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Oral Delivery of Insulin: Meeting Challenges Through Nanoencapsulation and Emulsification/Internal Gelation

Por: | 26 de septiembre de 2013

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By Antonio José Ribeiro, University of Coimbra

Diabetes is one of the major causes of premature illness and death worldwide. For all people with type 1 diabetes and for some people with type 2 diabetes, insulin is essential to keep blood glucose levels under control. Insulin replacement therapy has been performed for decades by subcutaneous injections. The design of an alternative route for insulin delivery remains one of the most frustrating challenges in drug development. The oral route is potentially the most convenient delivery because it is non-invasive, decreases risk of contamination, and it is physiologically desirable, since the exogenous protein imitates the physiological pathway. However, it is limited by proteolytic degradation and the size of insulin, too large to transport across the intestinal mucosa.

A two-step strategy was adopted to overcome barriers. The first step was focused on insulin protection during manufacture and in the gut, and the second step, concentrated on promoting insulin absorption across the intestinal mucosa.

The technique chosen to protect insulin was encapsulation, which, in addition, allows us to produce a carrier with dimensions less than 1,000 µm (less than 1 millimeter). A mild encapsulation technique, called emulsification/internal gelation, was selected because it minimizes protein denaturation and the loss of biological activity, avoiding exposure to high temperatures and organic solvents. The relatively mild cross-linking conditions needed to obtain microparticles made of biopolymers, such as alginate and chitosan, as well as the minimal toxicity and good biodegradability of these two naturally occurring biopolymers, have enabled them to be used for the encapsulation of insulin.

Analysis of microparticles before and after a passage through simulated gastrointestinal conditions (in vitro assays) showed insulin activity was preserved. However, in the presence of gastric enzymes, a significant loss of insulin was found. We observed that microparticles with cores made of alginate and reinforced with dextran sulphate — to avoid gastric enzyme penetration into the microparticle network — could still protect insulin from these enzymes. These microparticles were coated first with chitosan/polyethylene glycol (PEG) to promote insulin passage through intestinal epithelium, then with a protein that could function as a trap for the gastric enzymes; the protein albumin revealed to be the best coating, protecting more than 80% of insulin. This protection effect of albumin drove our experiments to the second step.

By performing absorption studies of insulin-loaded microparticles into Caco-2 cells (human cells cultured under specific conditions; a method that resembles the processes of actual cells lining the small intestine), we observed values that referred to a very low passage of insulin but revealed an enhancing effect of the chitosan coating. We also learned that in vitro absorption assays may not produce reliable results and, instead, decided to make in vitro/in vivo correlation studies.

It should be emphasized that collaboration among the research groups of Coimbra, Kingston and Strasbourg allowed us to keep our minds focused. At this stage in our research, two paths were possible:

  • Improve the studies on the correlation of in vitro/in vivo intestinal uptake studies by using our particles, collect more data, and consequently design a better strategy
  • Take into account our results and results from other experiments related to intestinal uptake of similar proteins and particles, particularly how they reflect on the function of a nanoparticle’s biopolymers, and perform in vivo studies.

Our results together with the available data on intestinal uptake of particulates suggested a path towards decreasing the particle’s size below 10 µm and incorporating polyethylene glycol 4000 (PEG 4000) — a polymer described as a membrane permeation enhancer and a particle-shielding effector against enzymes, proteins and cells. Thus, we followed the second path. 

Novel insulin-loaded nanoparticles with a small size (50% less than 812 nm; note: 1,000 nm = 1 µm) and an encapsulation efficiency of 85% were produced by emulsification/internal gelation for the first time (consequently, an European Patent was requested). These nanoparticles were strengthened first by a PEG/chitosan coating, and then with albumin, which protected the insulin during production as well as from proteolytic degradation in the gut. The blood glucose reduction following the oral administration of these nanoparticles was greater than 70% of the basal value, while empty nanoparticles or nonencapsulated insulin were ineffective in producing a hypoglycaemic response. Interestingly, nanoparticles lacking albumin and PEG in the coating material were also ineffective, as it appears that albumin prevents a protease attack on the insulin, and PEG serves as a nanoparticle stabilizer. In contrast, the chitosan-PEG-albumin-coated particles increased blood insulin level by a factor of seven, demonstrating that exogenous insulin administered by nanoparticles were efficient, and significantly improved the response to the glucose oral tolerance test. Furthermore, insulin-loaded nanoparticles exert an anti-diabetogenic effect when administered orally in diabetic rats and show high intestinal uptake in different parts of the intestine.

These excipients we used are already well accepted in the pharmaceutical field, thus, we hope this formulation will be able to markedly improve the intestinal absorption of insulin and will be of interest in the treatment of diabetes with oral insulin. The benefits are considerable for insulin-dependent diabetics, particularly since our nanoparticles did not exhibit any toxicity in haematological parameters and organ histology.


Antonio José Ribeiro
University of Coimbra

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