Elsevier

Biomaterials

Volume 30, Issue 12, April 2009, Pages 2329-2339
Biomaterials

In vivo evaluation of safety and efficacy of self-assembled nanoparticles for oral insulin delivery

https://doi.org/10.1016/j.biomaterials.2008.12.066Get rights and content

Abstract

A variety of approaches have been studied in the past to overcome the problems encountered with the oral delivery of insulin, but with little success. In this study, self-assembled nanoparticles (NPs) with a pH-sensitive characteristic were prepared by mixing the anionic poly-γ-glutamic acid solution with the cationic chitosan solution in the presence of MgSO4 and sodium tripolyphosphate. The in vitro results found that the transport of insulin across Caco-2 cell monolayers by NPs appeared to be pH-dependent; with increasing pH, the amount of insulin transported decreased significantly. An in vivo toxicity study was performed to establish the safety of the prepared NPs after oral administration. Additionally, the impact of orally administered NPs on the pharmacodynamics (PD) and pharmacokinetics (PK) of insulin was evaluated in a diabetic rat model. The in vivo results indicated that the prepared NPs could effectively adhere on the mucosal surface and their constituted components were able to infiltrate into the mucosal cell membrane. The toxicity study indicated that the NPs were well tolerated even at a dose 18 times higher than that used in the PD/PK study. Oral administration of insulin-loaded NPs demonstrated a significant hypoglycemic action for at least 10 h in diabetic rats and the corresponding relative bioavailability of insulin was found to be 15.1 ± 0.9%. These findings suggest that the NPs prepared in the study are a promising vehicle for oral delivery of insulin.

Introduction

Currently, multiple daily injections of insulin are the standard treatment for insulin-dependent diabetic patients. However, inadequate control of blood glucose level and poor patient compliance are associated drawbacks with this treatment [1], [2]. Oral administration of insulin may be beneficial to such patients as it can mimic the physiological fate of insulin and might provide a better glucose homeostasis [3]. Nevertheless, orally administered insulin is encountered with many difficulties such as rapid degradation and poor intestinal absorption [4]. Therefore, a delivery system is needed to protect the drug from the harsh environment in the gastrointestinal (GI) tract. An ideal delivery system for oral administration of insulin should prolong its intestinal residence time, reversibly increase the permeability of the mucosal epithelium to enhance the absorption of drug and provide the intact drug to the systemic circulation [1]. Additionally, this delivery system must be safe after oral administration.

Chitosan (CS), a natural-origin polymer, is non-toxic and soft-tissue compatible [5]. It has been widely used as a food additive and as a weight-loss product and been developed as a safe excipient in drug formulations [6]. Additionally, CS, an excellent mucoadhesive agent, has been widely used as a paracellular permeability enhancer for increasing the absorption of hydrophilic drugs [6], [7].

In the study, a nanoparticle (NP) delivery system self-assembled by the positively charged CS and the negatively charged poly-γ-glutamic acid (γ-PGA) for oral administration of insulin was developed. The pKa of CS is 6.5 [8]; thus, the amine groups on CS are deprotonated at pH values above 6.5, leading to the disintegration of NPs. It is known that the natural pH environment in the GI tract varies from acidic in the stomach to slightly alkaline in the small intestine [9], [10]. To improve their stability in a broader pH range, magnesium sulfate (MgSO4) and tripolyphosphate sodium (TPP) were introduced in the preparation of NPs. Evaluation of the prepared NPs in enhancing the intestinal paracellular transport of insulin was investigated in vitro in Caco-2 cell monolayers. An in vivo toxicity study was performed to evaluate the safety of the NP delivery system. Additionally, the efficacy of NPs for oral delivery and intestinal absorption of insulin was investigated in a diabetic rat model.

Section snippets

Preparation and characterization of test NPs

CS (MW 80 kDa) with a degree of deacetylation of approximately 85% was acquired from Koyo Chemical Co. Ltd. (Japan), while γ-PGA (MW 60 kDa) was purchased from Vedan Co. Ltd. (Taichung, Taiwan). A sample of 100 mg of insulin (from bovine pancreas, 27.4 IU/mg, Sigma–Aldrich, St. Louis, MO, USA) was dissolved in 10 mL of 0.01 N HCl and this solution was neutralized with 0.1 N NaOH [11]. The insulin solution was then diluted with deionized (DI) water to make a stock solution of 1 mg/mL insulin. Test NPs

Results

The luminal pH of the proximal small intestine (duodenum and jejunum) is approximately 6.0–7.0, while the pH of the body fluid underneath the epithelial cells is about 7.4, leading to a pH gradient between the intestinal lumen and the basolateral sides of epithelial cells [9], [24]. Thus, the hypothesis of the study is that the orally administered NPs with excessive mucoadhesive CS on their surfaces may adhere and infiltrate into the mucus of the intestinal tract. The infiltrated NPs can

Discussion

The efficacy of oral delivery of peptides and proteins is often limited because of their inherent instability in the GI tract and their low permeability across the epithelial membranes. The high molecular weight of this class of drugs coupled with their hydrophilic nature restricts their transcellular permeation [4]. Thus, enhancement of the paracellular permeation is a more feasible alternative for oral delivery of peptides and proteins [26]. CS is a well-known mucoadhesive agent with the

Conclusions

In this study, self-assembled NPs with a pH-sensitive characteristic were prepared for oral delivery of insulin. The in vivo toxicity study demonstrated the safety of the prepared NPs after oral administration. Additionally, the pharmacodynamic and pharmacokinetic evaluation of orally administered NPs in diabetic rats indicated that the intestinal absorption of insulin was significantly enhanced. These results suggested that the NPs developed in the study might be employed as a potential

Acknowledgment

This work was supported by a grant from the National Science Council (NSC 97-2120-M-007-001), Taiwan, Republic of China.

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