Patent CA-2437250-A1 (CA)
Inventor: LEBLOND FRANCOIS
IPC: A61L27/20
Filed: 2003-08-14
Abstract: Immunoisolation of cells in semipermeable membranes has been proposed as a means to prevent their immune destruction following transplantation[1, 2].  However, several fundamental issues remain to be addressed before considering clinical application of this method. One such issue is the resistance of microcapsules to chemical and mechanical degradation. Strong microcapsules will obviously increase the durability of the transplant. It is also likely to improve long-term biocompatibility of microcapsules, since a strong pericapsular reaction always develops around broken or damaged capsules[3, 4]. Moreover, the strength and durability of microcapsule membrane are critical issues for the transplantation of virus-transfected bioengineered cells. These virus-transfected bioengineered cells potentially include insulin-producing cells as well as cells producing molecules, which promote islet cell survival/replication/transdifferentiation or insure islet and microcapsule immunoprotection. Finally, considering the limited supply of human islets, the most promising approach to .beta.-cell replacement is the transdifferentiation of stem cells into islets[5] (not necessarily embryonic stem cells, which arise controversy, but stem cells of many other origins, including autologous cells). Note that even autologous cells would require immunoprotection against the recurrence of the autoimmune process. The major concern related to this approach is the risk of malignant transformation of immature stem cells. Microcapsules that can hardly be destroyed in conditions compatible with life would provide a safe method of transplanting stem cell derived cells, bioengineered cells or immortalized cells.   Complexation between alginate and polycations such as poly-L-lysine(PLL) is the most widely used method to microencapsulate cells[2]. Microcapsules are constructed by a simple three steps procedure. First, the calls are entrapped in a bead formed by the ionic cross-linking of alginate by a divalent ration such as calcium[6].  The second step comprises coating the alginate beads with a polycation such as PLL, which forms a peripheral membrane ensuring a good control of the molecular weight cut-off and increasing membrane stability. Finally, the third step consists to coat the microcapsules with a dilute alginate solution for insuring biocompatibility.  The two latter reactions rely on the ionic interactions between the polyanionic alginate and the polycationic polymer of lysine[7, 8].   In order to improve microcapsule strength, we[9] and others[7, 8, 10, 11], have evaluated the effect on microcapsule strength of modulating intrinsic parameters PLL molecular weight, concentration and incubation time and the mannuronic acid/guluronic acid ratio of alginate. The formation of neutral capsules by the introduction of a new coating agent[12] has also been investigated. Following these experiments, tighter binding between PLL and alginate was obtained. Nevertheless, poly-L-lysine still competes with other positively charged molecules in the environment to bind to the alginate beads. A prolonged incubation in solutions with high concentrations of Ca2+ or Sr2+ has showed a displacement of the alginate-poly-L-lysine bounds[13, 14].  In addition, Thu et al. have observed the progressive loss of the external sheet of alginate from microcapsules within days or weeks[7].     To prevent this competition with charged molecules in the environment, a new concept has been proposed: the introduction of covalent links into the membrane of the microcapsule[15-17]. Covalent links are known to be more stable than electrostatic interactions. The challenge is that most methods to create or to break a covalent link are incompatible with the survival of living cells. The objective of the present work was to develop and validate a method, compatible with encapsulated cell survival, to create a covalent link between the PLL layer and both alginate from the microcapsule core and from the outer sheet. It would be unlikely that such a microcapsules would be destroyed in conditions found in the living body.   Here we show that it is possible to microencapsulate living cells in membranes composed of covalently linked alginate-PLL-alginate membranes, without affecting cell viability. The formation of a covalent link between PLL and alginate from both the alginate core and the outer alginate shoes considerably increased microcapsule resistance to chemical and mechanical stress. Standard APA microcapsules dissolved within seconds when they were incubated in an alkaline solution whereas covalently linked APA microcapsules remained unaltered after two years in the same alkaline solution Covalently linked microcapsules were 22 times more resistant than standard microcapsules when they were submitted to a mechanical stress. The process is based on the chemical derivation of poly-L-lysine (PLL) with a photoactivatable heterobifunctional cross-linker, N-5-Azido-2-nitrobenzoyloxysuccinimide (ANB-NOS), which is able to covalently bind PLL to alginate when energized by UVA light illumination[18, 19]. The N-hydroxysuccinimid of the cross-linked was fist covalently linked to PLL in conditions that would damage living cells, but before the colts are involved in the procedure. Then islet cells encapsulated in calcium alginate microcapsules were incubated with the arose-linker derived PLL, than in alginate again to form the outer biocompatible sheet. When the preparation is illuminated with a UVA     lamp the photoactivatable residue reacts with the phenyl azide residue on alginate creating a covalent link between PLL and alginate. The latter reaction is not harmful for living cells. All reactions involving the crosslinker-derived PLL were performed in a dark room, until UVA illumination, to prevent activation of the photoactivatable residue.   We, herein, present a proof that a covalent link was formed providing considerable improvement of microcapsule resistance to chemical and mechanical degradation.  The results of the study showed that the procedure did not modify microcapsule membrane permeability and did not affect in vitro and in viva encapsulated cell survival.