Smadar Cohen

Controlled Delivery Systems for Proteins Based on Poly(Lactic/Glycolic Acid) Microspheres

This paper describes an investigation of the use of poly(lactic/glycolic acid) polymers for long-term delivery of high molecular weight, water-soluble proteins. Poly(lactic/glycolic acid) (PLGA) microspheres, containing (fluorescein isothiocyanate)-labeled bovine serum albumin and (fluorescein isothiocyanate)-labeled horseradish peroxidase, were prepared by a modified solvent evaporation method using a double emulsion. The microspheres were spherical with diameters of 55–95 µm and encapsulated more than 90% of the protein. The preparation method was gentle and maintained enzyme activity and protein solubility. Stability studies showed that the encapsulation of an enzyme inside PLGA microspheres can protect them from activity loss. When not placed inside PLGA microspheres, (fluorescein isothiocyanate)-labeled horseradish peroxidase lost 80% of its activity in solution at 37°C in a few days, whereas inside the PLGA microspheres it retained more than 55% of its activity after 21 days of incubation at 37°C. In vitro release studies revealed that different release profiles (i.e., near-constant or biphasic) and release rates can be achieved by simply modifying factors in the preparation procedure such as mixing rate and volume of inner water and organic phases. Degradation studies by scanning electron microscopy and gel-permeation chromatography suggested that the mechanism responsible for protein release is mainly through matrix erosion.

Novel alginate sponges for cell culture and transplantation

This paper describes the preparation and characterization of a three-dimensional, porous sponge made from the marine polysaccharide alginate for creating a cell-matrix transplant to replace damaged organs or tissues. The sponge is prepared by a three-step procedure: first gelation of the alginate with bivalent cations, followed by freezing of the hydrogel and finally lyophilization to produce a porous sponge. The pattern and the extent of sponge porosity, as well as its mechanical properties, were influenced by the concentration and the type of alginate (guluronic to mannuronic ratio and viscosity), the type and concentration of the cross-linkers and the freezing regime. By controlling these variables, macroporous sponges (pore size of 70-300 microns) that are suitable for cell culture and neovascularization were achieved. Fibroblasts seeded within the sponges preferred the pores, where they maintained a spherical shape. The alginate sponges conserved their initial volume for at least 3 months. It appears that alginate sponges may provide an excellent support for cell transplantation.

Effect of Injectable Alginate Implant on Cardiac Remodeling and Function After Recent and Old Infarcts in Rat

Background— Adverse cardiac remodeling and progression of heart failure after myocardial infarction are associated with excessive and continuous damage to the extracellular matrix. We hypothesized that injection of in situ-forming alginate hydrogel into recent and old infarcts would provide a temporary scaffold and attenuate adverse cardiac remodeling and dysfunction. Methods and Results— We developed a novel absorbable biomaterial composed of calcium-crosslinked alginate solution, which displays low viscosity and, after injection into the infarct, undergoes phase transition into hydrogel. To determine the outcome of the biomaterial after injection, calcium-crosslinked biotin-labeled alginate was injected into the infarct 7 days after anterior myocardial infarction in rat. Serial histology studies showed in situ formation of alginate hydrogel implant, which occupied up to 50% of the scar area. The biomaterial was replaced by connective tissue within 6 weeks. Serial echocardiography studies before and 60 days after injection showed that injection of alginate biomaterial into recent (7 days) infarct increased scar thickness and attenuated left ventricular systolic and diastolic dilatation and dysfunction. These beneficial effects were comparable and sometimes superior to those achieved by neonatal cardiomyocyte transplantation. Moreover, injection of alginate biomaterial into old myocardial infarction (60 days) increased scar thickness and improved systolic and diastolic dysfunction. Conclusions— We show for the first time that injection of in situ-forming, bioabsorbable alginate hydrogel is an effective acellular strategy that prevents adverse cardiac remodeling and dysfunction in recent and old myocardial infarctions in rat.

Hepatocyte behavior within three-dimensional porous alginate scaffolds

A potential approach to facilitate the performance of implanted hepatocytes is to enable their aggregation and re-expression of their differentiated function prior to implantation. Here we examined the behavior of freshly isolated rat adult hepatocytes seeded within a novel three-dimensional (3-D) scaffold based on alginate. The attractive features of this scaffold include a highly porous structure (sponge-like) with interconnecting pores, and pore sizes with diameters of 100-150 microm. Due to their hydrophilic nature, seeding hepatocytes onto the alginate sponges was efficient. DNA measurements showed that the total cell number within the sponges did not change over 2 weeks, indicating that hepatocytes do not proliferate under these culture conditions. Nearly all seeded cells maintained viability, according to the MTT assay. Within 24 h post-seeding, small clusters of viable cells, were seen scattered within the sponge. More than 90% of the seeded cells participated in the aggregation; the high efficiency is attributed to the non-adherent nature of alginate. The spheroids had smooth boundaries and by day 4 in culture reached an average diameter of 100 microm, which is at the same magnitude of the sponge pore size. The cells appeared to synthesize fibronectin which was deposited on the spheroids. No laminin or collagen type IV were detected in the deposit. The 3-D arrangement of hepatocytes within the alginate sponges promoted their functional expression; within a week the cells secreted the maximal albumin secretion rate of 60 microg albumin/10(6) cells/day. Urea secretion rate did not depend on cell aggregation and was similar to that obtained when hepatocytes were cultured on collagen type I coated dishes (100 microg/10(6) cells/day). Our studies show that alginate sponges can provide a conducive environment to facilitate the performance of cultured hepatocytes by enhancing their aggregation.

Prevascularization of cardiac patch on the omentum improves its therapeutic outcome

The recent progress made in the bioengineering of cardiac patches offers a new therapeutic modality for regenerating the myocardium after myocardial infarction (MI). We present here a strategy for the engineering of a cardiac patch with mature vasculature by heterotopic transplantation onto the omentum. The patch was constructed by seeding neonatal cardiac cells with a mixture of prosurvival and angiogenic factors into an alginate scaffold capable of factor binding and sustained release. After 48 h in culture, the patch was vascularized for 7 days on the omentum, then explanted and transplanted onto infarcted rat hearts, 7 days after MI induction. When evaluated 28 days later, the vascularized cardiac patch showed structural and electrical integration into host myocardium. Moreover, the vascularized patch induced thicker scars, prevented further dilatation of the chamber and ventricular dysfunction. Thus, our study provides evidence that grafting prevascularized cardiac patch into infarct can improve cardiac function after MI.

Tailoring the pore architecture in 3-D alginate scaffolds by controlling the freezing regime during fabrication.

The pore architecture in 3-D polymeric scaffoldings plays a critical role in tissue engineering as it provides the framework for the seeded cells to organize into a functioning tissue. In the present paper, we investigate the effect of freezing regime on the pore microstructure in 3-D alginate scaffolds, fabricated by the freeze-dry method. The scaffolds have shown isotropic pore structure, when the calcium crosslinked alginate solutions were slowly frozen at -20 degrees C, in a nearly homogenous cold atmosphere; the pores were spherical and interconnected. In contrast, when the cooling process was performed in liquid nitrogen or oil bath, where a temperature gradient was formed along the freezing solution, two main regions of pore structure were noted; at the interface with the cooling medium, small spherical pores were seen and above them a region with elongated pores. The different pore shape affected the compressibility of the scaffolds, while it had no effect on albumin diffusion. Rat hepatocytes seeded within the scaffolds were arranged according to the their pore shape. In scaffolds with elongated pores, the cells were lining along the pores, thus forming lines of interacting cells. In the scaffolds with the isotropic spherical pores, the hepatocytes clustered into spheroid-like aggregates. Thus, it appears that pore shape can modulate hepatocyte morphogenesis.

A NOVEL IN SITU-FORMING OPHTHALMIC DRUG DELIVERY SYSTEM FROM ALGINATES UNDERGOING GELATION IN THE EYE

Poor bioavailability of ophthalmic solutions caused by dilution and drainage from the eye can be overcome by using in-situ-forming ophthalmic drug delivery systems prepared from polymers that exhibit reversible liquid-gel phase transition. In the present paper, we demonstrate that an aqueous solution of sodium alginate can gel in the eye, without the addition of external calcium ions or other bivalent/polyvalent cations. The extent of alginate gelation and consequently the release of pilocarpine, depended on the percent guluronic acid (G) residues in the polymer backbone. Alginates with G contents of more than 65%, such as Manugel DMB, instantaneously formed gels upon their addition to simulated lacrimal fluid, while those having low G contents, such as Kelton LV, formed weak gels at a relatively slow rate. In vitro studies indicated that pilocarpine is released slowly from alginate gels, over a period of 24 h, and the release occurs mostly via diffusion from the gels. Dissolution of the hydrogels in the releasing media was negligible for the first 12 h of incubation at 37°C. Intraocular pressure (IOP) measurements of rabbit eyes treated with 2% (w/v) pilocarpine nitrate in solution, or in the in-situ gel forming formulation composed of the high G content DMB alginate, indicated that DMB significantly extended the duration of the pressure reducing effect of pilocarpine, to 10 h, as compared to the 3 h when pilocarpine nitrate was delivered as a solution. In contrast, there was no apparent difference in the duration and extent of IOP decrease between rabbits treated with pilocarpine in solution or in the Kelton LV alginate eye drop formulations. The overall results of this study indicate that the in situ-gelling alginate system, based on polymers with high G contents, is an excellent drug carrier for the prolonged delivery of pilocarpine.

Intracoronary injection of in situ forming alginate hydrogel reverses left ventricular remodeling after myocardial infarction in Swine.

OBJECTIVES This study sought to determine whether alginate biomaterial can be delivered effectively into the infarcted myocardium by intracoronary injection to prevent left ventricular (LV) remodeling early after myocardial infarction (MI). BACKGROUND Although injectable biomaterials can improve infarct healing and repair, the feasibility and effectiveness of intracoronary injection have not been studied. METHODS We prepared a calcium cross-linked alginate solution that undergoes liquid to gel phase transition after deposition in infarcted myocardium. Anterior MI was induced in swine by transient balloon occlusion of left anterior descending coronary artery. At 4 days after MI, either alginate solution (2 or 4 ml) or saline was injected selectively into the infarct-related coronary artery. An additional group (n = 19) was treated with incremental volumes of biomaterial (1, 2, and 4 ml) or 2 ml saline and underwent serial echocardiography studies. RESULTS Examination of hearts harvested after injection showed that the alginate crossed the infarcted leaky vessels and was deposited as hydrogel in the infarcted tissue. At 60 days, control swine experienced an increase in left ventricular (LV) diastolic area by 44%, LV systolic area by 45%, and LV mass by 35%. In contrast, intracoronary injection of alginate (2 and 4 ml) prevented and even reversed LV enlargement (p < 0.01). Post-mortem analysis showed that the biomaterial (2 ml) increased scar thickness by 53% compared with control (2.9 +/- 0.1 mm vs. 1.9 +/- 0.3 mm; p < 0.01) and was replaced by myofibroblasts and collagen. CONCLUSIONS Intracoronary injection of alginate biomaterial is feasible, safe, and effective. Our findings suggest a new percutaneous intervention to improve infarct repair and prevent adverse remodeling after reperfused MI.

Determinants of release rate of tetanus vaccine from polyester microspheres

Controlled-release formulations based on poly(lactic) (PLA) and poly(lactic/glycolic) acid (PLGA) microspheres containing tetanus vaccine were designed. The polymers forming the microspheres were L-PLA of different molecular weights and DL-PLGA, 50:50. These microspheres were prepared by two solvent elimination procedures, both using a double emulsion, and were characterized for size, morphology, and toxoid release kinetics. The influence of formulation variables such as polymer type, vaccine composition, and vaccine/polymer ratio was also investigated. Both techniques yielded microspheres with similar size, morphology, and release properties. Microsphere size was dependent on the type of polymer and the presence of the surfactant L-α-phosphatidylcholine, which led to a reduction in microsphere size. On the other hand, the release kinetics of encapsulated protein were affected by the polymer properties (ratio lactic/glycolic acid and molecular weight) as well as by the vaccine composition, vaccine loading, and microsphere size. Moreover, for some formulations, a decrease in microsphere size occurred simultaneously, with an increase in porosity leading to an augmentation of release rate. The changes in the PLA molecular weight during in vitro release studies indicated that release profiles of tetanus toxoid from these microspheres were only marginally influenced by polymer degradation. A significant fraction of protein (between 15 and 35%) was initially released by diffusion through water-filled channels. In contrast, the decrease in the PLGA molecular weight over the first 10 days of incubation suggested that erosion of the polymer matrix substantially affects protein release from these microspheres. Among all formulations developed, two differing in microsphere size, polymer hydrophobicity, and release profile were selected for in vivo administration to mice. Administration of both formulations resulted in tetanus neutralizing antibody levels that were higher than those obtained after administration of the fluid toxoid.

Human embryonic stem cell transplantation to repair the infarcted myocardium

Objective: To test the hypothesis that human embryonic stem cells (hESCs) can be guided to form new myocardium by transplantation into the normal or infarcted heart, and to assess the influence of hESC-derived cardiomyocytes (hESCMs) on cardiac function in a rat model of myocardial infarction (MI). Methods: Undifferentiated hESCs (0.5–1×106), human embryoid bodies (hEBs) (4–8 days; 0.5–1×106), 0.1 mm pieces of embryonic stem-derived beating myocardial tissue, and phosphate-buffered saline (control) were injected into the normal or infarcted myocardium of athymic nude rats (n = 58) by direct injection into the muscle or into preimplanted three-dimensional alginate scaffold. By 2–4 weeks after transplantation, heart sections were examined to detect the human cells and differentiation with fluorescent in situ hybridisation, using DNA probes specific for human sex chromosomes and HLA-DR or HLA-ABC immunostaining. Results: Microscopic examination showed transplanted human cells in the normal, and to a lesser extent in the infarcted myocardium (7/7 vs 2/6; p<0.05). The transplanted hESCs and hEBs rarely created new vessels and did not form new myocardium. Transplantation of hESCM tissue into normal heart produced islands of disorganised myofibres, fibrosis and, in a single case, a teratoma. However, transplantation of hESCMs into the infarcted myocardium did prevent post-MI dysfunction and scar thinning. Conclusions: Undifferentiated hESCs and hEBs are not directed to form new myocardium after transplantation into normal or infarcted heart and may create teratoma. Nevertheless, this study shows that hESC-derived cardiomyocyte transplantation can attenuate post-MI scar thinning and left ventricular dysfunction.