Solution self-assembly of hybrid block copolymers containing poly(ethylene glycol) and amphiphilic peptide sequences

The self-assembly in aqueous solution of hybrid block copolymers consisting of amphiphilic ?-strand peptide sequences flanked by one or two PEG chains was investigated by means of circular dichroism spectroscopy, small-angle X-ray scattering, and transmission electron microscopy. In comparison with the native peptide sequence, it was found that the peptide secondary structure was stabilized against pH variation in the di- and tri-block copolymers with PEG. Small-angle X-ray scattering indicated the presence of fibrillar structures, the dimensions of which are comparable to the estimated width of a ?-strand (with terminal PEG chains in the case of the copolymers). Transmission electron microscopy on selectively stained and dried specimens shows directly the presence of fibrils. It is proposed that these fibrils result from the hierarchical self-assembly of peptide ?-strands into helical tapes, which then stack into fibrils. © 2005 American Chemical Society.

Solution behavior of peptide and peptide-hybrid materials

Harald Nuhn

Peptide based and peptide hybrid materials represent two interesting and challenging classes of biomaterials. Both classes consist of and contain, respectively, one or more polypeptide blocks, endowing the resulting polymers with enhanced self-assembly properties, biocompatibility or novel characteristics. Peptide based materials, solely comprised of amino acids, benefit from their advanced self-assembly properties. In the case of peptide hybrid materials, being conjugates of peptides and synthetic polymers, either the polypeptide or the synthetic polymer block can drive the self-assembly process. Furthermore, the latter class benefits from combining the advantages of peptides and synthetic polymers, thus overcoming limitations related to the use of the individual components. The application of these materials is still challenging because it is not possible to predict their final self-assembly behavior based on their design. In order to facilitate a target-oriented design of novel materials for application such as drug delivery, tissue engineering, or self-healing materials, a detailed understanding of the complex interactions within and between these molecules is essential. The work presented in this thesis addresses these fundamental questions. Over the last decades, a plethora of peptide and peptide hybrid materials has been reported. Chapter 1 gives an overview of selected articles describing different synthesis routes for peptide as well as peptide hybrid materials, showing examples of molecules and their self-assembly into superstructures, and highlighting recent applications. Chapter 2 describes the synthesis and characterization of different peptide hybrid diblock oligomers composed of two classes of peptide sequences covalently attached to a poly(ethylene glycol) (PEG) block. The first class of peptide sequences contains the intrinsic propensity to form coiled coils, whereas the second class is composed of "switch" peptides and is able to adopt both amphiphilic α-helical and β-strand conformations. Upon dissolution, the first class yielded superstructures comprised of dimeric and tetrameric coiled coil aggregates, whereas the second class formed fibrillar assemblies. In both cases, the self-assembly properties were driven by the peptide block, and the final superstructure consisted of a peptidic core wrapped by the synthetic polymer block. Chapter 3 presents a detailed study on the aqueous solution self-assembly of two poly(styrene)n-b-poly(L-lysine)m peptide hybrid oligomers. The aim was to clarify whether the observed rod-like micelles, obtained by direct dissolution of the diblock oligomers in aqueous solution, reflect intrinsic self-assembly properties of the hybrid diblock oligomers or whether they present structures that are energetically trapped due to the glassy nature of the polystyrene block. To address this question, the influence of a variety of sample preparation techniques, including the use of organic solvents, dialysis from an organic solvent and several thermal treatments, was investigated. All treatments resulted in solution aggregates that differed in size and shape from samples prepared by direct dissolution in aqueous solution. Thus, it seems likely that direct dissolution of polystyrene-b-poly(L-lysine) diblock oligomers in aqueous solution results in kinetically trapped, rod-like aggregates, which can be transformed into non-spherical micelles by the appropriate treatment. Chapter 4 contains a comprehensive study on peptide-based materials, investigating four different series of short elastin-like peptides (ELPs) based on the GVGVP motif. The objective was to test whether and how defined changes in the primary structure of short ELPs affect the secondary structure and the lower critical solution temperature (LCST) behavior. Therefore, the number of repeats was altered, and within one peptide of constant length, all valines were successively substituted by the more hydrophobic amino acids isoleucine, leucine and phenylalanine, respectively. In parallel, a theoretical approach based on the partition coefficient log P was used to predict the shift of the LCST as function of chain length and composition. For short ELPs, the LCST is strongly affected by changes in the primary structure. This has also been predicted by calculating log P. An increase in the hydrophobicity resulted in a shift of the LCST towards lower temperatures. Furthermore, it was shown for the first time that the sensitivity of short ELPs towards external factors, such as salt concentration, is determined by the intrinsic propensity of the incorporated amino acids to form either α-helices or β-strands. In summary, the obtained results on peptide based and peptide-hybrid materials provides deeper insights into their self-assembly characteristics and highlights the potential of these materials to be used for technical and medical applications.

EPFL2008

Combating HIV Type 1 with Peptide - Synthetic Polymer Conjugates

Maarten Danial

Polymer therapeutics is a class of (bio)hybrid materials that include a plethora of biologically active (synthetic) macromolecules that either consist of polymeric drugs, drug-polymer conjugates, peptide/protein-polymer conjugates or of drug, peptide or proteins covalently linked to self-assembled entities, such as polymer micelles and liposomes. Interest in polymer therapeutics has increased in the last decades particularly as anti-cancer therapeutics, influenza therapeutics, non-viral alternatives to gene delivery and in the advent of biological terrorism as anthrax inoculates. The human immunodeficiency virus (HIV) is one of the most deadly diseases known and despite improvement in education, preventative methods and drug therapies, the virus still affects approximately 33 million people worldwide. Classical therapeutics against HIV consist of low molecular weight drugs that inhibit various viral enzymes in the cell, including reverse transcriptase, integrase and protease. More recently, drugs that target the entry and fusion of the virus have formed a new class of inhibitors, some of which are effective (candidate) drugs. One of the fusion inhibitors that were approved is the drug T-20. T-20 is a 36-amino acid peptide derived from the heptad repeat 2 (HR2) of the HIV-1 glycoprotein gp41and acts as a competitive inhibitor by preventing the fusion of the viral and host cell membrane. Despite the effectiveness of T-20, one major drawback is associated with this drug namely low plasma lifetime. One of the goals of this Thesis is to design and synthesize effective HR2 derived fusion inhibitors through conjugation with synthetic polymers, while extending the lifetime. In addition, a novel concept for macromolecular HIV entry inhibition known as polyvalent inhibition is assessed in this Thesis. This Thesis consists of five chapters in which polymer therapeutics as carriers and polyvalent entities are explored against HIV-1. With the state of the art of HIV-1 therapeutics including polymer therapeutics summarized in Chapter 1, the remainder of the Thesis expands and defines the terrain with regard to novel (bio)hybrid materials as therapeutics against HIV-1. Very few polymer therapeutics that target HIV before cellular entry have been described. Even fewer polymer-based therapeutics inhibit HIV-1 entry via the specific interaction to the HIV-1 envelope proteins. In Chapter 2, novel polyvalent copolymers that specifically target the HIV-1 envelope protein gp120 were generated using a living polymerization technique known as ‘reversible addition-fragmentation chain transfer’ (RAFT), followed by a post-polymerization modification strategy. The strategy involves two synthetic steps, namely an ester/amide exchange followed by a thiol-ene addition of peptide ligands to generate a library of polyvalent copolymers, with varying peptide ligand, 2-hydroxypropyl methacrylamide and allyl methacrylamide content. These novel polyvalent polymers were shown to be effective HIV-1 entry inhibitors. Fusion inhibitors derived from the HR2 domain of gp41 such as T-20, act as competitive inhibitors by binding to the HR1 domain on the envelope glycoprotein gp41, which is involved in HIV-1 – host cell membrane fusion. However, the peptidic nature of these fusion inhibitors restricts their lifetime because they are prone to proteolytic degradation. Chapter 3 explores the effect of attaching synthetic polymer poly(ethylene glycol) (PEG) site-specifically to a HR2 derived peptide fusion inhibitor. The PEGylated peptides were assessed for their biophysical properties, efficacy as membrane fusion inhibitors and stability towards trypsin that was used as a model to show the effectiveness of the PEG to protect the peptide from proteolytic degradation. Although PEGylation causes a reduction in efficacy, this Chapter demonstrates that judicial PEGylation of the peptide can minimize the detrimental effects on inhibitory efficacy, while increasing lifetime against a protease. Conjugation of PEG and the site of PEG conjugation both have a profound effect on the efficacy of HR2 derived membrane fusion inhibitors. However, the effects on the mechanism of HIV-1 infectivity inhibition by polymer conjugated HR2 peptide inhibitors are not known. Chapter 4 investigates the effect of architecturally different synthetic linear PEG, v-shaped PEG or branched poly(glycerol) polymers conjugated to HR2 derived peptides. The peptide-polymer conjugates were then assessed for their inhibition efficacy, affinity and kinetics of binding towards a model of gp41, known as 5-Helix. The results indicate that attachment of a synthetic linear or branched polymer, despite causing a reduction in HIV-1 infectivity inhibition, does not significantly affect affinity towards 5-Helix. However, the rates of association and dissociation towards and from 5-Helix were reduced. Furthermore, synthetic polymer conjugation to HR2 derived peptides prolong their lifetime bound to gp41 and thus potentially reduces HIV-1 inhibition escape. Therefore, this Chapter demonstrates a previously unknown advantage of polymer conjugation to HIV-1 fusion peptide inhibitors. Chapter 5 systematically explores the modification of hydrophilic residues on the hydrophobic face of a HR2 derived peptide. The peptide mutants were assessed for their efficacy against membrane fusion inhibition as well as against fusion of HIV-1 wild type and T-20 resistant variants. Although the peptide mutants did not exhibit any improvement towards the inhibition of wild type membrane fusion, selected peptide mutants with point mutations were shown to be effective inhibitors towards T-20 resistant HIV-1 strains. In conclusion, peptide-polymer hybrid materials can make excellent candidates as long lasting therapeutics against wild type HIV-1 but also against emergent HIV-1 mutant variants. The knowledge from these Chapters will aid the design of innovative, next generation of peptide-polymer hybrid therapeutics.

EPFL2011

Water-soluble, unimolecular containers based on amphiphilic multiarm star block copolymers

Harm-Anton Klok, Jan-Anders Månson, John Christopher Plummer, Céline Ternat

A new class of water-soluble, amphiphilic star block copolymers with a large number of arms was prepared by sequential atom transfer radical polymerization (ATRP) of n-butyl methacrylate (BMA) and poly-(ethylene glycol) methyl ether methacrylate (PEGMA). As the macroinitiator for the ATRP, a 2-bromoisobutyric acid functionalized fourth-generation hyperbranched polyester (Boltorn H40) was used, which allowed the preparation of star polymers that contained on average 20 diblock copolymer arms. The synthetic concept was validated by AFM experiments, which allowed direct visualization of single molecules of the multiarm star block copolymers. DSC and SAXS experiments on bulk samples suggested a microphase-separated structure, in agreement with the core-shell architecture of the polymers. SAXS experiments on aqueous solutions indicated that the star block copolymers can be regarded as unimolecular micelles composed of a PBMA core and a diffuse PPEGMA corona. The ability of the polymers to encapsulate and release hydrophobic guests was evaluated using 1H NMR spectroscopy. In dilute aqueous solution, these polymers act as unimolecular containers that can be loaded with up to 27 wt % hydrophobic guest molecules. © 2006 American Chemical Society.