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.

Macrophage membrane-reversibly camouflaged nanotherapeutics accelerate fracture healing by fostering MSCs recruitment and osteogenic differentiation.

The fracture healing outcome is largely dependent on the quantities as well as osteogenic differentiation capacities of mesenchymal stem cells (MSCs) at the lesion site. Herein, macrophage membrane (MM)-reversibly cloaked nanocomplexes (NCs) are engineered for the lesion-targeted and hierarchical co-delivery of short stromal derived factor-1α peptide (sSDF-1α) and Ckip-1 small interfering RNA (Ckip-1 siRNA, siCkip-1) to promote bone repair by concurrently fostering recruitment and osteogenic differentiation of endogenous MSCs. To construct the NCs, a membrane-penetrating α-helical polypeptide first assembles with siCkip-1, and the cationic NCs are sequentially coated with catalase and an outer shell of sSDF-1α-anchored MM. Due to MM-assisted inflammation homing, intravenously injected NCs could efficiently accumulate at the fractured femur, where catalase decomposes the local hydrogen peroxide to generate oxygen bubbles that drives the shedding of sSDF-1α-anchored MM in the extracellular compartment. The exposed, cationic inner core thus enables robust trans-membrane delivery into MSCs to induce Ckip-1 silencing. Consequently, sSDF-1α-guided MSCs recruitment cooperates with siCkip-1-mediated osteogenic differentiation to facilitate bone formation and accelerate bone fracture healing. This study provides an enlightened strategy for the hierarchical co-delivery of macromolecular drugs into different cellular compartments, and it also renders a promising modality for the management of fracture healing.

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.

Engineering Nucleotidoproteins for Base-Pairing-Assisted Cytosolic Delivery and Genome Editing.

Protein therapeutics targeting intracellular machineries hold profound potential for disease treatment, and hence robust cytosolic protein delivery technologies are imperatively demanded. Inspired by the super-negatively charged, nucleotide-enriched structure of nucleic acids, adenylated pro-proteins (A-proteins) with dramatically enhanced negative surface charges have been engineered for the first time via facile green synthesis. Then, thymidine-modified polyethyleneimine is developed, which exhibits strong electrostatic attraction, complementary base pairing, and hydrophobic interaction with A-proteins to form salt-resistant nanocomplexes with robust cytosolic delivery efficiencies. The acidic endolysosomal environment enables traceless restoration of the A-proteins and consequently promotes the intracellular release of the native proteins. This strategy shows high efficiency and universality for a variety of proteins with different molecular weights and isoelectric points in mammalian cells. Moreover, it enables highly efficient delivery of CRISPR-Cas9 ribonucleoproteins targeting fusion oncogene EWSR1-FLI1, leading to pronounced anti-tumor efficacy against Ewing sarcoma. This study provides a potent and versatile platform for cytosolic protein delivery and gene editing, and may benefit the development of protein pharmaceuticals.

ROS-Responsive Selenopolypeptide Micelles: Preparation, Characterization, and Controlled Drug Release.

Sulfur-containing polypeptides, capable of reactive oxygen species (ROS)-responsive structural change, are one of the most important building blocks for the construction of polypeptide-based drug delivery systems. However, the relatively low ROS sensitivity of side-chain thioethers limits the biomedical applications of these polypeptides because they usually require a high concentration of ROS beyond the pathological ROS level in the tumor microenvironment. Herein, we report the design and synthesis of a selenium-containing polypeptide, which undergoes random coil-to-extended helix and hydrophobic-to-hydrophilic transitions in the presence of 0.1% H2O2, a concentration that is much lower than the ROS requirement for thioether. ROS-responsive micelles were thus prepared from the amphiphilic copolymer consisting of the hydrophilic poly(ethylene glycol) (PEG) segment and hydrophobic selenopolypeptide segment and were used to encapsulate doxorubicin (DOX). The micelles could be sensitively dissociated inside tumor cells in consequence of ROS-triggered oxidation of side-chain selenoether and structural change of the micelles, thereby efficiently and selectively releasing the encapsulated DOX to kill cancer cells. This work provides an alternative design of ROS-responsive polypeptides with higher sensitivity than that of the existing sulfur-containing polypeptides, which may expand the biomedical applications of polypeptide materials.

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.

Recent advances of octenyl succinic anhydride modified polysaccharides as wall materials for nano-encapsulation of hydrophobic bioactive compounds.

Polysaccharides can be esterified with octenyl succinic anhydride (OSA) to form derivatives with amphiphilic properties. The general preparation methods of OSA-polysaccharides are described, especially the aqueous method. The new hydrophobic groups introduced result in OSA-polysaccharides showing higher interfacial properties, better emulsifying stability, higher viscosity, and lower digestibility. There have been advances in the development of OSA-polysaccharides-based nano-encapsulation systems for hydrophobic bioactive compounds in recent years. Nano-encapsulation systems are formed through nanoemulsions, nanocapsules, nanoparticles, micelles, vesicles, molecular inclusion complexes, and so on. This review aims to describe the preparation methods, the structure characterizations, and the physicochemical properties of OSA-polysaccharides as encapsulating agents. In addition, the focus is on the different nano-encapsulation systems based on OSA-polysaccharides as wall materials. Future perspectives will concern OSA-polysaccharides-based nano-encapsulation systems with optimized functional properties for providing higher bioavailability and targeted delivery of various hydrophobic bioactive compounds. © 2022 Society of Chemical Industry.

Fabrication and characterization of octenyl succinic anhydride modified pullulan micelles for encapsulating curcumin.

BACKGROUND: Curcumin has become increasingly popular in functional foods and beverages field as a result of its high biological activity. Nevertheless, the application of curcumin is usually limited by its poor water solubility, low absorption, rapid metabolism and instability. Accordingly, the development of an appropriate wall material is crucial for its effective use. In the present study, curcumin-octenyl succinic anhydride modified pullulan (Cur-OSAP) micelles were successfully prepared by an anti-solvent co-precipitation method. RESULTS: Octenyl succinic anhydride modified pullulan (OSAP) micelles exhibited the highest encapsulation efficiency (57.31%) and loading capacity (5.73%) of curcumin when the mass ratio of OSAP to curcumin was 10:1 and the degree of substitution of OSAP was 0.0469, at which point Cur-OSAP micelles formed via hydrogen binding and hydrophobic interactions, as confirmed by Fourier transform infrared and fluorescence techniques. The transmission electron microscopy results showed that the Cur-OSAP micelles were roughly spherical in shape with diameters in the approximate range 30-60 nm. CONCLUSION: The encapsulation of OSAP greatly improved photostability and sustained release properties of curcumin in Cur-OSAP micelles. These findings suggest that OSAP can be used as a carrier to encapsulate and protect hydrophobic food ingredients. © 2021 Society of Chemical Industry.

Single-Chain Nanoparticle-Based Coatings with Improved Bactericidal Activity and Antifouling Properties.

Dual-function antibacterial surfaces have exhibited promising potential in addressing implant-associated infections. However, both bactericidal and antifouling properties need to be further improved prior to practical uses. Herein, we report the preparation and properties of a linear block copolymer coating (LP-KF) and a single-chain nanoparticle coating (NP-KF) with poly(ethylene glycol) (PEG) and cationic polypeptide segments. NP-KF with cyclic PEG segments and densely charged polypeptide segments was expected to display improved bactericidal and antifouling properties. LP-KF was prepared by the combination of ring-opening polymerization of N-carboxyanhydride (NCA) monomers and subsequent deprotection. NP-KF was prepared by intramolecular cross-linking of LP-KF in diluted solutions. Both LP-KF- and NP-KF-coated PDMS surfaces were prepared by dipping with polydopamine-coated surfaces. They showed superior in vitro bactericidal activity against both Staphylococcus aureus and Escherichia coli with >99.9% killing efficacy, excellent protein adsorption resistance, antibacterial adhesion, and low cytotoxicity. The NP-KF coating showed higher bactericidal activity and antifouling properties than its linear counterpart. It also showed significant anti-infective property and histocompatibility in vivo, which makes it a good candidate for implants and biomedical device applications.

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.

Fluorinated α-Helical Polypeptides Synchronize Mucus Permeation and Cell Penetration toward Highly Efficient Pulmonary siRNA Delivery against Acute Lung Injury.

The mucus layer and cell membrane are two major barriers against pulmonary siRNA delivery. Commonly used polycationic gene vectors can hardly penetrate the mucus layer due to the adsorption of mucin glycoproteins that trap and destabilize the polyplexes. Herein, guanidinated and fluorinated bifunctional helical polypeptides were developed to synchronizingly overcome these two barriers. The guanidine domain and α-helix facilitated trans-membrane siRNA delivery into macrophages, whereas fluorination of the polypeptides dramatically enhanced the mucus permeation capability by ∼240 folds, because incorporated fluorocarbon segments prevented adsorption of mucin glycoproteins onto polyplexes surfaces. Thus, when delivering TNF-α siRNA intratracheally, the top-performing polypeptide P7F7 provoked highly efficient gene knockdown by ∼96% at 200 µg/kg siRNA and exerted pronounced anti-inflammatory effect against acute lung injury. This study thus provides an effective strategy for transmucosal gene delivery, and it also renders promising utilities for the noninvasive, localized treatment of inflammatory pulmonary diseases.

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.

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.

Hypoxia-Induced Pro-Protein Therapy Assisted by a Self-Catalyzed Nanozymogen.

The success of intracellular protein therapy demands efficient delivery and selective protein activity in diseased cells. Therefore, a cascaded nanozymogen consisting of a hypoxia-activatable pro-protein, a hypoxia-inducing protein, and a hypoxia-strengthened intracellular protein delivery nanovehicle was developed. RPAB, an enzymatically inactive pro-protein of RNase, reversibly caged with hypoxia-cleavable azobenzene, was delivered with glucose oxidase (GOx) using hypoxia-responsive nanocomplexes (NCs) consisting of azobenzene-cross-linked oligoethylenimine (AOEI) and hyaluronic acid (HA). Upon NC-mediated delivery into cancer cells, GOx catalyzed glucose decomposition and aggravated tumoral hypoxia, which drove the recovery of RPAB back to the hydrolytically active RNase and expedited the degradation of AOEI to release more protein cargoes. Thus, the catalytic reaction of the nanozymogen was self-accelerated and self-cycled, ultimately leading to a cooperative anti-cancer effect between GOx-mediated starvation therapy and RNase-mediated pro-apoptotic therapy.

Recent Advances in Anti-cancer Protein/Peptide Delivery.

Protein/peptide drugs possess unique advantages, such as high pharmacological potency, molecular specificity, multifunctionality, and low toxicity, and thus hold great potential for use in cancer therapy. In the past decades, great achievements have been made in protein delivery systems, which can protect cargo proteins against detrimental physiological environments and efficiently deliver proteins into tumor sites and cells. In this Review, we first summarize the existing protein/peptide drugs used for cancer treatment, illustrate their anti-tumor mechanisms, and point out the potential challenges/barriers against their medical utility. We then discuss the existing strategies for protein encapsulation/conjugation and survey recent advances in the development of protein delivery vehicles, including lipid-based membrane nanocarriers, polymeric carriers, metal-organic frameworks, inorganic carriers, protein/peptide-based nanocarriers, and DNA nanostructures. The design strategies, advantages in potentiating protein delivery efficiencies, and possible limitations of these delivery systems are also discussed. Finally, future opportunities and challenges in anti-cancer protein/peptide delivery are indicated.

Selective in vivo metabolic cell-labeling-mediated cancer targeting.

Distinguishing cancer cells from normal cells through surface receptors is vital for cancer diagnosis and targeted therapy. Metabolic glycoengineering of unnatural sugars provides a powerful tool to manually introduce chemical receptors onto the cell surface; however, cancer-selective labeling still remains a great challenge. Herein we report the design of sugars that can selectively label cancer cells both in vitro and in vivo. Specifically, we inhibit the cell-labeling activity of tetraacetyl-N-azidoacetylmannosamine (Ac4ManAz) by converting its anomeric acetyl group to a caged ether bond that can be selectively cleaved by cancer-overexpressed enzymes and thus enables the overexpression of azido groups on the surface of cancer cells. Histone deacetylase and cathepsin L-responsive acetylated azidomannosamine, one such enzymatically activatable Ac4ManAz analog developed, mediated cancer-selective labeling in vivo, which enhanced tumor accumulation of a dibenzocyclooctyne-doxorubicin conjugate via click chemistry and enabled targeted therapy against LS174T colon cancer, MDA-MB-231 triple-negative breast cancer and 4T1 metastatic breast cancer in mice.

Recent Advances on Reactive Oxygen Species-Responsive Delivery and Diagnosis System.

Reactive oxygen species (ROS) play crucial roles in biological metabolism and intercellular signaling. However, ROS level is dramatically elevated due to abnormal metabolism during multiple pathologies, including neurodegenerative diseases, diabetes, cancer, and premature aging. By taking advantage of the discrepancy of ROS levels between normal and diseased tissues, a variety of ROS-sensitive moieties or linkers have been developed to design ROS-responsive systems for the site-specific delivery of drugs and genes. In this review, we summarized the ROS-responsive chemical structures, mechanisms, and delivery systems, focusing on their current advances for precise drug/gene delivery. In particular, ROS-responsive nanocarriers, prodrugs, and supramolecular hydrogels are summarized in terms of their application for drug/gene delivery, and common strategies to elevate or diminish cellular ROS concentrations, as well as the recent development of ROS-related imaging probes were also discussed.

Cancer-Selective Bioreductive Chemotherapy Mediated by Dual Hypoxia-Responsive Nanomedicine upon Photodynamic Therapy-Induced Hypoxia Aggravation.

Stimuli-responsive drug delivery has rendered promising utilities in cancer treatment. Nevertheless, cancer selectivity as well as sensitivity still remains critical challenges that would undermine the therapeutic efficacy of chemodrugs and cause undesired systemic toxicity. Herein, a dual hypoxia-responsive drug delivery system was developed to enable photodynamic therapy (PDT)-induced drug release and drug activation intermediated via PDT-induced hypoxia. Particularly, tumor-targeting and hypoxia-dissociable nanoparticles (NPs) were self-assembled from the amphiphilic polyethylenimine-alkyl nitroimidazole [PEI-ANI, (PA)] and hyaluronic acid-chlorin e6 (HA-Ce6) to encapsulate bioreductive chemodrug, tirapazamine (TPZ). After systemic administration, the obtained PA/HA-Ce6@TPZ NPs enabled effective tumor accumulation due to HA-mediated cancer targeting. Upon receptor-mediated endocytosis, light irradiation (660 nm, 10 mW/cm2) produced high levels of reactive oxygen species to mediate PDT and generated a severe local hypoxic environment to dissociate the NPs and selectively release TPZ, as a consequence of hypoxia-triggered hydrophobic-to-hydrophilic transformation of ANI. In the meantime, TPZ was activated under hypoxia, finally contributing to a synergistic anticancer treatment between PDT and hypoxia-strengthened bioreductive chemotherapy. This study, therefore, demonstrates a suitable strategy for cancer-selective drug delivery as well as programmed combination therapy.

High Drug Loading and Sub-Quantitative Loading Efficiency of Polymeric Micelles Driven by Donor-Receptor Coordination Interactions.

Polymeric micelles are extensively used for the delivery of hydrophobic drugs, which, however, suffer from unsatisfactory drug loading, colloidal uniformity, formulation stability, and drug release. Herein, we demonstrate a convenient strategy to prepare micelles with ultrahigh drug loading via the incorporation of polymer-drug coordination interactions. An amphiphilic copolymer containing pendant phenylboronic acid as electron acceptor unit was synthesized, which afforded donor-acceptor coordination with doxorubicin to obtain micelles with ultrahigh drug loading (∼50%), nearly quantitative loading efficiency (>95%), uniform size, and colloidal stability. Besides, the encapsulated drug can be effectively and selectively released in response to the high reactive oxygen species levels in cancer cells, which potentiated the anticancer efficacy and reduced systemic toxicity. Apart from doxorubicin, the current platform could be extended to other drugs with electron-donating groups (e.g., epirubicin and irinotecan), rendering a simple and robust strategy for enabling high drug loading in polymeric micelles and cancer-specific drug release.

Photoresponsive Drug/Gene Delivery Systems.

Light as an external stimulus can be precisely manipulated in terms of irradiation time, site, wavelength, and density. As such, photoresponsive drug/gene delivery systems have been increasingly pursued and utilized for the spatiotemporal control of drug/gene delivery to enhance their therapeutic efficacy and safety. In this review, we summarized the recent research progress on photoresponsive drug/gene delivery, and two major categories of delivery systems were discussed. The first category is the direct responsive systems that experience photoreactions on the vehicle or drug themselves, and different materials as well as chemical structures responsive to UV, visible, and NIR light are summarized. The second category is the indirect responsive systems that require a light-generated mediator signal, such as heat, ROS, hypoxia, and gas molecules, to cascadingly trigger the structural transformation. The future outlook and challenges are also discussed at the end.