Tuning the membrane properties of functional polymersomes developed by aqueous polymerization-induced self-assembly (PISA)

Lipid bilayer membranes with precisely programmed properties play a vital role in most biological processes, enabling structural organization, confinement and communication on both a cellular and subcellular level. Self-assembled synthetic analogues, such as liposomes and polymersomes, have been widely investigated as simplified biomimetic models, providing a deeper understanding of fundamental membrane functions. The primary aim of this thesis was to explore novel methodologies for tuning the physicochemical membrane characteristics of functional polymersomes developed via aqueous polymerization-induced self-assembly (PISA) and highlight their potential application in biomimicry. In particular, PISA was employed for in situ preparation of block copolymer nano-objects at high concentrations throughout this thesis, owing to its robustness, versatility, and high reproducibility. Importantly, highly efficient strategies were established herein that afforded fine control over various properties of the prepared nanostructures, including the thickness, hydrophobicity, permeability, rigidity, and functionality of their membranes. Moreover, polymersomes of controllable size and shape could be also obtained depending on the experimental procedure followed. The outlined findings and identified trends were used for studying different communication and transport mechanisms within or between these nanocompartments, mediated either via passive diffusion of small molecules across selectively permeable membranes of catalytic nanoreactors or via intervesicular fusion events, and are expected to set the groundwork for future studies in biocatalysis and therapeutic delivery

Protein-, (Poly)peptide-, and Amino Acid-Based Nanostructures Prepared via Polymerization-Induced Self-Assembly

Proteins and peptides, built from precisely defined amino acid sequences, are an important class of biomolecules that play a vital role in most biological functions. Preparation of nanostructures through functionalization of natural, hydrophilic proteins/peptides with synthetic polymers or upon self-assembly of all-synthetic amphiphilic copolypept(o)ides and amino acid-containing polymers enables access to novel protein-mimicking biomaterials with superior physicochemical properties and immense biorelevant scope. In recent years, polymerization-induced self-assembly (PISA) has been established as an efficient and versatile alternative method to existing self-assembly procedures for the reproducible development of block copolymer nano-objects in situ at high concentrations and, thus, provides an ideal platform for engineering protein-inspired nanomaterials. In this review article, the different strategies employed for direct construction of protein-, (poly)peptide-, and amino acid-based nanostructures via PISA are described with particular focus on the characteristics of the developed block copolymer assemblies, as well as their utilization in various pharmaceutical and biomedical applications

Poly(Sarcosine)-Based Nano-Objects with Multi-Protease Resistance by Aqueous Photoinitiated Polymerization-Induced Self-Assembly (Photo-PISA)

Poly­(sarcosine) (PSar) is a non-ionic hydrophilic polypeptoid with numerous biologically relevant properties, making it an appealing candidate for the development of amphiphilic block copolymer nanostructures. In this work, the fabrication of poly­(sarcosine)-based diblock copolymer nano-objects with various morphologies via aqueous reversible addition–fragmentation chain-transfer (RAFT)-mediated photoinitiated polymerization-induced self-assembly (photo-PISA) is reported. Poly­(sarcosine) was first synthesized via ring-opening polymerization (ROP) of sarcosine N-carboxyanhydride, using high-vacuum techniques. A small molecule chain transfer agent (CTA) was then coupled to the active ω-amino chain end of the telechelic polymer for the synthesis of a poly­(sarcosine)-based macro-CTA. Controlled chain-extensions of a commercially available water-miscible methacrylate monomer (2-hydroxypropyl methacrylate) were achieved via photo-PISA under mild reaction conditions, using PSar macro-CTA. Upon varying the degree of polymerization and concentration of the core-forming monomer, morphologies evolving from spherical micelles to worm-like micelles and vesicles were accessed, as determined by dynamic light scattering and transmission electron microscopy, resulting in the construction of a detailed phase diagram. The resistance of both colloidally stable empty vesicles and enzyme-loaded nanoreactors against degradation by a series of proteases was finally assessed. Overall, our findings underline the potential of poly­(sarcosine) as an alternative corona-forming polymer to poly­(ethylene glycol)-based analogues of PISA assemblies for use in various pharmaceutical and biomedical applications

Permeable protein-loaded polymersome cascade nanoreactors by polymerization-induced self-assembly

Enzyme loading of polymersomes requires permeability to enable them to interact with the external environment, typically requiring addition of complex functionality to enable porosity. Herein, we describe a synthetic route toward intrinsically permeable polymersomes loaded with functional proteins using initiator-free visible light-mediated polymerization-induced self-assembly (photo-PISA) under mild, aqueous conditions using a commercial monomer. Compartmentalization and retention of protein functionality was demonstrated using green fluorescent protein as a macromolecular chromophore. Catalytic enzyme-loaded vesicles using horseradish peroxidase and glucose oxidase were also prepared and the permeability of the membrane toward their small molecule substrates was revealed for the first time. Finally, the interaction of the compartmentalized enzymes between separate vesicles was validated by means of an enzymatic cascade reaction. These findings have a broad scope as the methodology could be applied for the encapsulation of a large range of macromolecules for advancements in the fields of nanotechnology, biomimicry, and nanomedicine

Confinement of Therapeutic Enzymes in Selectively Permeable Polymer Vesicles by Polymerization-Induced Self-Assembly (PISA) Reduces Antibody Binding and Proteolytic Susceptibility

Covalent PEGylation of biologics has been widely employed to reduce immunogenicity, while improving stability and half-life in vivo. This approach requires covalent protein modification, creating a new entity. An alternative approach is stabilization by encapsulation into polymersomes; however this typically requires multiple steps, and the segregation requires the vesicles to be permeable to retain function. Herein, we demonstrate the one-pot synthesis of therapeutic enzyme-loaded vesicles with size-selective permeability using polymerization-induced self-assembly (PISA) enabling the encapsulated enzyme to function from within a confined domain. This strategy increased the proteolytic stability and reduced antibody recognition compared to the free protein or a PEGylated conjugate, thereby reducing potential dose frequency and the risk of immune response. Finally, the efficacy of encapsulated l-asparaginase (clinically used for leukemia treatment) against a cancer line was demonstrated, and its biodistribution and circulation behavior in vivo was compared to the free enzyme, highlighting this methodology as an attractive alternative to the covalent PEGylation of enzymes

Predicting monomers for use in aqueous ring-opening metathesis polymerization-induced self-assembly

Aqueous polymerization-induced self-assembly (PISA) is a well-established methodology enabling <i>in situ</i> synthesis of polymeric nanoparticles of controllable morphology. Notably, PISA <i>via</i> ring-opening metathesis polymerization (ROMPISA) is an emerging technology for block copolymer self-assembly, mainly due to its high versatility and robustness. However, a limited number of monomers suitable for core-forming blocks in aqueous ROMPISA have been reported to date. In this work, we identified seven monomers for use as either corona- or core-forming blocks during aqueous ROMPISA by <i>in silico</i> calculation of relative hydrophobicity for corresponding oligomeric models. The predicted monomers were validated experimentally by conducting ROMPISA using our previously reported two-step approach. In addition to predictive data, our computational model was exploited to identify trends between polymer hydrophobicity and the morphology of the self-assembled nano-objects they formed. We expect that this methodology will greatly expand the scope of aqueous ROMPISA, as monomers can be easily identified based on the structure–property relationships observed herein

pH-Sensitive nanogates based on poly(l-histidine) for controlled drug release from mesoporous silica nanoparticles

The design and synthesis of novel poly(l-histidine)-grafted mesoporous silica nanoparticles (MSNs) by a surface-initiated ring-opening polymerization process (ROP) is reported. Using (3-aminopropyl)triethoxysilane (APTES) to introduce primary amino groups onto the MSN outer surface that work as ROP initiators, the nanoparticles were decorated with a uniform pH-sensitive poly(l-histidine) (PHis) shell. The method applied for the MSN functionalization, guaranteed that PHis chains were not grafted inside the MSNs&apos; nanochannels. Successful grafting of the PHis chains was confirmed by FT-IR spectroscopy, TEM and TGA, while the controlled character of the polymerization was monitored by SEC analysis. Dynamic light scattering (DLS) and zeta potential analysis were used to reveal the pH-responsive nature of the polypeptide-gated MSNs. The role of the grafted PHis chains as pH-sensitive nanogates for the MSN pores was verified by drug loading and release studies, using the model anticancer drug doxorubicin (DOX). DOX was efficiently loaded within the nanochannels of the hybrid MSN@PHis nanostructures (approximately 90%), and was released in a relatively controlled pH-triggered manner. Overall, the described materials are promising candidates as nanocarriers for potential drug delivery applications. © The Royal Society of Chemistry 2016