Nucleation and Growth of Amino Acid and Peptide Supramolecular Polymers through Liquid-Liquid Phase Separation.

The transition of peptides and proteins from the solution phase into fibrillar structures is a general phenomenon encountered in functional and aberrant biology and is increasingly exploited in soft materials science. However, the fundamental molecular events underpinning the early stages of their assembly and subsequent growth have remained challenging to elucidate. Here, we show that liquid-liquid phase separation into solute-rich and solute-poor phases is a fundamental step leading to the nucleation of supramolecular nanofibrils from molecular building blocks, including peptides and even amphiphilic amino acids. The solute-rich liquid droplets act as nucleation sites, allowing the formation of thermodynamically favorable nanofibrils following Ostwald's step rule. The transition from solution to liquid droplets is entropy driven while the transition from liquid droplets to nanofibrils is mediated by enthalpic interactions and characterized by structural reorganization. These findings shed light on how the nucleation barrier toward the formation of solid phases can be lowered through a kinetic mechanism which proceeds through a metastable liquid phase.

Multiscale simulations for understanding the evolution and mechanism of hierarchical peptide self-assembly.

Hierarchical self-assembly, abundant in biological systems, has been explored as an effective bottom-up method to fabricate highly ordered functional superstructures from elemental building units. Biomolecules, especially short peptides consisting of several amino acids, are a type of elegant building blocks due to their advantages of structural, mechanical, and functional diversity as well as high biocompatibility and biodegradability. The hierarchical self-assembly of peptides is a spontaneous process spanning multiple time and length scales under certain thermodynamics and kinetics conditions. Therefore, understanding the mechanisms of dynamic processes is crucial to directing the construction of complicated biomimetic systems with multiple functionalities. Multiscale molecular simulations that combine and systematically link several hierarchies can provide insights into the evolution and dynamics of hierarchical self-assembly from the molecular level to the mesoscale. Herein, we provided an overview of the simulation hierarchies in the general field of peptide self-assembly modeling. In particular, we highlighted multiscale simulations for unraveling the mechanisms underlying the dynamic self-assembly process with an emphasis on weak intermolecular interactions in the process stages and the energies of different molecular alignments as well as the role of thermodynamic and kinetic factors at the microscopic level.

Nanoengineering of stimuli-responsive protein-based biomimetic protocells as versatile drug delivery tools.

We present a general strategy to nanoengineer protein-based colloidal spheres (biomimetic protocells) as versatile delivery carriers with stimuli responsiveness by the electrostatic assembly of binary components (proteins and polypeptides) in association with intermolecular disulfide cross-linking. The size of the colloidal spheres, ranging from nanoscale to microscale, is readily tuned through parameters like protein and polypeptide concentration, the ratio between both, pH, and so on. Moreover, such colloidal spheres show versatile encapsulation of various guest molecules including small organic molecules and biomacromolecules. The pH and redox dual-responsiveness facilitates the rapid release of the payload in an acidic and reductant-enriched ambient such as in lysosomes. Thus, nanoengineering of protein-based biomimetic protocells opens a new alternative avenue for developing delivery vehicles with multifunctional properties towards a range of therapeutic and diagnostic applications.

Novel active stealth micelles based on ß2M achieved effective antitumor therapy.

Micelles have been extensively investigated as drug delivery systems for loading of antitumor drugs with the advantages of good dispersibility, controllable size distribution, and high loading capacity. However, phagocytic clearance by the mononuclear phagocyte system remains a major impediment that inhibits blood circulation and thus tumor accumulation of micelles. Inspired by the antiphagocytic properties of ß2-microglobulin (ß2M), here we developed a ß2M-derived peptide for the surface functionalization of micelles. A ß2M-derived sequence was integrated with a matrix metalloproteinase-2 (MMP-2) cleavable sequence and then modified on the surface of poly(ethylene glycol)-b-poly(caprolactone) (PEG-PCL) micelles, endowing the micelles with both antiphagocytic and MMP-2-responsive properties. Compared to pristine PEG-PCL micelles, micelles modified with the dual-functional peptide exhibited higher inhibition of phagocytosis by macrophages in the absence of MMP-2, and enhanced internalization by tumor-associated macrophages in the presence of MMP-2, leading to enhanced tumor accumulation of the loaded photosensitizer, thus enabling antitumor therapy. These results demonstrated that antiphagocytic peptides derived from endogenous proteins are promising for functionalization of micelles in smart drug delivery.

Polyethylene glycol-functionalized benzylidene cyclopentanone dyes for two-photon excited photodynamic therapy.

A series of polyethylene glycol-functionalized benzylidene cyclopentanone dyes with varying lipid/water partition coefficients were synthesized in high yields by a simple process. Detailed characterization and systematic studies of these molecules, including linear and nonlinear photophysical properties, reactive oxygen yields, and in vitro photodynamic therapy (PDT) activities, were conducted. Four of these dyes exhibited good solubility in PBS (>2 mg ml(-1), which is sufficient for clinical venous injection), high reactive oxygen yields, large two-photon absorption and low dark toxicity, under the therapy dosage. Among them, two dyes could be absorbed efficiently by human rectal cancer 1116 cells, and presented strong two-photon excited PDT activity in in vitro cell experiments.

Water-soluble benzylidene cyclopentanone based photosensitizers for in vitro and in vivo antimicrobial photodynamic therapy.

Antimicrobial photodynamic therapy (aPDT) has been proposed to cope with the increasing antibiotic resistance among pathogens. As versatile pharmacophores, benzylidene cyclopentanone based photosensitizers (PSs) have been used in various bioactive materials. However, their reports as aPDT agents are very limited, and relationships between their chemical structures and antibacterial abilities have not been systematically discussed. Here, nine water-soluble benzylidene cyclopentanone PSs modified by polyethylene glycol (PEG), carboxylate anionic or pyridyl cationic agents are studied for aPDT. It is found that the binding/uptake abilities and aPDT effects of these PSs toward bacterial cells vary significantly when adjusting the number and position of their terminal charged groups. Though the comparable (also best) binding/uptake amounts are achieved by both cationic PS P3 and anionic PS Y1, only Y1 exhibits much more excellent aPDT activities than other PSs. Antibacterial mechanisms reveal that, relative to the favorable cell wall-binding of cationic PS P3, the anionic PS Y1 can accumulate more in the spheroplast/protoplast of methicillin-resistant Staphylococcus aureus (MRSA), which ensures its high efficient aPDT abilities both in vitro and in vivo. This study suggests the great clinical application potential of Y1 in inactivation of MRSA.

Effective Two-Photon Excited Photodynamic Therapy of Xenograft Tumors Sensitized by Water-Soluble Bis(arylidene)cycloalkanone Photosensitizers.

A series of bis(arylidene)cycloalkanone photosensitizers modified by polyethylene glycol (PEG) have been studied for two-photon excited photodynamic therapy (2PE-PDT). As compared with their prototype compounds, these PEGylated photosensitizers show enhanced water solubilities while their photophysical and photochemical properties, including linear absorption, two-photon absorption, fluorescence, and singlet oxygen quantum yield, remain unaltered. In vitro behaviors (cellular uptake, subcellular localization, photocytotoxicity in both PDT and 2PE-PDT) of these photosensitizers reveal that an optimized lipid-water partition coefficient can be obtained by adjusting the length and position of the PEG chains. Among them, the photosensitizer modified asymmetrically by two tetraethylene glycol chains presents the best performance as a 2PE-PDT candidate. Selective blood-vessel closure and obvious therapeutic effect in inhibiting the growth of tumors are confirmed by in vivo 2PE-PDT after intravenous injection of this photosensitiezer. The survival periods of treated tumor-bearing mice are significantly prolonged. This study demonstrates the feasibility of using a simple molecule to construct a potential candidate for 2PE-PDT.