Triggered release of insulin from glucose-sensitive enzyme multilayer shells

A glucose-sensitive multilayer shell, which was fabricated by the layer-by-layer (LbL) assembly method, can be used as a carrier for the encapsulation and controlled release of insulin. In the present report, glucose oxidase (GOD) and catalase (CAT) were assembled on insulin particles alternately via glutaraldehyde (GA) cross-linking. The resulting core–shell system has been proven to be glucose-sensitive. When the external glucose was introduced, the release ratio of insulin from the protein multilayer can be increased observably. This is likely attributed to the catalysis interaction of CAT/GOD shells to glucose, which leads to the production of H⁺ and thus drops the pH of the microenvironment. Under the acidic conditions, on the one hand, a part of CN bond formed from Schiff base reaction can be broken and thus increasing the permeability of the capsule wall. On the other hand, the solubility of insulin can also be increased. The above factors may be the key control to increase the release of insulin from the multilayer. Therefore, such CAT/GOD multilayer may have a great potential as a glucose-sensitive release carrier for insulin, and may open the way for the further application of LbL capsules in the drug delivery and controlled release, etc.     

Controlled release of vascular endothelial growth factor from alginate hydrogels nano-coated with polyelectrolyte multilayer films

Alginate (ALG) hydrogels incorporating vascular endothelial growth factor (VEGF) were nano-coated with polyelectrolyte multilayer (PEM) films composed of chitosan (CT) and dextran sulfate (DEX) in order to control the VEGF release from the hydrogels. When non- and nano-coated ALG hydrogels containing VEGF were incubated in phosphate-buffered saline (PBS) at 37 degrees C for the prescribed times, the nano-coated hydrogels were stable, even after incubation for a week, whereas the non-coated hydrogels collapsed after 6 h. The release profile of VEGF from the non- and nano-coated ALG hydrogels was evaluated by an enzyme-linked immunosorbent assay (ELISA). Although all of the VEGF incorporated into the non-coated ALG hydrogels was released within 6 h by the collapse, only a few percent of the VEGF incorporated in the nano-coated hydrogels was released. Furthermore, the incorporated VEGF in the nano-coated hydrogels was released continuously, even after a month, without any initial burst release. The release percentage of VEGF was easily controlled by the PEM film thickness on the surface of the ALG hydrogels. The VEGF released from the nano-coated hydrogels retained its activity without denaturation. Consequently, the nano-coating of hydrogel surfaces with PEM films may be useful for controlled and sustained drug-delivery systems.     

Controlled release from multilayer silk biomaterial coatings to modulate vascular cell responses

A multilayered silk fibroin protein coating system was employed as a drug carrier and delivery system to evaluate vascular cell responses to heparin, paclitaxel, and clopidogrel. The results demonstrated that the silk coating system was an effective system for drug-eluting coatings, such as for stent applications, based on its useful micromechanical properties and biological outcomes. Cell attachment and viability studies with human aortic endothelial cells (HAECs) and human coronary artery smooth muscle cells (HCASMCs) on the drug-incorporated silk coatings demonstrated that paclitaxel and clopidogrel inhibited smooth muscle cell (SMC) proliferation and retarded endothelial cell proliferation. Heparin-loaded silk multilayers promoted HAEC proliferation while inhibiting HCASMC proliferation, desired outcomes for the prevention of restenosis. The preservation of the phenotype of endothelial cells on silk and heparin-loaded silk coatings was confirmed with the presence of endothelial markers CD-31, CD-146, vWF and VE-Cadherin using immunocytochemistry assays. A preliminary in-vivo study in a porcine aorta showed integrity of the silk coatings after implantation and the reduction of platelet adhesion on the heparin-loaded silk coatings.     

Controlled release of NFkappaB decoy oligonucleotides from biodegradable polymer microparticles

The objective of this study was to evaluate a poly(DL-lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG) delivery system for nuclear factor-kappa B (NFkappaB) decoy phosphorothioated oligonucleotides (ODNs). PLGA/PEG microparticles loaded with ODNs were fabricated with entrapment efficiencies up to 70%. The effects of PEG contents (0, 5, and l0 wt%), ODN loading densities (0.4, 4, and 40 microg/mg), and pH of the incubation medium (pH 5, 7.4. and 10) on ODN release kinetics from the PLGA/PEG microparticles were investigated in vitro for up to 28 days. The release profiles in pH 7.4 phosphate buffered saline (PBS) were characterized by an initial burst during the first 2 days, a linear release phase until day 18, and a final release phase for the rest of the period. Up to 85% of the ODNs were released after 28 days in pH 7.4 PBS regardless of the ODN loading density and PEG content. Higher ODN loading densities resulted in lower entrapment efficiencies and greater initial burst effects. The bulk degradation of PLGA was not significantly affected by the PEG content and ODN loading density, but significantly accelerated at acidic buffer pH. Under acidic and basic conditions, the aggregation of microparticles resulted in significantly lower cumulative mass of released ODNs than that released at neutral pH. The effects of pH were reduced by the incorporation of PEG into PLGA microparticles. Since the PLGA degradation products are acidic, PLGA/PEG microparticles might provide a better ODN delivery vehicle than PLGA microparticles. These results suggest that PLGA/PEG microparticles are useful as delivery vehicles for controlled release of ODNs and merit further investigation in cell culture and animal models of glioblastoma.     

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.     

Sustained release of complexed and naked DNA from polymer films

Controlled release studies of DNA from polymers have been limited, with most studies concentrating on microsphere formulations. This report details a study done on the release and transfection efficiencies of pDNA-lipofectamine complex (lipoplex), from selected polymeric films in which it was dispersed; the release and transfection efficiency was compared with that of naked pDNA. A biodegradable and a biostable polymer were compared. A sustained release profile was obtained from both the polymeric films. For the release of pDNA (naked DNA), a burst effect was always seen, and was suppressed using additives; for complexed pDNA (lipoplex), the release was slow, but could be accelerated using additives. The compositions of the released lipoplexes were also quantified in terms of the fraction that was complexed. In addition, the transfection efficiency of the released complexes and of the naked pDNA was determined in vitro using COS 7 cells. The results also demonstrated that bioactivity of the released complexed pDNA was superior to that of the released naked DNA. Such formulations may be useful for local sustained delivery of lipoplexes from implanted films.     

Controlled release of transforming growth factor beta1 from biodegradable polymer microparticles

Recombinant human transforming growth factor beta1 (TGF-beta1) was incorporated into biodegradable microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) at 6 ng/1 mg microparticles. Fluorescein isothiocynate labeled bovine serum albumin (FITC-BSA) was coencapsulated as a porogen at 4 microg/1 mg of microparticles. The effects of PEG content (0, 1, or 5 wt %) and buffer pH (3, 5, or 7.4) on the protein release kinetics and the degradation of PLGA were determined in vitro for up to 28 days. The entrapment yield of TGF-beta1 was 83.4 +/- 13.1 and 54.2 +/- 12.1% for PEG contents of 0 and 5%, respectively. The FITC-BSA and TGF-beta1 were both released in a multiphasic fashion including an initial burst effect. Increasing the PEG content resulted in the decreased cumulative mass of released proteins. By day 28, 3.8 +/- 0. 1 and 2.8 +/- 0.3 microg (based on 1 mg microparticles) of loaded FITC-BSA and 3.4 +/- 0.2 and 2.2 +/- 0.3 ng of loaded TGF-beta1 were released into pH 7.4 phosphate buffered saline (PBS) from microparticles with 0 and 5% PEG, respectively. Aggregation of FITC-BSA occurred at lower buffer pH, which led to decreased release rates of both proteins. For microparticles with 5% PEG, 2.3 +/- 0.1 microg of FITC-BSA and 2.0 +/- 0.2 ng of TGF-beta1 were released in pH 7.4 buffer after 28 days, while only 1.7 +/- 0.3 microg and 1.3 +/- 0.4 ng of the corresponding proteins were released in pH 3 buffer. The degradation of PLGA was also enhanced at 5% PEG content, which was significantly accelerated at acidic pH conditions. The calculated half-lives of PLGA were 20.3 +/- 0.9 and 15.9 +/- 1.2 days for PEG contents of 0 and 5%, respectively, in pH 7.4 PBS and 14.8 +/- 0.4 and 5.5 +/- 0.1 days for 5% PEG in pH 7.4 and 3 buffers, respectively. These results suggest that PLGA/PEG blend microparticles are useful as delivery vehicles for controlled release of growth factors.     

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.     

Controlled release of vancomycin from thin sol-gel films on titanium alloy fracture plate material

Risk of infection is considerable in open fractures and its management is challenging, especially when fracture fixation material is used. Thus, it may be desirable to use a device from which antibiotics can be released in a controlled way. Room temperature processed silica sol-gels are novel, resorbable and biocompatible, controlled release materials. Vancomycin, a potent antibiotic used in treating osteomyelitis, can be released from silica sol-gels. Herein, we describe the synthesis of thin, resorbable, controlled release bactericidal sol-gel films on a Ti-alloy substrate and determine the effect of processing parameters on its degradation and vancomycin release. A close correlation between release and degradation rates suggests that film degradation is the main mechanism underlying the control of release. Using a multi-layer process and various concentrations of vancomycin, released concentrations exceed the minimal inhibitory concentration (MIC) of vancomycin against Staphylococcus aureus. The findings enable the tailoring of release and degradation properties of the films to therapeutic needs by controlling sol-gel processing parameters. Given the bactericidal properties of released vancomycin, and the biocompatibility of the sol-gel films, the present data suggest great promise to prevent and treat bone infections in a clinical setting.     

Controlled release of doxorubicin from graphene oxide based charge-reversal nanocarrier

A number of anticancer drugs, such as doxorubicin (DOX), operate only after being transported into the nucleus of cancer cells. Thus it is essential for the drug carriers to effectively release the anticancer drugs into the cytoplasm of cancer cells and make them move to nucleus freely. Herein, a pH-responsive charge-reversal polyelectrolyte and integrin α_Ⅴβ₃ mono-antibody functionalized graphene oxide (GO) complex is constituted as a nanocarrier for targeted delivery and controlled release of DOX into cancer cells. The DOX loading and releasing in vitro demonstrates that this nanocarrier cannot only load DOX with high efficiency, but also effectively release it under mild acidic pH stimulation. Cellular toxicity assay, confocal laser scanning microscopy and flow cytometer analysis results together confirm that with the targeting nanocarrier, DOX can be selectively transported into the targeted cancer cells. Then they will be effectively released from the nanocarriers in cytoplasm and moved into the nucleus subsequently, stimulating by charge-reverse of the polyelectrolyte in acidic intracellular compartments. The effective delivery and release of the anticancer drugs into nucleus of the targeted cancer cells will lead to a high therapeutic efficiency. Hence, such a targeting nanocarrier prepared from GO and charge-reversal polyelectrolytes is likely to be an available candidate for targeted drug delivery in tumor therapy.     

Controlled release of 5-fluorouridine from radiation-crosslinked poly(ethylene-co-vinyl acetate) films

The effect of γ-radiation doses of 12.5–380 kGy on the infrared spectra, gel content, mechanical properties, and the release of oxobutyl-5-fluoro-2′-deoxyuridine (OfdUrd, an antitumor agent) from poly(ethylene-co-vinyl acetate) (EVA) films was studied. The results showed that the application of radiation doses produced a crosslinking reaction leading to a maximum gel content of about 85% in the case of 150 kGy. Higher doses did not increase the gel content in EVA films. The mechanical properties (tensile strength, percentage elongation at break and Young’s modulus) of all studied EVA matrices were affected by the exposure to γ-radiation. Irradiation doses over 50 kGy caused an increase in the Young’s modulus of EVA and at the same time a decrease in the strain per cent. Moreover, the network structure formed after irradiation reduced significantly the OFdUrd release from EVA films. In this manner, the radiation dose applied to the polymeric matrix modulated the release of OFdUrd, avoiding the high concentrations that may cause severe systemic toxicity. The loading of OFdUrd to EVA film triggered a slight hyperemia after implantation, while the inflammatory reaction was only observed during the first two days.     

Syntheses and properties of films from polymer gels based on polymer-metal complexes

Films of poly(2-vinylpyridine) (1) , poly(allylamine hydrochloride) (2) , poly(5-methyl-2-vinylpyridine) (3) and poly(ethyleneimine) (4) with Ag⁺, Cu²⁺, Li⁺ and Ca²⁺ salts as well as complex transition metal ions [Fe(Cn)₆]^3? are prepared at the interface boundaries of immiscible solvents. The polymer/metal salt complexes exhibit highly ordered structures and high thermal stabilities as revealed by reduced viscosity, X-ray diffraction, infrared spectrometry, thermogravimetry, differential thermal analysis as well as measurements of swelling coefficients and electroconductivity. Mole ratios of complex formation of the neat polymers as well as the polymer gels with these salts were found at [polymer] : [salt] = 1, 2, 3 and 4 referring to the repeating units of the polymers. It is suggested that the properties of the films are due to ionic bonds, ion-coordination bonding, coordination bonds and ion-dipole interactions between the functional groups of the polymeric hydro- and organic gels and the metal ions.     

Controlled release of ethacrynic acid from poly(lactide-co-glycolide) films for glaucoma treatment

Ethacrynic acid (ECA) is a potential glaucoma drug that can reduce intraocular pressure. However, conventional methods of ECA administration may cause toxicity to normal eye tissues and are inconvenient to patients. Therefore, we developed and characterized an ECA loaded poly(lactide-co-glycolide) (PLGA) copolymer film, and quantified the therapeutic efficacy of the film implanted in the rabbit eye. In the aqueous medium, the release of ECA from the PLGA50:50 film was time dependent and more than 90% of ECA was released within a week. This release profile was consistent with the kinetics of water uptake and microstructural changes of PLGA50:50 films as revealed by an electron microscopy examination. ECA release and PLGA degradation caused a gradual pH decrease in the release medium. The total pH decrease was 0.4 unit in 3 days. We also observed that the initial rate of ECA release was positively correlated with the weight ratio of ECA versus PLGA and inversely correlated with the molar ratio of lactide versus glycolide in PLGA films. At the end of a 3-day incubation, the cumulative release of ECA from PLGA50:50, PLGA85:15 and PLGA100:00 films were 78.8%, 9.35% and 3.60%, respectively. When the PLGA50:50 film loaded with ECA was implanted into the sclera of rabbit eyes, the intraocular pressure was significantly reduced and the reduction was maintained for at least 10 days. These data indicate that PLGA films have a potential to be used as a controlled ECA release device for glaucoma treatment.     

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.     

High-throughput cellular screening of engineered ECM based on combinatorial polyelectrolyte multilayer films

The capacity to engineer the extracellular matrix is critical to better understand cell function and to design optimal cellular environments to support tissue engineering, transplantation and repair. Stacks of adsorbed polymers can be engineered as soft wet three dimensional matrices, with properties tailored to support cell survival and growth. Here, we have developed a combinatorial method to generate coatings that self assemble from solutions of polyelectrolytes in water, layer by layer, to produce a polyelectrolyte multilayer (PEM) coating that has enabled high-throughput screening for cellular biocompatibility. Two dimensional combinatorial PEMs were used to rapidly identify assembly conditions that promote optimal cell survival and viability. Conditions were first piloted using a cell line, human embryonic kidney 293 cells (HEK 293), and subsequently tested using primary cultures of embryonic rat spinal commissural neurons. Cell viability was correlated with surface energy (wettability), modulus (matrix stiffness), and surface charge of the coatings.Our findings indicate that the modulus is a crucial determinant of the capacity of a surface to inhibit or support cell survival.     

Photochromic polymer films based on a 14-F bacteriorhodopsin derivative

Spectral and kinetic characteristics were measured for polymer (gelatin) films based on 14-F bacteriorhodopsin (BR), both wild-type (WT) and D96N mutant, to study the peculiarities of photo-induced transformation of the samples. It was demonstrated earlier that incorporation of 14-F retinal into the apomembrane of both 14-F WT and 14-F D96N produces pigments with drastically different photo-induced behavior, regarding the formation and decay of a red-shifted species at 660 nm. However, similar fundamental differences between 14-F WT and 14-F D96N, if embedded into gelatin matrix, were not observed. Results are discussed in the frame of the relationship between the kinetic rates of two photo-induced processes that occur in suspensions and gelatin films of corresponding pigments. These experimental results can possibly be explained by the difference in kinetics for gelatin films and water suspensions. The main factor in altering the kinetics is the relative humidity of gelatin samples. Therefore, the effect of relative humidity on performance of 14-F BR gelatin films, both WT and D96N, was studied. A range of humidity has been defined for each pigment, in which spectral and kinetic characteristics were changed in the desired direction. It was shown that 14-F WT gelatin films may offer a technological advantage relative to those based on 14-F D96N.     

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 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-beta1 (TGF-beta1) 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-beta1 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-beta1 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.     

Development of a spiral piezoelectric immunosensor based on thiol self-assembled monolayers for the detection of insulin

A spiral piezoelectric (Pz) immunosensor based on thiol self-assembled monolayers (SAMs) and fabricated with a screw clamp apparatus was developed for quantitative detection of insulin in serum. Anti-human insulin antibody was first modified with sulfosuccinimidyl 6-[3'-(2-pyridyldithio) propionamido] hexanoate (Sulfo-LC-SPDP) to graft the active sulfhydryl groups into antibody molecules. Then, modified antibody molecules were assembled on a gold electrode via covalent bonding to form SAMs as the recognition element of the insulin immunosensor. The response characteristics of the Pz immunosensor, including buffer solution, time-costs, reproducibility and specificity, were also investigated in detail. Coating a Au electrode with SAMs gave better results within 40 min, with a detection range of 2.5 to 80 mIU/L and a coefficient of variance (CV) less than 5%. The stability and reusability of the immunosensor was improved with a mild eluting reagent which successfully removed the bound insulin molecules from the antibody-coated crystal without affecting the immobilized antibody. A comparison study revealed that analytical results of insulin in samples obtained with this immunosensor were in good agreement with those given by the radioimmunoassay. Thus, the proposed Pz immunosensor provided a rapid, sensitive, specific, reusable and reliable alternative for the detection of insulin in clinical laboratory.