Insulin nanocomplexes formed by self-assembly from amine-modified poly(vinyl alcohol)-graft-poly(L-Lactide) for non-invasive mucosal delivery: Preparation, characterization and in vivo investigations

In this work biodegradable DEAPA-PVAL-g-PLLA nanocomplexes were investigated as a colloidal peptide carrier system for non-invasive transmucosal insulin delivery. Chapter 1 describes the basic fundamentals of insulin therapy, current status, problems and future trends. The pathogenesis of diabetes m...

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Bibliographic Details
Main Author: Simon, Michael
Contributors: Kissel, Thomas (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2005
Pharmazeutische Technologie und Biopharmazie
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Summary:In this work biodegradable DEAPA-PVAL-g-PLLA nanocomplexes were investigated as a colloidal peptide carrier system for non-invasive transmucosal insulin delivery. Chapter 1 describes the basic fundamentals of insulin therapy, current status, problems and future trends. The pathogenesis of diabetes mellitus and the different treatment options are discussed to give an understanding of the necessity for alternative non-invasive application systems. It was emphasized that epithelial barriers and enzymatic degradation prevent effective transmucosal insulin delivery. The potential of colloidal bioadhesive polymeric carriers for oral, pulmonal or nasal application of insulin were discussed. The factors bioadhesivity and complexing-capability of polymers were considered with regard to literature. Reference was made to other fields where charge modified grafted polyester of theses novel class were already succesfully utilized for drug delivery. In Chapter 2 the DEAPA-PVAL-g-PLLA polymers were characterized for their substitution degree of amine-groups and the extent of lactide grafting. It was possible to manipulate the hydrophilic-hydrophobic balance of these polyesters by factors such as molecular weight, PLLA chain lengths and degree of amine-substitution. Water solubility of these polymers allowed the spontaneous self-assembling with insulin to small nano-sized complexes with narrow size distribution. The formulation of nanocomplexes made of polymers with varying structural composition were characterized for various physico-chemical characteristics, such as size distribution, surface charge and insulin association efficiency. Studies with isothermal titration calorimetry demonstrated the influence of the structural architecture on binding constants. An increase in binding affinity, drug loading, zeta potential and a decrease of complex size could be correlated with a higher substitution degree of DEAPA groups and a higher lactic acid grafting of the backbone. Systemic investigations of the complexation process, using nephelometric and light scattering techniques, allowed the evaluation of an optimal ratio of the binding partners. The complexes visualized by atomic force microscopy revealed a homogeneous particle collective with spheroidal complexes exhibiting an entangled internal structure. In Chapter 3 the suitability of insulin containing DEAPA-PVAL-g-PLLA nanocomplexes as a nasal insulin delivery system was studied under in-vivo conditions at wistar rats. Changes in blood glucose and insulin concentration were monitored in anaesthetized rats using a glucose meter and ELISA. The blood glucose response revealed a significant decrease of glucose concentration, which was accompanied by an increase of exogenous insulin concentration in rat blood. From the investigated two different NC options, the more amphiphilic variant with grafted lactic acid side chains was superior to the more hydrophilic backbone with no additional grafting. The pharmacological response observed in healthy rats was reproduced on streptozotocin induced diabetic rats, demonstrating in-vivo effectivness in diseased subjects respectively. A significant increase in relative bioavailability was observed at higher polymer concentrations for the grafted variant. The histological examination of the nasal mucosa with light microscopy showed no signs of toxicity on nasal morphology at the site of application after a single administration procedure. In Chapter 4 the interaction of DEAPA-PVAL-g-PLLA nanocomplexes with enterocyte-like Caco-2 cells in terms of cytotoxicity, transport through and uptake in the cell layers was investigated. The NC were compared against a nanoparticle formulation based on a higher grafted variant of DEAPA- PVAL-g-PLGA and prepared by a solvent displacement technique. The preformulated NP were loaded with insulin by surface adsorption. The comparison of permeability enhancing effects of nanocomplexes with the nanoparticle formulation, showed no significant differences. However, nanocomplexes physico-chemical properties and interaction with Caco- 2 monolayers varied strongly as a function of the polymer structural composition. The most effective carrier posses a combination of a hydrophilic backbone and hydrophobic side chains. On the other hand, this combination led to a higher amphiphilic, surfactant-like character of the polymer, which probably caused damages to membrane and tight junctions, as indicated by lactate dehydrogenase release and decrease of transepithelial resistance. However, since these effects seemed to be reversible and NC with a higher lactide-grafting showed the best protection capabilities against trypsinic degradation, the highest internalization and transport through Caco-2 monolayers, it may be assumed that these NC are suitable carriers for insulin to overcome mucosal absorption barriers.
DOI:https://doi.org/10.17192/z2006.0076