Biomimetic artificial organelles with in vitro and in vivo activity triggered by reduction in microenvironment

Despite tremendous efforts to develop stimuli-responsive enzyme delivery systems, their efficacy has been mostly limited to in vitro applications. Here we introduce, by using an approach of combining biomolecules with artificial compartments, a biomimetic strategy to create artificial organelles (AOs) as cellular implants, with endogenous stimuli-triggered enzymatic activity. AOs are produced by inserting protein gates in the membrane of polymersomes containing horseradish peroxidase enzymes selected as a model for natures own enzymes involved in the redox homoeostasis. The inserted protein gates are engineered by attaching molecular caps to genetically modified channel porins in order to induce redox-responsive control of the molecular flow through the membrane. AOs preserve their structure and are activated by intracellular glutathione levels in vitro. Importantly, our biomimetic AOs are functional in vivo in zebrafish embryos, which demonstrates the feasibility of using AOs as cellular implants in living organisms. This opens new perspectives for patient-oriented protein therapy

Polymeric Nanocarriers and Nanoreactors : a Survey of Possible Therapeutic Applications

There is, today, great need for new systems and strategies for therapeutic applications that will lead to improvements in patient conditions and prognoses, especially in complex diseases such as neurodegenerative diseases and cancer. Recently, polymer nanocarriers have been developed to protect and transport active compounds to pathological sites more efficiently than free compounds in terms of stability, amount required, localization and efficacy. There are two strategies to deliver active compounds: conventional drug delivery systems based on transport and release of active compounds in biological compartments and nanoreactors that transport active compounds and permit them to act in situ, behaving like rudimentary artificial organelles. Here, we present both strategies with their advantages and limitations, and indicate how they can contribute to therapeutic improvement. We focus on presenting the design and development of polymer nanocarriers and nanoreactors as an essential stage of conceiving therapeutic approaches. The properties of the polymer carrier and its behavior under biological conditions dramatically influence the efficacy of the active compound, and thus of the treatment scheme. The key contributions that nanocarriers and nanoreactors could make include protecting active compounds from degradation in biological compartments other than those desired, and concentrating such compounds within their assemblage to allow for multiple deliveries in one single polymer assembly. To efficiently cope with the challenges of complex pathological conditions it is necessary to go one step beyond conventional drug delivery systems by designing and developing nanocarriers that mimic organelles, by combining various active molecules in a single carrier, and even by combining therapeutic agents along with agents for detection, as in a theragnostic approach

Polymer Nanoreactors

This article introduces protein-polymer systems that serve as chemical reaction spaces at the nanoscale. Such polymeric nanoreactors accommodate enzymatic reactions either inside polymer supramolecular assemblies or at their interfaces with the environment, or through a combination of both. We provide an overview of polymer structures that include micelles, vesicles (primarily polymersomes, but also PICsomes and peptosomes), dendrimers, layer-by-layer capsules, and capsids-all of which can accommodate sensitive biomolecules to mimic natural systems and functions. This article further deals with the functionalization of polymer membranes and the analytical techniques that apply to nanoreactors. In the last section, we highlight the most relevant applications of such polymeric nanoreactors in the domains of medicine, ecology, and biotechnology

Proteins 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

Selective and Responsive Nanoreactors

Chemical reactions can be confined to nanoscale compartments by encapsulating catalysts in hollow nanoobjects. Such reaction compartments effectively become nanoreactors when substrate and product are exchanged between bulk solution and cavity. A key issue, thereby, is control of shell permeability. Nanoreactors exhibit selectivity and responsiveness if their shells discriminate among molecules and if access can be modulated by external triggers. Here, we review natural nanoreactors that include protein-based bacterial microcompartments, protein cages, and viruses. Artificial nanoreactors based on dendrimers, layer-by-layer capsules, and amphiphilic block copolymer polymersomes are also discussed. Selectivity in these nanoreactors is either due to intrinsic reactor-shell semipermeability or can be engineered using smart polymers to gate the reactors. Moreover, a rich repertoire of pores and channels are already provided in nature, e. g., in protein-based nanoreactors or in trans-membrane channel proteins. The latter can be reconstituted in polymersomes, resulting in gated vesicles. Nanoreactors hold promise for applications ranging from selective and size-constrained organic synthesis to biomedical advances (e.g., artificial organelles, biosensing) and as analytical tools to study reaction mechanisms

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. (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

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

Protein-polymer nanoreactors for medical applications

Major challenges that confront nanoscience in medicine today include the development of efficacious therapies with minimum side effects, diagnostic methods featuring significantly higher sensitivities and selectivities, and personalized diagnostics and therapeutics for theragnostic approaches. With these goals in mind, combining biological molecules and synthetic carriers/templates, such as polymer supramolecular assemblies, represents a very promising strategy. In this critical review, we present protein-polymer systems as reaction spaces at the nano-scale in which the enzymatic reactions take place inside polymer supramolecular assembly, at its interface with the environment or in a combination of both. The location of the protein(s) with respect to the polymer assembly generates a diversity of systems ranging from nanoreactors to active enzymatic polymer surfaces. We describe these both in terms of general modelling and addressing the specific conditions and requirements related to the medical domain. We will particularly present protein-polymer nanoreactors that provide protected spaces for enzymatic reactions. We also show how polymer supramolecular structures, such as micelles, capsules, dendrimers and vesicles, can accommodate sensitive biomolecules to mimic natural systems and functions, and to serve as avenues for new medical approaches. Even though not yet on the market, we will emphasize possible applications of protein-polymer systems that generate reaction nanospaces-as novel ways to advanced medicine (264 references)

How to reduce superoxide anion concentration using antioxidant nanoreactors

In order to fight oxidative stress we developed antioxidant nanoreactors based on encapsulation of Cu/Zn superoxide dismutase or its mimics in amphiphilic copolymer nanovesicles whose membranes are oxygen permeable. The nanovesicles, made of poly-(2methyloxazoline)poly(dimethylsiloxane)-poly(2-methyloxazoline), successfully encapsulated the protein/mimics during their self-assembly process and were stable for more than one month. Testing with superoxide anions generated by pulse radiolysis demonstrated that the antioxidant agent remains active inside the nanovesicles and detoxifies the superoxide radical in situ. The triblock copolymer membrane of our nanovesicle plays a double role: both to shield the sensitive protein and to selectively allow superoxide and dioxygen to penetrate to the inner space of the vesicle. A simple and robust system, the antioxidant nanoreactor represents a new direction for developing medical applications for the treatment of oxidative stress