Cornelia G. Palivan

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 Polymers and Their Applications in Nanomedicine

This review focuses on smart nano-materials built of stimuli-responsive (SR) polymers and will discuss their numerous applications in the biomedical field. The authors will first provide an overview of different stimuli and their corresponding, responsive polymers. By introducing myriad functionalities, SR polymers present a wide range of possibilities in the design of stimuli-responsive devices, making use of virtually all types of polymer constructs, from self-assembled structures (micelles, vesicles) to surfaces (polymer brushes, films) as described in the second section of the review. In the last section of this review the authors report on some of the most promising applications of stimuli-responsive polymers in nanomedicine. In particular, we will discuss applications pertaining to diagnosis, where SR polymers are used to construct sensors capable of selective recognition and quantification of analytes and physical variables, as well as imaging devices. We will also highlight some examples of responsive systems used for therapeutic applications, including smart drug delivery systems (micelles, vesicles, dendrimers …) and surfaces for regenerative medicine.

Biocompatible Functionalization of Polymersome Surfaces: A New Approach to Surface Immobilization and Cell Targeting Using Polymersomes

Vesicles assembled from amphiphilic block copolymers represent promising nanomaterials for applications that include drug delivery and surface functionalization. One essential requirement to guide such polymersomes to a desired site in vivo is conjugation of active, targeting ligands to the surface of preformed self-assemblies. Such conjugation chemistry must fulfill criteria of efficiency and selectivity, stability of the resulting bond, and biocompatibility. We have here developed a new system that achieves these criteria by simple conjugation of 4-formylbenzoate (4FB) functionalized polymersomes with 6-hydrazinonicotinate acetone hydrazone (HyNic) functionalized antibodies in aqueous buffer. The number of available amino groups on the surface of polymersomes composed of poly(dimethylsiloxane)-block-poly(2-methyloxazoline) diblock copolymers was investigated by reacting hydrophilic succinimidyl-activated fluorescent dye with polymersomes and evaluating the resulting emission intensity. To prove attachment of biomolecules to polymersomes, HyNic functionalized enhanced yellow fluorescent protein (eYFP) was attached to 4FB functionalized polymersomes, resulting in an average number of 5 eYFP molecules per polymersome. Two different polymersome-antibody conjugates were produced using either antibiotin IgG or trastuzumab. They showed specific targeting toward biotin-patterned surfaces and breast cancer cells. Overall, the polymersome-ligand platform appears promising for therapeutic and diagnostic use.

Metal-Peptide Frameworks (MPFs) : Bioinspired Metal Organic Frameworks

Chiral metal-organic frameworks (MOFs) have attracted a growing interest for their potential use in energy technologies, asymmetric catalysis, chiral separation, and on a more basic level, the creation of new topologies in inorganic materials. The current paper is the first report on a peptide-based MOF, a metal peptide framework (MPF), constructed from an oligovaline peptide family developed earlier by our group (Mantion, A.; et al. Macromol. Biosci. 2007, 7, 208). We have used a simple oligopeptide, Z-(L-Val)2-L-Glu(OH)-OH, to grow porous copper and calcium MPFs. The MPFs form thanks to the self-assembling properties of the peptide and specific metal-peptide and metal-ammonia interactions. They are stable up to ca. 250 degrees C and have some internal porosity, which makes them a promising prototype for the further development of MPFs.

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.

Antioxidant Nanoreactor Based on Superoxide Dismutase Encapsulated in Superoxide-Permeable Vesicles

We designed and tested an antioxidant nanoreactor based on encapsulation of Cu,Zn superoxide dismutase in amphiphilic copolymer nanovesicles, the membranes of which are oxygen permeable. The nanovesicles, made of poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline), successfully encapsulated the protein during their self-assembling process, as proved by confocal laser-scanning microscopy and fluorescence-correlation spectroscopy. Electron paramagnetic resonance spectroscopy and circular dichroism analyses showed that no structural changes appeared in the protein molecules once inside the inner space of the nanovesicles. The function of this antioxidant nanoreactor was tested by pulse radiolysis, which demonstrated that superoxide dismutase remains active inside the nanovesicles and detoxifies the superoxide radical in situ. The membrane of our triblock copolymer nanovesicles plays a double role, both to shield the sensitive protein and to selectively let superoxide and dioxygen penetrate to its inner space. This simple and robust hybrid system provides a selective shielding of sensitive enzymes from proteolytic attack and therefore a new direction for developing drug delivery applications.

Photoresponsive polymersomes as smart, triggerable nanocarriers

The need for sophisticated systems to improve drug delivery to the body is growing, as is the complexity of therapeutic agents available to treat a variety of conditions. Among the requirements for intelligent drug delivery systems (DDS), responsiveness is highly desirable as a means to control pharmacokinetics and pharmacodynamics. Here, we study the potential of polymeric vesicles obtained from the self-assembly of a photocleavable amphiphilic block copolymer as a light-triggered DDS. The vesicles disintegrate upon UV irradiation, yielding small micellar-like structures, and simultaneously releasing their payload. The versatility of our system is tested both for low molecular weight molecules (fluorescein and ATTO655 dye), and for proteins (enhanced green fluorescent protein). By varying the UV intensity, the payload is released in a controlled manner, as established by fluorescence spectroscopy and fluorescence correlation spectroscopy. Therefore, these responsive polymer vesicles serve as smart, triggerable nanocarriers that can be applied to a variety of payloads, ranging from conventional drug molecules to proteins, enzymes, or DNA.

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.