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