Chronic delivery of α-melanocyte-stimulating hormone in rat hypothalamus using albumin-alginate microparticles: effects on food intake and body weight.

Chronic delivery of neuropeptides in the brain is a useful experimental approach to study their long-term effects on various biological parameters. In this work, we tested albumin-alginate microparticles, as a potential delivery system, to study if continuous release in the hypothalamus of α-melanocyte-stimulating hormone (α-MSH), an anorexigenic neuropeptide, may result in a long-term decrease in food intake and body weight. The 2-week release of α-MSH from peptide-loaded particles was confirmed by an in vitro assay. Then, daily food intake and body weight were studied for 18 days in rats injected bilaterally into the paraventricular hypothalamic nucleus with particles loaded or not with α-MSH. A decrease in body weight gain, persisting throughout the study, was found in rats injected with α-MSH-charged particles as compared with rats receiving non-charged particles and with rats injected with the same dose of α-MSH in solution. Food intake was significantly decreased for 3 days in rats receiving α-MSH-loaded particles and it was not followed by the feeding rebound effect which appears after food restriction. The presence of α-MSH-loaded particles in the hypothalamus was confirmed by immunohistochemistry. In conclusion, our study validates albumin-alginate microparticles as a new carrier system for long-term delivery of neuropeptides in the brain and demonstrates that chronic delivery of α-MSH in the hypothalamus results in a prolonged suppression of food intake and a decrease of body weight gain in rats.

Comparison between measurements of elasticity and free amino group content of ovalbumin microcapsule membranes: discrimination of the cross-linking degree.

An inverse method is used to characterize the membrane mechanical behavior of liquid filled microcapsules. Cross-linked ovalbumin microcapsules are flowed and deformed into a cylindrical microchannel of comparable size. The deformed shape is compared to predictions obtained numerically when modeling a capsule under the same flow conditions. The unknown shear modulus value corresponds to the best fit. The degree of reticulation is estimated in parallel by determining the free amino groups remaining on the microcapsules after the cross-linking reaction. We characterize microcapsule populations fabricated at different reaction pH (5-8) and times (5-30 min) to study different cross-linking degrees. The capsule shear modulus and the amino groups are nearly constant with the reaction pH for the capsules fabricated after 5 min of reticulation. The shear modulus increases with the reaction time, while the NH(2) content decreases with it. A global increase in shear modulus with pH is also observed, together with an unexpected increase in NH(2) content. The study shows that the inverse method is capable of discriminating between various cross-linking degrees of microcapsules. Moreover, for this type of microcapsules, the mechanical method appears more reliable than the chemical one to obtain an estimation of their cross-linking degree.

Preparation and characterization of an electrodeposited calcium phosphate coating associated with a calcium alginate matrix.

A new way of optimizing osteoconduction of biomaterials is to bring to them biological properties. In this work, we associated a novel release system with an electrodeposited calcium phosphate (CaP) coated titanium alloy Ti6Al4V. The characterization of this material was performed by means of light microscopy, scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM) and X-ray energy dispersive spectroscopy (EDXS). The electrodeposited CaP coating was a tricalcium phosphate, and the release system was composed of microcapsules entrapped in an alginate film. We observed that the alginate matrix had a close contact with the coating. An intermediate layer containing calcium and phosphorus appeared at the interface between the alginate matrix and the CaP coating. These results allowed us to conclude that the association of two techniques, i.e. electrodeposition followed by deposition of a calcium alginate matrix, led to the elaboration of a new biomaterial.

Rates of transport through a capsule membrane to attain Donnan equilibrium.

The swelling of a capsule consisting of salt solution and polyelectrolyte, surrounded by a membrane, is studied. The membrane allows salt and water to pass, but is impermeable to polyelectrolyte molecules. Equilibrium swelling of the capsule is governed by Donnan equilibrium. Transport rates of a salt and water through the membrane are expressed in terms of a Darcy permeability and a salt diffusivity. The governing equations predict that the rate at which equilibrium is attained as the external salt concentration varies is controlled by the timescale for diffusion of salt, rather than by that for Darcy flow. Experiments were performed using capsules with membranes made of covalently linked HSA and alginate. The capsule volume varied with a single relaxation rate when the external salt concentration was changed, as predicted by theory. This constitutes the first step toward a simple method for determining the membrane properties of capsules by measuring rates of change of capsule volume.

Cross-linked beta-cyclodextrin microcapsules. II. Retarding effect on drug release through semi-permeable membranes.

Microcapsules were prepared by interfacial cross-linking of beta-cyclodextrins (beta-CD) with terephthaloylchloride (TC) as described previously. Complexation assays were conducted with propranolol HCl. After 1 h incubation of 50 mg lyophilized microcapsules in 10 ml propranolol solution, the amounts of fixed drug were 507.5+/-8.6 micromol and 811.2+/-16.0 micromol per g lyophilized microcapsules with 1 mM and 2 mM solutions, respectively. A dialysis experiment was then performed. After 1 h incubation of microcapsules (10 or 50 mg) in 10 ml of 2 mM propranolol solution, the suspension was dialysed against a phosphate buffer pH 7.4 at 37 degrees C. The drug diffusion was all the more retarded that the amount of added beta-CD microcapsules was higher. Finally, double microcapsules were prepared using a suspension of beta-CD microcapsules (10-100 mg) in a solution of methylene blue in an acetate buffer pH 7.4. After adding human serum albumin (HSA), the suspension was emulsified in cyclohexane and double microcapsules were obtained by cross-linking the HSA with TC. In vitro release studies showed that the incorporation of beta-CD microcapsules resulted in a decrease in release rate of methylene blue, the decrease being related to the amount of encapsulated beta-CD microcapsules. The study then suggests interesting applications of beta-CD microcapsules for modulating the release rate of drugs through semi-permeable membranes.

Cross-linked beta-cyclodextrin microcapsules: preparation and properties.

Microcapsules were prepared by interfacial cross-linking of beta-cyclodextrins (beta-CD) with terephthaloyl chloride (TC). Batches were prepared from beta-CD solutions in 1 M NaOH, using 5% TC and a 30 min reaction time. Microcapsules were studied with respect to morphology (microscopy), size (laser diffraction technique) and, for selected batches, IR spectroscopy, determination of beta-CD content (polarimetry after alkaline dissolution of microcapsules) and complexing properties, evaluated using p-nitrophenol (pNP) as the guest molecule. Well-formed microcapsules were obtained from 5, 7.5, and 10% beta-CD solutions. The mean size of all batches was in the 10-35 microm range. The IR spectrum showed bands at 1724, 1280 and 731 cm(-1), reflecting the formation of esters. The beta-CD contents were 46, 56-58 or 60-66% for batches prepared from 5, 7.5 or 10% beta-CD solutions, respectively. The experiments conducted with 1 mM pNP showed a rapid complexation reaching a maximum within 1 h. When incubating 50 mg lyophilized microcapsules in 10 ml pNP solution, the maximal fixation (97.8 micromol/g microcapsules) was observed for small-sized particles ( approximately 11 microm) prepared from a 7.5% beta-CD solution. The method then appears as a simple and rapid procedure to provide stable microcapsules, having an interesting guest-binding ability.

Serum albumin-alginate coated beads: mechanical properties and stability.

According to a previously described method, alginate beads were prepared from a Na-alginate solution containing propylene glycol alginate (PGA) and human serum albumin (HSA). The solution was added dropwise to a CaCl2 solution. The beads were treated with NaOH, which started the formation of amide bonds between HSA and PGA at the periphery, giving a membrane. Batches of beads with increasingly thick membranes were prepared using growing concentrations of NaOH, and studied with a texture analyser. When raising NaOH concentration, the rupture strength progressively increased, and the resistance strength to a deformation of 50% of total height also increased before slightly decreasing for the highest NaOH concentration. Variations of bead elasticity were also observed. When the beads were prepared with saline reducing gelation time from 10 to 5 min, and reaction time from 15 to 5 min, mechanical properties varied more progressively with the NaOH concentration, while the results became more reproducible. A series of assays conducted with 0.01 M NaOH confirmed the importance of using a short gelation time, and saline rather than water. Stability assays were also performed. The results were compared to those of alginate-polylysine coated beads and showed the interest of the transacylation method.

In-vivo delivery of therapeutic proteins by genetically-modified cells: comparison of organoids and human serum albumin alginate-coated beads.

We have designed a self-assembling multimeric soluble CD4 molecule by inserting the C-terminal fragment of the alpha chain of human C4-binding protein (C4bp alpha) at the C-terminal end of human soluble CD4 genes. This CD4-C4bp alpha fusion protein (sMulti-CD4) and two other reference molecules, a fusion protein of human serum albumin (HSA) and the first two domains of CD4 (HSA-CD4) and monomeric soluble CD4 (sMono-CD4), were delivered in vivo by genetically modified 293 cells. These cells were implanted in mice as organoids and also encapsulated in HSA alginate-coated beads. sMulti-CD4 showed an apparent molecular weight of about 300-350 kDa, in accordance with a possible heptamer formula. sMulti-CD4 produced either in cell culture or in vivo in mice appeared to be a better invitro inhibitor of HIV infection than sMono-CD4. Plasma levels of sMulti-CD4, HSA-CD4, and sMono-CD4 reached approximately 2,300, 2,700, and 170 ng/mL, respectively, 13 weeks after in-vivo organoid implantation, which had formed tumours at that time. This suggests that the plasma half-life of sMulti-CD4 is much longer than that of sMono-CD4. The 293 xenogeneic cells encapsulated in HSA alginate-coated beads remained alive and kept secreting sMono-CD4 or HSA-CD4 continuously at significant levels for 18 weeks in nude mice, without tumour formation. When implanted in immunocompetent Balb/c mice, they were rejected two to three weeks after implantation. In contrast, encapsulated BL4 hybridoma cells remained alive and kept secreting BL4 anti-CD4 mAb for at least four weeks in Balb/c mice. These results suggest the clinical potential of the C4bp-multimerizing system, which could improve both the biological activity and the poor in-vivo pharmacokinetic performance of a monomeric functional protein like soluble CD4. These data also show that a systemic delivery of therapeutic proteins, including immunoglobulins, can be obtained by the in-vivo implantation of engineered allogeneic cells encapsulated in HSA alginate-coated beads.

Survival, proliferation, and functions of porcine hepatocytes encapsulated in coated alginate beads: a step toward a reliable bioartificial liver.

Orthotopic liver transplantation is the most effective treatment for fulminant hepatic failure. As an alternative treatment, an efficient extracorporeal bioartificial liver should contain a large yield of functional hepatocytes with an immunoprotective barrier, for providing temporary adequate metabolic support to allow spontaneous liver regeneration or for acting as a bridge toward transplantation. Survival, proliferation, and functions of porcine hepatocytes were evaluated in primary cultures and after embedding in alginate beads, which were subsequently coated with a membrane made by a transacylation reaction between propylene glycol alginate and human serum albumin. Disruption of total pig livers by collagenase perfusion/recirculation allowed the obtention of up to 10(11) hepatocytes with a viability greater than 95%. Hepatocytes in conventional cultures or embedded in coated alginate beads survived for about 10 days, secreted proteins, particularly albumin, and maintained several phase I and II enzymatic activities, namely ethoxyresorufin-O-deethylase, oxidation of nifedipine to pyridine, phenacetin deethylation to paracetamol, glucuroconjugation of paracetamol, and N-acetylation of procainamide. Typical features of mitosis and [3H]thymidine incorporation indicated that porcine hepatocytes proliferated in both conventional cultures and alginate beads. The efficacy of the membrane surrounding alginate beads for protecting cells from immunoglobulins was tested by embedding HLA-typed human lymphocytes, which were subsequently incubated with specific anti-HLA immunoglobulin G and complement. These data show that large yields of porcine hepatocytes that are embedded in coated alginate beads remain functional and are isolated from large molecular weight molecules, such as immunoglobulins. This system represents a promising tool for the design of an extracorporeal bioartificial liver, containing xenogeneic hepatocytes, to treat acute liver disease in humans.

Coating alginate beads with cross-linked biopolymers: a novel method based on a transacylation reaction.

Stable membranes were formed around alginate beads using a transacylation reaction between polysaccharidic esters, namely propylene glycol alginate (PGA) or pectin, and various proteins (human serum albumin (HSA), ovalbumin, bovine hemoglobin, lactoserum proteins). In a standard procedure, two reagents (PGA or pectin+protein) were added to a Na-alginate solution: beads were formed by dropwise addition into a calcium solution. Then the transacylation reaction was started by alkalinization of the bead suspension. A membrane was formed around the beads, made of a protein directly bound to a polysaccharide through amide linkages. The thickness of the membranes and the lysis time in trypsin were increased by raising the amount of NaOH used for the transacylation step. In a modified procedure, coated beads were obtained, incorporating PGA in the initial Na-alginate solution, and HSA in the transacylation bath. Activated charcoal was encapsulated in HSA-PGA beads, giving particles with adsorption properties towards creatinine. Assays were performed using PGA associated with alkaline phosphatase as the membrane-forming protein. Stable beads were obtained having a relative activity of 39.3%, as compared with free enzyme.