Investigation Of Vitamin B12-Modified Amphiphilic Sodium Alginate Derivatives For Enhancing The Oral Delivery Efficacy Of Peptide Drugs

Abstract

Purpose: Peptide drugs have been used in therapy various diseases. However, the poor bioavailability of peptide drugs for oral administration has limited their clinical applications, on account of the acidic environment and digestive enzymes inside the human gastrointestinal tract. To enhance stability in the human gastrointestinal tract, bioavailability, and targeted drug delivery of peptide drugs through oral administration, a vitamin B₁₂-modified amphiphilic sodium alginate derivative (CSAD-VB₁₂) was synthesized.

Materials and methods: A vitamin B₁₂-modified amphiphilic sodium alginate derivative (CSAD-VB₁₂) was synthesized via the N,N'-dicyclohexylcarbodiimide active method at room temperature, and then characterized using FTIR and ¹H NMR spectroscopy. Insulin was used as a model peptide drug and the insulin-loaded CSAD-VB₁₂ (CSAD-VB₁₂/insulin) nanoparticles with negative zeta potentials were prepared in PBS (pH=7.4). Scanning electron microscopy was used to observe CSAD-VB₁₂/insulin as spherical nanoparticles. The CSAD-VB₁₂ derivatives and CSAD-VB₁₂/insulin nanoparticles displayed nontoxicity towards the human colon adenocarcinoma (Caco-2) cells by CCK-8 test. Caco-2 cell model was used to measure the apparent permeability (Papp) of insulin, CSAD/insulin and CSAD-VB₁₂/insulin. Furthermore, confocal was used to confirm the endocytosis of intestinal enterocytes. Type 1 diabetes mice were used to evaluate the intestinal absorption and retention effect of test nanoparticles.

Results: They were observed as spherical nanoparticles in the size of 30-50 nm. The CSAD-VB₁₂ derivatives and CSAD-VB₁₂/insulin nanoparticles displayed nontoxicity towards the human colon adenocarcinoma (Caco-2) cells. Comparing with insulin and the CSAD/insulin nanoparticles, the CSAD-VB₁₂/insulin nanoparticles exhibited higher permeation ability through intestinal enterocytes in the Caco-2 cell model. Oral administration of the CSAD-VB₁₂/insulin nanoparticles to Type 1 diabetic mice yields higher intestinal retention effect, targeted absorption, and outstanding efficacy.

Conclusion: CSAD-VB₁₂ derivatives enhance the small intestinal absorption efficacy and retention of peptide by oral administration, which indicated that it could be a promising candidate for oral peptide delivery in the prospective clinical application.

Keywords: human colon adenocarcinoma cell; insulin; nanoparticle; small intestinal absorption efficacy.

Scheme 1

(A) Chemical structures of the CSAD derivative and CSAD-VB₁₂ derivative and (B) proposed self-assembly mechanism of CSAD-VB₁₂/insulin nanoparticles.

Scheme 2

Synthesis of the CSAD and CSAD-VB₁₂ derivatives.

Figure 1

FTIR spectra of (A) the CSAD derivative and (B) sodium alginate.

Figure 2

¹H NMR spectra (400 MHz, D₂O, 25ºC) of (A) sodium alginate, (B) the CSAD derivative, (C) the CSAD-VB₁₂ derivative, and (D) vitamin B₁₂ (a–c: the signal at δ 4.67 ppm for HDO was suppressed).

Figure 3

UV-vis spectra of VB₁₂ and the CSAD-VB₁₂ derivative.

Figure 4

(A) Photo of the solution of CSAD-VB₁₂/insulin nanoparticles, (B) SEM image of CSAD-VB₁₂/insulin nanoparticles, (C) hydrodynamic diameter distribution of CSAD-VB₁₂/insulin nanoparticles.

Figure 5

In vitro release profiles of insulin from CSAD-VB₁₂/insulin nanoparticles in the simulated gastrointestinal fluids: (A) the simulated stomach fluid, (B) the duodenum fluid, and (C) the small intestinal fluid.

Figure 6

Influence of cell viability of Caco-2 cells incubated with the CSAD and CSAD-VB₁₂ derivatives, CSAD/Insulin and CSAD-VB_12/Insulin nanoparticles on the concentration of CSAD and CSAD-VB₁₂ (n = 3, mean ± standard deviation, P > 0.05).

Figure 7

Permeability and cellular uptake study. (A) Papp of different insulin formulations: insulin, the CASD/Insulin nanoparticles and the CASD-VB₁₂/Insulin nanoparticles (n = 3; data shown are mean ± SD, *P < 0.05 in comparison with insulin group). (B) Confocal laser scanning microscopic images of Caco-2 cells incubated with the CSAD-VB₁₂/FITC-insulin nanoparticles at 37°C for 2 hrs, and their overlay at the 0, 1, and 2 hrs after incubation. (C) The quantification of FITC in cells (values represented as the mean ± standard deviation (n = 3), **p <0.01, compared to 0 hr).

Figure 8

The retention effect and absorption of FITC-insulin, CSAD/FITC-insulin, CSAD-VB₁₂/FITC-insulin in intestine. (A) In vivo animal image system showed the fluorescent signal of FITC-insulin (A1) , CSAD/FITC-insulin (A2), CSAD- VB₁₂/FITC-insulin (A3) at the 1 h after oral administration in T1D mice. The fluorescent intensity in GI of each group was quantified in (E). (B) Representative fluorescence imaging of small intestine from mice treated with FITC-insulin (B1), CSAD/FITC-insulin (B2), CSAD- VB₁₂/FITC-insulin (B3). The fluorescent intensity in isolated small intestine was quantified in (F). (C) Intestinal absorption of FITC-insulin (C1), CSAD/FITC-insulin (C2), CSAD- VB₁₂/FITC-insulin (C3) after removal of mucus. The fluorescent intension of FITC-insulin in intestinal villus cells was qualified as the rate of FITC/DAPI in (G). (D) Blood glucose change of T1D mice after oral administration of CSAD/insulin, CSAD- VB₁₂/insulin (insulin, 70 IU/kg, free INS, and saline as control). Subcutaneous (s.c.) injection with insulin at a dose of 5 IU/kg was viewed as a positive control. ***p < 0.001 for comparisons with other group.

Scheme 3

Proposed schematic presentation of CASD-VB₁₂/Insulin nanoparticles permeating through monolayer of intestinal enterocytes.