Novel enzymatic cross-linking-based hydrogel nanofilm caging system on pancreatic β cell spheroid for long-term blood glucose regulation

Abstract

Pancreatic β cell therapy for type 1 diabetes is limited by low cell survival rate owing to physical stress and aggressive host immune response. In this study, we demonstrate a multilayer hydrogel nanofilm caging strategy capable of protecting cells from high shear stress and reducing immune response by interfering cell-cell interaction. Hydrogel nanofilm is fabricated by monophenol-modified glycol chitosan and hyaluronic acid that cross-link each other to form a nanothin hydrogel film on the cell surface via tyrosinase-mediated reactions. Furthermore, hydrogel nanofilm formation was conducted on mouse β cell spheroids for the islet transplantation application. The cytoprotective effect against physical stress and the immune protective effect were evaluated. Last, caged mouse β cell spheroids were transplanted into the type 1 diabetes mouse model and successfully regulated its blood glucose level. Overall, our enzymatic cross-linking-based hydrogel nanofilm caging method will provide a new platform for clinical applications of cell-based therapies.

Fig. 1. Representative scheme of β cell spheroid transplantation with an enzymatic cross-linking–based LbL hydrogel nanofilm caging system.

Fig. 2. Synthesis and characterization of GC-T and HA-T, with cross-linking reaction by SA-Ty.

(A) Synthesis of GC-T and HA-T by conjugation of monophenols to GC and HA. (B) ζ potential of 0.1% (w/v) GC-T and 0.1% (w/v) HA-T. (C) Enzymatic reactivity profile of SA-Ty reacted with 0.1% (w/v) GC-T and 0.1% (w/v) HA-T. (D) FTIR spectra of fully oxidized GC-T and HA-T by SA-Ty. (E) Schematic illustration of the enzymatic two-step oxidative reaction mediated by tyrosinase and nonenzymatic cross-linking reactions of o-quinone. (F) Images of 5% (w/v) GC-T, 5% (w/v) HA-T, and 2.5% (w/v) GC-T and 2.5% (w/v) HA-T hydrogel cross-linked by tyrosinase at day 0 and day 1 in PBS. (G) Comparison of swelling ratio between 5% (w/v) GC-T, 5% (w/v) HA-T, and 2.5% (w/v) GC-T and 2.5% (w/v) HA-T hydrogel (N = 6). (H) Schematic illustration of the experimental setup and the diffusion profile of FITC-dextran (20 and 70 kDa) across six layers of GC-T and HA-T (L6) cross-linked by SA-Ty (N = 3). Error bars denote means ± SD. *P < 0.05; **P < 0.01.

Fig. 3. LbL hydrogel nanofilm on the single-cell surface.

(A) The effect of SA-Ty on GC-T and HA-T coating on the cell surface (N = 3). (B) The efficiency difference of HA-T-FA coating on different cell surface charges (N = 3). (C) Quantitative analysis of LbL deposition of GC-T and HA-T on the cell surface. (D) Schematic image of single-cell LbL hydrogel nanofilm formation. (E) Changes in ζ potential of the encapsulated cell surface by the increment of layers (N = 5). (F) Flow cytometry analysis of encapsulated cells based on the increasing numbers of layers. GC-T-RITC and HA-T-FA were detected using the PE channel and FITC channel, respectively. (G) Confocal laser microscopic images of native and L6-encapsulated cells. Scale bars, 10 μm. (H) TEM images of native and L6-encapsulated cell surface. Error bars denote means ± SD.

Fig. 4. Encapsulation of β cell spheroids by L6 hydrogel nanofilm.

(A) Schematic and fluorescence images of β cell spheroid encapsulated with hydrogel nanofilm (HA-T-FA in green and GC-T-RITC in red). (B) Live/Dead assay images of native and L6-encapsulated β cell spheroids. Live cells are represented in green, and dead cells are represented in red. Scale bars, 100 μm. (C) The difference in insulin level between native and L6-encapsulated β cell spheroids at low- and high-glucose solutions (N = 4). (D) Comparison of SI of native and L6-encapsulated β cell spheroids calculated by dividing the insulin level at a high-glucose solution by the insulin level at a low-glucose solution. For reference, 25 clusters equate to ~10⁶ cells. Error bars denote means ± SD. *P < 0.05.

Fig. 5. Hydrogel nanofilm as a physical barrier to β cell spheroids.

(A) Stability test of hydrogel nanofilm until 1 week. Treatment of SA-Ty prolonged the stability of hydrogel nanofilm. (B) Morphological changes of spheroids with/without hydrogel nanofilm against external stress. (C) Schematic images of immune protection by the L6 hydrogel nanofilm. (D) Confocal microscopic images of β cell spheroids with/without hydrogel nanofilm cocultured with NK-92 cell. (E) Comparison of the coverage fraction of NK cells on each β cell spheroid group (**P < 0.01, N = 3). (F) Percentage reduction of β cell spheroid area by NK cells dissociating β cells from the spheroid (N = 3). Scale bars, 200 μm. Error bars denote means ± SD.

Fig. 6. β cell spheroids encapsulated with SA-Ty–mediated hydrogel nanofilm sustain normoglycemia in streptozotocin-treated BALB/C mice.

(A) Schematic image of transplantation of β cell spheroids into the kidney capsule of BALB/C mice. Nonfasting blood glucose level (B) and body weight (C) of mice in each group were monitored periodically (N = 4). (D) Measurement of the blood glucose level of mice that were subjected to IPGTT after implantation (N = 4). (E) Histological analysis of implants retrieved 30 days after implantation from the streptozotocin-treated BALB/C mice, represented with hematoxylin and eosin (H&E) staining and immunostaining of insulin in each group. Scale bars, 200 μm (top images) and 100 μm (bottom images). Error bars denote means ± SD.