individual subjects. Surprisingly, the assembly of an ELP block copolymer (78 kD) into nanoparticles (R(h) = 37.5 nm) minimally influences pharmacokinetics or tumor accumulation compared to a free ELP of similar length (74 kD). Instead, ELP molecular weight is the most important factor controlling the fate of these polymers, whereby long ELPs (74 kD) have a heart activity half-life of 8.7 hours and short ELPs (37 kD) have a half-life of 2.1 hours. These results suggest that ELP-based protein polymers may be a viable platform for the development of multifunctional therapeutic nanoparticles that can be imaged using clinical PET scanners.

Helix self-assembly through the coiling of cylindrical micelles

Both single and double helical superstructures have been created through solution self-assembly of cylindrical micelles for the first time. Helical micelles were formed from the co-assembly of poly(acrylic acid)-block-poly(methyl acrylate)-block-polystyrene (PAA-b-PMA-b-PS) triblock copolymers with different multiamines. The helix pitch could be adjusted by adjusting the amount and type of multiamine added. The helical structure exhibits unprecedented regularity for a nanostructure self-assembled from solution indicating the presence of strong, synergistic forces underlying the helix formation.

Spontaneous and x-ray-triggered crystallization at long range in self-assembling filament networks.

X-rays to Order Self-assembly of molecules is often irreversible. Cui et al. (p. 555, published online 17 December; see the Perspective by Safinya and Li) examined the ordering of a short peptide sequence (Ala6Glu3) terminated with an alkyl chain. Aqueous solutions of this molecule could form hexagonally ordered filaments, but more dilute solutions were disordered. However, prolonged x-ray exposure caused these arrays to become ordered. These arrays were stable for several hours but eventually returned to a disordered state; the addition of salts slowed the ordering processes. It is possible that during the ordering process, X-ray–induced charging affected the repulsive forces that balance tension within the filament. Dilute solutions of alkyl-terminated peptide filaments can undergo ordering upon x-ray exposure. We report here crystallization at long range in networks of like-charge supramolecular peptide filaments mediated by repulsive forces. The crystallization is spontaneous beyond a given concentration of the molecules that form the filaments but can be triggered by x-rays at lower concentrations. The crystalline domains formed by x-ray irradiation, with interfilament separations of up to 320 angstroms, can be stable for hours after the beam is turned off, and ions that screen charges on the filaments suppress ordering. We hypothesize that the stability of crystalline domains emerges from a balance of repulsive tensions linked to native or x-ray–induced charges and the mechanical compressive entrapment of filaments within a network. Similar phenomena may occur naturally in the cytoskeleton of cells and, if induced externally in biological or artificial systems, lead to possible biomedical and lithographic functions.

Self-Assembled Tat Nanofibers as Effective Drug Carrier and Transporter

Cell penetrating peptides (CPPs) have been extensively explored as molecular vectors through covalent linkage to anticancer drugs to improve the drugs water solubility and to help overcome multidrug resistance. We report here the use of the Tat CPP as a molecular building unit to construct well-defined supramolecular nanofibers that can be utilized as a nanoscale vector to encapsulate the hydrophobic drug paclitaxel (PTX) (loading efficiency: 89.7 ± 5.0%) with a high loading capacity (6.8 ± 0.4%). Notably, our TEM imaging results reveal that nanofibers containing a higher PTX content tend to be more flexible than those with a lower PTX content. Fluorescence and confocal microscopy imaging show that the Tat nanofibers can effectively transport encapsulated molecules into the cells through an adsorptive-mediated endocytosis pathway. Cytotoxicity experiments and flow cytometry measurements demonstrate that PTX loaded in the nanofibers exerts its cytotoxicity against cancer cells by arresting the cells at the G2/M phase, the same working mechanism as free PTX.