Brigitte Städler

A Microreactor with Thousands of Subcompartments: Enzyme‐Loaded Liposomes within Polymer Capsules

Fully loaded: Noncovalent anchoring of liposomes into polymer multilayered films with cholesterol-modified polymers allows the preparation of capsosomes-liposome-compartmentalized polymer capsules (see picture). A quantitative enzymatic reaction confirmed the presence of active cargo within the capsosomes and was used to determine the number of subcompartments within this novel biomedical carrier system.

Liposome and Lipid Bilayer Arrays Towards Biosensing Applications

Sensitive and selective biosensors for high-throughput screening are having an increasing impact in modern medical care. The establishment of robust protein biosensing platforms however remains challenging, especially when membrane proteins are involved. Although this type of proteins is of enormous relevance since they are considered in >60% of the pharmaceutical drug targets, their fragile nature (i.e., the requirement to preserve their natural lipid environment to avoid denaturation and loss of function) puts strong additional prerequisites onto a successful biochip. In this review, the leading approaches to create lipid membrane-based arrays towards the creation of membrane protein biosensing platforms are described. Liposomes assembled in micro- and nanoarrays and the successful set-ups containing functional membrane proteins, as well as the use of liposomes in networks, are discussed in the first part. Then, the complementary approaches to create cell-mimicking supported membrane patches on a substrate in an array format will be addressed. Finally, the progress in assembling free-standing (functional) lipid bilayers over nanopore arrays for ion channel sensing will be reported. This review illustrates the rapid pace by which advances are being made towards the creation of a heterogeneous biochip for the high-throughput screening of membrane proteins for diagnostics, drug screening, or drug discovery purposes.

Engineering Advanced Capsosomes: Maximizing the Number of Subcompartments, Cargo Retention, and Temperature-Triggered Reaction

Advanced mimics of cells require a large yet controllable number of subcompartments encapsulated within a scaffold, equipped with a trigger to initiate, terminate, and potentially restart an enzymatic reaction. Recently introduced capsosomes, polymer capsules containing thousands of liposomes, are a promising platform for the creation of artificial cells. Capsosomes are formed by sequentially layering liposomes and polymers onto particle templates, followed by removal of the template cores. Herein, we engineer advanced capsosomes and demonstrate the ability to control the number of subcompartments and hence the degree of cargo loading. To achieve this, we employ a range of polymer separation layers and liposomes to form functional capsosomes comprising multiple layers of enzyme-loaded liposomes. Differences in conversion rates of an enzymatic assay are used to verify that multilayers of intact enzyme-loaded liposomes are assembled within a polymer hydrogel capsule. The size-dependent retention of the cargo encapsulated within the liposomal subcompartments during capsosome assembly and its dependence on environmental pH changes are also examined. We further show that temperature can be used to trigger an enzymatic reaction at the phase transition temperature of the liposomal subcompartments, and that the encapsulated enzymes can be utilized repeatedly in several subsequent conversions. These engineered capsosomes with tailored properties present new opportunities en route to the development of functional artificial cells.

Capsosomes: Subcompartmentalizing Polyelectrolyte Capsules Using Liposomes

Next-generation therapeutic approaches are expected to rely on the engineering of multifunctional particle carriers that can mimic specific cellular functions. The key features of such particles are the semipermeable nature of the shell for communication with the external environment and multiple nanosized individual subcompartments confined within a micron-sized structurally stable scaffold for conducting specific reactions. Herein, we report the formation of capsosomes, a new class of polyelectrolyte capsules containing structurally intact liposomes as cargo. The multilayer film assembly of polyelectrolytes (poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH)) and liposomes (50 nm 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) was characterized on planar substrates using quartz crystal microbalance with dissipation monitoring, and these findings were then correlated to the film growth of the polyelectrolytes and structurally intact liposomes on silica particles. Upon removal of the silica template core, stable capsosomes, containing one or two layers of intact liposomes as cargo, were obtained. This novel platform, capsosomes, which combines the advantages of two systems, liposomes and polyelectrolyte capsules, is expected to find diverse applications in biomedicine, in particular for the creation of artificial cells or organelles where the performance of reactions within a confined environment is a prerequisite.

Low-fouling, biofunctionalized, and biodegradable click capsules.

We report the synthesis of covalently stabilized hollow capsules from biodegradable materials using a combination of click chemistry and layer-by-layer (LbL) assembly. The biodegradable polymers poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) were modified with alkyne and azide moieties. Linear film buildup was observed for both materials on planar surfaces and colloidal silica templates. A variation of the assembly conditions, such as an increase in the salt concentration and variations in pH, was shown to increase the individual layer thickness by almost 200%. The biodegradable click capsules were analyzed with optical microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM). Capsules were uniform in size and had a regular, spherical shape. They were found to be stable between pH 2 and 11 and showed reversible, pH-responsive shrinking/swelling behavior. We also show that covalently stabilized PLL films can be postfunctionalized by depositing a monolayer of heterobifunctional poly(ethylene glycol) (PEG), which provides low-fouling properties and simultaneously enhances specific protein binding. The responsive, biodegradable click films reported herein are promising for a range of applications in the biomedical field.

Cholesterol-mediated anchoring of enzyme-loaded liposomes within disulfide-stabilized polymer carrier capsules

Polymer capsules containing multiple liposomes, termed capsosomes, are a promising new concept toward the design of artificial cells. Herein, we report on the fundamental aspects underpinning the assembly of capsosomes. A stable and high loading of intact liposomal cargo into a polymer film was achieved by non-covalently sandwiching the liposomes between a tailor-made cholesterol-modified poly(L-lysine) (PLL(c)) precursor layer and a poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMA(c)) capping layer. The film assembly, optimized on planar surfaces, was successfully transferred onto colloidal substrates, and a polymer membrane was subsequently assembled by the alternating adsorption of poly(N-vinyl pyrrolidone) (PVP) and thiol-modified poly(methacrylic acid) (PMA(SH)) onto the pre-adsorbed layer of liposomes. Upon removal of the silica template, stable capsosomes encapsulating the enzyme luciferase or beta-lactamase within their liposomal sub-compartments were obtained at both assembly (pH 4) and physiological conditions (pH 7.4). Excellent retention of the liposomes and the enzymatic cargo within the polymer carrier capsules was observed for up to 14 days. These engineered capsosomes are particularly attractive as autonomous microreactors, which can be utilized to repetitively add smaller reactants to cause successive distinct reactions within the capsosomes and simultaneously release the products to the surrounding environment, bringing these systems one step closer toward constructing artificial cells.

Stabilization of Polymer‐Hydrogel Capsules via Thiol–Disulfide Exchange

Polymer hydrogels are used in diverse biomedical applications including drug delivery and tissue engineering. Among different chemical linkages, the natural and reversible thiol-disulfide interconversion is extensively explored to stabilize hydrogels. The creation of macro-, micro-, and nanoscale disulfide-stabilized hydrogels commonly relies on the use of oxidizing agents that may have a detrimental effect on encapsulated cargo. Herein an oxidization-free approach to create disulfide-stabilized polymer hydrogels via a thiol-disulfide exchange reaction is reported. In particular, thiolated poly(methacrylic acid) is used and the conditions of polymer crosslinking in solution and on colloidal porous and solid microparticles are established. In the latter case, removal of the core particles yields stable, hollow, disulfide-crosslinked hydrogel capsules. Further, a procedure is developed to achieve efficient disulfide crosslinking of multilayered polymer films to obtain stable, liposome-loaded polymer-hydrogel capsules that contain functional enzymatic cargo within the liposomal subcompartments. This approach is envisaged to facilitate the development of biomedical applications of hydrogels, specifically those including fragile cargo.

Assembly of Poly(dopamine) Films Mixed with a Nonionic Polymer

Poly(dopamine) (PDA) coatings have recently attracted considerable interest for a variety of applications. Here, we investigate the film deposition of dopamine mixed with a nonionic polymer (i.e., poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), and poly(N-vinyl pyrrolidone) (PVP)) onto silica substrates using X-ray photoelectron spectroscopy and quartz crystal microbalance. Furthermore, we assess the possibility of coating silica colloids to yield polymer capsules and liposomes with these mixtures. We found that mixed PDA/PEG and PDA/PVA films are deposited without the need for a covalent linker such as an amine or thiol. We also discovered the first material, namely, PVP, that can suppress PDA film assembly. These fundamental findings give further insight into PDA film properties and contribute to establish PDA as a widely applicable coating.

Monitoring ion-channel function in real time through quantum decoherence

In drug discovery, there is a clear and urgent need for detection of cell-membrane ion-channel operation with wide-field capability. Existing techniques are generally invasive or require specialized nanostructures. We show that quantum nanotechnology could provide a solution. The nitrogen-vacancy (NV) center in nanodiamond is of great interest as a single-atom quantum probe for nanoscale processes. However, until now nothing was known about the quantum behavior of a NV probe in a complex biological environment. We explore the quantum dynamics of a NV probe in proximity to the ion channel, lipid bilayer, and surrounding aqueous environment. Our theoretical results indicate that real-time detection of ion-channel operation at millisecond resolution is possible by directly monitoring the quantum decoherence of the NV probe. With the potential to scan and scale up to an array-based system, this conclusion may have wide-ranging implications for nanoscale biology and drug discovery.

Triggered cargo release by encapsulated enzymatic catalysis in capsosomes.

We report the coencapsulation of glutathione reductase and disulfide-linked polymer-oligopeptide conjugates into capsosomes, polymer carrier capsules containing liposomal subcompartments. The architecture of the capsosomes enables a temperature-triggered conversion of oxidized glutathione to its reduced sulfhydryl form by the encapsulated glutathione reductase. The reduced glutathione subsequently induces the release of the encapsulated oligopeptides from the capsosomes by reducing the disulfide linkages of the conjugates. This study highlights the potential of capsosomes to continuously generate a potent antioxidant while simultaneously releasing small molecule therapeutics.