Rona Chandrawati

Colloidal nanoparticles as advanced biological sensors

Background Nanoparticle biosensors have the potential to enhance or supersede current analytical techniques, and their introduction could have a great impact in research and clinical practice. The unique optical properties of many nanomaterials make them ideal for biosensing. Colloidal fluorescent and plasmonic nanoparticles are particularly interesting, as they produce intense responses to incident light, and linking this response to the presence of a target analyte yields extremely sensitive detection in solution. Optimization of nanoparticle biosensors by considering the nanoparticle core, surface, and sensing properties, with an eye toward their application in research and as clinical tools. This translational process relies on the availability of high-quality nanoparticles with precisely engineered surfaces and biosensing mechanisms that allow detection with high sensitivity and specificity. Advances Much research has centered on fluorescent quantum dots (QDs) and plasmonic gold nanoparticles (AuNPs) and has expanded to include carbon dots, silicon dots, upconverting nanoparticles, alloyed plasmonic nanoparticles, and gold and silver nanoclusters, among a constantly growing repertoire. New tools for probing the nucleation and growth of nanoparticles, such as in situ synchrotron x-ray irradiation, liquid cell transmission electron microscopy, and computer simulation, are revealing ever more about fundamental processes—for example, the importance of particle coalescence and surface ligand conformation during particle growth. Precision engineering of particle surfaces is required to construct advanced nanoparticle biosensors, and this is being facilitated by a new generation of modular small-molecule and polymeric capping agents, the incorporation of new high-yield bio-orthogonal “click”-like functional groups, and advanced engineering of biomolecular sensing constructs (e.g., by recombinant protein engineering). Notable successes in the field include the precision synthesis of nanoparticles in microfluidic systems; ultrasensitive detection of cancer biomarkers in human serum with time-gated QD fluorescence; multiplexed intracellular sensing of mRNA using superquenching AuNPs; multiplexed detection of analytes with simple technologies such as smartphones; in vivo sensing of reactive oxygen species and methyl mercury; and the integration of nanoparticle biosensors with advanced DNA/RNA target amplification protocols. Outlook Many advanced applications are anticipated for nanoparticle biosensors, including as research analytical tools, intracellular sensors, and in vivo sensors for real-time detection and visualization of analytes and structures inside the body. There has been great success in synthesizing nanoparticles with excellent physical and chemical properties and in demonstrating their applications as biosensors. Looking toward high-impact applications, the main challenges are to develop techniques to synthesize reproducible high-quality nanoparticle biosensors on a mass scale and to maximize performance in physiological conditions. By drawing on many fields, including molecular biology, bioinformatics, computer modeling, bioengineering, and applied physics, we can compile the diverse toolset required to overcome these challenges and move toward high-impact applications of nanoparticle biosensors. Biological sensing using nanoparticles Colloidal fluorescent and plasmonic nanoparticles yield intense responses to incident light, making them useful as sensors or probes for sensitive detection in solution. Howes et al. review the potential uses of nanoparticle biosensors in research and diagnostics. A range of methods allow for the chemical modification of the particle surfaces so that they can be tuned for specific analytes and give optical signals for a range of biological conditions of interest. Signals can be detected in complex media or in vivo making the particles of interest for both laboratory research and in clinical settings. Science, this issue 10.1126/science.1247390 Colloidal nanoparticle biosensors have received intense scientific attention and offer promising applications in both research and medicine. We review the state of the art in nanoparticle development, surface chemistry, and biosensing mechanisms, discussing how a range of technologies are contributing toward commercial and clinical translation. Recent examples of success include the ultrasensitive detection of cancer biomarkers in human serum and in vivo sensing of methyl mercury. We identify five key materials challenges, including the development of robust mass-scale nanoparticle synthesis methods, and five broader challenges, including the use of simulations and bioinformatics-driven experimental approaches for predictive modeling of biosensor performance. The resultant generation of nanoparticle biosensors will form the basis of high-performance analytical assays, effective multiplexed intracellular sensors, and sophisticated in vivo probes.

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.

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.

Biomimetic Liposome- and Polymersome-Based Multicompartmentalized Assemblies

Liposomes and polymersomes have attracted significant attention and have emerged as versatile materials for therapeutic delivery and in the design of artificial cells and organelles. Through the judicious choice of building blocks, these synthetic carriers can be readily engineered with tailored interfacial properties, offering new possibilities for the design of advanced assemblies with specific permeability, stability, stimuli response, and targeting capabilities. In this feature article, we highlight recent studies on biomimetic liposome- and polymersome-based multicompartmentalized assemblies en route toward the development of artificial cells, microreactors, and therapeutic delivery carriers. The strategies employed to produce these carriers are outlined, and the properties that contribute to their performance are discussed. Applications of these biomimetic assemblies are highlighted, and finally, areas that require additional investigation for the future development of these assemblies as next-generation therapeutic systems are outlined.

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.

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.

Capsosomes with “Free‐Floating” Liposomal Subcompartments

Biological cells are able to perform multiple complex reactions within confi ned environments, owing to their structures comprising internal subcompartments (e.g., cell organelles). [ 1 ] Artifi cial cells, [ 2–4 ] although far less complex than their biological counterparts, can exhibit a hierarchical structure with a large number of subcompartments confi ned within a structurally stable scaffold. Such systems are engineered for therapeutic cell mimicry via the encapsulation and/or conversion of biologically active materials. [ 5 ]

Noncovalent Liposome Linkage and Miniaturization of Capsosomes for Drug Delivery

We report the synthesis of poly(methacrylic acid)-co-(oleyl methacrylate) with three different amounts of oleyl methacrylate and compare the ability of these polymers with that of poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMA(c)) to noncovalently anchor liposomes to polymer layers. We subsequently assembled ∼1 μm diameter PMA(c)-based capsosomes, polymer hydrogel capsules that contain up to ∼2000 liposomal subcompartments, and investigate the potential of these carriers to deliver water-insoluble drugs by encapsulating two different antitumor compounds, thiocoraline or paclitaxel, into the liposomes. The viability of lung cancer cells is used to substantiate the cargo concentration-dependent activity of the capsosomes. These findings cover several crucial aspects for the application of capsosomes as potential drug delivery vehicles.