Enhanced resistance of polyelectrolyte multilayer microcapsules to pepsin erosion and release properties of encapsulated indomethacin

Polyelectrolyte multilayer films were prepared through layer-by-layer (LbL) self-assembly using polysaccharide sodium alginate (ALG) and chitosan (CHI). After incubation in an enzyme pepsin solution, the multilayer film was partially destroyed as detected by the decrease in fluorescent intensity because of the enzymatic degradation of CHI. The enzymatic desorption was also observed from the microcapsule wall made of the ALG/CHI multilayer film directly deposited on indomethacin (IDM) microcrystals through LbL self-assembly. After pepsin erosion, the IDM release from the microcapsules monitored by UV absorbance was obviously accelerated because of desorption. To enhance the stability of the ALG/CHI multilayer film to the enzymatic erosion, some physical and chemical methods were established to increase film thickness or to cross-link the polysaccharides within the film. Increasing the layer number and raising the deposition temperature effectively slowed down the enzymatic desorption and release rate. Especially, increasing deposition temperature was more effective because of producing a more perfect structure in the ALG/CHI multilayer film. Cross-linking the neighboring layers of ALG and CHI with 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide in the ALG/CHI multilayer film significantly reduced the enzymatic desorption and release rate. Therefore, increasing deposition temperature and cross-linking neighboring layers are effective methods to protect the multilayer film fabricated using LbL assembly from the enzymatic erosion and to prolong the release of the encapsulated drug.     

Encapsulation of rat hepatocyte spheroids for the development of artificial liver

Hepatocyte spheroids and hepatocyte were immobilized in chitosan/alginate capsules formed by the electrostatic interactions between chitosan and alginate. After encapsulation, there was a 10% decrease in the viability of spheroids due to the exposure of the cells to a pH 6 during the encapsulation process. However, the encapsulated hepatocyte spheroids maintained over 50% viability and liver specific functions for 2 weeks while the encapsulated hepatocytes, free hepatocytes and free hepatocyte spheroids showed low viability and liver specific functions. Therefore, encapsulated hepatocyte spheroid might be applied to the development of a bioartificial liver.     

Coating and selective deposition of nanofilm on silicone rubber for cell adhesion and growth

A recently developed method for surface modification, layer-by-layer (LbL) assembly, has been applied to silicone, and its ability to encourage endothelial cell growth and control cell growth patterns has been examined. The surfaces studied consisted of a precursor, with alternating cationic polyethyleneimine (PEI) and anionic sodium polystyrene sulfonate (PSS) layers followed by alternating gelatin and poly-d-lysine (PDL) layers. Film growth increased linearly with the number of layers. Each PSS/PEI bilayer was 3 nm thick, and each gelatin/PDL bilayer was 5 nm thick. All layers were more hydrophilic than the unmodified silicone rubber surface, as determined from contact angle measurements. The contact angle was primarily dictated by the outermost layer. Of the coatings studied, gelatin was the most hydrophilic. A film of (PSS/PEI)₄/(gelatin/PDL)₄/ gelatin was highly favorable for cell adhesion and growth, in contrast to films of (PSS/PEI)₈ or (PSS/PEI)₈/PSS. Cell growth patterns were successfully controlled by selective deposition of microspheres on silicone rubber, using microcontact printing with a silicone stamp. Cell adhesion was confined to the region of microsphere deposition. These results demonstrate that the LbL self-assembly technique provides a general approach to coat and selectively deposit films with nanometer thickness on silicone rubber. Furthermore, they show that this method is a viable technique for controlling cellular adhesion and growth.     

Layer-by-Layer Assembly of Food-Grade Alginate/Chitosan Nanolaminates: Formation and Physicochemical Characterization

The alternate deposition of oppositely charged materials (layer-by-layer technique) is an effective approach to functionalize materials. Biopolymer-based nanolaminates obtained by the layer-by-layer technique can also be used to change the surface properties of food products or food contact materials. However, the final properties of nanolaminates may be affected by the conditions of the adsorbing solutions. The objective of this study was to form and characterize the physicochemical properties of nanolaminates assembled from alginate and chitosan solutions. The effect of pH, ionic strength and polysaccharide concentration on the properties of the adsorbing solutions was also evaluated. The ζ-potential, viscosity and whiteness index of the solutions were assessed before the assembly. Alginate/chitosan nanolaminates were characterized in terms of UV-visible spectroscopy, surface ζ-potential, contact angle, DSC analysis and SEM. The absorbance increased as a function of the number of polysaccharide layers on the substrate, suggesting an increase in the mass adsorbed. The surface ζ-potential of nanolaminates changed depending on the last polysaccharide deposited. Alginate layers were negatively charged, whereas chitosan layers were positively charged. Contact angles obtained in alginate layers were ≈ 10°, being mostly hydrophilic. Chitosan layers showed higher contact angle values (80°), indicating a more hydrophobic behavior. Microscopic examinations revealed the presence densely packed structures that corresponded to alginate/chitosan nanolaminates, having an estimated thickness of 700 nm. The results obtained in this work lay the basis for the rational design of polysaccharide-based nanolaminates in the food sector.     

Functionalization of magnetic nanowires by charged biopolymers

We report on a facile method for the preparation of biocompatible and bioactive magnetic nanowires. The method consists of the direct deposition of polysaccharides by layer-by-layer (LbL) assembly onto a brush of metallic nanowires obtained by electrodeposition of the metal within the nanopores of an alumina template supported on a silicon wafer. Carboxymethylpullulan (CMP) and chitosan (CHI) multilayers were grown on brushes of Ni nanowires; subsequent grafting of an enzyme was performed by conjugating free amine side groups of chitosan with carboxylic groups of the enzyme. The nanowires are finally released by a gentle ultrasonic treatment. Transmission electron microscopy, electron energy-dispersive loss spectroscopy, and x-ray photoelectron spectroscopy indicate the formation of an homogeneous coating onto the nickel nanowires when one, two, or three CMP/CHI bilayers are deposited. This easy and efficient route to the biochemical functionalization of magnetic nanowires could find widespread use for the preparation of a broad range of nanowires with tailored surface properties.     

Locally controlled release of basic fibroblast growth factor from multilayered capsules

Biodegradable multilayered capsules encapsulating basic fibroblast growth factor (bFGF) were developed as a cytokine release carrier for drug delivery systems. The multilayered hollow capsules were fabricated via the layer-by-layer (LbL) assembly of chitosan (CT) and dextran sulfate (Dex). The bFGF was encapsulated into the CT/Dex multilayered capsules by controlling the membrane permeability, and the local and sustained release of bFGF from the capsules was examined. At pH < 8.0, the capsule membrane tightened, and FITC-dextran ( Mw = 4000) could not enter the capsules. However, FITC-dextran ( M w = 250000) easily entered the capsules at pH > 8.0, which can be attributed to the electrostatic repulsion of Dex caused by the deprotonation of the amine group in CT. After treatment with acetic acid buffer (pH 5.6), FITC-dextran or bFGF was successfully encapsulated into the capsules. The amount of encapsulated bFGF was approximately 34 microg/1 mg of capsule. Initially, about 30% of the encapsulated bFGF was released in serum-free medium within a few hours, however, the release was sustained over 70 h. When the bFGF encapsulating capsules were added to cell culture medium (serum-free), the mouse L929 fibroblast cells proliferated well for 2 weeks as compared to cultures, where bFGF was added to the medium or where bFGF and empty hollow capsules were added separately. The proliferation is due to the local and sustained release of bFGF from the adsorbent capsule to the cell surface.     

Intumescent multilayer nanocoating, made with renewable polyelectrolytes, for flame-retardant cotton

Thin films of fully renewable and environmentally benign electrolytes, cationic chitosan (CH) and anionic phytic acid (PA), were deposited on cotton fabric via layer-by-layer (LbL) assembly in an effort to reduce flammability. Altering the pH of aqueous deposition solutions modifies the composition of the final nanocoating. CH-PA films created at pH 6 were thicker and had 48 wt % PA in the coating, while the thinnest films (with a PA content of 66 wt %) were created at pH 4. Each coating was evaluated at both 30 bilayers (BL) and at the same coating weight added to the fabric. In a vertical flame test, fabrics coated with high PA content multilayers completely extinguished the flame, while uncoated cotton was completely consumed. Microcombustion calorimetry confirmed that all coated fabric reduces peak heat release rate (pkHRR) by at least 50% relative to the uncoated control. Fabric coated with pH 4 solutions shows the greatest reduction in pkHRR and total heat release of 60% and 76%, respectively. This superior performance is believed to be due to high phosphorus content that enhances the intumescent behavior of these nanocoatings. These results demonstrate the first completely renewable intumescent LbL assembly, which conformally coats every fiber in cotton fabric and provides an effective alternative to current flame retardant treatments.     

Alginate-chitosan coacervation in production of artificial seeds

Survival of secondary embryoids of winter oilseed rape (Brassica napus ssp. oleifera cv. Primor) has been used as an assay for the development of artificial seeds involving complex coacervation of alginate (polyanion) with chitosan (polycation). Germination frequency of 100% was achieved for encapsulated embryoids when alginate formed the inner matrix and chitosan the outer layer. When the matrix makeup was reversed, there was no germination of embryoids. The artificial seeds produced were hardened in dilute alkaline solutions of NaOH and Ca(OH)(2). An optimum setting time could be selected based on a quantitative measurement of resistance of hardened capsules to compression and the germination frequency of the encapsulated embryoids. (c) 1993 John Wiley & Sons, Inc.     

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose,Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.     

Graphene Multilayer Supported Gold Nanoparticles for Efficient Electrocatalysts Toward Methanol Oxidation

This study reports a simple method of integrating electroactive gold nanoparticles (Au NPs) with graphene oxide (GO) nanosheet support by layer‐by‐layer (LbL) assembly for the creation of 3‐dimensional electrocatalytic thin films that are active toward methanol oxidation. This approach involves the alternating assembly of two oppositely charged suspensions of Au NPs with GO nanosheets based on electrostatic interactions. The GO nanosheets not only serve as structural components of the multilayer thin film, but also potentially improve the utilization and dispersion of Au NPs by taking advantages of the high catalytic surface area and the electronic conduction of graphene nanosheets. Furthermore, it is found that the electrocatalytic activity of the multilayer thin films of Au NPs with graphene nanosheet is highly tunable with respect to the number of bilayers and thermal treatment, benefiting from the advantageous features of LbL assembly. Because of the highly versatile and tunable properties of LbL assembled thin films coupled with electrocatalytic NPs, we anticipate that the general concept presented here will offer new types of electroactive catalysts for direct methanol fuel cells.     

Deconvolution of images from 3D printed cells in layers on a chip

Layer‐by‐layer cell printing is useful in mimicking layered tissue structures inside the human body and has great potential for being a promising tool in the field of tissue engineering, regenerative medicine, and drug discovery. However, imaging human cells cultured in multiple hydrogel layers in 3D‐printed tissue constructs is challenging as the cells are not in a single focal plane. Although confocal microscopy could be a potential solution for this issue, it compromises the throughput which is a key factor in rapidly screening drug efficacy and toxicity in pharmaceutical industries. With epifluorescence microscopy, the throughput can be maintained at a cost of blurred cell images from printed tissue constructs. To rapidly acquire in‐focus cell images from bioprinted tissues using an epifluorescence microscope, we created two layers of Hep3B human hepatoma cells by printing green and red fluorescently labeled Hep3B cells encapsulated in two alginate layers in a microwell chip. In‐focus fluorescent cell images were obtained in high throughput using an automated epifluorescence microscopy coupled with image analysis algorithms, including three deconvolution methods in combination with three kernel estimation methods, generating a total of nine deconvolution paths. As a result, a combination of Inter‐Level Intra‐Level Deconvolution (ILILD) algorithm and Richardson‐Lucy (RL) kernel estimation proved to be highly useful in bringing out‐of‐focus cell images into focus, thus rapidly yielding more sensitive and accurate fluorescence reading from the cells in different layers. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:445–454, 2018     

Sulfasalazine release from alginate-N,O-carboxymethyl chitosan gel beads coated by chitosan

In the present study, spherical beads were prepared from a water-soluble chitosan (N,O-carboxymethyl chitosan, NOCC) and alginate with ionic gelation method. Then, swollen calcium–alginate–NOCC beads were coated with chitosan. To prepare drug loaded beads, sulfasalazine (SA) was added to the initial aqueous polymer solution. The effect of coating, as well as drying procedure, on the swelling behavior of unloaded beads and SA release of drug loaded ones were evaluated in simulated gastrointestinal tract fluid. The rate of swelling and drug release were decreased for air-dried and coated beads in comparison with freeze-dried and uncoated ones, respectively. No burst release of drug was observed from whole tested beads. Chitosan coated beads released approximately 40% of encapsulated drug in simulated gastric and small intestine tract fluid. Based on these results, the chitosan coated alginate–NOCC hydrogel may be used as potential polymeric carrier for colon-specific delivery of sulfasalazine.     

Matrices (re)loaded: Durability,viability, and fermentative capacity of yeast encapsulated in beads of different composition during long-term fed-batch culture

Encapsulated microbes have been used for decades to produce commodities ranging from methyl ketone to beer. Encapsulated cells undergo limited replication, which enables them to more efficiently convert substrate to product than planktonic cells and which contributes to their stress resistance. To determine how encapsulated yeast supports long-term, repeated fed-batch ethanologenic fermentation, and whether different matrices influence that process, fermentation and indicators of matrix durability and cell viability were monitored in high-dextrose, fed-batch culture over 7 weeks. At most timepoints, ethanol yield (g/g) in encapsulated cultures exceeded that in planktonic cultures. And frequently, ethanol yield differed among the four matrices tested: sodium alginate crosslinked with Ca²⁺ and chitosan, sodium alginate crosslinked with Ca²⁺, Protanal alginate crosslinked with Ca²⁺ and chitosan, Protanal alginate crosslinked with Ca²⁺, with the last of these consistently demonstrating the highest values. Young's modulus and viscosity were higher for matrices crosslinked with chitosan over the first week; thereafter values for both parameters declined and were indistinguishable among treatments. Encapsulated cells exhibited greater heat shock tolerance at 50°C than planktonic cells in either stationary or exponential phase, with similar thermotolerance observed across all four matrix types. Altogether, these data demonstrate the feasibility of re-using encapsulated yeast to convert dextrose to ethanol over at least 7 weeks.     

Titanium Surface Priming with Phase-Transited Lysozyme to Establish a Silver Nanoparticle-Loaded Chitosan/Hyaluronic Acid Antibacterial Multilayer via Layer-by-Layer Self-Assembly

Objectives

The formation of biofilm around implants, which is induced by immediate bacterial colonization after installation, is the primary cause of post-operation infection. Initial surface modification is usually required to incorporate antibacterial agents on titanium (Ti) surfaces to inhibit biofilm formation. However, simple and effective priming methods are still lacking for the development of an initial functional layer as a base for subsequent coatings on titanium surfaces. The purpose of our work was to establish a novel initial layer on Ti surfaces using phase-transited lysozyme (PTL), on which multilayer coatings can incorporate silver nanoparticles (AgNP) using chitosan (CS) and hyaluronic acid (HA) via a layer-by-layer (LbL) self-assembly technique.

Methods

In this study, the surfaces of Ti substrates were primed by dipping into a mixture of lysozyme and tris(2-carboxyethyl)phosphine (TCEP) to obtain PTL-functionalized Ti substrates. The subsequent alternating coatings of HA and chitosan loaded with AgNP onto the precursor layer of PTL were carried out via LbL self-assembly to construct multilayer coatings on Ti substrates.

Results

The results of SEM and XPS indicated that the necklace-like PTL and self-assembled multilayer were successfully immobilized on the Ti substrates. The multilayer coatings loaded with AgNP can kill planktonic and adherent bacteria to 100% during the first 4 days. The antibacterial efficacy of the samples against planktonic and adherent bacteria achieved 65%-90% after 14 days. The sustained release of Ag over 14 days can prevent bacterial invasion until mucosa healing. Although the AgNP-containing structure showed some cytotoxicity, the toxicity can be reduced by controlling the Ag release rate and concentration.

Conclusions

The PTL priming method provides a promising strategy for fabricating long-term antibacterial multilayer coatings on titanium surfaces via the LbL self-assembly technique, which is effective in preventing implant-associated infections in the early stage.     

Layer-by-layer assembly to modify poly(l-lactic acid) surface toward improving its cytocompatibility to human endothelial cells

A novel technique to introduce free amino groups onto polyester scaffolds via aminolyzing the ester groups with diamine has been developed recently. Positively charged chitosan was then deposited onto the aminolyzed poly(l-lactic acid) (PLLA) membrane surface in a layer-by-layer assembly manner using poly(styrene sulfonate, sodium salt) (PSS) as a negatively charged polyelectrolyte. The layer-by-layer deposition process of PSS and chitosan was monitored by UV-vis absorbance spectroscopy, energy transfer by fluorescence spectroscopy, and advancing contact angle measurements. The existed chitosan obviously improved the cytocompatibility of PLLA to human endothelial cells. The cell attachment, activity, and proliferation on the PLLA membranes assembled with three or five bilayers of PSS/chitosan with chitosan as the outermost layer were better than those with one bilayer of PSS/chitosan or the control PLLA. The cells also showed morphology of an elongated shape with abundant cytoplasm, and a confluent cell layer was reached after being cultured for 4 days. Measurement of von Willebrand factor secreted by these endothelial cells (ECs) verified the endothelial function. Hence, better ECs compatible PLLA were produced.     

海藻酸钠/壳聚糖微胶囊固定化大肠杆菌的研究

本文以大肠杆菌ＤＨ5α为模型体系 ,探索了大肠杆菌ＤＨ5α用海藻酸钠 壳聚糖 (ＡＣＡ)微胶囊培养的可行性 ,并观察了微囊化大肠杆菌ＤＨ5α细胞生长与物料渗透性能 ,通过将ＡＣＡ微胶囊移植到实验组小鼠体内 ,考察了ＡＣＡ微胶囊作为口服药物载体的可能性。1　材料和方法1.1　材料壳聚糖 ,本实验室改性所得 ;海藻酸钠 ,ＫｅｌｃｏＤｉｖｏｆＭｅｒ ｃｋＣｏ .Ｉｎｃ .ＵＳＡ ;其它试剂均为国产分析纯。大肠杆菌ＤＨ5α ,长春生物制品所 ;ＬＢ培养基 ,华美生物制品公司提供。昆明系小白鼠 18～ 2 0ｇ ,解放军大连高等医学专科学校实验动物中…     

Fine structure and molecular content of human chondrocytes encapsulated in alginate beads

Preservation of the chondrocytic phenotype in vitro requires a 3D (three‐dimensional) culture model. Diverse biomaterials have been tested as scaffolds for culture of animal chondrocytes; however, to date, none is considered a gold standard in regenerative medicine. Here, we studied the fine structure and the GAGs (glycosaminoglycans) content of human chondrocytes encapsulated in alginate beads by using electron microscopy and radioactive sulfate [³⁵S] incorporation, respectively. Cells were obtained from human cartilage, encapsulated in alginate beads and cultured for 28 days. [³⁵S]Na₂SO₄ was added to the culture media and later isolated for quantification of the sulfated GAGs found in three compartments: IC (intracellular), IB (intra‐bead) and EB (extra‐bead). Round cells were seen isolated or forming small groups throughout the alginate. Human chondrocytes presented the features of active cells such as euchromatic nuclei, abundant RER (rough endoplasmic reticulum) and many transport vesicles. We observed an extracellular matrix rich in collagen fibres and electrondense material adjacent to the cells. Most of the GAGs produced (74%) were found in the culture medium (EB), indicating that alginate has a limited capacity to retain the GAGs. CS (chondroitin sulfate), the major component of aggrecan, was the most prominent GAG produced by the encapsulated cells. Human chondrocytes cultured in alginate can sustain their phenotype, confirming the potential application of this biomaterial for cartilage engineering.     

Chitosan Enriched Three-Dimensional Matrix Reduces Inflammatory and Catabolic Mediators Production by Human Chondrocytes

This in vitro study investigated the metabolism of human osteoarthritic (OA) chondrocytes encapsulated in a spherical matrix enriched of chitosan. Human OA chondrocytes were encapsulated and cultured for 28 days either in chitosan-alginate beads or in alginate beads. The beads were formed by slowly passing dropwise either the chitosan 0.6%–alginate 1.2% or the alginate 1.2% solution through a syringe into a 102 mM CaCl2 solution. Beads were analyzed histologically after 28 days. Interleukin (IL)-6 and -8, prostaglandin (PG) E₂, matrix metalloproteinases (MMPs), hyaluronan and aggrecan were quantified directly in the culture supernatant by specific ELISA and nitric oxide (NO) by using a colorimetric method based on the Griess reaction. Hematoxylin and eosin staining showed that chitosan was homogeneously distributed through the matrix and was in direct contact with chondrocytes. The production of IL-6, IL-8 and MMP-3 by chondrocytes significantly decreased in chitosan-alginate beads compared to alginate beads. PGE₂ and NO decreased also significantly but only during the first three days of culture. Hyaluronan and aggrecan production tended to increase in chitosan-alginate beads after 28 days of culture. Chitosan-alginate beads reduced the production of inflammatory and catabolic mediators by OA chondrocytes and tended to stimulate the synthesis of cartilage matrix components. These particular effects indicate that chitosan-alginate beads are an interesting scaffold for chondrocytes encapsulation before transplantation to repair cartilage defects.