Controlled release of C-type natriuretic peptide by microencapsulation dampens proinflammatory effects induced by IL-1ß in cartilage explants.

C-type natriuretic peptide (CNP) exhibits potent anti-inflammatory effects in chondrocytes that have the potential to repair cartilage damage observed in osteoarthritis (OA). However, treatments for OA have been challenging due to poor targeting and delivery of therapeutics. The present study fabricated polyelectrolyte microcapsules loaded with CNP and examined whether the layer-by-layer (LbL) approach could have protective effects in cartilage explants treated with the pro-inflammatory cytokine, interleukin-1ß (IL-1ß). SEM showed uniform, 2 to 3 µm spherical microcapsules with morphological characteristic similar to templates loaded with or without CNP. The protein was localized around the external surface of the microcapsules with encapsulation efficiencies >82.9%. CNP release profiles were broadly similar following 9 days of culture. The presence of CNP microcapsules did not significantly affect cell viability (80%) with DNA values that remained stable throughout the culture conditions. Confocal imaging showed clustering of microcapsules in chondrocytes to natriuretic peptide receptor (Npr) 2 and 3. Treatment of cartilage explants with CNP microcapsules led to concentration-dependent inhibition of NO release in response to IL-1ß and restoration of matrix synthesis. In summary, we demonstrate controlled delivery of CNP to dampen pro-inflammatory effects induced by IL-1ß in cartilage explants. The LbL approach has the potential to promote cartilage repair in vivo.

Lessons in microcapsule assembly from imaging delivery of a bioluminescent enzyme.

Layer-by-layer assembled microcapsules have potential applications as delivery and biosensing systems, which make them attractive tools for use in various aspects of nanomedicine. We examined the effect of microcapsule location on activity of the bioluminescent enzyme luciferase in both intact capsules and following cell uptake. In intact capsules, the rate of reaction of luciferase was greatest for luciferase in the outer layer and least in the core. Following cell uptake, luciferase in the outer layer was rapidly reactive, and a similar rate of reaction and activity was observed for luciferase placed in capsule interior (core). By contrast, there was minimal activity detected when microcapsules with luciferase sandwiched between polyelectrolytes in a middle layer were delivered to cells. This study informs us of the availability of bioactive molecules located in different positions within microcapsules and will enable better microcapsule construction in line with the intended application, particularly delivery of functional proteins to cells.

Chemosensors and biosensors based on polyelectrolyte microcapsules containing fluorescent dyes and enzymes.

The concept of enzyme-assisted substrate sensing based on use of fluorescent markers to detect the products of enzymatic reaction has been investigated by fabrication of micron-scale polyelectrolyte capsules containing enzymes and dyes in one entity. Microcapsules approximately 5 µm in size entrap glucose oxidase or lactate oxidase, with peroxidase, together with the corresponding markers Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride (Ru(dpp)) complex and dihydrorhodamine 123 (DHR123), which are sensitive to oxygen and hydrogen peroxide, respectively. These capsules are produced by co-precipitation of calcium carbonate particles with the enzyme followed by layer-by-layer assembly of polyelectrolytes over the surface of the particles and incorporation of the dye in the capsule interior or in the multilayer shell. After dissolution of the calcium carbonate the enzymes and dyes remain in the multilayer capsules. In this study we produced enzyme-containing microcapsules sensitive to glucose and lactate. Calibration curves based on fluorescence intensity of Ru(dpp) and DHR123 were linearly dependent on substrate concentration, enabling reliable sensing in the millimolar range. The main advantages of using these capsules with optical recording is the possibility of building single capsule-based sensors. The response from individual capsules was observed by confocal microscopy as increasing fluorescence intensity of the capsule on addition of lactate at millimolar concentrations. Because internalization of the micron-sized multi-component capsules was feasible, they could be further optimized for in-situ intracellular sensing and metabolite monitoring on the basis of fluorescence reporting.

SERS Assays Based on Electrospun Nanofibers: Preparation and Analytical Applications.

Surface-enhanced Raman spectroscopy (SERS) is a powerful tool and an up-to-date method of analytical chemistry due to its high sensitivity and fingerprint recognition capabilities. Nowadays SERS due to its label-free detection capabilities is being actively developed in medical fields, for example in the analysis of biologically important substances in different matrixes, for potential on-site detection of toxic substances, food safety, and so on. To get the SERS signal, it is necessary the presence of plasmonic nanostructures in the SERS substrates. Electrospun nanofibers have been an attractive alternative to SERS-platforms due to the diversity of advantages, including ease of preparation, structure flexibility, and others. In this review, we summarized the methods of plasmonic nanostructures incorporating substrate based on electrospun nanofibers. Also, the analytical application of SERS-active electrospun nanofibers with embedded nanostructures focused on biologically significant molecules is observed in detail. Finally, the future outlook in the application of these substrates in bioanalysis as the most promising area in analytical chemistry is presented.

CaCO3-based carriers with prolonged release properties for antifungal drug delivery to hair follicles.

Superficial fungal infections are of serious concern worldwide due to their morbidity and increasing distribution across the globe in this era of growing antimicrobial resistance. The delivery of antifungals to the target regions of the skin and sustaining the effective drug concentration are essential for successful treatment of such mycoses. Topical formulations get extra benefits here if they penetrate into the hair follicles since fungal hyphae can proliferate and produce spores in such reservoirs. We designed a novel particulate system for the encapsulation and intrafollicular delivery of griseofulvin (Gf) antifungal drug, which is water-insoluble and currently commercially available in oral dosage forms. Micron-sized calcium carbonate (vaterite) carriers containing 25 ± 3% (w/w) of Gf were prepared via the wet chemical method. The successful in vivo transportation of the carriers into the hair follicles of rats was demonstrated using scanning electron and confocal laser scanning microscopy. In addition, we introduced an approach toward Gf release prolongation for the proposed system. The stabilizing coatings were formed on the surface of the obtained particles via the layer-by-layer technique. The formulations displayed sufficient biocompatibility and good cellular uptake in contact with fibroblast cells in vitro. Four different coatings were tested for their preserving ability in the course of continued carrier incubation in the model media. The best release prolonging formulation liberated 38% of the loaded Gf during 5 days, while the uncoated carriers demonstrated more than 50% drug release within the first 24 h in water. To assess the in vivo release properties, free Gf drug and Gf-loaded carriers (uncovered and covered with the stabilizing shell) were administered topically in rats and the drug excretion profiles were further studied. By comparing the daily Gf levels in urine, we verified the sustained effect (longer than a week) of the stabilizing shell formed on the carrier surface. Conversely, the application of the free drug did not provide reliable Gf detection for this period. These findings open new prospects for the efficiency enhancement of topical therapeutics. Importantly, the elaborated system could be adapted for the dermal delivery of various water-insoluble drugs beyond the scope of antifungal therapy.

Recrystallization of CsPbBr3 Nanoparticles in Fluoropolymer Nonwoven Mats for Down- and Up-Conversion of Light.

Inorganic halides perovskite CsPbX3 (X = Cl, Br, and I or mixed halide systems Cl/Br and Br/I) nanoparticles are efficient light-conversion objects that have attracted significant attention due to their broadband tunability over the entire visible spectral range of 410-700 nm and high quantum yield of up to 95%. Here, we demonstrate a new method of recrystallization of CsPbBr3 nanoparticles inside the electrospun fluoropolymer fibers. We have synthesized nonwoven tetrafluoroethylene mats embedding CsPbBr3 nanoparticles using inexpensive commercial precursors and syringe electrospinning equipment. The fabricated nonwoven mat samples demonstrated both down-conversion of UV light to 506 nm and up-conversion of IR femtosecond laser radiation to 513 nm green photoluminescence characterized by narrow emission line-widths of 35 nm. Nanoparticle formation inside nonwoven fibers was confirmed by TEM imaging and water stability tests controlled by fluorimetry measurements. The combination of enhanced optical properties of CsPbBr3 nanoparticles and mechanical stability and environmental robustness of highly deformable nonwoven fluoropolymer mats is appealing for flexible optoelectronic applications, while the industry-friendly fabrication method is attractive for commercial implementations.

Biodegradable Microcapsules Loaded with Nerve Growth Factor Enable Neurite Guidance and Synapse Formation.

Neurological disorders and traumas often involve loss of specific neuronal connections, which would require intervention with high spatial precision. We have previously demonstrated the biocompatibility and therapeutic potential of the layer-by-layer (LbL)-fabricated microcapsules aimed at the localized delivery of specific channel blockers to peripheral nerves. Here, we explore the potential of LbL-microcapsules to enable site-specific, directional action of neurotrophins to stimulate neuronal morphogenesis and synaptic circuit formation. We find that nanoengineered biodegradable microcapsules loaded with nerve growth factor (NGF) can guide the morphological development of hippocampal neurons in vitro. The presence of NGF-loaded microcapsules or their clusters increases the neurite outgrowth rate while boosting neurite branching. Microcapsule clusters appear to guide the trajectory of developing individual axons leading to the formation of functional synapses. Our observations highlight the potential of NGF-loaded, biodegradable LbL-microcapsules to help guide axonal development and possibly circuit regeneration in neuropathology.

Novel type of hollow hydrogel microspheres with magnetite and silver nanoparticles.

A novel type of microcontainers based on hollow silver alginate microspheres and magnetite nanoparticles is reported as development of recently published technology. Magnetite nanoparticles were incorporated by two methods - co-precipitation with porous calcium carbonate during template formation and adsorption onto CaCO3 particles or microcontainers' shell. Amount of magnetite loaded and microshells size (4.6 to 6.9 µm) were found to depend on the chosen method for magnetite nanoparticles incorporation. Stability of hollow microshells in saline, phosphate buffer and culturing media was studied. Microcontainers' susceptibility to magnetic field was investigated in solutions of varied viscosity, and their group movement velocity under constant magnetic field was evaluated by sequential optical microscopy imaging. Cell viability tests with prepared microshells were performed that demonstrated negligible cytotoxicity effect on human dermal fibroblasts cells. With HeLa cells moderate viability inhibition was found at high carriers:cells ratio at early time points which is attributed to more active and receptor-mediated endocytosis of carriers as well as known cytotoxicity of magnetite in some cancer cells. At 24 and 48 h time points HeLa cells proliferation fully recovers. Reported data opens perspectives for further biomedical-oriented studies and application of this novel kind of microcontainers with a number of techniques applicable for imaging, control and triggered cargo release provided by presence of silver and magnetite nanoparticles in the carriers and their suitability for further versatile functionalization by traditional LbL approach if needed.

Improved and targeted delivery of bioactive molecules to cells with magnetic layer-by-layer assembled microcapsules.

Despite our increasing knowledge of cell biology and the recognition of an increasing repertoire of druggable intracellular therapeutic targets, there remain a limited number of approaches to deliver bioactive molecules to cells and even fewer that enable targeted delivery. Layer-by-layer (LbL) microcapsules are assembled using alternate layers of oppositely charged molecules and are potential cell delivery vehicles for applications in nanomedicine. There are a wide variety of charged molecules that can be included in the microcapsule structure including metal nanoparticles that introduce physical attributes. Delivery of bioactive molecules to cells with LbL microcapsules has recently been demonstrated, so in this study we explore the delivery of bioactive molecules (luciferase enzyme and plasmid DNA) to cells using biodegradable microcapsules containing a layer of magnetite nanoparticles. Interestingly, significantly improved intracellular luciferase enzyme activity (25 fold) and increased transfection efficiency with plasmid DNA (3.4 fold) was observed with magnetic microcapsules. The use of a neodymium magnet enabled efficient targeting of magnetic microcapsules which further improved the delivery efficiency of the cargoes as a consequence of increased microcapsule concentration at the magnetic site. Microcapsules were well tolerated by cells in these experiments and only displayed signs of toxicity at a capsule : cell ratio of 100 : 1 and with extended exposure. These studies illustrate how multi-functionalization of LbL microcapsules can improve and target delivery of bioactive molecules to cells.

Alpha-2-macroglobulin loaded microcapsules enhance human leukocyte functions and innate immune response.

Synthetic microstructures can be engineered to deliver bioactive compounds impacting on their pharmacokinetics and pharmacodynamics. Herein, we applied dextran-based layer-by-layer (LbL) microcapsules to deliver alpha-2-macroglobulin (α2MG), a protein with modulatory properties in inflammation. Extending recent observations made with dextran-microcapsules loaded with α2MG in experimental sepsis, we focused on the physical and chemical characteristics of these microstructures and determined their biology on rodent and human cells. We report an efficient encapsulation of α2MG into microcapsules, which enhanced i) human leukocyte recruitment to inflamed endothelium and ii) human macrophage phagocytosis: in both settings microcapsules were more effective than soluble α2MG or empty microcapsules (devoid of active protein). Translation of these findings revealed that intravenous administration of α2MG-microcapsules (but not empty microcapsules) promoted neutrophil migration into peritoneal exudates and augmented macrophage phagocytic functions, the latter response being associated with alteration of bioactive lipid mediators as assessed by mass spectrometry. The present study indicates that microencapsulation can be an effective strategy to harness the complex biology of α2MG with enhancing outcomes on fundamental processes of the innate immune response paving the way to potential future development in the control of sepsis.

Multilayer Capsules of Bovine Serum Albumin and Tannic Acid for Controlled Release by Enzymatic Degradation.

With the purpose to replace expensive and significantly cytotoxic positively charged polypeptides in biodegradable capsules formed via Layer-by-Layer (LbL) assembly, multilayers of bovine serum albumin (BSA) and tannic acid (TA) are obtained and employed for encapsulation and release of model drugs with different solubility in water: hydrophilic-tetramethylrhodamine-isothiocyanate-labeled BSA (TRITC-BSA) and hydrophobic 3,4,9,10-tetra-(hectoxy-carbonyl)-perylene (THCP). Hydrogen bonding is proposed to be predominant within thus formed BSA/TA films. The TRITC-BSA-loaded capsules comprising 6 bilayers of the protein and polyphenol are benchmarked against the shells composed of dextran sulfate (DS) and poly-l-arginine (PARG) on degradability by two proteolytic enzymes with different cleavage site specificity (i.e., α-chymotrypsin and trypsin) and toxicity for murine RAW264.7 macrophage cells. Capsules of both types possess low cytotoxicity taken at concentrations equal or below 50 capsules per cell, and evident susceptibility to α-chymotrypsin resulted in release of TRITC-BSA. While the BSA/TA-based capsules clearly display resistance to treatment with trypsin, the assemblies of DS/PARG extensively degrade. Successful encapsulation of THCP in the TRITC-BSA/TA/BSA multilayer is confirmed, and the release of the model drug is observed in response to treatment with α-chymotrypsin. The thickness, surface morphology, and enzyme-catalyzed degradation process of the BSA/TA-based films are investigated on a planar multilayer comprising 40 bilayers of the protein and polyphenol deposited on a silicon wafer. The developed BSA/TA-based capsules with a protease-specific degradation mechanism are proposed to find applications in personal care, pharmacology, and the development of drug delivery systems including those intravenous injectable and having site-specific release capability.

Microparticle alpha-2-macroglobulin enhances pro-resolving responses and promotes survival in sepsis.

Incorporation of locally produced signaling molecules into cell-derived vesicles may serve as an endogenous mediator delivery system. We recently reported that levels alpha-2-macroglobulin (A2MG)-containing microparticles are elevated in plasma from patients with sepsis. Herein, we investigated the immunomodulatory actions of A2MG containing microparticles during sepsis. Administration of A2MG-enriched (A2MG-E)-microparticles to mice with microbial sepsis protected against hypothermia, reduced bacterial titers, elevated immunoresolvent lipid mediator levels in inflammatory exudates and reduced systemic inflammation. A2MG-E microparticles also enhanced survival in murine sepsis, an action lost in mice transfected with siRNA for LRP1, a putative A2MG receptor. In vitro, A2MG was functionally transferred onto endothelial cell plasma membranes from microparticles, augmenting neutrophil-endothelial adhesion. A2MG also modulated human leukocyte responses: enhanced bacterial phagocytosis, reactive oxygen species production, cathelicidin release, prevented endotoxin induced CXCR2 downregulation and preserved neutrophil chemotaxis in the presence of LPS. A significant association was also found between elevated plasma levels of A2MG-containing microparticles and survival in human sepsis patients. Taken together, these results identify A2MG enrichment in microparticles as an important host protective mechanism in sepsis.

Location of molecules in layer-by-layer assembled microcapsules influences activity, cell delivery and susceptibility to enzyme degradation.

Layer-by-layer assembled microcapsules have the potential to be versatile cell delivery systems incorporating multiple activities and functions. However, it is necessary to determine the influence that different capsule locations have on activity of bioactive molecules in order to optimise delivery and for generation of multifunctional capsules. In this study we examine the influence that locating the bioluminescent enzyme luciferase in different microcapsule locations has on activity in intact synthetic and biodegradable microcapsules before and after cell delivery as well as its susceptibility to protease degradation. We also examine the effect of microcapsule position on cell transfection with plasmid DNA. Based on the findings of experiments in this study we also demonstrate co-delivery of luciferase protein and plasmid DNA encoding a fluorescent protein from two different locations within the same microcapsule. Our studies confirm that, the core, subouter layer, and outer layer are optimal for cell delivery but these positions offer least protection from protease activity. By contrast middle layer molecules remain entangled with capsule layers preventing their release which is inefficient for cell delivery but this provides better protection from protease degradation. The findings of this study will enable more rationale layer-by-layer assembly of microcapsules containing biologically active molecules for cell delivery and aid in the generation of multifunctional microcapsules.

Magnetically engineered microcapsules as intracellular anchors for remote control over cellular mobility.

Living cells are anchored with magnetic microcapsules that allow in vitro manipulation via a magnetic field.

NIR-light triggered delivery of macromolecules into the cytosol.

Light-responsive microcapsules constructed by layer-by-layer self-assembly are used as microcarriers to deliver different macromolecules inside cells. The microcapsules carry the macromolecules as cargo in their cavity, while their walls are modified with agglomerated gold nanoparticles. Microcapsules are incorporated by living cells and are then located in lysosomal compartments. Controlled release of the encapsulated material from the interior of the capsule to the cytosol is possible upon NIR-light irradiation. This is based on local heating of the gold nanoparticles upon NIR light and disruption of the capsule wall, what results on release of encapsulated materials. We illustrate several key advances in controlled release induced by light. First, we demonstrate that capsules can be opened individually, which allows for sequentially releasing cargo from different capsules within one single cell. Second, by using a pH-indicator as cargo the claim of release from the acidic lysosomal compartments to the neutral cytosol is experimentally evident which until now has been only speculated. Third, green fluorescent protein (GFP) is released to the cytosol while retaining its functionality. This demonstrates that proteins can be released without destruction by the local heating. Fourth, GFP is also administered in biodegradable capsules, which leads to a different release mechanism compared to externally triggering for light-responsive microcapsules.

Neuron cells uptake of polymeric microcapsules and subsequent intracellular release.

Neuron cells uptake of biodegradable and synthetic polymeric microcapsules functionalized with aggregates of gold nanoparticles incorporated into their shells is demonstrated in situ. In addition to traditionally used optical microscopy, electron microscopy is used both for higher-resolution imaging and for confirming the uptake by focused ion beam cross-sectioning of specific cells in situ. Subsequently, physical methods of release are compared to chemical methods wherein laser-induced intracellular release of dextran molecules into the cytosol of hippocampal neuron cells is studied in comparison to biodegradation. Implications of this work for neuroscience, bio-medicine and single cell studies are discussed.

Magnetic/gold nanoparticle functionalized biocompatible microcapsules with sensitivity to laser irradiation.

Nanocomposite microcapsules with both gold and magnetite nanoparticles in the shell were prepared in a layer-by-layer procedure using biocompatible polyelectrolytes and nanoparticles. The process of a nanocomposite multilayer formation was investigated using a quartz crystal microbalance (QCM). In addition, nanocomposite microcapsules were characterized by atomic force microscopy (AFM), transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDX). It is found that the amount of adsorbed nanoparticles is similar for nanoparticles of various sizes, while the concentration of gold nanoparticles in the shell is higher for smaller nanoparticles. Adsorption of gold nanoparticles is found to be more effective than adsorption of magnetic nanoparticles. Multifunctionality of microcapsules is manifested by dual: magnetic and optical responses. Iron oxide nanoparticles embedded in the microcapsule shell allowed for control over capsules positioning by external magnetic fields. Furthermore, the nanocomposite microcapsules could be opened by laser irradiation; these results are of interest for medical and biological applications.