Patterning alginate hydrogels using light-directed release of caged calcium in a microfluidic device

This paper describes a simple reversible hydrogel patterning method for 3D cell culture. Alginate gel is formed in select regions of a microfluidic device through light-triggered release of caged calcium. In the pre-gelled alginate solution, calcium is chelated by DM-nitrophen (DM-n) to prevent cross-linking of alginate. After sufficient UV exposure the caged calcium is released from DM-n causing alginate to cross-link. The effect of using different concentrations of calcium and chelating agents as well as the duration of UV exposure is described. Since the cross-linking is based on calcium concentration, the cross-linked alginate can easily be dissolved by EDTA. We also demonstrate application of this capability to patterned microscale 3D co-culture using endothelial cells and osteoblastic cells in a microchannel.     

Photo-crosslinkable,thermo-sensitive and biodegradable Pluronic hydrogels for sustained release of protein

—Thermo-sensitive and biodegradable hydrogels based on Pluronic tri-block copolymers were prepared by a photo-polymerization method. Two terminal hydroxyl groups in Pluronic F-127 were acrylated to form a Pluronic macromer. Photo-cross-linked Pluronic hydrogels prepared by UV radiation showed a gradually decreased swelling ratio with increasing temperature and exhibited a thermally-responsive change in the swelling ratio when the temperature was cycled between 10°C and 37°C. These hydrogels degraded slowly due to the cleavage of ester linkage in the acrylated Pluronic terminal end. When lysozyme, a model protein drug, was loaded in the hydrogels, bi-phasic protein release profiles were attained: a burst-free and rapid controlled release profile was initially observed for a one week period and a much slower sustained release was followed thereafter. The release rates could be controlled by varying the amount of Pluronic macromer for photo-polymerization.     

Photo-crosslinkable, thermo-sensitive and biodegradable pluronic hydrogels for sustained release of protein

Thermo-sensitive and biodegradable hydrogels based on Pluronic tri-block copolymers were prepared by a photo-polymerization method. Two terminal hydroxyl groups in Pluronic F-127 were acrylated to form a Pluronic macromer. Photo-cross-linked Pluronic hydrogels prepared by UV radiation showed a gradually decreased swelling ratio with increasing temperature and exhibited a thermally-responsive change in the swelling ratio when the temperature was cycled between 10 degrees C and 37 degrees C. These hydrogels degraded slowly due to the cleavage of ester linkage in the acrylated Pluronic terminal end. When lysozyme, a model protein drug, was loaded in the hydrogels, bi-phasic protein release profiles were attained: a burst-free and rapid controlled release profile was initially observed for a one week period and a much slower sustained release was followed thereafter. The release rates could be controlled by varying the amount of Pluronic macromer for photo-polymerization.     

FMLP-induced enzyme release from neutrophils: a role for intracellular calcium

The ability of the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) to stimulate beta-glucuronidase release and 45Ca2+ release from rabbit neutrophils was studied. FMLP stimulated enzyme release from cytochalasin B-treated cells either in the presence or the absence of extracellular calcium. Depletion of cell calcium, by exposure to either ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid or the calcium ionophore A23187, blocked the ability of FMLP to stimulate enzyme release and 45Ca2+ release in the absence of extracellular calcium. The ability of A23187 to lower the 45Ca2+ content of neutrophils, to block FMLP-stimulated 45Ca2+ release, and to inhibit FMLP-stimulated enzyme release in the absence of calcium was dose dependent over the same concentration range (10(-8) to 10(-6) M A23187) for all three actions. In contrast, FMLP stimulated enzyme release from A23187-treated cells, provided that extracellular calcium was present. This secretory response was normal as judged by cell ultrastructure and FMLP dose-response relationships. It is concluded that A23187 depletes a pool of intracellular calcium usually released by FMLP and that release of calcium from this pool is necessary for initiation of enzyme secretion in the absence of extracellular calcium.     

Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides

Biomaterials for the precise control of protein release are important to the development of new strategies for treating human diseases. This study aimed to fundamentally understand aptamer--protein dissociation triggered by complementary oligonucleotides, and to apply this understanding to develop affinity hydrogels for controlled protein release. The results showed that the oligonucleotide tails of the aptamers played a critical role in inducing intermolecular hybridization and triggering aptamer--protein dissociation. In addition, the attachment of the oligonucleotide tails to the aptamers and the increase of hybridizing length could produce a synergistic effect on the dissociation of bound proteins from their aptamers. More importantly, pegylated complementary oligonucleotides could successfully trigger protein release from the aptamer-functionalized hydrogels at multiple time points. Based on these results, it is believed that aptamer-functionalized hydrogels and complementary oligonucleotides hold great potential of controlling the release of protein drugs to treat human diseases.     

Crosslinked hyaluronic acid hydrogels: a strategy to functionalize and pattern

The physiological activity of hyaluronic acid (HA) polymers and oligomers makes it a promising material for a variety of applications. The development of HA-hydrogel scaffolds with improved mechanical stability against degradation and biochemical functionality may enhance their application to tissue engineering. In this report, a crosslinking strategy targeting the alcohol groups via a poly(ethylene glycol) diepoxide crosslinker was investigated for the generation of degradable HA hydrogels. To provide support for cell adhesion in vitro, collagen was incorporated into the HA solution prior to the crosslinking process. The hydrogels have a continuous exterior and a porous interior, with pore diameters ranging from 6 to 9 microm. HA and HA-collagen hydrogels degrade in the presence of hyaluronidase and collagenase enzymes, indicating that the chemical modification does not prevent biodegradation. Complete degradation of the hydrogels occurred within 14 days in hyaluronidase (100 U/ml) and 3 days in collagenase (66 U/ml). Pattern transfer was employed to introduce a surface topography onto the hydrogel, which was able to orient cell growth. Furthermore, the hydrogels could be functionalized with the biomolecule neutravidin by incorporation of biotin along the HA backbone. This biotinylation approach may allow attachment of bioactive molecules that are conjugated to avidin.     

Preserved bioactivity and tunable release of a SDF1-GPVI bi-specific protein using photo-crosslinked PEGda hydrogels

Chemokine-induced stem cell recruitment is a promising strategy for post myocardial infarction treatment. Injection of stromal cell-derived factor 1 (SDF1) has been shown to attract bone marrow-derived progenitor cells (BMPCs) from the blood that have the potential to differentiate into cardiovascular cells, which support angiogenesis, enabling the improvement of myocardial function. SDF1-GPVI bi-specific protein contains a glycoprotein VI (GPVI)-domain that serves as an anchor for collagen type I (Col I) and III, which are exposed in the wall of injured vasculature. In this study, we generated a cytocompatible hydrogel via photo-crosslinking of poly(ethylene glycol) diacrylate that serves as a reservoir for SDF1-GPVI. Controlled and sustained release of SDF1-GPVI was demonstrated over a period of 7 days. Release features were modifiable depending on the degree of the crosslinking density. Functionality of the GPVI-domain was investigated using a GPVI-binding ELISA to Col I. Activity of the SDF1-domain was tested for its CXCR4 binding potential. Preserved functionality of SDF1-GPVI bi-specific protein after photo-crosslinking and controllable release was successfully demonstrated in vitro supporting the implementation of this drug delivery system as a powerful tool for therapeutic protein delivery in the treatment of cardiovascular ischemic disease.     

Cyclodextrin-containing hydrogels for contact lenses as a platform for drug incorporation and release

Poly(2-hydroxyethyl methacrylate) hydrogels containing β-cyclodextrin (pHEMA/β-CD) have been investigated as a platform for sustained release of ophthalmic drugs. First of all, pHEMA/β-CD hydrogel membranes and contact lenses were prepared by photopolymerization of HEMA, mono-methacrylated β-CD (mono-MA-β-CD) and trimethylolpropane trimethacrylate using a cast molding process. The hydrogels were characterized by Fourier transform infrared spectroscopy, equilibrium swelling ratio (ESR) and tensile tester. The results showed that the incorporation of β-CD in the hydrogels increased the ESR and tensile strength. Then, puerarin was used as a model to evaluate drug loading and in vitro and in vivo release behavior of the pHEMA/β-CD hydrogels. It was revealed that puerarin loading and in vitro release rate were dependent on β-CD content in the pHEMA/β-CD hydrogels. In rabbit eyes the pHEMA/β-CD hydrogel contact lenses exhibited longer mean residence times (MRT_F) of puerarin in tear fluid than that of pHEMA contact lenses and 1% puerarin eye drops. The puerarin concentration in the aqueous humor of rabbit reached a maximum of 0.81 μg ml^?1 after wearing the pHEMA/β-CD contact lens, which had been presoaked in 0.802 mg ml^?1 puerarin solution for 4.81 h. Also, the pHEMA/β-CD contact lenses had a higher drug bioavailability in aqueous humor than puerarin eye drops. The data demonstrate that pHEMA/β-CD hydrogel contact lenses can effectively deliver puerarin through the cornea.     

Mesoporous calcium silicate for controlled release of bovine serum albumin protein

The purpose of this study is to synthesize mesoporous calcium silicate (CS or wollastonite, CaSiO₃) and evaluate its possible application in protein/drug delivery. First, calcium silicate was synthesized by wet chemical method and then mesoporosity was created by acid modification of the synthesized CS particle using hydrochloric acid at pH 7, 4.5, and 0.5. The results showed that a hydrated silica gel with abundant Si–OH functional group formed on the surface of calcium silicate due to acid modification. This surface layer had mesoporous structure, with pore diameter between 4 and 5 nm. BET specific average surface area increased to 221, 333, and 356 m² g^?1 due to acid modification at pH 7, 4.5, and 0.5, respectively, whereas the surface area for unmodified CS particles was 65 m² g^?1. Protein adsorption studies indicated that mesoporous CS has higher ability to adsorb bovine serum albumin and lysozyme compared to unmodified particles. The release kinetics showed that proteins on mesoporous CS released sequentially over one week, whereas the proteins on unmodified particle followed burst release kinetics within a few hours. Human osteoblast cell–material interaction study showed that these materials were biocompatible and promoted excellent bone cell proliferation. In summary, this work has demonstrated the potential to produce mesoporous CS as a carrier for protein/drug delivery for bone regeneration and other biomedical applications.     

Stimulation of pepsinogen release from chief cells byHelicobacter pylori: evidence for a role of calcium and calmodulin

To define the mechanisms by whichHelicobacter pyloristimulates pepsinogen secretion, thein vitrorelease of pepsinogen was studied using a preparation of pig chief cell monolayers.Helicobacter pyloriinduced a time- and concentration-dependent release of pepsinogen into the medium, with about a three-fold increase in pepsinogen secretion over controls found after 45 min of incubation. 3×10⁷H. pylori produced 50% of the maximal response found at aH. pyloricount of 2×10⁸. The action ofH. pyloridid not depend on the presence of the vacuolating toxin (vacA) and the cytotoxin-associated protein (cagA). Dibutyryl-cAMP and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate also markedly stimulated pepsinogen secretion and enhanced the stimulatory effect ofH. pylori. Helicobacter pylori-stimulated pepsinogen release was inhibited by lanthanum and the calmodulin antagonist W-7, but not by the L-type Ca²⁺channel blocker nifedipine, TMB-8, an agent that blocks the release of Ca²⁺from intracellular stores, the protein kinase C inhibitor staurosporine and the protein kinase A inhibitor H-8. It is suggested thatH. pyloridirectly stimulates pepsinogen release from gastric chief cells and that this effect is mediated via the calcium/calmodulin messenger branch.     

Regulation of depolarization-induced calcium release from skeletal muscle triads by cyclic AMP-dependent protein kinase

cAMP activated Ca2+ release from the sarcoplasmic reticulum (SR) triggered by transverse tubular membrane depolarization monitored in a triadic vesicle prepared from rabbit skeletal muscle. A kinetic analysis showed that the activation was in the amounts of released Ca2+ and not in the release rates. This activation was found to be depolarization-dependent, that is, the activation effect of cAMP decreased with an increase in the magnitude of depolarization. Because the activation was abolished by a protein kinase inhibitor, protein phosphorylation by the endogenous cAMP-dependent protein kinase (PKA) was thought to be in effect. On the contrary, caffeine-induced Ca2+ release was not activated by cAMP; however, the exogenous PKA catalytic subunit activated Ca2+ release, and this effect was probably through the phosphorylation of the SR Ca2+ release channel as described elsewhere. These results suggest that the protein, except for the SR Ca2+ release channel, is phosphorylated by endogenous PKA and plays a modulatory role in depolarization-induced Ca2+ release, that is, excitation-contraction coupling of the skeletal muscle.     

Gelatin hydrogels cross-linked by gamma-ray irradiation: materials for absorption and release of dye

Gelatin hydrogels cross-linked by y-ray irradiation using 60Co as gamma-ray source were prepared. As a model of controlled release of low-molecular-weight compounds, absorption and release of methylene blue, a water-soluble cationic dye, was investigated. Irradiated gelatin hydrogels did not redissolve at temperatures over 40 degrees C, while unirradiated gels were thermoplastic and reversibly changed the stage between gel and sol. Measurement of both the wet weight after swelling in distilled water and dry weight after freeze-drying showed that the higher-dose irradiation gave stiffer and more compact gels with the lower specific water content, irrespective of the absorbed dose rate. The time-course of absorption and release of methylene blue in aqueous solution was measured. Since absorption of dye into gelatin gels was much affected by liquid phase pH, amount of absorption was higher in pH above an IEP of gelatins. Moreover, the absorption and release of methylene blue with Type-B gelatin were higher than with Type-A gelatin, respectively. Therefore, absorption and release of the dye depend on the electrostatic interaction between the dye molecule and gelatin.     

Extended and sequential delivery of protein from injectable thermoresponsive hydrogels

Thermoresponsive hydrogels are attractive for their injectability and retention in tissue sites where they may serve as a mechanical support and as a scaffold to guide tissue remodeling. Our objective in this report was to develop a thermoresponsive, biodegradable hydrogel system that would be capable of protein release from two distinct reservoirs--one where protein was attached to the hydrogel backbone, and one where protein was loaded into biodegradable microparticles mixed into the network. Thermoresponsive hydrogels consisting of N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacrylate (HEMA), and biodegradable methacrylate polylactide were synthesized along with modified copolymers incorporating 1 mol % protein-reactive methacryloxy N-hydroxysuccinimide (MANHS), hydrophilic acrylic acid (AAc), or both. In vitro bovine serum albumin (BSA) release was studied from hydrogels, poly(lactide-co-glycolide) microparticles, or microparticles mixed into the hydrogels. The synthesized copolymers were able to gel below 37°C and release protein in excess of 3 months. The presence of MANHS and AAc in the copolymers was associated with higher loaded protein retention during thermal transition (45% vs. 22%) and faster release (2 months), respectively. Microspheres entrapped in the hydrogel released protein in a delayed fashion relative to microspheres in saline. The combination of a protein-reactive hydrogel mixed with protein-loaded microspheres demonstrated a sequential release of specific BSA populations. Overall the described drug delivery system combines the advantages of injectability, degradability, extended release, and sequential release, which may be useful in tissue engineering applications.     

Biomimetic hydrogels for enhanced loading and extended release of ocular therapeutics

We have applied the principles of biomimesis by incorporating a natural receptor-based rational design strategy in the synthesis of novel recognitive soft contact lenses. We have demonstrated the potential of biomimetic carriers to load significant amounts of ocular medication such as H(1)-antihistamines, as well as to release a therapeutic dosage of drug in vitro in a controlled fashion for 5 days, with an even further extension in the presence of protein. Gels of multiple complexation points with varying functionalities outperformed gels formed with less diverse functional monomers and showed superior loading with a six-fold difference over control gels and a three-fold difference over less biomimetic gels. Moreover, mechanical and optical properties of these hydrogels agreed with conventional lenses, and increased loading was reflected in a reduced propagation of polymer chains. This approach can be extended to a wider biological spectrum in the design of novel, controlled and modulated delivery devices to alleviate ocular disorders and provide an alternative to topical therapy.     

Biological reactivity of nanoparticles: mosaics from optical microscopy videos of giant lipid vesicles

Emerging fields such as nanomedicine and nanotoxicology, demand new information on the effects of nanoparticles on biological membranes and lipid vesicles are suitable as an experimental model for bio-nano interaction studies. This paper describes image processing algorithms which stitch video sequences into mosaics and recording the shapes of thousands of lipid vesicles, which were used to assess the effect of CoFe(2)O(4) nanoparticles on the population of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine lipid vesicles. The applicability of this methodology for assessing the potential of engineered nanoparticles to affect morphological properties of lipid membranes is discussed.     

Building a polysaccharide hydrogel capsule delivery system for control release of ibuprofen

Development of a delivery system which can effectively carry hydrophobic drugs and have pH response is becoming necessary. Here we demonstrate that through preparation of β-cyclodextrin polymer (β-CDP), a hydrophobic drug molecule of ibuprofen (IBU) was incorporated into our prepared β-CDP inner cavities, aiming to improve the poor water solubility of IBU. A core-shell capsule structure has been designed for achieving the drug pH targeted and sustained release. This delivery system was built with polysaccharide polymer of Sodium alginate (SA), sodium carboxymethylcellulose (CMC) and hydroxyethyl cellulose (HEC) by physical cross-linking. The drug pH-response control release is this hydrogel system’s chief merit, which has potential value for synthesizing enteric capsule. Besides, due to our simple preparing strategy, optimal conditions can be readily determined and the synthesis process can be accurately controlled, leading to consistent and reproducible hydrogel capsules. In addition, phase-solubility method was used to investigate the solubilization effect of IBU by β-CDP. SEM was used to prove the forming of core and shell structure. FT-IR and ¹H-NMR were also used to perform structural characteristics. By the technique of UV determination, the pH targeted and sustained release study were also performed. The results have proved that our prepared polysaccharide hydrogel capsule delivery system has potential applications as oral drugs delivery in the field of biomedical materials.     

Physically crosslinked alginate/N,O-carboxymethyl chitosan hydrogels with calcium for oral delivery of protein drugs

In the study, a complex composed of alginate blended with a water-soluble chitosan (N,O-carboxymethyl chitosan, NOCC) was prepared to form microencapsulated beads by dropping aqueous alginate-NOCC into a Ca(2+) solution. These microencapsulated beads were evaluated as a pH-sensitive system for delivery of a model protein drug (bovine serum albumin, BSA). The main advantage of this system is that all procedures used were performed in aqueous medium at neutral environment, which may preserve the bioactivity of protein drugs. The swelling characteristics of these hydrogel beads at distinct compositions as a function of pH values were investigated. It was found that the test beads with an alginate-to-NOCC weight ratio of 1:1 had a better swelling characteristic among all studied groups. With increasing the total concentration of alginate-NOCC, the effective crosslinking density of test beads increased significantly and a greater amount of drug was entrapped in the polymer chains (up to 77%). The swelling ratios of all test groups were approximately the same ( approximately 3.0) at pH 1.2. At pH 7.4, with increasing the total concentration of alginate-NOCC, the swelling ratios of test beads increased significantly (20.0-40.0), due to a larger swelling force created by the electrostatic repulsion between the ionized acid groups (-COO(-)). It was shown that BSA was uniformly distributed in all test beads. At pH 1.2, retention of BSA in hydrogels may be improved by rinsing test beads with acetone (the amount of BSA released was below 15%). At pH 7.4, the amounts of BSA released increased significantly ( approximately 80%) as compared to those released at pH 1.2. With increasing the total concentration of alginate-NOCC, the release of encapsulated proteins was slower. Thus, the calcium-alginate-NOCC beads with distinct total concentrations developed in the study may be used as a potential system for oral delivery of protein drugs to different regions of the intestinal tract.     

Controlled release of EGF and bFGF from dextran hydrogels in vitro and in vivo

In the present study, dextran-epichlorohydrin hydrogels were employed as carriers for the controlled release of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). The hydrogels were synthesized from 50% (by weight) monomeric cross-linker, epichlorohydrin, containing dextran mixtures by intermolecular side-chain reaction of dextran-hydroxyl groups with epichlorohydrin-epoxy groups. The hydrogel disks of 3-mm diameter and 1.5-mm thickness have a high swelling capacity (EWC = 650%) and enough mechanical stability for the studies in vivo. Impregnation of EGF and bFGF into the dried hydrogels was carried out by use of phosphate buffered saline solution (PBS, pH = 7.4) containing 0.5 microg mL(-1) EGF and 0.1 microg mL(-1) bFGF, respectively. The in vitro release of growth factors was detected by fluorescence spectroscopy. The prolonged release of EGF is continued up to the 14th day, in comparison with a 26-day release of bFGF. The in vivo studies were realized with subcutaneously implanted hydrogels in Wistar albino rats. The rate of neovascularization was analyzed statistically using one-way analysis of significance with EGF and bFGF incorporated hydrogels. In conclusion, dextran-epichlorohydrin hydrogels were shown to be an alternative delivery system for the release of growth factors.