Multilayered Freestanding Porous Polycarbonate Nanosheets with Directed Protein Permeability for Cell-Encapsulated Devices

Abstract

Implantable pancreatic β cell-encapsulated devices are required for the treatment of type 1 diabetes. Such devices should enable a semipermeable membrane to release insulin in response to glucose levels while avoiding immune reactions. Micrometer-thick track-etched porous polycarbonate (PC) membranes have been used for this purpose. However, the immediate release of insulin remains a challenge in the development of such semipermeable membranes. Herein, we attempted to develop a freestanding polymeric ultrathin film (nanosheet) with a porous structure that can be used in a cell-encapsulated device. Specifically, we fabricated a nonbiodegradable, porous PC nanosheet to enhance molecular permeability. The nanosheet was multistacked to ensure the controlled permeability of proteins of various molecular weights, such as insulin and IgG. The porous PC nanosheet was prepared by gravure coating using a blend solution comprising PC and polystyrene (PS) to induce macro-phase separation of the PC and PS. When the PC:PS weight ratio of the mixture was reduced to 3:1, we succeeded in fabricating a porous PC nanosheet (thickness: 100 nm, diameter: < 2.5 μm). A triple layer of such porous nanosheets with various pore sizes demonstrated 10 times less protein clogging, 10 times higher insulin permeability, and comparable IgG-blocking capability compared with commercially available porous PC membranes (thickness: 10 μm). Finally, we demonstrated that a cell-encapsulated device equipped with the multilayered porous PC nanosheet as a permeable membrane preserved the glucose response level of insulin-producing cells before, during, and after the cell-encapsulation process. We believe that cell-encapsulated devices equipped with such porous PC nanosheets will enable immediate insulin release in response to changes in glucose levels.

Keywords: cell-encapsulated device; insulin; phase separation; polycarbonate; polymeric ultra-thin film (nanosheet).

Figure 1

Design of multilayered porous PC nanosheets for a cell-encapsulated device for directed insulin release. (PC = polycarbonate).

Figure 2

Fabrication and characterization of porous PC nanosheets by phase separation and solvent etching. (a) AFM images of a PC/PS nanosheet surface before and after removal of the PS regions by etching with cyclohexane. (b) ATR-FTIR spectra obtained before and after etching with cyclohexane. (c) AFM images of porous PC nanosheets fabricated from various PC/PS weight ratios (PC:PS = 1:1, 2:1, and 3:1). (PC = polycarbonate; AFM = atomic force microscopy; PS = polystyrene; ATR-FTIR = attenuated total reflection–Fourier-transform infrared spectroscopy).

Figure 3

Evaluation of semipermeability of porous PC nanosheets with regard to insulin and IgG. (a) Setup for evaluation of semipermeability using a Transwell insert. (b) Fluorescence images of Transwell inserts with PC nanosheets of various porosities and a commercial PC membrane after permeation of FITC-labeled proteins at 60 min. The images were captured by focusing on the nanosheets attached to the Transwell inserts. (c) FITC-Insulin and (d) FITC-IgG accumulated on the various semipermeable membranes. Cumulative release amounts of (e) insulin and (f) IgG permeated through the various semipermeable membranes. We named the multilayered porous nanosheets according to their porosity numbers (i.e., 21.1% as 21, and 40.3% as 40) in the order of protein permeation, as shown in (a). Statistical analysis was performed by one-way ANOVA multiple comparison (n = 3, **p < 0.01).

Figure 4

Morphology of the insulin-producing cells encapsulated in the device. (a) Bright-field and (b) Live/Dead stained fluorescence images of insulin-producing cells encapsulated by different semipermeable membranes (a triple-layered porous PC nanosheet and a commercial PC membrane). The Live/Dead staining of the MIN6 and islet cells was conducted after GSIS assays at 24 and 7 h of encapsulation, respectively. (PC = polycarbonate; GSIS = glucose-stimulated insulin secretion).

Figure 5

(a) SI values (insulin secretion at high versus low glucose concentrations) in the GSIS assay of MIN6 and islet cells under normal culture conditions; the C-peptide release of (b) MIN6 and (c) islet cells (low: 3 mM glucose, high: 20 mM glucose); and the SI values in the GSIS assay of (d) MIN6 and (e) islet cells. Statistical analysis was performed by t test (n = 3). (SI = glucose stimulating index; GSIS = glucose-stimulated insulin secretion).