Preparation and Evaluation of Porous Multiparticulate Systems for Controlled Drug Delivery

Abstract

The wide achievements in the development of therapeutic macromolecules as insulin increased the demand for creating suitable delivery systems for such molecules. Parenteral administration is considered the most common route of administration of such molecules. Although many alternative delivery routes were investigated, oral route remains the most preferred. That’s because it offers superior patient compliance and decrease the adverse effects from parenteral route, in addition, oral insulin is mandatory because it mimics its normal physiological pathway. However, oral route suffers a lot of barriers in order for the macromolecule drug to pass to the blood stream represented in chemical, enzymatic and, most important, absorption barriers. In order to outweigh these challenges, many approaches were adopted to increase oral bioavailability of such large hydrophilic molecules. The most common approach used is microencapsulation that would not only offer protection for macromolecules but may also aid in their absorption through the GIT. Nevertheless, several parameters in the process of microencapsulation may affect macromolecule stability as the use of organic solvents, shear forces or pH/ temperature variations. Accordingly, scientists adopted an alternative approach by loading macromolecules in prefabricated porous microparticles (MPs). Porous MPs can be synthesized by different methods depending on the type of material used. Both polymeric and inorganic porous MPs could be synthesized yielding various pore sizes and porosity patterns. In our study, we used freeze-drying as an alternative technique for loading insulin in porous poly(lacticide-co-glycolide) (PLGA) and mesoporous silica (SBA15) MPs and we tried to achieve high loading capacityand controlled release by coating these particles with chitosan.

The work in this thesis was divided into three chapters: Chapter I: Preparation and characterization of insulin loaded porous PLGA and mesoporous silica MPs Chapter II: Preparation and characterization of chitosan coated porous PLGA and mesoporous silica MPs Chapter III: In-vitro and in-vivo studies on insulin structure integrity and activity in porous PLGA and mesoporous silica MPs

Chapter I: Preparation and characterization of insulin loaded porous PLGA and mesoporous silica microparticles This chapter dealt with the preparation of porous PLGA MPs and comparing them to SBA15 MPs in terms of insulin loading and release.

The work in this chapter included the following: 1- Preparation of mesoporous silica MPs using surfactant assisted soft template method. 2- Preparation of porous PLGA by double emulsion solvent evaporation methodusing three PLGA molecular weights and two types of porogens; hydroxyl propyl-β-cyclodextrin and pluronic F-127. 3- Characterization of MP size and porosity using SEM and He pycnometry. 4- Loading of insulin was performed by either immersion or freeze-drying onto preformed porous MPs followed by the determination of both loading capacity and actual loading capacity of insulin loaded in the MPs. 5- Confocal microscopy was used to determine the distribution of insulin inside the MPs. 6- In-vitro release study was performed to examine the release pattern from selected formulae.

The results of this work revealed the following: 1- PLGA MPs formed using intermediate molecular weight (15 kDa) and hydroxyl propyl-β-cyclodextrin10% w/w had more surface and internal pores. 2- Insulin was loaded by an alternative method; freeze-drying and this method was compared by the usual immersion methods. Higher LC% values were noticed for SBA15 relative to PLGA MPs, however, the desorption rate was also high leading to low ALC%. 3- Loading in a pH close to the peptide iso-electric point significantly increased loading capacity. Insulin loading by freeze-drying was slightly higher at pH 3.5. 4- The effect of insulin concentration showed that PLGA MPs reach saturation at lower concentrations indicating the higher adsorption power of mesoporous silica. 5- Insulin was found as accumulations in porous PLGA MPs but it was uniformly distributed in SBA15MPs. 6- SBA15 MPs showed less burst release and slower pattern relative to porous PLGA.

Chapter II: Preparation and characterization of chitosan coated porous PLGA and mesoporous silica microparticles In this chapter, chitosan (CS) coating was done simultaneously with insulin loading using an alternative double freeze-drying technique. The work in this chapter included the following: 1- CS was used to coat porous PLGA and SBA15 MPs using two CS molecular weights (high-low) as well as serial CS concentrations. 2- Different coating techniques were compared in terms of insulin ALC% 3- SEM was used to visualize the coating layer deposition onto the particles 4- Confocal microscopy was similarly used to determine the distribution of insulin inside the MPs and the coating layer. 5- In-vitro release experiments were performed in pH 7.2 and pH 1.2 to mimic GIT different pH regions. The results of this work revealed the following: 1- CS coating on Porous MPs by double freeze-drying significantly (P<0.001) increased loading capacity to 20% in case of PLGA and 15 % for SBA15 using initial insulin concentration as low as 5 mg/mL. 2- CS with low molecular weight showed higher loading efficiencies, similarly, CS concentration at 5 mg/mL. 3- From confocal images, insulin could be found both in the outer coating shell as well as the inner compartment of porous PLGA MPs. 4- The burst release was significantly decreased in case of PLGA relative to mesoporous silica.

Chapter III: In-vitro and in-vivo studies on insulin structure integrity and activity in porous PLGA and mesoporous silica microparticles We investigated in this chapter the integrity of insulin structure using both in-vitro techniques as fluorescent spectroscopy and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and in-vivo techniques by oral administration to rats. The work in this chapter included the following: 1- Insulin released from selected MP formulae was scanned by fluorescent spectrophotometer to determine the emission maximum compared to that of standard and denatured insulin samples. 2- Insulin samples were run by gel electrophoresis and compared to that of standard insulin samples 3- In-vivo pharmacodynamics were studied in order to confirm the maintenance of biological activity of insulin after oral absorption 4- For in-vivo studies rats were divided into 5 groups as follows: Group I: Injection insulin suspension SC 5IU/KG GroupII: Oral administration insulin suspension 50IU/kg GroupIII: Oral administration insulin loaded PLGA MPs equivalent to 50IU/kg GroupIV: Oral administration insulin loaded CS-PLGA MPs equivalent to 50IU/kg GroupV: Oral administration insulin loaded SBA15 MPs equivalent to 50IU/kg GroupVI: Oral administration insulin loaded CS-SBA15 MPs equivalent to 50IU/kg