A new method for microcapsule characterization

Numerous microcapsule systems have been developed for a wide range of applications, including the sustained release of drugs, cell transplantation for therapy, cell immobilization, and other biotechnological applications. Despite the fact that microcapsule membrane is a dominant factor governing overall microcapsule performance, its characterization is challenging. We report a new method for characterizing microcapsule membranes, using the most common alginate-poly-l-lysine-alginate (APA) microcapsule as an example. Our data demonstrate that genipin, a naturally derived reagent extracted from gardenia fruits, interacts with poly-l-lysine (PLL) and generates fluorescence. This fluorescence allows clear visualization and easy analysis of the PLL membrane in the APA microcapsules using confocal laser scanning microscopy. The results also show that PLL binding correlates to the reaction variables during PLL coating such as PLL concentration and coating time. In addition, five other different microcapsule formulations consisting of PLL and/or chitosan membranes were examined, and the results imply that this method can be extended to characterize a variety of microcapsule membranes. These findings suggest that genipin can serve as a fluorogenic marker for rapid characterization of microcapsule membranes, a superior method that would have important implications for microcapsule research and potential in many other applications.     

Proliferation, viability, and metabolism of human tumor and normal cells cultured in microcapsule

In this study, we investigated the effect of the microenvironment provided by alginate-poly-l-lysine-alginate (APA) microcapsule with liquefied or gelled core on the proliferation, viability, and metabolism of human cells, including anchorage-dependent MCF-7 breast cancer cells and primary fibroblasts, and anchorage-independent K-562 leukemia cells; cells in conventional culture were used as control. The growth pattern of cells in microcapsule was examined by phase-contrast micrography. The cell viability, proliferation, organization, and gene expression were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, hematoxylin and eosin staining, live/dead staining, 5-bromo-20-deoxyuridine labeling, and immunohistochemistry, respectively. Cell metabolism was determined by measuring glucose and lactate concentrations in medium. The results demonstrate that APA microcapsule with liquefied core provides a microenvironment for both anchorage-dependent and anchorage-independent cells to grow into a large cell aggregate and maintain cell viability at a constant level for a period of time. In conclusion, cells in APA microcapsule are alive and have proliferation potential with lower metabolism rate. APA microcapsule may be a useful tool for in vitro tumor cell modeling and anticancer drug screening as well as for cancer gene therapy. In addition, it lays a solid foundation for the use of microencapsulation in cell culture in vitro and cell implantation in vivo.     

Single-particle light scattering study of polyethyleneglycol-grafted poly(ureaurethane) microcapsule in ethanol

Microcapsules having polyethyleneglycol-grafted poly(ureaurethane) (PUU) membrane and di-2ethylhexyl phthalate core have been prepared, and the structure when they were suspended in dispersing ethanol have been studied by means of single-particle light scattering method. The PUU membrane was synthesized from monomers with aromatic functional groups (microcapsule MC110) and hexamethylene functional groups (microcapsule MC160). Because the outer and inner solvent passed through the membrane easily, the outside and inside of the membrane were the same at the equilibrium state. The thickness of the wall membrane was significantly smaller than that calculated from the overall weight ratio of the wall-forming material and the core solvents. It was attributed to low affinity of PUU membranes and ethanol.     

Viscoelastic properties of hydrogel microcapsule membranes

The viscoelastic properties of poly(L-lysine-alt-terephthalic acid) (PPL) microcapsules were studied as a function of medium pH. An abrupt increase in the apparent relative viscosity of PPL microcapsule suspensions was observed in the pH range between 4.0 and 7.0 due to the increased total particle volume concentration caused by a sudden increase in microcapsule size. Above pH 7.0, the relative viscosity decreased, being indicative of a deformability of the microcapsules. The adiabatic compressibility of PPL microcapsule membranes was the lowest at pH 4.0 and increased remarkably when the pH value departed from 4.0. On the contrary, the membrane density was the highest at pH 4.0 and decreased as medium pH shifted to either side of this value, implying that the microcapsules behave as compact particles at low pH and loose particles at high pH. The amount of hydrated water also showed a similar change when the pH of the medium was altered.     

Using NaCl particles as porogen to prepare a highly adsorbent chitosan membranes

Macroporous chitosan membranes were prepared by using NaCl particles porogen and genipin as cross-linking agent. For characterization and sorption behavior comparison, other genipin cross-linked chitosan membranes were prepared by either freeze drying or by using silica particles as porogen. The mean pore diameter, the porosity, the crystallinity index (CrI) as well as the effect of the drying procedures of these chitosan membranes were examined. NaCl reduced the CrI of the chitosan membrane. The oven drying (OD) procedure decreased the mean pore diameter, the porosity, and increased the CrI of the chitosan membranes when compared with the vacuum drying (VD) procedure. The heat treatment of chitosan membrane in aqueous NaOH to attract silica porogen increased the CrI of the membrane. Under the same conditions, the membranes prepared with NaCl had better sorption performance on RR 189 and Cu²⁺ than other membranes. The maximum sorption capacity (q_e) reached 1836.17 mg RR 189/g chitosan and 151.98 mg Cu²⁺/g chitosan. The pore diameter (d_pore) of the membranes was much larger than the diameter of the adsorbate molecules (d_adsorbate), such that the ratio of d_pore/d_adsorbate had little influence on q_e. The porosity and the amorphous extent of the membranes were almost the same on q_e. When using tyrosinase catalyzing, the hydrocaffeic acid (HCA) grafted on the NaCl treated chitosan membrane was almost 10 times more than on chitosan beads. The chitosan membrane prepared with NaCl can be used as a good adsorbent with high loading capacity for implanting molecules (such as ligands, enzymes, etc.) on.     

APA微胶囊扩散数学模型的改进

在膜阻力系数和膜内基质的分配系数两个新特性的基础上,重新建立了蛋白质在APA微胶囊体系上的扩散数学模型,该模型更为准确地反映出蛋白质在微胶囊上的扩散特性     

Preparation of Antibacterial Gelatin/Genipin Nanofibrous Membrane for Tympanic Membrane Repair

Tympanic membrane perforation (TMP), a common disease, often needs a scaffold as the patch to support surgery. Due to the environment of auditory meatus, the patch can be infected by bacteria that results in failure; therefore, the ideal scaffold may combine biomimetic and antibacterial features. In this work, gelatin was used as the electrospinning framework, genipin as the crosslinking agent, and levofloxacin as an antibacterial in order to prepare the scaffold for TMP. Different contents of levofloxacin have been added to gelatin/genipin. It was found that, with the addition of levofloxacin, the gelatin/genipin membranes exhibit improved hydrophilia and enhanced tensile strength. The antibacterial and cell-cultured experiments showed that the prepared antibacterial membranes had excellent antibacterial properties and good biocompatibility, respectively. In summary, levofloxacin is a good group for the gelatin/genipin scaffold because it improves the physical properties and antibacterial action. Compared with different amounts of levofloxacin, a gelatin/genipin membrane with 1% levofloxacin is more suitable for a TM.     

Embedded silver ions-containing liposomes in polyelectrolyte multilayers: cargos films for antibacterial agents

A new antibacterial coating made of poly(L-lysine)/hyaluronic acid (PLL/HA) multilayer films and liposome aggregates loaded with silver ions was designed. Liposomes filled with an AgNO 3 solution were first aggregated by the addition of PLL in solution. The obtained micrometer-sized aggregates were then deposited on a PLL/HA multilayer film, playing the role of a spacer with the support. Finally, HA/PLL/HA capping layers were deposited on top of the architecture to form a composite AgNO 3 coating. Release of encapsulated AgNO 3 from this composite coating was followed and triggered upon temperature increase over the transition temperature of vesicles, found to be equal to 34 degrees C. After determination of the minimal inhibitory concentration (MIC) of AgNO 3 in solution, the antibacterial activity of the AgNO 3 coating was investigated against Escherichia coli. A 4-log reduction in the number of viable E. coli cells was observed after contact for 120 min with a 120 ng/cm (2) AgNO 3 coating. In comparison, no bactericidal activity was found for PLL/HA films previously dipped in an AgNO 3 solution and for PLL/HA films with liposome aggregates containing no AgNO 3 solution. The strong bactericidal effect could be linked to the diffusion of silver ions out of the AgNO 3 coating, leading to an important bactericidal concentration close to the membrane of the bacteria. A simple method to prepare antibacterial coatings loaded with a high and controlled amount of AgNO 3 is therefore proposed. This procedure is far superior to that soaking AgNO 3 or Ag nanoparticles into a coating. In principle, other small bactericidal chemicals like antibiotics could be encapsulated by this method. This study opens a new route to modify surfaces with small solutes that are not permeating phospholipid membranes below the phase transition temperature.     

Fluorescent lipids: functional parts of fusogenic liposomes and tools for cell membrane labeling and visualization

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.     

Flow-induced shear stress and deformation of a core–shell-structured microcapsule in a microchannel

A numerical model was developed and validated to investigate the fluid–structure interactions between fully developed pipe flow and core–shell-structured microcapsule in a microchannel. Different flow rates and microcapsule shell thicknesses were considered. A sixth-order rotational symmetric distribution of von Mises stress over the microcapsule shell can be observed on the microcapsule with a thinner shell configuration, especially at higher flow rate conditions. It is also observed that when being carried along in a fully developed pipe flow, the microcapsule with a thinner shell tends to accumulate stress at a higher rate compared to that with a thicker shell. In general, for the same microcapsule configuration, higher flow velocity would induce a higher stress level over the microcapsule shell. The deformation gradient was used to capture the microcapsule's deformation in the present study. The effect of Young's modulus on the microcapsule shell on the microcapsule deformation was investigated as well. Our findings will shed light on the understanding of the stability of core–shell-structured microcapsule when subjected to flow-induced shear stress in a microfluidic system, enabling a more exquisite control over the breakup dynamics of drug-loaded microcapsule for biomedical applications.     

Porous microcapsule formation with microsieve emulsification

A simple route is presented to prepare core-shell Eudragit microcapsules through a solvent extraction method with the use of microsieve emulsification. Droplets from a solution of Eudragit FS 30D (a commercial copolymer of poly(methyl acrylate-co-methyl methacrylate-co-methacrylic acid) 7:3:1) and hexadecane in dichloromethane are dispersed into water, using a micro-engineered membrane with well-defined pores, in a cross-flow setting. The dichloromethane is extracted from the droplets, which induces demixing in the droplets, leading to a hexadecane-rich core, and an Eudragit-rich shell. The obtained microcapsules have a narrow size distribution due to the microsieve emulsification process. The capsules have a porous shell as shown by SEM and AFM measurements. Their porosity and pore size is dependent on the ratios of Eudragit and hexadecane in the dispersed phase. At pH 7.1 and above Eudragit (FS 30D) dissolves in water; this pH change is used to release the contents of the microcapsule.     

An approach to membrane fouling characterization by confocal scanning laser microscopy

Confocal scanning laser microscopy (CSLM) is an optical microscopic technique that, among other advantages, can provide high-resolution images from different depths of a three-dimensional object, therefore rendering invasive techniques unnecessary for sample preparation. CSLM in fluorescence mode is a powerful technique in biological applications and in the microscopy of food materials. The main goal of the present study is to develop the appropriate strategies so that CSLM can be used for membrane fouling characterization during the filtration of protein solutions. Single and binary solutions of BSA–fluorescein and ovalbumin–Texas red conjugates were filtered using 0.8 μm polycarbonate membranes. Samples of the membranes at the end of the filtration runs were analyzed by CSLM. A standardized protocol for sample analysis by CSLM was developed and applied in this study. The most significant results show that CSLM can be used to visualize BSA–fluorescein and ovalbumin–Texas red conjugates on top of and inside the membranes, and that they can be distinguished when they jointly foul the membrane. Finally, if the appropriate sectioning is applied a 3D reconstruction of the membrane and the adsorbed/deposited protein can be obtained which give information on the fouling morphology.     

Interaction between charged soft microcapsules and red blood cells: effects of PEGylation of microcapsule membranes upon their surface properties

Four types of hydrophilic gel microcapsules containing water have been prepared by an interfacial polymerization method. Each type of microcapsules has a membrane of different composition. Using three kinds of monomers, N,N-dimethylacrylamide (DMAAm), 4-(aminomethyl)styrene (AmSt), and N,N-dimethylaminopropylacrylamide (DMAPAA), one type of aqueous copolymer having primary and tertiary amino groups was obtained. By the polymerization of three kinds of monomers, DMAAm, AmSt, and 2-[(methacryloyloxy)ethyl] trimethylammoniumchloride (METAC), another type of aqueous copolymer having primary and quaternary ammonium groups was also obtained. Two more types of copolymers were synthesized by copolymerization of -acryloxy-ω-methoxy-poly(ethylene glycol) (a-PEG) with the above two kinds of monomer mixture. These copolymers were polymerized with terephthaloyldichloride at the water/oil interface to prepare four types of microcapsules containing water, i.e., poly(DMAAm-co-DMAPAA-co-AmSt-alt-terephthalic acid) microcapsules, poly(DMAAm-co-DMAPAA-co-AmSt-co-PEG-alt-terephthalic acid) microcapsules, poly (DMAAm-co-METAC-co-AmSt-alt-terephthalic acid) microcapsules, and poly (DMAAm-co-METAC-co-AmSt-co-PEG-alt-terephthalic acid) microcapsules, which will be abbreviated to MC 1, MC 2, MC 3, and MC 4, respectively. It has been predicted that the microcapsule membranes are hydrophilic and soft and have two-sublayer structures from electrophoretic mobility measurements and from the analysis of the data with Ohshima’s electrokinetic theory for soft particles. The outer sublayers of MC 1 and MC 2 are negatively charged and those of MC 3 and 4 are slightly positively charged. Also, the surfaces of MC 1 and MC 2 are harder than those of MC 3 and 4. By PEGylation, the surface charge density in the membranes decreases and the surface becomes softer. It has been found that the membrane of red blood cells (RBC) is also soft and is composed of two-sublayers, the outer sublayer of which is negatively charged and the inner one is positively charged. The interaction of four types of microcapsules with RBC has been studied. It was found that microcapsules with soft surfaces (MC 3 and MC 4) do not interact with RBC, even though the microcapsule surfaces are positively charged and the surface of RBC is negatively charged. On the other hand, microcapsules with negatively charged but harder surfaces (MC 1) interact with RBC to introduce hemolysis. The membrane surface of MC 2, which is obtained by PEGylation of MC 1, becomes softer than that of MC 1 so that the interaction with RBC was weakly suppressed. From these, it was concluded that the dominant factor to control the interaction between synthetic polymer surfaces and biological cell surfaces is not the surface charges carried by the polymer surfaces but the softness of the polymer surfaces.     

Quantitative characterization of membrane formation process of alginate–chitosan microcapsules by GPC

The semi-permeable membrane of alginate–chitosan (AC) microcapsules has been proven important to control the microcapsule stability and selective substance diffusion rate. Therefore, a novel and operable methodology based on gel permeation chromatography (GPC) was established for quantitative characterization of the membrane formation process, so as to provide guidance on performance improvement of AC microcapsules in biomedical applications. Not only the molecular weight (M_w) and its distribution of chitosan can be obtained by GPC, but also the area integral of molecular weight peaks can be linearly correlated to chitosan concentration in certain range. The dynamic membrane formation process was then obtained by quantitatively analyzing reaction amount of chitosan with time, which showed that for chitosan molecules with wide M_w distribution, only parts of molecules bind with alginate to form microcapsule membrane. Moreover, the contribution of chitosan molecules participating in the membrane formation process was also different. These new findings, therefore, are helpful for adjusting and controlling the membrane formation process and properties of microcapsule membrane.     

Phase transition of microcapsule membranes induced by pH changes

The phase transition of poly(L-lysine-alt-terephthalic acid) (PPL) microcapsules induced by changes in the pH of the medium was investigated. The total number of ionizable groups in one capsule or the charge density of the microcapsule membrane was found to be important for the phase transition phenomenon to occur. Thus, the existence of a minimum was suggested, for the total number of ionizable groups in one capsule or for the charge density of the microcapsule membrane, if a pH-induced phase transition of PPL microcapsules is to be caused.The presence of SDS at low concentrations brought about the phase transition of PPL microcapsules, even when the microcapsules were poorly charged at low pH values, through enhanced adsorption of the surfactant unions to the constituent polymers of the microcapsules at high ionic strengths of the medium. Addition of 1,4-dioxane to the microcapsule suspension prevented the phase transition phenomenon from taking place.     

Re-entrant volume phase transition of hydrogel membrane of microcapsule

Re-entrant volume phase transition of hydrogel wall membrane of microcapsules (MC) was first observed using MC suspensions consisting of poly (L-lysine-alt-terephthalic acid) wall and aqueous inner and outer solutions with different pHs. To analyze the dynamics of the re-entrant phase transition, we extended the theory for the swelling and the shrinking dynamics of the microcapsule gel [T. Narita, T. Yamamoto, D. Suzuki, T. Dobashi, Langmuir 19 (2004) 4051]. In the theory, the microcapsule size and the force constant for the driving force which gives rise to the size relaxation were chosen as the thermodynamic variables. The time course of the cross-sectional area of the microcapsules fitted well to the theoretical equations, and the time constants determined as the fitting parameters were discussed in terms of the force constant relaxation and the size relaxation.     

Dielectric measurement of a single sub-millimeter size microcapsule

A new technique is described for measuring dielectric dispersion of a single microcapsule in suspension over a frequency range from 1 kHz to 10 MHz. It was applied to polystyrene microcapsules which showed a two-step dielectric dispersion, that is, a superposition of two Debye type dispersions. The dielectric dispersion was analyzed by an electrical model in which a spherical core covered with a shell is immersed in a continuous phase, yielding the phase parameters related to the microcapsule: the wall thickness, the permittivity and conductivity of the core phase. The advantage of this technique is that it can characterize individual microcapsules, whereas the conventional method provides average properties of many microcapsules. Hence, the technique enables us to directly determine the distributions of the phase parameters and to exactly examine the relationship between the dielectric behavior and the microcapsule structure simultaneously observed by microscopy.     

Novel nanocomposite membranes from cellulose acetate and clay‐silica nanowires

In this study, a new class of heterogeneous membranes based on cellulose acetate (CA) polymer and a complex filler clay‐silica nanowires (SiO₂NWs) was investigated for potential biomedical applications. SiO₂NWs were synthesized using natural clay through a facile sol–gel method and were dispersed in the polymer solution by sonication in the 1.25, 2.5, and 5% weight ratio to the CA acetate polymer. Membranes were subsequently prepared via phase inversion by precipitation of the CA polymer in water. The pristine CA membrane and SiO₂NWs based nanocomposites membranes were characterized using different characterization techniques. The presence of the SiO₂NWs in the CA membrane was found to significantly enhance the protein retention, water wettability and thermal as well as mechanical properties in comparison to the pristine CA membrane. Water flows studies at different temperatures and the retention of bovine serum albumin have been studied and the nanocomposite membranes were found to exhibit superior performances compared with the pristine CA membranes. SiO₂NWs‐CA membranes showed a much higher stability to the water temperature change during separation than CA membranes. Morphological changes clearly revealed that the composite membrane were much more compact than the pristine CA membranes. The rabbit dermal fibroblasts cell viability in cultures after 72 hr of incubation was found to be greater than 80%. These newly synthesized composite membranes exhibit a high potential to be used for various medical applications because of their non‐cytotoxic characteristics. Copyright © 2016 John Wiley & Sons, Ltd.     

Kinetic analysis of distribution of weak acid in an oil-gelatin microcapsule/water system studied by microcapillary injection and microabsorption methods.

The distribution of anthracene-9-carboxylic acid across dibutyl phthalate/gelatin-membrane/water interface of a single microcapsule was analyzed using microcapillary manipulation and microabsorption methods. The partitioning ratio and the distribution rate in the microcapsule/water system were measured for various pH values in the water phase. Results were compared with those in the dibutyl phthalate/water system in the absence of the gelatin membrane. The distribution rate could be analyzed on the basis of a first-order type reaction. The observed rate constant was linearly proportional to the inverse of the microcapsule radius, indicating that the distribution rate is limited by interfacial mass transfer. Analysis of the pH dependence of the interfacial mass transfer rate suggests that the mass transfer of the neutral species of anthracene-9-carboxylic acid (AnH) competes with the ion transfer of the dissociated species (An-) at the liquid/liquid interface in the gelatin membrane of the microcapsule.