Synthesis of multilayered alginate microcapsules for the sustained release of fibroblast growth factor-1

Abstract

Alginate microcapsules coated with a permselective poly-L-ornithine (PLO) membrane have been investigated for the encapsulation and transplantation of islets as a treatment for type 1 diabetes. The therapeutic potential of this approach could be improved through local stimulation of microvascular networks to meet mass transport demands of the encapsulated cells. Fibroblast growth factor-1 (FGF-1) is a potent angiogenic factor with optimal effect occurring when it is delivered in a sustained manner. In this article, a technique is described for the generation of multilayered alginate microcapsules with an outer alginate layer that can be used for the delivery of FGF-1. The influence of alginate concentration and composition (high mannuronic acid (M) or guluronic acid (G) content) on outer layer size and stability, protein encapsulation efficiency, and release kinetics was investigated. The technique results in a stable outer layer of alginate with a mean thickness between 113 and 164 μm, increasing with alginate concentration and G-content. The outer layer was able to encapsulate and release FGF-1 for up to 30 days, with 1.25% of high G alginate displaying the most sustained release. The released FGF-1 retained its biologic activity in the presence of heparin, and the addition of the outer layer did not alter the permselectivity of the PLO coat. This technique could be used to generate encapsulation systems that deliver proteins to stimulate local neovascularization around encapsulated islets.

Figure 1

Schematic of the procedure for synthesizing multilayered alginate microcapsules. The inner alginate core is formed by crosslinking 1.5% LVM alginate in 2mM CaCl₂. A PLO coating is then generated on the bead surface before the addition of alginate and crosslinking with CaCl₂ to form the outer layer.

Figure 2

(A) PLO coated microbeads prior to formation of the outer layer. (B) A multilayer alginate microbead with an outer alginate layer that is distinctly visible. (C) FITC-BSA (green) can be seen in the outer layer immediately after synthesis, indicating that this region can be used for protein encapsulation.

Figure 3

The size of the outer layer of multilayered alginate microbeads varied with the composition and concentration of alginate used. At equal concentrations, it was found that LVG alginate formed larger outer layers than LVM. For both LVM and LVG alginate, the size of the outer layer increased with increasing alginate concentration. All groups were statistically different (p < 0.05) from each other.

Figure 4

The size of the outer alginate layer remained stable over a period of thirty days when incubated in a 2mM solution of CaCl₂ at 37°C. * indicates that all groups are statistically different from one another, and # denotes that all groups are statistically different from each other except 1% LVM from 1.25% LVG.

Figure 5

Release of FGF-1 from the outer alginate layer varied based on the concentration of LVM and LVG alginate used. (A) and (B) show percent release, and (C) and (D) denote the corresponding mass release of FGF-1 versus time for different outer layer formulations. There is a burst release exhibited for all conditions within the initial 5 hours (A and C), and low-dose continuous release for up to thirty days (B and D).

Figure 6

Heparin (5 U/mL) was added to the outer layer composed of 1.25% LVG, and FGF-1 release is compared with results in the absence of heparin.

Figure 7

Intensity profile of a cross-section of multilayered alginate microbeads incubated with solutions containing fluorescently labeled IgG (A, B) or BSA (C, D). The IgG molecule was not present within the inner alginate core at either (A) 5 hours or (B) 3 days. BSA rapidly diffused into the bead and was present throughout the core at (A) 5 hours and (B) 3 days. Vertical lines designate the position of the PLO coating of the multilayered microbeads.

Figure 8

FGF-1 encapsulated with heparin (5 U/mL) in the outer layer released after 12 hours stimulated the proliferation of HUVECs, but FGF-1 without heparin did not. Proliferation data are presented normalized to the positive control (stimulation with 20% FBS). * indicates statistical significance (p < 0.05) in comparison to the negative control (HBSS).