## 3 Results and Discussion
### Dissolution Studies  
The dissolution curves (i.e., the graphs of the percentage of insulin released from each insulin microparticle formulation plotted as a function of time) are shown in Figure 2.  
The AVP emulsion microparticles exhibited the highest release of insulin over the first 5 min reaching 84.8%, which increased to 92.2% at 10 min. A plateau was then reached, and the maximum insulin released from this microparticle formulation fluctuated around 92% for the duration of the dissolution test. This relatively fast release from the microparticles is desirable for the delivery of insulin into the brain after intranasal administration to achieve a quick pharmacological action.  
The E + AVP emulsion microparticles also showed relatively fast insulin release, with 73.6% insulin released over the first 5 min. Thereafter, the insulin release increased to 88.7% at 10 min and fluctuated around this value for the duration of this dissolution study. The inclusion of Eudragiti(r) L100 with the _A. vera_ polysaccharides in the microparticle formulation showed a very slight reduction in the rate and extent of insulin release in comparison to the microparticle formulation consisting of _A. vera_ polysaccharides only.  
The AVP ionic gelation microparticles showed a much slower rate and extent of an insulin release than the other two microparticle formulations, which were prepared by means of the emulsion-solvent evaporation method. At 5 min, the percentage of insulin release was only 31.1%, and a maximum insulin release of 45.9% was reached at 90 min. This slower release of insulin from these microparticles may be explained by the larger  
Figure 1: Confocal laser scanning microscope images of the three microparticle formulations, namely: (**A**) E + AVP emulsion, (**B**) AVP emulsion, and (**C**) AVP ionic gelation.  
Figure 2: Dissolution curves displaying the percentage of insulin released as a function of time for the different microparticle formulations. (\(\lozenge\)) E + AVP emulsion = microparticles prepared by emulsion-solvent evaporation consisting of Eudragit® L100 and _A. vera_ polysaccharides, (\(\square\)) AVP emulsion = microparticles prepared by emulsion-solvent evaporation consisting of _A. vera_ polysaccharides and (\(\Circinus\)) AVP ionic gelation = microparticles prepared by ionic gelation consisting of _A. vera_ polysaccharides.  
particle size of this formulation as well as possible ionic interactions of the cross-linker ions (Ca\({}^{2+}\)) between the insulin molecules and the _A. vera_ polysaccharide molecules. The cross-linker ions may, therefore, have formed bridges between the insulin molecules and the _A. vera_ polysaccharide molecules, which could have potentially decreased the release rate of the insulin molecules from the microparticles. However, this should be further investigated to be conclusive.  
Although immediate drug release from the dosage form is desirable for the intranasal route of drug administration to ensure quick drug uptake, the slower release of insulin from this microparticle formulation may provide an advantage in terms of insulin delivery across the nasal epithelium. This advantage can be explained as follows: the _A. vera_ polysaccharide molecules in the microparticles get the opportunity to first interact with the nasal epithelial tissue in order to open the tight junctions between epithelial cells, and thereafter, when the insulin molecules are released slowly, they can permeate through the intercellular spaces via opened tight junctions. This concept was previously shown for a biphasic intestinal insulin delivery system [40]. However, mucociliary clearance may prevent complete absorption, and therefore, the clinical effectiveness of insulin delivery from these microparticles should be investigated in vivo before final conclusions can be made.