Deterioration of polyamino acid-coated alginate microcapsules in vivo.

The implantation of immuno-isolated recombinant cell lines secreting a therapeutic protein in alginate microcapsules presents an alternative approach to gene therapy. Its clinical efficacy has recently been demonstrated in treating several genetic diseases in murine models. However, its application to humans will depend on the long-term structural stability of the microcapsules. Based on previous implantations in canines, it appears that survival of alginate-poly-L-lysine-alginate microcapsules in such large animals is short-lived. This article reports on the biological factors that may have contributed to the degradation of these microcapsules after implantation in dogs. Alginate microcapsules coated with poly-L-lysine or poly-L-arginine were implanted in subcutaneous or intraperitoneal sites. The retrieved microcapsules showed a loss of mechanical stability, as measured by resistance to osmotic stress. The polyamino acid coats were rendered fragile and easily lost, particularly when poly-L-lysine was used for coating and the intraperitoneal site was used for implantation. Various plasma proteins were associated with the retrieved microcapsules and identified with western blotting to include Factor XI, Factor XII, prekallikrein, HMWK, fibrinogen, plasminogen, ATIII, transferrin, alpha-1-antitrypsin, fibronectin, IgG, alpha-2-macroglobulin, vitronectin, prothrombin, apolipoprotein A1, and particularly albumin, a major Ca-transporting plasma protein. Complement proteins (C3, Factor B, Factor H, Factor I) and C3 activation fragments were detected. Release of the amino acids from the microcapsule polyamino acid coats was observed after incubation with plasma. indicating the occurrence of proteolytic degradation. Hence, the loss of long-term stability of the polyamino acid-coated alginate microcapsules is associated with activation of the complement system, degradation of the polyamino acid coating, and destabilization of the alginate core matrix, probably through loss of calcium-mediated ionic cross-linking of the guluronic acid polymers in the alginate. These destructive forces may be slightly mitigated by using poly-L-arginine instead of poly-L-lysine for coating and by implanting in a subcutaneous instead of an intraperitoneal site. However, the long-term stability of such devices may require significant improvements in the microcapsule polymer chemistry to withstand such biological impediments.

Treatment of hemophilia B in mice with nonautologous somatic gene therapeutics.

The implantation of nonautologous cells encapsulated in immunoprotective microcapsules provides an alternative nonviral method for gene therapy. This strategy was successful in reversing the disease phenotypes of dwarfism and a lysosomal storage disease, mucopolysaccharidosis VII, in murine models. In this article we implanted transgenic hemophilic B mice with microcapsules enclosing factor IX-secreting C2C12 myoblasts to study the clinical potential of this approach in the treatment of hemophilia. Treated mice showed increased plasma factor IX levels as high as 28 ng of human factor IX per milliliter of plasma and decreased activated thromboplastin times (reduced by 20% to 29%). However, the level of factor IX decreased to baseline levels by day 7, coinciding with emergence of anti-human factor IX antibody, the titer of which increased greater than 10-fold by day 28. Monoclonal anti-CD4 antibodies were used to deplete CD4+ T cells to suppress the immune response against the recombinant factor IX. In the treated hemophilic mice, the anti-factor IX antibody response was totally suppressed to beyond day 28 accompanied by a significant decrease in activated thromboplastin time compared with that seen in untreated hemophilic mice. When the microcapsules were recovered from the intraperitoneal cavity after 38 days of implantation, the encapsulated cells continued to secrete factor IX at preimplantation levels, but both cell viability and microcapsule mechanical stability were reduced. Hence although the polymer chemistry of the microcapsules and cell viability may need to be improved for long-term delivery, nonautologous gene therapy with microencapsulated cells has been shown to be effective, at least for the short-term, in alleviating the hemophilic hemostatic anomaly. Coadministration of an immunosuppressant is effective in inhibiting antibody development against the delivered factor IX and should be considered for recipients at risk of inhibitor development.

Osmotic pressure test: a simple, quantitative method to assess the mechanical stability of alginate microcapsules.

Implantation of microencapsulated, nonautologous cells and tissues is an effective method to deliver therapeutic proteins in vivo. Its success depends on the maintenance of the immunoisolating barrier provided by the microcapsule. Thus, one goal in the development of this technology is to create mechanically stable microcapsules. We have developed an osmotic pressure test to quantify the strength of microcapsules by exposing alginate microcapsules to a graded series of hypotonic solutions and quantifying the percentage of broken microcapsules. The test was validated by confirming the relative strengths of different types of alginate capsules, previously known from implantation in dogs to have differing mechanical stability in vivo. Thus, solid alginate microcapsules crosslinked with Ba(2+) were shown to be stronger than those crosslinked with Ca(2+), which in turn were shown to be stronger than the corresponding hollow alginate microcapsules. The incorporation of cells was demonstrated to reduce the mechanical stability of the microcapsules significantly. Hence, this test provides a simple and quantitative method for rapidly determining the strength of a large number of microcapsules. Thus, it is suitable for monitoring the mechanical stability of various types of microcapsules, predicting the performance of microcapsules in vivo, and for quality control of microcapsules during scale-up productions.

The in vivo delivery of heterologous proteins by microencapsulated recombinant cells.

The microencapsulation of recombinant cells is a novel and potentially cost-effective method of heterologous protein delivery. A 'universal' cell line, genetically modified to secrete any desired protein, is immunologically protected from tissue rejection by enclosure in microcapsules. The microcapsule can then be implanted in different recipients to deliver recombinant proteins in vivo.