Study On Nanoparticles Co-modified With Cell Penetrating Peptides And Engrailed Secretion Peptide For Oral Insulin Delivery

In recent years,oral delivery of peptide and protein drugs has remained to be a great challenge owing to their poor stability in the gastrointestinal tract and low permeability through the intestinal epithelium membrane caused by their high molecular weights and hydrophilicity inherently.To improve bioavailability of these drugs by oral administration,various promising strategies have been utilized to circumvent these obstacles.Biodegradable polymer nanoparticle drug carriers have been considered as highly promising tools to improve oral bioavailability of peptide and protein drugs,as nanoparticles（NPs）possess the ability not only to improve the stability of encapsulated drugs but also to increase their retention time in the gastrointestinal tract.However,the ability of nanoparticles to cross the intestinal epithelium membrane is largely limited.Cell-penetrating peptides（CPPs）have received considerable interest as attractive delivery vehicles owing to their ability to cross the cellular membrane and mediate the uptake of cargoes into cells.CPPs have been harnessed to traverse epithelial barriers for transepithelial drug delivery.It was reported that CPPs could be applied for enhanced intestinal insulin absorption.The Sec peptide（QSLAQELGLNERQIKI）,a sixteen amino acid sequence derived from the second and third helix of the Engrailed homeodomain,was essential for Engrailed secretion.Based on such a background,we speculated that transcellular transport of peptide and protein drugs in intestinal enterocytes might be improved by using the Sec peptide together with CPPs,since Sec might promote the exocytosis of the cargoes after internalization.Therefore,in the present study,CPPs and Sec with N-terminal stearylation were introduced to modify nanoparticles on the surface to improve oral bioavailability of peptide and protein drugs.Insulin was selected as the representative model drug.Three different cationic CPPs,including penetratin,octaarginine（R8）and Tat,were used here since they are the most extensively utilized CPPs.Insulin loaded nanoparticles modified with only penetratin（Pen-NPs）and modified together with Sec and penetratin（Sec-Pen-NPs）were prepared by using the emulsion solvent evaporation method.A low molecular weight emulsifier was used for the preparation of modified nanoparticles.As a control group,unmodified nanoparticles were prepared using PVA as the emulsifier.The encapsulated Insulin-phospholipid（Ins-SPC）complex was formulated by an anhydrous co-solvent lyophilization method in order to improve drug liposolubility and thus the encapsulation efficiency.The transmission electron microscopy（TEM）images of Pen-NPs and Sec-Pen-NPs demonstrated that the nanoparticles had regular spherical shapes and similar particle size in aqueous solution.The mean diameters of Sec-Pen-NPs and Pen-NPs exhibited around 147.5 nm and 163.1 nm,respectively.Unmodified nanoparticles displayed the negative surface charge（-15.6±2.3 m V）.In contrast,Pen-NPs and Sec-Pen-NPs were positively charged between 20 and 30 m V.The encapsulation efficiency and drug loading of insulin loaded nanoparticles were around 60%and 2.5%,respectively.The release profiles of insulin from nanoparticles in simulated intestinal medium（PBS,p H 6.8）without trypsin was investigated.Insulin was released about 50%from unmodified nanoparticles for 8 h.The amount of insulin released from Pen-NPs and Sec-Pen-NPs was around 35%for 8 h.The transcellular transport,cellular uptake and cytotoxicity studies were conducted in human colon carcinoma cells（Caco-2 cells）.Coumarin-6 was selected as the representative fluorescence probe for assessing in vitro behavior of cellular uptake and transport of nanoparticles.The results showed the value of the apparent permeability coefficient(P_app)of the nanoparticles co-modified with Sec and penetratin（Sec-Pen-NPs）was about 2-times greater than that of the nanoparticles modified with only penetratin（Pen-NPs）,while the increase of transcellular transport of nanoparticles modified together with Sec and R8（Sec-R8-NPs）,or Sec and Tat（Sec-Tat-NPs）,was not significant compared with nanoparticles modified with only R8（R8-NPs）or Tat（Tat-NPs）.The pharmacokinetic and pharmacodynamic studies were further conducted on normal male Sprague Dawley rats by ileal segments administration to evaluate the efficacy of modified nanoparticles loaded with insulin.Sec-Pen-NPs and Pen-NPs were used for further in vivo studies by loading insulin according to the results of in vitro studies.The relative bioavailability（BA%）of insulin for unmodified nanoparticles,Pen-NPs and Sec-Pen-NPs were 6.01±2.07%,11.17±2.05%,19.10±2.58%.The pharmacological bioavailability（PA%）of insulin for unmodified nanoparticles,Pen-NPs and Sec-Pen-NPs were 5.61±1.59%,9.18±1.32%,14.72±2.73%.These results indicated that compared to Pen-NPs,the relative bioavailability of insulin for Sec-Pen-NPs was 1.71-times increased after ileal segments administration.Stronger hypoglycemic effects was also observed.Therefore,the nanoparticle-based drug carrier co-modified with penetratin and Sec provides insight into the oral delivery of insulin.