Patric Baumann

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.

pH-Responsive PDMS-b-PDMAEMA Micelles for Intracellular Anticancer Drug Delivery

A series of poly(dimethysiloxane)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMS-b-PDMAEMA) block copolymers were synthesized with atom transfer radical polymerization (ATRP). In aqueous solution the polymers self-assembled into micelles with diameters between 80 and 300 nm, with the ability to encapsulate DOX. The polymer with the shortest PDMAEMA block (5 units) displayed excellent cell viability, while micelles containing longer PDMAEMA block lengths (13 and 22 units) led to increased cytotoxicity. The carriers released DOX in response to a decrease in pH from 7.4 to 5.5. Confocal laser scanning microscopy (CLSM) revealed that nanoparticles were taken up by endocytosis into acidic cell compartments. Furthermore, DOX-loaded nanocarriers exhibited intracellular pH-response as changes in cell morphology and drug release were observed within 24 h.

Poly(N-vinylpyrrolidone)-poly(dimethylsiloxane)-based polymersome nanoreactors for laccase-catalyzed biotransformations.

Laccases (Lac) are oxidizing enzymes with a broad range of applications, for example, in soil remediation, as bleaching agent in the textile industry, and for cosmetics. Protecting the enzyme against degradation and inhibition is of great importance for many of these applications. Polymer vesicles (polymersomes) from poly(N-vinylpyrrolidone)-block-poly(dimethylsiloxane)-block-poly(N-vinylpyrrolidone) (PNVP-b-PDMS-b-PNVP) triblock copolymers were prepared and investigated as intrinsically semipermeable nanoreactors for Lac. The block copolymers allow oxygen to enter and reactive oxygen species (ROS) to leave the polymersomes. EPR spectroscopy proved that Lac can generate ROS. They could diffuse out of the polymersome and oxidize an aromatic substrate outside the vesicles. Michaelis-Menten constants Km between 60 and 143 μM and turn over numbers kcat of 0.11 to 0.18 s(-1) were determined for Lac in the nanoreactors. The molecular weight and the PDMS-to-PNVP ratio of the block copolymers influenced these apparent Michaelis-Menten parameters. Encapsulation of Lac in the polymersomes significantly protected the enzyme against enzymatic degradation and against small inhibitors: proteinase K caused 90% less degradation and the inhibitor sodium azide did not affect the enzymes activity. Therefore, these polymer nanoreactors are an effective means to stabilize laccase.

Water-soluble Co(III) complexes of substituted phenanthrolines with cell selective anticancer activity.

Transition metal complexes with substituted phenanthrolines as ligands represent potential anticancer products without the drawbacks of platinum complexes that are currently marketed. Here, we report the synthesis and cell selective anticancer activity of five new water-soluble Co(III) complexes with methyl substituted phenanthroline ligands. The complexes were characterized by elemental analysis, NMR, FAB-mass spectrometry, FTIR, electronic spectroscopy, and single crystal X-ray diffraction. Possible interaction of these complexes with DNA was assessed by a combination of circular dichroism, UV-vis spectroscopy titration, and ethidium bromide displacement assay, and the results indicated that DNA interaction is weak for these complexes. Cellular uptake and cytotoxicity of complexes at low concentrations were assessed by flow cytometry on PC-3 cells, while their effect on intracellular mitochondrial function was measured by MTS assay on HeLa and PC-3 cell lines. These complexes showed selective cytotoxicity with a significantly higher effect on intracellular mitochondrial function in PC-3 cells than in HeLa cells. At low concentrations, complex 2 had the highest cytotoxic effect on PC-3 cells, inducing around 38% cell death, and the correlation of cytotoxicity of these complexes to their hydrophobicity indicates that an appropriate value of the hydrophobicity is essential for high antitumor activity.

Reconstitution of OmpF membrane protein on bended lipid bilayers: perforated hexagonal mesophases

Membrane proteins have been reconstituted on lipid bilayers with zero mean-curvature (cubic phases or vesicles). Here we show that reconstitution of pore-forming membrane proteins can also occur on highly curved lipidic bilayers of reverse hexagonal mesophases, for which the mean-curvature is significantly different from zero. We further show that the membrane protein provides unique topological interconnectivities between the aqueous nanochannels, significantly enhancing mesophase transport properties.

The Amine Content of PEGylated Chitosan Bombyx mori Nanoparticles Acts as a Trigger for Protein Delivery

In modern medicine, effective protein therapy is a major challenge to which a significant contribution can be expected from nanoscience through the development of novel delivery systems. Here we present the effect of the amine content of nanoparticles based on PEGylated chitosan Bombyx mori (PEG-O-ChsBm) copolymers on the entrapment of molecules in a search for highly efficient nanocarriers. PEG-O-ChsBm copolymers were synthesized with amine contents from 1.12% to 0.70%, and nanoparticles were generated by self-assembly in dilute aqueous solutions. These nanoparticles successfully entrapped molecules with a wide range of sizes, the efficiency of which was dependent on their amine contents. While hydrophobic molecules were entrapped with high efficiency in all types of nanoparticle, hydrophilic molecules were entrapped only in those with low amine content. Bovine serum albumin, selected as a model protein, was entrapped in nanoparticles and efficiently released in acidic conditions. The triggered entrapment of molecules in PEG-O-ChsBm nanoparticles by selection of the appropriate amine content represents a straightforward way to modulate their delivery by fine changes in the properties of nanocarriers.

Investigation of Horseradish Peroxidase Kinetics in an “Organelle‐Like” Environment

In order to mimic cell organelles, artificial nanoreactors have been investigated based on polymeric vesicles with reconstituted channel proteins (outer membrane protein F) and coencapsulated enzymes horseradish peroxidase (HRP) along with a crowding agent (Ficoll or polyethylene glycol) inside the cavity. Importantly, the presence of macromolecules has a strong impact on the enzyme kinetics, but no influence on the integrity of vesicles up to certain concentrations. This particular design allows for the first time the determination of HRP kinetics inside nanoreactors with crowded milieu. The values of the Michaelis-Menten constant (K m ) measured for HRP in a confined space (encapsulated in nanoreactors) in the absence of macromolecules are ≈50% lower than in free conditions, and the presence of a crowding agent results in a further pronounced decrease. These results clearly suggest that activities of enzymes in confined spaces can be tuned by varying the concentrations of crowding compounds. The present investigation represents an advance in nanoreactor design by considering the influence of environmental factors on enzymatic performance, and it demonstrates that both encapsulation and the presence of a crowding environment increase the enzyme-substrate affinity.

Polymeric particulates for subunit vaccine delivery

Vaccines still represent the best long-term treatment option for reducing many infectious diseases, including acquired immune deficiency syndrome (AIDS), malaria, and tuberculosis. Therefore, to effectively combat these severe diseases, it is of utmost importance to develop and explore novel and more efficient delivery modalities and administration routes. In this context, new polymeric nano- and microparticulate delivery platforms may represent an alternative and/or complementary therapeutic option. With the help of modern polymer chemistry, an increased number of sophisticated architectures have been developed, although these materials are in terms of bio applications still in relatively early stages. Therefore, a lot of recent attention has been dedicated to designing and tailoring novel particulates delivery systems with focus to create more efficient delivery platform. Various structures, including nanogels, nanocapsules, nano- and microparticles, dendrimers, and different hierarchical assemblies in solution have been studied in vaccine delivery. However, none of these explored platforms until now fully complies with basic delivery requirements like biocompatibility, non-toxicity, high encapsulation efficiency, and the ability to induce prolonged immune responses. In general, the unique structural and mechanical properties of polymers and their abilities to create three-dimensional structures or hybrid systems is under intensive investigation and hold a great promise in vaccine delivery.