Ozana Onaca

Polymeric Vesicles : from Drug Carriers to Nanoreactors and Artificial Organelles

One strategy in modern medicine is the development of new platforms that combine multifunctional compounds with stable, safe carriers in patient-oriented therapeutic strategies. The simultaneous detection and treatment of pathological events through interactions manipulated at the molecular level offer treatment strategies that can decrease side effects resulting from conventional therapeutic approaches. Several types of nanocarriers have been proposed for biomedical purposes, including inorganic nanoparticles, lipid aggregates, including liposomes, and synthetic polymeric systems, such as vesicles, micelles, or nanotubes. Polymeric vesicles--structures similar to lipid vesicles but created using synthetic block copolymers--represent an excellent candidate for new nanocarriers for medical applications. These structures are more stable than liposomes but retain their low immunogenicity. Significant efforts have been made to improve the size, membrane flexibility, and permeability of polymeric vesicles and to enhance their target specificity. The optimization of these properties will allow researchers to design smart compartments that can co-encapsulate sensitive molecules, such as RNA, enzymes, and proteins, and their membranes allow insertion of membrane proteins rather than simply serving as passive carriers. In this Account, we illustrate the advances that are shifting these molecular systems from simple polymeric carriers to smart-complex protein-polymer assemblies, such as nanoreactors or synthetic organelles. Polymeric vesicles generated by the self-assembly of amphiphilic copolymers (polymersomes) offer the advantage of simultaneous encapsulation of hydrophilic compounds in their aqueous cavities and the insertion of fragile, hydrophobic compounds in their membranes. This strategy has permitted us and others to design and develop new systems such as nanoreactors and artificial organelles in which active compounds are simultaneously protected and allowed to act in situ. In recent years, we have created a variety of multifunctional, proteinpolymersomes combinations for biomedical applications. The insertion of membrane proteins or biopores into the polymer membrane supported the activity of co-encapsulated enzymes that act in tandem inside the cavity or of combinations of drugs and imaging agents. Surface functionalization of these nanocarriers permitted specific targeting of the desired biological compartments. Polymeric vesicles alone are relatively easy to prepare and functionalize. Those features, along with their stability and multifunctionality, promote their use in the development of new theranostic strategies. The combination of polymer vesicles and biological entities will serve as tools to improve the observation and treatment of pathological events and the overall condition of the patient.

Stimuli-responsive polymersomes as nanocarriers for drug and gene delivery.

Polymeric formulations (micelles, vesicles etc.) have emerged as versatile drug carriers due to their increased stability, site specificity, blood circulation resistance and thus overall potential therapeutic effects compared to liposomes. Furthermore, stimuli-responsive systems have been developed whose properties change after applying certain external triggers. Polymersomes are mainly composed of amphiphilic block copolymers that are held together in water due to strong physical interactions between the insoluble hydrophobic blocks, thus forming a bilayer morphology or, in the case of triblock copolymers, a bilayer-like morphology. Formation and destabilization of these assemblies is a consequence of external stimuli (temperature, pH, oxidation/reduction conditions etc.). This review focuses on recent developments concerning stimuli- responsive polymersomes made of amphiphilic block copolymers and their potential applications within the biomedical field.

Enzymatic Cascade Reactions inside Polymeric Nanocontainers: A Means to Combat Oxidative Stress

Oxidative stress, which is primarily due to an imbalance in reactive oxygen species, such as superoxide radicals, peroxynitrite, or hydrogen peroxide, represents a significant initiator in pathological conditions that range from arthritis to cancer. Herein we introduce the concept of enzymatic cascade reactions inside polymeric nanocontainers as an effective means to detect and combat superoxide radicals. By simultaneously encapsulating a set of enzymes that act in tandem inside the cavities of polymeric nanovesicles and by reconstituting channel proteins in their membranes, an efficient catalytic system was formed, as demonstrated by fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy. Superoxide dismutase and lactoperoxidase were selected as a model to highlight the combination of enzymes. These were shown to participate in sequential reactions in situ in the nanovesicle cavity, transforming superoxide radicals to molecular oxygen and water and, therefore, mimicking their natural behavior. A channel protein, outer membrane protein F, facilitated the diffusion of lactoperoxidase substrate/products and dramatically increased the penetration of superoxide radicals through the polymer membrane, as established by activity assays. The system remained active after uptake by THP-1 cells, thus behaving as an artificial organelle and exemplifying an effective approach to enzyme therapy.

Can polymeric vesicles that confine enzymatic reactions act as simplified organelles

In various pathological conditions an advantage may be gained by reinforcing an intrinsic organismal response. This can be achieved, for example, by enzyme replacement therapy, which can amplify specific, intrinsic activities of the organelles. In this respect, polymeric nanoreactors composed of vesicles that encapsulate an enzyme or a combination of enzymes in their cavities represent a novel approach in therapeutic applications because they behave like simplified organelles. As compartments, polymeric vesicles possess a membrane that is more stable than the corresponding lipid membrane of liposomes, with the dual role of protecting enzymes and simultaneously allowing them to act in situ. A complex scenario of requirements must be fulfilled by enzyme‐containing polymeric nanoreactors if they are to function under biological conditions and serve to model organelles. Nanoreactors are described here in terms of the existing models and the challenges faced in developing artificial organelles for therapeutic applications. We will focus on describing how polymeric vesicles can be used to provide a protected compartment for enzymatic reactions, and serve as simplified organelles inside cells.

SOD antioxidant nanoreactors: influence of block copolymer composition on the nanoreactor efficiency.

The bioavailability limitations of proteins make them difficult to be directly delivered, particularly in diseases caused by insufficient amounts or inactive variants of those proteins. Nanoreactors represent a new promising approach to overcome these limitations because they serve both to protect the protein in their aqueous interior, and simultaneously to allow the protein to act in situ. Here we examine an antioxidant nanoreactor based on SOD encapsulated in amphiphilic block copolymer nanovesicles, and analyze its behavior as a function of the copolymer composition. The membrane of the triblock copolymer nanovesicles plays a double role, both to shield the sensitive protein and selectively to let superoxide and dioxygen penetrate to its inner space. The encapsulation efficiency for different triblock copolymer vesicles was quantified by fluorescence correlation spectroscopy using a fluorescently labeled SOD. Pulse radiolysis experiments and an enzymatic assay were used to compare the permeability of the wall-forming membranes towards superoxide anions. While the encapsulation efficiency mainly depends on the vesicle dimensions, the membrane permeability is mainly affected by the length of the hydrophobic PDMS middle blocks of our polymers. For polymers with very long PDMS chains superoxide anion transport across the membranes was too slow to be detected by our experiments.

Protein delivery: from conventional drug delivery carriers to polymeric nanoreactors

Due to their low bioavailability, many naturally occurring proteins can not be used in their native form in diseases caused by insufficient amounts or inactive variants of those proteins. The strategy of delivering proteins to biological compartments using carriers represents the most promising approach to improve protein bioavailability. A large variety of systems have been developed to protect and deliver proteins, based on lipids, polymers or conjugates. Here we present the current progress of the carriers design criteria with the help of recent specific examples in the literature ranging from conventional liposomes to polymeric nanoreactors, with sizes from micrometer to nanometer scale, and having various morphologies. The design and optimisation of carriers in the dual way of addressing questions of a particular application and of keeping them very flexible and reliable for general applications represent an important step in protein delivery approaches, which influence considerably the therapeutic efficacy. We examine several options currently under exploration for creating suitable protein carriers, discuss their advantages and limitations that induced the need to develop alternative ways to deliver proteins to biological compartments. We consider that only tailored systems can serve to improve proteins bioavailability, and thus solve specific pathological situations. This can be accomplished by developing nanocarriers and nanoreactors based on biocompatible, biodegradable and non-toxic polymer systems, adapted sizes and surface properties, and multifunctionality to cope with the complexity of the in-vivo biological conditions.

Functionalized Nanocompartments (Synthosomes) with a Reduction‐Triggered Release System

Biologically derived compartments are constrained in design by their biological functions to ensure life at ambient temperature. Polymer vesicles can be designed to match application demands, such as mechanical stability, organic solvent, substrate and product tolerance, and permeation resistance, that are out of reach for biologically derived vesicles. Synthosomes use, in contrast to polymersomes, a transmembrane channel for controlling the in and out compound fluxes. The block copolymers in synthosomes prevent compound penetration through the polymer shell, whereas polymersomes depend on the diffusion of substrate and product molecules through the polymer shell. The main advantage of synthosomes over polymersomes is that, through protein engineering, it is possible to design functionalized protein channels. A protein channel that can function as an on/off switch offers opportunities for the design of functional nanocompartments with potential applications in synthetic biology (pathway engineering), medicine (drug release), and industrial biotechnology (chiral nanoreactors, multistep syntheses, bioconversions in nonaqueous environments, and selective product recovery). The channel proteins FhuA, OmpF, and Tsx have been incorporated, in functional active form, into blockcopolymer membranes. FhuA, ferric hydroxamate uptake protein component, is a large monomeric transmembrane protein of 714 amino acids folded into 22 antiparallel b strands and made up of two domains. Crystal structures of FhuA have been resolved, and a large passive diffusion channel (FhuA D1–160) was designed by removing a capping globular domain (deletion of amino acids 5–160). FhuA and Tsx were crystallized as monomers and OmpFas a trimer. FhuA and its engineered variants have a significantly wider channel than OmpF (OmpF 27–38 5, FhuA 39–46 5) and this allows even the translocation of single-stranded DNA. The aim and novelty of our work is the introduction of a triggering system, by means of a reduction-triggered “release switch” based on an engineered FhuA channel variant. To the best of our knowledge, in none of the reported triggered systems, was a channel protein employed as a switch. In fact, for polymersomes, a pH trigger, a temperatureassisted pH trigger, and a combined pH/salt trigger have been developed. Furthermore, hydrogen peroxide generation was used for polymer-vesicle degradation by glucose oxidase catalyzing glucose oxidation, and a pH-triggered release system for a polypeptide vesicle has been reported. For synthosomes, the activation of an encapsulated phosphatase after a change in the pH value has been reported. To build up a reduction-triggered release system in synthosomes, the amino-group-labeling agents 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (pyridyl label) and (2-[biotinamido]ethylamido)-3,3’-dithiodipropionic acid N-hydroxysuccinimide ester (biotinyl label) were selected, due to size considerations and the presence of a cleavable disulfide bond within the labeling reagents. Reagents for the specific labeling of amino, hydroxy, carboxyl, and sulfhydryl groups have been well studied and are routinely used for protein modifications. The synthosome calcein release system proposed herein is a triggered release system in which the entrapped compound (calcein) is liberated through an engineered transmembrane channel (FhuA D1–160) upon addition of a reducing agent. Interestingly, label size played an important role in calcein release. A detection protocol for calcein release from liposomes through wild-type FhuA and FhuA D1–160 has been reported. The liposomes were loaded with calcein at a selfquenching concentration (50 mm) and calcein release was achieved by addition of wild-type FhuA and FhuA D1–160. The fluorescence generation upon calcein release was used to record the release kinetics. In order to build a reduction-triggered release system, the amino groups of lysine residues in FhuA D1–160 were modified with either a pyridyl or a biotinyl label (see above). Figure 1 illustrates the reactions for FhuA D1–160 with eight lysine residues (L167, L226, L344, L364, L455, L537, L556, and L586) chemically modified with pyridyl (left) or biotinyl labels (right). Upon disulfide-bond reduction with DTT, a 3-thiopropionic amide group remains on the lysine residues of the FhuA D1–160 with both labels (Figure 1, upper part). Details [*] Dr. O. Onaca, P. Sarkar, Dr. D. Roccatano, A. G ven, Dr. M. Fioroni, Prof. Dr. U. Schwaneberg School of Engineering and Science, Jacobs University Bremen Campus Ring 8, 28759 Bremen (Germany) Fax: (+49)421-200-3543 E-mail: u.schwaneberg@jacobs-university.de

A surprising system: polymeric nanoreactors containing a mimic with dual-enzyme activity†

Reactive oxygen species have been implicated in various diseases, but attempts to find efficient antioxidant treatments for such conditions have met with only limited success. Here, we have developed an antioxidant nanoreactor by encapsulating a dual-enzyme mimic of superoxide dismutase and catalase, in polymeric nanovesicles and examined how this nanoreactor combats oxidative stress. The mimic (CuIIENZm) is encapsulated inside poly-(2-methyloxazoline)–poly-(dimethylsiloxane)–poly(2-methyloxazoline) polymer vesicles that feature membranes permeable to superoxide, enabling the enzyme mimic to act in situ. We ensured that the size and shape of polymeric vesicles were not changed during the encapsulation procedure by analysis with light scattering and transmission electron microscopy, and that the structural geometry of CuIIENZm was preserved, as demonstrated by electron paramagnetic resonance and UV-vis spectroscopy. Due to its bi-functionality, CuIIENZm detoxified both superoxide radicals and related H2O2. The intracellular localization of the nanoreactor in THP-1 cells was established using confocal laser scanning microscopy and flow cytometry. No evident toxicity was found using MTS and LDH assays. As CuIIENZm remained active inside the vesicles therefore, these CuIIENZm-containing nanoreactors exhibited efficient antioxidant activity in THP-1 cells. Development of this simple, robust antioxidant nanoreactor represents a new direction in efficiently fighting oxidative stress.

Nanocompartments with a pH release system based on an engineered OmpF channel protein

Synthosomes are a subclass of Polymersomes with a block copolymer membrane (PMOXA–PDMS–PMOXA) and a modified embedded transmembrane channel protein which acts as a selective gate. A synthosome based pH release system functioning increasing the pH from 5 to 7 was developed by introducing six histidine mutations to the OmpF constriction site (OmpF 6His). The pH-dependent compound release (acridine orange, positively charged between 5 ≤ pH ≤ 7) has been found to be tuned by the constriction site electrostatics and size by local structural modifications.