Synthetic Biology: Bottom-Up Assembly of Molecular Systems.

The bottom-up assembly of biological and chemical components opens exciting opportunities to engineer artificial vesicular systems for applications with previously unmet requirements. The modular combination of scaffolds and functional building blocks enables the engineering of complex systems with biomimetic or new-to-nature functionalities. Inspired by the compartmentalized organization of cells and organelles, lipid or polymer vesicles are widely used as model membrane systems to investigate the translocation of solutes and the transduction of signals by membrane proteins. The bottom-up assembly and functionalization of such artificial compartments enables full control over their composition and can thus provide specifically optimized environments for synthetic biological processes. This review aims to inspire future endeavors by providing a diverse toolbox of molecular modules, engineering methodologies, and different approaches to assemble artificial vesicular systems. Important technical and practical aspects are addressed and selected applications are presented, highlighting particular achievements and limitations of the bottom-up approach. Complementing the cutting-edge technological achievements, fundamental aspects are also discussed to cater to the inherently diverse background of the target audience, which results from the interdisciplinary nature of synthetic biology. The engineering of proteins as functional modules and the use of lipids and block copolymers as scaffold modules for the assembly of functionalized vesicular systems are explored in detail. Particular emphasis is placed on ensuring the controlled assembly of these components into increasingly complex vesicular systems. Finally, all descriptions are presented in the greater context of engineering valuable synthetic biological systems for applications in biocatalysis, biosensing, bioremediation, or targeted drug delivery.

Multicomponent Copolymer Planar Membranes with Nanoscale Domain Separation.

Domain separation is crucial for proper cellular function and numerous biomedical technologies, especially artificial cells. While phase separation in hybrid membranes containing lipids and copolymers is well-known, the membranes' overall stability, limited by the lipid part, is hindering the technological applications. Here, we introduce a fully synthetic planar membrane undergoing phase separation into domains embedded within a continuous phase. The mono- and bilayer membranes are composed of two amphiphilic diblock copolymers (PEO45-b-PEHOx20 and PMOXA10-b-PDMS25) with distinct properties and mixed at various concentrations. The molar ratio of the copolymers in the mixture and the nature of the solid support were the key parameters inducing nanoscale phase separation of the planar membranes. The size of the domains and resulting morphology of the nanopatterned surfaces were tailored by adjusting the molar ratios of the copolymers and transfer conditions. Our approach opens new avenues for the development of biomimetic planar membranes with a nanoscale texture.

Block Length-Dependent Protein Fouling on Poly(2-oxazoline)-Based Polymersomes: Influence on Macrophage Association and Circulation Behavior.

Polymersomes are vesicular structures self-assembled from amphiphilic block copolymers and are considered an alternative to liposomes for applications in drug delivery, immunotherapy, biosensing, and as nanoreactors and artificial organelles. However, the limited availability of systematic stability, protein fouling (protein corona formation), and blood circulation studies hampers their clinical translation. Poly(2-oxazoline)s (POx) are valuable antifouling hydrophilic polymers that can replace the current gold-standard, poly(ethylene glycol) (PEG), yet investigations of POx functionality on nanoparticles are relatively sparse. Herein, a systematic study is reported of the structural, dynamic and antifouling properties of polymersomes made of poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA). The study relates in vitro antifouling performance of the polymersomes to atomistic molecular dynamics simulations of polymersome membrane hydration behavior. These observations support the experimentally demonstrated benefit of maximizing the length of PMOXA (degree of polymerization (DP) > 6) while keeping PDMS at a minimal length that still provides sufficient membrane stability (DP > 19). In vitro macrophage association and in vivo blood circulation evaluation of polymersomes in zebrafish embryos corroborate these findings. They further suggest that single copolymer presentation on polymersomes is outperformed by blends of varied copolymer lengths. This study helps to rationalize design rules for stable and low-fouling polymersomes for future medical applications.

Tailoring a Solvent-Assisted Method for Solid-Supported Hybrid Lipid-Polymer Membranes.

Combining amphiphilic block copolymers and phospholipids opens new opportunities for the preparation of artificial membranes. The chemical versatility and mechanical robustness of polymers together with the fluidity and biocompatibility of lipids afford hybrid membranes with unique properties that are of great interest in the field of bioengineering. Owing to its straightforwardness, the solvent-assisted method (SA) is particularly attractive for obtaining solid-supported membranes. While the SA method was first developed for lipids and very recently extended to amphiphilic block copolymers, its potential to develop hybrid membranes has not yet been explored. Here, we tailor the SA method to prepare solid-supported polymer-lipid hybrid membranes by combining a small library of amphiphilic diblock copolymers poly(dimethyl siloxane)-poly(2-methyl-2-oxazoline) and poly(butylene oxide)-block-poly(glycidol) with phospholipids commonly found in cell membranes including 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, sphingomyelin, and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl). The optimization of the conditions under which the SA method was applied allowed for the formation of hybrid polymer-lipid solid-supported membranes. The real-time formation and morphology of these hybrid membranes were evaluated using a combination of quartz crystal microbalance and atomic force microscopy. Depending on the type of polymer-lipid combination, significant differences in membrane coverage, formation of domains, and quality of membranes were obtained. The use of the SA method for a rapid and controlled formation of solid-supported hybrid membranes provides the basis for developing customized artificial hybrid membranes.

Inverting glucuronidation of hymecromone in situ by catalytic nanocompartments.

Glucuronidation is a metabolic pathway that inactivates many drugs including hymecromone. Adverse effects of glucuronide metabolites include a reduction of half-life circulation times and rapid elimination from the body. Herein, we developed synthetic catalytic nanocompartments able to cleave the glucuronide moiety from the metabolized form of hymecromone in order to convert it to the active drug. By shielding enzymes from their surroundings, catalytic nanocompartments favor prolonged activity and lower immunogenicity as key aspects to improve the therapeutic solution. The catalytic nanocompartments (CNCs) consist of self-assembled poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) diblock copolymer polymersomes encapsulating ß-glucuronidase. Insertion of melittin in the synthetic membrane of these polymersomes provided pores for the diffusion of the hydrophilic hymecromone-glucuronide conjugate to the compartment inside where the encapsulated ß-glucuronidase catalyzed its conversion to hymecromone. Our system successfully produced hymecromone from its glucuronide conjugate in both phosphate buffered solution and cell culture medium. CNCs were non-cytotoxic when incubated with HepG2 cells. After being taken up by cells, CNCs produced the drug in situ over 24 hours. Such catalytic platforms, which locally revert a drug metabolite into its active form, open new avenues in the design of therapeutics that aim at prolonging the residence time of a drug.

Fully amorphous atactic and isotactic block copolymers and their self-assembly into nano- and microscopic vesicles.

The introduction of chirality into aqueous self-assemblies by employing isotactic block copolymers (BCPs) is an emerging field of interest as it promises special membrane properties of polymersomes not accessible by atactic BCPs. However, isotactic BCPs typically exhibit crystalline behaviour, inducing high membrane stiffness and limiting their applicability in systems involving membrane proteins or sensitive cargo. In this study, an isotactic yet fully amorphous BCP is introduced which overcomes these limitations. Three BCPs composed of poly(butylene oxide)-block-poly(glycidol) (PBO-b-PG), differing solely in their tacticities (R/S, R and S), were synthesised and characterised regarding their structural, optical and thermal properties. Their self-assembly into homogenous phases of nanoscopic polymersomes (referred to as small unilamellar vesicles, SUVs) was analysed, revealing stability differences between SUVs composed of the different BCPs. Additionally, microscopic giant unilamellar vesicles (GUVs) were prepared by double emulsion microfluidics. Only the atactic BCP formed GUVs which were stable over several hours, whereas GUVs composed of isotactic BCPs ruptured within several minutes after formation. The ability of atactic PBO-b-PG to form microreactors was elucidated by reconstituting the membrane protein OmpF in the GUV membrane by microfluidics and performing an enzyme reaction inside its lumen. The system presented here serves as platform to design versatile vesicles with flexible membranes composed of atactic or isotactic BCPs. Hence, they allow for the introduction of chirality into nano- or microreactors which is a yet unstudied field and could enable special biotechonological applications.

Expanding the Potential of the Solvent-Assisted Method to Create Bio-Interfaces from Amphiphilic Block Copolymers.

Artificial membranes, as materials with biomimetic properties, can be applied in various fields, such as drug screening or bio-sensing. The solvent-assisted method (SA) represents a straightforward method to prepare lipid solid-supported membranes. It overcomes the main limitations of established membrane preparation methods, such as Langmuir-Blodgett (LB) or vesicle fusion. However, it has not yet been applied to create artificial membranes based on amphiphilic block copolymers, despite their enhanced mechanical stability compared to lipid-based membranes and bio-compatible properties. Here, we applied the SA method on different amphiphilic di- and triblock poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA) copolymers and optimized the conditions to prepare artificial membranes on a solid support. The real-time membrane formation, the morphology, and the mechanical properties have been evaluated by a combination of atomic force microscopy and quartz crystal microbalance. Then, selected biomolecules including complementary DNA strands and an artificial deallylase metalloenzyme (ADAse) were incorporated into these membranes relying on the biotin-streptavidin technology. DNA strands served to establish the capability of these synthetic membranes to interact with biomolecules by preserving their correct conformation. The catalytic activity of the ADAse following its membrane anchoring induced the functionality of the biomimetic platform. Polymer membranes on solid support as prepared by the SA method open new opportunities for the creation of artificial membranes with tailored biomimetic properties and functionality.

The influence of decision-making in tree ring-based climate reconstructions.

Tree-ring chronologies underpin the majority of annually-resolved reconstructions of Common Era climate. However, they are derived using different datasets and techniques, the ramifications of which have hitherto been little explored. Here, we report the results of a double-blind experiment that yielded 15 Northern Hemisphere summer temperature reconstructions from a common network of regional tree-ring width datasets. Taken together as an ensemble, the Common Era reconstruction mean correlates with instrumental temperatures from 1794-2016 CE at 0.79 (p < 0.001), reveals summer cooling in the years following large volcanic eruptions, and exhibits strong warming since the 1980s. Differing in their mean, variance, amplitude, sensitivity, and persistence, the ensemble members demonstrate the influence of subjectivity in the reconstruction process. We therefore recommend the routine use of ensemble reconstruction approaches to provide a more consensual picture of past climate variability.

From spherical compartments to polymer films: exploiting vesicle fusion to generate solid supported thin polymer membranes.

Solid supported polymer membranes as scaffold for the insertion of functional biomolecules provide the basis for mimicking natural membranes. They also provide the means for unraveling biomolecule-membrane interactions and engineering platforms for biosensing. Vesicle fusion is an established procedure to obtain solid supported lipid bilayers but the more robust polymer vesicles tend to resist fusion and planar membranes rarely form. Here, we build on vesicle fusion to develop a refined and efficient way to produce solid supported membranes based on poly(dimethylsiloxane)-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA) amphiphilic triblock copolymers. We first create thiol-bearing polymer vesicles (polymersomes) and anchor them on a gold substrate. An osmotic shock then provokes polymersome rupture and drives planar film formation. Prerequisite for a uniform amphiphilic planar membrane is the proper combination of immobilized polymersomes and osmotic shock conditions. Thus, we explored the impact of the hydrophobic PDMS block length of the polymersome on the formation and the characteristics of the resulting solid supported polymer assemblies by quarz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM) and spectroscopic ellipsometry (SE). When the PDMS block is short enough, attached polymersomes restructure in response to osmotic shock, resulting in a uniform planar membrane. Our approach to rapidly form planar polymer membranes by vesicle fusion brings many advantages to the development of synthetic planar membranes for bio-sensing and biotechnological applications.

Stabilizing Enzymes within Polymersomes by Coencapsulation of Trehalose.

Enzymes are essential biocatalysts and very attractive as therapeutics. However, their functionality is strictly related to their stability, which is significantly affected by the environmental changes occurring during their usage or long-term storage. Therefore, maintaining the activity of enzymes is essential when they are exposed to high temperature during usage or when they are stored for extended periods of time. Here, we stabilize and protect enzymes by coencapsulating them with trehalose into polymersomes. The anhydrobiotic disaccharide preserved up to about 81% of the enzyme's original activity when laccase/trehalose-loaded nanoreactors were kept desiccated for 2 months at room temperature and 75% of its activity when heated at 50 °C for 3 weeks. Moreover, the applicability of laccase/trehalose-loaded nanoreactors as catalysts for bleaching of the textile dyes orange G, toluidine blue O, and indigo was proven. Our results demonstrate the advantages of coencapsulating trehalose within polymersomes to stabilize enzymes in dehydrated state for extended periods of time, preserving their activity even when heated to elevated temperature.

Metal cation responsive anionic microgels: behaviour towards biologically relevant divalent and trivalent ions.

Anionic poly(vinylcaprolactam-co-itaconicacid-co-dimethylitaconate) microgels were synthesized via dispersion polymerization and their responsiveness towards cations, namely Mg2+, Sr2+, Cu2+ and Fe3+, was investigated. The itaconic moieties chelate the metal ions which act as a crosslinker and decrease the electrostatic repulsion within the network, leading to a decrease in the gel size. The responsiveness towards the metal ion concentration has been studied via dynamic light scattering (DLS) and the number of ions bonded within the network has been quantified with ion chromatography. Through the protonation of the carboxylate groups in the gel network, their interaction with the cations is significantly lowered, and the metals are consequently released back in solution. The number of ions released was assessed also via ion chromatography for all four ions, whilst Mg2+ was also used as a model ion to display the reversibility of the system. The microgels can bond and release divalent cations over multiple cycles without undergoing any loss of functionality. Moreover, these gels also selectively entrap Fe3+ with respect to the remaining divalent cations, opening the possibility of using the proposed gels in the digestive tract as biocompatible chelating agents to fight iron overaccumulation.

Combinatorial Strategy for Studying Biochemical Pathways in Double Emulsion Templated Cell-Sized Compartments.

Cells rely upon producing enzymes at precise rates and stoichiometry for maximizing functionalities. The reasons for this optimal control are unknown, primarily because of the interconnectivity of the enzymatic cascade effects within multi-step pathways. Here, an elegant strategy for studying such behavior, by controlling segregation/combination of enzymes/metabolites in synthetic cell-sized compartments, while preserving vital cellular elements is presented. Therefore, compartments shaped into polymer GUVs are developed, producing via high-precision double-emulsion microfluidics that enable: i) tight control over the absolute and relative enzymatic contents inside the GUVs, reaching nearly 100% encapsulation and co-encapsulation efficiencies, and ii) functional reconstitution of biopores and membrane proteins in the GUVs polymeric membrane, thus supporting in situ reactions. GUVs equipped with biopores/membrane proteins and loaded with one or more enzymes are arranged in a variety of combinations that allow the study of a three-step cascade in multiple topologies. Due to the spatiotemporal control provided, optimum conditions for decreasing the accumulation of inhibitors are unveiled, and benefited from reactive intermediates to maximize the overall cascade efficiency in compartments. The non-system-specific feature of the novel strategy makes this system an ideal candidate for the development of new synthetic routes as well as for screening natural and more complex pathways.

Recent Advances in Hybrid Biomimetic Polymer-Based Films: from Assembly to Applications.

Biological membranes, in addition to being a cell boundary, can host a variety of proteins that are involved in different biological functions, including selective nutrient transport, signal transduction, inter- and intra-cellular communication, and cell-cell recognition. Due to their extreme complexity, there has been an increasing interest in developing model membrane systems of controlled properties based on combinations of polymers and different biomacromolecules, i.e., polymer-based hybrid films. In this review, we have highlighted recent advances in the development and applications of hybrid biomimetic planar systems based on different polymeric species. We have focused in particular on hybrid films based on (i) polyelectrolytes, (ii) polymer brushes, as well as (iii) tethers and cushions formed from synthetic polymers, and (iv) block copolymers and their combinations with biomacromolecules, such as lipids, proteins, enzymes, biopolymers, and chosen nanoparticles. In this respect, multiple approaches to the synthesis, characterization, and processing of such hybrid films have been presented. The review has further exemplified their bioengineering, biomedical, and environmental applications, in dependence on the composition and properties of the respective hybrids. We believed that this comprehensive review would be of interest to both the specialists in the field of biomimicry as well as persons entering the field.

Polymer-Lipid Hybrid Membranes as a Model Platform to Drive Membrane-Cytochrome c Interaction and Peroxidase-like Activity.

Controllable attachment of proteins to material surfaces is very attractive for many applications including biosensors, bioengineered scaffolds or drug screening. Especially, redox proteins have received considerable attention as a model system not only to understand the mechanism of electron transfer in biological systems, but also the development of novel biosensors. However, current research attempts suffer from denaturation of the protein after its attachment to solid substrates. Here, we present how lipid, polymer and hybrid membranes based on mixtures of lipids and copolymers on a solid support provide a more favorable environment to drive selective and functional attachment of a model redox protein, cytochrome c (cyt c). Polymer membranes provided chemical versatility to support covalent attachment of cyt c, whereas lipid membranes provided flexibility and biocompatibility to support insertion of cyt c through its hydrophobic part. Hybrid membranes combine the most promising characteristics of both lipids and polymers and allowed attachment of cyt c with both covalent attachment and insertion driven by hydrophobic interactions. We then investigated the effect of different attachment strategies on the accessibility and peroxidase-like activity of cyt c, in the presence of different membranes. The real-time combination of cyt c with the planar membranes was investigated by quartz crystal microbalance with dissipation. It was possible to selectively drive the insertion of cyt c into a specific lipid domain of hybrid membranes. In addition, protein accessibility and its functionality were dependent on the specificity of the combination strategy: covalent conjugation of cyt c to polymer and hybrid membranes promoted higher accessibility and supported higher peroxidase-like activity. Taking together, the combination of biomolecules with planar membranes can be modulated in such a way to improve the accessibility of the biomolecules and their resulting functionality for the development of efficient "active surfaces".

Bioinspired Molecular Factories with Architecture and In Vivo Functionalities as Cell Mimics.

Despite huge need in the medical domain and significant development efforts, artificial cells to date have limited composition and functionality. Although some artificial cells have proven successful for producing therapeutics or performing in vitro specific reactions, they have not been investigated in vivo to determine whether they preserve their architecture and functionality while avoiding toxicity. Here, these limitations are overcome and customizable cell mimic is achieved-molecular factories (MFs)-by supplementing giant plasma membrane vesicles derived from donor cells with nanometer-sized artificial organelles (AOs). MFs inherit the donor cell's natural cytoplasm and membrane, while the AOs house reactive components and provide cell-like architecture and functionality. It is demonstrated that reactions inside AOs take place in a close-to-nature environment due to the unprecedented level of complexity in the composition of the MFs. It is further demonstrated that in a zebrafish vertebrate animal model, these cell mimics show no apparent toxicity and retain their integrity and function. The unique advantages of highly varied composition, multicompartmentalized architecture, and preserved functionality in vivo open new biological avenues ranging from the study of biorelevant processes in robust cell-like environments to the production of specific bioactive compounds.

Deepening the insight into poly(butylene oxide)-block-poly(glycidol) synthesis and self-assemblies: micelles, worms and vesicles.

Aqueous self-assembly of amphiphilic block copolymers is studied extensively for biomedical applications like drug delivery and nanoreactors. The commonly used hydrophilic block poly(ethylene oxide) (PEO), however, suffers from several drawbacks. As a potent alternative, poly(glycidol) (PG) has gained increasing interest, benefiting from its easy synthesis, high biocompatibility and flexibility as well as enhanced functionality compared to PEO. In this study, we present a quick and well-controlled synthesis of poly(butylene oxide)-block-poly(glycidol) (PBO-b-PG) amphiphilic diblock copolymers together with a straight-forward self-assembly protocol. Depending on the hydrophilic mass fraction of the copolymer, nanoscopic micelles, worms and polymersomes were formed as well as microscopic giant unilamellar vesicles. The particles were analysed regarding their size and shape, using dynamic and static light scattering, TEM and Cryo-TEM imaging as well as confocal laser scanning microscopy. We have discovered a strong dependence of the formed morphology on the self-assembly method and show that only solvent exchange leads to the formation of homogenous phases. Thus, a variety of different structures can be obtained from a highly flexible copolymer, justifying a potential use in biomedical applications.

Decorating Nanostructured Surfaces with Antimicrobial Peptides to Efficiently Fight Bacteria.

With conventional antibiotic therapies being increasingly ineffective, bacterial infections with subsequent biofilm formation represent a global threat to human health. Here, an active and a passive strategy based on polymeric micelles were combined to fight bacterial growth. The passive strategy involved covalent immobilization of polymeric micelles through Michael addition between exposed maleimide and thiol functionalized surfaces. Compared to the bare surface, micelle-decorated surfaces showed reduced adherence and survival of bacteria. To extend this passive defense against bacteria with an active strategy, the immobilized micelles were equipped with the antimicrobial peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR). The peptide interacted nonspecifically with the immobilized micelles where it retained its antimicrobial property. The successful surface decoration with KYE28 was demonstrated by a combination of X-ray photoelectron spectroscopy and quartz crystal microbalance with dissipation monitoring. The initial antimicrobial activity of the nanostructured surfaces against Escherichia coli was found to be increased by the presence of KYE28. The combination of the active and passive strategy represents a straightforward modular approach that can easily be adapted, for example, by exchanging the antimicrobial peptide to optimize potency against challenging bacterial strains, and/or to simultaneously achieve antimicrobial and anti-infection properties.

Self-Assembled Polymeric Membranes and Nanoassemblies on Surfaces: Preparation, Characterization, and Current Applications.

Biomembranes play a crucial role in a multitude of biological processes, where high selectivity and efficiency are key points in the reaction course. The outstanding performance of biological membranes is based on the coupling between the membrane and biomolecules, such as membrane proteins. Polymer-based membranes and assemblies represent a great alternative to lipid ones, as their presence not only dramatically increases the mechanical stability of such systems, but also opens the scope to a broad range of chemical functionalities, which can be fine-tuned to selectively combine with a specific biomolecule. Tethering the membranes or nanoassemblies on a solid support opens the way to a class of functional surfaces finding application as sensors, biocomputing systems, molecular recognition, and filtration membranes. Herein, the design, physical assembly, and biomolecule attachment/insertion on/within solid-supported polymeric membranes and nanoassemblies are presented in detail with relevant examples. Furthermore, the models and applications for these materials are highlighted with the recent advances in each field.

How Do the Properties of Amphiphilic Polymer Membranes Influence the Functional Insertion of Peptide Pores?

Pore-forming peptides are of high biological relevance particularly as cytotoxic agents, but their properties are also applicable for the permeabilization of lipid membranes for biotechnological applications, which can then be translated to the more stable and versatile polymeric membranes. However, their interactions with synthetic membranes leading to pore formation are still poorly understood, hampering the development of peptide-based nanotechnological applications, such as biosensors or catalytic compartments. To elucidate these interactions, we chose the model peptide melittin, the main component of bee venom. Here, we present our systematic investigation on how melittin interacts with and inserts into synthetic membranes, based on amphiphilic block copolymers, to induce pore formation in three different setups (planar membranes and micrometric and nanometric vesicles). By varying selected molecular properties of block copolymers and resulting membranes (e.g., hydrophilic to hydrophobic block ratio, membrane thickness, surface roughness, and membrane curvature) and the stage of melittin addition to the synthetic membranes, we gained a deeper understanding of melittin insertion requirements. In the case of solid-supported planar membranes, melittin interaction was favored by membrane roughness and thickness, but its insertion and pore formation were hindered when the membrane was excessively thick. The additional property provided by micrometric vesicles, curvature, increased the functional insertion of melittin, which was evidenced by the even more curved nanometric vesicles. Using nanometric vesicles allowed us to estimate the pore size and density, and by changing the stage of melittin addition, we overcame the limitations of peptide-polymer membrane interaction. Mirroring the functionality assay of planar membranes, we produced glucose-sensing vesicles. The design of synthetic membranes permeabilized with melittin opens a new path toward the development of biosensors and catalytic compartments based on pore-forming peptides functionally inserted in synthetic planar or three-dimensional membranes.

Porphyrin Containing Polymersomes with Enhanced ROS Generation Efficiency: In Vitro Evaluation.

Porphyrins are molecules possessing unique photophysical properties making them suitable for application in photodynamic therapy. The incorporation of porphyrins into natural or synthetic nano-assemblies such as polymersomes is a strategy to improve and prolong their therapeutic capacities and to overcome their limitations as therapeutic and diagnostic agents. Here, 5,10,15,20-tetrakis(1-(6-ethoxy-6-oxohexyl)-4-pyridin-1-io)-21H,23H-porphyrin tetrabromide porphyrin is inserted into polymersomes in order to demonstrate that the encapsulation enhances its ability to generate highly reactive singlet oxygen (1 O2 ) upon irradiation in vitro. The photoactivation of the free and polymersome-encapsulated porphyrin is evaluated by electron spin resonance and cell viability assays on three different mammalian cell lines. The results indicate that by encapsulating the porphyrin, a controlled ROS delivery within the cells is achieved, at the same time avoiding side effects such as dark toxicity, non-specific porphyrin release and over time decreased activity in vitro. This work focuses on showing a not-toxic model system for modern therapeutic nanomedicine, which works under mild irradiation and dosage conditions.