Mesoporous Protein Particles Through Colloidal CaCO3 Templates

Porous colloidal particles can be tailored using templating techniques to maximize their effectiveness for a wide range of applications, including separation, catalysis, and drug delivery. However, templating usually involves harsh and complex preparation conditions, thereby complicating the fabrication of sensitive bio‐functionalized particles. Here a simple, yet versatile and mild approach us used to create porous protein particles using mesoporous CaCO₃ colloids as sacrificial templates. The three‐step preparation procedure involves infiltrating the colloidal templates with the protein by solvent evaporation, protein crosslinking, and removal of CaCO₃. Using this method one can obtain porous particles consisting of virtually any protein. To explore the applicability of the particles for various scenarios particles composed of different proteins are fabricated focusing on hemoglobin and trypsin and particle morphology, porosity, mechanical properties, the protein redox state, and enzymatic activity are determined. The results show that the nanoporous template structure is replicated and that the proteins are fully functional. By varying preparation conditions such as crosslinker concentration and protein content the elastic modulus is adjusted in the range of red blood cells. This ensures high deformability upon flow in microchannels and makes the porous protein particles a versatile platform for biomedical applications.     

Preparation of Protamine–Titania Microcapsules Through Synergy Between Layer‐by‐Layer Assembly and Biomimetic Mineralization

A novel approach combining layer‐by‐layer (LbL) assembly with biomimetic mineralization is proposed to prepare protamine–titiania hybrid microcapsules. More specifically, these microcapsules are fabricated by alternative deposition of positively charged protamine layers and negatively charged titania layers on the surface of CaCO₃ microparticles, followed by dissolution of the CaCO₃ microparticles using EDTA. During the deposition process, the protamine layer induces the hydrolysis and condensation of a titania precursor, to form the titania layer. Thereafter, the negatively charged titania layer allows a new cycle of deposition step of the protamine layer, which ensures a continuous LbL process. The morphology, structure, and chemical composition of the microcapsules are characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared, and X‐ray photoelectron spectroscopy. Moreover, these protamine–titania hybrid microcapsules are first employed as the carrier for the immobilization of yeast alcohol dehydrogenase (YADH), and the encapsulated YADH displays enhanced recycling stability. This approach may open a facile, general, and efficient way to prepare organic–inorganic hybrid materials with different compositions and shapes.     

Mosaic,Single‐Crystal CaCO3 Thin Films Fabricated on Modified Polymer Templates

Mosaic, single‐crystal CaCO₃ thin films have been prepared on modified poly(ethylene terephthalate) (PET) templates. Surface modification of PET through the introduction of carboxylic acid groups (COOH‐PET), and the subsequent physical and chemical adsorption of poly(allylamine hydrochloride) (PAH) at pH 8 (PAH8‐PET) and pH 11 (PAH11‐PET), afford template surfaces that influenced the phase transition of an amorphous CaCO₃ (ACC) films during crystallization in air. Macroscopic ACC thin films are prepared on modified PET films in the presence of poly(acrylic acid). Polycrystalline, spherulitic vaterite (CaCO₃) films are observed to form on native PET and PAH11‐PET, while mosaic, single‐crystal calcitic (CaCO₃) films form on COOH‐PET and PAH8‐PET templates. These results confirm that single‐crystal CaCO₃ growth patterns are dependent on the surface characteristics of the PET template. We infer therefore, that the nucleation and growth of ceramic films on polymeric templates can be controlled by chemical modification of the polymeric template surface, and by the subsequent attachment of ionic polyelectrolytes.     

Fabrication of Gelatin Microgels by a “Cast” Strategy for Controlled Drug Release

In this paper, we propose a “casting” strategy to prepare intrinsically fluorescent, uniform and porous gelatin microgels with multi‐responsiveness. Gelatin microgels with tunable size were obtained by copying the structure of a porous CaCO₃ template. The diameter of the gelatin microgels was sensitive to salt concentration and pH. Doxorubicin and Rhodamine B as model drugs were loaded into the microgels via electrostatic interaction and release of the payload was triggered by changing the salt concentration and pH, respectively. Cell experiments demonstrated that the gelatin microgels had an excellent biocompatibility and biodegradability. The merits of gelatin microgels such as tunable size, biocompatibility, and stimulus responsive upload and release of positively charged small molecules will permit the microgels as excellent carriers for drug delivery. The whole manufacturing process is furthermore environmental‐friendly involving no organic solvents and surfactants.     

White‐Emitting Protein Nanoparticles for Cell‐Entry and pH Sensing

Facile synthesis of white‐emitting, protein‐based, metal‐free, stable, nontoxic, and pH sensitive, advanced functional nanoparticles (GlowDots), as alternatives to quantum dots, is reported here. Controlled cross‐linking of bovine serum albumin resulted in facile formation of spherical nanoparticles of 35 nm in diameter with a sharp size distribution (±10 nm), which were then conjugated with specific dyes to produce white‐emitting particles with tunable excitation wavelengths. Chemical novelty is that the particle size, size distribution, stability, surface chemistry, and emission properties are under full chemical control where the size and absorption/emission properties are independently tuned. Up to 100 dye molecules were attached to each particle, on an average, and hence, particles acquired strong absorption cross‐sections as well as high brightness. White fluorescence of GlowDots is strongly sensitive to pH over a range of pH 2–11, and pH‐induced emission changes are fully reversible. The particles readily entered HeLa cells and emission color depended on particle location in the live cells, which is most likely due to the local environment surrounding the particles. These are the very first reports of white‐emitting advanced functional nanoparticles that are biodegradable, sensitive to pH, and amenable for live cell imaging to probe the subcellular compartments.     

Targeted Single‐Cell Therapeutics with Magnetic Tubular Micromotor by One‐Step Exposure of Structured Femtosecond Optical Vortices

Selective manipulation of specific single cells for therapeutics is important and highly desirable in biomedical research. As a simple and maneuverable tool, tubular micromotors have displayed appealing applications in encapsulation and transportation of cells. However, so far there are no reports on the simultaneous transportation of target single cells and the drugs with microtubes in a custom arrayed environment for targeted therapeutics. Moreover, fabrication of microtubes with 3D features in a reproducible and single‐step fashion, while, endowing them with the ability of remote control, remains challenging. In this study, a novel method for one‐step fabrication of magnetic 3D tubular micromotors by single exposure of structured optical vortices in a magnetic photoresist is presented. The size and geometry of fabricated microtubes are flexibly controlled in three dimensions. Precise propelling of the tubular micromotors and precise capture, targeted delivery, and release of SiO₂ microparticles are realized. Finally, as a proof‐of‐concept demonstration, in situ observation of the development of doxorubicin in Hela cells for therapeutic study is performed by targeted delivery of single cells and drug particles. The technology is simple and stable, which has promising applications in targeted cell therapy, drug screening, single cell studies, and other biomedical areas.     

Photoinduced Tetrazole‐Based Functionalization of Off‐Stoichiometric Clickable Microparticles

We report the preparation of tetrazole‐containing step‐growth microparticles and the subsequent use of photoinduced nitrile imine‐mediated tetrazole‐ene cycloaddition (NITEC) reactions on the particles with spatiotemporal control. Microparticles with an average diameter of 4.1 µm and with inherent tetrazole‐ene dual functionality are prepared by a one‐pot off‐stoichiometric thiol‐Michael addition dispersion polymerization. The NITEC reaction is performed efficiently in the solid phase by UV irradiation, leading to the formation of fluorescent pyrozoline adducts, with an estimated quantum yield of 0.7. Particle concentration‐independent reaction kinetics are observed and full conversion is reached within 10 min of UV exposure at an intensity of 8 mW cm^?2. Temporal control is demonstrated with either UV or rooftop sunlight irradiation of variable duration. By using two‐photon writing with a laser centered around 700 nm wavelength, spatial control is demonstrated with micrometer‐scale resolution via surface patterning of the microparticles. Further, microparticles with exclusive tetrazole functionality are prepared by a one‐pot, two‐step thiol‐Michael addition dispersion polymerization. The NITEC reaction between tetrazole‐functional particles and acrylates in solution is examined at various tetrazole/alkene molar ratios, and a 10:1 excess of alkenes in solution is found necessary for efficient functionalization.     

From Fragility to Flexibility: Construction of Hydrogel Bridges toward a Flexible Multifunctional Free‐Standing CaCO3 Film

Free‐standing CaCO₃ materials are an important member in biological systems because of their existence in many natural organisms such as nacre, shell, and crustacean cuticle. However, toughness of those artificial mineral films is sacrificed once their inorganic content is up to 90%, thus free‐standing characteristics have seldom been achieved for CaCO₃ films, let alone their real applications. Herein a fast and simple method for constructing hydrogel “bridges” for CaCO₃ microparticles is presented, developing highly flexible free‐standing CaCO₃ films with only 5% organic content. Such integrated films have underwater superoleophobicity and self‐cleaning function, which guarantee their repeated application in oil/water separation. Furthermore, heavy metal ions can be efficiently removed by simple filtration with the films. Because of the self‐similar structure, the films are able to resist mechanical abrasion without losing the anti‐wetting property and separation efficiency. The free‐standing CaCO₃ films are put forward for the first time to practical application, demonstrating the strategy can bring a brilliant prospect to artificial biomineral materials.     

Polymer Microparticles Exhibit Size and Shape Dependent Accumulation around the Nucleus after Endocytosis

Particulate drug delivery has received significant attention in the last few decades. The effect of particle properties such as size, shape and surface properties on particle‐cell interaction has been studied. Here, intracellular accumulation and subsequent spatial segregation of spherical and rod shaped microparticles is investigated. It is observed that both spherical and rod shaped particles exhibit perinuclear accumulation. However, when the cells are fed with binary mixtures of particles, they spatially segregate in the cytoplasm based on their shape and size. Larger particles exhibit preferential accumulation closer to the nucleus. These results have potential implications in understanding the biophysical forces operating in the cells that impact the intracellular organization of drug carriers as well as organelles.     

Protein Particles Formed by Protein Activation and Spontaneous Self‐Assembly

In this article, a non‐chemical crosslinking method is used to produce pure protein microparticles with an innovative approach, so‐called protein activation spontaneous and self‐assembly (PASS). The fabrication of protein microparticles is based on the idea of using the internal disulfide bridges within protein molecules as molecular linkers to assemble protein molecules into a microparticle form. The assembly process is triggered by an activating reagent–dithiothreitol (DTT), which only involved in the intermediate step without being incorporated into the resulting protein microparticles. Conventional protein microparticle fabrication methods usually involve emulsification process and chemical crosslink reactions using amine reactive reagents such as glutaraldehdye or EDC/NHS. The resulting protein microparticles are usually having various size distributions. Most importantly crosslinking reactions using amine reactive reagents will result in producing protein microparticles with undesired properties such as auto‐fluorescence and high toxicity. In contrast to the conventional methods, our technology provides a simple and robust method to produce highly homogeneous, stable and non‐fluorescence pure protein microparticles under mild conditions at physiological pH and temperature. The protein microparticles are found to be biodegradable, non‐toxic to MDCK cells and with preserved biological activities. Results on the cytotoxcity study and enzyme function demonstrate the potential applications of the protein microparticles in the area of pharmaceutics and analytical chemistry.     

Hemoglobin‐CdTe‐CaCO3@Polyelectrolytes 3D Architecture: Fabrication,Characterization, and Application in Biosensing

A Hemoglobin‐CdTe‐CaCO₃@polyelectrolyte 3D architecture is synthesized by a stepwise layer‐by‐layer method and is further used to fabricate an electrochemistry biosensor. While the calcium carbonate (CaCO₃) microsphere acts as an effective host for the loading of cadmium telluride (CdTe) quantum dots due to its channel‐like structure, the polyelectrolyte layers further increase the loading amount and help in the formation of a thick and uniform quantum‐dot “shell”, which not only improves the stability of the spheres in water, but also contributes to the fast and effective direct electron transfer between the protein redox center and the macroscopic electrode. The materials are characterized and compared, and the possible mechanism for the direct electrochemistry phenomenon is hypothesized. Our work not only provides a facile and effective route for the preparation of quantum‐dot‐loaded spheres, but also sets an example of how the structure of functional materials can be tuned and related to their applications. In addition, it is one of the few examples of using CaCO₃ microspheres in quantum‐dot loading and biosensing.     

A Novel Mesoporous CaO‐Loaded In2O3 Material for CO2 Sensing

A mesoporous CaO‐loaded In₂O₃ material (with Ca/In₂O₃ ratios ranging from 2.5 to 8.5 at %) has been synthesized and used as resistive gas sensor for the detection of CO₂. A nanostructured In₂O₃ matrix has been obtained by hard template route from the SBA‐15 silica template. Additive presence does not distort the lattice of In₂O₃, which crystallizes in the Ia3 cubic space group. It has been proved by XRD, HRTEM, Raman and XPS measurements that samples contain not only CaO but also CaCO₃ in calcite phase as a consequence of CaO carbonation. Pure In₂O₃ based sensors are low sensitive to CO₂, whereas those containing the additive show an important response in the 300–5000 ppm range of gas concentrations. As seen by DRIFTS, the electrical response arises from the interaction between CO₃^2– and CO₂, yielding bicarbonates products. The reaction is water‐assisted, so that hydration of the sensing material ensures sensor reliability whilst its dehydration would inhibit sensor response. The use of CaCO₃ instead of CaO does not cause significant differences in electrical and DRIFTS data, which corroborates the important role played by carbonate species in the sensing mechanism.     

Synthesis of Optically Complex,Porous, and Anisometric Polymeric Microparticles by Templating from Liquid Crystalline Droplets

It is demonstrated that aqueous dispersions of micrometer‐sized liquid crystal (LC) droplets provide the basis of a general and facile methodology for the templated synthesis of spherical and nonspherical polymeric microparticles with complex internal structure and porosity. Specifically, nematic droplets of reactive (RM257)/nonreactive mesogens with distinct internal configurations are prepared using a range of approaches, the reactive mesogens are photopolymerized, and then the nonreactive mesogens are extracted to yield polymeric particles. It is found that LC droplets exhibiting bipolar, radial, axial or preradial configurations template the formation of spindle‐shaped, spherical, spherocylindrical or tear‐shaped polymeric microparticles, respectively. Each type of microparticle exhibits distinct optical signatures indicating the presence of an internal LC‐templated, anisotropic polymer network. In addition, by using a microfluidic system to generate monodisperse LC droplets containing 10%–40% wt/wt of RM257, spindle‐shaped microparticles with tailored aspect ratios ranging from 2.4 to 1.2 are formed. The mass density of spherical microparticles templated from radial LC droplets can be tuned to range from 0.2 to 0.6 g cm^?3, revealing the introduction of porosity (confirmed by electron microscopy) with a volume‐average pore diameter of 39 ± 16 nm (obtained from nitrogen sorption isotherms).     

Glucose‐Responsive Bioinorganic Nanohybrid Membrane for Self‐Regulated Insulin Release

A bioinorganic nanohybrid glucose‐responsive membrane is developed for self‐regulated insulin delivery analogous to a healthy human pancreas. The application of MnO₂ nanoparticles as a multifunctional component in a glucose‐responsive, protein‐based membrane with embedded pH‐responsive hydrogel nanoparticles is proposed. The bio‐nanohybrid membrane is prepared by crosslinking bovine serum albumin (BSA)–MnO₂ nanoparticle conjugates with glucose oxidase and catalase in the presence of poly(N‐isopropyl acrylamide‐co‐methacrylic acid) nanoparticles. The preparation and performance of this new nanocomposite material for a glucose‐responsive insulin release system is presented. The activity and stability of immobilized glucose oxidase and the morphology and mechanical properties of the membrane are investigated. The enzymatic activity is well preserved in the membranes. The use of MnO₂ nanoparticles not only reinforces the mechanical strength and the porous structure of the BSA‐based membrane, but enhances the long‐term stability of the enzymes. The in vitro release of insulin across the membrane is modulated by changes in glucose concentration mimicking possible fluctuations of blood‐glucose level in diabetic patients. A four‐fold increase in insulin permeation is observed when the glucose concentration is increased from normal to hyperglycemic levels, which returns to the baseline level when the glucose concentration is reduced to a normal level.     

Responsive Macroscopic Materials From Self‐Assembled Cross‐Linked SiO2‐PNIPAAm Core/Shell Structures

A way to obtain macroscopic responsive materials from silicon‐oxide polymer core/shell microstructures is presented. The microparticles are composed of a 60 nm SiO₂‐core with a random copolymer corona of the temperature responsive poly‐N‐isopropylacrylamide (PNIPAAm) and the UV‐cross‐linkable 2‐(dimethyl maleinimido)‐N‐ethyl‐acrylamide. The particles shrink upon heating and form a stable gel in both water and tetrahydrofuran (THF) at 3–5 wt% particle content. Cross‐linking the aqueous gel results in shrinkage when the temperature is increased above the lower critical solution temperature and it regains its original size upon cooling. By freeze drying with subsequent UV irradiation, thin stable layers are prepared. Stable fibers are produced by extruding a THF gel into water and subsequent UV irradiation, harnessing the cononsolvency effect of PNIPAAm in water/THF mixtures. The temperature responsiveness translates to the macroscopic materials as both films and fibers show the same collapsing behavior as the microcore/shell particle. The collapse and re‐swelling of the materials is related to the expelling and re‐uptake of water, which is used to incorporate gold nanoparticles into the materials by a simple heating/cooling cycle. This allows for future applications, as various functional particles (antibacterial, fluorescence, catalysis, etc.) can easily be incorporated in these systems.     

Electric Field Assisted Microfluidic Platform for Generation of Tailorable Porous Microbeads as Cell Carriers for Tissue Engineering

Injection of cell‐laden scaffolds in the form of mesoscopic particles directly to the site of treatment is one of the most promising approaches to tissue regeneration. Here, a novel and highly efficient method is presented for preparation of porous microbeads of tailorable dimensions (in the range ≈300–1500 mm) and with a uniform and fully interconnected internal porous texture. The method starts with generation of a monodisperse oil‐in‐water emulsion inside a flow‐focusing microfluidic device. This emulsion is later broken‐up, with the use of electric field, into mesoscopic double droplets, that in turn serve as a template for the porous microbeads. By tuning the amplitude and frequency of the electric pulses, the template droplets and the resulting porous bead scaffolds are precisely produced. Furthermore, a model of pulsed electrodripping is proposed that predicts the size of the template droplets as a function of the applied voltage. To prove the potential of the porous microbeads as cell carries, they are tested with human mesenchymal stem cells and hepatic cells, with their viability and degree of microbead colonization being monitored. Finally, the presented porous microbeads are benchmarked against conventional microparticles with nonhomogenous internal texture, revealing their superior performance.     

Synthesis and Optical Properties of KYF4/Yb,Er Nanocrystals,and their Surface Modification with Undoped KYF4

KYF₄/Yb³⁺, Er³⁺ nanocrystals with a mean diameter of approximately 13 nm were synthesized at 200 °C in the high boiling organic solvent N‐(2‐hydroxyethyl)ethylenediamine (HEEDA). The particles crystallize in the cubic phase known from α‐NaYF₄ and form transparent colloidal solutions in tetraethylene glycol (TEG) or propanol. Solutions containing 1 wt % of the nanocrystals in TEG display visible upconversion emission upon continuous wave (CW) excitation at 978 nm. Growing undoped KYF₄ on the surface of the KYF₄/Yb³⁺, Er³⁺ nanocrystals increases the upconversion efficiency by more than a factor of 20. The XRD data of these particles, display a slight increase in the mean particle size from 13 to 15.5 nm, indicating that only a part of the subsequently added KYF₄ shell material is deposited onto the particle surface. Nevertheless the performed surface modification obviously leads to core/shell structured particles.     

Recent Progress in the Synthesis of Spherical Titania Nanostructures and Their Applications

Recent progress in the fabrication and application of diverse spherical titania nanostructures, including mesoporous spheres, spherical flaky assemblies, and dendritic particles of variable diameter and monodispersity in size, is summarized in this article. Utilizing different synthesis strategies, spherical titania nanostructures with tailored polymorphs (including amorphous, anatase, rutile, brookite and TiO₂‐B), particle sizes (from tens of nanometers to millimeters), monodispersity, porosity, and variable surface properties have been produced. Such spherical titania nanostructures show realized and potential applications in the areas of chromatographic separation, lithium‐ion batteries, dye‐sensitized solar cells, photocatalytic oxidation and water splitting, photoluminescence, electrorheological fluids, catalysis, gas sensing, and anticancer intracellular drug delivery. Gaining further understanding of both synthesis design and application of these materials will promote the commercialization of such spherical titania nanostructures in the future.     

Hexahistidine‐Tagged Single‐Walled Carbon Nanotubes (His6‐tagSWNTs): A Multifunctional Hard Template for Hierarchical Directed Self‐Assembly and Nanocomposite Construction

While a hexahistidine affinity tag can be introduced at protein termini or internal sites by standard molecular biology procedures for purification, immobilization, or labeling of proteins, here the versatility of this concept is exploited for the chemical preparation of novel hexahistidine‐tagged single‐walled carbon nanotubes (His₆‐tagSWNTs), a novel hard template useful for solubilizing, assembling, processing, and interfacing SWNTs in aqueous conditions. Water‐soluble and exfoliated His₆‐tagSWNTs are prepared and fully characterized. This functional molecular module is able to interact via robust physisorption (π?π stacking) with the sidewall of SWNTs and combines the versatility of small, water‐soluble reporters (His₆) for hierarchical directed self‐assembly (HDSA) and construction of nanocomposites. It is demonstrated that metal coordination bonds with Ni(II) can be used for the supramolecular self assembly of His₆‐tagSWNTs, generating complex reticulated networks and architectures. The His₆‐tagSWNTs hard template nanohybrid is further utilized for directed self‐assembly with silica nanoparticles. The versatility of the novel hybrids opens a new era for the rational design, smart (bio)functionalization, processing, interfacing, and self assembling of carbon nanotubes for the construction of multicomposites and more complex systems with controllable spatial organization and programmable properties for a wide range of applications in biology, nanoelectronics, and catalysis.     

The Coral Protein CARP3 Acts from a Disordered Mineral Surface Film to Divert Aragonite Crystallization in Favor of Mg‐Calcite

Stony corals construct their aragonite skeleton by calcium carbonate precipitation, in a process recently suggested to be biologically controlled. Amorphous calcium carbonate and small amounts of calcite are also reported recently, however, their functional role is unknown. Coral acid‐rich proteins (CARPs) are extracted from the coral skeleton and are shown to be active in calcium carbonate precipitation in vitro. However, individual function of these proteins in coral mineralization is not known. Here, the regulatory activity of the aspartate‐rich CARP3 protein is examined. The whole protein and two peptides representing its acidic domain and its variable domain are used in CaCO₃ precipitation reactions from Mg‐rich solutions. The biomolecules alter crystallization pathways, promoting Mg‐calcite in place of aragonite, with the acidic peptide capable of eradicating aragonite formation. The activity of CARP3 and its representative peptides is exerted from disordered CaCO₃ mineral phases, coating the crystals formed, as shown by 2D ¹H–¹³C heteronuclear correlation nuclear magnetic resonance (NMR) measurements, localizing organic protons in atomic proximity to disordered carbonate carbons. The structures of the protein and individual domains as derived from NMR measurements and folding calculations and their amino acid compositions are discussed in the context of their observed activity and its implication to mineralization in hard corals.