Synthesis and characterization of a novel peptide-grafted Cs and evaluation of its nanoparticles for the oral delivery of insulin, in vitro, and in vivo study

Abstract

Background: Despite years of experience and rigorous research, injectable insulin is the sole trusted treatment method to control the blood glucose level in diabetes type 1 patients, but injection of insulin is painful and poses a lot of stress to the patients, especially children, therefore, development of a non-injectable formulation of insulin is a major breakthrough in the history of medicine and pharmaceutical sciences.

Methods: In this study, a novel peptide grafted derivative of chitosan (CPP-g- chitosan) is synthesized and its potential for oral delivery of proteins and peptides is evaluated. Drug-loaded nanoparticles were developed from this derivative using ionic gelation method with application of sodium tripolyphosphate (TPP) as a cross-linking agent. Human insulin was used as the model protein drug and release kinetic was studied at gastrointestinal pH. Finally the developed nanoparticles were filled into very tiny enteric protective capsules and its effects on blood glucose level are evaluated in laboratory animals.

Results: Presence of the positively charged cell-penetrating peptide moiety in the structure of chitosan polymer had slight inhibitory effects on the release of insulin from the nanoparticles in simulated gastric fluid (pH 1.2) comparing to native chitosan. The nanoparticles were positively charged in gastrointestinal pH with size ranging from 180 nm to 326 nm. The polypeptide grafted to chitosan is a novel analog of Penetratin, presenting both the hydrophilic and hydrophobic characteristics altering the release behavior of the nanoparticles and significantly increase the absorption of insulin into the rat epithelium comparing to nanoparticles from simple chitosan. In-vivo results in diabetic rat proved that this nanoparticulate system can significantly lower the blood glucose levels in diabetic rats and remain effective for a duration of 9-11 hours.

Conclusion: The results indicate that nanoparticles developed from this new peptide conjugated derivative of chitosan are very promising for oral delivery of proteins and peptides.

Keywords: cell-penetrating peptide; oral delivery; penetratin; peptide grafted.

Figure 1

Chemical structure of chitosan (A), the novel peptide-grafted derivative of chitosan (B) and the schematic representation for the chemical attachment of CPP sequence to the amine groups of chitosan resulting in a novel peptide-grafted derivative of chitosan. Abbreviations: CPP, cell-penetrating peptide; EDC, N-ethyl-N′-(3-dimethylam-inopropyl) carbodiimide.

Figure 2

FTIR spectra of native chitosan (A) and the new peptide-grafted derivative of chitosan (B). Abbreviation: FTIR, Fourier transform infrared.

Figure 3

¹H NMR spectra of both Cs and CPP-PEG-Cs confirming successful covalent conjugation with CPP; (A) native Cs, (B) peptide-grafted derivative of Cs (CPP-PEG-Cs). Notes: The chemical shift at δ 6.7–8.2 belongs to the aromatic protons of the phenyl alanine moiety, which is present in the structure of CPP conjugated to Cs (B); multiple peaks of oxymethyl groups in PEG at δ 3.3–3.7 cover the signals of protons related to pyranose ring of Cs in spectra (A). The characteristic peak at δ 2.05 is related to protons of methoxy groups of Cs as seen in both spectra. The multiple peaks at δ 1.1–1.4 in spectra (B) are from the −CH₂−CH₂−CH₂−NH−NH−NH₂ in arginine amino acid in the CPP. Abbreviations: CPP, cell-penetrating peptide; Cs, chitosan; ¹H NMR, ¹H nuclear magnetic resonance; PzEG, polyethylene glycol.

Figure 4

SEM image of insulin-loaded nanoparticles from native chitosan; size distribution is between 100 and 450 nm. Abbreviation: SEM, scanning electron microscopy.

Figure 5

SEM image of insulin-loaded nanoparticles from peptide-grafted derivative of chitosan; size distribution is between 120 and 250 nm. Abbreviation: SEM, scanning electron microscopy.

Figure 6

Insulin release profile of nanoparticles fabricated from native chitosan (Cs) and the new peptide-grafted derivative of Cs (CPP-g-Cs) at three different pH values. Note: ●: Nanoparticles from Cs, ▲: nanoparticles from CPP-g-Cs. Abbreviations: CPP, cell-penetrating peptide; Cs, chitosan.

Figure 7

Glucose level changes (% base level) vs time profiles in diabetic rats following the administration of different formulations. Note: ■: Insulin-loaded NPs from native Cs in enteric capsules (oral, 30 IU/kg); ▲: insulin-loaded NPs from CPP-g-Cs in enteric capsules (oral, 30 IU/kg); ●: simple insulin powder in enteric capsules (oral, 30 IU/kg); ×: simple insulin solution (SC, 5 IU/kg). Abbreviations: CPP, cell-penetrating peptide; Cs, chitosan; CPP-g-Cs, peptide-grafted derivative of chitosan; NPs, nanoparticles; SC, subcutaneous.

Figure 8

Plasma insulin level vs time profiles (μIU/mL) following the administration of different formulations. Note: ×: Simple insulin solution (SC, 5 IU/kg); ■: insulin-loaded NPs from native Cs in enteric capsules (oral, 30 IU/kg); ▲: insulin-loaded NPs from CPP-g-Cs in enteric capsules (oral, 30 IU/kg); ●: simple insulin powder in enteric capsules (oral, 30 IU/kg). Abbreviations: CPP, cell-penetrating peptide; Cs, chitosan; CPP-g-Cs, peptide-grafted derivative of chitosan; NPs, nanoparticles; SC, subcutaneous.