Toward Synthetic Intrinsically Disordered Polypeptides (IDPs): Controlled Incorporation of Glycine in the Ring-Opening Polymerization of N-Carboxyanhydrides.

Intrinsically disordered proteins (IDPs) do not have a well-defined folded structure but instead behave as extended polymer chains in solution. Many IDPs are rich in glycine residues, which create steric barriers to secondary structuring and protein folding. Inspired by this feature, we have studied how the introduction of glycine residues influences the secondary structure of a model polypeptide, poly(l-glutamic acid), a helical polymer. For this purpose, we carried out ring-opening copolymerization with γ-benzyl-l-glutamate and glycine N-carboxyanhydride (NCA) monomers. We aimed to control the glycine distribution within PBLG by adjusting the reactivity ratios of the two NCAs using different reaction conditions (temperature, solvent). The relationship between those conditions, the monomer distributions, and the secondary structure enabled the design of intrinsically disordered polypeptides when a highly gradient microstructure was achieved in DMSO.

Polypeptide- and Protein-Based Conjugate Nanoparticles via Aqueous Ring-Opening Polymerization-Induced Self-Assembly (ROPISA).

Protein-polymer conjugates and polymeric nanomaterials hold great promise in many applications including biomaterials, medicine, or nanoelectronics. In this work, the first polymerization-induced self-assembly (PISA) approach performed in aqueous medium enabling protein-polymer conjugates and nanoparticles entirely composed of amino acids is presented by using ring-opening polymerization (ROP). It is indeed shown that aqueous ring-opening polymerization-induced self-assembly (ROPISA) can be used with protein or peptidic macroinitiators without prior chemical modification and afford the simple preparation of nanomaterials with protein-like property, for example, to implement biomimetic thermoresponsivity in drug delivery.

New Insights on the Chemical Origin of Life: The Role of Aqueous Polymerization of N-carboxyanhydrides (NCA).

At the origin, the emergence of proteins was based on crucial prebiotic stages in which simple amino acids-based building blocks spontaneously evolved from the prebiotic soup into random proto-polymers called protoproteins. Despite advances in modern peptide synthesis, these prebiotic chemical routes to protoproteins remain puzzling. We discuss in this perspective how polymer science and systems chemistry are reaching a point of convergence in which simple monomers called N-carboxyanhydrides would be able to form such protoproteins via the emergence of a protometabolic cycle involving aqueous polymerization and featuring macromolecular Darwinism behavior.

Efficient Gene Delivery of Tailored Amphiphilic Polypeptides by Polyplex Surfing.

Within this study, an amphiphilic and potentially biodegradable polypeptide library based on poly[(4-aminobutyl)-l-glutamine-stat-hexyl-l-glutamine] [P(AB-l-Gln-stat-Hex-l-Gln)] was investigated for gene delivery. The influence of varying proportions of aliphatic and cationic side chains affecting the physicochemical properties of the polypeptides on transfection efficiency was investigated. A composition of 40 mol% Hex-l-Gln and 60 mol % AB-l-Gln (P3) was identified as best performer over polypeptides with higher proportions of protonatable monomers. Detailed studies of the transfection mechanism revealed the strongest interaction of P3 with cell membranes, promoting efficient endocytic cell uptake and high endosomal release. Spectrally, time-, and z-resolved fluorescence microscopy further revealed the crucial role of filopodia surfing in polyplex-cell interaction and particle internalization in lamellipodia regions, followed by rapid particle transport into cells. This study demonstrates the great potential of polypeptides for gene delivery. The amphiphilic character improves performance over cationic homopolypeptides, and the potential biodegradability is advantageous toward other synthetic polymeric delivery systems.

Memory Effect in Thermoresponsive Proline-based Polymers.

We report that synthetic polymers consisting of L-proline monomer units exhibit temperature-driven aggregation in water with unprecedented hysteresis. This protein-like behavior is robust and governed by the chirality of the proline units. It paves the way to new processes, driven by either temperature or ionic strength changes, such as a simple "with memory" thermometer.

Self-assembled PEGylated amphiphilic polypeptides for gene transfection.

In the present study, three biodegradable block copolymers composed of a poly(ethylene glycol) block and a copolypeptide block with varying compositions of cationic L-lysine (L-Lys) and hydrophobic benzyl-L-glutamate (Bzl-L-Glu) were designed for gene delivery applications. The polypeptides were synthesized by ring opening polymerization (ROP) and after orthogonal deprotection of Boc-L-Lys side chains, the polymer exhibited an amphiphilic character. To bind or encapsulate plasmid DNA (pDNA), different formulations were investigated: a nanoprecipitation and an emulsion technique using various organic solvents as well as an aqueous pH-controlled formulation method. The complex and nanoparticle (NP) formations were monitored by dynamic light scattering (DLS), and pDNA interaction was shown by gel electrophoresis and subsequent controlled release with heparin. The polypeptides were further tested for their cytotoxicity as well as biodegradability. The complexes and NPs presenting the most promising size distributions and pDNA binding ability were subsequently evaluated for their transfection efficiency in HEK293T cells. The highest transfection efficiencies were obtained with an aqueous formulation of the polypeptide containing the highest L-Lys content and lowest proportion of hydrophobic, helical structures (P1*), which is therefore a promising candidate for efficient gene delivery by biodegradable gene delivery vectors.

Synthetic Polypeptide Polymers as Simplified Analogues of Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are naturally occurring macromolecules made of amino acids that are potent broad-spectrum antibiotics with potential as novel therapeutic agents. This review aims to summarize the fundamental principles concerning the structure and mechanism of action of these AMPs, in order to guide the design of polymeric analogues that organic chemistry can generate. Among those simplified analogues, this review particularly focuses on those made of amino acids called polypeptide polymers: they are showing great potential by providing one of the best biomimetic and bioactive structures for further biomaterials science applications.

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.

Bidimensional lamellar assembly by coordination of peptidic homopolymers to platinum nanoparticles.

A key challenge for designing hybrid materials is the development of chemical tools to control the organization of inorganic nanoobjects at low scales, from mesoscopic (~µm) to nanometric (~nm). So far, the most efficient strategy to align assemblies of nanoparticles consists in a bottom-up approach by decorating block copolymer lamellae with nanoobjects. This well accomplished procedure is nonetheless limited by the thermodynamic constraints that govern copolymer assembly, the entropy of mixing as described by the Flory-Huggins solution theory supplemented by the critical influence of the volume fraction of the block components. Here we show that a completely different approach can lead to tunable 2D lamellar organization of nanoparticles with homopolymers only, on condition that few elementary rules are respected: 1) the polymer spontaneously allows a structural preorganization, 2) the polymer owns functional groups that interact with the nanoparticle surface, 3) the nanoparticles show a surface accessible for coordination.

Combination of photodynamic therapy and gene silencing achieved through the hierarchical self-assembly of porphyrin-siRNA complexes.

In this work, we implemented a supramolecular approach in order to combine photodynamic therapy (PDT) with gene therapy. We made use of a simple cationic guanidylated porphyrin (H2­PG) with the hypothesis that porphyrin aggregates should be capable of complexing siRNA through multivalent interactions and thus contribute to its intracellular delivery, while remaining active photosensitizers for PDT. The PDT effect of H2­PG was shown by incubating human breast cancer cells (MDA-MB-231) with H2­PG followed by light-irradiation at 405 nm. On the other hand, while siRNA do not enter cells alone, we showed, by fluorescence confocal microscopy and flow cytometry, that H2­PG promotes the internalization of Atto-488 siRNA. Finally, studying the combined PDT and delivery of siRNA directed against inhibitory apoptotic protein (IAP) family, we found an additive effect of the two therapies, thereby demonstrating that H2­PG is capable of acting both as a photosensitizer and supramolecular siRNA vector.

Antitrypanosomatid Pharmacomodulation at Position 3 of the 8-Nitroquinolin-2(1H)-one Scaffold Using Palladium-Catalysed Cross-Coupling Reactions.

An antikinetoplastid pharmacomodulation study at position 3 of the recently described hit molecule 3-bromo-8-nitroquinolin-2(1H)-one was conducted. Twenty-four derivatives were synthesised using the Suzuki-Miyaura cross-coupling reaction and evaluated in vitro on both Leishmania infantum axenic amastigotes and Trypanosoma brucei brucei trypomastigotes. Introduction of a para-carboxyphenyl group at position 3 of the scaffold led to the selective antitrypanosomal hit molecule 3-(4-carboxyphenyl)-8-nitroquinolin-2(1H)-one (21) with a lower reduction potential (-0.56 V) than the initial hit (-0.45 V). Compound 21 displays micromolar antitrypanosomal activity (IC50 =1.5 µm) and low cytotoxicity on the human HepG2 cell line (CC50 =120 µm), having a higher selectivity index (SI=80) than the reference drug eflornithine. Contrary to results previously obtained in this series, hit compound 21 is inactive toward L. infantum and is not efficiently bioactivated by T. brucei brucei type I nitroreductase, which suggests the existence of an alternative mechanism of action.

Ionic Polypeptide Polymers with Unusual ß-Sheet Stability.

Incorporating charged amino acid side chains in polypeptide polymer backbones to improve solubility usually leads to reduced secondary structuring. Here we show that highly water soluble (>15 mg.mL-1) ß-sheets can be obtained via nucleotide monophosphate grafting onto simple poly(γ-propargyl- L-glutamate) backbone. This synthetic methodology has been applied to the synthesis of thymidine-based nucleopolypeptides presenting stable ß-sheet conformation in aqueous solutions with pH values comprised between 4 and 8. These polymeric analogues of nucleoproteins exhibited selective interaction with simple DNA sequences displaying adenine.

Nucleopolypeptides with DNA-triggered α helix-to-ß sheet transition.

Synthetic polypeptides are versatile polymers outstandingly relevant to prepare bioinspired materials. In this work, we present a new class of smart polypeptide polymers, called nucleopolypeptides, having lateral chains functionalized with thymidine nucleobases. Structural studies performed by circular dichroism have revealed that the secondary structure of the polymers was influenced by nucleotide interaction and DNA sequence variation affording a selective helix-to-beta sheet transition with oligo(AAAAA)6.

Smart Poly(imidazoyl-l-lysine): Synthesis and Reversible Helix-to-Coil Transition at Neutral pH.

Polypeptide polymers can adopt natural protein secondary structures such as α-helices or ß-sheets, and this unique feature is at the origin of some intriguing physico⁻chemical properties. In this work, we present how side chain imidazoylation of a poly(l-lysine) scaffold affords the preparation of poly(histidine) counterparts exhibiting α-helix conformation. This structuring behavior is reversible and can be controlled by means of pH and or temperature changes.

Functionalization of alkyne-terminated thermally hydrocarbonized porous silicon nanoparticles with targeting peptides and antifouling polymers: effect on the human plasma protein adsorption.

Porous silicon (PSi) nanomaterials combine a high drug loading capacity and tunable surface chemistry with various surface modifications to meet the requirements for biomedical applications. In this work, alkyne-terminated thermally hydrocarbonized porous silicon (THCPSi) nanoparticles were fabricated and postmodified using five bioactive molecules (targeting peptides and antifouling polymers) via a single-step click chemistry to modulate the bioactivity of the THCPSi nanoparticles, such as enhancing the cellular uptake and reducing the plasma protein association. The size of the nanoparticles after modification was increased from 176 to 180-220 nm. Dextran 40 kDa modified THCPSi nanoparticles showed the highest stability in aqueous buffer. Both peptide- and polymer-functionalized THCPSi nanoparticles showed an extensive cellular uptake which was dependent on the functionalized moieties presented on the surface of the nanoparticles. The plasma protein adsorption study showed that the surface modification with different peptides or polymers induced different protein association profiles. Dextran 40 kDa functionalized THCPSi nanoparticles presented the least protein association. Overall, these results demonstrate that the "click" conjugation of the biomolecules onto the alkyne-terminated THCPSi nanoparticles is a versatile and simple approach to modulate the surface chemistry, which has high potential for biomedical applications.

Iminosugar-based glycopolypeptides: glycosidase inhibition with bioinspired glycoprotein analogue micellar self-assemblies.

Biomimetic nanoparticles prepared by self-assembly of iminosugar-based glycopolypeptides evidenced remarkable multivalency properties when inhibiting α-mannosidase activity. This approach paves the way to obtain biologically active drug delivery systems having glycosidase inhibition potency.

Synthesis and self-assembly of branched glycopolypeptides: effect of topology and conformation.

Block copolymers combining peptide and saccharide moieties may play a significant role in future applications of polymers in biology, as they can be viewed as simplified synthetic analogues of glycosylated proteins, which display a wide range of biological functions in nature. While a small number of oligosaccharides containing synthetic polypeptides have been described so far, here we present the efficient preparation of a small library of tree-like glycopolypeptides incorporating poly(gamma-benzyl-L-glutamate) having different molecular weights. A comprehensive study of the self-assembly of these unique grafted macromolecular structures is detailed. All the copolymers presented the ability to spontaneously self-assemble into spherical micelles in water; a property assigned both to the spontaneous curvature of the grafted oligosaccharide segments and to the favorable hydrophilic to hydrophobic volume ratios. These hydrophilic to hydrophobic volume ratios were efficiently counterbalanced by the self-assembly of blends incorporating poly(gamma-benzyl-L-glutamate) homopolymers - this simple approach minimized the influence of the spontaneous curvature of the oligosaccharides' dendrons and afforded a transition to lamellar structures in solution.

Synthesis and self-assembly of "tree-like" amphiphilic glycopolypeptides.

Novel synthetic tree-like oligosaccharides-grafted-polypeptides were prepared by using Huisgen 1,3-dipolar cycloaddition between poly(γ-benzyl-L-glutamate)-block-poly(propargylglycine) and two different oligosaccharides, dextran or hyaluronan. By direct solubilisation in water, these tree-like glycopeptides spontaneously form very small assemblies with sizes below 50 nm and low polydispersity.

Biologically active polymersomes from amphiphilic glycopeptides.

Polypeptide block copolymers with different block length ratios were obtained by sequential ring-opening polymerization of benzyl-L-glutamate and propargylglycine (PG) N-carboxyanhydrides. Glycosylation of the poly(PG) block was obtained by Huisgens cycloaddition "click" reaction using azide-functionalized galactose. All copolymers were self-assembled using the nanoprecipitation method to obtain spherical and wormlike micelles as well as polymersomes depending on the block length ratio and the nanoprecipitation conditions. These structures display bioactive galactose units in the polymersome shell, as proven by selective lectin binding experiments.

Preparation of protein- and cell-resistant surfaces by hyperthermal hydrogen induced cross-linking of poly(ethylene oxide).

The functionalization of surfaces with poly(ethylene oxide) (PEO) is an effective means of imparting resistance to the adsorption of proteins and the attachment and growth of cells, properties that are critical for many biomedical applications. In this work, a new hyperthermal hydrogen induced cross-linking (HHIC) method was explored as a simple one-step approach for attaching PEO to surfaces through the selective cleavage of C-H bonds and subsequent cross-linking of the resulting carbon radicals. In order to study the effects of the process on the polymer, PEO-coated silicon wafers were prepared and the effects of different treatment times were investigated. Subsequently, using an optimized treatment time and a modified butyl polymer with increased affinity for PEO, the technique was applied to butyl rubber surfaces. All of the treated surfaces exhibited significantly reduced protein adsorption and cell growth relative to control surfaces and compared favorably with surfaces that were functionalized with PEO using conventional chemical methods. Thus HHIC is a simple and effective means of attaching PEO to non-functional polymer surfaces.