Controlled Release of Insulin Based on Temperature and Glucose Dual Responsive Biomicrocapsules

Abstract

The treatment of diabetes lies in developing novel functional carriers, which are expected to have the unique capability of monitoring blood glucose levels continuously and dispensing insulin correctly and timely. Hence, this study is proposing to create a smart self-regulated insulin delivery system according to changes in glucose concentration. Temperature and glucose dual responsive copolymer microcapsules bearing N-isopropylacrylamide and 3-acrylamidophenylboronic acid as main components were developed by bottom-spray coating technology and template method. The insulinoma β-TC6 cells were trapped in the copolymer microcapsules by use of temperature sensitivity, and then growth, proliferation, and glucose-responsive insulin secretion of microencapsulated cells were successively monitored. The copolymer microcapsules showed favorable structural stability and good biocompatibility against β-TC6 cells. Compared with free cells, the biomicrocapsules presented a more effective and safer glucose-dependent insulin release behavior. The bioactivity of secreted and released insulin did not differ between free and encapsulated β-TC6 cells. The results demonstrated that the copolymer microcapsules had a positive effect on real-time sensing of glucose and precise controlled release of insulin. The intelligent drug delivery system is supposed to mimic insulin secretion in a physiological manner, and further provide new perspectives and technical support for the development of artificial pancreas.

Keywords: copolymer microcapsule; fluidized bed reactor; insulin delivery system; insulinoma β-TC6 cells; poly(N-isopropylacrylamide).

Figure 1

(A) Schematic diagram of copolymer microcapsule formation. (B) Schematic illustration for the synthesis of p(N-isopropylacrylamide-co-3-acrylamidophenylboronic acid-co- hydroxypropyl methacrylate-co-3-trimethoxysilylpropyl methacrylate), p(NIPAAm-co-AAPBA-co-HPM-co-TMSPM) copolymers, and the formation of copolymer film with clear indication of three possible types of methanol removal when annealed. (C) Experimental rig of bottom-spray fluidized bed reactor system: 1 fixed support, 2 main reactor, 3 atomizing nozzle, 4 air distribution plate, 5 reservoir, 6 air inlet chamber, 7 volume flowmeter, 8 control device, 9 auxiliary equipment. (D) Flow diagram of bottom-spray coating technology: 1 fan, 2 anemometer, 3 bottom-spray fluidized bed reactor, 4 diaphragm metering pump, 5 volume flowmeter, 6 atomizing nozzle, 7 air distribution plate, 8 glass mirospheres, 9 superfine mesh.

Figure 2

Attenuated total reflection–Fourier transform infrared (ATR–FTIR) spectra of copolymer microcapsules (A) and nanoparticles (B).

Figure 3

Swelling ratio (A), size distribution (B), and surface topology (C) of copolymer microcapsules in different solutions. (a) Dry state; (b) deionized water at 20 °C; (c) deionized water at 40 °C; (d) phosphate buffered solution (PBS) at 20 °C, pH 7.4; (e) 5.0 mg∙mL⁻¹ glucose solution at 20 °C, pH 7.4; (f) 5.0 mg∙mL⁻¹ glucose solution at 40 °C, pH 7.4. ** p < 0.05 is considered to be statistically significant.

Figure 4

Poly(N-isopropylacrylamide) (PNIPAAm) and its mechanism of thermosensitivity (A), phenylboronic acid (PBA) and its mechanism of sugar-sensitivity (B).

Figure 5

Degradation of copolymer microcapsules in different solutions including deionized water, phosphate buffered solution (PBS) with pH 7.4 as well as 5.0 mg∙mL⁻¹ glucose solution under 20 °C (A) and 40 °C (B) at regular intervals.

Figure 6

Schematic diagram of encapsulation of β-TC6 cells, and glucose-responsive insulin secretion and release of cells entrapped in copolymer microcapsules.

Figure 7

Growth (A) and viability (B) of β-TC6 cells cultured in copolymer microcapsules on Day 7. The red rectangle marked some representative small clusters of β-TC6 cells.

Figure 8

Growth curves of β-TC6 cells in different culture manners.

Figure 9

Glucose stimulated insulin secretion and release by microencapsulated β-TC6 cells under the same glucose concentration of 1.0 mg∙mL⁻¹ at regular intervals.

Figure 10

Glucose stimulated insulin secretion and release of β-TC6 cells with gradient increased glucose concentration (0~5.0 mg∙mL⁻¹).

Figure 11

Circular dichroism spectra (A) and fluorescence emission spectra (B) of released insulin derived from free and microencapsulated β-TC6 cells.