A novel nanoemulsion-based method to produce ultrasmall, water-dispersible nanoparticles from chitosan, surface modified with cell-penetrating peptide for oral delivery of proteins and peptides

Abstract

A simple and reproducible water-in-oil (W/O) nanoemulsion technique for making ultrasmall (<15 nm), monodispersed and water-dispersible nanoparticles (NPs) from chitosan (CS) is reported. The nano-sized (50 nm) water pools of the W/O nanoemulsion serve as "nano-containers and nano-reactors". The entrapped polymer chains of CS inside these "nano-reactors" are covalently cross-linked with the chains of polyethylene glycol (PEG), leading to rigidification and formation of NPs. These NPs possess excessive swelling properties in aqueous medium and preserve integrity in all pH ranges due to chemical cross-linking with PEG. A potent and newly developed cell-penetrating peptide (CPP) is further chemically conjugated to the surface of the NPs, leading to development of a novel peptide-conjugated derivative of CS with profound tight-junction opening properties. The CPP-conjugated NPs can easily be loaded with almost all kinds of proteins, peptides and nucleotides for oral delivery applications. Feasibility of this nanoparticulate system for oral delivery of a model peptide (insulin) is investigated in Caco-2 cell line. The cell culture results for translocation of insulin across the cell monolayer are very promising (15%-19% increase), and animal studies are actively under progress and will be published separately.

Keywords: Caco-2 cell; cell-penetrating peptide; chitosan; nanoemulsion; oral insulin; ultrasmall.

Figure 1

Schematic representation of (A) covalent cross-linking of CS with PEG and (B) covalent conjugation of CPP sequence with the cross-linked NPs. Abbreviations: CPP, cell-penetrating peptide; CS, chitosan; EDC, N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide; NPs, nanoparticles; PEG, polyethylene glycol.

Figure 2

Schematic representation of NP fabrication and drug loading process. Abbreviations: CPP, cell-penetrating peptide; CS, chitosan; NPs, nanoparticles; PEG, polyethylene glycol.

Figure 3

Particle size and shape of CS-PEG NPs in dry (not swollen) state taken by SEM. Abbreviations: CS, chitosan; NPs, nanoparticles; PEG, polyethylene glycol; SEM, scanning electron microscopy.

Figure 4

Particle size and shape of CS-PEG-CPP NPs in dry (not swollen) state taken by SEM. Abbreviations: CPP, cell-penetrating peptide; CS, chitosan; NPs, nanoparticles; PEG, polyethylene glycol; SEM, scanning electron microscopy.

Figure 5

¹H NMR studies of CPP-conjugated NPs confirming successful cross-linking with PEG and covalent conjugation with CPP. Notes: A, simple CS; B, PEGylated CS (CS-N-PEG); C, CPP-tagged PEGylated CS (PEG-N-CS-N-CPP). The chemical shift at δ 6.7–7.8 belongs to the aromatic protons of the phenylalanine moiety, which is present in the spectra (C). In spectra, (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 all spectra: A, B and C. The multiple peaks at δ 1.3–1.7 in spectra (C) are from the –CH₂–CH₂–CH₂–NH–NH–NH₂ in arginine amino acid in the CPP. Abbreviations: CPP, cell-penetrating peptide; CS, chitosan; NMR, nuclear magnetic resonance; NPs, nanoparticles; PEG, polyethylene glycol.

Figure 6

TEM image of cross-linked NPs. Note: Before loading with insulin, the size distribution is between 10 and 20 nm. Abbreviations: NPs, nanoparticles; TEM, transmission electron microscopy.

Figure 7

TEM image of the insulin-loaded NPs. Note: The size distribution is between 234 and 367 nm. Abbreviations: NPs, nanoparticles; TEM, transmission electron microscopy.

Figure 8

Insulin release profile from CPP-conjugated CS NPs in the following: A, phosphate buffer (pH 7.4); B, SIF (pH 6.8). Abbreviations: CPP, cell-penetrating peptide; CS, chitosan; NPs, nanoparticles; SIF, simulated intestinal fluid.

Figure 9

Effect of different formulations on TEER. Notes: A, simple solution of regular human insulin (1.6 mg/mL); B, simple solution of aspart insulin (1.6 mg/mL); C, dispersion of plain CS NPs (10 mg/mL); D, CPP-conjugated CS NPs loaded with regular human insulin (10 mg/mL); E, CPP-conjugated CS NPs loaded with aspart insulin (10 mg/mL); F, simple solution of CPP (PenetraMax; 10 µM/mL). Abbreviations: CPP, cell-penetrating peptide; CS, chitosan; NPs, nanoparticles; TEER, transepithelial electrical resistance.

Figure 10

Cumulative transported insulin from different formulations. Notes: A, CPP-conjugated CS NPs loaded with regular human insulin (10 mg/mL); B, CPP-conjugated CS NPs loaded with aspart insulin (10 mg/mL); C, dispersion of plain CS NPs loaded with regular human insulin (10 mg/mL); D, simple solution of regular human insulin (1.6 mg/mL); E, simple solution of aspart insulin (1.6 mg/mL); F, dispersion of plain CS NPs loaded with aspart insulin (10 mg/mL). Abbreviations: CPP, cell-penetrating peptide; CS, chitosan; NPs, nanoparticles.