Florence Edwards-Levy

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.

Arteriogenic Therapy by Intramyocardial Sustained Delivery of a Novel Growth Factor Combination Prevents Chronic Heart Failure

Background— Therapeutic angiogenesis is a promising approach for the treatment of cardiovascular diseases, including myocardial infarction and chronic heart failure. We aimed to improve proangiogenic therapies by identifying novel arteriogenic growth factor combinations, developing injectable delivery systems for spatiotemporally controlled growth factor release, and evaluating functional consequences of targeted intramyocardial growth factor delivery in chronic heart failure. Methods and Results— First, we observed that fibroblast growth factor and hepatocyte growth factor synergistically stimulate vascular cell migration and proliferation in vitro. Using 2 in vivo angiogenesis assays (n=5 mice per group), we found that the growth factor combination results in a more potent and durable angiogenic response than either growth factor used alone. Furthermore, we determined that the molecular mechanisms involve potentiation of Akt and mitogen-activated protein kinase signal transduction pathways, as well as upregulation of angiogenic growth factor receptors. Next, we developed crosslinked albumin-alginate microcapsules that sequentially release fibroblast growth factor-2 and hepatocyte growth factor. Finally, in a rat model of chronic heart failure induced by coronary ligation (n=14 to 15 rats per group), we found that intramyocardial slow release of fibroblast growth factor-2 with hepatocyte growth factor potently stimulates angiogenesis and arteriogenesis and prevents cardiac hypertrophy and fibrosis, as determined by immunohistochemistry, leading to improved cardiac perfusion after 3 months, as shown by magnetic resonance imaging. These multiple beneficial effects resulted in reduced adverse cardiac remodeling and improved left ventricular function, as revealed by echocardiography. Conclusion— Our data showing the selective advantage of using fibroblast growth factor-2 together with hepatocyte growth factor suggest that this growth factor combination may constitute an efficient novel treatment for chronic heart failure.

Flow of artificial microcapsules in microfluidic channels: A method for determining the elastic properties of the membrane

The paper deals with a method to characterize the membrane mechanical properties of microcapsules. The technique consists in flowing microcapsules into a microchannel of comparable dimensions, observing the deformation as a function of the flow rate, and deducing the membrane elastic modulus by means of an inverse method based on a numerical model of the flowing capsule. The method is tested on liquid-filled microcapsules (average diameter of 67 μm) with a membrane made of crossed-linked ovalbumin flowing inside a cylindrical channel. For a neo-Hookean constitutive law, the method yields a constant value for the membrane shear elastic modulus independently of capsule size or deformation. When the capsules are flowed into a square-section microchannel, an approximate analysis of the deformation yields the same value of the membrane shear modulus provided that the size ratio between the capsule and the channel is of order unity.

Determination of free amino group content of serum albumin microcapsules using trinitrobenzenesulfonic acid: effect of variations in polycondensation pH

Microcapsules were prepared from human serum albumin (HSA) through an interfacial cross-linking process using terephthaloyl chloride at various pH values (5.9–11). Determination of free amino groups was performed on lyophilized microcapsules by means of a back titration method using trinitrobenzenesulfonic acid (TNBS). Increasing reaction pH was shown to result in a progressive decrease of microcapsule free amino groups. Moreover, two groups of microcapsules could be distinguished with respect to their -NH2 content: those obtained at low pH values (8 and < 8) which contained more than 400 μmolg dry weight, and those prepared from pH 9, whose -NH2 content did not exceed 110 μmolg dry weight. These results were compared with previous findings concerning the involvement of hydroxy and carboxy groups of HSA microcapsules as a function of pH, that had been obtained from FT-IR spectroscopic studies.

Selective Stimulation of Cardiac Lymphangiogenesis Reduces Myocardial Edema and Fibrosis Leading to Improved Cardiac Function Following Myocardial Infarction.

Background— The lymphatic system regulates interstitial tissue fluid balance, and lymphatic malfunction causes edema. The heart has an extensive lymphatic network displaying a dynamic range of lymph flow in physiology. Myocardial edema occurs in many cardiovascular diseases, eg, myocardial infarction (MI) and chronic heart failure, suggesting that cardiac lymphatic transport may be insufficient in pathology. Here, we investigate in rats the impact of MI and subsequent chronic heart failure on the cardiac lymphatic network. Further, we evaluate for the first time the functional effects of selective therapeutic stimulation of cardiac lymphangiogenesis post-MI. Methods and Results— We investigated cardiac lymphatic structure and function in rats with MI induced by either temporary occlusion (n=160) or permanent ligation (n=100) of the left coronary artery. Although MI induced robust, intramyocardial capillary lymphangiogenesis, adverse remodeling of epicardial precollector and collector lymphatics occurred, leading to reduced cardiac lymphatic transport capacity. Consequently, myocardial edema persisted for several months post-MI, extending from the infarct to noninfarcted myocardium. Intramyocardial-targeted delivery of the vascular endothelial growth factor receptor 3–selective designer protein VEGF-CC152S, using albumin-alginate microparticles, accelerated cardiac lymphangiogenesis in a dose-dependent manner and limited precollector remodeling post-MI. As a result, myocardial fluid balance was improved, and cardiac inflammation, fibrosis, and dysfunction were attenuated. Conclusions— We show that, despite the endogenous cardiac lymphangiogenic response post-MI, the remodeling and dysfunction of collecting ducts contribute to the development of chronic myocardial edema and inflammation-aggravating cardiac fibrosis and dysfunction. Moreover, our data reveal that therapeutic lymphangiogenesis may be a promising new approach for the treatment of cardiovascular diseases.

Cross-linked β-cyclodextrin microcapsules : preparation and properties

Abstract Microcapsules were prepared by interfacial cross-linking of β-cyclodextrins (β-CD) with terephthaloyl chloride (TC). Batches were prepared from β-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 β-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% β-CD solutions. The mean size of all batches was in the 10–35 μm range. The IR spectrum showed bands at 1724, 1280 and 731 cm −1 , reflecting the formation of esters. The β-CD contents were 46, 56–58 or 60–66% for batches prepared from 5, 7.5 or 10% β-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 μmol/g microcapsules) was observed for small-sized particles (≈11 μm) prepared from a 7.5% β-CD solution. The method then appears as a simple and rapid procedure to provide stable microcapsules, having an interesting guest-binding ability.

Cross-linked β-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.

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.

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.

Free amino group content of serum albumin microcapsules. III. A study at low pH values

Abstract Microcapsules were prepared by interfacial cross-linking of human serum albumin (HSA) with terephthaloyl chloride (TC). Determination of free amino groups was performed on lyophilized microcapsules with trinitrobenzene-sulfonic acid (TNBS). In two series of assays conducted with phosphate buffers, pH 7.4 and 8, respectively, microcapsules exhibited a high −NH 2 content (> 330 μmol/g dry weight), with only a slight decrease when raising TC concentration from 2.5 to 5% TC (w/v) and/or reaction time from 30 to 60 min. When the phosphate buffer, pH 7.4 (ionic strength (μ): 0.40), was replaced by an acetate buffer, pH 7.4 (μ: 0.66), supplemented with 0.125 or 0.25 M CaCl 2 , microcapsule −NH 2 content was reduced from 346 to 134 and 168 μmol/g, respectively. Control batches prepared with the acetate buffer without adding CaCI 2 also had a reduced −NH 2 content (182, μmol/g), while a higher value (302 μmol/g) was found for microcapsules prepared using a phosphate buffer with a 0.66 ionic strength. Finally, a sharp decrease in −NH 2 , content was also found for microcapsules prepared from acetate buffers at pH 5.9 (174 μmol/g) and 6.8 (158 μmol/g), as compared with microcapsules prepared from phosphate buffers at the same pH values (438 and 388 μmol/g, respectively).