Conformation-Switchable Polypeptides as Molecular Gates for Controllable Drug Release.

The control over secondary structure has been widely studied to regulate the properties of polypeptide materials, which is used to change their functions in situ for various biomedical applications. Herein, we designed and constructed enzyme-responsive polypeptides as gating materials for mesoporous silica nanoparticles (MSNs), which underwent a distorted structure-to-helix transition to promote the release of encapsulated drugs. The polypeptide conjugated on the MSN surface adopted a negatively charged, distorted, flexible conformation, covering the pores of MSN to prevent drug leakage. Upon triggering by alkaline phosphatase (ALP) overproduced by tumor cells, the polypeptide transformed into positively charged, α-helical, rigid conformation with potent membrane-penetrating capabilities, which protruded from the MSN surface to uncover the pores. Such a transition thus enabled cancer-selective drug release and cellular internalization to efficiently kill tumor cells. This study highlights the important role of chain flexibility in modulating the biological function of polypeptides and provides a new application paradigm for synthetic polypeptides with secondary-structure transition.

Rational Construction of Protein-Mimetic Nano-Switch Systems Based on Secondary Structure Transitions of Synthetic Polypeptides.

The manipulation of the flexibility/rigidity of polymeric chains to control their function is commonly observed in natural macromolecules but largely unexplored in synthetic systems. Herein, we construct a series of protein-mimetic nano-switches consisting of a gold nanoparticle (GNP) core, a synthetic polypeptide linker, and an optically functional molecule (OFM), whose biological function can be dynamically regulated by the flexibility of the polypeptide linker. At the dormant state, the polypeptide adopts a flexible, random-coiled conformation, bringing GNP and OFM in close proximity that leads to the "turn-off" of the OFM. Once treated with alkaline phosphatase (ALP), the nano-switches are activated due to the increased separation distance between GNP and OFM driven by the coil-to-helix and flexible-to-rigid transition of the polypeptide linker. The nano-switches therefore enable selective fluorescence imaging or photodynamic therapy in response to ALP overproduced by tumor cells. The control over polymer flexibility represents an effective strategy to manipulate the optical activity of nano-switches, which mimics the delicate structure-property relationship of natural proteins.

Modeling and Designing Particle-Regulated Amyloid-like Assembly of Synthetic Polypeptides in Aqueous Solution.

In cells, actin and tubulin polymerization is regulated by nucleation factors, which promote the nucleation and subsequent growth of protein filaments in a controlled manner. Mimicking this natural mechanism to control the supramolecular polymerization of macromolecular monomers by artificially created nucleation factors remains a largely unmet challenge. Biological nucleation factors act as molecular scaffolds to boost the local concentrations of protein monomers and facilitate the required conformational changes to accelerate the nucleation and subsequent polymerization. An accelerated assembly of synthetic poly(l-glutamic acid) into amyloid fibrils catalyzed by cationic silica nanoparticle clusters (NPCs) as artificial nucleation factors is demonstrated here and modeled as supramolecular polymerization with a surface-induced heterogeneous nucleation pathway. Kinetic studies of fibril growth coupled with mechanistic analysis demonstrate that the artificial nucleators predictably accelerate the supramolecular polymerization process by orders of magnitude (e.g., shortening the assembly time by more than 10 times) when compared to the uncatalyzed reaction, under otherwise identical conditions. Amyloid-like fibrillation was supported by a variety of standard characterization methods. Nucleation followed a Michaelis-Menten-like scheme for the cationic silica NPCs, while the corresponding anionic or neutral nanoparticles had no effect on fibrillation. This approach shows the effectiveness of charge-charge interactions and surface functionalities in facilitating the conformational change of macromolecular monomers and controlling the rates of nucleation for fibril growth. Molecular design approaches like these inspire the development of novel materials via biomimetic supramolecular polymerizations.

Synthesis of polypeptides via bioinspired polymerization of in situ purified N-carboxyanhydrides.

Ribozymes synthesize proteins in a highly regulated local environment to minimize side reactions caused by various competing species. In contrast, it is challenging to prepare synthetic polypeptides from the polymerization of N-carboxyanhydrides (NCAs) in the presence of water and impurities, which induce monomer degradations and chain terminations, respectively. Inspired by natural protein synthesis, we herein report the preparation of well-defined polypeptides in the presence of competing species, by using a water/dichloromethane biphasic system with macroinitiators anchored at the interface. The impurities are extracted into the aqueous phase in situ, and the localized macroinitiators allow for NCA polymerization at a rate which outpaces water-induced side reactions. Our polymerization strategy streamlines the process from amino acids toward high molecular weight polypeptides with low dispersity by circumventing the tedious NCA purification and the demands for air-free conditions, enabling low-cost, large-scale production of polypeptides that has potential to change the paradigm of polypeptide-based biomaterials.

Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide.

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications.

Unimolecular Polypeptide Micelles via Ultrafast Polymerization of N-Carboxyanhydrides.

Polypeptide micelles are widely used as biocompatible nanoplatforms but often suffer from their poor structural stability. Unimolecular polypeptide micelles can effectively address the structure instability issue, but their synthesis with uniform structure and well-controlled and desired sizes remains challenging. Herein we report the convenient preparation of spherical unimolecular micelles through dendritic polyamine-initiated ultrafast ring-opening polymerization of N-carboxyanhydrides (NCAs). Synthetic polypeptides with exceptionally high molecular weights (up to 85 MDa) and low dispersity (D < 1.05) can be readily obtained, which are the biggest synthetic polypeptides ever reported. The degree of polymerization was controlled in a vast range (25-3200), giving access to nearly monodisperse unimolecular micelles with predictable sizes. Many NCA monomers can be polymerized using this ultrafast polymerization method, which enables the incorporation of various structural and functional moieties into the unimolecular micelles. Because of the simplicity of the synthesis and superior control over the structure, the unimolecular polypeptide micelles may find applications in nanomedicine, supermolecular chemistry, and bionanotechnology.

Super-swelled lyotropic single crystals.

Lipids self-assemble into diverse supramolecular structures that exhibit thermotropic and/or lyotropic behavior. Lyotropic mesophases, where membranes conform to periodic minimal surfaces dividing two nonpenetrating aqueous subspaces, are arguably one of the most intriguing phases of lipid materials. Traditional 3D bicontinuous cubic lipid materials appear as a polycrystal of varying degrees of order. When exposed to water, the properties of the molecular building blocks of the membrane determine specific swelling limits setting the lattice dimensions at about 15 nm. This limited swelling severely impairs their application as delivery vehicles of large drugs or as matrices for guiding protein crystallization. We report the discovery of self-assembly strategies leading to the emergence of lipid bicontinuous single crystals with unprecedented swelling capacity. The conventional strategy to increase unit cell size is tweaking membrane composition to include charged building blocks, a process to achieve electrostatic-driven swelling. In this paper, we demonstrate that controlling self-assembly external conditions when coupled to membrane composition yields 3D bicontinuous cubic phases that swell up to lattice dimensions of 68 nm. Importantly, and contrary to what is perceived for soft lyotropic materials in general, the self-assembly methodology enables the development of large super-swelled monocrystals. Utilizing small-angle X-ray scattering and cryoelectron microscopy, we underpin three crucial factors dictating the stabilization of super-swelled lipid bicontinuous cubic single crystals: (i) organic solvent drying speed, (ii) membrane charge density, and (iii) polyethylene glycol-conjugated lipids amount.

Selective killing of Helicobacter pylori with pH-responsive helix-coil conformation transitionable antimicrobial polypeptides.

Current clinical treatment of Helicobacter pylori infection, the main etiological factor in the development of gastritis, gastric ulcers, and gastric carcinoma, requires a combination of at least two antibiotics and one proton pump inhibitor. However, such triple therapy suffers from progressively decreased therapeutic efficacy due to the drug resistance and undesired killing of the commensal bacteria due to poor selectivity. Here, we report the development of antimicrobial polypeptide-based monotherapy, which can specifically kill H. pylori under acidic pH in the stomach while inducing minimal toxicity to commensal bacteria under physiological pH. Specifically, we designed a class of pH-sensitive, helix-coil conformation transitionable antimicrobial polypeptides (HCT-AMPs) (PGA)m-r-(PHLG-MHH)n, bearing randomly distributed negatively charged glutamic acid and positively charged poly(γ-6-N-(methyldihexylammonium)hexyl-l-glutamate) (PHLG-MHH) residues. The HCT-AMPs showed unappreciable toxicity at physiological pH when they adopted random coiled conformation. Under acidic condition in the stomach, they transformed to the helical structure and exhibited potent antibacterial activity against H. pylori, including clinically isolated drug-resistant strains. After oral gavage, the HCT-AMPs afforded comparable H. pylori killing efficacy to the triple-therapy approach while inducing minimal toxicity against normal tissues and commensal bacteria, in comparison with the remarkable killing of commensal bacteria by 65% and 86% in the ileal contents and feces, respectively, following triple therapy. This strategy renders an effective approach to specifically target and kill H. pylori in the stomach while not harming the commensal bacteria/normal tissues.

Proximity-Induced Cooperative Polymerization in "Hinged" Helical Polypeptides.

Cooperative interactions and transitions are among the most important strategies utilized by biological systems to regulate a variety of physical and chemical processes. We report herein an auto-accelerated, rapid cooperative polymerization of N-carboxyanhydrides (NCAs) with initiators structurally as simple as linear aliphatic diamines for the synthesis of polypeptides. The polymerization initiated by diamines proceeds via the formation of "hinged" polypeptides, which are two blocks of helical chains connected head-to-head by the diamine molecules in the polymerization solution. The reactions follow a two-stage, cooperative polymerization kinetic; the cooperative interactions between the macrodipoles of the two hinged helical polypeptides dramatically accelerate the polymerization. Compared to the NCA polymerization initiated by the hexylamine (CH3(CH2)5NH2), the chain propagation rate of the NCA polymerization is increased by more than 600 times when initiated by its diamine analogue (1,6-diaminohexane, NH2(CH2)6NH2). This proximity-induced cooperative polymerization showcases the single helix as a remarkable cooperativity-enabling motif in synthetic chemistry.

Pamidronate functionalized nanoconjugates for targeted therapy of focal skeletal malignant osteolysis.

Malignant osteolysis associated with inoperable primary bone tumors and multifocal skeletal metastases remains a challenging clinical problem in cancer patients. Nanomedicine that is able to target and deliver therapeutic agents to diseased bone sites could potentially provide an effective treatment option for different types of skeletal cancers. Here, we report the development of polylactide nanoparticles (NPs) loaded with doxorubicin (Doxo) and coated with bone-seeking pamidronate (Pam) for the targeted treatment of malignant skeletal tumors. In vivo biodistribution of radiolabeled targeted Pam-NPs demonstrated enhanced bone tumor accumulation and prolonged retention compared with nontargeted NPs. In a murine model of focal malignant osteolysis, Pam-functionalized, Doxo-loaded NPs (Pam-Doxo-NPs) significantly attenuated localized osteosarcoma (OS) progression compared with nontargeted Doxo-NPs. Importantly, we report on the first evaluation to our knowlege of Pam-Doxo-NPs in dogs with OS, which possess tumors of anatomic size and physiology comparable to those in humans. The repeat dosing of Pam-Doxo-NPs in dogs with naturally occurring OS indicated the therapeutic was well tolerated without hematologic, nonhematologic, and cardiac toxicities. By nuclear scintigraphy, the biodistribution of Pam-Doxo-NPs demonstrated malignant bone-targeting capability and exerted measurable anticancer activities as confirmed with percent tumor necrosis histopathology assessment.

Secondary structures in synthetic polypeptides from N-carboxyanhydrides: design, modulation, association, and material applications.

Synthetic polypeptides derived from the ring-opening polymerization of N-carboxyanhydrides can spontaneously fold into stable secondary structures under specific environmental conditions. These secondary structures and their dynamic transitions play an important role in regulating the properties of polypeptides in self-assembly, catalysis, polymerization, and biomedical applications. Here, we review the current strategies to modulate the secondary structures, and highlight the conformation-specific dynamic properties of synthetic polypeptides and the corresponding materials. A number of mechanistic studies elucidating the role of secondary structures are discussed, aiming to provide insights into the new designs and applications of synthetic polypeptides. We aim for this article to bring to people's attention synthetic polymers with ordered conformations, which may exhibit association behaviors and material properties that are otherwise not found in polymers without stable secondary structures.

Helical antimicrobial polypeptides with radial amphiphilicity.

α-Helical antimicrobial peptides (AMPs) generally have facially amphiphilic structures that may lead to undesired peptide interactions with blood proteins and self-aggregation due to exposed hydrophobic surfaces. Here we report the design of a class of cationic, helical homo-polypeptide antimicrobials with a hydrophobic internal helical core and a charged exterior shell, possessing unprecedented radial amphiphilicity. The radially amphiphilic structure enables the polypeptide to bind effectively to the negatively charged bacterial surface and exhibit high antimicrobial activity against both gram-positive and gram-negative bacteria. Moreover, the shielding of the hydrophobic core by the charged exterior shell decreases nonspecific interactions with eukaryotic cells, as evidenced by low hemolytic activity, and protects the polypeptide backbone from proteolytic degradation. The radially amphiphilic polypeptides can also be used as effective adjuvants, allowing improved permeation of commercial antibiotics in bacteria and enhanced antimicrobial activity by one to two orders of magnitude. Designing AMPs bearing this unprecedented, unique radially amphiphilic structure represents an alternative direction of AMP development; radially amphiphilic polypeptides may become a general platform for developing AMPs to treat drug-resistant bacteria.

Synthetic polypeptides: from polymer design to supramolecular assembly and biomedical application.

Synthetic polypeptides from the ring-opening polymerization of N-carboxyanhydrides (NCAs) are one of the most important biomaterials. The unique features of these synthetic polypeptides, including their chemical diversity of side chains and their ability to form secondary structures, enable their broad applications in the field of gene delivery, drug delivery, bio-imaging, tissue engineering, and antimicrobials. In this review article, we summarize the recent advances in the design of polypeptide-based supramolecular structures, including complexes with nucleic acids, micelles, vesicles, hybrid nanoparticles, and hydrogels. We also highlight the progress in the chemical design of functional polypeptides, which plays a crucial role to manipulate their assembly behaviours and optimize their biomedical performances. Finally, we conclude the review by discussing the future opportunities in this field, including further studies on the secondary structures and cost-effective synthesis of polypeptide materials.

Bacteria-Assisted Activation of Antimicrobial Polypeptides by a Random-Coil to Helix Transition.

The application of antimicrobial peptides (AMPs) is largely hindered by their non-specific toxicity against mammalian cells, which is usually associated with helical structure, hydrophobicity, and charge density. A random coil-to-helix transition mechanism has now been introduced into the design of AMPs, minimizing the toxicity against mammalian cells while maintaining high antimicrobial activity. By incorporating anionic phosphorylated tyrosine into the cationic polypeptide, the helical structure of AMPs was distorted owing to the side-chain charge interaction. Together with the decreased charge density, the AMPs exhibited inhibited toxicity against mammalian cells. At the infectious site, the AMPs can be activated by bacterial phosphatase to restore the helical structure, thus contributing to strong membrane disruptive capability and potent antimicrobial activity. This bacteria-activated system is an effective strategy to enhance the therapeutic selectivity of AMPs.

Integrating Display and Delivery Functionality with a Cell Penetrating Peptide Mimic as a Scaffold for Intracellular Multivalent Multitargeting.

The construction of a multivalent ligand is an effective way to increase affinity and selectivity toward biomolecular targets with multiple-ligand binding sites. Adopting this strategy, we used a known cell-penetrating peptide (CPP) mimic as a scaffold to develop a series of multivalent ligand constructs that bind to the expanded dCTG (CTG(exp)) and rCUG nucleotide repeats (CUG(exp)) known to cause myotonic dystrophy type I (DM1), an incurable neuromuscular disease. By assembling this polyvalent construct, the hydrophobic ligands are solubilized and delivered into cell nuclei, and their enhanced binding affinity leads to the inhibition of ribonuclear foci formation and a reversal of splicing defects, all at low concentrations. Some of the multivalent ligands are shown to inhibit selectively the in vitro transcription of (CTG·CAG)74, to reduce the concentration of the toxic CUG RNA in DM1 model cells, and to show phenotypic improvement in vivo in a Drosophila model of DM1. This strategy may be useful in drug design for other trinucleotide repeat disorders and more broadly for intracellular multivalent targeting.

Dimeric drug polymeric nanoparticles with exceptionally high drug loading and quantitative loading efficiency.

Encapsulation of small-molecule drugs in hydrophobic polymers or amphiphilic copolymers has been extensively used for preparing polymeric nanoparticles (NPs). The loadings and loading efficiencies of a wide range of drugs in polymeric NPs, however, tend to be very low. In this Communication, we report a strategy to prepare polymeric NPs with exceptionally high drug loading (>50%) and quantitative loading efficiency. Specifically, a dimeric drug conjugate bearing a trigger-responsive domain was designed and used as the core-constructing unit of the NPs. Upon co-precipitation of the dimeric drug and methoxypoly(ethylene glycol)-block-polylactide (mPEG-PLA), NPs with a dimeric drug core and a polymer shell were formed. The high-drug-loading NPs showed excellent stability in physiological conditions. No premature drug or prodrug release was observed in PBS solution without triggering, while external triggering led to controlled release of drug in its authentic form.

Polypeptide vesicles with densely packed multilayer membranes.

Multilamellar membranes are important building blocks for constructing self-assembled structures with improved barrier properties, such as multilamellar lipid vesicles. Polymeric vesicles (polymersomes) have attracted growing interest, but multilamellar polymersomes are much less explored. Here, we report the formation of polypeptide vesicles with unprecedented densely packed multilayer membrane structures with poly(ethylene glycol)-block-poly(γ-(4,5-dimethoxy-2-nitrobenzyl)-l-glutamate) (PEG-b-PL), an amphiphilic diblock rod-coil copolymer containing a short PEG block and a short hydrophobic rod-like polypeptide segment. The polypeptide rods undergo smectic ordering with PEG buried between the hydrophobic polypeptide layers. The size of both blocks and the rigidity of the hydrophobic polypeptide block are critical in determining the membrane structures. Increase of the PEG length in PEG-b-PL results in the formation of bilayer sheets, while using random-coil polypeptide block leads to the formation of large compound micelles. UV treatment causes ester bond cleavage of the polypeptide side chain, which induces helix-to-coil transition, change of copolymer amphiphilicity, and eventual disassembly of vesicles. These polypeptide vesicles with unique membrane structures provide a new insight into self-assembly structure control by precisely tuning the composition and conformation of polymeric amphiphiles.

Self-assembly of α-helical polypeptides driven by complex coacervation.

Reported is the ability of α-helical polypeptides to self-assemble with oppositely-charged polypeptides to form liquid complexes while maintaining their α-helical secondary structure. Coupling the α-helical polypeptide to a neutral, hydrophilic polymer and subsequent complexation enables the formation of nanoscale coacervate-core micelles. While previous reports on polypeptide complexation demonstrated a critical dependence of the nature of the complex (liquid versus solid) on chirality, the α-helical structure of the positively charged polypeptide prevents the formation of ß-sheets, which would otherwise drive the assembly into a solid state, thereby, enabling coacervate formation between two chiral components. The higher charge density of the assembly, a result of the folding of the α-helical polypeptide, provides enhanced resistance to salts known to inhibit polypeptide complexation. The unique combination of properties of these materials can enhance the known potential of fluid polypeptide complexes for delivery of biologically relevant molecules.

Polypeptides with quaternary phosphonium side chains: synthesis, characterization, and cell-penetrating properties.

Polypeptides bearing quaternary phosphonium side chains were synthesized via controlled ring-opening polymerization of chlorine-functionalized amino acid N-carboxyanhydride monomers followed by one-step nucleophilic substitution reaction with triethylphosphine. The conformation of the resulting polypeptides can be controlled by modulating the side-chain length and α-carbon stereochemistry. The phosphonium-based poly(l-glutamate) derivatives with 11 σ-bond backbone-to-charge distance adopt stable α-helical conformation against pH and ionic strength changes. These helical, quaternary phosphonium-bearing polypeptides exhibit higher cell-penetrating capability than their racemic and random-coiled analogues. They enter cells mainly via an energy-independent, nonendocytic cell membrane transduction mechanism and exhibit low cytotoxicity, substantiating their potential use as a safe and effective cell-penetrating agent.

Combination of ATRA and ATRC for the synthesis of periodic vinyl copolymers.

A new approach to periodic vinyl copolymers via combination of atom transfer radical addition (ATRA) and atom transfer radical coupling (ATRC) is reported. The two examples are methyl methacrylate (MMA) and styrene (St) periodic copolymer (P(SMMS)) and acrylonitrile (AN) and St periodic copolymer (P(SAAS)). First, two monomer sequence units (MSU) with built-in sequence, SMMS and SAAS, are synthesized through the controlled ATRA of two ATRP initiators with St. Then, the ATRC of SMMS and SAAS are conducted at high radical conditions to generate two types of high-molecular-weight copolymers, P(SMMS) and P(SAAS). Though side reactions can not be totally avoided, characterizations of the polymer structure with a variety of means confirm that the main chain structures of P(SMMS) and (PSAAS) are predominantly with the periodic sequences from the MSUs. Attempts to suppress the side reactions are successful via the MNP-mediated ATRC of SMMS and SAAS.