Glucose- and temperature-sensitive nanoparticles for insulin delivery

Abstract

Glucose- and temperature-sensitive polymers of a phenylboronic acid derivative and diethylene glycol dimethacrylate (poly(3-acrylamidophenyl boronic acid-b-diethylene glycol methyl ether methacrylate); p(AAPBA-b-DEGMA)) were prepared by reversible addition-fragmentation chain transfer polymerization. Successful polymerization was evidenced by ¹H nuclear magnetic resonance and infrared spectroscopy, and the polymers were further explored in terms of their glass transition temperatures and by gel permeation chromatography (GPC). The materials were found to be temperature sensitive, with lower critical solution temperatures in the region of 12°C-47°C depending on the monomer ratio used for reaction. The polymers could be self-assembled into nanoparticles (NPs), and the zeta potential and size of these particles were determined as a function of temperature and glucose concentration. Subsequently, the optimum NP formulation was loaded with insulin, and the drug release was studied. We found that insulin was easily encapsulated into the p(AAPBA-b-DEGMA) NPs, with a loading capacity of ~15% and encapsulation efficiency of ~70%. Insulin release could be regulated by changes in temperature and glucose concentration. Furthermore, the NPs were non-toxic both in vitro and in vivo. Finally, the efficacy of the formulations at managing blood glucose levels in a murine hyperglycemic diabetes model was studied. The insulin-loaded NPs could reduce blood glucose levels over an extended period of 48 h. Since they are both temperature and glucose sensitive and offer a sustained-release profile, these systems may comprise potent new formulations for insulin delivery.

Keywords: 3-acrylamidophenylboronic acid; diethylene glycol methyl ether methacrylate; glucose sensitive; insulin delivery; nanoparticle; thermosensitive.

Figure 1

FT-IR spectra of AAPBA, DEGMA, p(AAPBA), and PAD-5-1. Note: PAD-5-1, p(AAPBA-b-DEGMA) (pAAPBA:DEGMA =1:5). Abbreviations: AAPBA, 3-acrylamidophenylboronic acid; DEGMA, diethylene glycol methyl ether methacrylate; p(AAPBA), poly(3-acrylamidophenylboronic acid); FTIR, Fourier transform infrared spectroscopy.

Figure 2

¹H-NMR spectra of (A) DEGMA, (B) AAPBA, (C) p(AAPBA), and (D) PAD-5-1. Notes: PAD-5-1, p(AAPBA-b-DEGMA) (pAAPBA:DEGMA =1:5). Peaks crossed through correspond to the solvents used for NMR. Abbreviations: ¹H-NMR, ¹H nuclear magnetic resonance; AAPBA, 3-acrylamidophenylboronic acid; DEGMA, diethylene glycol methyl ether methacrylate; p(AAPBA), poly(3-acrylamidophenylboronic acid).

Figure 3

Thermal analysis of p(AAPBA) and PAD-5-1, showing (A) DTG and (B) TG curves. Note: PAD-5-1, p(AAPBA-b-DEGMA) (pAAPBA:DEGMA =1:5). Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; DTG, derivative thermogravimetric; p(AAPBA), poly(3-acrylamidophenylboronic acid); TG, thermogravimetric.

Figure 4

Images of the p(AAPBA-b-DEGMA) NP suspensions (1) and TEM micro-graphs of the dried NPs (2). Notes: (A) PAD-50-1, (B) PAD-20-1, (C) PAD-10-1, (D) PAD-5-1, and (E) PAD-2-1. PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y. Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; NP, nanoparticle; p(AAPBA), poly(3-acrylamidophenylboronic acid); TEM, transmission electron microscopy.

Figure 5

The hydrodynamic diameters of copolymer NPs at different (A) pH and (B) temperatures. Note: PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y. Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; NP, nanoparticle; p(AAPBA), poly(3-acrylamidophenylboronic acid); T, temperature.

Figure 6

Changes in the size of the p(AAPBA-b-DEGMA) NPs as a function of immersion time (t) in pH 7.4 PBS solutions of glucose. Notes: (A) PAD-50-1; (B) PAD-20-1; (C) PAD-10-1; (D) PAD-5-1; and (E) PAD-2-1 are shown. The left images show the hydrodynamic diameters, while those on the right give I/I₀ (the light scattering intensity of the glucose-treated NP suspensions divided by the scattering intensity of the particles without glucose treatment). I/I₀ values reflected the extent of swelling. PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y. Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; NP, nanoparticle; p(AAPBA), poly(3-acrylamidophenylboronic acid); PBS, phosphate-buffered saline.

Figure 6

Changes in the size of the p(AAPBA-b-DEGMA) NPs as a function of immersion time (t) in pH 7.4 PBS solutions of glucose. Notes: (A) PAD-50-1; (B) PAD-20-1; (C) PAD-10-1; (D) PAD-5-1; and (E) PAD-2-1 are shown. The left images show the hydrodynamic diameters, while those on the right give I/I₀ (the light scattering intensity of the glucose-treated NP suspensions divided by the scattering intensity of the particles without glucose treatment). I/I₀ values reflected the extent of swelling. PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y. Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; NP, nanoparticle; p(AAPBA), poly(3-acrylamidophenylboronic acid); PBS, phosphate-buffered saline.

Figure 7

In vitro release of the insulin-loaded I-L-4 particles in PBS (pH 7.4) at different (A) temperatures and (B) glucose concentrations. Note: I-L-4, insulin-loaded NPs prepared from a 1 mg·mL⁻¹ insulin solution. Abbreviations: NP, nanoparticle; PBS, phosphate-buffered saline.

Figure 8

Cell viability as a function of the concentration of p(AAPBA-b-DEGMA) NPs, as assessed using the MTT assay at 37°C after 24 h exposure. Notes: The data are expressed as mean ± SD from five independent experiments, with three replicates per experiment. PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y. The concentration refers to the initial concentration of the NP suspension; the final concentration in culture is one-third of this value. Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; NP, nano-particle; p(AAPBA), poly(3-acrylamidophenylboronic acid); SD, standard deviation.

Figure 9

Representative HE staining images (200× magnification) from the (A) liver, (B) lung, (C) spleen, (D) kidney, and (E) heart with images marked (1) denoting the negative control group, and the observation groups were given (2) 10, (3) 20, and (4) 40 mg·kg⁻¹·d⁻¹ of PAD-5-1 by IP injection. Note: PAD-5-1, p(AAPBA-b-DEGMA) (pAAPBA:DEGMA =1:5). Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; HE, hematoxylin–eosin; IP, intraperitoneal; p(AAPBA), poly(3-acrylamidophenylboronic acid).

Figure 10

Blood glucose concentrations after the treatment of diabetes mellitus mice with insulin formulations. Notes: The control group comprised normal mice, and the NPs group animals treated with insulin-loaded PAD-5-1 [I-L-4]. I-L-4 nanoparticles were prepared using a 1 mg·mL⁻¹ insulin solution; PAD-5-1, p(AAPBA-b-DEGMA) (pAAPBA:DEGMA =1:5). Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; NP, nanoparticle; p(AAPBA), poly(3-acrylamidophenylboronic acid).