Stimuli-responsive Polypeptide Research Articles

Supramolecular assembly of multifunctional protein gels via an N-glycosylation consensus sequence fusion domain

Stimuli-responsive polypeptide tags direct the assembly of active proteins into supramolecular assemblies. This allows for recombinant expression of soluble fusion protein and subsequent user-controlled formation of functional biomaterials.

Construction of nano-drug delivery and antitumor system of stimuli-responsive polypeptides

The size of the nanoparticles is moderate and the dispersion is well, which will not be recognized nonspecifically and clearance by the endothelial reticular system. In this study, stimuli-responsive polypeptides nano-delivery system has been constructed, which can realize the response to various stimuli in the tumor microenvironment. Tertiary amine groups are grafted to the side chain of polypeptides as the point of charge reversal and particle expansion. In addition, a new kind of liquid crystal monomer was prepared by substituting cholesterol-cysteamine, which can promote polymers to realize the transformation of spatial conformation by adjusting the ordered arrangement of macromolecules. The introduction of hydrophobic elements greatly enhanced the self-assembly performance of polypeptides, which could effectively improve the drug loading and encapsulation rate of nanoparticles. Nanoparticles could achieve targeted aggregation in tumor tissues, and there were no toxicity and side effects on normal bodies during treatment, with good safety in vivo.

Stimuli-Responsive Polypeptide Nanoparticles for Enhanced DNA Delivery

The development of non-viral delivery systems for effective gene therapy is one of the current challenges in modern biomedicinal chemistry. In this paper, the synthesis of pH- and redox-responsive amphiphilic polypeptides for intracellular DNA delivery is reported and discussed. Two series of polypeptides consisting of L-lysine, L-phenylalanine, L-histidine, and L-cysteine as well as the same amino acids with L-glutamic acid were synthesized by a combination of copolymerization of N-carboxyanhydrides of α-amino acids and post-polymerization modification of the resulting copolymers. The presence of histidine provided pH-sensitive properties under weakly acidic conditions specific to endosomal pH. In turn, the presence of cysteine allowed for the formation of redox-responsive disulfide bonds, which stabilized the self-assembled nanoparticles in the extracellular environment but could degrade inside the cell. The formation of intraparticle disulfide bonds resulted in their compactization from 200-250 to 55-100 nm. Empty and pDNA-loaded cross-linked nanoparticles showed enhanced stability in various media compared to non-crosslinked nanoparticles. At the same time, the addition of glutathione promoted particle degradation and nucleic acid release. The delivery systems were able to retain their size and surface charge at polypeptide/pDNA ratios of 10 or higher. GFP expression in HEK 293 was induced by the delivery of pEGFP-N3 with the developed polypeptide nanoparticles. The maximal transfection efficacy (70%) was observed when the polypeptide/pDNA ratio was 100.

Preparation of Synthetic Polypeptide–PolyHIPE Hydrogels with Stimuli-Responsive Behavior

In this work, biodegradable macroporous stimuli-responsive polypeptide hydrogels based on l-glutamic acid (Glu) and its copolymers with equimolar amounts of l-phenylalanine (Phe) or l-lysine (Lys) were prepared by deprotection of the corresponding organogels under acidic conditions. The organogels were synthesized by ring-opening polymerization of N-carboxyanhydride (NCA) derivatives of the corresponding α-amino acids in oil-in-oil high-internal phase emulsions (HIPEs) using the di-NCA derivative of l-cystine as a cross-linker. The organogels exhibit the typical interconnected porous polyHIPE morphology, which is completely preserved in the hydrogels after removal of the protecting groups of the Glu and Lys repeating units. The pH-dependent behavior and mechanical properties of the obtained hydrogels were studied in buffer solutions with different pH values. At pH 7.5, P(Glu) and P(Glu-co-Phe) can be compressed to half their original height and both return to their initial state after unloading. By lowering the pH to 5.5, P(Glu) remains soft, while P(Glu-co-Phe) already becomes much stiffer. In contrast, for the P(Glu-co-Lys) hydrogel, high buffer uptake was observed only at high or low pH values, whereas at intermediate pH values, the low buffer uptake and the impaired ability to return to the original height are attributed to the attractive ionic interaction between the oppositely charged side groups. We have shown that by tuning the chemical composition of the polypeptides, the uptake, in vitro enzymatic degradation, and compression behavior of the hydrogels can be modulated.

Stimuli-responsive polypeptide nanoassemblies: Recent progress and applications in cancer nanomedicine.

Stimuli-responsive polypeptide nanoassemblies exhibit great potentials for cancer nanomedicines because of desirable biocompatibility and biodegradability, unique secondary conformations, varying functionalities, and especially the stimuli-enhanced therapeutic efficacy and reduced side effect. This review introduces the design and fabrication of stimuli-responsive polypeptide nanoassemblies that exhibit endogenous stimuli (e.g., pH, reduction, reactive oxygen species, adenosine triphosphate and enzyme, etc.) and exogenous light stimuli (e.g., UV and near-infrared light), which are biologically related or applied in the clinic. We also discuss the applications and prospects of those stimuli-responsive polypeptide nanoassemblies that might overcome the biological barriers of cancer nanomedicines for in vivo administration. Much more effort is needed to accelerate the second-generation stimuli-responsive polypeptide nanomedicines for clinical transition and applications. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Stimuli-responsive polypeptides for controlled drug delivery.

Controlled drug delivery systems, which could release loaded therapeutics upon physicochemical changes imposed by physiological triggers in the desired zone and during the required period of time, offer numerous advantages over traditional drug carriers including enhanced therapeutic effects and reduced toxicity. A polypeptide is a biocompatible and biodegradable polymer, which can be conveniently endowed with stimuli-responsiveness by introducing natural amino acid residues with innate stimuli-responsive characteristics or introducing responsive moieties to its side chains using simple conjugating methods, rendering it an ideal biomedical material for controlled drug delivery. This feature article summarizes our recent work and other relevant studies on the development of polypeptide-based drug delivery systems that respond to single or multiple physiological stimuli (e.g., pH, redox potential, glucose, and hypoxia) for controlled drug delivery applications. The material designs, synthetic strategies, loading and controlled-release mechanisms of drugs, and biomedical applications of these stimuli-responsive polypeptides-based drug delivery systems are elaborated. Finally, the challenges and opportunities in this field are briefly discussed.

Synthesis and Properties of Mono- or Diamine-Initiated Imidazolium-Based Cationic Polypeptides.

A series of cationic polypeptide imidazolium conjugates were prepared by ring-opening polymerization (ROP) of γ-4-(3-chloropropoxycarbonyl)benzyl-L- glutamic acid-based N-carboxyanhydride (CPBLG-NCA) initiated by various mono- or diamine initiators and subsequent side-chain modification with high grafting efficiency. Rapid and controlled ROP was achieved by polymerizing CPBLG-NCA in a dichloromethane/NaHCO3/H2O solvent mixture with the amine initiators. The resulting polypeptides bearing imidazolium iodide pendants showed reversible upper critical solution temperature (UCST)-type thermoresponsive properties in both ethanol and DI water while the polypeptides with tetrafluoroborate counter-anions showed a UCST in phosphate buffer saline (PBS). The cloud point temperature (Tcp) in ethanol and aqueous solutions can be tuned by both molecular weight and the end- or linkage-groups in the main chain. The cationic polypeptides showed good antibacterial activity against Staphylococcus aureus and low hemolysis. Our results provide a facile and rapid ROP strategy to develop new families of stimuli-responsive polypeptides with tunable properties as well as antibacterial polypeptides with optimized selectivity.

Dissipative Self-Assembly of Dynamic Multicompartmentalized Microsystems with Light-Responsive Behaviors

Summary Living cells are compartmentalized systems operating far from thermodynamic equilibrium and consume energy from the environment to carry out their functions. The construction of biomimetic synthetic compartments has both scientific and technological value. Despite many advances, the assembly of synthetic compartments that present nonequilibrium behaviors based on dissipative (or transient) self-assembly remains a grand challenge. Here, we describe the design and synthesis of multicompartmental systems with an active microstructure and complex behaviors. By including an artificial pathway for energy dissipation, we demonstrate the autonomous generation of active polymeric systems using a strategy based on polymerization-induced self-assembly (PISA), driven by chemical fuel. Remarkably, these self-organized synthetic systems exhibit a dynamic change in their multicompartmental structures when exposed to light. This work introduces a strategy toward the design and construction of synthetic compartments with a dynamic structure and also presents pathways to engineer materials with spatially controllable structures and functionalities.

AIE/FRET-based versatile PEG-Pep-TPE/DOX nanoparticles for cancer therapy and real-time drug release monitoring.

On account of the biological significance of self-assembling peptides in blocking the cellular mass exchange as well as impeding the formation for actin filaments resulting in program cell death, stimuli-responsive polypeptide nanoparticles have attracted more and more attention. In this work, we successfully fabricated doxorubicin-loaded polyethylene glycol-block-peptide (FFKY)-block-tetraphenylethylene (PEG-Pep-TPE/DOX) nanoparticles, where the aggregation-induced emission luminogens (AIEgen, TPE-CHO) can become a fluorescence resonance energy transfer (FRET) pair with the entrapped antitumor drug DOX to detect the release of drugs dynamically. This is the first successful attempt to detect and quantify the change of FRET signals in A549 cells via three methods to monitor the cellular uptake of nanoprobes and intracellular drug molecule release intuitively. As we proposed here, the combination of free DOX and the self-assembling peptide could achieve the synergistic anticancer efficacy. The multifunctional PEG-Pep-TPE/DOX nanoparticles may provide a new opportunity for combination cancer therapy and real-time detection of the drug release from stimuli-responsive nanomedicine.

Synthesis of polypeptide bearing 1,4-dithiane pendants for ROS-responsive drug release

Stimuli-responsive polypeptides have been intensively investigated for controlled drug release, owing to their favorable biocompatibility and biodegradability. In this work, we designed and synthesized a new kind of polypeptide bearing 1,4-dithiane pendants for reactive oxygen species (ROS)-responsive drug release. The polypeptide-based block copolymer was facilely synthesized by ring-opening polymerization (ROP) of 1,4-dithian-substituted l-glutamate N-carboxyanhydride (DTG-NCA) monomer using an amino-terminated poly(ethylene glycol) methyl ether (mPEG-NH2) as the macromolecular initiator. The resultant block copolymer, mPEG-b-PDTG, could self-assemble into uniform micelles in aqueous medium owing to its amphiphilic structure. Then, the H2O2-triggered oxidation behaviors of the mPEG-b-PDTG micelles were studied by dynamic light scattering (DLS), FT-IR and turbidimetric assay. It was revealed that the oxidation of thioether into sulfoxide in the side chains would result in disassembly of the micelles. Furthermore, the ROS-responsive drug release behavior of the mPEG-b-PDTG micelles was verified by using Nile Red as a model drug. MTT assay also proved that mPEG-b-PDTG was non-toxic in B16F10 and L929 cells. Therefore, such a new class of oxidation-responsive polypeptide might provide a promising platform for ROS-responsive drug delivery.

Preparation and properties of a temperature- and pH- responsive polypeptide hydrogel

Stimuli-responsive polypeptide hydrogel has attracted a great deal of interests because of the biodegradability, biocompatibility and the response-ability to external stimuli. Here we synthesized triblock copolymers methoxy-poly (ethylene glycols)-b-poly (L-lysine)-b-poly (L-valine) (mPEG-PLysx-PVy) by sequential ring opening polymerization. Through adjusting the length of hydrophilic and hydrophobic segments, we constructed a temperature- and pH- responsive hydrogel system using polymer mPEG-PLys2-PV7. The phase diagram from test tube inversion method showed that the gel transition temperature could cover the body temperature (37 °C) and decreased with the elevated pH value. TEM and FTIR measurements proved that the secondary structure of the polymer solution was mainly β–sheet structure, and the sol-gel transition process was caused by both the dehydration of mPEG and the change of the secondary structure. After loading DOX in hydrogels, we investigated the drug release behavior in vitro at different pH values. The results showed that the drug release at pH5.5 was faster than that at pH 7.4. About 36% and 100% of loaded drug was respectively released from hydrogels at 37 °C within 48 h at pH 7.4 and pH 5.7. This polypeptide hydrogel material might be used as drug targeting release system and does not affect normal tissues due to its significant pH sensitivity.

Programmable stimuli-responsive polypeptides for biomimetic synthesis of silica nanocomposites and enzyme self-immobilization

Bioinspired silicification is an attractive route for achieving unique silica nanocomposites. Herein, a novel, facile and inexpensive route for biosilica synthesis is developed using the stimuli-responsive elastin-like polypeptide (ELP). The ELP is precisely tailored to a silica-mineralizing peptide by programming it with lysine residues. The resulting cationic ELP[KV8F-40] is purified in ultrahigh yield using a chromatography-free ITC purification technique based on thermal-responsive property. Excitingly, the specific activity of ELP is 40-fold higher than that of silaffin. Besides, efficient and strong entrapment of ELP is achieved with over 98% of immobilization yield and less than 2% of leakage. These imply that cationic ELP may be used as a bifunctional tag (purification and immobilization) for fusion protein. An enzyme (xylanase) is therefore chosen to genetically fuse to ELP. The ELP-fused xylanase is purified by ELP with high purity (~98%) and enables the rapid (within minutes) self-immobilization. The immobilization yield was greater than 95%, and the immobilized xylanases hardly leaked from the silica matrix, demonstrating high efficiency of the self-immobilization process. The strategy developed here may provide a new opportunity for fabricating functional silica nanocomposites in a feasible and inexpensive pathway, which will have great potentials in the field of biotechnology.

Stimuli-Responsive Polypeptides for Biomedical Applications.

Stimuli-responsive polypeptides have gained attention because desirable bioactive properties can be easily imparted to them while keeping their biocompatibility and biodegradability intact. In this review, we summarize the most recent advances in various stimuli-responsive polypeptides (pH, reduction, oxidation, glucose, adenosine triphosphate (ATP), and enzyme) over the past five years. Various synthetic strategies exploited for advanced polypeptide-based materials are introduced, and their applicability in biomedical fields is discussed. The recent polypeptides imparted with new stimuli-responsiveness and their novel chemical and physical properties are explained in this review.

Cd2+ coordination: an efficient structuring switch for polypeptide polymers

Stimuli-responsive polypeptides have practical applications in devices and therapeutic delivery. Here we report that coordination to Cd2+ metal species can be used as a robust stimulus to control both, α-helix and β-sheet secondary structuring of polypeptide polymers.

Morphological variation of stimuli-responsive polypeptide at air–water interface

The morphological variation of stimuli-responsive polypeptide molecules at the air–water interface as a function of temperature and compression was described. The surface pressure–area (π–A) isotherms of an elastin-like polypeptide (ELP) monolayer were obtained under variable external conditions, and Langmuir–Blodgett (LB) monolayers were deposited onto a mica substrate for characterization. As the compression of the ELP monolayer increased, the surface pressure increased gradually, indicating that the ELP monolayer could be prepared with high stability at the air–water interface. The temperature in the subphase of the ELP monolayer was critical in the preparation of LB monolayers. The change in temperature induced a shift in the π–A isotherms as well as a change in ELP secondary structures. Surprisingly, the compression of the ELP monolayer influenced the ELP secondary structure due to the reduction in the phase transition temperature with decreasing temperature. The change in the ELP secondary structure formed at the air–water interface was investigated by surface-enhanced Raman scattering. Moreover, the morphology of the ELP monolayer was subsequently imaged using atomic force microscopy. The temperature responsive behavior resulted in changes in surface morphology from relatively flat structures to rugged labyrinth structures, which suggested conformational changes in the ELP monolayers.

Biodegradable stimuli-responsive polypeptide materials prepared by ring opening polymerization.

The stimuli-responsive polypeptides have drawn extensive attention because of their promising applications in biotechnology considering their biocompatibility, biodegradability, and bioactivity. In this tutorial review, we summarize the most recent progress in this area, including thermo-, redox-, photo-, and biomolecule responsive polypeptides over the past decade. The design and synthesis of stimuli-responsive polypeptides will be briefly introduced. The correlation between the structure and properties, particularly the effects of polypeptide conformation, will be emphasized here. In addition, the applications of stimuli-responsive polypeptides in controlled drug release and tissue engineering are briefly discussed.

Stimuli responsive synthetic polypeptides derived from N-carboxyanhydride (NCA) polymerisation

The progress in NCA polymerisation combined with advanced orthogonal functionalization techniques as well as the integration with other controlled polymerisation techniques significantly widened the scope of polypeptide building blocks in a variety of material designs. Well-defined synthetic stimuli-responsive polypeptides ("smart" polypeptides) with incorporated different functionalities have been extensively explored over the past decades. Their significant potential lies in the fact that they combine natural and synthetic elements both contributing to their properties. These novel materials have potential applications in biomedicine and biotechnology including tissue engineering, drug delivery and biodiagnostics. Responsive polypeptides are capable of undergoing conformational changes and phase transition accompanied by variations in the chemical and physical changes of the polypeptides in response to an external stimulus such as biologically relevant species (i.e. biomolecules), the environment (i.e. temperature, pH), irradiation with light or exposure to a magnetic field. In this review, the recent developments including synthetic strategies and applications of synthetic stimuli-responsive homo- and block polypeptides are reviewed.

Synthesis and micellization behavior of stimuli-responsive polypeptide hybrid triblock copolymer

Polypeptide hybrid triblock copolymer, poly(L-glutamic acid)-b-poly(propylene oxide)-b-poly (L-glutamic acid) (PLGA-b-PPO-b-PLGA), was synthesized by the ring-opening polymerization of benzyl-L-glutamic N-carboxyanhydride (BLG-NCA) using poly(propylene glycol) bis(2-aminopropyl ether) as initiator, followed by the subsequent deprotection step. The obtained double hydrophilic triblock copolymer exhibits “schizophrenic” micellization behavior in aqueous solution upon dually playing with solution pH and temperature. The multi-responsive micellization behavior of this polypeptide hybrid triblock copolymer has been thoroughly investigated by 1H NMR, laser light scattering (LLS), temperature-dependent optical transmittance, and circular dichroism spectroscopy (CD).

Controlled Molecular Organization of Surface Macromolecular Assemblies Based on Stimuli-Responsive Polypeptide Brushes

End-tethered cationic polypeptide brushes of poly(L-lysine) (t-PLL) were combined with three anionic polymers, poly(acrylic acid) (PAA), poly(L-glutamic acid) (PLGA), and poly(L-aspartic acid) (PLAA), to form reversible polyelectrolyte complex films at surfaces at neutral pH. The polyelectrolyte complex formation was confirmed by an in situ zeta-potential study and by positive fluorescent images after adding prelabeled anionic polymers. The secondary conformations of the t-PLL complex films depend upon the specific polyelectrolyte with which t-PLL was coupled as studied by circular dichroism and FTIR. Specifically, the random coil chain configuration of the t-PLL film was converted to an alpha-helical, beta-sheet, or random coil structure after forming complexes with PAA, PLGA, or PLAA, respectively. Each of these complexes could be returned to the original random coil t-PLL structure by a dilute acid rinse. Additional thickness and morphological studies from ellipsometry and atomic force microscopy have further shown that the corresponding film thicknesses of the individual solvated films were affected more by the secondary structures in films than by the adsorbed mass or surface net charges. The solvated thickness was reduced significantly after the random coil t-PLL film was coupled with polyanions in forming compact regulated structures in films. This biomimetic approach provides a new opportunity for controlling the molecular organization in surface macromolecular assemblies and may provide a model for structural study of protein complexes on a chip.

Hydration and conformational mechanics of single, end-tethered elastin-like polypeptides.

We investigated the effect of temperature, ionic strength, solvent polarity, and type of guest residue on the force-extension behavior of single, end-tethered elastin-like polypeptides (ELPs), using single molecule force spectroscopy (SMFS). ELPs are stimulus-responsive polypeptides that contain repeats of the five amino acids Val-Pro-Gly-Xaa-Gly (VPGXG), where Xaa is a guest residue that can be any amino acid with the exception of proline. We fitted the force-extension data with a freely jointed chain (FJC) model which allowed us to resolve small differences in the effective Kuhn segment length distributions that largely arise from differences in the hydrophobic hydration behavior of ELP. Our results agree qualitatively with predictions from recent molecular dynamics simulations and demonstrate that hydrophobic hydration modulates the molecular elasticity for ELPs. Furthermore, our results show that SMFS, when combined with our approach for data analysis, can be used to study the subtleties of polypeptide-water interactions and thus provides a basis for the study of hydrophobic hydration in intrinsically unstructured biomacromolecules.