Controlled release of growth factors based on biodegradation of gelatin hydrogel

To develop a carrier for the controlled release of biologically-active growth factors, biodegradable hydrogels were prepared through glutaraldehyde cross-linking of gelatin with isoelectric points (IEP) of 5.0 and 9.0, i.e. 'acidic' and 'basic' gelatins, respectively. Radioiodinated growth factors were used to investigate their sorption and desorption from the hydrogel of both types of gelatin. Basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) were well sorbed with time to the acidic gelatin hydrogel, while less sorption was observed for the basic gelatin hydrogel. This could be explained in terms of the electrostatic interaction between the growth factors and the acidic gelatin. However, bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF), though their IEPs are higher than 7.0, were sorbed to the acidic gelatin hydrogel to a smaller extent than the two other growth factors. Under in vitro non-degradation conditions, approximately 20% of the incorporated bFGF and TGF-β1 was desorbed from the hydrogels within the initial 40 min, followed by no further substantial desorption, whereas large initial desorption was observed for BMP-2 and VEGF. When implanted in the back subcutis of mice, gelatin hydrogels were degraded over time. Each growth factor was retained in vivo being incorporated in the acidic gelatin hydrogel: the smaller the in vitro desorption amount from the hydrogel, the longer the in vivo retention time. The in vivo profile of bFGF and TGF-β1 retention was in good accordance with that of the hydrogel. These findings indicate that the growth factor immobilized to the acidic gelatin hydrogel through ionic interaction was released in vivo as a result of hydrogel degradation.     

Controlled release systems for proteins based on gelatin microspheres

The preparation and characterization of biodegradable gelatin microspheres for the controlled release of peptides and proteins has been investigated. Bovine serum albumin (BSA) was chosen for incorporation into the gelatin microspheres and the spheres were characterized for the in vitro release of BSA and other properties. BSA was labelled with fluorescein isothiocyanate (FITC) for easy analysis. FITC-BSA was entrapped into the gelatin microspheres using a polymer dispersion technique developed in our earlier studies. The morphological characteristics of microspheres were analysed by optical and scanning electron microscopy (SEM). The optical and SEM photographs of FITC-BSA microspheres showed the solid spherical nature of the spheres. The entrapment efficiency of FITC-BSA was about 62%. The in vitro release pattern of FITC-BSA showed that 51 % of the entrapped drug was released during the first day and the release followed approximate zero order kinetics from day 2 onwards. The total release of FITC-BSA lasted for about 8 days. SDS-PAGE analysis revealed that BSA was not degraded by this preparation of microspheres.     

Controlled release of lysozyme from succinylated gelatin microspheres

Gelatin was anionized to increase the carboxylic acid groups through succinylation. Succinylation of gelatin was performed using varying amounts of succinic anhydride. This gave various percentages of substitution. Lysozyme, a cationic antibacterial enzyme, which has important applications in the reduction of prosthetic valve endocarditis, was chosen as a model protein drug. Microspheres were prepared using unmodified gelatin and succinylated gelatin (SG) and lysozyme was incorporated into them. The percentage loading and release profiles of lysozyme for gelatin and SG microspheres were evaluated and compared. It was found that the SG microspheres exhibited higher loading efficiency for lysozyme (50%) than the unmodified gelatin microspheres. The in vitro release of lysozyme from SG microspheres occurred up to 122 h, compared to 96 h for gelatin microspheres, for the release of most of the lysozyme incorporated. This prolonged release of lysozyme from SG microspheres was attributed to the electrostatic interaction between the cationic lysozyme and the anionic SG microsphere carrier.     

Controlled release of lysozyme from succinylated gelatin microspheres

Gelatin was anionized to increase the carboxylic acid groups through succinylation. Succinylation of gelatin was performed using varying amounts of succinic anhydride. This gave various percentages of substitution. Lysozyme, a cationic antibacterial enzyme, which has important applications in the reduction of prosthetic valve endocarditis, was chosen as a model protein drug. Microspheres were prepared using unmodified gelatin and succinylated gelatin (SG) and lysozyme was incorporated into them. The percentage loading and release profiles of lysozyme for gelatin and SG microspheres were evaluated and compared. It was found that the SG microspheres exhibited higher loading efficiency for lysozyme (50%) than the unmodified gelatin microspheres. The in vitro release of lysozyme from SG microspheres occurred up to 122 h, compared to 96 h for gelatin microspheres, for the release of most of the lysozyme incorporated. This prolonged release of lysozyme from SG microspheres was attributed to the electrostatic interaction between the cationic lysozyme and the anionic SG microsphere carrier.     

Preparation of gelatin hydrogel sponges incorporating bioactive glasses capable for the controlled release of fibroblast growth factor-2

Gelatin hydrogel sponges incorporating bioactive glasses (Gel-BG) were fabricated. We evaluated the characteristics of Gel-BG as scaffolds from the perspective of their mechanical properties and the formation of hydroxyapatite by the incorporation of bioactive glasses (BG). In addition, the Gel-BG degradation and the profile of fibroblast growth factor-2 (FGF-2) release from the Gel-BG were examined. Every Gel-BG showed an interconnected pore structure with the pore size range of 180–200?µm. The compression modulus of sponges incorporating BG increased. The time profiles of degradation for the 72-h crosslinked gelatin hydrogel sponges incorporating 10?wt% BG (Gel-BG(10)) and 50?wt% BG (Gel-BG(50)) were analogous to that of the 24-h crosslinked gelatin hydrogel sponge without BG (Gel-BG(0)). In measuring the release of FGF-2 from Gel-BG, the Gel-BG(10) and Gel-BG(50) showed almost analogous 100% cumulative release within 28?days in vivo. Additionally, a bioactivity evaluation showed that the presence of gelatin does not affect the in vitro bioactivity of Gel-BG. These sponges showed mechanical and chemical functionality as scaffolds, featuring both the controlled release of FGF-2 and the induction of hydroxyapatite crystallization.     

Controlled release from a mechanically-stimulated thermosensitive self-heating composite hydrogel

Temperature has been extensively explored as a trigger to control the delivery of a payload from environment-sensitive polymers. The need for an external heat source only allows limited spatiotemporal control over the delivery process. We propose a new approach by using the dissipative properties of a hydrogel matrix as an internal heat source when the material is mechanically loaded. The system is comprised of a highly dissipative hydrogel matrix and thermo-sensitive nanoparticles that shrink upon an increase in temperature. Exposing the hydrogel to a cyclic mechanical loading for a period of 5 min leads to an increase of temperature of the nanoparticles. The concomitant decrease in the volume of the nanoparticles increases the permeability of the hydrogel network facilitating the release of its payload. As a proof-of-concept, we showed that the payload of the hydrogel is released after 5–8 min following the initiation of the mechanical loading. This delivery method would be particularly suited for the release of growth factor as it has been shown that cell receptor to growth factor is activated 5–20 min following a mechanical loading.     

Controlled release from a composite silicone/hydrogel membrane

To enhance the drug uptake and release capacity of silicone rubber (SR), N-isopropylacrylamide (NIPA) hydrogel particles have been incorporated into a SR membrane. The NIPA particles were thoroughly blended with uncured SR with a certain ratio at room temperature. The mixture was then cast in a Petri dish to 1 mm thickness and cured 10 hours at 90 degrees C. The SR/NIPA composite gel can absorb water approximately equal to its dry weight. Brilliant blue, used as a mock drug, was loaded into the composite gel. Drug release increased exponentially to a final value that is temperature dependent: low at T> =34 degrees C, and high at T< 34 degrees C. This finding is because the hydrophobicity of NIPA changes with temperature. Pulsed release in response to temperature switching between 20 and 39 degrees C has been achieved. Drug uptake and release capability strongly depends upon the structure of the composite gel. The optimal range of NIPA composition is between 75 and 87% by volume. In the cited range, the NIPA particles form an interconnected network that provides a channel for diffusion of drug solution. The SR/NIPA composite gel has promising attributes as a wound dressing and other uses.     

Controlled drug release from multilayered phospholipid polymer hydrogel on titanium alloy surface

Here we describe the functionalization of a multilayered hydrogel layer on a Ti alloy with an antineoplastic agent, paclitaxel (PTX). The multilayered hydrogel was synthesized via layer-by-layer self-assembly (LbL) using selective intermolecular reactions between two water-soluble polymers, phospholipid polymer (PMBV) containing a phenylboronic acid unit and poly(vinyl alcohol) (PVA). Reversible covalent bonding between phenylboronic acid and the polyol provided the driving force for self-assembly. Poorly water-soluble PTX dissolves in PMBV aqueous solutions because PMBV is amphiphilic. Therefore, our multilayered hydrogel could be loaded with PTX at different locations to control the release profile and act as a drug reservoir. The amount of PTX incorporated in the hydrogel samples increased with the number of layers but was not directly proportional to the number of layers. However, as the step for making layers was repeated, the concentration of PTX in the PMBV layers increased. The different solubilities of PTX in PMBV and PVA aqueous solutions allow for the production of multilayered hydrogels loaded with PTX at different locations. In vitro experiments demonstrated that the location of PTX in the multilayered hydrogel influences the start and profile of PTX release. We expect that this rapid and facile LbL synthesis of multilayered hydrogels and technique for in situ loading with PTX, where the location of loading controls the release pattern, will find applications in biomedicine and pharmaceutics as a promising new technique.     

Controlled release of bioactive doxorubicin from microspheres embedded within gelatin scaffolds

We have encapsulated the chemotherapeutic agent doxorubicin into biodegradable polymer microspheres, and incorporated these microspheres into gelatin scaffolds, resulting in a controlled delivery system. Doxorubicin was encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) using a double emulsion/solvent extraction method. Characterization of the microspheres including diameter, surface morphology, and in vitro drug release was determined. The release of doxorubicin up to 30 days in phosphate buffered solution was assessed by measuring the absorbance of the releasate solution. Gelatin scaffolds were crosslinked using glutaraldehyde and microspheres were added to gelatin during gelation. The murine mammary mouse tumor cell line, 4T1, was treated with various doses of doxorubicin. A propidium iodide assay was utilized to visualize dead cells. Using a Transwell basket assay, PLGA microspheres and gelatin constructs were suspended above 4T1 cells for 48 h. Viable cells were determined using the CyQUANT cell proliferation assay. Results indicate that the release was controlled by the incorporation of PLGA microspheres into gelatin constructs. A significant difference was seen in the cumulative release over days 5-16 (p < 0.05). The bioactivity of doxorubicin released from the microspheres and scaffolds was maintained as proven by significant reduction in viable cells after treatment with PLGA microspheres as well as with the gelatin constructs (p < 0.001). The drug-polymer conjugate can be used as a controlled drug delivery system in a biocompatible scaffold that could potentially promote preservation of soft tissue contour.     

Controlled release of fibroblast growth factor-2 from an injectable 6-O-desulfated heparin hydrogel and subsequent effect on in vivo vascularization

We prepared a 6-O-desulfated (DS-) heparin (Hep) hydrogel as an excellent carrier for the controlled release of Hep-binding growth factors, such as fibroblast growth factor (FGF)-2. This material, which is partially derived from photoreactive groups, such as cinnamate, is easily crosslinked upon ultraviolet light (UV)-irradiation, resulting in a water-insoluble, viscous, and injectable hydrogel. In the present study, we examined the capacity of 6-O-DS-Hep hydrogel to immobilize FGF-2, as well as the controlled release of FGF-2 molecules from this hydrogel in vitro and in vivo. Only 10% of FGF-2 was gradually released from the FGF-2-containing 6-O-DS-Hep hydrogel (photocrosslinked 6-O-DS-Hep (4%; w/w) hydrogel containing 50 microg/mL FGF-2) into PBS (phosphate-buffered saline) within first 7 days. The 6-O-DS-Hep hydrogel in vitro maintained the original form through 1 weeks incubation in PBS, but it was gradually fragmented and could not maintain the original form by 2-3 week-washing. When the FGF-2-containing 6-O-DS-Hep hydrogel was subcutaneously injected into the back of rats, significant neovascularization and fibrous tissue formation were induced near the injected site from day 3 after the injection. And, the hydrogel had been biodegraded and completely disappeared from the injected sites in vivo within about 15-20 days after the injection. These findings indicate a controlled release of biologically active FGF-2 molecules together with fragmentation and biodegradation of 6-O-DS-Hep hydrogel and the subsequent induction of neovascularization in vivo.     

Controlled release of sphingosine-1-phosphate agonist with gelatin hydrogels for macrophage recruitment

The objective of this study is to design a drug delivery system (DDS) for the in vivo promotion of macrophage recruitment. As the drug, a water-insoluble agonist of sphingosine-1-phosphate type 1 receptor (SEW2871) was selected. SEW2871 (SEW) was water-solubilized by micelle formation with gelatin grafted by l-lactic acid oligomer. SEW micelles were mixed with gelatin, followed by dehydrothermal crosslinking of gelatin to obtain gelatin hydrogels incorporating SEW micelles. SEW was released from the hydrogels incorporating SEW micelles in vitro and in vivo. The water-solubilized SEW showed in vitro macrophage migration activity. When implanted into the back subcutis or the skin wound defect of mice, the hydrogel incorporating SEW micelles promoted macrophage migration toward the tissue around the implanted site to a significantly great extent compared with SEW-free hydrogel and that mixed with SEW micelles. The hydrogel is a promising DDS to enhance macrophage recruitment in vivo.     

Evaluation of an in situ forming hydrogel wound dressing based on oxidized alginate and gelatin

Wound dressings that can be formed in situ offer several advantages over the use of preformed dressings such as conformability without wrinkling or fluting in the wound bed, ease of application and improved patient compliance and comfort. Here we describe such an in situ forming hydrogel wound dressing from gelatin, oxidized alginate and borax. Periodate oxidized alginate rapidly cross-links proteins such as gelatin in the presence of borax to give in situ forming hydrogels that are both non-toxic and biodegradable. The composite matrix has the haemostatic effect of gelatin, the wound healing-promoting feature of alginate and the antiseptic property of borax to make it a potential wound dressing material. The hydrogel was found to have a fluid uptake of 90% of its weight which would prevent the wound bed from accumulation of exudates. The water vapour transmission rate (WVTR) of the hydrogel was found to be 2686+/-124 g/m2/day indicating that the hydrogel can maintain a moist environment over wound bed in moderate to heavily exuding wound which would enhance epithelial cell migration during the healing process. The wound healing efficacy of hydrogel was evaluated in experimental full thickness wounds using a rat model which demonstrated that within 2 weeks, the wound covered with gel was completely filled with new epithelium without any significant adverse reactions. These in situ forming hydrogels fulfil many critical elements desirable in a wound dressing material.     

Controlled release of tethered molecules via engineered hydrogel degradation: model development and validation

A statistical-co-kinetic model has been developed to predict effects of hydrolytic or enzymatic degradation on the macroscopic properties of hydrogels formed through Michael-type addition reactions. Important parameters accounted for by the theoretical calculations are bond cleavage kinetics, microstructural network characteristics such as macromer functionality and crosslinking efficiency, and detailed analysis of degradation products. Previous work indicated the validity of this modeling approach for predicting swelling behavior of hydrolytically degradable gels during early stages of degradation and the quantitative dependence of gel degradation on kinetic and structural parameters. The theoretical methodology is extended in the current work to predict release of covalently bound proteins from the network via labile bonds. Release studies of a network-bound fluoroscopic probe allow validation of model degradation parameters and indicate that macromer functionalization and network crosslinking efficiency can be appropriately tailored to achieve desired swelling profiles and protein release rates over the lifetime of the degradable gel. The effects of these network parameters on the timing of gel dissolution and the protein release that occurs during this phase of degradation are also identified, highlighting the utility of the developed model as a comprehensive tool for optimizing degradable hydrogels as matrices for drug delivery and tissue regeneration.     

Studies on biodegradation and release of gentamicin sulphate from interpenetrating network hydrogels based on poly(acrylic acid) and gelatin: in vitro and in vivo

Interpenetrating network hydrogels (IPNs) based on poly(acrylic acid) and gelatin (Ge) were evaluated for in vitro and in vivo biodegradation and in vivo release of gentamicin sulphate. In vitro and in vivo degradation studies demonstrated that with the increase of acrylic acid content in the polymer, the rate of degradation decreases, and a reverse phenomenon was observed with increasing Ge content in the hydrogel. The rate of in vivo degradation was much lower than in vitro degradation. Incorporation of gentamicin sulphate in hydrogel further reduces their degradation. In vitro and in vivo drug release profile showed a burst effect, followed by controlled release. Drug concentration was measured in the local skin tissue, blood serum, kidney, liver and spleen. The local skin tissue concentration of 50% and 100% gentamicin sulphate, loaded full IPNs (i.e., Ax-1 and Ax-2), was found to be higher (20+/-2mug/g) than the minimum bactericidal concentration for Staphylococcus aureus (1.2mug/g) and Pseudomonas aeruginosa (10mug/g), respectively, for a study time of 60 days.     

Effect of gelatin hydrogel incorporating fibroblast growth factor 2 on human meniscal cells in an organ culture model

Efforts to use growth factors to enhance the healing potential of the meniscus have been impeded because their half-lives are too short to maintain the biological activity. The thread was coated with gelatin hydrogel and fibroblast growth factor 2 (FGF 2) was biologically stabilized by incorporating in a gelatin hydrogel-coated thread. The purpose of this study is to investigate the effect of gelatin hydrogel-coated thread incorporating FGF 2 on human meniscal cells in an organ culture. Twenty-five menisci were cut into small pieces, and selected pieces were sutured with gelatin hydrogel-coated thread incorporating FGF 2 (FGF(+) group) or physiologic saline (FGF(−) group), followed by organ culture. The meniscal samples histologically evaluated 4, 7, and 14 days later. The cell density and the number of PCNA-positive cells for the FGF(+) group were higher than those of the FGF(−) group, while the number of TUNEL-positive cells was lower. These results suggest that FGF 2 stimulates the proliferation of meniscal cells and inhibits meniscal cell death. Gelatin hydrogel-coated threads releasable FGF 2 may be useful to promote repairing of human meniscus.     

Controlled release of nerve growth factor from fibrin gel

Nerve growth factor (NGF) is known to promote the axonal regeneration in injured nerve system. Delivery of NGF for a long period in a controlled manner may enhance the regeneration efficacy. In this study, we investigated whether NGF can be released from fibrin gel for a long period in a controlled manner. We also investigated whether sustained delivery of NGF using fibrin gel can enhance the efficacy of NGF in vitro. The addition of heparin to fibrin gel decreased the rate of NGF release from the fibrin gel. As the concentrations of thrombin and fibrinogen in fibrin gel increased, the NGF release rate decreased significantly, and the initial release burst decreased. NGF was released for up to 14 days in vitro. The bioactivity of NGF released from fibrin gel was assessed by morphological changes of pheochromocytoma (PC12) cells cultured in the presence of NGF-containing fibrin gel. NGF released from fibrin gel exhibited significantly higher degrees of PC12 cell viability and differentiation than NGF added in a free form daily into the culture medium. This study demonstrates that fibrin gel can release NGF in a sustained, controlled manner and in a bioactive form.     

Controlled release of fibroblast growth factors and heparin from photocrosslinked chitosan hydrogels and subsequent effect on in vivo vascularization

Application of ultraviolet (UV) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted within 10 s in an insoluble, flexible hydrogel. A low molecular weight acidic molecule like trypan blue and various high molecular weight molecules such as bovine serum albumin (BSA), heparin and protamine were all retained within the hydrogel, while a low molecular weight basic molecule like toluidine blue was rapidly released from the hydrogel. In the present work, we examined the retaining capability of the chitosan hydrogel for growth factors and controlled release of growth factors from the chitosan hydrogel in vitro and in vivo. Fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor(165) (VEGF(165)), heparin-binding epidermal growth factor (HB-EGF) in phosphate buffered saline (PBS) were mixed with Az-CH-LA aqueous solution to form growth factor-incorporated chitosan hydrogels. About 10-25% of the growth factor was released from a growth factor-incorporated chitosan hydrogel into PBS within the first day, after which no further substantial release took place. The growth factors interacted with Az-CH-LA molecules poly-ion complexation, and probably were unable to be released after the first day under the in vitro nondegradation conditions of the hydrogel. Although the FGF-1, FGF-2, and VEGF(165)-incorporated chitosan hydrogels on a culture plate significantly stimulated HUVEC growth, the stimulating activity of the growth factor-incorporated chitosan hydrogel was completely cancelled out by washing the hydrogel with PBS solution for 3 days or more. The stimulating activity on the HUVEC growth were however highly recovered by treating the washed growth factor-incorporated chitosan hydrogel during 7 days with chitinase and chitosanase to partly degrade the hydrogel, strongly suggesting that the growth factors within the hydrogel retained their biologically active forms. The chitosan hydrogel (100 microl) when implanted into the back of a mouse was biodegraded in about 10-14 days. When FGF-1- and FGF-2-incorporated chitosan hydrogels were subcutaneously implanted into the back of a mouse, significant neovascularization was induced near the implanted site of the FGF-1- and FGF-2-incorporated chitosan hydrogels. Furthermore, addition of heparin with either FGF-1 or FGF-2 into the hydrogel resulted in a significantly enhanced and prolonged vascularization effect. These results indicate that the controlled release of biologically active FGF-1 and FGF-2 with heparin is caused by biodegradation of the chitosan hydrogel, and subsequent induction of vascularization.     

Tissue regeneration based on growth factor release

Tissue engineering is an emerging biomedical field intended to assist the regeneration of body tissue defects too large to self-repair as well as to substitute for the biological functions of damaged and injured organs by using cells with proliferative and differentiative potential. In addition to basic research on such cells, it is undoubtedly indispensable for successful tissue engineering to create an artificial environment enabling cells to induce tissue regeneration. Such an environment can be achieved by making use of a scaffold for cell proliferation and differentiation and for growth factors, as well as their combination. Growth factors are often required to promote tissue regeneration, as they can induce angiogenesis, which supplies oxygen and nutrients to cells transplanted for organ substitution to maintain their biological functions. However, the biological effects of growth factors cannot always be expected because of their poor in vivo stability, unless a drug delivery system is contrived. In this article, tissue regeneration based on the release of growth factors is reviewed to emphasize the significance of drug delivery systems in tissue engineering.     

Controlled and modulated release of basic fibroblast growth factor.

Basic fibroblast growth factor has multivariate effects in stimulating cell growth and the processes that surround tissue repair. Pathophysiologic studies have been hampered by the stability of the compound. Though very potent, basic fibroblast growth factor is rapidly degraded when injected or ingested. Controlled release of basic fibroblast growth factor would allow for examination of the chronic effects of this compound. Conventional matrix polymer-based release devices were fabricated and basic fibroblast growth factor released in a sustained fashion, but 99% of basic fibroblast growth factor mitogenic activity was lost. The source of these losses was identified and preventative measures examined. Preservation and stabilization of basic fibroblast growth factor was accomplished by binding the factor to heparin-Sepharose beads. This permitted prolonged storage, repeated handling, and the encapsulation of basic fibroblast growth factor within a microspherical controlled-release device using a naturally occurring polymer material, alginate. Encapsulation was accomplished with 77% efficiency and 87.5 +/- 12% of the basic fibroblast growth factor was released in a biologically active form. Release activation and regulation was achieved when cleavage of the basic fibroblast growth factor-heparin bonds was enhanced (e.g. by enzymatic bond cleavage with heparinase). Kinetic profiles were identified for a variety of experimental conditions and the effects of the controlled release of basic fibroblast growth factor on BALBc/3T3 fibroblasts examined.     

Controlled drug release from implantable matrices based on hydrophobic polymers.

Reports on the controlled release of drugs, including macromolecular drugs, from silicone elastomers and ethylene-vinyl acetate copolymers, based on the formation of channels and cracks in the polymer, are reviewed. Aqueous interconnected pores are produced by osmotically active additives or by using loads of water-soluble drugs exceeding the percolation threshold. The release is generally proportional to the square root of time (t1/2). Nevertheless, pseudo-zero-order release kinetics can be obtained by adequately controlling the formulation variables. The factors controlling the release pattern and rate are discussed. In vivo applications of these types of systems are also considered.