Improving Hydrophilic Barriers of Encapsulated Compounds in Ca-Alginate Microgel Particles through a New Ionotropic Gelation Method for Double Emulsion Droplets

The ability of encapsulation to protect hydrophilic–bioactive food compounds from harsh environments can be improved by strengthening the hydrophilic barriers of encapsulated food compounds in Ca-alginate microgel particles via the integration of oil into the microgels. This study introduces a one-step procedure to integrate water-in-oil (W/O) emulsion droplets directly into Ca-alginate microgels during the production using the impinging aerosols system. A water-in-oil-in-water (20 kg m⁻³ alginate solution) (W₁/O/W₂) double emulsion was prepared using a high speed homogeniser followed by a microfluidiser. The microstructure of the W₁/O/W₂ emulsion was analysed using optical and fluorescence microscopy. The mean diameters of the W₁/O/W₂ emulsion droplets and resultant microgels were in the range of 27.8–65.4 μm and 160–420 μm, respectively. Food dye was used as a proxy for a hydrophilic food compound and its release from the microgels was significantly decreased when it was encapsulated in the W/O emulsion droplets. Based on the numerical analysis, the presence of the W/O emulsion droplets in the gel network reduced the degree of gelation of the microgel because the diffusion rate of Ca²⁺ cation in the microgel is reduced. The degree of gelation of the W/O emulsion droplets encapsulated microgel is 0.6 when the diameter of the droplet is reduced to 77.5 μm and the concentration of CaCl₂ solution is doubled to 22 kg m⁻³. The potentiality of the impinging aerosol system to produce Ca-alginate microgels to encapsulate hydrophilic compounds with improved barriers is presented in this work.

     

Surface‐directed assembly of cell‐laden microgels

Cell‐laden microscale hydrogels (microgels) can be used as tissue building blocks and assembled to create 3D tissue constructs with well‐defined microarchitecture. In this article, we present a bottom‐up approach to achieve microgel assembly on a patterned surface. Driven by surface tension, the hydrophilic microgels can be assembled into well‐defined shapes on a glass surface patterned with hydrophobic and hydrophilic regions. We found that the cuboidic microgels (～100–200 µm in width) could self‐assemble into defined shapes with high fidelity to the surface patterns. The microgel assembly process was improved by increasing the hydrophilicity of the microgels and reducing the surface tension of the surrounding solution. The assembled microgels were stabilized by a secondary crosslinking step. Assembled microgels containing cells stained with different dyes were fabricated to demonstrate the application of this approach for engineering microscale tissue constructs containing multiple cell types. This bottom‐up approach enables rapid fabrication of cell‐laden microgel assemblies with pre‐defined geometrical and biological features, which is easily scalable and can be potentially used in microscale tissue engineering applications. Biotechnol. Bioeng. 2010; 105: 655–662. © 2009 Wiley Periodicals, Inc.     

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e. low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.     

Injectable doubly cross-linked microgels for improving the mechanical properties of degenerated intervertebral discs

The use of injectable pH-responsive doubly cross-linked microgels (DX microgels) to improve the mechanical properties of degenerated intervertebral discs is demonstrated for the first time. The microgel comprised methyl methacrylate (MMA), methacrylic acid (MAA), ethyleneglycol dimethacrylate (EGD) and glycidyl methacrylate (GM) and was poly(MMA/MAA/EGD)-GM. The GM facilitated covalent interparticle cross-linking. The DX microgels are shown to have tunable mechanical properties. Degeneration of model bovine intervertebral discs (IVDs) was induced using collagenase. When injected into degenerated IVDs the DX microgels were shown to improve the strain, modulus, toughness and resilience. The extent of mechanical property improvement was an increasing function of DX microgel concentration, suggesting tunability. Cytotoxicity studies showed that the DX microgel was biocompatible under the conditions investigated. The results of this study imply that injectable DX microgels have good potential as a future regenerative medicine strategy for restoring the mechanical properties of degenerated load-bearing soft tissue, such as IVDs.     

Functional finishing of aminated polyester using biopolymer-based polyelectrolyte microgels

This study focuses on a microgel-based functionalization method applicable to polyester textiles for improving their hydrophilicity and/or moisture-management properties, eventually enhancing wear comfort. The method proposed aims at achieving pH-/temperature-controlled wettability of polyester within a physiological pH/temperature range. First, primary amine groups are created on polyester surfaces using ethylenediamine; second, biopolymer-based polyelectrolyte microgels are incorporated using the natural cross-linker genipin. The microgels consist of the pH-responsive natural polysaccharide chitosan and pH/thermoresponsive poly(N-isopropylacrylamide-co-acrylic acid) microparticles. Scanning electron microscopy confirmed the microgel presence on polyester surfaces. X-ray photoelectron spectroscopy revealed nitrogen concentration, supporting increased microscopy results. Electrokinetic analysis showed that functionalized polyester surfaces have a zero-charge point at pH 6.5, close to the microgel isoelectric point. Dynamic wetting measurements revealed that functionalized polyester has shorter total water absorption time than the reference. This absorption time is also pH dependent, based on dynamic contact angle and micro-roughness measurements, which indicated microgel swelling at different pH values. Furthermore, at 40 °C functionalized polyester has higher vapor transmission rates than the reference, even at high relative humidity. This was attributed to the microgel thermoresponsiveness, which was confirmed through the almost 50% decrease in microparticle size between 20 and 40 °C, as determined by dynamic light scattering measurements.     

Viscoelastic behavior and in vivo release study of microgel dispersions with inverse thermoreversible gelation

The dispersion of microgels with two interpenetrating polymer networks of poly( N-isopropylacrylamide) and poly(acrylic acid) (PNIPAM-IPN-PAAc) has been studied for its viscoelastic behavior, biocompatibility, and in vivo release properties. The IPN microgels in water had an average hydrodynamic radius of about 85 nm at 21 degrees C, measured by dynamic light scattering method. The atomic force microscope image showed that the particles were much smaller after they were dried but remained spherical shape. The storage and loss moduli ( G' and G') of dispersions of IPN microgels were measured in the linear stress regime as functions of temperature and frequency at various polymer concentrations using a stress-controlled rheometer. For dispersions with polymer concentrations of 3.0 and 6.0 wt % above 33 degrees C, the samples behave as viscoelastic solids and the storage modulus was larger than the loss modulus over the entire frequency range. The loss tangent was measured at various frequencies as a function of temperature. The gelation temperature was determined to be 33 degrees C at the point where a frequency-independent value of the loss tangent was first observed. At pH 2.5, when heated above the gelation temperature, IPN microgels flocculate by pumping a large amount of water from the gel. When the pH value was adjusted to neutral, deprotonation of -COOH groups on PAAc made the microgel keep water even above the gelation temperature. Using an animal implantation model, the biocompatibility and drug release properties of the IPN microgel dispersion were evaluated. Fluorescein as a model drug was mixed into an aqueous microgel dispersion at ambient temperature. This drug-loaded liquid was then injected subcutaneously in Balb/C mice from Taconic Farms. The test results have shown that the IPN microgels did not adversely promote foreign body reactions in this acute implantation model and the presence of gelled microgel dispersion substantially slowed the release of fluorescein.     

Effects of peptide secondary structure on the interaction with oppositely charged microgels

The importance of peptide secondary structure on the interaction between antimicrobial peptides and oppositely charged poly(acrylic acid-co-acrylamide) microgels of various charge density was investigated for EFKRIVQRIKDFLRNLV (EFK17). Through D-enantiomer (EFK17-d/a; E(dF)KR(dI)VQR(dI)KD(dF)LRNLV) or tryptophan (EFK17-W/a; EWKRWVQRWKDFLRNLV) substitutions, both conformation-dependent and -independent amphiphilicity of this peptide could be precisely controlled. Peptide secondary structure was investigated by circular dichroism, whereas microgel deswelling and reswelling in response to peptide binding and release were studied by micromanipulator-assisted light and fluorescence microscopy, and peptide uptake in the microgels was determined from solution depletion measurements. Results show that peptide binding to the microgel is highly influenced by peptide secondary structure. EFK17-a, characterized by an idealized helix with all polar/charged amino acids located at one side of the helix, and all nonpolar/hydrophobic residues on the other, displays pronounced α-helix induction on peptide binding to the microgels. EFK17-d/a, on the other hand, displays no such amphiphilic helix induction. Mirroring this, EFK17-a displays substantially higher binding to the microgels than EFK17-d/a as well as much larger peptide-induced microgel deswelling. For EFK17-W/a, both conformation-dependent and -independent amphiphilicity effects were demonstrated. Overall, the results show that peptide conformational aspects need to be considered in peptide/microgel interactions, for example, in the design of microgel carrier systems for peptide drugs.     

Growth of fibroblasts on linear and planar anchorages of limiting dimensions

BHK and 3T3 cells were grown on small glass fibres and platelets (size range 20–250 μm) in agar gel, over a layer of mouse primary feeder cells. Under these conditions the growth of colonies (4–20 cells) from single control cells, suspended freely in the gel, was less than 1 %, while cells attached to the larger platelets yielded more than 95 % colonies. Fibres (average diameter 0.625 μm) induced somewhat fewer colonies (45–70 %). However, there was a significant induction of single divisions by fibres as short as 30 μm, while on 60 μm fibres more than half the cells had gone through at least one division, compared with 4–12% of freely suspended controls. Longer fibres promoted multiple divisions. Colony formation increased sigmoidally with fibre length from 30 to 250 μm, with a d₅₀ around 115 μm. These figures resembled the observed range of lengths of freely growing BHK and 3T3 fibroblasts in tissue culture dishes. In another experiment, finer fibres (average diameter 0.075 μm) performed almost as well as platelets of equal length but of width 20–30 μm.Thus linear extension provides a stimulus for division of anchorage-dependent fibroblasts. It is suggested that this stimulus may arise from a change in conformation or tension in the cell membrane, rather than from increased surface, cell motility or interaction with substrate surface.The present results are discussed in relation to recent findings on the induction of tumours by solid particles.     

Characterization of Different Substituted Carboxymethyl Starch Microgels and Their Interactions with Lysozyme

A carboxymethyl starch (CMS) microgel system was prepared for the control of uptaking and releasing proteins (lysozyme). The physicochemical properties of microgels in various degrees of substitution (DS) were determined by thermal gravimetric analysis (TGA), swelling degree, and rheological analysis. The microgel particle size mostly ranged from 25 µm to 45 µm. The result obtained from the TGA studies indicated that carboxymethylation decreased the thermal stability of starch, but crosslinking increased the thermal stability of CMS. The CMS microgels showed typical pH sensitivity, and the swelling degree of microgel increased with the increasing of DS and pH, because of the large amounts of carboxyl group ionization. The samples (2.25%) could behave as viscoelastic solids since the storage modulus was larger than the loss modulus over the entire frequency range. The protein uptake increased with increasing pH and DS at low salt concentration. The optimal pH shifted to lower pH with increasing ionic strength. The saturated protein uptake decreased with increasing ionic strength at each pH. The protein was easily released from the microgel with high pH and high salt concentration.     

Doxorubicin uptake and release from microgel thin films

We report investigations on the thermally regulated uptake and release of the chemotherapeutic drug doxorubicin from microgel thin films. A spin coating, layer-by-layer (scLbL) assembly approach was used to prepare thin films composed of thermoresponsive poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-AAc) microgels by alternatively exposing a 3-aminopropyltrimethoxysilane (APTMS) functionalized glass substrate to polyanionic pNIPAm-AAc microgels and polycationic poly(allylamine hydrochloride) (PAH). Using this method, 10, 20, and 30 microgel layer films were constructed with uniform layer buildup, as confirmed by quartz crystal microgravimetry (QCM). The films were subsequently loaded with doxorubicin by cycling the temperature of the film in an aqueous doxorubicin solution between 25 and 50 degrees C. Release characteristics were then examined using UV-vis spectroscopy, which revealed temperature-dependent release properties.     

Ultrastructure of the labial tip sensilla of the tarnished plant bug,Lygus lineolaris (P. de Beauvois) (Hemiptera : Miridae)

Sensilla on the labial tip of the tarnished plant bug, Lygus lineolaris, were examined with scanning and transmission electron microscopy in order to provide morphological evidence indicative of their function. The tripartate apex of the labium consists of 2 lateral lobes and an apical plate. Each lateral lobe possesses a field of 11 thick-walled, uniporous peg sensilla, 5–6 μm long and a thick-walled, nonporous hair sensillum, 18–22 μm long. The uniporous peg sensilla are innervated by 3 or 5 bipolar neurons. The nonporous hair sensillum has no dendrites within its lumen. The apical plate is a noninnervated structure which possesses terminal cuticular projections 5–8 μm long. Morphological evidence supports previously reported physiological evidence that the uniporous peg sensilla have a chemosensory function.     

Biodegradable and biocompatible polyampholyte microgels derived from chitosan, carboxymethyl cellulose and modified methyl cellulose

Two biocompatible and biodegradable polyampholyte microgels, namely chitosan-carboxymethyl cellulose (CS-CMC) and chitosan-modified methyl cellulose (CS-ModMC) were synthesized by an inverse microemulsion technique. The CS-CMC microgel system was pH-responsive while the CS-ModMC system possessed both pH and thermo-responsive properties. For CS-CMC system, the number of -OCH₂COOH and -NH₂ groups was determined to be 1.5 and 1.1 meq/g of microgel, respectively. In the pH range of 4-9, the zeta potential values varied from +10 to −40 mV, while the hydrodynamic radius varied from 160 nm in the swollen state (acidic and basic pH) to 110 nm in the “collapse” state (neutral pH). Furthermore, TEM micrographs confirmed the swelling/deswelling behaviour of CS-CMC microgel particles at acidic, neutral and basic conditions. For CS-ModMC system, the number of -OCH₂COOH and -NH₂ groups was determined to be 0.8 and 0.6 meq/g microgel, respectively. In the pH range of 4-9, the surface charge on the microgels varied from +25 to −60 mV and the hydrodynamic radii were 190 nm at low pH, 80 nm at neutral pH, to 120 nm at a high pH. In vitro drug release studies confirmed that CS-CMC microgels could encapsulate and release a model drug, thus they could potentially be used as biocompatible and biodegradable drug carriers.     

External morphology of sensilla in the sacculus of an antennal flagellum of the fruit fly Drosophila melanogaster Meigen (Diptera : Drosophilidae)

Sensilla in the sacculus of an antennal 3rd segment, a funiculus, of the fruit fly, Drosophila melanogaster (Diptera : Drosophilidae) have been examined by scanning electron microscopy. The sacculus was divided into 3 cavities in its interior. A morphologically distinct group of sensilla was present in each cavity. Grooved sensillum (GS), found in the largest cavity, was further subclassified on the basis of the side wall sculpture into 3 subgroups: GS Ia, GS Ib and GS II. GS Ia was 4 μm long and had 10–12 grooves (0.25 μm wide) and GS Ib was 3.8 μm long and had 6–9 grooves (0.25-0.4 μm wide). GS 1a and GS Ib were inferred to be olfactory and thermoreceptive, and olfactory, respectively. GS II was 3.2 μm long, and had 4–5 grooves. Basiconic sensillum (BS), found in the smallest cavity, was 4.5 μm long and had an irregularly sculptured side wall, suggesting the presence of numerous irregular-shaped olfactory pores. Blunt-tipped sensillum (BTS), found in the middle-sized cavity, was 1.9 μm long and had a smooth-surfaced side wall and a button-like structure on its apex. These features suggested that BTS was hygro- and thermoreceptive.     

Architecture of chitin gel as examined by scanning electron microscopy

An architecture of the solid phase of chitin gel was examined by scanning electron microscopy. The ultrastructure of the xerogel was microporous with parallel channels surrounded with membranous walls. The pore shapes at cross section were polyhedral with three walls at the junctions. The pore was 30–50 μm in diameter and 80–300 μm in length, and the thickness of the walls was less than 1.5 μm. The gel is considered to be a polyphasic gel, consisting of small droplets of water held up in these pores.     

Cell adaptations of Rhodococcus rhodochrous IEGM 66 to betulin biotransformation

The findings of a comprehensive study on R. rhodochrous IEGM 66 and triterpenoid betulin interactions during its biotransformation were reported. In the presence of betulin, rhodococci were shown to form heterogeneous cell aggregates. The enhanced size of the aggregates from 12–15 μm to 25– 35 μm was consistent with the increase in betulin concentration from 0.5 to 3.0 g/L. The confocal laser scanning microscopy indicated a high (80.0%) level of rhodococcal viability during betulin biotransformation regardless of the betulin concentration. Experiments employing the combined confocal laser scanning and atomic force microscopy system confirmed that interactions between actinobacterial cells and betulin occur by direct contact. Transforming activities of the crude cell extracts from R. rhodochrous IEGM 66 were compared, and localization of enzymes catalyzing betulin oxidation to betulone was determined. Additionally the effects of betulin on fatty acid composition of rhodococci and their morphometric and morphofunctional characteristics during biotransformation were studied. Our findings could be used to develop approaches for enhanced betulin bioavailability, thus leading to improved biotransformation efficiency.     

Fabrication and biocompatibility of a porous bioglass ceramic in a Na2OCaOSiO2P2O5 system

A porous bioglass ceramic was prepared from a finely pulverized bioglass powder mixed with particles of two sizes (5 and 500 μm) of 30% by weight with the foaming agent polyethylene glycol 4000 (HO (C₂H₄O) nH). The batch composition of the bioglass was Na₂O 12%, CaO 28%, SiO₂ 50% and P₂O₅ 10% by weight. The specimens, formed by pressing, were sintered in a high temperature furnace. In this study we are concerned with the preparation and microstructure of the material and its performance in biological tests. The microstructure and crystalline phases of the material were investigated by differential thermal analysis, X-ray diffraction analysis, transmission electron microscopy and scanning electron microscopy. In a biomedical examination, it was shown that the porous material was compatible with animal tissues. The microstructure of the implant indicated that newly grown bone interlocked well with the glass ceramic and that macropores and micropores were distributed uniformly in the material, which provided channels for bone ingrowth and improved the microscopic bioresorption.     

Ultrastructure of the sperm of Dolania americana Edmunds and Traver (Ephemeroptera : Behningiidae)

The ultrastructure of the mature sperm of the mayfly, Dolania americana Edmunds and Traver (Ephemeroptera : Behningiidae), is described from scanning and transmission electron microscopy. The head is 0.7–1 μm wide and 4.6–6.9 μm long, rodlike, and topped by a short, rounded acrosome 0.4 μm long and 0.6 μm wide. The flagellum is 5–6 times the head length and is flattened, except for a thin, tubelike terminal portion. The axoneme pattern is 9-9-1 (9 outer singlet microtubules, 9 doublet microtubules, and a central dark element) and is new for Ephemeroptera. The inner dynein arms are conspicuous and outer arms are lacking, and radial spokes and a central sheath are prominent. A densely-staining and bi-lobed accessory body lies adjacent to the axoneme. A mitochondrial derivative with regularly arranged transverse-to-oblique cristae lies adjacent to the accessory body.     

Charge-switching, amphoteric glucose-responsive microgels with physiological swelling activity

Amphoteric, poly(N-isopropylacrylamide)-based microgels are functionalized with aminophenylboronic acid (PBA) functional groups to produce colloidally stable, glucose-responsive gel nanoparticles that exhibit glucose-dependent swelling responses at physiological temperature, pH, and ionic strength. Up to 2-fold volumetric swelling responses are observed in response to physiological glucose concentrations, the first such physiological response reported for a colloidally stable microgel. Amphoteric microgels can also be designed to both swell and deswell in response to glucose according to the pH of the medium, the concentration of PBA groups grafted to the microgel, and the relative concentrations of the cationic and anionic functional groups in the platform microgel. The increasing anionic charge density on the microgels observed at higher glucose binding fractions can be applied to switch the net charge of the microgels from cationic to anionic as the glucose concentration increases. Preliminary experiments suggest that such amphoteric PBA-microgels have a high capacity for insulin uptake and can selectively release more insulin at higher glucose concentrations under physiological conditions via glucose-induced, "on-off" switching of electrostatic attractions between insulin and the microgel.     

Design and Concept of Polyzwitterionic Copolymer Microgel Drug Delivery Systems <Emphasis Type="Italic">In Situ</Emphasis> Loaded with Non-steroidal Anti-inflammatory Ibuprofen

Nowadays, the modern pharmaceutical investigations are directed toward obtaining of new polymer micro- and nano-sized drug delivery carriers. In this respect, the use of hydrogel carriers based on polyzwitterions (PZIs) is an opportunity in the preparation of polymer drug delivery systems with desired characteristics. This paper describes the synthesis and characterization of micro-structured p(VA-co-DMAPS) systems with different compositions in situ loaded with Ibuprofen by emulsifier-free emulsion copolymerization (EEC) in water. The mean size of the prepared microparticles was measured by SEM and particles have been visualized by AFM. The inclusion of Ibuprofen in the polyzwitterionic copolymer microgel systems was established by using DSC. In vitro drug release experiments were carried out in order to estimate the ability of the obtained microgels to modify the release of water-insoluble Ibuprofen.