Liposome-based nanocapsules

Here we present three different types of mechanically stable nanometer-sized hollow capsules. The common point of the currently developed systems in our laboratory is that they are liposome based. Biomolecules can be used to functionalize lipid vesicles to create a new type of intelligent material. For example, insertion of membrane channels into the capsule wall can modify the permeability. Covalent binding of antibodies allows targeting of the capsule to specific sites. Liposomes loaded with enzymes may provide an optimal environment for them with respect to the maximal turnover and may stabilize the enzyme. However, the main drawback of liposomes is their instability in biological media as well as their sensitivity to many external parameters such as temperature or osmotic pressure. To increase their stability we follow different strategies: 1) polymerize a two-dimensional network in the hydrophobic core of the membrane; 2) coat the liposome with a polyelectrolyte shell; or 3) add surface active polymers to form mixed vesicular structures.     

Mechanical property of lipid-coated polyelectrolyte microcapsules

The deformations of lipid coated polyelectrolyte capsules induced by osmotic pressure were determined in poly(styrene sulfonate, sodium salt) (PSS) solution by making use of the fact that PSS molecules with a molecular weight 70000 will not penetrate into the capsules. At a critical osmotic pressure the initial spherical capsules changed their shape and became an invagination. The measurements of single particle light scattering provided the wall thickness of the lipid-coated capsules with 25.6 nm while the thickness for pure polyelectrolyte capsules in solution is about 21.6 nm, indicating that the coating lipid layer has about 4 nm thickness. It demonstrates that DMPA forms a bilayer on the surface of polyelectrolyte capsules. With these data, that the elasticity coefficient of the lipid-coated capsules is about 426 Mpa can be obtained.     

Redox-controlled molecular permeability of composite-wall microcapsules

Many smart materials in bioengineering, nanotechnology and medicine allow the storage and release of encapsulated drugs on demand at a specific location by an external stimulus. Owing to their versatility in material selection, polyelectrolyte multilayers are very promising systems in the development of microencapsulation technologies with permeation control governed by variations in the environmental conditions. Here, organometallic polyelectrolyte multilayer capsules, composed of polyanions and polycations of poly(ferrocenylsilane) (PFS), are introduced. Their preparation involved layer-by-layer self-assembly onto colloidal templates followed by core removal. PFS polyelectrolytes feature redox-active ferrocene units in the main chain. Incorporation of PFS into the capsule walls allowed us to explore the effects of a new stimulus, that is, changing the redox state, on capsule wall permeability. The permeability of these capsules could be sensitively tuned via chemical oxidation, resulting in a fast capsule expansion accompanied by a drastic permeability increase in response to a very small trigger. The substantial swelling could be suppressed by the application of an additional coating bearing common redox-inert species of poly(styrene sulfonate) (PSS(-)) and poly(allylamine hydrochloride) (PAH(+)) on the outer wall of the capsules. Hence, we obtained a unique capsule system with redox-controlled permeability and swellability with a high application potential in materials as well as in bioscience.     

Hydrogen-bonding layer protecting polyelectrolyte capsules and its mechanical property study with atomic force microscope

The hydrogen-bonding multilayered polyelectrolyte capsules with sizes around 6 microm were fabricated by layer-by-layer self-assembly method. The morphology of the obtained capsules was observed with Scanning Electron Microscope (SEM), Confocal Laser Scanning Microscope (CLSM) and Atomic Force Microscope (AFM), respectively. The elastic properties of the capsules were studied with AFM. The capsule was pressed by cantilever with different lengths, a glass bead glued at the end of the cantilever. The force curves were measured on the capsule in air. The Young's modulus of the capsule was obtained (E = 170 MPa for the loading). Results show that this model can predict the elastic deformation of the microcapsule. The accuracy of the elastic deformation of polymer capsule can be ensured using a cantilever of mediate stiffness. Our results show that the existence of the hydrogen-bonding layer makes the multilayered polyelectrolyte harder in comparison with the pure multilayered polyelectrolyte capsules.     

以MnCO3为模板的聚电解质胶囊的制备

通过沉淀反应制备MnCO3，以乙醇为添加剂成功制备出分散性好的、具有所需尺寸和很窄的粒径分布的球形MnCO3微粒。采用MnCO3粒子为模板吸附相反电荷聚电解质再除去核来制备纯净中空聚电解质胶囊。对组装到MnCO3上制备出来的中空聚电解质胶囊作了研究。结果表明：这种胶囊代表了仅含有所需物质的微米尺度的独立式的聚电解质膜。粒子表面结构决定了所制备的胶囊的形态和胶囊壁厚度。     

Synthesis and characterization of ratiometric ion-sensitive polyelectrolyte capsules

Micrometer-sized polyelectrolyte capsules are synthesized, which have ion-sensitive fluorophores embedded in their cavities. As the membranes of the capsules are permeable to ions, the fluorescence of the capsules changed with the ion concentration. In particular, capsules sensitive to protons, sodium, potassium, and chloride ions are fabricated and their fluorescence response analyzed. In order to allow for ratiometric measurements, additional fluorophores whose emission do not depend on the ion concentration and which emit a different wavelength are co-embedded in the capsule cavities.     

Spontaneous Structuration in Coacervate‐Based Protocells by Polyoxometalate‐Mediated Membrane Assembly

Molecularly crowded, polyelectrolyte/ribonucleotide‐enriched membrane‐free coacervate droplets are transformed into membrane‐bounded sub‐divided vesicles by using a polyoxometalate‐mediated surface‐templating procedure. The coacervate to vesicle transition results in reconstruction of the coacervate micro‐droplets into novel three‐tiered micro‐compartments comprising a semi‐permeable negatively charged polyoxometalate/polyelectrolyte outer membrane, a sub‐membrane coacervate shell, and an internal aqueous lumen. We demonstrate that organic dyes, ssDNA, magnetic nanoparticles and enzymes can be concentrated into the interior of the micro‐compartments by sequestration into the coacervate micro‐droplets prior to vesicle formation. The vesicle‐encapsulated proteins are inaccessible to proteases in the external medium, and can be exploited for the spatial localization and coupling of two‐enzyme cascade reactions within single or between multiple populations of hybrid vesicles dispersed in aqueous media.     

Synthetic ion channels via self-assembly: a route for embedding porous polyoxometalate nanocapsules in lipid bilayer membranes

Porous polyoxometalate nanocapsules of Keplerate type are known to exhibit the functionality of biological ion channels; however, their use as an artificial ion channel is tempered by the high negative charge of the capsules, which renders their spontaneous incorporation into a lipid bilayer membrane unlikely. In this Letter we report coarse-grained molecular dynamics simulations that demonstrate a route for embedding negatively charged nanocapsules into lipid bilayer membranes via self-assembly. A homogeneous mixture of water, cationic detergent, and phospholipid was observed to spontaneously self-assemble around the nanocapsule into a layered, liposome-like structure, where the nanocapsule was enveloped by a layer of cationic detergent followed by a layer of phospholipid. Fusion of such a layered liposome with a lipid bilayer membrane was observed to embed the nanocapsule into the lipid bilayer. The resulting assembly was found to remain stable even after the surface of the capsule was exposed to electrolyte. In the latter conformation, water was observed to flow into and out of the capsule as Na(+) cations entered, suggesting that a polyoxometalate nanocapsule can form a functional synthetic ion channel in a lipid bilayer membrane.     

Silver nanoparticle synthesis: novel route for laser triggering of polyelectrolyte capsules

We have demonstrated the synthesis of light-sensitive polyelectrolyte capsules (PECs) by utilizing a novel polyol reduction method and investigated its applicability as photosensitive drug delivery vehicle. The nanostructured capsules were prepared via layer by layer (LbL) assembly of poly(allylamine hydrochloride) (PAH) and dextran sulfate (DS) on silica particles followed by in-situ synthesis of silver nanoparticles (NPs). Capsules without silver NPs were permeable to low molecular weight (M(w), 479 g/mol) rhodamine but impermeable to higher molecular weight fluorescence labeled dextran (FITC-dextran). However, capsules synthesized with silver NPs showed porous morphology and were permeable to higher molecular weight (M(w) 70 kDa) FITC-dextran also. These capsules were loaded with FITC-dextran using thermal encapsulation method by exploiting temperature induced shrinking of the capsules. During heat treatment the porous morphology of the capsules transformed into smooth pore free structure which prevents the movement of dextran into bulk during the loading process. When these loaded capsules are exposed to laser pulses, the capsule wall ruptured, resulting in the release of the loaded drug/dye. The rupture of the capsules was dependent on particle size, laser pulse energy and exposure time. The release was linear with time when pulse energy of 400 μJ was used and burst release was observed when pulse energy increased to 600 μJ.     

Fusion of enveloped virus nanoparticles with polyelectrolyte-supported lipid membranes for the design of bio/nonbio interfaces

Fusion of lipid-enveloped viruses with endosomal membranes triggered by low pH in the endosome is a key step in the course of viral infection. This ubiquitous mechanism can be used to integrate functional nanoparticles of viral origin into composite materials consisting of a polyelectrolyte multilayer with an adsorbed lipid membrane in a natural and biomimetic way. Polyelectrolyte multilayers as the support for the lipid membrane are a versatile means to combine the biological functions of the viral surface with the multiplicity of polyelectrolyte borne functions into a novel bio/nonbio composite material.     

A novel flow-cytometry-based assay for cellular uptake studies of polyelectrolyte microcapsules

A flow-cytometry-based assay is presented with which the uptake of polyelectrolyte capsules can be quantified. The cavity of the capsules is loaded with the pH-sensitive dye SNARF, which emits in the red and green in alkaline and acidic environments, respectively. By recording the fluorescence intensities in the red and green channels, the localization of capsules associated with cells can be determined. Capsules adherent to the outer cell membrane fluoresce in the red due to the alkaline pH of the cell medium, whereas capsules internalized by cells fluoresce in the green due to the acidic pH in the endosomal/lysosomal/phagosomal compartments in which incorporated capsules are located. Adding the SNARF readout to the scattering signal typically derived with flow cytometry analysis allows for a more detailed quantitative analysis of particle uptake, which can also distinguish between adherent and ingested particles.     

Freestanding polyelectrolyte multilayers as functional and construction elements

This article reviews the progress in the field of polyelectrolyte multilayer membranes with special attention to freestanding membranes. These can be prepared both in the form of hollow capsules and as flat membrane sheets. While (bio) functionality, or bioactivity as it is known, from solid supported multilayers is maintained, additional applications arise for the freestanding membranes in the fields of encapsulation, separation and micromechanics. The production processes and functionalities achieved for capsules and flat sheets. The integration of membranes into larger scale structures is essential for their use and an overview of existing strategies is given. In particular, the way in which arrays of micro-compartments can be built up is shown, and their potential for sensing and combinatorial chemistry discussed. Recent results on the applications of such systems as membrane sensors in the case of flat membrane sheets are also discussed.     

“Smart” Surface Capsules for Delivery Devices

This review describes emerging trends, basic principles, applications, and future challenges for designing next generation responsive “smart” surface capsules. Advances and importance of “surface” capsules which are not deposited onto the surface but are built into the surface are highlighted for selective applications with specific examples of surface sponge structures formed by high intensity ultrasonic surface treatment (HIUS). Surface capsules can be adapted for biomedical applications, membrane materials, lab‐on‐chip, organ‐on‐chip, and for template synthesis. They provide attractive self‐healing anticorrosion and antifouling prospects. Nowadays delivery systems are built from inorganic, organic, hybrid, biological materials to deliver various drugs from low molecular weight substances to large protein molecules and even live cells. It is important that capsules are designed to have time prolonged release features. Available stimuli to control capsule opening are physical, chemical and biological ones. Understanding the underlying mechanisms of capsule opening by different stimuli is essential for developing new methods of encapsulation, release, and targeting. Development of “smart” surface capsules is preferable to respond to multiple stimuli. More and more often a new generation of “smart” capsules is designed by a bio‐inspired approach.     

Nanocapsules: coating for living cells

One of the most promising tools for future applications in science and medicine is the use of nanotechnologies. Especially self-assembly systems, e.g., polyelectrolyte (PE) capsules prepared by means of the layer-by-layer technique with tailored properties, fulfill the requirements for nano-organized systems in a satisfactory manner. The nano-organized shells are suitable as coating for living cells or artificial tissue to prevent immune response. With these shells, material can be delivered to predefined organs. In this paper, some preliminary results are presented, giving a broad overview over the possibilities to use nano-organized capsules. Based on the observations that the cells while duplicating break the capsule a mutant yeast strain (Saccharomyces cerevisiae), which express GFP-tubulin under galactose promotion, was investigated by means of confocal laser scanning microscopy. The measurements reveal an increased surface charge in the region of buds developed prior encapsulation. In order to test the used PE pair for cytotoxicity, germinating conidia of the fungi Neurospora crassa were coated. The investigation with fluorescence microscopy shows a variation in the surface charge for the growing region and the conidium poles. The capsules exhibit interesting properties as valuable tool in science and a promising candidate for application in the field of medicine.     

A Facile Method to Coat Nanoparticles with Lipid Bilayer Membrane: Hybrid Silica Nanoparticles Disguised as Biomembrane Vesicles by Particle Penetration of Concentrated Lipid Layers

Natural membrane vesicles, including extracellular vesicles and enveloped viruses, participate in various events in vivo. To study and manipulate these events, biomembrane-coated nanoparticles inspired by natural membrane vesicles are developed. Herein, an efficient method is presented to prepare organic–inorganic hybrid materials in high yields that can accommodate various lipid compositions and particle sizes. To demonstrate this method, silica nanoparticles are passed through concentrated lipid layers prepared using density gradient centrifugation, followed by purification, to obtain lipid membrane-coated nanoparticles. Various lipids, including neutral, anionic, and cationic lipids, are used to prepare concentrated lipid layers. Single-particle analysis by imaging flow cytometry determines that silica nanoparticles are uniformly coated with a single lipid bilayer. Moreover, cellular uptake of silica nanoparticles is enhanced when covered with a lipid membrane containing cationic lipids. Finally, cell-free protein expression is applied to embed a membrane protein, namely the Spike protein of severe acute respiratory syndrome coronavirus 2, into the coating of the nanoparticles, with the correct orientation. Therefore, this method can be used to develop organic–inorganic hybrid nanomaterials with an inorganic core and a virus-like coating, serving as carriers for targeted delivery of cargos such as proteins, DNA, and drugs.     

Femtomolar electrochemical detection of DNA hybridization using hollow polyelectrolyte shells bearing silver nanoparticles

The preparation, and use as electrochemical labels, of polyelectrolyte shells bearing Ag nanoparticles is described. Their potential for highly sensitive detection is demonstrated. The shells are prepared by layer-by-layer self-assembly around templates (500 nm diameter) which are then dissolved. The shells can be opened and closed by adjustment of solution pH, and this process is utilized to encapsulate Ag nanoparticles, chiefly by adsorption to the inner walls of the capsules. Based on absorbance, TEM and voltammetric measurements, the highest loading achieved is approximately 78 Ag particles per capsule. The Ag capsules are used via biotin-avidin binding as labels for the detection of DNA hybridization, following acid dissolution and then detection of the Ag (+) by ASV. A 30-mer sequence specific to Escherichia coli is measured at DNA-modified screen-printed electrodes with a detection limit of approximately 25 fM, which corresponds to the detection of 4.6 fg ( approximately 3 x 10 (5) molecules) in the 20 microL analyte sample. A 200 fM target containing a single mismatch gives a significantly (<74%) lower response than 200 fM of complementary target; 60 pM of noncomplementary target gives a negligible response.     

The role of metal nanoparticles in remote release of encapsulated materials

Laser mediated remote release of encapsulated fluorescently labeled polymers from nanoengineered polyelectrolyte multilayer capsules containing gold sulfide core/gold shell nanoparticles in their walls is observed in real time on a single capsule level. We have developed a method for measuring the temperature increase and have quantitatively investigated the influence of absorption, size, and surface density of metal nanoparticles using an analytical model. Experimental measurements and numerical simulations agree with the model. The treatment presented in this work is of general nature, and it is applicable to any system where nanoparticles are used as absorbing centers. Potential biomedical applications are highlighted.     

Microscopical characterization of nanocapsules templated on ionic crystals and biological cells toward biomedical applications

Nanocapsules, fuzzy assemblies of polyelectrolyte, represent a comparatively new class of colloids with controlled nanostructure and tunable properties. Due to the fact that the core as well as the dissolution influences the wall texture and the properties of the hollow capsules, the use of carbonate crystals as template is most convenient. Yeast cells constitute as a core candidate as well. They are a good system for testing the protective ability of shells and the permeability of the walls with respect to the needs of biological systems, namely, feeding and stability against attacks. The main features of the nanocapsules have been studied by two-photon, confocal, and atomic force microscopy. Nanocapsules are of biomedical interest because they can be used, for example, for the controlled release and targeting of drugs as well as for the protection of enzymes, proteins, and foreign cells.     

Experiment on formulation and drug release behavior of porosity asymmetric membrane capsules in vitro

Porosity asymmetric membrane capsules were prepared to study the relationship between the capsule formulation and drug release. Cellulose acetate (CA) and pore formers were used in the capsule shell formulation as the main semipermeable membrane material. The capsules were permeable to both water and dissolved solutes. Using sparingly soluble drug acetaminophen as a model, cumulative release was calculated. The slope of the release profile from the distilled water had good relationship with the concentration of the pore formers F68. The release of acetaminophen was independent to the pH, osmotic pressure of dissolution medium, but influenced by intensity of agitation. When the concentration of pore former was low, zero-order release behavior was observed within 24?h which was consistent with Fickian diffusion model. When the concentration of pore former was high, however, Higuchi model release was found which is caused by Fickian diffusion and osmotic pressure release. With scanning electron microscope (SEM), the surface structure and cross-section of the capsule shell were also studied before and after drug delivery. With simple preparation and broad scope of drug application, porosity asymmetric membrane capsules can give desired drug extended release and show more convenience than controlled tablets with laser drilling.     

Self-assembled virus-membrane complexes

Anionic polyelectrolytes and cationic lipid membranes can self-assemble into lamellar structures ranging from alternating layers of membranes and polyelectrolytes to 'missing layer' superlattice structures. We show that these structural differences can be understood in terms of the surface-charge-density mismatch between the polyelectrolyte and membrane components by examining complexes between cationic membranes and highly charged M13 viruses, a system that allowed us to vary the polyelectrolyte diameter independently of the charge density. Such virus-membrane complexes have pore sizes that are about ten times larger in area than DNA-membrane complexes, and can be used to package and organize large functional molecules; correlated arrays of Ru(bpy)(3)(2+) macroionic dyes have been directly observed within the virus-membrane complexes using an electron-density reconstruction. These observations elucidate fundamental design rules for rational control of self-assembled polyelectrolyte-membrane structures, which have applications ranging from non-viral gene therapy to biomolecular templates for nanofabrication.