The long-term viability of transplanted encapsulated islets is dependent, in part, on its ability to acquire oxygen and nutrients from a vascular blood supply. A biomaterial system that can serve simultaneously as an encapsulation system for islets as well as a sustained delivery system for angiogenic proteins may stimulate vascular growth around the transplanted cells, resulting in improved graft viability. We have shown that in order to achieve persistent neovascularization, a sustained release of FGF-1 is required, in contrast to a single bolus delivery. 20
In this paper, we present an approach for generating multilayered alginate microbeads. The outer layer can be used for the encapsulation and sustained release of FGF-1 while the inner layer can be used for islet encapsulation.
The size of the outer layer ranged from 113 µm to 164 µm. In our studies, the outer layer thickness increased with increasing alginate concentration. In addition, LVG alginate formed thicker outer layers than LVM alginate at equal concentrations. Other researchers have reported a similar relationship between alginate microbead size and alginate composition. 24,25
The di-axial configuration of G units allow for a more favorable interaction with the cation, which results in a greater degree of gelation. 10,26
As described by the ‘egg-box’ model, 27
when sufficient Ca2+
ions are present, it can be expected that the outer layer created using LVG alginate will be thicker than LVM owing to the presence of more G-unit cavities. Properties such as porosity and gel strength differ for LVM and LVG alginates based on the nature and the degree of crosslinking that occurs. A larger fraction of G-units decreases the space available (porosity) for molecules to diffuse, resulting in greater diffusive resistance in alginate with high G content. These differences may help explain the slow rate of release for LVG than LVM alginate at equal concentrations. 10
In addition, high M content results in more rigid gels that may influence the inflammatory response to the implanted material.
Any degradation or swelling of the layer may alter the release kinetics of the entrapped protein. Previous studies have shown that alginate beads without a permselective coating or an additional outer alginate layer undergo significant swelling due to ion exchange. 28
Alginate hydrogels are not degraded by bond cleavage, but instead erode due to ion exchange resulting from calcium and sodium ions in the local environment. Depending on the ionic strength of solutions used to study alginate-based materials, degradation may occur in days or months. 29
In our study, microbeads were incubated in a solution that reflected physiological calcium concentrations in order to approximate in vivo
conditions. For both LVG and LVM alginate, the outer layer did not shrink or swell over the course of thirty days, which suggests that the outer layer will be maintained under in vivo
conditions. Animal studies must be conducted to gain further insight into the stability of the outer layer in vivo
Our previous studies have shown that the PLO coating serves as a permselective membrane, which prevents the passage of molecules greater than 120 kDa in size into the inner alginate bead. 23
It is essential that the microcapsules retain this selectivity in the presence of the outer layer. The PLO polymer coating was effective in preventing antibody, but not BSA, diffusion into the inner alginate bead. These results are consistent with prior studies using PLO-coated alginate microbeads for the encapsulation of cells. It is also possible that FGF-1 will diffuse through the PLO membrane and into the inner alginate matrix. The effect of FGF-1 on encapsulated islets is unclear, but FGF-1 is known to activate fibroblast growth factor receptor-1 (FGFR1) on β-cells, which regulates function, survival, and proliferation. 30
The permselectivity of PLO seems to suggest that cells would be protected from molecules that contribute to rejection upon transplantation. However, our results provide only a simple evaluation of PLO as a permselective membrane. Additional studies are required to determine the potential immunological response to cells encapsulated in PLO-coated multilayered alginate microbeads.
We have shown in this study that proteins can be encapsulated and released from the outer alginate layer. FGF-1 in the outer layer of the microbeads was released for over 30 days under experimental infinite sink conditions. The long-term delivery of FGF-1 may stimulate the formation of new vasculature directly towards the transplant site, and in turn, potentially improve the viability of transplanted islets. Previous studies have shown that the concentration of FGF-1 plays an important role in determining the morphology of microvascular networks. We have found that 0.5 ng/day of FGF-1 results in persistent neovascularization.19
In our study, all four batches of microbeads provided a burst release that could stimulate the formation of new vasculature, while the lower doses delivered at later times may lead to persistent neovascularization. Outer layers made with 1.25% LVG but provided a daily release > 0.5 ng/day out to 26 days, which suggests that this may be the optimal condition for use in vivo
Previous studies have shown that protein diffusion is more restricted by alginate with high G content. 31,32
In addition, the 1.25% G conditions resulted in the thickest outer layer, which would contribute to longer transport times. Diffusion, however, is not likely to be the primary mechanism of release. Release would be complete in a few hours if the release were strictly via ordinary diffusion. Diffusion may contribute to the initial burst release, but other interactions need to occur in order to delay release to the time scale on the order of 30 days. Electrostatic interactions between FGF-1 and negatively charged alginate are unlikely because of FGF-1’s negative charge at neutral pH. 29
Instead, it is possible that FGF-1 interacts with the positively charged PLO layer, which could delay release. The extent of this interaction may be determined in part by the composition of the alginate in the outer layer and how its interactions with the PLO alters availability for interactions with FGF-1. Further studies are needed to better understand the mechanism determining release from the outer layer.
FGF-1 released from the outer layer was active only when it was co-encapsulated with heparin in the outer layer. Immobilization of heparin, or heparin analogs, to biomaterials has been used as a method for prolonging the release of a number of heparin-binding growth factors. 33–35
However, the presence of heparin in the outer alginate layer did not greatly affect the rate of release of FGF-1 in our system. In our experimental approach, heparin was not covalently attached to the alginate, but was incorporated in a form that it could freely diffuse. Heparin facilitates FGF-1 dimerization and activation of its cell surface receptors, and also protects the protein from denaturation by proteolysis or heat inactivation. 36,37
Heparin binding to FGF-1 can increase its half-life 100-fold. 38
The release solution contained heparin in both cases (with or without heparin in the outer layer), so the benefit of facilitating dimerization and receptor interaction upon addition to ECs was always present. In this case, it is likely that the improved activity was due to the improved stability and increased half-life of heparin-bound FGF-1. Biologically enhanced chimeras of FGF-1 could allow for the release of FGF-1 that is active in the absence of exogenous heparin. Heparin-binding growth-associated molecule fibroblast growth factor-1 (HB-GAM/FGF-1) chimeric protein has previously been shown to retain FGF-1’s normal mitogenic properties while exhibiting heparin-independent behavior. 39,40
In conclusion, we have developed a technique for the synthesis of multilayered alginate microcapsules with an outer alginate layer that can be used as a region for the encapsulation and release of FGF-1. Our studies have shown that the outer layer provides a long-term sustained release of FGF-1. The release of FGF-1 can be influenced by the composition and concentration of alginate used to make the outer layer. Indeed, multilayered alginate microbeads have promise for the simultaneous encapsulation of islets in the inner core and FGF-1 in the outer alginate layer, in order to enhance the viability of transplanted islets due to local stimulation of neovascularization.