Development of a pH-Sensitive Nanoparticle via Self-Assembly of Fucoidan and Protamine for the Oral Delivery of Insulin

Abstract

Objectives: Oral insulin delivery has received much attention over the past 20 years due to its high compliance. The aim of this study is to prepare nanoparticles for the oral delivery of insulin; Methods: Fucoidan and protamine were used to prepare a pH-sensitive nanoparticle via self-assembly. The secondary structure and in vitro stability of the nanoparticles were characterized using FTIR, XRD, ITC, and TEM. the nanoparticles had a controlled release effect on insulin in simulated intestinal fluid. The pre-liminary therapeutic effect on high-fat-fed type 2 diabetic mice; Results: When the fucoidan/protamine mass ratio was 10:3 (w/w), the particle size and zeta potential were 140.83 ± 1.64 nm and -48.13 ± 0.61 mV.The encapsulation efficiency of insulin was 62.97 ± 0.59%. The preliminary therapeutic effect on type 2 diabetic mice showed that the fasting blood glucose of diabetic mice decreased from 10.28 ± 0.88 mmol/L to 9.22 ± 0.64 mmol/L, the area under the curve value of oral glucose tolerance test was reduced by 11.70%, and the insulin se-cretion of diabetic mice was increased by 13.3%; Conclusions: The nanoparticles were prepared successfully by self-assembly. The empty and insulin-loaded nanoparticles remained stable in simulated gastric fluid, and the nanoparticles had a controlled release effect on insulin in simulated intestinal fluid. Moreover, insulin-loaded nanoparticles could relieve on type 2 diabetic mice.

Keywords: diabetic mice; drug delivery; in vitro stability; oral drug; thermodynamic property.

Figure 1

Spectroscopic analysis of fucoidan/protamine nanoparticles. (A) Fourier transform infrared spectroscopy (FTIR) spectra of fucoidan, protamine, fucoidan/protamine physical mixture, and fucoidan/protamine nanoparticles; (B) circular dichroism (CD) spectra; (C) X-ray diffraction (XRD) spectra of fucoidan, protamine, and fucoidan/protamine nanoparticles; (D) isothermal titration calorimetry (ITC) data for titration of protamine solution with fucoidan solution.

Figure 2

The in vitro stability of fucoidan/protamine nanoparticles in different digestive fluids. (A) The particle size and (B) zeta potential of fucoidan/protamine nanoparticles after incubation in simulated gastric fluid (SGF, pH 1.2), simulated intestinal fluid (SIF, pH 6.8), and simulated body fluid (SBF, pH 7.4), respectively. (C) The FTIR spectra of original fucoidan/protamine nanoparticles and the nanoparticles incubated in SGF, SIF, and SBF for 3 h, 12 h, and 12 h, respectively. (D) The micro morphologies of fucoidan/protamine nanoparticles. The scanning electron microscope (SEM) images of (a) original fucoidan/protamine nanoparticles and the nanoparticles incubated in (b) SGF for 3 h, (c) SIF for 12 h, and (d) SBF for 12 h. The different lowercase letters above the columns indicate statistical differences in the same digestive liquid at p  <  0.05.

Figure 3

In vitro stability of insulin-loaded fucoidan/protamine nanoparticles in different simulated digestive fluids. (A) The particle size and (B) zeta potential of insulin-loaded fucoidan/protamine nanoparticles after incubation in simulated gastric fluid (SGF, pH 1.2) for 3 h and simulated intestinal fluid (SIF, pH 6.8) for 12 h, respectively. (C) The FTIR spectra of insulin-loaded fucoidan/protamine nanoparticles incubated in SGF and SIF for 3 h and 12 h, respectively. (D) The micro morphologies of the insulin-loaded fucoidan/protamine nanoparticles. The transmission electron microscope (TEM) images of (a) original insulin-loaded fucoidan/protamine nanoparticles and the nanoparticles incubated in (b) SGF for 3 h and (c) SIF for 12 h. The different lowercase letters above the columns indicate statistical differences at p <  0.05 in the same digestive liquid.

Figure 4

In vitro release ratio of insulin encapsuled in nanoparticles in (A) simulated gastric fluid (SGF, pH 1.2) in 3 h and (B) simulated intestinal fluid (SIF, pH 6.8) in 12 h.

Figure 5

The various indexes of the type 2 diabetic mice treated with high-fat feeding (HFD) and HFD + insulin-loaded fucoidan/protamine nanoparticles (insulin-loaded NPs) for 21 d, respectively, n = 6. (A) Changes in body weight. (B) Fasting blood glucose before/after savage insulin-loaded NPs. (C) Oral glucose tolerance test (OGTT). (D) Area under the curve (AUC) value. (E) Serum insulin content. (F) Total triglyceride content.