Small Intestine pH 7.0 0.15 mg/ml tripsin

1. Summer Research Experience Scholarship Program 2005-2006 Process Development for the Formation of a Plant Made Measles Vaccine into Biodegradable Particles Alia M. Al-Lawati1, Rajprathab Gopiraj1, Elena Virtue2, Diane E. Webster2 and Gareth M. Forde1. 1: Bio Engineering Laboratory (BEL), Department of Chemical Engineering, Monash University, VIC 3800, Australia 2: Macfarlane Burnet Institute for Medical Research and Public Health, Alfred Hospital, VIC 3001, Australia. The particle size distribution of milled plant material and microparticles was determined using a Malvern Mastersizer-E 5. Particle Size Analysis 1. Background Despite the existence of a safe and effective vaccine (LAV), the measles virus (MV) remains a serious childhood illness. Each year 30 million cases and over 600,000 deaths are reported, due to short-comings of the LAV, which requires refrigeration and must be administrated by injection. Plants are feasible alternatives for the production and delivery of a MV vaccine. A plant-made vaccine, which is temperature stable, orally delivered and inexpensive has the potential to improve vaccine coverage. Figure 3: Particle size frequency distribution for hammer milled plant material. The final mass moment average (D[4,3]) was found to be 77.58 μm. 6. Biocompatible Microparticle Creation Plant material was encapsulated in the biocompatible / biodegradable polymer poly-ε-caprolactone (PCL) via the use of an ultrasonic atomizer (40 kHz). The particle size (D[4,3]) of the plant material-loaded microparticles was found to be 98.79m when a volume of 0.2 mL of a 100:2 solution of PCL in acetone and PVA in plant material was atomized at a flow rate of 36mL/hr into a 50mL hardening agent of 10mM SDS and 20mM EDTA. Samples were measured after 15 minutes of stirring. 2. Particle Analysis • 2. The Objective • This project aims to develop a method and hence study the: • processing: particularizing of freeze-dried plant material, • formulating: encapsulation in biocompatible polymer, and • in vitro delivery using a gastro-intestinal (GI) model • of a plant-made MV vaccine suitable for oral administration. 3. Target Protein Extraction and Analysis Freeze-dried transgenic root cultures, produced by the vaccine development group at the Burnet Institute, was lysed and the total protein extracted for analysis. Use of Bradford reagent assays and Western Blots (Figure 1) enabled calculation of the percentage of MV hemagglutinin (HMV) protein in the freeze-dried samples. This analysis showed approximately 0.21 mg HMV was present per 1.0 g of dried plant material. 7. GI Model A key element was to see whether the HMV protein microparticles survived the human digestive system to be absorbed by the Peyer’s patch in the upper small intestine, and hence into the blood stream. Figure 4 is a process flow diagram of the GI model used as an in vitro replication of the digestive system. Figure 1:Western Blot Transfer of positive and negative controls, and 7 freeze dried samples of protein extracted from freeze dried root cultures. MV-H (MW ~ 75 kDa) is present in all of the 7 freeze-dried samples. • Ingestion • Plant material • 20 mM phosphate buffer • 1.75 mg/ml • α-amylase • Stomach • pH 2.5 • 0.15 mg/ml • pepsin • Small Intestine • pH 7.0 • 0.15 mg/ml • tripsin Figure 4: GI model for the in vitro analysis of HMV delivery. This system models the passage of the encapsulated plant material from ingestion, into the gut then into the upper small intestine. 4. Grinding / Milling Batches of the freeze-dried root culture was ground for direct encapsulation. Particle size distribution was determined using a Malvern Mastersizer-E (Figure 2). 8. GI Model Results A non-encapsulated sample was run through the GI Model along with an encapsulated sample. As expected, the encapsulated sample had lower protein reading since the majority of the protein would have been encapsulated by PCL. Figure 5: Variations in concentration readings between encapsulated and non-encapsulated samples through Bradford reagent assay. 12.3 m Acknowledgement: The work for this project was funded by the Monash Engineering Faculty Small Grant Scheme. Figure 2: Microscopy image of freeze-dried root culture material processed via a laboratory blender or small-scale hammer mill.