Cenderitide-Eluting Film for Potential Cardiac Patch Applications

Cenderitide, also known as CD-NP, is a designer peptide developed by combining native mammalian c-type natriuretic peptide (CNP) and the C-terminus isolated from the dendroapis natriuretic peptide (DNP) of the venom from the green mamba. In early studies, intravenous and subcutaneous infusion of cenderitide was reported to reduce left ventricular (LV) mass and ameliorate cardiac remodelling. In this work, biodegradable polymeric films encapsulating CD-NP were developed and were investigated for their in vitro release and degradation characteristics. Subsequently, the bioactivity of released peptide and its effects on human cardiac fibroblast (HCF) were explored. We achieved sustained release from three films with low, intermediate and high release profiles for 30 days. Moreover, the bioactivity of released peptide was verified from the elevated production of cyclic guanosine monophospate (cGMP). The CD-NP released from films was able to inhibit the proliferation of hypertrophic HCF as well as suppress DNA synthesis in HCF. Furthermore, the sustained delivery from films showed comparable or superior suppressive actions on hypertrophic HCF compared to daily infusion of CD-NP. The results suggest that these films could be used to inhibit fibrosis and reduce cardiac remodelling via local delivery as cardiac patches.     

Chitosan film as rhBMP2 carrier: delivery properties for bone tissue application

Tissue engineering approaches need biomaterials with suitable properties to provide an appropriate environment for cell attachment and growth. The performance of these biomaterials can be greatly enhanced through the incorporation of bioactive agents. For this reason, we developed chitosan films with cell-attachment ability, rhBMP-2 carrier capacity, and good in vivo performance, and we employ them as covering for implantable materials. In this work, we have tried to explain how the rh-BMP2 is delivered to the surroundings from the development chitosan films. Protein diffusion from film, film stability versus in vitro dissolution, and biodegradation were evaluated to study rhBMP-2 delivery. Our results show that chitosan film has sufficiently good features to be used as an rhBMP-2 carrier. A low diffusion rate was observed, which was sufficient to quickly induce an in vitro differentiation stimulus, although heavily activated films retain more than 80-85% of the protein on the film. On the other hand, we estimated that chitosan film dissolution due to initial acidification in the wound environment is no more than 15-20%. We also estimated chitosan film response to lysozyme and concluded that degradation via this process proceeded at a slow kinetic rate. In addition, rhBMP-2 in vitro activity after film processing, as well as in vivo film behavior, were studied. We confirm that rhBMP-2 remains active on the film and after release, both in vitro and in vivo. These results support the conclusion that the developed chitosan film allows sustained release of the rhBMP-2 osteoinductive protein and could be used as an activated coat for implant and surgical prosthesis.     

Opiate-prostaglandin interactions in the regulation of insulin secretion from rat islets of Langerhans in vitro

The inadequate insulin secretory response to glucose stimulation in non-insulin dependent diabetes has been attributed to many factors including high PGE2 levels blunting the secretory response, and to the existence of inhibitory opiate activity in vivo. The purpose of the present work was to see if there was a connection between these two independent theories. Radioimmunoassayable PGE2 in islets of Langerhans was found to be proportional to islet number and protein content and was typically 4 to 5pg/micrograms islet protein. Indomethacin (2.8 X 10(-5) M), sodium salicylate (1.25 X 10(-3) M) and chlorpropamide (7.2 X 10(-5) M) all lowered islet PGE2 levels and stimulated insulin release in vitro. Dynorphin (1-13), stimulated insulin release at a concentration of 6 X 10(-9) M, while lowering islet PGE2. Conversely, at a higher concentration, (6 X 10(-7) M), dynorphin had no stimulatory effect on insulin secretion and did not lower PGE2 levels in islets or in the incubation media. The stimulatory effects of dynorphin and sodium salicylate on insulin secretion were blocked by exogenous PGE2 (10(-5) M). PGE2 at a lower concentration (10(-9) M) did not exert any inhibitory effect on dynorphin- or sodium salicylate-induced insulin release. This concentration of exogenous PGE2 stimulated insulin release in the presence of 6mM glucose. Results from these experiments suggest that since an opioid peptide can lower endogenous PGE2 production in islets and since the stimulatory effects of the opioid peptide are reversed by exogenous PGE2 there may be interactions between these two modulators of insulin secretion.     

Effects of porcine diazepam-binding inhibitor on insulin and glucagon secretion in vitro from the rat endocrine pancreas.

Porcine diazepam-binding inhibitor (pDBI) is a novel peptide that has been isolated from the small bowel of the pig, and that occurs also in the islet D-cells. We have studied its effects on hormone release in vitro from the endocrine pancreas of the rat. In isolated islets, pDBI (10(-9)-10(-6)M) did not affect basal insulin release at 3.3 mM glucose, whereas stimulated release at 8.3 mM glucose was dose-dependently suppressed by 32-69% (P less than 0.01). Furthermore, insulin secretion stimulated by either 16.7 mM glucose or 1 mM IBMX (3-isobutyl-1-methylxanthine) or 1 micrograms/ml glibenclamide was suppressed by pDBI at 10(-8) M (by 28-30%, P less than 0.05) and 10(-7) M (by 43-47%, P less than 0.01). In contrast, islet insulin secretion induced by 20 mM arginine was unaffected by these concentrations of pDBI. In the perfused rat pancreas, pDBI (10(-8) M) enhanced by 30% (P less than 0.05) the first phase (0-5 min) of arginine-stimulated insulin release, whereas the second phase (5-20 min) was unchanged. Moreover, pDBI suppressed by 28% (P less than 0.05) the second phase of arginine-induced glucagon release. Arginine-induced somatostatin release was not significantly affected by the peptide. Since pDBI immunoreactivity has been localized also to islet D-cells, the present results suggest that pDBI may act as a local modulator of islet hormone release.     

1H NMR investigation of thermally triggered insulin release from poly(N-isopropylacrylamide) microgels

We describe investigations of insulin release from thermoresponsive microgels using variable temperature (1)H NMR. Microgel particles composed of poly(N-isopropylacrylamide) were loaded with the peptide via a swelling technique, and this method was compared to simple equilibrium partitioning. Variable temperature (1)H NMR studies suggest that the swelling loading method results in enhanced entrapment of the peptide versus equilibrium partitioning. A centrifugation-loading assay supports this finding. Pseudo-temperature jump (1)H NMR measurements suggest that the insulin release rate is partially decoupled from microgel collapse. These types of direct release investigations could prove to be useful methods in the future design of controlled macromolecule drug delivery devices.     

Inherent charge-shifting polyelectrolyte multilayer blends: a facile route for tunable protein release from surfaces

Recent research has highlighted degradable multilayer films that enable the programmed release of different therapeutics. Multilayers constructed by the layer-by-layer (LbL) deposition that can undergo disassembly have been demonstrated to be of considerable interest, particularly for biomedical surface coatings due to their versatility and mild aqueous processing conditions, enabling the inclusion of biologic drugs with high activity. In this study, we examine the controlled release of a protein using a different mechanism for film disassembly, the gradual dissociation of film interactions under release conditions. Poly(β-amino ester)s and poly(L-lysine) (PLL) were used as the positively charged multilayer components coassembled with a model negatively charged antigen protein, ovalbumin (Ova). The release of the protein from these multilayer films is dominated by the slow shift in the charge of components under physiological pH conditions rather than by hydrolytic degradative release. The time scale of release can be varied over almost 2 orders of magnitude by varying the ratio of the two polyamines in the deposition solution. The highly versatile and tunable properties of these films form a basis for designing controlled and sequential delivery of drug coatings using a variety of polyions.     

Insulin-releasing properties of the frog skin peptide pseudin-2 and its [Lys18]-substituted analogue

Abstract Pseudin-2 is a cationic alpha-helical peptide that was first isolated from the skin of the paradoxical frog Pseudis paradoxa on the basis of its antimicrobial activity. We have investigated the insulin-releasing properties and cytotoxicity of the peptide, together with selected analogues with increased cationicity and hydrophobicity. At concentrations in the range 10(-9)-10(-6) m, pseudin-2, and its [Lys18], [Phe8], and [d-Lys3,d-Lys10,d-Lys14] derivatives, stimulated insulin release from the BRIN-BD11 clonal beta-cell line without increasing release of lactate dehydrogenase. The [Lys18] analogue was the most potent (46% increase in insulin release at 10(-9) m) and the most effective (215% increase in insulin release at 10(-6) m). The more cationic [Lys3,Lys10,Lys14] and [Lys3,Lys10,Lys14,Lys21] analogues lacked insulinotropic action and the more hydrophobic [Phe16] analogue was cytotoxic at concentrations > or =10(-7) m. Pseudin-2 and [Lys18]-pseudin-2 had no effect on intracellular calcium concentrations and stimulated insulin release in the absence of external calcium. [Lys18]-pseudin-2 (10(-8) m) stimulated insulin release in the presence of diazoxide and verapamil. Our results demonstrate that pseudin-2 stimulates insulin secretion from BRIN-BD11 cells by a mechanism involving Ca2+-independent pathways and identify [Lys18]-pseudin-2 as a peptide that may have potential for development as a therapeutically valuable insulinotropic agent for the treatment of type 2 diabetes.     

Addition of biological functionality to poly(epsilon-caprolactone) films

Biodegradable polyesters such as poly(epsilon-caprolactone) (PCL) have a number of biomedical applications; however, their usage is often limited by a lack of biological functionality. In this paper, a PCL-based polymer containing pendent groups activated by 4-nitrophenyl chloroformate (NPC) and reactive toward primary amines has been cast into thin films. The reactivity of the films toward poly(l-lysine) and the cell adhesion peptide, GRGDS, was assessed, and their cell adhesive capabilities were characterized. ATR-FTIR analysis found that NPC functional groups were present on the surface of the cast film, and the synthesis, conjugation, and visualization of a fluorescent molecule on these films further demonstrated the success of this functionalization methodology. The immersion of these films into a solution of either poly(l-lysine) (PLL) or GRGDS in PBS (pH 7.4) and subsequent 3T3 fibroblast adhesion studies demonstrated significant improvement in cell adhesion and spreading over films cast from unmodified PCL. This investigation has shown that this novel NPC-containing polymer can be utilized in many applications where increased cellular adhesion is required, or the coupling of specific molecules to polymer surfaces is of interest.     

Antagonism by GRP(18-27) and substance P analogues on insulin release stimulated by GRP(18-27).

The antagonistic effects of [D-Phe25]gastrin-releasing peptide (GRP)(18-27) and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]substance P (SP) on the stimulation of insulin release by GRP(18-27) from isolated canine pancreas were compared with that of [Ala23]GRP(18-27). The stimulation of insulin release by 1 nM GRP(18-27) was reduced to 24.1% and 15.4% by the prior infusion of 1 microM of [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP and 10 microM of [D-Phe25]GRP(18-27), respectively. Glucagon release by GRP(18-27) was not affected by these peptides using the above concentrations. The results indicate that these peptides are antagonists of bombesin-like peptide receptors on pancreatic B-cells, although the inhibitory activities are lower than that of [Ala23]GRP(18-27).     

Inhibition of insulin release by galanin and gastrin-releasing peptide in the anaesthetized rat

The present study was designed to determine the effects of intravenously administered galanin or gastrin-releasing peptide (GRP) on glucose- and/or glucose-dependent insulinotropic peptide (GIP)-stimulated insulin release in the anaesthetized rat. Galanin inhibited glucose-stimulated insulin responses in a dose-related manner. Galanin also inhibited insulin release in response to glucose administered with GIP; this effect was due largely to inhibition of the glucose-stimulated component since galanin did not inhibit GIP-stimulated insulin release. Galanin also inhibited insulin responses to ingestion of a mixed meal. GRP inhibited glucose-stimulated insulin responses, and the insulin responses to glucose plus GIP; unlike galanin, GRP inhibited both glucose- and GIP-stimulated insulin release. GRP also inhibited insulin release following ingestion of a mixed meal. The results suggest a possible modulatory role for these neuropeptides in regulation of insulin secretion.     

Glucagon-like peptide-1 and islet lipolysis.

A role for glucagon-like peptide 1 (GLP-1) has been suggested in stimulating beta-cell lipolysis via elevation of cAMP and activation of protein kinase A, which in turn may activate hormone-sensitive lipase (HSL), thereby contributing to fatty acid generation (FFA) from intracellular triglyceride stores. FFAs may then be metabolized to a lipid signal, which is required for optimal glucose-stimulated insulin secretion. Since HSL is expressed in islet beta-cells, this effect could contribute to the stimulation of insulin secretion by GLP-1, provided that a lipid signal of importance for insulin secretion is generated. To examine this hypothesis, we have studied the acute effect of GLP-1 on isolated mouse islets from normal mice and from mice with high-fat diet induced insulin resistance. We found, however, that although GLP-1 (100 nM) markedly potentiated glucose-stimulated insulin secretion from islets of both feeding groups, the peptide was not able to stimulate islet palmitate oxidation or increase lipolysis measured as glycerol release. This indicates that a lipid signal does not contribute to the acute stimulation of insulin secretion by GLP-1. To test whether lipolysis might be involved in the islet effects of long-term GLP-1 action, mice from the two feeding groups were chronically treated with exendin-4, a peptide that lowers blood glucose by interacting with GLP-1 receptors, in order to stimulate insulin secretion, for 16 days before isolation of the islets. The insulinotropic effects of GLP-1 and forskolin were exaggerated in isolated islets from exendin-4 treated mice given a high-fat diet, with a augmented palmitate oxidation as well as islet lipolysis at high glucose levels in these islets. Exendin-4 treatment had less impact on mice fed a normal diet. From these results we conclude that while GLP-1 does not seem to induce beta-cell lipolysis acutely in mouse islets, the peptide affects beta-cell fat metabolism after long-term adaptation to GLP-1 receptor stimulation.     

Circular dichroism and Fourier transform infrared spectroscopic studies on self-assembly of tetrapeptide derivative in solution and solvated film.

Aggregation of the hydrophobic peptide derivative Boc-Ala-Ile-Ile-Gly-OMe (1) was examined in methanol solution and in solvated film states. Formation of the peptide by self-assembly was evidenced using fluorescence [Mg salt of 8-anilino-naphthalenesulfonic acid (ANS) as an external probe] and circular dichroism (CD) spectroscopic techniques. In solution, peptide 1 formed as a stable aggregate at a concentration around 3 x 10(-4)m. The peptide gelled into a thin film for which we carried out CD and Fourier transform infrared (FTIR) measurements. Our spectroscopic study on peptide films at differing methanol concentrations indicates that the helical content of the peptide decreases with decreasing methanol concentration in solvated films. However, by reducing the methanol concentration we were able to observe a conformational transition from a predominantly helical turn to a beta-sheet structure via a random coil conformation. Our study focused on the aggregation of the alpha-helical turn-forming peptide derivative, which shows conformational transition on changing solvent concentration in the film form.     

Morphological and optical characterization of polyelectrolyte multilayers incorporating nanocrystalline cellulose

Aqueous layer-by-layer (LbL) processing was used to create polyelectrolyte multilayer (PEM) nanocomposites containing cellulose nanocrystals and poly(allylamine hydrochloride). Solution-dipping and spin-coating assembly methods gave smooth, stable, thin films. Morphology was studied by atomic force microscopy (AFM) and scanning electron microscopy (SEM), and film growth was characterized by X-ray photoelectron spectroscopy (XPS), ellipsometry, and optical reflectometry. Relatively few deposition cycles were needed to give full surface coverage, with film thicknesses ranging from 10 to 500 nm. Films prepared by spin-coating were substantially thicker than solution-dipped films and displayed radial orientation of the rod-shaped cellulose nanocrystals. The relationship between film color and thickness is discussed according to the principles of thin film interference and indicates that the iridescent properties of the films can be easily tailored in this system.     

DNA-collagen complex as a carrier for Ag+ delivery

The possibility of DNA-collagen complex as a drug carrier was investigated. The interaction between DNA and silver ions was proved by CD spectra. The release property of the complex of DNA-Ag+ was measured through turbidity of PBS solution to indicate that silver ions could coordinate with base pairs of DNA, and be released slowly from the complex of DNA-Ag+. Collagen film, collagen-Ag+ film, DNA-collagen film and DNA-collagen-Ag+ film were prepared, and studied through SEM. Particles were found present in DNA-collagen-Ag+ film by SEM. These show that silver ions may be enclosed inside these particles, which led to the slow release of Ag+ to the environments. Two bacteria, Escherichia coli and Staphylococcus aureus, were used to study the antibiotic properties of the complex films. The growth of E. coli and S. aureus could be inhibited by these films. It indicates that DNA-collagen may be a good drug carrier for the drug-controlled release.     

Drug release from and hydrolytic degradation of a poly(ethylene glycol) grafted poly(3-hydroxyoctanoate)

Monoacrylate-poly(ethylene glycol)-grafted poly(3-hydroxyoctanoate) (PEGMA-g-PHO) copolymers were synthesized to develop a swelling-controlled release delivery system for ibuprofen as a model drug. The in vitro hydrolytic degradation of and the drug release from a film made of the PEGMA-g-PHO copolymer were carried out in a phosphate buffer saline (pH 7.4) medium. The hydrolytic degradation of the copolymer was strongly dependent on the degree of grafting (DG) of the PEGMA group. The degradation rate of the copolymer films in vitro increased with increasing DG of the PEGMA group on the PHO chain. The copolymer films showed a controlled delivery of ibuprofen to the medium in periods of time that depend on the composition, hydrophilic/hydrophobic characteristics, initial drug loading amount and film thickness of the graft copolymer support. The drug release rate from the grafted copolymer films was faster than the rate of weight loss of the films themselves. In particular, a combination of the low DG of the PEGMA group in the PHO chains with the low ibuprofen solubility in water led to long-term constant release from these matrices in vitro.     

Polypeptide multilayer film co-delivers oppositely-charged drug molecules in sustained manners

The current state-of-the-art for drug-carrying biomedical devices is mostly limited to those that release a single drug. Yet there are many situations in which more than one therapeutic agent is needed. Also, most polyelectrolyte multilayer films intended for drug delivery are loaded with active molecules only during multilayer film preparation. In this paper, we present the integration of capsules as vehicles within polypeptide multilayer films for sustained release of multiple oppositely charged drug molecules using layer-by-layer nanoassembly technology. Calcium carbonate (CaCO(3)) particles were impregnated with polyelectrolytes, shelled with polyelectrolyte multilayers, and then assembled onto polypeptide multilayer films using glutaraldehyde. Capsule-integrated polypeptide multilayer films were obtained after decomposition of CaCO(3) templates. Two oppositely charged drugs were loaded into capsules within polypeptide multilayer films postpreparation based on electrostatic interactions between the drugs and the polyelectrolytes impregnated within capsules. We determined that the developed innovative capsule-integrated polypeptide multilayer films could be used to load multiple drugs of very different properties (e.g., opposite charges) any time postpreparation (e.g., minutes before surgical implantation inside an operating room), and such capsule-integrated films allowed simultaneous delivery of two oppositely charged drug molecules and a sustained (up to two weeks or longer) and sequential release was achieved.     

Palmitoylation of a pulmonary surfactant protein C analogue affects the surface associated lipid reservoir and film stability

Surfactant protein C (SP-C) is a lipopeptide that contains two thioester-linked palmitoyl groups and is considered to be important for formation of the alveolar surface active lipid film. Here, a non- or dipalmitoylated SP-C analogue (SP-C(Leu)), in which all helical Val residues were replaced with Leu and Cys-5 and Cys-6 were replaced with Ser, was tested for surface activity in a captive bubble system (CBS). SP-C(Leu), either palmitoylated at Ser-5 and Ser-6 or non-palmitoylated, was added to mixtures of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidyl glycerol (PG)/palmitic acid (PA), 68:22:9, (by mass) at a concentration of 2 and 5%. With 2% peptide, surface film formation was rapid, reaching a surface tension below 25 mN/m within 5 s, but the samples with 5% SP-C(Leu) required more than 20 s to reach values below 25 mN/m. Minimum surface tension for the samples with dipalmitoylated SP-C(Leu) was below 1.5 mN/m and very stable, as the surface tension increased by less than 0.5 mN/m within 10 min at constant bubble volume. Minimum surface tension for the non-palmitoylated SP-C(Leu) was approximately 2 and 5 mN/m for 2 and 5% peptide, respectively, but the films were less stable as seen by frequent bubble clicking at low surface tensions. Films with dipalmitoylated SP-C(Leu) that were dynamically cycled at 20-30 cycles/min were substantially less compressible at a surface tension of 20 mN/m (0.007 m/mN) than those that contained the non-palmitoylated peptide (0.02 m/mN). After subphase depletion, the incorporation of lipids into the surface active film during initial bubble expansion occurred at a relatively low surface tension (about 35 mN/m) for the samples with dipalmitoylated SP-C(Leu) compared to approximately 45 mN/m for those containing the non-palmitoylated peptide. Furthermore, for samples that contained non-palmitoylated SP-C(Leu), the ability to reach near zero stable surface tension was lost after a few adsorption steps, whereas with the dipalmitoylated peptide the film quality did not deteriorate even after more than 10 expansion steps and the incorporation of reservoir material equivalent to more than two monolayers. It appears that the covalently linked palmitoyl groups of the SP-C analogue studied are important for the mechanical stability of the lipid film, for the capacity to incorporate material from the reservoir into the surface active film upon area expansion, and for the low film compressibility of dynamically cycled films.     

Dimeric N-terminal segment of human surfactant protein B (dSP-B(1-25)) has enhanced surface properties compared to monomeric SP-B(1-25)

Surfactant protein B (SP-B) is a 17-kDa dimeric protein produced by alveolar type II cells. Its main function is to lower the surface tension by inserting lipids into the air/liquid interface of the lung. SP-B's function can be mimicked by a 25-amino acid peptide, SP-B(1-25), which is based on the N-terminal sequence of SP-B. We synthesized a dimeric version of this peptide, dSP-B(1-25), and the two peptides were tested for their surface activity. Both SP-B(1-25) and dSP-B(1-25) showed good lipid mixing and adsorption activities. The dimeric peptide showed activity comparable to that of native SP-B in the pressure-driven captive bubble surfactometer. Spread surface films led to stable near-zero minimum surface tensions during cycling while protein free, and films containing SP-B(1-25) lost material from the interface during compression. We propose that dimerization of the peptide is required to create a lipid reservoir attached to the monolayer from which new material can enter the surface film upon expansion of the air/liquid interface. The dimeric state of SP-B can fulfill the same function in vivo.     

Effects of galnon, a non-peptide galanin-receptor agonist, on insulin release from rat pancreatic islets

Galanin is a neurotransmitter peptide that suppresses insulin secretion. The present study aimed at investigating how a non-peptide galanin receptor agonist, galnon, affects insulin secretion from isolated pancreatic islets of healthy Wistar and diabetic Goto-Kakizaki (GK) rats. Galnon stimulated insulin release potently in isolated Wistar rat islets; 100 microM of the compound increased the release 8.5 times (p<0.001) at 3.3 mM and 3.7 times (p<0.001) at 16.7 mM glucose. Also in islet perifusions, galnon augmented several-fold both acute and late phases of insulin response to glucose. Furthermore, galnon stimulated insulin release in GK rat islets. These effects were not inhibited by the presence of galanin or the galanin receptor antagonist M35. The stimulatory effects of galnon were partly inhibited by the PKA and PKC inhibitors, H-89 and calphostin C, respectively, at 16.7 but not 3.3 mM glucose. In both Wistar and GK rat islets, insulin release was stimulated by depolarization of 30 mM KCl, and 100 microM galnon further enhanced insulin release 1.5-2 times (p<0.05). Cytosolic calcium levels, determined by fura-2, were increased in parallel with insulin release, and the L-type Ca2+-channel blocker nimodipine suppressed insulin response to glucose and galnon. In conclusion, galnon stimulates insulin release in islets of healthy rats and diabetic GK rats. The mechanism of this stimulatory effect does not involve galanin receptors. Galnon-induced insulin release is not glucose-dependent and appears to involve opening of L-type Ca2+-channels, but the main effect of galnon seems to be exerted at a step distal to these channels, i.e., at B-cell exocytosis.