In Vitro and In Vivo Biocompatibility Evaluation of Polyallylamine and Macromolecular Heparin Conjugates Modified Alginate Microbeads

Abstract

Host reactivity to biocompatible immunoisolation devices is a major challenge for cellular therapies, and a human screening model would be of great value. We designed new types of surface modified barium alginate microspheres, and evaluated their inflammatory properties using human whole blood, and the intraperitoneal response after three weeks in Wistar rats. Microspheres were modified using proprietary polyallylamine (PAV) and coupled with macromolecular heparin conjugates (Corline Heparin Conjugate, CHC). The PAV-CHC strategy resulted in uniform and stable coatings with increased anti-clot activity and low cytotoxicity. In human whole blood, PAV coating at high dose (100 µg/ml) induced elevated complement, leukocyte CD11b and inflammatory mediators, and in Wistar rats increased fibrotic overgrowth. Coating of high dose PAV with CHC significantly reduced these responses. Low dose PAV (10 µg/ml) ± CHC and unmodified alginate microbeads showed low responses. That the human whole blood inflammatory reactions paralleled the host response shows a link between inflammatory potential and initial fibrotic response. CHC possessed anti-inflammatory activity, but failed to improve overall biocompatibility. We conclude that the human whole blood assay is an efficient first-phase screening model for inflammation, and a guiding tool in development of new generation microspheres for cell encapsulation therapy.

Figure 1

LBL binding of CHC to alginate microbeads by cationic linkers. Representative confocal images of fluorescently labelled CHC (36 µg/ml) binding to alginate microbeads via polycationic linkers PAV, PLL and PLO at varied concentrations of 100 µg/ml (A,D,G), 10 µg/ml (B,E,H) and 3 µg/ml (C,F,I). Negative control (J) is CHC added to alginate microbeads without linkers (Number of separate experiments was 3, and number of replicates in each group was 3).

Figure 2

Confocal imaging of LBL modified microbeads. Representative confocal images of LBL modified alginate microbeads containing high (100 µg/ml) or low (10 µg/ml) concentrations of fluorescently labelled PAV (PAV-Cy5.5; red) and 36 µg/ml CHC (CHC-Alexa488; green) cultured for 1, 7, 14 and 21 days in phosphate buffered saline post-heparinization (n = 3 for each group). The figures above are representative confocal images of LBL modified beads taken at day 21 post-heparinization. Arrows point to intermittent areas of excess deposition of PAV which are not completely masked by CHC (seen in higher magnification images of PAV_(high) + CHC microbeads) and hence remain exposed to initiate an immune response.

Figure 3

Cytotoxicity of LBL modified microbeads. Direct contact (A) and indirect extract (B) cytotoxicity assay of PAV_(high) + CHC and PAV_(low) + CHC microbeads compared to non-coated barium alginate microbeads using mouse fibroblast L929 cells after 24 hr in culture. A cut-off value of <70% was considered cytotoxic as seen with the positive control 5% DMSO and a value >70% was considered non-toxic as seen with the negative control 5% PBS and SFM reference control. Values = mean ± SEM (n = 3 for each group).

Figure 4

Effect of LBL modified microbeads on complement activation (Terminal Complement Complex [TCC]). Relative TCC formation compared to saline after incubation in whole blood with various coated and non-coated alginate microbeads for 30 (A), 120 (B) and 240 (C) min. Values are mean ± SEM (n = 4–5 of separate experiments with different donors); *p < 0.0001 for microbeads versus saline control (ANOVA, with posthoc Duncan’s Multiple-Comparison test) and ^#p < 0.0001 between PAV_(high) and PAV_(high) + CHC (Student’s t-test). Values (AU/ml) are normalized to saline based on data obtained from two separate experiments with different donors each study, given in Supplementary Figure 4.

Figure 5

Effect of LBL modified microbeads on leucocyte activation. Leucocyte activation as measured by CD11b expression on both granulocytes (A) and monocytes (B) relative to saline after incubation of whole blood with various coated and non-coated alginate microbeads for 240 min. Values are mean ± SEM (n = 3 of separate experiments with different donors); **p < 0.0001 and *p < 0.001 for PAV_(high) and PAV_(high) + CHC versus the saline control (ANOVA, with posthoc Duncan’s Multiple-Comparison test), and ^#p < 0.0001 and <0.001 between PAV_(high) and PAV_(high) + CHC for granulocytes and monocytes respectively (Student’s t-test). Values (mean fluorescence intensity; MFI) are normalized to saline from data of two different experiments with different donors. MFI values from the two separate experiments are shown in Supplementary Figure 5.

Figure 6

Effect of LBL modified microbeads on TNF-α and IL-8 response. PAV _(high), PAV_(high) + CHC, PAV _(low), PAV_(low) + CHC and non-coated Ba-alg microbeads incubated in lepirudin anti-coagulated whole blood for 240 min. Values are mean ± SEM (n = 4–5 of separate experiments with different donors); *p < 0.01 and **p < 0.0001 for microbeads versus saline control (ANOVA, with posthoc Duncan’s Multiple-Comparison test) and ^#p < 0.05 between PAV_(high) and PAV_(high) + CHC (Student’s t-test). Values (measured as pg/ml) are normalized to saline and measured in plasma obtained from four to five independent donors. Raw data values for plasma baseline, saline, LBL modified microbeads and the positive control zymosan measured are shown in Supplementary Figure 6.

Figure 7

Effect of LBL modified microbeads containing PAV_(high) on the cytokine response. PAV _(high), PAV_(high) + CHC or non-coated Ba-alg microbeads incubated in lepirudin anti-coagulated whole blood for 240 min. Values are mean ± SEM (n = 5); *p < 0.05 for PAV_(high) > saline, non-coated, PAV_(high) + CHC microbeads and ^#p < 0.05 for PAV_(high) > PAV_(high) + CHC (ANOVA with posthoc Duncan’s Multiple-Comparison test). Values (measured as pg/ml) are normalized to saline and measured in plasma obtained from five independent donors. Raw data values for plasma baseline, saline, LBL modified microbeads and the positive control zymosan measured are shown in Supplementary Figure 7.

Figure 8

Assessment of PFO on retrieved LBL modified microbeads. The fibrotic scores (A) and cell adhesion scores (B) on retrieved microbeads transplanted into peritoneal cavity of Wistar rats. Extent of host cell adhesion on retrieved LBL modified microbeads is represented by a cell adhesion score, on a scale of 0 (no cell adhesion) to 16 (complete host cell adhesion). Values are mean ± SEM (n = 4–8 animals per group); *p < 0.0001 for cell adhesion score where PAV_(high)and PAV_(high) + CHC > saline, non-coated, PAV_(low) and PAV_(low) + CHC microbeads and ^#p < 0.0001 for cell adhesion score where PAV_(high) > PAV_(high) + CHC microbeads (ANOVA with posthoc Duncan’s Multiple-Comparison test).