Glucose-Sensitive Nanoparticles Based On Poly(3-Acrylamidophenylboronic Acid-Block-N-Vinylcaprolactam) For Insulin Delivery

Abstract

Background: Compared with random copolymers, block copolymerization is easier to prepare for nanoparticles with core-shell structure, and they will have better glucose sensitivity and higher insulin loading.

Purpose: In our study, insulin-loaded poly (3-acrylamidophenylboronic acid-block-N-vinyl caprolactam) p(AAPBA-b-NVCL) nanoparticles were successfully prepared and were glucose-sensitive, which could effectively lower the blood sugar levels within 72 hrs.

Methods: The polymer of p(AAPBA-b-NVCL) was produced by reversible addition-fragmentation chain transfer polymerization based on different ratios of 3-acrylamidophenylboronic acid (AAPBA) and N-vinylcaprolactam (NVCL), and its structure was discussed by Fourier transform infrared spectroscopy and 1H-nuclear magnetic resonance . Next, the polymer was manufactured into the nanoparticles, and the characteristics of nanoparticles were detected by dynamic light scattering, lower critical solution temperature, and transmission electron microscopy. After that, the cell and animal toxicity of nanoparticles were also investigated.

Results: The results demonstrated that p(AAPBA-b-NVCL) was successfully synthesized, and can be easily self-assembled to form nanoparticles. The new nanoparticles included monodisperse submicron particles, with the size of the nanoparticle ranged between 150 and 300nm and are glucose- and temperature-sensitive. Meanwhile, insulin can be easily loaded by p(AAPBA-b-NVCL) nanoparticles and an effective sustained release of insulin was observed when the nanoparticles were placed in physiological saline. Besides, MTT assay revealed that cell viability was more than 80%, and mice demonstrated no negative impact on blood biochemistry and heart, liver, spleen, lung, and kidney after intraperitoneal injection of 10 mg/kg/d of nanoparticles. This suggested that the nanoparticles were low-toxic to both cells and animals. Moreover, they could lower the blood sugar level within 72h.

Conclusion: Our research suggested that these p(AAPBA-b-NVCL) nanoparticles might have the potential to be applied in a delivery system for insulin or other hypoglycemic proteins.

Keywords: 3-acrylamidophenylboronic acid; AAPBA; N-vinylcaprolactam; NVCL; glucose-sensitive; nanoparticles; temperature-sensitive.

Scheme 1

Schematic representation of temperature- and glucose-sensitive poly (3-acrylamidophenylboronic acid-block-N-vinylcaprolactam) p(AAPBA-b-NVCL) nanoparticles.

Scheme 2

Synthesis of p(AAPBA).

Scheme 3

Synthesis of p(AAPBA-b-NVCL).

Figure 1

FT-IR spectra of NVCL, AAPBA, pAAPBA, and p(AAPBA-b-NVCL)3.

Figure 2

¹H-NMR spectra of (A) NVCL, (B) AAPBA, (C) pAAPBA, and (D) p(AAPBA-b-NVCL)3.

Figure 3

Hydrodynamic diameters at different: (A) pH, (B) temperatures, (C) glucose concentration, and (D) reversible glucose sensitivity.

Figure 4

TEM of p(AAPBA-b-NVCL)3 nanoparticles (A) before and (B) after treatment with 3 mg/mL glucose.

Figure 5

In vitro cumulative release of insulin at pH 7.4 in PBS from (A) was p(AAPBA), p(AAPBA-b-NVCL)1, p(AAPBA-b-NVCL)2, p(AAPBA-b-NVCL)3, and p(AAPBA-b-NVCL)4 at 0 mg/mL glucose concentration; (B) was p(AAPBA), p(AAPBA-b-NVCL)1, p(AAPBA-b-NVCL)2, p(AAPBA-b-NVCL)3, and p(AAPBA-b-NVCL)4 at 1 mg/mL glucose concentration; (C) was p(AAPBA), p(AAPBA-b-NVCL)1, p(AAPBA-b-NVCL)2, p(AAPBA-b-NVCL)3, and p(AAPBA-b-NVCL)4 at 3 mg/mL glucose concentration; and (D) was p(AAPBA-b-NVCL)3 nanoparticles at 0, 1, and 3 mg/mL glucose concentrations.

Figure 6

Cell viability of p(AAPBA-b-NVCL) nanoparticles by MTT assay at 37°C after incubation for 24 hrs. All values are presented as mean±SD (n = 5).

Figure 7

Representative images of HE staining results from (A) heart, (B) liver, (C) spleen, (D) lung, and (E) kidney with 1) control group (0mg); 2) p(AAPBA-b-NVCL)1 group (10mg); 3) p(AAPBA-b-NVCL)2 group (10mg); 4) p(AAPBA-b-NVCL)3 group (10mg); and 5) p(AAPBA-b-NVCL)4 group (10mg).

Figure 8

Blood glucose concentration after injection (A over 96 hrs, B over 6 hrs, because A did not show hypoglycemic effect within the first 6 hrs, and so we placed it separately, that is as B).