Triblock Copolypeptide Research Articles

Injectable Hydrogels of Stimuli-Responsive Elastin and Calmodulin-Based Triblock Copolypeptides for Controlled Drug Release.

A variety of block copolypeptides with stimuli responsiveness have been of growing interest for dynamic self-assembly. Here, multistimuli-responsive triblock copolypeptides composed of thermosensitive elastin-based polypeptides (EBP) and ligand-responsive calmodulin (CalM) were genetically engineered, over-expressed, and nonchromatographically purified by inverse transition cycling. Diluted EBP-CalM-EBP (ECE) triblock copolypeptides under physiological conditions self-assembled into vesicles at the nanoscale by temperature-triggered aggregation of the EBP block with lower critical solution temperature behaviors. Furthermore, concentrated ECE triblock copolypeptides under identical conditions exhibited thermally induced gelation, resulting in physically crosslinked hydrogels. They showed controlled rheological and mechanical properties depending on the conformational change of the CalM middle block induced by binding either Ca2+ or Ca2+ and trifluoperazines (TFPs) as ligands. In addition, both Ca2+-free and Ca2+-bound ECE triblock copolypeptide hydrogels exhibited biocompatibility, while those bound to both Ca2+ and TFPs showed severe cytotoxicity because of controlled TFP release of the CalM blocks. The ECE triblock hydrogels with stimuli responsiveness would be useful as injectable drug delivery depots for biomedical applications.

Facile Synthesis of Imidazolium-Based Block Copolypeptides with Excellent Antimicrobial Activity.

Antimicrobial polypeptides are promising mimics of antimicrobial peptides (AMPs) with low risks of antimicrobial resistance (AMR). Polypeptides with facile and efficient production, high antimicrobial activity, and low toxicity toward mammalian cells are highly desirable for practical applications. Herein, triblock copolypeptides with chloro groups (PPGn-PCPBLGm) and different main-chain lengths were synthesized via an ultrafast ring-opening polymerization (ROP) using a macroinitiator, namely poly(propylene glycol) bis(2-aminopropyl ether), and purified or nonpurified monomer (i.e., CPBLG-NCA). PPGn-PCPBLGm with 90 amino acid residues can be readily prepared within 300 s. Imidazolium-based block copolypeptides (PPGn-PILm) were facilely prepared via nucleophilic substitution of PPGn-PCPBLGm with NaN3 and subsequent "click" chemistry. α-Helical PPGn-PILm can self-assemble into nanostructured and cationic micelles which displayed highly potent antimicrobial activity and low hemolysis. The top-performing material, namely PPG34-PIL70, showed low minimum inhibitory concentration (MIC) against both Gram-positive S.aureus and Gram-negative E.coli (25 μg mL-1). It also displayed low toxicity against mouse embryonic fibroblast (NIH 3T3) and human embryonic kidney (293T) cells at 2× MIC.

Three-dimensionally printable shear-thinning triblock copolypeptide hydrogels with antimicrobial potency.

Through rational design, block sequence controlled triblock copolypeptides comprising cysteine and tyrosine as well as a lysine or glutamic acid central block are devised. In these copolypeptides, each block contributes a specific property to the hydrogels to render them extrusion printable and antimicrobial. Three-dimensional (3D) printing of complex hydrogel structures with high shape retention is demonstrated. Moreover, composition dependent potent antimicrobial activity in contact-killing assays is elucidated.

Amphiphilic Nucleobase-Containing Polypeptide Copolymers-Synthesis and Self-Assembly.

Nucleobase-containing polymers are an emerging class of building blocks for the self-assembly of nanoobjects with promising applications in nanomedicine and biology. Here we present a macromolecular engineering approach to design nucleobase-containing polypeptide polymers incorporating thymine that further self-assemble in nanomaterials. Diblock and triblock copolypeptide polymers were prepared using sequential ring-opening polymerization of γ-Benzyl-l-glutamate N-carboxyanhydride (BLG-NCA) and γ-Propargyl-l-glutamate N-carboxyanhydride (PLG-NCA), followed by an efficient copper(I)-catalyzed azide alkyne cycloaddition (CuAAc) functionalization with thymidine monophosphate. Resulting amphiphilic copolymers were able to spontaneously form nanoobjects in aqueous solutions avoiding a pre-solubilization step with an organic solvent. Upon self-assembly, light scattering measurements and transmission electron microscopy (TEM) revealed the impact of the architecture (diblock versus triblock) on the morphology of the resulted nanoassemblies. Interestingly, the nucleobase-containing nanoobjects displayed free thymine units in the shell that were found available for further DNA-binding.

Synthesis and Characterization of a Leucine-Based Block Co-Polypeptide: The Effect of the Leucine Zipper on Self-Assembly.

The self-assembly behavior of an ABC triblock copolypeptide consisting of poly(ethylene oxide-b-(leucine-s-valine)-b-lysine) (PEO-PLV-PK) was examined via dynamic light scattering in dilute aqueous solution. Leucine is a hydrophobic, α-helix forming polypeptide that exhibits a "zipper effect" in coiled-coil dimers. We hypothesize that the specific interaction afforded by the leucine zipper dominates the thermodynamics of self-assembly through the side-by-side ordering of α-helices, which drives vesicle formation in a polymer with only 6 wt % hydrophobic content. Additionally, a multitude of assembly sizes and morphologies were attainable from a single polymer, depending on the solution processing method. Thermodynamic effects of the leucine zipper can be interpreted, in part, from solubility parameters determined from molecular modeling. The combination of synthesis, solvent processing, and computational studies helps to elucidate the thermodynamic effects of this unique assembly motif on classical self-assembly processes.

Small-Sized and Robust Chimaeric Lipopepsomes: A Simple and Functional Platform with High Protein Loading for Targeted Intracellular Delivery of Protein Toxin in Vivo

How to chaperone protein drugs into target tumor cells in vivo is a big challenge. Here, we report on small-sized and robust chimaeric vesicles (lipopepsomes) constructed with asymmetric poly(ethylene glycol)-b-poly(α-aminopalmitic acid)-b-poly(l-aspartic acid) triblock copolypeptide as a simple and functional platform for high loading and targeted intracellular delivery of saporin, a protein toxin, in vivo. Cyclic RGD peptide-decorated chimaeric lipopepsomes (cRGD-CLP) following loading 2.0–9.4 wt % of model protein FITC-labeled cytochrome C showed a small hydrodynamic size of 81–86 nm, enhanced internalization by αvβ3-overexpressing A549 lung tumor cells, as well as remarkable accumulation of 7.73% ID/g in the cancerous lung in mice. Saporin-loaded cRGD-CLP displayed a low half-maximal inhibitory concentration of 16.3 nM to A549 cancer cells. Intriguingly, saporin-loaded cRGD-CLP at 16.7 nmol saporin equiv/kg showed a high potency in treating orthotopically xenografted A549 lung tumors, suppressed tumor...

Synthesis of triblock amphiphilic copolypeptides with excellent antibacterial activity

Antibacterial peptides as a novel antimicrobial agent have overwhelmingly caught researchers’ attention with unique superiority. Here, we report a series of triblock copolypeptides K10-b-Fn-b-K10 synthesized by sequential NCA ring-opening polymerization. The obtained triblock copolypeptides have good antibacterial efficacy against both S. aureus and E. coli. The hemolysis test has been conducted and it suggests that the triblock copolypeptides have low cytotoxicity and good biocompatibility. Furthermore, we present a systematic study on their self-assembly behavior and confirm that they can self-assemble into vesicles. Hence, such triblock copolypeptides have potential applications in antibacterial therapy and biomedical application field.

Metal Ion-Induced Self-Assembly and Packaging of CCMV Nanocapsules.

The controlled self-assembly of protein cages is vital for the use of these nanocompartments in biomedical and nanotechnological applications. Recently, we showed that by combining different structural peptide elements, it is possible to assemble viral capsid proteins in distinct well-defined morphologies. In this chapter, a triblock copolypeptide is discussed, consisting of a metal ion-coordinating hexahistidine tag, a stimulus-responsive elastin-like polypeptide and a pH-responsive self-assembling viral capsid protein. This protein is able to form two different types of capsids, depending on the assembly pathway that is followed. Here, we focus on the metal ion-induced assembly process and describe the relevant experimental procedures to induce and utilize this assembly behavior.

Injectable Hydrogel: Amplifying the pH Sensitivity of a Triblock Copolypeptide by Conjugating the N-Termini via Dynamic Covalent Bonding.

We explore the self-assembly behavior of aqueous solutions of an amphiphilic, pH-sensitive poly(l-alanine)-b-poly(l-glutamic acid)-b-poly(l-alanine), (A5E11A5) triblock copolypeptide, end-capped by benzaldehyde through Schiff base reaction. At elevated concentrations and under physiological pH (7.4) and ionic strength (0.15M), the bare copolypeptide aqueous solutions underwent a sol-gel transition after heating and slow cooling thermal treatment, forming opaque stiff gels due to a hierarchical self-assembly that led to the formation of β-sheet-based twisted super fibers (Popescu et al. Soft Matter 2015, 11, 331-342). The conjugation of the N-termini with benzaldehyde (Bz) through a Schiff base reaction amplifies the copolypeptide pH-sensitivity within a narrow pH window relevant for in vivo applications. Specifically, the dynamic character of the imine bond allowed coupling/decoupling of the Bz upon switching pH. The presence of Bz conjugates to the N-termini of the copolypeptide resulted in enhanced packing of the elementary superfibers into thick and short piles, which inhibited the ability of the system for gelation. However, partial cleavage of Bz upon lowering pH to 6.5 prompted recovery of the hydrogel. The sol-gel transition triggered by pH was reversible, due to the coupling/decoupling of the benzoic-imine dynamic covalent bonding, endowing thus the gelling system with injectability. Undesirably, the gelation temperature window was significantly reduced, which however can be regulated at physiological temperatures by using a suitable mixture of the bare and the Bz-conjugated coplypeptide. This triblock copolypeptide gelator was investigated as a scaffold for the encapsulation of polymersome nanocarriers, loaded with a hydrophilic model drug, calcein. The polymersome/polypeptide complex system showed prolonged probe release in pH 6.5, which is relevant to extracellular tumor environment, rendering the system potentially useful for sustained delivery of anticancer drugs locally in the tumor.

Metal Ion-Induced Self-Assembly of a Multi-Responsive Block Copolypeptide into Well-Defined Nanocapsules.

Protein cages are an interesting class of biomaterials with potential applications in bionanotechnology. Therefore, substantial effort is spent on the development of capsule-forming designer polypeptides with a tailor-made assembly profile. The expanded assembly profile of a triblock copolypeptide consisting of a metal ion chelating hexahistidine-tag, a stimulus-responsive elastin-like polypeptide block, and a pH-responsive morphology-controlling viral capsid protein is presented. The self-assembly of this multi-responsive protein-based block copolymer is triggered by the addition of divalent metal ions. This assembly process yields monodisperse nanocapsules with a 20 nm diameter composed of 60 polypeptides. The well-defined nanoparticles are the result of the emergent properties of all the blocks of the polypeptide. These results demonstrate the feasibility of hexahistidine-tags to function as supramolecular cross-linkers. Furthermore, their potential for the metal ion-mediated encapsulation of hexahistidine-tagged proteins is shown.

Poly(PEGA)-b-poly(L-lysine)-b-poly(L-histidine) Hybrid Vesicles for Tumoral pH-Triggered Intracellular Delivery of Doxorubicin Hydrochloride.

A series of poly(ethylene glycol) methyl ether acrylate-block-poly(L-lysine)-block-poly(L-histidine) [p(PEGA)30-b-p(Lys)25-b-p(His)n] (n = 25, 50, 75, 100) triblock copolypeptides were designed and synthesized for tumoral pH-responsive intracellular release of anticancer drug doxorubicin hydrochloride (Dox). The tumoral acidic pH-responsive hybrid vesicles fabricated were stable at physiological pH 7.4 and could gradually destabilize in acidic pH as a result of pH-induced swelling of the p(His) block. The blank vesicles were nontoxic over a wide concentration range (0.01-100 μg/mL) in normal cell lines. The tumor acidic pH responsiveness of these vesicles was exploited for intracellular delivery of Dox. Vesicles efficiently encapsulated Dox, and pH-induced destabilization resulted in the controlled and sustained release of Dox in CT26 murine cancer cells, and dose-dependent cytotoxicity. The tumor-specific controlled release Dox from vesicles demonstrates this system represents a promising theranostic agent for tumor-targeted delivery.

Stimuli responsive fibrous hydrogels from hierarchical self-assembly of a triblock copolypeptide.

In this work, the self-assembly behavior and pH responsiveness of a triblock copolypeptide in aqueous media are demonstrated. The copolypeptide was composed of a central pH responsive poly(l-glutamic acid) (PGA), flanked by two hydrophobic poly(l-alanine) blocks (PAla) (PAla5-PGA11-PAla5). This system showed a pH-responsive transition from short tapes to spherical aggregates by increasing the pH, as a result of deprotonation of the PGA block and a conformational change from α-helix to random coil. Increasing the ionic strength to physiological conditions (0.15 M) has triggered fibrillar self-assembly through intermolecular hydrogen bonding of PAla end-blocks that form β-sheet nanostructures, in conjunction with charge screening of the central random coil PGA segments. At elevated concentrations a thermo-responsive free supporting hydrogel was obtained, consisting of rigid β-sheet based twisted superfibers, resulting from hierarchical self-assembly of the copolypeptide. Yet, morphological transformation of this nanostructure was observed upon switching the pH from physiological conditions to pH 4. An unexpected morphology constituted of α-helix-based giant nanobelts was observed as a consequence of the secondary peptide transitions.

Blending of Diblock and Triblock Copolypeptide Amphiphiles Yields Cell Penetrating Vesicles with Low Toxicity

We prepared dual hydrophilic triblock copolypeptide vesicles that form both micron and nanometer scale vesicles in aqueous media. The incorporation of terminal homoarginine segments into methionine sulfoxide-based vesicles was found to significantly enhance their cellular uptake compared to a non-ionic control. We also demonstrated that diblock and triblock copolypeptides with similar hydrophobic domains were found to mix well and form vesicle populations with uniform compositions. Blending of amphiphiles in vesicle nanocarriers was found to impart these materials with many advantageous properties, including good cellular uptake while maintaining minimal toxicity, as well as biological responsiveness to promote vesicle disruption and release of encapsulated cargos.

Fine Tuning of Vesicle Assembly and Properties Using Dual Hydrophilic Triblock Copolypeptides

The design, synthesis, and self-assembly of the first dual hydrophilic triblock copolypeptide vesicles, R(H)(m)E(n)L(o) and K(P)(m)R(H)(n)L(o), is reported. Variation of the two distinct hydrophilic domains is used to tune cellular interactions without disrupting the self-assembled structure. The aqueous self-assemblies of these triblock copolypeptides in water are characterized using microscopy and DLS. Cell culture studies are used to evaluate cytotoxicity as well as intracellular uptake of the vesicles. The ability of polypeptides to incorporate ordered chain conformations that direct self-assembly, combined with the facile preparation of functional, multiblock copolypeptide sequences of defined lengths, allow the design of vesicles attractive for development as drug carriers.

Synthesis of polypeptide conjugated with near infrared fluorescence probe and doxorubicin for pH-responsive and image-guided drug delivery

A near infrared fluorescent polymeric drug delivery system (NIRF DDS) with pH-responsive drug release properties has been designed and developed. This material was prepared by chemical conjugation of the anticancer drug doxorubicin and hydrophobic aminocyanine dye to triblock copolypeptide via hydrazone and amide bonds, respectively. Conjugation with aminocyanine shows almost no toxicity of the material, while conjugation with doxorubicin induces pronounced toxicity on the original biocompatible material. The pH sensitive drug release nature of the near infrared fluorescent polymeric drug (NIRF prodrug) was confirmed by accelerated drug release at pH of 5.0 via an in vitro drug release experiment and gradual drug cleavage from the NIRF prodrug during a confocal laser scanning microscopic (CLSM) experiment. The CLSM experiment also reveals that the released drug subsequently migrated to the nucleus, while the polymeric residue still remained in cytoplasm, indicating that the as-prepared polymer can be a promising candidate for theranosis of cancer.

Twofold pH and temperature stimuli-responsive behaviour in block copolypeptide-decorated single wall carbon nanotubes

Temperature- and pH-responsive dispersions of carbon nanotubes in water have been obtained by using di- and tri-block copolypeptides, in which the temperature responsive behaviour is provided by a poly(N-isopropylacrylamide) block, while the pH responsiveness is provided by two different polypeptides with an ampholytic behaviour, poly(L-glutamic acid) and poly(L-lysine). The molecular architectures of the block copolypeptides can be engineered to fine-tune the stimuli-responsive behaviour.

Supramolecular structures from self-assembled poly(γ-benzyl- l-glutamate)–polydimethylsiloxane–poly(γ-benzyl- l-glutamate) triblock copolypeptides in thin films

We describe the self-assembly of A–B–A triblock copolymers in thin films composed of a soft polydimethylsiloxane (PDMS) central block (B) and two polypeptidic (A) blocks, poly(γ-benzyl)- l-glutamate (PBLG). The PBLG segment exhibits depending on the chain length two distinct secondary conformations either a β-sheet or a α-helical conformation. The direct relationship between the surface morphology and the secondary conformation of the polypeptide segment has been evidenced by atomic force microscopy. For chain lengths below 20 U the polypeptide segments adopt preferentially a β-sheet secondary structure and the triblock copolymer self-assembled in fibers. Moreover, the fiber diameters increased with the chain length of the triblock copolymer. For chain lengths above 20, the α-helical structure is stabilized and a lamellar morphology is formed driven by rod–rod interactions in spite of the very asymmetric composition of the triblock copolymer. However, decreasing the film thickness from 25 to 8 nm, i.e., below the L/2 and due to the preferential attraction of the polypeptide block for the hydrophilic substrate employed, instead of a lamellar morphology a rod-like morphology could be found. Thus, the use of hybrid block copolymer containing polypeptides with particular secondary structures offers novel alternatives to control the self-assembly in thin films compared to traditional amorphous block copolymers.

Solid state nanofibers based on self-assemblies: from cleaving from self-assemblies to multilevel hierarchical constructs

Self-assemblies and their hierarchies are useful to construct soft materials with structures at different length scales and to tune the materials properties for various functions. Here we address routes for solid nanofibers based on different forms of self-assemblies. On the other hand, we discuss rational "bottom-up" routes for multi-level hierarchical self-assembled constructs, with the aim of learning more about design principles for competing interactions and packing frustrations. Here we use the triblock copolypeptide poly(L-lysine)-b-poly(gamma-benzyl-L-glutamate)-b-poly(L-lysine) complexed with 2'-deoxyguanosine 5'-monophosphate. Supramolecular disks (G-quartets) stabilized by metal cations are formed and their columnar assembly leads to a packing frustration with the cylindrical packing of helical poly(gamma-benzyl-L-glutamate), which we suggest is important in controlling the lateral dimensions of the nanofibers. We foresee routes for functionalities by selecting different metal cations within the G-quartets. On the other hand, we discuss nanofibers that are cleaved from bulk self-assemblies in a "top-down" manner. After a short introduction based on cleaving nanofibers from diblock copolymeric self-assemblies, we focus on native cellulose nanofibers, as cleaved from plant cell wall fibers, which are expected to have feasible mechanical properties and to be templates for functional nanomaterials. Long nanofibers with 5-20 nm lateral dimensions can be cleaved within an aqueous medium to allow hydrogels and water can be removed to allow highly porous, lightweight, and flexible aerogels. We further describe inorganic/ organic hybrids as prepared by chemical vapour deposition and atomic layer deposition of the different nanofibers. We foresee functional materials by selecting inorganic coatings. Finally we briefly discuss how the organic template can be removed e.g., by thermal treatments to allow completely inorganic hollow nanofibrillar structures.

Architecturally Induced Multiresponsive Vesicles from Well-Defined Polypeptides. Formation of Gene Vehicles

A series of novel, partially labeled amphiphilic triblock copolypeptides, PLL-b-PBLG-d7-b-PLL, has been synthesized, where PLL and PBLG-d7 are poly(L-lysine hydrochloride) and poly(gamma-benzyl-d7-L-glutamate), respectively. The synthetic approach involved the sequential ring-opening polymerization (ROP) of gamma-benzyl-L-glutamate and epsilon-Boc-L-lysine N-carboxy anhydrides by a diamino initiator using high-vacuum techniques, followed by the selective deprotection of the Boc groups. Combined characterization results showed that the copolypeptides exhibit high degrees of molecular and compositional homogeneity. The synthesized copolypeptides had similar molecular weights, while the composition of the middle block ranged between 19 and 74% with respect to the monomeric units. Due to the macromolecular architecture of the copolypeptide and the rigid nature of the middle block, the formation of monolayers was favored, and, surprisingly, vesicles were formed in water at neutral pH over the entire compositional range. The vesicular structures were extensively characterized by static and dynamic light scattering, small-angle neutron scattering, atomic force microscopy, cryo-transmission electron microscopy, scanning electron microscopy, UV and Fourier transform infrared spectroscopy, and circular dichroism. In contrast to other vesicular structures derived from conventional polymers, the formed polypeptidic vesicles possess the unique feature of being stimuli-responsive to pH and temperature. When the copolypeptides were mixed with plasmid DNA (pDNA), large vesicular structures were also formed. The molecular characterization of the vectors was performed with most of the methods mentioned above, and indicated that the pDNA is both partially condensed on the PLL phase and partially encapsulated inside the vesicle. Consequently, the synthesized vectors combine the advantages of the polylysine-DNA systems to condense large amounts of genes, as well as those of the liposome-DNA systems to better protect the encapsulated DNA. These vectors are expected to present better gene transfection efficiency to the cell nucleus.

Formation of Microphase-Separated Structure in a Triblock Copolypeptide LB Film

Formation of Microphase-Separated Structure in a Triblock Copolypeptide LB Film