Oligoarginine Modified Biodegradable Nanoparticles Improve Intestinal Absorption Of Insulin

During the past few decades, an increasing number of peptide-and protein-based Pharmaceuticals have been discovered, which were often administered parenterally because of insufficient oral bioavailability, owing to their low permeability through the intestinal mucosa associated with their hydrophilicity, high molecular weight, and susceptibility to enzymatic degradation. Numerous efforts have so far been made in order to reach an adequate bioavailability of these bioactivity macromolecules with the aid of an oral delivery system. Encapsulating bioactive macromolecules into nanocarriers has been widely used in pharmaceutics, which could protect drug from the harsh environment and enzymatic degradation in the gastrointestinal tract. However, theit transduction efficiency has yet to be improved.Cell-penetrating peptides (CPPs) are a class of transporters which consist of a group of cationic amino acid sequences with a significant capability for membrane translocation, which was found to be capable of translocating cell membranes and gaining intracellular access. Since then, CPPs has been widely used in enhancing intestinal drug absorption of peptide-and protein-based pharmaceuticals, such as a physical mixture between CPPs and drug without intermolecular cross-linking and covalently modifying drug with CPPs. However, the application of physical mixture is restricted only to negatively charged macromolecules and the bioactivity of macromolecules will be affected by covalently modification. Therefore, we devised an approach using CPPs modified nanoparticles to enhance the absorption of bioactive macromolecules, and hence to improve their oral bioavilibility. This nanoparticle delivery system has prospects to extend the application field of CPPs in oral drug delivery of biological macromolecules.As a model drug, insulin was incorporated in the poly(lactide-co-glycolide acid)(PLGA) nanoparticles, which surface was covalently decorated with CPPs via polyethylene glycol (PEG) bridge. The stability of drug in the gastrointestinal tract and increased membrane permeability benefit from the formulation contribute enough to improve oral bioavailabilities of peptide-and protein-based pharmaceuticals. CPPs composed of natural L-form amino acids are fragile when exposed on the nanoparticle surface, and liable to break down in the presence of gastrointestinal degradation enzymes. Whereas the D-form peptides are far more resistant to enzymatic degradation than their L-form enantiomers. In this study, poly(arginine)8(R8) enantiomers, both L-R8and D-R8were evaluated to compare the capacity of improving intestinal absorption of insulin.In the first part, the peptides of L/D-R8-FAM and L/D-R8-Cys were prepared by a solid phase peptide synthesis method using HBTU activation/DIEA in situ neutralization protocol for t-butoxycarbonyl chemistry. The purity of L/D-R8-FAM and L/D-R8-Cys determined by HPLC was above95%. Insulin-loaded PLGA nanoparticles (INS-NP) were prepared by a double emulsion-solvent evaporation method, and R8was subsequently conjugated to the surface of the INS-NP via polyethylene glycol bridge (R8-INS-NP). The morphology of INS-NP was spherical with a uniform size, ranging from about180nm to200nm. The entrapment efficiency of R8-INS-NP was (29.10±2.59)%and the drug loading capacity was (5.05±0.50)%. The Zeta potential of the NPs exhibited a significant change, from-34mV to about+15mV after the L/D-R8-Cys were decorated on their surface, for the reason that L/D-R8is positively charged under the normal physiological condition. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) results validated that L/D-R8-Cys had covalently conjugated to Mal-PEG-PLGA nanoparticles and covered on the surface of nanoparticles. The in vitro release behavior of insulin from the NPs in three differfent media showed a slow release in Kreb-Ringers solution, faster in artificial gastric juice and fastest in artificial stomach juice. The obtained INS-NPs were subjected to preliminary stability tests. Cryprotectants,3%sucrose, are needed in the freeze-dried PEG-PLGA nanoparticles. High temperature and illumination severely affected the stability of INS-NPs, so the preparation should be stored in4℃and dark condition.In the second part, a Caco-2cell monolayer model was established to evaluate the cellular uptake and transportation capabilities of L/D-R8and the nanoparticles with or without R8modification. The results manifested that L/D-R8-C6-NP was internalized by Caco-2cells more efficiently than unmodified C6-NP and showed mild cytotoxicity and reversible effect toward tight junctions. With R8modification, the percentages of internalized nanoparticles by Caco-2cell were1.5times higher than that of unmodified one and the percentage of cellular transportation was up to3.3times higher than the unmodified one. D-R8-NP exhibited a stronger internalizing ability than L-R8-NP, owing to D-form peptides are far more resistant to enzymatic degradation. In the third part, the results of in vivo pharmacokinetic and pharmacodynamic studies showed that more intestinal insulin absorption and more pronounced hypoglycemic effects were observed compared to the unmodified nanoparticles. With20%R8modification on the surface of INS-NP, the relative bioavailabilities of insulin were1.24%,3.87%,10.31%, and the pharmacological bioavailabilities were2.36%,3.96%,8.24%, for INS-NP, L-R8-INS-NP and D-R8-INS-NP, respectively. With50%R8modification, the relative bioavailabilities of insulin were3.13%,10.18%,13.91%, and the pharmacological bioavailabilities were3.02%,7.56%,11.49%, for INS-NP, L-R8-INS-NP and D-R8-INS-NP, respectively. The data showed that more obvious effects were found with increasing the surface modification of L/D-R8. Consistent with the results of in vitro cellular uptake and transportation, D-form R8was more powerful in promoting insulin absorption than its L-form enantiomer. LDH leakage and the microscopic observations studies showed that the R8modified nanoparticles were almost as safe as the unmodified PEG-PLGA nanoparticles.Therefore, nanoparticles decorated with oligoarginine, especially its D-form enantiomer, was an effective delivery system for improving oral bioavailability of macromolecules without causing detectable damage in intestinal tissues.