... containing alginate and fish oil for spray drying 2) To optimize spray drying conditions for the production of microspheres containing alginate as wall material 3) To investigate the effect of alginate. .. hypothesized that the use of alginates as wall material can enhance the oil- loading capacities of microspheres produced by spray drying In addition, the use of different alginate grades will affect... of alginate as wall material for fish oil encapsulation by spray drying A4 Methods of oil encapsulation Many methods have been explored for the purpose of oil encapsulation They can generally
DEVELOPMENT OF OIL-LOADED ALGINATE-COMPOSITE MICROSPHERES BY SPRAY DRYING TAN LAY HUI (B Sc (Pharm.) (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2008 i ACKNOWLEDGEMENTS I wish to express my heartfelt gratitude to my supervisors, A/P Paul Heng Wan Sia and A/P Chan Lai Wah for their guidance and support for both my research and personal life I am also grateful for their effort put into reading and giving comments and suggestions for improvements on my manuscripts I also sincerely thank them for giving me the opportunity to discover and explore the various aspects of research work I also deeply appreciate the moral support given to me when I took time off research to start my family I also wish to thank the Head of the Department of Pharmacy, A/P Chan Sui Yung, for the support she has given me throughout my years as a graduate student I also thank her for the use of the departmental facilities for my research project I would also like to acknowledge the research scholarship awarded by the National University of Singapore Many thanks also to my fellow laboratory mates, especially Wai See, Sze Nam, Huey Ying, Kang Teng, Ai Ling, Josephine and Qiyun for guiding and helping me in my research and for being good role models for me to emulate I also thank Teresa, Mei Yin and Peter for the technical assistance provided for my research work My sincere appreciation also goes to Dr Anton Dolzhenko for his assistance in performing the NMR studies I would also like to express my heartfelt thanks to my mother, sister and auntie for their unfailing support and faith in me Without their moral and financial support, I would not have been able to embark on and complete my undergraduate and ii graduate studies I also wish to thank my husband for his love and encouragement through these years and my children, Amanda, Bethany and Christian, for enriching my life in a way that no others could Last but not least, I would like to dedicate this work to my late father, for without him, I would not be what I am today Thank you Lay Hui July 2008 iii CONTENTS ACKNOWLEDGEMENTS ii CONTENTS iv SUMMARY ix LIST OF TABLES xi LIST OF FIGURES xii I INTRODUCTION A Microencapsulation A1 Significance of microencapsulation A2 Microencapsulation of oils A3 Fish oils and polyunsaturated fatty acids (PUFAs) A3.1 Microencapsulation of fish oils A4 Methods of oil encapsulation A4.1 Chemical processes A4.1.1 Complex coacervation A4.1.2 Other processes A4.2 Physical processes A4.2.1 Spray drying A5 Wall materials for oil encapsulation by spray drying 9 11 A5.1 Starches and sugars 12 A5.2 Gums 13 A5.3 Proteins 14 A5.4 Alginates 15 B Evaluation of oil-loaded microspheres B1 Mechanical strength 18 19 iv B1.1 Single-microparticle studies 19 B1.2 Bulk-microparticle studies 22 C Process analytical technology (PAT) 23 C1 Definition of PAT 23 C2 PAT tools 24 C3 PAT in particle sizing 25 C3.1 Focused beam reflectance measurement (FBRM) 25 C3.2 Laser diffraction 27 C3.3 Imaging methods 28 C3.4 Other methods 29 C4 PAT for microsphere sizing during spray drying 29 II HYPOTHESES AND OBJECTIVES 31 III EXPERIMENTAL 32 A Materials 32 A1 Alginates 32 A2 Starch 32 A3 Fish oil 33 A4 Maltodextrin 33 A5 Materials used in microsphere characterization 33 A5.1 Stainless steel shots 33 A5.2 Ferronyl powder 33 B Methods 34 B1 Viscosity reduction of alginate solutions 34 B2 Viscometry of alginate solutions 34 v B3 Nuclear magnetic resonance (NMR) studies 34 B4 Emulsion preparation 35 B5 Emulsion oil droplet size analysis 36 B6 Spray drying of emulsions 37 B7 Yield 37 B8 Size determination 38 B8.1 Light microscopy 38 B8.2 In-line and at-line laser diffraction 38 B8.3 Off-line laser diffraction 40 B9 Microsphere morphology 41 B9.1 Scanning electron microscopy (SEM) studies 41 B9.2 Microsphere roundness 41 B10 Microsphere true density determination 42 B11 Flow determination 42 B11.1 Poured bulk density 43 B11.2 Tapped bulk density 43 B11.3 Carr’s index 44 B12 Determination of microsphere surface area 44 B12.1 BET specific surface area 44 B12.2 Theoretical specific surface area 45 B13 Determination of microencapsulation efficiency (ME) 46 B13.1 Surface oil 46 B13.2 Total oil content, microencapsulated oil and ME 47 B14 Determination of oil content and stability on storage 48 vi B14.1 Sample preparation 48 B14.2 Gas chromatography 48 B14.3 Calculation of EPA and DHA content 49 B14.4 Degradation kinetics 50 B15 Microsphere mechanical strength 51 B15.1 Microsphere compression 51 B15.2 Determination of oil leakage after compression 51 B16 Statistical analysis IV RESULTS AND DISCUSSION A Formulation and production of microspheres A1 Emulsion formulation A1.1 Pre-treatment of alginate solutions A1.1.1 Autoclaving of alginate solutions A1.2 Emulsion stability A1.2.1 Effect of homogenization conditions A2 Optimization of spray drying conditions 52 53 53 53 53 55 58 58 64 B Microsphere characterization 69 B1 Physical properties 69 B1.1 Microencapsulation efficiency (ME) 69 B1.2 Microsphere morphology 71 B1.3 Microsphere size 77 B1.4 Microsphere roundness 80 B1.5 Specific surface area 82 B1.6 Microsphere true density 86 vii B1.7 Bulk and flow properties 88 B1.8 Yield 90 B1.9 Summary 92 B2 Oil content and stability on storage 93 B3 Mechanical properties 107 B3.1 Method development 108 B3.2 Oil leakage studies 111 C PAT for microsphere sizing 113 C1 In-line monitoring of real-time changes 114 C2 Microsphere sizing 118 C2.1 In-line laser diffraction 120 C2.2 At-line laser diffraction 129 C2.2.1 Optimization of sizing conditions 129 C2.2.2 Microsphere sizing 133 C2.3 Off-line laser diffraction 136 C2.4 Light microscopy 136 C3 Summary 139 V CONCLUSION 140 VI REFERENCES 142 VII APPENDIX 177 VIII PUBLICATIONS / PRESENTATIONS ARISING FROM THIS STUDY 183 viii SUMMARY Microencapsulation is a method that is commonly used in the food and pharmaceutical industries for various purposes that include controlled release and protection of sensitive materials from degradation It has been found to be a useful way to retard the oxidation process and improve the handling properties of ω-3 polyunsaturated fatty acids present in fish oils Various wall materials and methods have been used for the microencapsulation of fish oils Although alginates have wide pharmaceutical applications as excipients and formulation aids in many drug delivery systems, little information is available on its use as a wall material for oil encapsulation, especially by spray drying This provides the impetus for the present study Microencapsulated products generally need to be intact to carry out their functions However, the mechanical properties of oilloaded microspheres are not well characterized This warrants further investigations to be conducted Process analytical technology (PAT) has been applied to pharmaceutical processes for the purposes of quality improvement and improved process understanding The particle size distribution of a pharmaceutical product is an important quality characteristic, and PAT has been applied to milling and crystallization processes for real-time monitoring of particle size However, little scientific literature is available on its application to particle sizing during spray drying It was therefore of interest to explore the feasibility of applying an in-process particle sizer as a PAT tool to the spray drying operation during microsphere production ix Fish oil-containing emulsions consisting of blends of alginate and modified starch as wall material were spray dried at conditions optimized to produce microspheres with the highest microencapsulation efficiencies and yield The properties of the microspheres, such as size and morphology, true density, flow and specific surface area, were evaluated In addition, storage stability studies were carried out to assess the protective capability of the microsphere matrices composed of different alginate type and content The mechanical properties of the microspheres were further investigated through compression studies Partial substitution of modified starch with alginate produced microspheres which generally performed better in terms of oil holding and oxidative protective capabilities This could be due to the formation of larger and microspheres with the incorporation of alginate into the microsphere wall matrix It also resulted in a product with better flow and yield However, between the grades of alginate studied, Manucol LB appeared to perform better in these aspects The addition of alginate also made the microspheres more resistant to compression Application of an in-process particle size analyzer during the spray drying process allowed the elucidation of real-time information regarding microsphere size changes especially during process start-up and shut-down For highly agglomerated products like the microspheres produced in the present study, an at-line set-up was found to be more useful for the determination of individual microsphere size x Rehor, A., Canaple, L., Zhang, Z., Hunkeler, D The compressive deformation of multicomponent microcapsules: Influence of size, membrane thickness, and compression speed J Biomater Sci Polym Ed 12(2) (2001) 157-170 Ribeiro, A., Arnaud, P., Frazao, S., Venâncio, F., Chaumeil, J C Development of vegetable extracts by microencapsulation J Microencapsul 14(6) (1997) 735-742 Rosenberg, M., Kopelman, I J., Talmon, Y Factors affecting retention in spraydrying microencapsulation of volatile materials J Agric Food Chem 38(5) (1990) 1288-1294 Rosenberg, M., Sheu, T Y Microencapsulation of volatiles by spray-drying in whey protein-based wall systems Int Dairy J 6(3) (1996) 273-284 Rosiński, S., Grigorescu, G., Lewińska, D., Ritzén, L G., Viernstein, H., Teunou, E., Poncelet, D., Zhang, Z., Fan, X., Serpұ, D., Marisonұ, I., Hunkeler, D Characterization of microcapsules: Recommended methods based on round-robin testing J Microencapsul 19(5) (2002) 641-659 Rowley, J A., Madlambayan, G., Mooney, D J Alginate hydrogels as synthetic extracellular matrix materials Biomaterials 20(1) (1999) 45-53 Scheffold, F Particle sizing with diffusing wave spectroscopy J Dispersion Sci Technol 23(5) (2002) 591-599 170 Schürks, N., Wingender, J., Flemming, H C., Mayer, C Monomer composition and sequence of alginates from Pseudomonas aeruginosa Int J Biol Macromol 30(2) (2002) 105-111 Shahidi, F., Han, X Q Encapsulation of food ingredients Crit Rev Food Sci Nutr 33(6) (1993) 501-547 Shahidi, F., Wanasundara, U N Omega-3 fatty acid concentrates: nutritional aspects and production technologies Trends Food Sci Technol 9(6) (1998) 230240 Shapiro, L., Cohen, S Novel alginate sponges for cell culture and transplantation Biomaterials 18(8) (1997) 583-590 Sheu, T Y., Rosenberg, M Microencapsulation by spray drying ethyl caprylate in whey protein and carbohydrate wall systems J Food Sci 60(1) (1995) 98-103 Sheu, T Y., Rosenberg, M Microstructure of microcapsules consisting of whey proteins and carbohydrates J Food Sci 63(3) (1998) 491-494 Shiga, H., Yoshii, H., Nishiyama, T., Forssele, P., Poutanen, K., Linko, P Flavour encapsulation and release characteristics of spray-dried powder by the blended encapsulant of cyclodextrin and gum Arabic Drying Technol 19(7) (2001) 13851395 171 Simopoulos, A P Omega-3 fatty acids in inflammation and autoimmune diseases J Am Coll Nutr 21(6) (2002) 495-505 Sistare, F., Berry, L S P., Mojica, C A Process analytical technology: an investment in process knowledge Org Process Res Dev 9(3) (2005) 332-336 Skibsted, E T S., Boelens, H F M., Westerhuis, J A., Witte, D T., Smilde, A K Simple assessment of homogeneity in pharmaceutical mixing processes using near-infrared reflectance probe and control charts J Pharm Biomed Anal 41(1) (2006) 26-35 Sontrop, J., Campbell, M K ω-3 polyunsaturated fatty acids and depression: A review of the evidence and a methodological critique Prev Med 42(1) (2006) 413 Soottitantawat, A., Yoshii, H., Furuta, T., Ohgawara, M., Forssell, P., Partanen, R., Poutanen, K., Linko, P Effect of water activity on the release characteristics and oxidative stability of D-limonene encapsulated by spray drying J Agric Food Chem 52(5) (2004) 1269-1276 Staniforth, J Powder flow In: Aulton, M E (Ed), Pharmaceutics: The science of dosage form design, Harcourt Publishers Limited, London (2002) 172 Su, K P., Huang, S Y., Chiu, C C., Shen, W W Omega-3 fatty acids in major depressive disorder: a preliminary double-blind, placebo-controlled trial Eur Neuropsychopharmacol 13(4) (2003) 267-271 Takeuchi, H., Sasaki, H., Niwa, T., Hino, T., Kawashima, Y., Uesugi, K., Ozawa, H Improvement of photostability of ubidecarenone in the formulation of a novel powdered dosage form termed redispersible dry emulsion Int J Pharm 86(1) (1992) 25-33 Tan, S., Sherman, R L., Ford, W T Nanoscale compression of polymer microspheres by atomic force microscopy Langmuir 20(17) (2004) 7015-7020 Tønnesen, H H., Karlsen, J Alginate in drug delivery systems Drug Dev Ind Pharm 28(6) (2002) 621-630 Van Raamsdonk, J M., Chang, P L Osmotic pressure test: A simple, quantitative method to assess the mechanical stability of alginate microcapsules J Biomed Mater Res 54(2) (2001) 264-271 Varavinit, S., Chaokasem, N., Shobsngob, S Studies of flavor encapsulation by agents produced from modified sago and tapioca starches Starch 53(6) (2001) 281-287 Walton, D E., Mumford, C J Spray-dried products – characterization of particle morphology Chem Eng Res Des 77(A1) (1999) 21-38 173 Walton, D E The morphology of spray-dried particles A qualitative view Drying Technol 18(9) (2000) 1943-1986 Wandrey, C., Espinosa, D., Rehor, A., Hunkeler, D Influence of alginate characteristics on the properties of multi-component microcapsules J Microencapsul 20(5) (2003) 597-611 Wang, C X, Cowen, C., Zhang, Z., Thomas, C R High-speed compression of single alginate microspheres Chem Eng Sci 60(23) (2005) 6649-6657 Wang, X Z., Roberts, K J., Ma, C Crystal growth measurement using 2D and 3D imaging and the perspectives for shape control Chem Eng Sci 63(5) (2008) 1173-1184 Watanabe, Y., Fang, X., Adachi, S., Fukami, H., Matsuno, R Oxidation of 6-Oarachidonoyl L-ascorbate microencapsulated with a polysaccharide by spraydrying Lebensm Wiss Technol 37(4) (2004) 395-400 Watano, S Direct control of wet granulation processes by image processing system Powder Technol 117(1-2) (2001) 163-172 Weiβ, G., Knoch, A., Laicher, A., Stanislaus, F., Daniels, R Simple coacervation of hydroxypropyl methylcellulose phthalate (HPMCP) II Microencapsulation of ibuprofen Int J Pharm 124(1) (1995) 97-105 174 Weibull, W A statistical distribution function of wide applicability J Appl Mech 18 (1951) 293-297 Weinbreck, F., Minor, M., de Kruif, C G Microencapsulation of oils using whey protein/gum Arabic coacervates J Microencapsul 21(6) (2004) 667-679 Wong, D Y T., Wright, P., Aulton, M E The deformation of alpha-lactose monohydrate and anhydrous alpha-lactose monocrystals Drug Dev Ind Pharm 14(15-17) (1988) 2109-2126 Xu, R Particle characterization: Light scattering methods Kluwer Academic Publishers (2000) Xu, R., Di Guida, O A Comparison of sizing small particles using different technologies Powder Technol 132(2-3) (2003) 145-153 Yan, N., Zhang, M., Ni, P Study on polyamide microcapsules containing oily liquids J Microencapsul 11(4) (1994) 365-372 Yilmaz, G., Jongboom, R O J., Feil, H., Hennink, W E Encapsulation of sunflower oil in starch matrices via extrusion: effect of the interfacial properties and processing conditions on the formation of dispersed phase morphologies Carbohydr Polym 45(4) (2001) 403-410 175 Zhang, Z., Saunders, R., Thomas, C R Mechanical strength of single microcapsules by a novel micromanipulation technique J Microencapsul 16(1) (1999) 117-124 Zhang, J Q., Yan, Y On-line continuous measurement of particle size using electrostatic sensors Powder Technol 135-136 (2003) 164-168 Zhao, L., Zhang, Z Mechanical characterization of biocompatible microspheres and microcapsules by direct compression Artif Cells Blood Substit Immobil Biotechnol 32(1) (2004) 25-40 176 VII APPENDIX Appendix I Properties of (A) blank microspheres and microspheres prepared with (B) 50 %, (C) 100 %, (D) 150 %, (E) 200 % and (F) 250 % oil loading (A) Formulation Mean particle size (µm) Roundnessa BET specific surface area (m2/g) Yield (%) True densityb (g/cm3) Carr’s index C 13.8 (0.0) 1.15 1.42 (0.12) 74.4 (5.0) 1.25 53.8 LB1 14.2 (0.1) 1.15 1.27 (0.08) 73.9 (5.2) 1.23 53.6 LB5 15.7 (0.3) 1.14 0.92 (0.04) 75.2 (4.3) 1.24 52.8 LB10 16.8 (0.3) 1.13 0.89 (0.03) 76.9 (4.3) 1.22 50.0 LBB1 14.0 (0.1) 1.15 1.26 (0.03) 74.5 (4.8) 1.27 53.5 LBB5 14.8 (0.2) 1.15 1.01 (0.05) 73.4 (4.9) 1.24 53.0 LBB10 15.6 (0.1) 1.14 0.96 (0.05) 73.8 (4.8) 1.24 50.2 177 (B) Formulation Mean particle size (µm) Roundnessa BET specific surface area (m2/g) Yield (%) ME (%) True densityb (g/cm3) Carr’s index C 15.7 (0.0) 1.14 0.78 (0.07) 43.8 (5.9) 92.1 (0.0) 1.10 54.6 LB1 15.9 (0.1) 1.14 0.75 (0.05) 45.6 (5.00 92.4 (0.1) 1.10 55.6 LB5 17.0 (0.2) 1.14 0.70 (0.03) 60.2 (5.1) 94.5 (0.0) 1.10 53.4 LB10 18.0 (0.3) 1.13 0.62 (0.03) 61.9 (4.4) 94.1 (0.1) 1.11 51.3 LBB1 15.9 (0.2) 1.15 0.75 (0.03) 43.9 (5.2) 92.0 (0.2) 1.12 55.5 LBB5 16.3 (0.2) 1.14 0.71 (0.05) 44.1 (5.1) 92.1 (0.0) 1.10 54.1 LBB10 17.7 (0.2) 1.13 0.67 (0.03) 50.3 (5.1) 93.0 (0.0) 1.10 52.0 178 (C) Formulation Mean particle size (µm) Roundnessa BET specific surface area (m2/g) Yield (%) ME (%) True densityb (g/cm3) Carr’s index C 19.5 (0.4) 1.13 0.68 (0.04) 68.6 (5.8) 89.1 (0.1) 1.10 52.1 LB1 19.2 (0.3) 1.13 0.63 (0.03) 69.4 (4.2) 90.3 (0.1) 1.10 52.7 LB5 19.3 (0.3) 1.12 0.59 (0.03) 73.5 (4.3) 94.2 (0.0) 1.10 50.6 LB10 19.3 (0.3) 1.13 0.56 (0.04) 75.6 (4.3) 94.1 (0.0) 1.10 48.8 LBB1 19.1 (0.3) 1.13 0.68 (0.03) 68.9 (4.8) 90.0 (0.1) 1.10 53.3 LBB5 19.2 (0.3) 1.13 0.68 (0.05) 69.2 (4.6) 91.9 (0.1) 1.11 51.9 LBB10 19.2 (0.3) 1.12 0.66 (0.02) 72.5 (4.1) 94.2 (0.2) 1.10 49.6 179 (D) Formulation Mean particle size (µm) Roundnessa BET specific surface area (m2/g) Yield (%) ME (%) True densityb (g/cm3) Carr’s index C 18.9 (0.4) 1.13 0.66 (0.04) 47.8 (7.5) 75.8 (2.9) 1.06 52.5 LB1 18.6 (0.3) 1.12 0.64 (0.02) 51.3 (5.8) 77.2 (2.0) 1.07 54.9 LB5 19.1 (0.2) 1.10 0.54 (0.05) 65.5 (4.3) 83.0 (2.7) 1.07 53.0 LB10 19.8 (0.3) 1.08 0.51 (0.05) 72.6 (4.5) 90.2 (2.1) 1.07 51.8 LBB1 18.8 (0.3) 1.12 0.66 (0.03) 49.2 (5.2) 76.1 (3.4) 1.08 54.8 LBB5 19.0 (0.4) 1.12 0.65 (0.00) 58.7 (4.9) 77.1 (2.8) 1.09 53.1 LBB10 19.2 (0.3) 1.10 0.63 (0.02) 68.6 (4.7) 83.0 (1.1) 1.07 51.6 180 (E) Formulation Mean particle size (µm) Roundnessa BET specific surface area (m2/g) Yield (%) ME (%) True densityb (g/cm3) Carr’s index C 19.2 (0.5) 1.13 0.65 (0.04) 30.2 (8.8) 70.5 (2.4) 1.05 57.0 LB1 19.3 (0.3) 1.12 0.65 (0.04) 32.7 (7.2) 72.6 (2.3) 1.05 57.1 LB5 19.6 (0.3) 1.1 0.53 (0.03) 41.8 (6.3) 78.0 (2.8) 1.05 55.9 LB10 20.1 (0.4) 1.07 0.51 (0.04) 50.3 (4.8) 86.3 (2.4) 1.05 53.8 LBB1 19.1 (0.3) 1.12 0.66 (0.05) 29.6 (7.4) 71.3 (3.1) 1.06 58.0 LBB5 19.3 (0.4) 1.12 0.66 (0.03) 32.0 (6.9) 72.3 (2.5) 1.06 55.5 LBB10 19.6 (0.3) 1.1 0.63 (0.02) 41.0 (6.1) 78.4 (2.7) 1.06 54.1 181 (F) a Formulation Mean particle size (µm) Roundnessa BET specific surface area (m2/g) Yield (%) ME (%) True densityb (g/cm3) Carr’s index C 19.3 (0.5) 1.13 0.65 (0.04) 16.7 (8.5) 57.4 (2.5) 1.04 65.2 LB1 19.3 (0.3) 1.12 0.65 (0.03) 20.1 (8.0) 59.4 (2.4) 1.04 64.8 LB5 19.8 (0.3) 1.1 0.52 (0.03) 33.4 (7.2) 67.3 (2.5) 1.04 60.1 LB10 20.4 (0.3) 1.07 0.50 (0.03) 40.9 (6.0) 76.6 (2.6) 1.04 56.2 LBB1 19.3 (0.4) 1.12 0.66 (0.04) 20.1 (8.1) 60.8 (2.8) 1.04 65.4 LBB5 19.5 (0.3) 1.11 0.66 (0.03) 21.8 (7.7) 66.2 (2.4) 1.04 61.0 LBB10 19.9 (0.4) 1.09 0.62 (0.02) 35.5 (7.0) 72.2 (2.5) 1.04 56.9 standard deviation < 0.01; bstandard deviation < 0.005; values in parentheses represent the standard deviation 182 VIII PUBLICATIONS / PRESENTATIONS ARISING FROM THIS STUDY Journal Publications: Tan, L H., Chan, L W., Heng, P W S Alginate/starch composites as wall material to achieve microencapsulation with high oil loading, (accepted, 26 June 2008) Chan, L W., Tan, L H., Heng, P W S Process analytical technology: Application to particle sizing in spray drying, AAPS Pharm Sci Tech 9(1) (2008) 259-266 Tan, L H., Chan, L W., Heng, P W S Effect of oil loading on microspheres produced by spray drying, J Microencapsul 22(3) (2005) 253-259 Conference Presentations: Tan, L H Using alginate composite as wall material to achieve microencapsulation with high oil loading 3rd American Association of Pharmaceutical Scientists – National University of Singapore (AAPS – NUS) Student Chapter Symposium, Singapore (Mar, 2007); oral presentation Tan, L H., Chan, L W., Heng, P W S Process analytical technology: Application to microsphere sizing in spray drying, Asian Pharmaceutics Graduate Congress, Singapore (Sept, 2006) Tan, L H., Heng, P W S., Chan, L W Application of process analytical technology to microsphere sizing during spray drying, 15th International Symposium on Microencapsulation, Italy (Sept, 2005) Heng, P W S., Chan, L W., Tan, L H Microencapsulation of PUFA by spray drying using alginate and/or starch, American Association of Pharmaceutical Scientists Annual Meeting and Exposition, USA (Nov, 2004) 183 Tan, L H Microencapsulation of PUFA by spray-drying using alginate and/or starch, Pharmacy Graduate Seminar IV, Singapore (Dec, 2004); oral presentation Tan, L H., Chan, L W., Heng, P W S Effect of oil loading on microspheres produced by spray drying, 14th International Symposium on Microencapsulation, Singapore (Sept, 2003) 184