Release Of Macromolecules Research Articles

Endosomolysis by masking of a membrane-active agent (EMMA) for cytoplasmic release of macromolecules.

Endosomolysis, a critical barrier to efficient delivery of macromolecules such as nucleic acids, has been breached using a novel approach: endosomolysis by masking of a membrane-active agent (EMMA). To demonstrate the concept of EMMA, a cationic membrane-active peptide, melittin, was reversibly inhibited using a maleic anhydride derivative. At neutral pH, the lysines of melittin are covalently acylated with the anhydride, thereby inhibiting melittin's membrane disruption activity. Under acidic conditions such as those present within endosomes, the amide bond of the maleamate is cleaved, thus unmasking melittin. The active melittin can then disrupt the endosomal membrane resulting in release of biologically active molecules into the cytoplasm. This approach avoids cellular toxicity by restricting melittin's activity until it reaches the endosomal compartment. The utility of this approach was demonstrated by delivery phosphorodiamidate morpholino oligonucleotides (PMOs).

Pancreatin enhanced erosion of and macromolecule release from 2,2-bis(2-oxazoline)-linked poly(ϵ-caprolactone)

The degradation and erosion of solvent cast films and injection molded bars prepared from poly(ϵ-caprolactone) (PCL) and 2,2′-bis(2-oxazoline) linked poly(ϵ-caprolactone) (PCL-O) were evaluated in simulated gastric fluid (SGF) (pH 1.2, pepsin present) and in simulated intestinal fluid (SIF) (pH 7.5, pancreatin present). After incubation of the polymer films (10 mg) and bars (70 mg) in the medium, the resulting decrease in molecular weight (degradation) was determined by size exclusion chromatography and the weight loss of the preparations was measured. In addition, the effect of pancreatin on FITC–dextran (MW 4400) release from PCL and PCL-O microparticles, prepared by w/o/w double emulsion technique, was studied. No degradation or weight loss was observed for either PCL or PCL-O films in SGF (12 h incubation, 37 °C). When compared to PBS pH 7.4, pancreatin hardly enhanced the weight loss of PCL films and bars. In contrast, pancreatin enhanced substantially erosion of PCL-O films and bars. Unlike PCL preparations, the PCL-O preparations showed surface erosion in SIF. Pancreatin increased considerably FITC–dextran release from both PCL and PCL-O microparticles. In conclusion, the present results demonstrate the enzyme sensitivity of the novel PCL-O polymer. In addition, the results show that pancreatin present in intestinal fluid may substantially affect drug release from PCL based preparations.

Ion channels in secretory granules of the pancreas and their role in exocytosis and release of secretory proteins.

Regulated secretion in exocrine and neuroendocrine cells occurs through exocytosis of secretory granules and the subsequent release of stored small molecules and proteins. The introduction of biophysical techniques with high temporal and spatial resolution, and the identification of Ca(2+)-dependent and -independent "docking" and "fusion" proteins, has greatly enhanced our understanding of exocytosis. The cloning of families of ion channel proteins, including intracellular ion channels, has also revived interest in the role of secretory granule ion channels in exocytotic secretion. Thus secretory granules of pancreatic acinar cell express a ClC-2 Cl(-) channel, a HCO-permeable member of the CLCA Ca(2+)-dependent anion channel family, and a KCNQ1 K(+) channel. Evidence suggests that these channels may facilitate the release of digestive enzymes and/or prevent exocytosed granules from collapsing during "kiss and run" recycling. In pancreatic beta-cells, a granular ClC-3 Cl(-) channel provides a shunt pathway for a vacuolar-type H(+)-ATPase. Acidification "primes" the granules for Ca(2+)-dependent exocytosis and release of insulin. In summary, secretory granules are equipped with specific sets of ion channels, which modulate regulated exocytosis and the release of macromolecules. These channels could represent excellent targets for therapeutic interventions to control exocytotic secretion in relevant diseases, such as pancreatitis, cystic fibrosis, or diabetes mellitus.

Thermosensitive chitosan-based hydrogel containing liposomes for the delivery of hydrophilic molecules

A novel injectable in situ gelling thermosensitive chitosan–β-glycerophosphate (C–GP) formulation has been recently proposed for tissue repair and drug delivery. The system can sustain the release of macromolecules over a period of several hours to a few days. However, with low-molecular-weight hydrophilic compounds, the release is generally completed within 24 h. In this study, liposomes were added to the C–GP solution and their effect on the viscoelastic properties of the system and release kinetics of encapsulated carboxyfluorescein was investigated. The gelation rate and gel strength were slightly increased by the presence of the liposomes. The in vitro release profiles demonstrated controlled delivery over at least 2 weeks. The release rate strongly depended on the liposome size and composition (i.e. addition of cholesterol), and on the presence of phospholipase in the release medium. The kinetics was not substantially modified when using liposomes prepared with a negatively-charged lipid or a lipid having a high phase transition temperature. These results indicate that the liposome–C–GP system rapidly gels at body temperature, and can sustain the delivery of low-molecular-weight hydrophilic compounds. A mathematical model was proposed to characterize the release kinetics.

Functional Characterization of an Endosome-disruptive Peptide and Its Application in Cytosolic Delivery of Immunoliposome-entrapped Proteins

Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e. peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH(2)-terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes.

The release of model macromolecules may be controlled by the hydrophobicity of palmitoyl glycol chitosan hydrogels

A non-covalently cross-linked palmitoyl glycol chitosan (GCP) hydrogel has been evaluated as an erodible controlled release system for the delivery of hydrophilic macromolecules. Samples of GCP with hydrophobicity decreasing in the order GCP12>GCP11>GCP21 were synthesised and characterised by 1H NMR. Hydrogels were prepared by freeze-drying an aqueous dispersion of the polymer in the presence or absence of either a model macromolecule fluorescein isothiocyanate-dextran (FITC-dextran, MW 4400), and/or amphiphilic derivatives Gelucire 50/13 or vitamin E d-α-tocopherol polyethylene glycol succinate. Gels were analysed for aqueous hydration, FITC-dextran release, and bioadhesion, and imaged by scanning electron microscopy. The gels were highly porous and could be hydrated to up to 95× their original weight without an appreciable volume change and most gels eventually eroded. Hydration and erosion were governed by the hydrophobicity of the gel and the presence of the amphiphilic additives. GCP gels could be loaded with up to 27.5% (w/w) of FITC-dextran by freeze-drying a dispersion of GCP in a solution of FITC-dextran. The controlled release of FITC-dextran was governed by the hydrophobicity of the gel following the trend GCP21>GCP11>GCP12. GCP gels were bioadhesive but less so than hydroxypropylmethylcellulose, Carbopol 974NF (7:3) tablets.

Concentration of collagen, aggrecan and cartilage oligomeric matrix protein (COMP) in synovial fluid from equine middle carpal joints.

The aim of the present investigation was to study the metabolic activity of the third carpal bone and the release of COMP, aggrecan and collagen type II molecules in the synovial fluid as a result of injury. Cartilage oligomeric matrix protein (COMP), aggrecan and collagen type II or fragments of these molecules released to the synovial fluid and serum (COMP) were quantified in samples from 73 left equine middle carpal joints from 2 breeds with different activity profiles (52 Standardbred trotters [STB] and 21 Swedish Warmblood riding horses [SWH]) and different articular cartilage lesions. Synovial and serum samples were analysed using inhibition ELISA for COMP and aggrecan. An ELISA that combines features of both the competitive and capture ELISAs was used for collagen type II. COMP and aggrecan concentrations decreased in synovial fluid from the joints with moderate lesions of STB compared with the normal joints; COMP from 16.6 to 12.0 microg/ml and aggrecan from 93.0 to 68.1 microg/ml. In serum, COMP concentrations were also lowered in the STB with moderate lesions compared with the normal joints, while in the SWH, the COMP concentration in synovial fluids from joints with moderate lesions was somewhat increased at 19.6 microg/ml compared with the normal joints (17.6 microg/ml). The ratio between aggrecan/COMP in the synovial fluid from joints with moderate lesions was higher in the STB (6.2) than in the SWH (3.4). The level of collagen type II in synovial fluid was higher in the SWH (8.8 microg/ml) than the STB (1.6 microg/ml), but there was no correlation between joint damage and collagen concentrations in synovial fluids (10.0 and 1.8 microg/ml in joints with moderate lesions from SWH and STB, respectively). A marked difference in COMP synthesised upon metabolic labelling between the normal and osteoarthritic cartilage was seen and the synthesis of COMP in the articular cartilage of the third carpal bone with moderate articular lesions (from an STB) was lower than in the joint with mild lesions. This difference between breeds may reflect different load characters, in release of macromolecules in osteoarthritic and normal joints. This a novel finding that should be considered in studies of equine traumatic arthritis.

PH-Controlled Macromolecule Encapsulation in and Release from Polyelectrolyte Multilayer Nanocapsules

pH-Controlled encapsulation in and release of macromolecules from polyelectrolyte capsules of a few microns in diameter is demonstrated. Capsules were prepared via alternating adsorption of the oppositely charged polymers poly(allylamine hydrochloride) and poly(styrene sulfonate) onto decomposable melamin formaldehyde cores. The capsules were open for macromolecules at pH values below 6 and closed at pH > 8.

On the release of proteins from degrading dextran methacrylate hydrogels and the correlation with the rheologic properties of the hydrogels.

To study the release of macromolecules of different sizes (bovine serum albumin, immunoglobulin G) from degrading (addition of dextranase) dextran methacrylate (dex-MA) hydrogels and to correlate the release with the evolution of the rehologic properties of the hydrogels during degradation. The size of the macromolecules, the degree of substitution (i.e., number of methacrylates per 100 glycopyranose residues) of the dex-MA and the dextranase concentration in the hydrogels was varied. The rheologic properties were measured with a controlled stress rheometer. The release from dex-MA hydrogels without dextranase was very small [7-20% (time frame up to 180 days)] showing that most of the molecules were entrapped within the hydrogel network. The release from degrading dex-MA hydrogels followed zero-order kinetics for all molecules during a substantial period of the release. This was explained by a liberation and an increasing diffusivity of the proteins in the course of the degradation. The total amount released and the release rates could be well correlated with the rheologically observed degradation rates. It was shown that rheology can be a useful tool to help explain the release from degrading hydrogels.

Controlled release of macromolecules from PLA microspheres: using porous structure topology

Release of hydrophilic macromolecules (FITC-dextran, M w=71 and 2000 kDa) from porous poly ( d, l-lactic acid), PLA, microspheres was studied by applying percolation theory. Microspheres were prepared by the double emulsion method using high molecular weight PLA. The microspheres showed a percolation threshold, ρ C , at porosity 0.34. From this parameter, the effective diffusion coefficients, D eff, and the accessible porosity (total releasable active ingredient), ρ A , of the microspheres were calculated using the Bethe lattice model with coordination number 4. Decreasing porosity of the microspheres decreased the release rate of the active ingredients and a long-term release was observed for the microspheres with porosity close to ρ C . The calculated ρ A agreed with the experimental data and also the calculated D eff for the microspheres with larger porosity (>0.4) was a good estimation to predict the experimentally determined release profiles by applying the continuum structure model (CSM). For microspheres with porosity larger than 0.4 which contained FITC-dextran M w=71 kDa, a lag time was observed, which was attributed to delayed saturation of the microspheres with release medium. For microspheres with porosity close to ρ C , the limited number of exit holes on the exterior surface changed the mechanism of release and controlled the release rate rather than the tortuosity of the porous structure of the microspheres.

Potential usage of thermosensitive liposomes for site-specific delivery of cytokines

Long-circulating liposomes reside long in the bloodstream, and transient swelling during phase transition of liposomes with hyper-osmotic internal aqueous phase causes release of macromolecules. Here we examined the applicability of long-circulating thermosensitive liposomes for delivery of tumor necrosis factor (TNF) by the heating of a local tumor-growing site after injection of TNF-loaded liposomes into tumor-bearing mice. Glucuronate modified thermosensitive liposomes with internal solution of two-fold higher osmotic pressure and sized through 200 nm-pore, released encapsulated [ 131I] human serum albumin at 42°C in vitro and showed long-circulating character in vivo. Cytotoxic action of TNF encapsulated in long-circulating thermosensitive-liposomes (LCTS-liposomes) against L929 fibrosarcoma cells was enhanced at 42°C in vitro. Furthermore, the tumor growth tended to be inhibited more by hyperthermia of mice bearing Meth A sarcoma than without heating after injection of TNF encapsulated in LCTS-liposomes. These results suggest that the cytokine can be released at the tumor site from the circulating CLTS-liposomes.

Biodegradable comb polyesters. Part II. Erosion and release properties of poly(vinyl alcohol)-g-poly(lactic-co-glycolic acid)

Poly(lactic acid) (PLA) and its random copolymers with glycolide (PLG) were modified by grafting onto hydrophilic macromolecular backbones, such as poly(vinyl alcohol) (PVA), to increase both hydrophilicity and to manipulate the polymer structure. The resulting branched PVA-g-PLG offers the possibility to manipulate physico-chemical properties, such as molecular weight and glass transition temperature. The degradation and erosion rates required for continuous release of hydrophilic macromolecules differs significantly from linear PLG. Microspheres were prepared to investigate the release of hydrophilic dextran as a function of polymer structure.A reduction of the poly(lactic-co-glycolic acid) chain lengths in PVA-g-PLG caused a change in erosion profiles from bulk erosion to a surface front erosion mechanism, when the molecular weight of the PLG side chains was below 1000g/mol, which is equal to water-solubility when cleaved from the backbone. Drug release rates from microspheres were significantly influenced by the polymer structure. A reduction of the PLG chain lengths led to increasing erosion controlled release rates, while an increase of the molecular weight of the core PVA resulted in a more diffusion controlled release mechanism. Release profiles could be adjusted over a broad range from ca. 14days to 3months. In combination with the possibility of avoiding accumulation of acidic breakdown products in the delivery device, PVA-g-PLG are of particular interest for parenteral delivery systems containing proteins, peptides or oligonucleotides.

'Tissue' transglutaminase release from apoptotic cells into extracellular matrix during human liver fibrogenesis.

Enhanced apoptosis characterizes several pathologies affecting human liver. This study sought to determine whether apoptosis is involved in the formation of fibrotic lesions occurring in hepatic disease. The expression of Bcl-2 was analysed, and of 'tissue' transglutaminase (tTG), a cross-linking enzyme which recent evidence suggests plays a role in the formation of fibrotic lesions in experimental settings. Regardless of the degree of liver injury, tTG abnormally accumulated in the liver cells adjacent to fibrotic tissue. Many cells showing DNA fragmentation and morphological features of apoptosis were also observed near fibrotic lesions. Bcl-2 was detected predominantly in infiltrating lymphocytes within the liver tissue. Marked staining for both tTG protein and chromatin was also observed in the acellular fibrotic tissue, which suggested an active release of intracellular macromolecules from the dying cells into the extracellular matrix. This study indicates that fibrogenesis in the liver is associated with the release of tTG from dying cells. By cross-linking extracellular matrix proteins, this enzyme might play a role in the formation of fibrotic lesions.

SERUM MARKERS OF ARTICULAR CARTILAGE DAMAGE AND REPAIR

Joint cartilage is a dynamic tissue that reacts to trauma, inflammation, and other insults by attempting to repair its matrix. This reaction results in the release of cartilage macromolecules into the body fluids. Analysis of these fluids has identified a limited number of at least somewhat tissue-specific markers of altered cartilage metabolism. Analyses of serum are less specific and less sensitive than analyses of synovial fluid, but their use as research tools in clinical studies, drug development, and experimental work in animal models is increasing.

Degradation of porous poly(anhydride- co-imide) microspheres and implications for controlled macromolecule delivery

The degradation properties of porous microspheres made using a new family of polyanhydride copolymers, the poly(anhydride- co-imides), were studied. Poly[trimellitylimido- l-tyrosine- co-sebacic acid- co-1,3-bis(carboxyphenoxy)propane] microspheres, with and without entrapped bovine serum albumin (BSA) as a model protein, were made using the double emulsion solvent evaporation process. Water penetration and anhydride bond cleavage (polymer degradation) occurred rapidly (<5 days) compared to the time scale of overall microsphere erosion (weeks to months) with most polymer compositions. Subsequent to bond cleavage, the ultimate erosion of the microsphere and release of entrapped BSA was due mainly to the slow dissolution of the individual hydrophobic monomers (TMA-Tyr, SA and CPP) from the microsphere surface. BSA was released at approximately the same rate as the polymer eroded. Due to the fast degradation of anhydride bonds relative to microsphere erosion, initial polymer molecular weight did not have a significant effect on macromolecule release rates. Instead, monomer solubility correlated well with polymer erosion and BSA release rates. This erosion mechanism leads to predictable drug release rates which may be appropriate for the delivery of many protein therapeutics, including vaccine antigens. The anhydride–imide copolymers were well tolerated in acute toxicity studies in rats and therefore show promise as biomaterials.

Desorption of organic macromolecules from activated sludge: Effect of ionic composition

Structural properties of activated sludge flocs were found to be sensitive to small changes in ionic strength and ionic composition. Removal of calcium from the bulk water by an ion exchange resin resulted in a release of calcium ions and organic macromolecules from the flocs. Dilution of the activated sludge with deionized water resulted in a release of calcium and organics to bulk water as well. In both types of experiments the small particles in the bulk water showed an increase in the negative surface charge density, as measured by zeta-potential or by colloidal titration. A change in the repulsive forces due to calcium desorption were possibly more important for the observed floc disintegration than were the effects of changes in the ionic strength. The kinetics of dissolution of calcium were shown to be controlled by Fickian diffusion. A model of a typical activated sludge floc, taking into account the colloidal stability, is suggested, where a “cloud” of organic macromolecules and single bacteria are attached with very weak forces to a more rigid backbone consisting of fibers, filamentous bacteria and bacterial colonies. The observed changes in stability of the flocs leading to increased turbidity have major practical implications in the solid-liquid separation, and effects on effluent quality and dewatering are discussed.

Controlled protein delivery from biodegradable tyrosine-containing poly(anhydride-co-imide) microspheres

Polymer microspheres capable of the controlled release of macromolecules for periods ranging from days to over a month were developed. The microspheres were made using a new family of anhydride polymers: tyrosine-containing poly(anhydride-co-imides), specifically poly[trimellitylimido- l-tyrosineco-sebacic acid-co-1,3-bis(carboxyphenoxy)propane] anhydrides [poly(TMA-Tyr:SA:CPP)]. These polymers may be of particular interest for controlled delivery of vaccine antigens due to the incorporation of an immunological adjuvant, l-tyrosine, into their backbone. Microspheres were produced from a variety of polymer compositions using a double-emulsion solvent-evaporation technique, and tested for their ability to provide controlled release of a model protein, bovine serum albumin, in vitro. The microspheres are spherical with smooth surfaces and encapsulate greater than 70% of the protein. Protein release rates from polymers of identical composition could be varied from 0.3 to over 125 μg per mg spheres per month by changing the amount of protein encapsulated. This effect can be magnified by using polymers with various monomer ratios. A close correlation between protein release and polymer weight loss was observed, suggesting a release mechanism controlled mainly by polymer erosion. Bovine serum albumin release from poly(TMA-Tyr:SA:CPP) microspheres is also pH sensitive, being enhanced at high pH and depressed under acidic conditions.

Controlled macromolecule release from poly(phosphazene) matrices

Hydrolytically unstable poly(phosphazene) PPHOS matrices with 50% ethyl glycinato/50% p-methylphenoxy substitution were investigated as vehicles for the controlled release of macromolecules. Specifically, the effects of matrix pH environment and macromolecule loading were studied. 14C-labeled inulin was incorporated into matrices by a solvent casting technique at 1, 10 and 40% loadings (w/w). Degradation and release studies were performed at 37°C at pH 2.0, 7.4 and 10.0. The PPHOS polymer degraded relatively slowly in neutral and basic solutions (pH 7.0 and pH 10.0). In contrast, significantly ( p < 0.01) higher levels of degradation were seen in acidic solutions (pH 2.0) after 35 days. The presence of the hydrophilic macromolecule inulin in the polymer matrix resulted in increased degradation of PPHOS with time. Inulin release, like polymer degradation, was highest at pH 2.0 followed by pH 10.0 and 7.4. Inulin release appeared to be dependent on polymer degradation and inulin diffusion. High inulin loading increased the levels of initial drug burst release and resulted in higher levels of ultimate drug release as measured at 25 days. Environmental scanning electron microscopy (ESEM) demonstrated smooth surfaces on matrices without drug, rough and granular surfaces on matrices loaded with inulin before release, and surfaces possessing micropores and macropores after inulin loading and release. PPHOS polymers can predictably release macromolecules such as inulin. Release can be modulated through changes in pH environment and drug loading.

Macroporous poly(sucrose acrylate) hydrogel for controlled release of macromolecules

We have studied the controlled release of proteins from poly(sucrose acrylate) hydrogels. The hydrogels were prepared by a two-step procedure in which sucrose was first acylated to sucrose-1′-acrylate followed by free radical polymerization. By adjusting the cross-link ratio and initial monomer concentration, the swelling ratio of the hydrogel was varied from five to 28. The mechanical strength of these hydrogels was comparable to that of other hydrogels with approximately the same swelling ratio. Scanning electron micrographs and mesh size calculations indicate that the hydrogel is macroporous, suggesting it may be suitable for a variety of biomedical applications. The release kinetics of β-lactoglobulin, bovine serum albumin and γ-globulin were studied as a function of initial monomer concentrations for the sucrose-based hydrogel. All of the release profiles were characterized by an initial burst of protein in the first 25 h followed by a long period of sustained release (s> 500 h). The magnitude of the initial burst was reduced by increasing the initial monomer concentration and by increasing the molecular weight of the protein. A quantitative model based on the heterogeneous nature of the hydrogel was developed to explain the observed release kinetics.

Recombinant Expression of Hepatitis A Virus Protein 3A: Interaction with Membranes

The function of hepatitis A virus (HAV) protein 3A and its structural requirements were studied in vitro and in a bacterial expression system by comparing the polypeptide precursor 3AB derived from a cytopathogenic strain with that of an attenuated strain. Although the precursor polypeptides 3AB of both HAV strains bind to microsomal membranes after translation in vitro they differ in inducing membrane permeability when expression is induced in bacteria. Intake and release of macromolecules was dramatically increased when 3AB of the cytopathogenic strain was expressed. Amino acid sequence alignments suggest that membrane binding might be due to a hydrophobic stretch near the C-terminus of 3A found in all picornaviruses whereas the ability to induce permeability of E. coli membranes is determined by an amphipathic helix formed at the N-terminus of 3A of HAV FG.