Design, Preparation, and Characterization of Nanoparticles as Delivery Systems for Bioactive Phenolic Phytochemicals

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Design, Preparation, and Characterization of Nanoparticles as Delivery Systems for Bioactive Phenolic Phytochemicals
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1 online resource (134 p.)
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english
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Li, Zheng
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University of Florida
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Gainesville, Fla.
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Degree:
Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Food Science and Human Nutrition
Committee Chair:
Gu, Liwei
Committee Members:
Gregory, Jesse F, Iii
Percival, Susan S
Powers, Kevin William

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Subjects / Keywords:
bsa -- egcg -- ellagitannins -- gelatin -- nanoparticles -- ovalbumin
Food Science and Human Nutrition -- Dissertations, Academic -- UF
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Food Science and Human Nutrition thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract:
Three types of nanoparticles were designed as delivery systems for ellagitannins or EGCG. In the first design, the PPE were obtained from pomegranate pericarp to contain punicalagin A (16.6%) and B (32.5%, w/w). Punicalagin and gelatin self-assembled into nanoparticles via hydrogen bonding and hydrophobic interactions. Nanoparticles formed at a mass ratio 5:5 had a size of 149 nm, zeta-potential of 17.8 mV, and loading capacity of punicalagin A and B of 14.8% and 25.7%. PPE-gelatin nanoparticles were less effective at inducing early stage apoptosis, but as effective as PPE solution at inducing late stage apoptosis and necrosis on HL-60 cells. PPE-to-gelatin mass ratios from 1:5-6:5 resulted in nanoparticles with sizes of 122-129 nm. Nanoparticles fabricated at pH 4.0-5.3 (gelatin solution) had particle sizes of 21-194 nm and zeta-potentials of 14.7-23.8 mV. Acidic or basic pH hindered particle formation,whereas pH 6-7 caused particle aggregation. Nanoparticles formed at 25-50oChad sizes below 500 nm. Nanoparticles remained stable for 4 days. In the second design, BSA-EGCG nanoparticles were coated with poly-?-lysine or chitosan with sizes of 100-200 nm. Loading efficiency and capacity of EGCG were 35.4% and32.7%, and 17.0% and 16.0%, respectively. Coatings prevented particle aggregation at pH 4.5-5.0 and changed particle zeta-potentials. Coatings delayed EGCG release from the nanoparticles in simulated fluids with or without digestive enzymes. EGCG in CBEN showed higher Papp on Caco-2 monolayers. In the third design, EGCG ovalbumin-dextran conjugate nanoparticles were self-assembled and further crosslinked by glutaraldehyde.These nanoparticles had sizes of 285 nm and 339 nm. Loading efficiency and capacity of EGCG was 23.4% and 30.0%, and 19.6% and 20.9%, respectively. They showed nano-scale sizes and similar zeta-potentials in the pH range 2.5-7.5.Good nanoparticle stabilities were observed in simulated fluids at 37oC.Digestive enzymes caused quicker release of EGCG from the nanoparticles. The conjugate nanoparticles significantly enhanced Papp of EGCG on Caco-2 monolayers.
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In the series University of Florida Digital Collections.
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Includes vita.
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by Zheng Li.
Thesis:
Thesis (Ph.D.)--University of Florida, 2013.
Local:
Adviser: Gu, Liwei.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-02-28

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1 DESIGN, PREPAR ATION, AND CHARACTERIZATION OF NANOPARTICLES AS DELIVERY SYSTEMS FOR BIOACTIVE PHENOLIC PHYTOCHEMICALS By ZHENG LI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FUL FILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013

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2 2013 Zheng Li

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3 To my family and friends

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4 ACKNOWLEDGMENTS I want to express my sincere appreciation to my m ajor advisor, Dr. Liwei Gu, for his patience, advice and mentorship. Without his guidance and support, this research could not be accomplished. I am grateful for my committee members, Dr. Susan S. Percival, Dr. Jesse F. Gregory III Dr. Brij M. Moudgil, an d Dr. Kevin W. Powers, for their valuable time and suggestions. I cherished the friendship with my lab members: Amandeep K. Sandhu, Bo Zhao, Haiyan Liu, Kaijie Song, Keqin Ou, Timothy Buran, Sara M. Marshall, Dr. Tao Zou, and Wei Wang. They were always wi lling to offer me helping hands and emotional support. Last and most important, I express my deepest gratitude to my parents for their constant love and commitment to my education. They were my first teachers and gave me many good lessons. They always enc ouraged me to pursue my dream. I would also like to extend my gratitude to my beloved girl friend Qiong Cheng, who is always proud of eve ry single achievement I obtain Because of her love, patience, and unconditional support, I gained the strength to com plete this program.

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5 TABLE OF CONTENTS p age ACKNO WLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVI ATIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 A REVIEW: USING NANOPARTICLES TO ENHANCE BIOAVAILBILITY AND BIOACTIVITY OF PHENOLIC PHYTOCHE MICALS ................................ .............. 16 Introduction ................................ ................................ ................................ ............. 16 Phenolic Phytochemicals ................................ ................................ ........................ 16 Health Bene fits ................................ ................................ ................................ ....... 17 Factors Affecting Oral Bioavailability of Phenolic Phytochemicals .......................... 17 Poor Stability ................................ ................................ ................................ .... 17 Lack of Absorptive Transporters ................................ ................................ ....... 18 Metabolism and Active Efflux in Epithelial Cells ................................ ............... 18 Nanopartic les for Phytochemical Delivery ................................ ............................... 19 Nanoparticle Fabrication ................................ ................................ ................... 19 Methods of Nanoparticle Analysis ................................ ................................ .... 20 Dynamic light scattering ................................ ................................ ............. 20 Electron microscopy ................................ ................................ ................... 20 Nanoparticles for Phytochemical Delivery ................................ ........................ 21 Nanoparticles enhance solubility of loaded phytochemicals ...................... 21 Nanoparticles enhance stability of phytochemicals ................................ .... 21 Nanoparticle transport on epithelium ................................ ......................... 21 Research Objectives ................................ ................................ ............................... 23 2 FABRICAT ION OF NANOPARTICLES USING PARTIALLY PURIFIED POMEGRANATE ELLAGITANNINS AND GELATIN AND THEIR APOPTOTIC EFFECTS ................................ ................................ ................................ ............... 25 Background ................................ ................................ ................................ ............. 25 Materials and Methods ................................ ................................ ............................ 26 Samples and Chemicals ................................ ................................ ................... 26 Extraction and Purification of Pomegranate Ellagitannins ................................ 26 HPLC ESI MS n Analyses ................................ ................................ ................. 27 Self assembly of PPE gelatin Nanoparticles ................................ .................... 27 Particle S ize and Zeta potential Measurement ................................ ................. 28

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6 Scanning Electron Microscopy (SEM) ................................ .............................. 28 Fourier Transform Infrared (FTIR) Spectroscopy ................................ ............. 28 Cell Apoptosis ................................ ................................ ................................ .. 29 Statistical Analyses ................................ ................................ .......................... 30 Results ................................ ................................ ................................ .................... 30 Ellagitannin Identification and Quantification ................................ .................... 30 Characteristics of Ellagitannin gelatin Nanoparticles ................................ ........ 30 Production Efficiency and Loading Capacity ................................ .................... 31 Apoptosis ................................ ................................ ................................ .......... 32 Discussion ................................ ................................ ................................ .............. 32 Summary ................................ ................................ ................................ ................ 35 3 EFFECT OF MASS RATIO, PH, TEMPERATURE, AND REACTION TIME ON FABRICATION OF PARTIALLY PURIFIED POMEGRANATE ELLAGITANNIN (PPE) GELATIN NAN OPARTICLES ................................ ................................ ...... 44 Background ................................ ................................ ................................ ............. 44 Materials and Methods ................................ ................................ ............................ 45 Chemicals ................................ ................................ ................................ ......... 45 Fabrication of PPE gelatin Nanoparticles ................................ ......................... 45 Transmission Electron Microscopy (TEM) ................................ ........................ 46 Particle Size and Zeta potential Measurements ................................ ............... 47 Loading Efficiency Assessment ................................ ................................ ........ 47 Data Analyses ................................ ................................ ................................ .. 48 Results ................................ ................................ ................................ .................... 48 Transmission Electron Microscopy (TEM) ................................ ........................ 48 Effects of PPE t o gelatin Mass Ratios ................................ .............................. 48 Effects of p H ................................ ................................ ................................ ..... 49 Effects of Temperatures ................................ ................................ ................... 50 Effects of Reaction Time ................................ ................................ .................. 51 Discussion ................................ ................................ ................................ .............. 52 Summary ................................ ................................ ................................ ................ 55 4 FABRICATION OF COATED BOVINE SERUM ALBUMIN (BSA) EPIGALLOCATECHIN GALLATE (EGCG) NANOPARTICLES AND THEIR TRANSPORT ACROSS MONOLAYERS OF HUMAN INTESTINAL EPITHELIAL CACO 2 CELLS ................................ ................................ ................. 61 Background ................................ ................................ ................................ ............. 61 Materials and Methods ................................ ................................ ............................ 62 Chemicals ................................ ................................ ................................ ......... 62 Fabrication of BSA Nanop articles ................................ ................................ ..... 63 Loading EGCG into BSA Nanoparticles ................................ ........................... 63 Coating the BSA EGCG Nanoparticles ................................ ............................ 63 Scanning Electron Microscopy (SEM) ................................ .............................. 63 Loading Efficiency and Loading Capacity Assessments ................................ .. 64 Nanopartic les Characterization under Different p H Conditions ........................ 64

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7 Release of EGCG in Simulated Digestive Fluids ................................ .............. 64 Storage Stability of Load ed EGCG in Nanoparticles ................................ ........ 65 EGCG Absorption on Caco 2 Cell Monolayers ................................ ................. 66 Confocal Laser Scanning Microscopy ................................ .............................. 67 Statistical Analyses ................................ ................................ .......................... 68 Results ................................ ................................ ................................ .................... 68 Particle Morphology, EGCG Loading Efficiency a nd Loading Capacity ............ 68 Impact of Coating on Particle Size and Zeta potential ................................ ...... 68 Impact of Nano encapsulation and Particle Coatin g on EGCG Release in Simulated Digestive Fluids ................................ ................................ ............ 69 Impact of Nano encapsulation and Particle Coating on EGCG Stability ........... 70 Impact of Nano encapsulation and Particle Coating on EGCG Transport on Caco 2 Monolayers ................................ ................................ ....................... 71 Impact of Nano encapsulation and Particle Coating on Nanoparticle Cellular Uptake by Caco 2 Cells ................................ ................................ ................ 71 Discussion ................................ ................................ ................................ .............. 72 Summary ................................ ................................ ................................ ................ 76 5 FABRICATION OF SELF ASSEMBLED ( ) EPIG ALLOCATECHIN GALLATE (EGCG) OVALBUMIN DEXTRAN CONJUGATE NANOPARTICLES AND THEIR TRANSPORT ACROSS MONOLAYERS OF HUMAN INTESTINAL EPITHELIAL CACO 2 CELLS ................................ ................................ ................. 85 Background ................................ ................................ ................................ ............. 85 Materials and Methods ................................ ................................ ............................ 86 Chemicals ................................ ................................ ................................ ......... 86 Preparations of Ovalbumin dextran Conjugates ................................ ............... 86 SDS PAGE Analysis of Ovalbumin dextran Conjugates ................................ .. 87 Preparations of EGCG Ovalbumin dextran Conjugate Nanoparticles and Crosslink ed EGCG Ovalbumin dextran Conjugate Nanoparticles ................. 87 Particle Size and Particle Morphology ................................ .............................. 88 Loading Efficiency and Loading Cap acity Assessments ................................ .. 88 Nanoparticle Ch aracterization under Different p H Conditions .......................... 88 Nanoparticle Stability in Simulated Gastric or Intestinal Fluid ........................... 89 Release of EGCG in Simulated Digestive Fluids ................................ .............. 89 EGCG Absorption on Caco 2 Cell Monolayers ................................ ................. 90 Statistical Analyses ................................ ................................ .......................... 91 Results ................................ ................................ ................................ .................... 92 Synthesis of Ovalbumin dextran Conjugate s ................................ .................... 92 Particle Size, Morphology, EGCG Loading Efficiency and Loading Capacity ... 92 Nanoparticle Ch aracterization under Different p H Conditions .......................... 93 Stability of Nanoparticles in Simulated Digestive Fluids ................................ ... 94 EGCG Release from Nanoparticles in Simulated Digestiv e Fluids ................... 95 EGCG Transport on Caco 2 Monolayers ................................ .......................... 95 Discussion ................................ ................................ ................................ .............. 96 Summary ................................ ................................ ................................ .............. 101

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8 6 CONCLUSIONS ................................ ................................ ................................ ... 111 LIST OF REFERENCES ................................ ................................ ............................. 11 3 BIOGRA PHICAL SKETCH ................................ ................................ .......................... 134

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9 LIST OF TABLES Table page 2 1 Particle size, zeta potential, and production efficiency of nanoparticles fabricated using three PPE to gelatin mass ratios ................................ .............. 37 2 2 Content and loading capacity of individual ellagitannin in nanoparticles fabricated using three PPE gelatin mass ratios ................................ .................. 38 4 1 Loading efficiency and loading capacity of EGCG in BSA EGCG nanoparticles (BEN), poly lysine coated BSA EGCG nanoparticles (PBEN), and chitosan coated BSA EGCG nanoparticles (CBEN) ................................ .... 78 5 1 Loading efficiency and loading capacity of EGCG in EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles ................................ ................................ .................... 103

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10 LIST OF F IGURES Figure page 2 1 HPLC chromatogra m of PPE solution ................................ ............................... 39 2 2 Product ion spectra (MS 2 ) of ellagitannins ................................ ........................ 40 2 3 Morphology of PPE gelatin nanoparticles using scanning electron microscopy (SEM). ................................ ................................ ............................. 41 2 4 Fourier Transform In frared (FTIR) spectra of P PE gelatin nanoparticles .......... 42 2 5 Apoptotic effect of PPE gelatin nanoparticles suspension and PPE solution on HL 60 cancer ce lls ................................ ................................ ........................ 43 3 1 Morphology of PPE gelatin nanoparticle using transmiss ion electron microscopy (TEM) ................................ ................................ ............................. 56 3 2 PPE gelatin nanoparticle characteristics under the conditions of PPE to gelatin mass ratio from 1:5 to 9:5 at 25 o C for 2 days ................................ ......... 57 3 3 PPE gelatin nanoparticle characteristics under the conditions of PPE to gelatin mass ratio at 1:5, 5:5, and 7:5, pH of gelatin solution were 1.0, 2.0, 3.0, 4.0, 5.3, 6.0, 7.0, and 11.0, temperature at 25 o C, and reaction time of 2 days ................................ ................................ ................................ ................... 58 3 4 PPE gelatin nanoparticle characteristics under PPE to gelatin mass ratio at 1:5, 5:5, and 7:5 at 5, 10, 15, 25, 40, or 50 o C for 2 days ................................ ... 59 3 5 PPE gelatin nanoparticle characteristics under PPE to gelatin mass ratio at 1:5, 5:5, and 7 :5, at 25 o C for 0 .5, 1, 1.5, 2, 3, or 4 days ................................ .... 60 4 1 Scanning electron micr oscopy images ................................ .............................. 79 4 2 Particles size and ze ta potential of BSA EGCG nanoparticles (BEN), poly lysine coated BSA EGCG nanoparticles (PBEN), and chitosan coated BSA EGCG nanoparticles (CBEN) i n the pH range from 1.5 to 7.0 ........................... 80 4 3 Cumulative release of EGCG from nanoparticles in s imulated fluids with and without digestive enzymes at 37 o C ................................ ................................ .... 81 4 4 Stability of EGCG, EGCG in dry BSA EGCG nanoparticles (BEN), poly lysine coated BSA EGCG nanoparticles (PBEN), and chi tosan coated BSA EGCG nanoparticles (CBEN) at 60 o C for 0, 3, 6, 9, 12, and 24 hours ............... 82 4 5 Apparent permeability coefficient ( P app ) and transepithelial electric resistance (TEER) of EGCG in solution, BSA EGCG nanoparticles (BEN), poly lysine

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11 coated BSA EGCG nanoparticles (PBEN), and chitosan coated BSA EGCG nanoparticles (CBEN) at 37 o C for 30, 60, 90, and 120 min ............................... 83 4 6 FITC labeled BSA EGCG nanoparticles (FITC labe led BEN), FITC labeled poly lysine coated BSA EGCG nanoparticles (FITC labeled PBEN), and FITC labeled chitosan coated BSA EGCG nanoparticles (FITC labeled CBEN) cellular uptake by Caco 2 cells for 60 min at 37 o C ................................ 84 5 1 SDS PAGE analysis of ovalbumin, dextran, ovalbumin/dextran physical mixture, and ovalbumin dextran conjuga tes ................................ .................... 104 5 2 Particle size of and particle mo rphology of EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticle s ................................ ................................ ................................ ... 105 5 3 Particle size and zeta potential of EGCG ovalbumin dextr an conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles in the pH range from 2.5 to 7.5 ................................ .................. 106 5 4 Particle size of EGCG ovalbumin dextran conjugate nanopa rticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles in simulated fluids at 37 o C for 0, 0.5, 1, 1.5, a nd 2 hour ................................ ...................... 107 5 5 Cumulative release of EGCG from EGCG ovalbumin d extran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles in simulated fluids without and with digestive enzymes at 37 o C 108 5 6 Transepithelial elect ric resistance (TEER) and apparent permeability coefficient ( P app ) of EGCG in solution and EGCG ovalbumin dextran conjugate nanoparticles at 37 o C for 30, 6 0, 90, and 120 min .......................... 109 5 7 Illu stration of formation of EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles. ............... 110

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12 LIST OF ABBREVIATIONS ANOVA Analysis of variance ATP Adenosine triphosphate BEN BSA EGCG nanoparticles BSA Bovine serum albumin CBEN Chitosan coated BSA EGCG nanoparticles CO 2 Carbon dioxide DAD Diode array detector DLS Dynamic light scattering EGCG ( ) Epigallocatechin gallate ESI Electrospray ionization FITC Fluorescein isothiocyanate FTIR Fourier transform infrared g gram HBSS HCl Hydrochloric acid HPLC High performance liquid chromatogram IMDM KBr Potassium bromide L Liter L DL Low density lipoprotein mg Milligram min Min ute(s) MS Mass spectrometer mV Millivolts

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13 g Microgram L Micro liter mL Milliliter NaOH Sodium hydroxide nm Nanometer m/z Mass to charge ratio P app Apparent permeability coefficient PBEN Poly lysine coated BSA EGCG nanoparticles PBS Phosphate buffered saline P L A Poly(D,L lactic acid) P L GA Poly(lactic co glycolic acid) PPE Partially purified ellagitannins rpm Revolutions per minute SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophor esis SEM Scanning electron microscopy SGF Simulated gastric fluid SIF Simulated intestinal fluid TEER Transepithelial electric resistance TEM Transmission electron microscopy UV Ultraviol e t

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14 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DESIGN, PREPARATION, AND CHARACTERIZATION OF NANOPARTICLES AS DELIVERY SYSTEMS FOR BIOACTIVE PHENOLIC PHYTOCHEMICALS By Zheng Li Augu st 2013 Chair: Liwei Gu Major: Food Science and Human Nutrition Three types of nanoparticles were designed as delivery systems for ellagitannins or EGCG. In the first design, the PPE were obtained from pomegranate pericarp to contain punicalagin A (16.6 %) and B (32.5%, w/w). P unic alagin and gelatin self assembled into nanoparticles via hydrogen bonding and hydrophobic interactions. Nanoparticles formed at a mass ratio 5:5 had a size of 149 nm, zeta potential of 17.8 mV, and loading capacity of punicalagi n A and B of 14.8% and 25.7%. PPE gelatin n an oparticles were less effective at inducing early stage apoptosis, but as effective as PPE solution at inducing late stage apoptosis and necrosis on HL 60 cells. PPE to gelatin mass ratio s from 1:5 6:5 resulted i n nanoparticles with sizes of 122 129 nm Nano particles fabricated at pH 4.0 5.3 (gelatin solution) had particle siz es of 21 194 nm and zeta potential s of 14.7 23.8 mV. Acidic or basic pH hindered particle formation, whereas p H 6 7 caused particle aggregat ion. Nanoparticles formed at 25 50 o C had size s below 500 nm Nanoparticles remained stable for 4 days In the second design, BSA EGCG nanoparticles were coated with poly lysine or chitos an with size s of 100 200 nm. L oading efficiency and capacity of EGCG w ere 35.4% and 32.7 %, and 17.0% and 16. 0% respectively Coatings prevented particle

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15 aggregation at pH 4.5 5.0 and changed particle zeta potentials. C oatings delayed EGCG release from the nanoparticles in simulated fluids with or without digestive enzymes E GCG in CBEN showed higher P app on Caco 2 monolayers. In the third design, E GCG ovalbumin dextran conjugate nanoparticles were self assembled and further crosslinked by glutaraldehyde. These nanoparticles had size s of 285 nm and 339 nm. Loading efficienc y and capacity of EGCG was 23.4% and 30.0%, and 19.6% and 20.9% respectively They showed nano scale sizes and similar zeta potentials in the pH range 2.5 7.5 Good nanoparticle stabilities were observed in simulated fluids at 37 o C. Digestive enzymes caus ed quicker release of EGCG from the nanoparticles. The c onjugate nanoparticles significantly enhanced P app of EGCG on Caco 2 mono layers.

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16 CHAPTER 1 A REVIEW: USING NANOPARTICLES TO ENHANCE BIOAVAILBILITY AND BIOACTIVITY OF PHENOLIC PHYTOCHEMICALS Introduct ion Phenolic phytochemicals are widely present in the plant kingdom ( 1 ) They are synthesized in plant s as secondary metabolites to modulate plant growth, development, and defense ( 2 5 ) Both epi demiologic and experimental studies have associated the consum ption of phenolic phytochemical rich foods with better health in human s ( 6 9 ) However, the low absorption rate of phenolic phytochemicals limits their health promoting properties ( 10 12 ) Nanoparticles are used to encapsulate and deliver drugs to the gastroinstestinal tract and result in enhanced absorption ( 13 14 ) In th is chapter, classification, health benefits, and absorption of phenolic phytochemicals will be outlined. Fabrication methods, analytical methods, and absorption of nanoparticles will be reviewed. Phenolic Phytochemicals Phenolic phytochemicals represent a variety of plant secondary metabolites consisting of at least one aromatic ring with attached polyhydroxyl groups. More than 8,000 phenolic phytochemicals have been reported to exist in different fruits, vegetables, and cereals ( 15 ) Phenoli c phytochemicals can be divided into flavonoids and non flavonoids according to chemical structure. Flavonoids consist of fifteen carbons, with two aromatic rings conjugated by a three carbon bridge (C 6 C 3 C 6 ) The m ajor flavonoids include flavonols, antho cyanidins, flavan 3 ols, and isoflavone s ( 3 4, 16 21 ) Non flavonoids include phenolic acid (C 6 C 1 ), hydroxycinammates and its derivates (C 6 C 3 ), and stilbenes (C 6 C 2 C 6 ) ( 2 2 26 )

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17 Health B enefits Consumption of pheno lic phytochemicals and phenolic rich foods showed anti cancer properties and offered benefits phenomenon suggests high wine consumption lowers the incidence of coronary hea rt disease in France despite a high i ntake of saturated fat ( 27 ) Resveratrol in red wine contributed to cardiopro tection, decreased platelet aggregation, suppression of prostaglandin biosynthesis, improvement of mitochondrial function, inhibition of LDL oxidation, cancer cell apoptosis, and extended life span ( 28 38 ) Similar ly, flavonols also showed anti cancer, anti inflammatory, and anti diabetic functions ( 39 42 ) Anthocyanidin and its glycosides induced apoptosis of cancer cells and inhibited cancer cell proliferation by regulatio n of gene expression ( 43 44 ) Anthocyanins also protected against UV induced oxidative damages, and inhibited diethylnitrosamine induced hepatocellular carcinogenesis ( 45 46 ) Soy isoflavones inhibited activity of transcription factors and genes essential for tumor cell proliferation, invasion, and neovascularization ( 47 49 ) EGCG prevented oxidative damage in health y cells, as well as provided antiangiogenic and antitumor properties ( 50 ) EGCG also induced apoptosis and caused cell growth arrest in cancer cells ( 51 53 ) Facto rs Affecting Oral Bioavailability of Phenolic P hytochemicals Poor stability, lack of absorptive transporters, metabolism and active efflux in epithelial cells contri buted to the low absorption of phenolic phytochemicals. Poor S tability The g astrointestinal tract has different pH segments and contains reactive oxygen species. Reactive oxygen species can oxidize phenolic phytochemicals. Phenolic phytochemicals are unst able in both low and neutral pH condition s For

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18 example anthocyanins ex perienced a rapid degradation at neutral pH ( 54 ) EGCG was degraded in a first order reaction in both acidic (pH below 2.0) and neutral pH conditions because of the gallate moiety ( 55 56 ) In addition, anaerobic bacteria in the colon can de grade phenolic phytochemicals to yield small molecular weight metabolites ( 57 58 ) Lack o f Absorptive T ransporters No absorptive transporters have been identified for phenolic phytochemicals. Passive diffusion was thought to be the major absorption route. Permeability of phenolic phytochemicals decreases when molecular weight increases. For example, proanthocyanidin dimers and trimer s were absorbed whereas polymers were n ot transported because of higher molecular w eight s ( 59 ) Flavonoid aglycones had much higher apparent permeability coefficient s ( P app ) compared to their glycosides on Caco 2 monolayers ( 60 63 ) For example, isoflavone and resveratrol showed higher P app compared to their glycosides ( 64 66 ) This is because glycoside conjugates are less lipophi lic than aglycones. Metabolism and Active Efflux in Epithelial C ells Once absorbed, p henolic phytochemicals are metaboli z diphospho glucuronosyltransferases (UGTs) and sulfotransferases (SULFs) to form conjugates in ephithelial cells Both absorbed phenolic phytochemicals and their conjugates are substrate s of efflux transporters. These efflux transporters include p glycoprotein, multidrug resistance associated protein 2 and breast cancer resistance protein on the apical cell membrance and multidrug resistance associated protein 3 on the basolateral memb rance ( 67 ) The m ajority of conjugates are pumped by efflux transporters to the lume n of intestine where they are excreted or degraded ( 66, 68 69 )

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19 A smaller portion of conjugate s may be absorbed via multidrug resistance associated protein 3 ( 67 ) Nanoparticles for Phytochemical D elivery In pharmaceutical science, particles with size s below 1000 nm are classified as nanoparticles because of their unique physicochemical properties compared to bulk materials. Nanoparticles have th e capacity to enhance the absorption of loaded drugs or other bioactive compounds. Nanoparticle F abrication Nanoparticles are fa bricate d using two basic approaches, i.e the ( 70 71 ) Bul k materials are shr unk to nano scale particles by a size reduction mechanical process, whereas small mol ecules are aggregated to yield na noparticles using a chemical process In a chemical process, the d e solvation method is a well established fabrication approach. This method uses t hre e important components, including a polymer, an internal solve nt, and an external solvent. P olymer molecules are dissolved in the internal solvent. Subsequently, the external s olvent is gradually added to the internal solvent. When the concentration of the external solvent reaches a certain threshold, p olymer molecules start to aggregate into nanoparticles ( 72 ) S elf assembly of polymers is often used in the chemical process. It take s advantage of direct a nd/or indirect interactions amo ng polymers and ions. Particle formation is affected by reaction conditions such as temperature and pH ( 73 76 )

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20 Methods of N anoparticle Analysis A number of technologies have been ap plied to characterize nanoparticles for their size, surface charge, and morphology. These methods are summarized in T able 1 1 Dynamic light scattering Dynamic light scatt ering (DLS) is a common analytical technique to evaluate the particle size distri buti on. The mechanism behind DLS measurement is particle motility due to Brownian motion. DLS measures the fluctuation intensity of scattered light and appl ies an algorithm to determine the size distribution ( 77 79 ) D LS can measure the particle size distribution quickly and give s a good st atistics. Also, the conversion from i ntensity distribution to volume and /or number weighted distribution can be achieved by M ie theory. Electron microscopy Electron microscopy meas ure s the size and morphology of dry particles Methods include transmission electron microscopy (TEM) and scanning electron microscopy (SEM) ( 77 ) A beam of electrons is transmitted through particle s interact ing with them ; as a result an image is formed from the interaction of the electrons transmitted through the particle. The image is magnified and focu sed onto an imaging device ( 80 ) The advantages of electron microscpy include the ability to measure particle size s below 1 n m and visual particle shape and morphology. However, samples need to be dried before th e electron microscopy measurements, the size distribution is difficult to analyze and electron beam may affect particle characterization ( 81 )

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21 Nanopa rticles for Phytochemical D elivery Nanoparticles enhance the absorp t i on of loaded drugs or phytochemicals by several mecha n i sms. Nanop articles enhance solubility of loaded phytochemicals Nan oparticles often have hydrophob ic groups inside and polar groups on the ir surface. Such structural feature s help to increa s e solubility of encapsulated c ompounds. For example, p roanthocyanidins have extremely low solubility in aqueous solution. After encapsulating them in zein nanoparticles, the solubility of proanthocyanidins increased significantly ( 82 ) Curcumin is insoluble in water. Nanoparticles made of PLGA, chitosan, and proteins were reported to improve the solubility of encapsulated curcumin in aqueous solution ( 83 9 0 ) Nanoparticles enhance stability of phytochemicals Phenolic phytochemicals are unstable in the gastrointestinal tract. For example, EGCG was rapidly degraded in both acidic (pH below 2) and neutral conditions ( 55 ) Encapsulating EGCG into particles, such as chitosan nanoparticles and Eudragit microparticles, significantly delayed its degradation in simulated digest ive fluids ( 55 56, 91 92 ) Nanoparticle transport on epithelium Nanoparticles can penetrate small intestinal epithelium by either the paracellular or transcellular pathway. Tight junctional proteins connect epithe lial cells to protect the integrity of epithelium. Paracellul ar transport refers to diffusion through intercellular spaces among epithelial cells ( 93 94 ) Nanoparticles with sizes below 20 nm can reversibly disrupt tight junctions and release bioactive compounds to facilitate the paracellular diffusion ( 95 97 ) Additionally, tight junction mediators, such as chitosan,

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22 thiolated chitosan, chitosan derivatives, and polyacrylat e derivatives were reported to improve the paracellular transport in the form of either nanoparticles or coatings on nanoparticles ( 13, 98 105 ) Small intenstine e pithe lium consists of goblet cells, enterocytes, M cells, and hormone secreting cells. Gobl et cells sec rete mucus for protection of the epithelium surfaces, whereas enterocytes and M cells internalize nanoparticles by cellul ar uptake. PLA nanoparticles, silica coated rhoadime 6G isothiocyanate nanoparticle s PLGA nanoparticles, and protein based nanoparticles showed much higher cellular uptake at 37 o C compared to 4 o C ( 106 114 ) This is because cellular uptake of nanoparticles requires energy in the form of ATP. N ano particles are taken up by cells via macropinocytosis or endocytosis. Macropinocytosis is an actin driven process without receptor mediation. Macropinosomes are formed on the surface of cells. Particles with size s below 2000 nm are enclosed by macropinosome s and internalized to cells. The internalized particles can be further translocated to endolysosome ( 115 117 ) Epithelial cells also take up nanoparticles via endo c ytosi s. Endocytosis includes clathrin mediated en docytosis, caveolae medated endocytosis, and clathrin and caveolae independent endocytosis ( 118 ) Clathrin mediated endocysotis requires clat hrin coated pits formed by assembly of clathrin. Coated pits develop to form vesicles of 120 nm, a process mediated by GTPase. These vesicles engulf nanoparticles into cells via low density lipoprotein receptor s or iron loaded transferrin receptors that in teract with clathrin adaptor proteins ( 119 ) Nanopartic les with an average size below 200 nm had the ability to stabilize the clathrin coated pits, and the internalization route

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23 was primarily clathrin mediated endocytosis ( 120 ) Nanoparticles internalized by clathrin mediate d endocytosis are translocated to lysosome s ( 119, 121 122 ) In caveolae mediated endocytosis, caveolae is formed by assembling caveolin protein, choloestrol, and sphingolipids on the cell membrane ( 121, 123 125 ) Nanoparticles with size s below 60 nm tend to be internalized by caveolae mediated endocytosis ( 126 ) However, 500 nm nanoparticles were also observed to be taken up in caveolae mediated pathway ( 127 ) After being interna lized in epithelial cells, nanoparticles are translocated to endo/lysosome for degradation. Nanopart icles also may remain intact in endo / lysosome and enter blood circulation by excytosis ( 128 130 ) Additionally, na nopart icles may be translocated to endoplasmic reticulum and Golgi complex and further undergo excytosis across the membrane ( 131 ) Research Objectives The overall objective of this research is to design, prepare, and characteri ze nanoparticles as delivery systems for phenolic phytochemicals. The goal is to improve the absorption and the bioactivity of loaded phytochemicals. This research has four specific aims: 1. To fabricate ellagitannin gelatin nanoparticles using self assembly method and to evaluate its bioactivity on HL 60 cells. 2. To investigate the effects of ellagitannin to gelatin mass ratio, pH, temperature, and reaction time on characterization of ellagitannin gelatin nanoparticles. 3. To fabricate coated BSA EGCG nanoparticle s using a desolvation method and to evaluate EGCG permeability on Caco 2 monolayer. 4. To fabricate EGCG ovalbumin dextran conjugate nanoparticles using self assembly and to investigate EGCG permeability on Caco 2 monolayer.

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24 Table 1 1. Analytical methods a nd technologies for nanoparticle characterization Technology Size range Sensitivity and reliability Suspension in vitro fluids Dynamic light scattering 1 1000 nm Accurate for monodisperse Yes Inaccurate for polydisperse samples Large influence on s ize accuracy and distribution Measurement fast (about 2 5 min per measurement) No sample visualization Approximate size distribution, intensity distribution Scanning mobility analyzing 2.5 1000 nm High resolution data No Fast measurements Discreet particle measurement Automatically flow control Nanoparticle tracking analysis 30 1000 nm Accurate for both mono and poly disperse samples Yes Peak resolution high Little influence on size accuracy and size distribution Measu rement slow (5 min to 1 hour ) per measurement Individual particle size, number distribution Sample visualization More reliable size distribution Electron microscopy 0.3 nm micron High resolution Maybe No calibration necessary Particles n eed to Particle sample shape (two dimension) be dried Samples preparation before measurement Poor statistics on size distribution High vacuum needed Atomic force microscopy 5 nm micron A three dimensional surface profile Maybe No vac uum, wrok well in ambient air or a liquid environment in ambient or Limitation on scanning speed Liquid condition Only see surface of sample

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25 CHAPTER 2 FABRICATION OF NANOPARTICLES USING PARTIALLY PURIFIED POMEGRANATE ELLAGITANNINS AND GELATIN AND THEIR APOPTOTIC EFFECTS Background Nanoparticles are defined in biological sciences as particles with at least one dimension less than 1000 nm ( 132 ) They possess many unique chemical, biological, and physical properties compared to bulk materials Drugs or other bioactive ingredients encapsulated in nanoparticles or other nanocarriers had drastically increased bioavailabi lity and bioactivity. For instance, epigallocathecin gallate (EGCG) encap sulated in nanoparticles was 10 fold as effective as EGCG that was not encapsulated ( 133 ) Carboplatin loaded chitosan alginate nano particles showed greater antiproliferative activities and apoptotic effects compared to the drug in solution ( 134 ) Cisplatin incorporated in gelatin nanoparticles provided stronger antitumor activities and was less toxic compared to free cisplatin in vivo ( 135 ) Pomegranate contains several types of ellagitannins, including punicalagin, punicalin, gallagic acid, ellagic acid, and ellagic acid glycosides ( 136 137 ) The se ellagitannins have been reported to prevent cancer and cardiovascular diseases, scavenge oxidative free radicals, and reduce the risk for atherosclerosis and other chronic diseases ( 138 141 ) The affinity betwee n tannins and proteins is a well known phenomenon ( 142 143 ) The present study takes advantage of such affinity to fabricate the self assembled nanoparticles using partially purified pomegranate ellagitannins (PPE) and gelatin. The PPE gelatin nanoparticles, to our knowledge, have not been reported before. Three different mass ratios of PPE and gelatin were applied and characteristics of nanoparticles including particle size, zeta potential, morphology, and

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26 interact ion binding modes were measured by Microtrac Nanotrac, ZetaPlus, Scanning Electron Microscopy, and Fourier Transform Infrared Spectroscopy, respectively Loading capacity of ellagitannins in the resultant nanoparticles were investigated using HPLC ESI MS n The Annexin V Staining Assay on a leukemic cell line HL 60 was selected to evaluate the apoptotic effect of PPE gelatin nanoparticles and compare that to the PPE solution. Materials and Methods Samples and C hemicals Pomegranates ( Punica granatum L.) were obtained from Publix Supermarket (Gainesville, FL). The pericarp of the fruits was manually separated. Pure punicalagin and ellagic acid was purchased from Quality Phytochemicals LLC (Edison, NJ) and Sigma Aldrich (St. Louis, MO), respectively. Ethanol, f ormic acid, methanol, gelatin type A, and other chemicals were products of Fisher Scientific (Pittsburg h PA). Amberlite XAD 16N resin was purchased from Dow Company (Piscataway, NJ) Extraction and Purification of Pomegranate E llagitannins Extraction and purification of pomegranate ellagitannins followed a published method with minor modifications ( 137 ) Frozen pomegranate pericarp (50 g) was blended in a kitchen blender and mixed with water (250 mL). The water suspension was sonicated for 5 min and k ept at 25 o C for 30 min. The sonication step was repeated for two more times. Water extract was obtained after filtration and then dried on an ISS110 Speed Vac evaporator (Fisher Scientific, Pittsburg h PA) at 25 o C. The resultant solid was re dissolved in 20 mL of water and loa ded on to a column (30 400 mm, ID Length) packed with Amberlite XAD 16 N resin. The column was eluted with 2 liters of

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27 water. E llagitannins were recovered from the column using 400 mL of ethanol. Ethanol elute was evaporated at 25 o C t o yiel d PPE as dry power (approximately 2.5 g). HPLC ESI MS n A nalyses An Agilent 1200 HPLC system consisting of an autosampler, a binary pump, a column compartment, a diode array detector (Agilent Technologies, Palo Alto CA) was interfaced to a HCT ion t rap mass spectrometer (Bruker Daltonics, Billerica, MA). PPE solution was filtered through 0.45 m filter units and 30 L was injected on an Agilent Zorbax ODS column (4.6 mm 25 c m, 5 m particle size) for separation of ellagitannins. The binary mobile p hase consisted of (A) formic acid: water (2:98 v/v) and (B) formic acid: methanol (2:98 v/v). G radient elution reported by Seeram et al was used with minor modifications ( 137 ) 20% B linear; 30 45 min, 20 40% B; 45 60 min, 40 95% B; 60 65 min, 95 1% B; followed by 5 min of re equilibration of the column before the next run. The detection wavelength on a diode array detector was 378 nm. Electrospray ionization in negative mode was performed using nebuliz er 65 psi, dry gas 11 L/min, drying temperature 350 o C and capillary 4000 V. The full scan mass spectra were measured at m/z 100 2000 Auto MS 2 was conducted with 50% compound sta bility and 60% trap drive level. Punicalagin A, B, and ellagic acid were used as external standard for quantification. Data was collected and calculated using Chemstation software (Version B. 01.03, Agilent Technologies, Palo Alto, CA). Self assembly of PPE gelatin N anoparticles Gelatin type A (0.5 g) and PPE powder (0.5 g) were dissolved in DI water (1000 mL) to a concentration of 0.5 mg/mL respectively. PPE solution with 1 mL, 5 mL, or 7 mL was mixed with 5 mL of gelatin solution which gave the PPE to gelatin mass ratio of

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28 1:5, 5:5, and 7:5, respectively. The mixture was incub ated at 25 o C for 48 h The nanoparticle suspensions were then centrifuged at 12,000 rpm for 5 min. The supernatant was removed from the suspension for calculations of loading capacity. The sediments were lyophilized for 48 h to form dry particles and store d at 20 o C for further analyses Particle Size and Zeta potential M easurement Mean particle size and size distribution was measured using Dynamic Light Scattering (DLS) on a Nanotrac ULTRA with an external probe (Microtrac Inc., Largo, FL). Each sa mple wa s analyzed in triplicate tests and each replicate was measured six times to yield the average particle size. Zeta potentials were determined using Brookhaven ZetaPlus (Brookhaven Instrument Corp., Holtsville, NY). Triplicate tests were conducted at a const ant temperature of 22 o C and each replicate was measured ten times to obtain the average zeta potential. Scanning Electron M icroscopy (SEM) Dry particle powders were used for morphology characterization using Field Emission Scanning Electron Microscope (Mo del JSM 633OF, JEOL Ltd, Tokyo, Japan). Fourier Transform Infrared (FTIR) S pectroscopy PPE gelatin nanoparticles were fabricated at PPE to gelatin mass ratio 5:5 Supernatant and nanoparticles were separated by centrifuge and freeze dried. Dry PPE, gelati n, PPE gelatin nanoparticles, and dried supernatant were mixed with p ure KBr powders using a ratio of 1: 100 sample: KBr (w/w) These mixtures were ground into fine powders and then analyzed using a Thermo Nicolet Magna 760 FTIR (Thermo Nicolet Corp., Madi son, WI) with a MCT A detector. Pure KBr powders were used as

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29 background. The FTIR spectra were obtained over the wave number range from 700 to 4000 cm 1 at a resolution of 2 cm 1 Cell A poptosis Annexin V s taining was used for cell apoptosis measurement s in the HL 60 cell line. Four PPE gelatin nanoparticle suspension samples were prepared, and each PPE gelatin nanoparticle suspension with the concentration of 0.0156, 0.0313, 0.0625, and 0.125 mg/mL was added to the appropriate wells i n triplicate PPE so lution with the concentration of 0.0156, 0.0313, 0.0625, and 0.125 mg/mL w as added to wells in triplicate HL into each well of a 48 well tissue culture plate (Costar, Corn ing, NY). Four hundred microliters of IMDM (Lonza, Basel, Switzerland) complete media (100,000 U/L penicillin; 100mg/L streptomycin; 0.25mg/L fungizone ; 50mg/L gentamicin) was added. The plate was incubated for 22 hours at 37 o C in a 7.5% CO 2 humidified atmosphere. Cell a poptosis was measured by following the Annexin V staining protocol (eBioscience, San Diego, CA). At 22 hours, the HL 60 c ells were harvested and washed once in PBS and diluted binding buffer, respectively. Then, cells were re suspended in diluted binding b conjugated Annexin V was added and incubated for 15 min at room temperature. Cells were washed again in diluted binding buffer and added to cells and the cells were then analyzed on a 3 color fluorescence FACsort Flow Cytometer (Becton Dickinson San Jose, CA ) at the UF ICBR Flow Cytometry core facility within 2 hours. Data was analyzed with FlowJo software (TreeStar, Inc. version 7.6).

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30 Statis tical Analyses Data was expressed as mean standard deviation. One way analyses of variance (ANOVA) with Tukey HSD comparison of the means were performed using JMP software (Version 8.0, SAS Institute Inc., Cary, NC). test was use to compare data from two independent groups. significant. Results Ellagitannin Identification and Q uantification The PPE were extracted and partially purified using porous adsorption resin. Chromatogram of PPE showed four pea ks that corresponded to four ellagitannins ( Figure 2 1 ). Peak identification ( Figure 2 2 ) was based on mass and product ion spectra ( 137 ) Peak A and B were identified as punicalagin A and B according to [M H] m/z 1083 and a product ion at m/z 781 whi ch suggested the existence of punicalin and ellagic acid moiety. Peak C had a deprontonated ion at m/z 463 and a product ion at m/z 301 formed by ellagic acid. This peak was identified as ellagic acid hexoside. Peak D had a deprontonated ion at m/z 301, wh ich was identified as ellagic acid Punicalagtin s were the major ellagitannins in PPE, and the content of punicalagin A and B was 166.2 and 324.6 mg/g. The content of ellagic acid hexoside and ellagic acid was 3.4 and 7.5 mg/g, respectively. Characteristic s of E llagitannin gelati n N anoparticles PPE gelatin nanoparticles were fabricated using three different PPE to gelatin mass ratios ( Table 2 1 ). The mean sizes of particles from the PPE to gelatin mass ratios at 1:5, 5:5, and 7:5, measured using dy namic li ght scattering (DLS), were 102, 149, and 229 nm, respectively. The particle size s increased significantly with the

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31 increase of the PPE to gelatin mass ratio. Scanning Electron Microscopy (SEM) image showed that PPE gelatin nanoparticles fabricated under th ese mass ratios had a spherical or approximately spherical morphology ( Figure 2 3 ). Mean zeta potentials of these formed nanoparticles were around +18 mV, indicating that PPE gelatin nanoparticles had a positive charge on the surface. Interaction bindings between PPE and gelatin were assessed with Fourier Transform Infrared (FTIR) spectra ( Figure 2 4 ). The FTIR spectra of gelatin ( Figure 2 4 A ) showed the typical amides I and II peaks at 1699 and 1558 cm 1 respectively. The amide I absorption was mainly due to carbonyl C=O stretching vibration whereas the amide II band consisted both C N stretching and C N H in plane bending ( 144 145 ) Peaks at 1728 and 1599 cm 1 in the FTIR spectra of PPE ( Figure 2 4B ) resulted from carbonyl stretching vibration ( 146 147 ) FTIR profile ( Figure 2 4 C ) of supernatant from PPE gelatin nanoparticle suspension system was similar to that of PPE ( Figure 2 4 B ). However, FTIR spectra ( Figure 2 4 D ) of PPE gelatin nanoparticles showed peaks at 1664 and 1535 cm 1 both of which resulted from peak shifts of PPE and gelatin toward lower wavenumbers. It suggested that both amide I and II and carbonyl C=O in PPE were involved in PPE helical configuration was formed in PPE gelatin nanoparticles ( 144 145, 148 ) The primary interacti on binding forces were hydrogen bonding and hyd rophobic interactions ( 149 ) Production Efficiency and Loading C apacity Among four ellagitannins identified from the PPE, only punicalagin s had the capacity to bind gelatin to form PPE gelatin nanoparticles ( Table 2 2 ). Produ ction efficiency (%) was the weight of dry nanoparticles divided by total weight of PPE and gelatin that were used. The production efficiency of nanoparticles at mass ratio 1:5, 5:5,

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32 and 7:5 was 18.5%, 53.0%, and 62.0 %, respectively. Loading capacity (% w/ w) is the content of ellagitannins in dry nanoparticles. The l oading capacity of punicalagin A under PPE to gelatin mass ratio at 1:5, 5:5, and 7:5 was 9.0%, 14.8%, and 10. 8 %, respectively. The l oading capacity of punicalagin B was 9.0 %, 25.7%, and 20.5%, respectively. Apoptosis Apoptotic properties of pomegranate PPE gelatin nanoparticles and PPE solution were evaluated using HL 60 cancer cell line at four concentrations Cells that are in the early stage of apoptosis stain positive for Annexin V but negat ive for propidium iodide. Necrotic cells and cells at the late stage of apoptosis stain positive for both Annexin V and propidium iodide. PPE solution was more effective than PPE gelatin nanoparticle suspension in inducing the early stage of apoptosis ( Fig ure 2 5A ). The differences were not significant at the lowest concentration but became significant as the concentrations increased. At concentrations from 0.0156 to 0.0625 mg /mL no significant differences were observed between PPE gelatin nanoparticle sus pension and PPE solution in inducing late stage of apoptosis and necrosis ( Figure 2 5 B ) However, at the higher concentration (0.125 mg/mL), PPE gelatin nanoparticles suspension was m ore effective than PPE solution (p=0.0144). Discussion PPE gelatin nanopa rticles formed spontaneously due to the affinity between ellagitannins and gelatin. Gelatin has an extended random coil conformation in aqueous solution because it contains higher amounts of proline residues than other proteins or polypeptides ( 150 ) Gelat in and tannin molecules bind to each other when they are mixed in solution ( 150 151 ) Magnitudes and selectivity of such binding a re determined

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33 by molecular weight and tertiary structures of tannins. It has been proposed that tannins of higher molecular weight have more hydroxyl groups and hydrophobic sites, and thus enhance the interaction with gelatin ( 151 152 ) In the present study, only punicalagin s were able to bind gelatin to form nanoparticles. Tannins of smaller molecular weight, such as ellagic acid and ellagic acid hexoside, lack tertiary structures and binding capacity ( 153 ) On the contrary, punicalagin, with higher molecular weight (MW 1084), has a larger and more complex molecular structure provi ding sufficient affinity toward gel atin to form nanoparticles. Conformation of tannin molecules also affects affinity fo r gelatin. It has been reported that ellagitannin, compared to gallotannins, had intramolecular biphenyl linkages that can constrain aromatic rings in hydroxydiphenoyl gro ups, leading to the loss of conformational freedom ( 151 ) Such str uctural characteristics enhance binding between ellagitannin and gelatin, however, they may negatively impact affinity for other proteins, such as bovine serum albumin ( 151 ) FTIR spectra of PPE gelatin nanoparticle showed that peaks at 1665 and 1535 cm 1 were shifted toward lower wavenumbers from correspondent peaks in gelatin and PPE. The intensity of peaks at 1699 and 1558 cm 1 in gelatin FTIR spectra was lower than those in PPE gelatin nanoparticle FTIR spectra, suggesting that carbonyl C=O stretching vibration in pomegranate ellagitannins was involved in PPE gelatin nanopa rticles. The major binding mechansim appeared to be hydrogen bonding, which was consistent with the appearance of peaks at 1665 and 1535 cm 1 and with the disappearance of hydroxyl groups (peak in the range of 3500 3600 cm 1 ) in gelatin FTIR spect ra ( 144 145, 149, 154 )

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34 Formation of PPE gelatin nanoparticles is also influenced by PPE to gelatin mass ratio ( 74, 155 ) Wh en a small amount of tannins is added into a gelatin solution, tannin molecules deposit onto gelatin molecules via hydrogen binding and hyd rophobic interactions. As the tannin content increases, single gelatin molecules adsorb more tannin molecules, which cause an increase in particle size. In this study, the particle size with PPE to gelatin mass ratios of 1:5, 5:5, and 7:5 was 102, 149, and 229 nm, respectively. The particle sizes measured by DLS appeared bigger than those in SEM photograph ( Figure 2 3 ). This was because lyophilized dry particles were used in SEM analysis ( 72 ) Stability of the nanoparticle system in suspension is impacted by zeta potential of nanoparticles ( 156 157 ) Nanoparticles with higher zeta potentials are more stable in solution due to static repulsion of particles. In this study, zeta potentials of the nanoparticle suspensions were around +18 mV. The resultant nanoparticles under these three mass ratios were stable opalescent suspensions in deionized water. Uptake of nanoparticles in vivo depends on particle size, zeta potential, and morphology of nanoparticles. In the gastrointestinal tract, nanopart icles are absorbed into epithelial cells by inter membranous diffusion and by endocytosis mediated by receptors on cell membranes ( 13, 118 ) Nanoparticle endocytosis was thought to be the major route. Nanoparticles with a particle size below 500 nm were effec tively absorbed by enterocytes in the gastrointestinal tract ( 118, 158 159 ) PPE gelatin nanoparticles fabricated in this study had a favorable particle size for absorption. Nanoparticles with spherical morphology w as found to be captured more easily by receptors on epithelial cells than particles of different shapes ( 160 ) Surface charge (zeta potent ial) of the nanoparticle is another factor that affects direct uptake ( 13, 118, 161 ) It was reported

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35 that the mucus layer attached to the s urface of epithelial cells has negative charges When positively charged na noparticles approach the mucus layer, weak ionic interaction is established, prolonging the retention time between nanoparticles and epithelial cells and eventually enhancing the entrapment of nanoparticles in epithelial cells ( 13 ) Nanoparticles in the present study had the positively charged surface (around +18 mV). Therefore, we anticipate that PPE gelatin nanoparticles may increase the absorption of ellagitannin in vivo This is of import ance because only 3 6% of ellagitannins were found to be absorbed from pomegranate juice ( 136, 162 ) Bioactivities of a component are often altered once it is embedded into nanoparticles ( 81 ) Pomegranate ellagitannins were known to induce cell cycle arrest and apoptosis of cancer cells ( 163 ) Punicalagin A and B were known to activate caspase 3 pa thway and cleave poly (ADP ribose) polymerases, resulting in DNA fragmentation in HL 60 cells and apoptosis ( 163 164 ) PPE gelatin nanoparticles were less effective than PPE in inducing the early stage of apoptosi s whereas they had similar effects in inducing the late stage of apoptosis and necrosis. We speculated that cell ular uptakes of nanoparticles may be a major reason for the observed di fferences in apoptotic effects. Our data suggested that PPE gelatin nano particles have lower toxicity than pomegranate ellagitannins. Summary The PPE obtained from pomegranate perica rp contained punicalagin A and B ellagic acid hexoside, and e llagic acid. Punicalagin s had the capacity of binding gelatin to form nanoparticles The nanoparticle s fabricated using three PPE to gelatin mass ratios had the particle size s < 250 nm, zeta potential s around +18 mv spherical

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36 morphology, and good loading capacity. PPE gelatin nanoparticl es had lower apoptotic effects o n HL 60 cells comp ared to PPE solution.

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37 Table 2 1. Particle size, zeta potential, and production efficiency of nanoparticles fabricated using three PPE to gelatin mass ratios Volume of PPE solution (mL) Volume of gelatin solution (mL) PPE to gelatin mass ratio Particle si ze (DLS, nm) Zeta potential (mV) Production efficiency (%) 1 5 1:5 102 9 c 18.3 1.2 a 18.5 4.9 b 5 5 5:5 149 2 b 17.8 0.9 a 53.0 4.2 a 7 5 7:5 229 5 a 18.0 1.3 a 62.0 2.8 a Data are mean standard deviation (n=3). Duplicate tests w ere done for production efficiency. Means

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38 Table 2 2. Content and loading capacity of individual ellagitannin in nanopart icles fabricated using three PPE gelatin mass ratios Ellagitannin compou nd Punicalagin A Punicalagin B Ellagic acid hexoside Ellagic acid Content in dry PPE powder (mg/g) 166.2 3.0 324.6 0.3 3.4 0.6 7.5 0.0 Loading capacity (%, w/w) 1:5 9.0 2.6 b 9.0 2.4 b ND ND 5:5 14.8 1.5 a 25.7 2.2 a ND ND 7:5 10.8 0.1 ab 20.5 0.1 a ND ND ND, not detected. Results are represented as mean standard deviation of duplicate tests.

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39 Figure 2 1 HPLC chromatogram of PPE solution A ) Punicalagin A. B ) Punicalagin B. C ) Ellagic acid hexoside. D ) Ellagic acid

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40 Figure 2 2. Product ion spectra (MS 2 ) of ellagitannins. A ) Punicalagin A. B ) Punicalagin B. C ) Ellagic acid hexoside. D ) E llagic acid

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41 Figure 2 3. Morphology of PPE gelatin nanoparticle s using scanning electron microscopy (SEM) A) PPE to gelatin mass ratio of 1:5. B) PPE to gelatin mass ratio of 5:5. C) PPE to gelatin mass ratio of 7:5

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42 Figure 2 4. Fourier Transform Infrared (FTIR) spectra of PPE gelatin nanoparticles. A) G elatin B ) PPE C) F reeze dried supernatant D) F reeze dried PPE gelatin nanoparticles.

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43 Figure 2 5 Apoptotic effect of PPE gelatin nanoparticles suspension and PPE solution on HL 60 cancer cells A) E arly stage of apoptosis B ) L ate stage of apoptosis and necr osis. PPE gelatin nanoparticle suspension was fabricated using PPE to gelatin mass ratio 5:5 Data are mean standa rd deviation of triplicate tests

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44 CHAPTER 3 EFFECT OF MASS RATIO, PH, TEMPERATURE, AND REACTION TIME ON FABRICATION OF PARTIALLY PURIFI ED P OMEGRANATE ELLAGITANNIN (PPE ) GELATIN NANOPARTICLES Background Nanoparticle fabrication is of particular interest for nutritional sciences due to the unique benefits of nanosized carriers to increase oral bioavailability of loaded bioactive ingredients ( 118, 165 ) In the gastrointestinal tract, enterocytes and microfold cell in epithelial wall absorb nanoparticles by endocytosis ( 13 ) Nanoparticle endocytosis was affected by particle size, zeta potential, and morphology It was reported that particle size s below 500 nm significantly improved the bioavailability of in gredients loaded on nanoparitlces ( 118, 158 159 ) Zeta potentials or surface char ge s of nanoparticles affect the stability of nanoparticle suspension system and their absorption in the gastrointestinal tract. It w as known that zeta potential above 30 mV enhanced the stability of nanoparticles in suspension ( 156, 166 167 ) Positive zeta potential stimulated the entrapment of nanoparticles in epithelial walls due to ionic in teractions between nanoparticles and the mucus layer on the surface of enterocytes and microfold cell, which prolongs retention time of nanoparticles on epithelial walls ( 13 ) Spherical nanoparticles can be recogni zed and captured by receptors on the surface of epithelial cells, causing increased absorption rate compared to nanoparticles of different morphologies ( 118, 168 ) ( 118, 169 ) The op isions as the energy sources to cut larger entities into n anoscale aggregates, whereas self assembly of smaller molecules is the primary mechanism for the

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45 Self assembly between molecules takes advantage of intermolecular affinity ( 74, 151 ) Particle size, zeta potential, and loading efficiency of self assembled particles are controlled by reaction conditions. In this study, pomegranate partially purified ellagitannin (PPE) gelatin nanoparticles were fabricated under different PPE t o gelatin mass ratios, pH conditions, temperatures, and reaction times to study their effects on particle characteristics, including particle size, zeta potential, and loading efficiency of ellagitannins. The o bjective of this study was to identify reactio n conditions that can result in smaller particles sizes, positive zeta potentials, and stable nanoparticle colloidal suspension. Materials and M ethods Chemicals Pomegranates were purchased from a local grocery store. Gelatin types A, formic acid, ethano l, and methanol were products of Fisher Scientific (Pittsburg, PA). Amberlite XAD 16N resin was a product of Rohn Haa s (Midland, Michigan). Punicalagin and ellagic acid was purchased from Quality Phytochemicals LLC (Edison, NJ) and Sigma Aldrich (St. Louis MO), respectively. Partially purified pomegranate ellagitannins (PPE) were prepared according to the published method s with minor modification ( 137, 170 ) It contained 16.6% of punicalagin A, 32.6% of punicalagin B, 0.3% of ellagic acid hexoside, and 0.8% of ellagic acid. Other compounds in PPE were not characterized. Fabrication of PPE gelatin N anoparticles Both gelatin type A (0.5 g) and PPE powder (0.5 g) were dissolved in de ionized water (1000 mL) to a concen tration of 0.5 mg/mL The pH value of de ionized water was 5.5 and it dropped to 5.3 and 4.2, respectively, after gelatin and PPE were dissolved. The PPE solution was mixed with the gelatin solution at different reaction conditions for

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46 the self assembly to occur. The resultant nanoparticle suspensions were centrifuged at 12,000 rpm for 5 min to separate suspension into supernatant and nanoparticles in sediment. Concentrations of punicalagin A and B in the supernatant were quantified using H PLC for loading e fficiency assessment To study the effects of the PPE to gelatin mass ratios on nanoparticle fabrication, 1, 2, 3, 4, 5, 6, 7, 8, or 9 mL of the PPE solution was mixed with 5 mL of the gelatin solution. This gave PPE to gelatin mass ratio 1:5 to 9:5. Nanop articles were fabricated at 25 o C for 2 days. To study the effects of pH on nanoparticle fabrication, HCl (3 mol/L) and NaOH (6 mol/L) solutions were added drop by drop to adjust pH value of gelatin solution to 1.0, 2.0, 3.0, 4.0, 5.3, 6.0, 7.0, or 11.0. P PE solution of 1, 5, or 7 mL was added into 5 mL of g elatin solution Size, zeta potentials, and loading efficiencies of resulting nanoparticles were measured after 2 days of reaction time. For the effects of temperature on nanopar ticle fabrication, PPE so lution of 1, 5, or 7 mL was mixed with 5 mL of gelatin solution. The suspensions were incubated at different temperatures from 5 o C to 50 o C for 2 days. Then, characteristics of these nanoparticles were measured. For the effects of reaction time PPE solutio n of 1, 5, or 7 mL was added into 5 mL of gelatin solution at 25 o C. The characteristics of the nanoparticles were measured at 0.5, 1, 1.5, 2, 3, and 4 days, respectively. Except for gelatin solutions which were used to study pH effects, the pH of gelatin a nd PPE solutions were not adjusted (5.3 and 4.2, respectively). Transmission Electron M icroscopy (TEM) PPE gelatin nanoparticles were fabricated using PPE to gelatin mass ratio 5:5 at 25 o C for 48 hours. The morphology of dry PPE gelatin nanoparticles was m easured on a JEOL 200CX TEM (JEOL Inc., Tokyo, Japan).

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47 Particle Size and Zeta potential M easurements Mean particle size and size distribution of fresh suspe nsions was measured by dynamic light s cattering (DLS) using Nanotrac ULTRA with an external probe ( Microtrac Inc., Largo, FL). Samples were measured in triplicate tests and six readings were obtained for each replicate to calculate the average particle size. Zeta potentials were determined using Brookhaven ZetaPlus (Brookhaven Instrument Corp., Holtsvil le, NY ). The following parameters were used for zeta po tential measurement: z eta potential model, Smoluchoswki; me dia, aqueous; temperature, 22.0 o C; viscosity, 0.955 cP; refractive index, 1.334; dielectric constant, 79.63. Triplicate tests were conducte d f or each sample and ten reading s were obtained for each replicate to calculate the average zeta potential. Loading Efficiency A ssessment An Agilent 1200 HPLC system consisting of an autosampler, a binary pump, a column compartment, a diode array dete ctor (A gilent Technologies, Palo Alto, CA) was interfaced to a HCT ion trap mass spectrometer (Bruker Daltonics, Billerica, M A). PPE solution and supernatant from PPE gelatin nanoparticle suspension were filtered th rough 0.45 m filter units and 3 0 L were inject ed without further purification, respectively. An Agilent Zorbax ODS column (4.6 mm 25 c m) was used for separati on of ellagitannins The binary mobile phase consisted of (A) formic acid: water (2:98 v/v) and (B) formic acid: methanol (2:98 v/v). A 65 min gradient 1 2 0% B l inear; 30 45 min, 20 40% B ; 45 60 min, 40 95% B; 60 65 min, 95 1% B; followed by 5 min of re equilibration of the column before the next run. The detection wavelengt h on the diode array detector was 378 nm Electrospray ionization in negative mode was performed using nebulizer 65 psi, dry gas 11 L/min, drying temperature

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48 350 o C, and capillary 4000 V. The full sc an mass spectra were measured from m/z 100 2000. The retention time for punicalagin A, punicalagin B, ellagic ac id hexoside, and ellagic acid were 14.48, 19.93, 42.50, and 49.83 min. Punicalagin A and B were quantified using external standa rd Data were collected and calculated using Chemstation software (Version B. 01.03, Agilent Technologies, Palo Alto, CA). The l oading efficiency was calculated as ( the amount of ellagitannin embedded into PPE gelatin nanoparticles ) / ( the amount of ellagitannin used for nanoparticle fabrication ) Data Analyses Samp les were analyzed in triplicate tests and the ave rage values were used. Data was expressed as mean standard deviation. Figures were drafted using Sigma Plot (Version 10.0, Systat Software, Chicago, IL). Results Transmission Electron M icroscopy (TEM) Self assembly between PPE and gelatin in aqueous solution resulted in the formation of nanoparticles. Nanoparticles fabricated using PPE to gelatin mass ratio 5:5 had a spherical morphology ( Figure 3 1 ). Effects of PPE to gelatin Mass R atios The average sizes of the nanoparticles fabricated using the PPE to gelatin mass rat ios from 1:5 to 6:5 were i n a range of 122 nm to 12 9 nm. A noticeable increase of nanoparticle size was observed as the PPE to gelatin mass ratio increas ed to 7:5 and 8:5, but the sizes were still below 273 nm. The average size dramatically increased to 62 1 nm at a mass ratio of 9:5. Turbidity in the suspension started to appear at this mass ratio. This observation suggested that there was a critical point when using PPE and gelatin to prepare nanoparticles. At such a point, the binding sites on the gelatin

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49 molecule were saturated by ellagitannins. Adding more tannin molecules caused precipitation due to the particle aggregation. As a result, the colloidal system lost stability and precipitation started to occur. The critical point for PPE and gelatin appear ed to be the mass ratio between 8:5 and 9:5. Zeta potentials of the nanoparticles increased and reached a peak of +21.7 mV at a PPE to gelatin mass ratio of 3:5. It dropped to +15.8 mV a nd +14.5 mV when the ratio increased to 8:5 and 9:5, respectively ( Fig ure 3 2 B ). Loading efficiency of punicalagin A was 39.3% at a mass ratio of 2:5 and increased to reach a peak of 78.2% at a mass ratio of 8:5 ( Figure 3 2C ). A similar trend was observed for punicalag in B. Its loading efficiency elevated from 29.6% to 74.5% when the ratio increased from 1:5 to 8:5 ( Figure 3 2D ). Effects of P H When gelatin s olution in the pH condition of 1.0, 2.0, 3.0 or 11.0 was used for fabrication, particle sizes around 0 nm were observed at three mass ratios. This indicated that no nanopa rticles were formed in these pH conditions and the PPE and gelatin existed in a true solution instead of a colloidal suspension ( Figure 3 3 A ). At pH 4.0, the size s of nanoparticles fabricated at the mass ratio 1:5, 5:5, and 7:5 were 21, 44, and 67 nm, resp ectively. At pH 5.3, the size s were 122, 129, and 194 nm, respectiv ely. However, the average sizes at the mass ratio 5 :5 and 7:5 dramatically increased to 752 nm and 13 07 nm at pH above 6.0 respectively. The pH range of 4.0 to 5.3 gave rise to nanoparticl es with smaller sizes. Zeta potential s of PPE gelatin nanoparticles were significantly affected by pH ( Figure 3 3 B ). Zeta potential s of the nanoparti cles using gelatin solution at pH 4.0 and 5.3 showed +26.1 and +14.7 mV at a mass ratio of 1:5, +23.8 and + 17.3 mV at a mass

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50 ratio of 5:5, and +15.0 and +17.0 mV at a mass ratio of 7:5, resp ectively. P articles fabricated at pH 6.0 and 7.0 had the zeta potential s of 1.5 and +1.1 mV at a mass ratio of 1:5, 1.3 and +1.0 mV at a mass ratio of 5: 5, and +0.9 and 0 .7 mV at a mass ratio of 7:5, respectively. Such low zeta potentials suggested that surface charges of nanoparticles were close to neutral. Low zeta potential decreases the stability of nanoparticles in colloidal system. Nanoparticles that were fabricated using gelatin solution with pH from 4.0 to 5.3 showed higher surface charges and better stability. In the pH range from 4.0 to 7.0 increases were observed in loading efficiency of punicalagin A and B from nanoparticles fabricated under these mass rat ios For punicalagin A, its loading efficiency in nanoparticles increased from 48.8% to 54.0% at a mass ratio of 1:5, from 29.5% to 84.3% at a mass ratio of 5:5, and from 44.1% to 70.7% at a mass ratio of 7:5, respectively. Punicalagin B was embedded into nanop articles, from 10.6% to 21.1%, from 23.1% to 73.9%, and from 33.9% to 63.5%, respectively. Effects of T emperatures At a mass ratio of 1:5, nanoparticles fabricated at different temperatures (25 50 o C) had similar particle sizes from 114 to 122 nm ( Figure 3 4 A ). Highest particle sizes were observed at 5 o C in the nanoparticles using mass ratios of 5:5 and 7:5. Th e sizes decreased to below 316 nm when the temperatures were 25 o C and higher. N anoparticle s fabricated below 25 o C at these mass ratios showed stable positive charge properties ( Figure 3 4 B ). Nanoparticle s formed at 5, 10, 15, and 25 o C had the zeta potential s from +14.7 to +17.2 mV at a mass ratio of 1:5, from +17.3 to +19.3 mV at a mass ratio of 5:5, and +14.2 to +17.0 mV at a mass ratio of 7:5. Howeve r, the fluctuations in zeta potentials were observed above 25 o C. Zeta potential s

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51 o f nanoparticles fabricated at a mass ratio of 7:5 dropped dramatically from +17.0 to 0.9 mV. Increase of temperature from 5 o C to 50 o C slightly increased loading eff iciency o f punicalagin A and B The increase was more pronounced for punicalagin A at a mass ratio of 1:5 Effects of Reaction T ime Nanoparticles were fa bricated at a mass ratio of 1:5 after 12 hours, and particle size s r emained similar to 4 days (138 nm) ( Figure 3 5 A ). At a mass ratio of 5:5, nanopart icles with a size of 75 nm were fabricated after 12 h of interaction, and size of 96 nm was observed by 4 days. Nanoparticles at a mass ratio o f 7:5 had a size of 125 nm after 12 h. However, it dramatically increased t o 194 nm after 2 days and 923 nm after 4 days, respectively. Particle surface charge showed little change at different reaction time ( Figure 3 5 B ). Zeta potentials of the nanoparticles, in reaction time from 0.5 to 4 days, fluctuated between +14.7 and +18. 4 mV at a mass ratio of 1:5, between +17.3 and +19.7 mV at a mass ratio of 5:5, and between +15.5 to +17.6 mV at a mass ratio of 7:5, respectively. In nanoparticles made at a mass ratio of 1:5, loading efficiency of punicalagin A and B remained 68.6% and 3 8.0% by 4 days, respectively Nanoparticles made at a mass ratio of 5:5 had a loading efficiency of 67.4% for punicalagin A and 57.2% for punicalagin B at 12 h of interaction, and loading efficiency of 88.3% and 78.9% was obtained after 4 days, respectivel y. From 78.3% to 85.8% of punicalagin A, and from 70.3% to 73.1% of punicalagin B was observed in loading efficiency from 0.5 to 4 day, respectively

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52 Discussion Fabrication of tannin protein nanoparticles dep ends on affinity between tannin and protein mole cules The structure of the protein molecule determines the degree of affinity ( 150 151, 171 ) It was known that the protein with compact tertiary structure provides less hydrophobic sites, constraining interaction with tannin molecules and thus resulting in poor affinity for tannin molecules ( 151 ) However, gelatin is a proline rich protein with extended random coil conformatio n. Hence, gelatin provides more interaction sites for tannin molecules, eventually promoting higher affinity for tannin molecules ( 150, 171 ) Assembl y of tannin protein particles is also directly determined by the structures of tannin molecules. Tannin molecular weight is a critical factor. Tannins with higher molecular weight s possess larger structure and more hydrophobic sites, which results in incre ased affinity toward protein ( 172 ) Among four ellagitannins were identified in PPE, punicalagin A and B were predominant in amount and also had much higher molecular weight (1084) than ellagic acid hexoside (464) and ellagic acid (302 ) ( 170 ) Punicalagins were able to bind with gelatin to form nanoparticles, whereas ellagic acid hexoside or ellagic acid could not. Self assembly of tannin and protein molecules is based on interaction among them via hydrogen bonding and hydrophobic interaction as driving for ces ( 173 174 ) The s ame amount of gelatin was used in the reaction system to provide the same number of hydrophobic sites. Different amount s of the PPE were added into the system to test effects of mass ratio. Whe n a small amount of tannins was added in the system, limited numbers of phenolic hydroxyl groups from tannin molecules were present to in teract with excessive amount of protein molecules. In this case, one tannin molecule can bind with two or more gelatin m olecules. After more PPE was added, more

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53 hydroxylic groups from the tannins were brought into the system, increasing interaction between tannin and gelatin molecules. As a result, a single protein molecule could carry more tannin molecules and higher l oadi ng efficiency was observed However, when the amount of PPE reached the critical point, interaction sites on gelatin molecules became saturated by tannin molecules. PPE that exceeded this stoichiometric value formed an extended network of hydrogen bonds am ong nanoparticles, which resu lted in their aggregation in suspension ( 74, 155 ) Therefore, fabrication of nanoparticles should be conducted below the critical point. Nano particles at mass ratios below 6:5 had similar particle sizes. However, lower loading efficiencies were observed in lower mass ratios, such as 2:5, 3:5, which indicated that one tannin molecule might assemble with two or three gelatin molecules during the fabrication process. Larger particle sizes were observed from the particles under the mass ratio s above 8:5, which suggests that the aggregation occurred when ellagitannin molecules exceeded the saturated value of the network of hydrogen bonds among the na noparticles. It was proposed that nanoparticles with zeta potential above 30 mV provided the best stability. Lower zeta potentials means weak repulsion between particles and can promote aggregation and precipitation o f nanoparticles ( 156 ) Particles fabricated using the mass ratio above 8:5 suggested n anoparticles had very low zeta potential s This may promote aggregation of p articles and increased particle sizes. The pH is a critical factor that affects the structures, the conformations, and charge properties of ge latins and tannins Ellagitannins can be hydrolyzed into ellagic acid in extreme pH conditions ( 175 176 ) Hydrolyzed ellagitannins will lose the capacity to bind with gelatin. This may explain the absence of nanoparticles when using gelatin

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54 s olution in the pH conditions of 1.0, 2.0, 3.0, and 11.0 Gelatins at different pH conditions possess differen t number of ionizable sites ( 177 ) Gelatin at its isoelectric point provides the most binding sites, which will promote self asse mbly between gelatin and tannin ( 74 ) However, stability of nanoparticle suspension relies critically on charge repulsions among nanoparticles. Zero repulsions among particles are anticipated when zeta potentials of nanoparticles are neutr al, promoting the aggregation of particles and thu s leading to precipitation ( 156, 166 167 ) The isoelectric point of gelatin is around pH 6.0 to 7.0. Our study showed that particle s fabric ated using gelatin solutio n in the pH conditions from 4.0 to 5.3 had small particle sizes and positive zeta potential. However, dramatic increases of particle size s were observed at pH 6.0 and 7.0. This was consistent with weak zeta potentials and the declined repulsion among the p articles. Excessively high temperature alters the structural conformations of ellagitannins and gelatins ( 177 178 ) In our study, PPE gelatin nanoparticles were able to be fabricated at 50 o C, sugge sting that this temperature has little negative influence on molecular binding. It was known that the coil conformation of gelatin became looser and more extended with the in crease of temperature ( 74 ) Higher temperature exposes more hydrophobic groups from gelatins to bind with more ellagitannin molecules. As a result, loading efficiency of tannin molecules was increased ( 74, 177 ) Particle s fabricated under the mass ratio s of 1:5, 5:5, and 7:5, within the temperature from 25 to 50 o C, had small particle size s and high loading efficiency, respectively. When interaction happens at lower temperatures, compact coil conformation of gelatin provides less hydrophobic sites for interaction with tannin mo lecules. As a result, larger

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55 particles were formed. Particles sizes prepared from the mass ratio s of 5:5 and 7:5 were above 500 nm when the temperature was below 25 o C When PPE and gelatin were mixed, interaction between them occurs rapidly till their hydr ophobic sites are saturated ( 151, 171 ) The suspension will be stable if no more tannins molecules participate in it to induce particle aggregation. In our study, nanoparticle suspensions at the mass ratio s of 1:5 and 5:5 had favorable nanoparticle charac teris tics after 0.5 day and remained stable un till 4 days. Nanoparticle s uspension from the mass ratio of 7:5 had constant particle characteristics by 2 day reaction, but increased particle size s with large variation were observed after 4 days It indicated tha t assembly of particles was promoted and eventually led to precipitation. Summary PPE to gelatin mass ratio determined characteristics and stability of nanoparticle suspension s The critical point for PPE gelatin nanoparticles was at a mass ratio of 8:5. N anoparticle s prepared in a pH range from 4 to 5 .3 had particle sizes and zeta potentials that favor higher bioavailability of loaded ellagitannins. Reaction temperature s from 25 to 50 o C had the positive influence on nanoparticle characteristics. Self assem bly occurred rapidly between gelatin and ellagitannin and fabrication was done after 12 hours and remained stable after 4 days.

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56 Figure 3 1. Morphology of PPE g elatin nanoparticle using t rans mission electron m icroscopy (TEM) PPE gelatin nanoparticles we re fabricated using PPE to gelatin mass ratio of 5:5 at 25 o C for 48 h. The pH of gelatin and PPE water solutions were 5.3 and 4.2, respectively. Nanoparticles in colloidal suspension are denoted by arrows.

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57 Figure 3 2. PPE gelatin n anoparticle characte ristics under the conditions of PPE to gelatin mass ratio from 1:5 to 9:5 at 25 o C for 2 days. A) Particle size. B) Zeta potential. C) Loading efficiency of punicalagin A. D) Loading efficiency of punicalagin B. The pH of gelatin and PPE water solutions wer e 5.3 and 4.2, respectively.

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58 Figure 3 3. PPE gelatin nanoparticle characteristics under the conditions of PPE to gelatin mass ratio at 1:5, 5:5, and 7:5, pH of gelatin solution were 1.0, 2.0, 3.0, 4.0, 5.3, 6.0, 7.0, and 11.0, temperature at 25 o C, and reaction time of 2 days. A) Particle size. B) Zeta potential. C) Loading efficiency of punicalagin A. D) Loading efficiency of punicalagin B. The pH of ge latin and PPE water solution were 5.3 and 4.2, respectively

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59 Figure 3 4. PPE gelatin nanoparticle characteri stics under PPE to gelatin mass ratio at 1:5, 5:5, and 7:5 at 5, 10, 15, 25, 40, or 50 o C for 2 days. A) Particle size. B) Zeta potential. C) Loading efficiency of punicalagin A. D) Loading efficiency of punicalagin B. The pH of gelatin and PPE wa ter solutions were 5.3 and 4.2, respectively

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60 Figure 3 5. PPE gelatin n anoparticle characteristics under PPE to gelatin mass ratio at 1:5, 5:5, and 7:5, at 25 o C for 0.5, 1, 1.5, 2, 3, or 4 days. A) Particle size. B) Zeta potential. C) Loading efficienc y of punicalagin A. D) Loading efficiency of punicalagin B. The pH of gelatin and PPE water solutions were 5.3 and 4.2, respectively

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61 CHAPTER 4 FABRICATION OF COATED BOVINE SERUM ALBUMIN (BSA) EPIGALLOCATECHIN GALLATE (EGCG ) NANOPARTICLES AND THEIR TRANS PORT ACROSS MONOLAYERS OF HUMAN INTESTINAL EPITHELIAL CACO 2 CELLS Background ( ) Epigallocatechin gallate (EGCG) is a major flavan 3 ol in green tea ( 179 ) EGCG has been reported to reduce plaques r elated to dementia and inhibit growth of brain, prostate, cervical, and bladder cancers by inducing apoptosis ( 180 184 ) The absorption rates of EGCG were less than 5% in human s and below 1% in rats after oral admin istration ( 185 188 ) Such low bioavailability of EGCG limits its bioactivity in vivo Nanoparticles are defined as particles below 1000 nm in size by biological sciences ( 132 ) Nanoparticles have the potential to increase absorption of bioactive compounds through several mechanisms. The low absorption rate of EGCG is attributed to its poor permeability across intestinal epithelium because passive diffusion is thought to be its only absorption route ( 68, 189 ) A number of nanoparticles, such as chitosan nanoparticles and poly(lactic co glycolic a cid) nanoparticles, are known to enhance absorption of bioactive compounds by opening tight junctions or improving their transcellular absorption by nanoparticle endocytosis ( 13, 118, 121 ) Secondly, low bioavailabi lity of EGCG is also caused by its poor stability in the gastrointestinal tract since EGCG was readily oxidized or degraded under acidic (pH below 1.5) or neutral pH conditions ( 55 ) It was suggested that chitosan nanoparticles protected EGCG from degradation in the gastrointestinal tract ( 190 191 ) Lastly, nanoparticles may increas e solubility of hydrophobic compounds to enhance the absorption in small intestine ( 192 ) For example, cyclodextrin has been widely used as a c arrier to increase the solubility of type II drugs for the enhancement of absorption ( 192 )

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62 Besides nanoparticles, absorption enhancers are als o suggested to improve absorption of bioactive compounds. Chitosan is an absorption enhancer that is known to promote absorption of drugs by disrupting tight junctions among epithelial cells or enhancing cellular uptake on epithelial cells ( 193 195 ) Poly lysine has a structure similar to many cell penetrating peptides and it has the capacity to increase endocytotic cellular uptake of drugs ( 196 199 ) The objectives of this st udy were to fabricate absorption enhancer coated nanoparticles and to investigate the impact of coating on size, surface chemistry, and zeta potential of nanoparticles. We hypothesized that coating the EGCG loaded BSA nanoparticles with absorption enhancer may improve the stability and/or the absorption of EGCG. Materials and M ethods Chemicals ( ) Epigallocatechin gallate (EGCG) was purchased from Quality Phytochemicals LLC (Edison, NJ) Poly lysine was a gift from Zhejiang Silver elephant Bioengineer ing Co (Taizhou, Zhejiang, China). Bovine serum albumin (BSA), chitosan ( 5 20mPa.s ), glutaraldehyde, simulated gastric fluid (SGF, pH adjusted to 4.5 to simulate fed state), simulated intestinal fluid (SIF, pH adjusted to 6.5) without pancreatin, SIF with pancreatin, and pepsin were purchased from Fisher Scientific (Pittsburg, PA). Fluorescein isothiocyanate (FITC) labeled BSA was purchased from Fisher Scientific (Pittsburg, PA). Alexa TM 594 concanavalin A was purchased from Molecular Probes (Grand Island, NY). Caco 2 cells were obtained from American Type Culture Collection (Manassas, VA).

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63 Fabrication of BSA N anoparticles BSA nanoparticles were fabricated using a desolvation method. Briefly, 5 mL of 5 mg/mL BSA water solution was transferred to a beaker u nder a stirring speed of 750 rpm at room temperature. Ten mL pure ethanol was added drop wise to BSA solution till a white milk suspension was formed. The suspension was stirred for additional 10 min before 200 L of glutaraldehyde was added drop wise to t he suspension. The suspension was stirred for 1 more hour. Afterwards, the suspension was centrifuged at 13,300 rpm for 10 min to remove the supernatant. The sediment was washed by deionized water three times and then re suspended in 5 mL of deionized wate r. Loading EGCG into BSA N anoparticles BSA nanoparticles suspension (5mL) was stirred at 750 rpm. EGCG water solution (15 mL, 1mg/mL) was added drop wise to the suspension and the suspension was stirred at the same speed for 30 min. Coating the BSA EGCG N anoparticles Poly lysine solution (2mL, 2 mg/mL) or chitosan solution (2 mL, 1 mg/mL) was added drop wise to 20 mL of BSA EGCG nanoparticles suspension at 750 rpm for 60 min. Scanning Electron M icroscopy (SEM) BSA EGCG nanoparticles (BEN), poly lysin e coated BSA EGCG nanoparticles (PBEN), and chitosan coated BSA EGCG nanoparticles (CBEN) suspensions were lyophilized for 24 hours. Morphology of dry BEN, PBEN, and CBEN were measured using Field Emission Scanning Electron Microscope (Model JSM 633OF, JEO L Ltd, Tokyo, Japan).

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64 Loading Efficiency and Loading Capacity A ssessments The nanoparticle suspensions were centrifuged at 13,300 rpm for 10 min to collect the supernatant. The supernatant was analyzed using HPLC to measure the EGCG content. The loading ef ficiency and the loading capacity were calculated using the equations below: L oading efficiency = (EGCG amount loaded into nanoparticles/EGCG amount used for nanoparticle fabrication) 100%. L oading capacity = (EGCG amount loaded into nanoparticles/Dry n anoparticle amount) 100%. Nanoparticles Ch aracterization under Different p H C onditions The nanoparticles were characterized under different pH conditions follow ing a published paper with minor modifications ( 200 ) Briefly, BEN, PBEN or CBEN water suspension was adjusted to pH of 1.5, 3.5, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 using a diluted HCl or NaOH solution. Mean par ticle size and zeta potential were measured on Zetatrac (Microtrac Inc., Largo, FL). Samp les were measured in duplicate tests at 25 o C and three reading s were ob tained for each replicate to calcul ate the average particle size and zeta potential. Release of EGCG in Simulated Digestive F luids The release profile of EGCG was measured in simulated gastric fluid (SGF) and in simulated intestinal fluid (SIF) with or wit hout digestive enzymes ( 201 ) Briefly, dry nanoparticles were suspended in 5 mL of SGF (pH 4.5) with or without 0.1% pepsin (w/v) and incubated at 37 o C for 0.5 h. Subsequently, 1 mL of the sample was centrifuged at 13,300 rpm for 10 min. The supernatant (0.5 mL) obtained was mixed with 0.05 mL of ascorbic acid solution (1% asc orbic acid in 0.28% H 3 PO 4 water solution)

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65 to reach pH 2.5, and then analyzed on HPLC. The dry nanoparticles were suspended in 5 mL of SIF (pH 6.5) without or with 1.0% pancreatin (w/v) and incubated at 37 o C for 2 hours. Then, 1 mL of the sample was centrif uged at 13,300 rpm for 10 min. The supernatant (0.5 mL) obtained was mixed with 0.4 mL of 1% ascorbic acid solution, followed by HPLC analysis. An Agilent Zorbax SB C18 column (4.6 mm 25 0 m m) was used to analyze EGCG The binary mobile phase consisted of 0.04 % acetic acid: water (A) and acetonitrile (B). A 22 min gradient was used 30 % B l inear; 20 22 min, 30 10% B linear; followed by 2 min of re equilibration of the column before the next run. The detection wavelengt h on the diod e array detector was 280 nm The injection volume was 20 L with a flow rate of 1 mL/min. Each s ample was analyzed in duplicate tests The cumulative EGCG release was calculated using the following equation: Cumulative EGCG release (%) = (EGCG amount in su pernatant/EGCG amount in dry nanoparticles) 100% Storage Stability of Loaded EGCG in N anoparticles EGCG (1 mg) or the dry nanoparticles with equal amounts of EGCG were kept for 0, 3, 6, 9, 12, or 24 hours at 60 o C. At each time point, the samples were mix ed with 1 mL of ethanol/water solution (9:1 v/v) to extract EGCG. After centrifuging at 13,300 rpm for 10 min, the supernatant was removed and analyzed on HPLC. Each treatme nt was carried out in duplicate tests EGCG remaining percentage was calculated usi ng the following equation: EGCG remaining percentage (%) = (EGCG amount in supernatant/Initial EGCG amount) 100%

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66 EGCG Absorption on Caco 2 Cell M onolayers Caco 2 cells were seeded at 6 well transwell plates with an internal diameter of 24 mm and membrane growth area of 4.67 cm 2 (Corning Inc, Lowell, MA). The cells ing 20% fetal bovine serum 1% penicillin streptomycin, and 1% non essential amino acids. The cells were grown to confluence and allowe d to mature for 17 days at 37 o C and 5% CO 2 The cell culture media was changed every 1 2 days. The permeability stud y was carried out in triplicate tests after the transepithelial electrical resistance (TEER) was above 600 cm 2 measured using an epithelial volt ohm meter (Millicell ERS 2, Millipore Corp, Billerica, MA). The permeability study followed a published paper with minor modifications ( 68 ) Dry EGCG, BEN, PBEN, or CBEN was added into salt solution ( HBSS, pH adjust ed to 6.0) to control EGCG concentration at 0.023 mg/mL One and half milliliter of each sample was transferred to the apical chamber of the tra nswells plate and 2.6 mL of the fresh buffer was added to the basolateral chamber. After incubation at 37 o C for 30, 60, 90, and 120 min, the transepithelial electric resistance (TEER) was measured. Subsequently, 0.5 mL of the samples was taken from the basolateral chamber, acidified with ascorbic acid solution to adjust to pH 2.5 and stored in 80 o C until analyses. The fresh buffer (0.5 mL) was re filled to the basolateral chamber. The samples were analyzed by HPLC ESI MS n using a published method wi th minor modifications ( 68 ) An Agilent Zorbax SB C18 column (4.6 mm 25 0 m m) was used The binary mobile phase consisted of 0.04 % acetic acid: water ( A) and acetonitrile (B). A 22 min gradient was used 30 % B l inear; 20 22 min, 30 10% B linear; followed by 2 min of re equilibration of the column before the next run. The injection volume was 20 L with a flow rate of 1 mL/min. Ele ctrospray

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67 ionization in negative mode was performed using nebulizer 65 psi, drying gas 11 L/min, drying temperature 350 o C, and capillary 4000 V. The MRM was used to monitor the transition of deprotonated EGCG m/z 457 [M H] to the product ion m/z 169 EG CG was used as an external standard. Quantification was conducted using QuantAnalysis ( Version 2.0 Bruker Daltonics Inc, Billerica, MA). The apparent permeability coefficient ( P app ) of EGCG was calculated using the following equation: P app = (dQ/dt) (1/ AC 0 ) W here dQ/dt is the permeability rate ( g/s), C 0 is the initial con centration in the apical chamber ( g/mL), and A is the surface area of filter (cm 2 ), which wa s 4.67 cm 2 in this study. Confocal Laser Scanning M icroscopy Fluorescein isothiocyanate ( FIT C ) lab eled BSA was used to fabricate FITC labeled BSA nanoparticles. The fabrication procedures followed the same procedure of fabrication of BEN, PBEN, and CBEN. Caco 2 cells were seeded on a four well chambered coverglass (Fisher Scientific Inc., Pit tsbu rg, PA) and incubated at 37 C in 95% air and 5% CO 2 environment until the monolayer was formed On the day of experiment, the growth medium was replaced by HBSS buffer (pH 6.0) After equilibration in the buffer at 37C for 30 mi n, the buffer was replaced with FITC labeled BEN FITC labeled PBEN, and FITC labeled CBEN suspension T hen the monolaye rs were further incubated for 60 min. At the end of experime nt, the cells were washed six times with the buffer to remove the excess nanoparticles Subsequently, the cells were fixed in 2% paraformaldehyde for 1 hour, and then washed with the buffer an additional three times. Afterward, Alexa TM 594 concanavalin A was used to stain the cell membrane for 30 min and then washed with buffer three times. Finally, the mo nolayers

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68 were kept in the buffer until they were examin ed on a confocal laser scanning microscope (Leica TCS SP2 AOBS system Germany) equ ipped with Leica LCS imaging software Statistical Analys e s Data was expressed as mean t test was used to compare mean values between two groups. One way analyses of variance with all pair ed Tukey HSD comparison of the means were performed using JMP software (Version 8.0, SAS Institute Inc., Cary, NC). A difference with p 0.05 was considere d significant. Results Particle Morphology, EGCG Loading Efficiency and Loading C apacity Lyophilized nanoparticles were measured by SEM ( Figure 4 1 ). BEN, PBEN, and CBEN showed a spherical morphology with the particle sizes between 100 and 200 nm. The load ing efficiency of EGCG on BEN was 32.3% ( Table 4 1 ). Poly lysine coating increased EGCG loading efficiency to 35.4%. Coating with chitosan did not change the loading efficiency. The loading capacity of EGCG in BEN was 18.9% (w/w) and remained unchanged after BEN was coated with poly lysine or chitosan (17.0% and 16.0%, respectively). Impact of Coating on Particle Size and Z eta potential The sizes of the nanoparticles in suspension appeared to be pH dependent ( Figure 4 2 ) The impact of coatings on particle size also depended on pH. BEN had an average size of 3 04 nm at pH 1.5 and 301 nm at pH 3.5 ( Figure 4 2A ). This particle size dramatically increased to 2828 nm at pH 4.5 and 2460 nm at pH 5.0 due to the par ticle aggregation. The particle size s dropped back to between 170 nm and 300 nm at

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69 a pH range 5.5 7.0. Po ly lysine coating prevented the particle aggregation at and below pH 6.0. The average sizes of PBEN at pH 1.5, 3.5, 4.5, 5.0, 5.5, and 6.0 were 297 nm, 264 nm, 286 nm, 296 nm, 265 nm, and 358 nm, respectively. At pH 6.5 and 7.0, the size of PBEN increase d to 5800 nm and 5330 nm, respectively. A similar phenomenon was also observed for CBEN. CBEN had an average size of 322 nm, 269 nm, 247 nm, 359 nm, 381 nm, and 383 nm at pH of 1.5, 3.5, 4.5, 5.0, 5.5, and 6.0, whereas the average size increased to 1315 nm and 2700 nm at pH 6.5 and 7.0, respectively. Coating also altered the zeta potential of the particles ( Figure 4 2B ). BEN showed positive surface charges over +20 mV at pH 1.5 and 3.5. The zeta potential s of BEN were 28.1 mV 11.5 mV 19.9 mV, 21.6 mV, 42.9 mV, and 34.2 mV at pH 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0, respectively. Poly lysine coating caused the nanoparticles to possess positive charge of 41.2 mV, 55.0 mV, 23.0 mV, 40.3 mV, 39.9 mV, and 38.5 mV at pH 1.5, 3.5, 4.5, 5.0, 5.5, and 6.0, resp ectively. However, the zeta potential s of PBEN drastically dropped to 4.4 mV and 4.0 mV at pH 6.5 and 7.0, respectively. CBEN showed the zeta potential s of 45.0 mV, 43.3 mV, 27.8 mV, 12.6 mV, 10.5 mV, and 14.6 mV at pH 1.5, 3.5, 4.5, 5.0, 5.5, and 6.0. How ever, adjusting pH to 6.5 and 7.0 reduced the zeta potential s of the particles to 2.4 mV and 2.8 mV, respectively. The decreases in zeta potential s coincided with particle aggregation and the increase in particle size s Impact of Nano encapsulation and Par ticle C oat ing on EGCG Release in Simulated Digestive F luids Release of EGCG from the nanoparticles was assessed in simulated gastrointestinal fluids with or without digestive enzymes ( Figure 4 3 ). After incubation of the nanoparticles in pepsin free simula ted gastric fluid for 0.5 hour, around 25% of

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70 EGCG was released from all tested nanoparticles with no significant differences on EGCG release observed among uncoated and coated nanoparticles ( Figure 4 3 A ). Adding pepsin in simulated gastric fluid caused mo re EGCG to be released from BEN, PBEN, and CBEN BEN released 52.7% of EGCG in SGF with pepsin after 0.5 hour, whereas the release percentage of EGCG from PBEN and CBEN were 33.0% and 37.1%, respectively. The release percentages of EGCG from the coated nan oparticles were significant lower than that from BEN. A similar phenomenon on EGCG release from the nanoparticles was observed in SIF with or without pancreatin ( Figur e 4 3 B ). BEN released 38.1% of EGCG in pancreatin free simulated intestine fluid after 2 hours. The release of EGCG from PBEN and CBEN was 29.1% and 24.3%, respectively. The releases of EGCG were significantly increased after pancreatin was added to intestinal fluid for BEN and CBEN Release of EGCG from PBEN in SIF with pancreatin was 44.6 % compared to 29.1% of EGCG release in SIF without pancreatin. However, no statistical difference was observed. Coating inhibited EGCG release in SIF with pancreatin. BEN released 68.5% of EGCG in 2 hours PBEN released 44.6% of EGCG, which was significantly less than that from BEN The release of EGCG from CBEN was 54.2%, which was not significantly different from BEN Impact of Nano encapsulation and Particle Coating on EGCG S tability EGCG loaded in the nanoparticles degraded faster than pure EGCG at 60 o C ( Figure 4 4 ). About 10% 20% of EGCG in all the nanoparticles was degraded at 3 hours, whereas pure EGCG showed no degradation. At 6 hours, about 40% of EGCG in BEN remained. Particle coating increased EGCG remaining percentage to 60%. In contrast, 91% of E GCG remained in pure EGCG sample. Such trend continued to 24

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71 hours. However, the positive effects of coating on EGCG stability were no longer significant after 6 hours. Impact of Nano encapsulation and Particle Coating on EGCG Transport on Caco 2 M onolayer s The apparent permeability coefficient ( P app ) of EGCG in the solution was 3.83 10 7 cm/s at 30 min and the P app of EGCG remained similar during the incubation period ( Figure 4 5 A ). However, the highest P app of EGCG in all the nanoparticles appeared at 3 0 min and then a decreasing trend of P app of EGCG was observed. P app of EGCG in CBEN ( 11.17 10 7 cm/s ) was significantly higher than that of EGCG solution at 30 min and it was similar to that of BEN and PBEN P app of EGCG in CBEN and PBEN were higher at 120 min compared with EGCG solution. The tran s epithelial electric resistance (TEER) of Caco 2 monolayer treated by EGCG solution did not significantly decrease during the incubation period ( Figure 4 5 B ). The TEER percentage of the init ial value was 95.6 % a t 30 min and re mained to be 93.0 % after 90 min. No significant differences on TEER percentage were observed in EGCG solution, BEN, or PBEN at 30, 60, or 90 min. However, CBEN significantly decrease d the TEER percent age of Caco 2 monolayer to 77.8 % at 30 mi n and to 70.2% at 120 min. Impact of Nano encapsulation and Particle Coating on Nanoparticle Cellular Uptake by Caco 2 C ells The cellular uptake of FITC labeled BEN, PBEN, and CBEN by Caco 2 cells was visualized using confocal laser scanning microscopy ( Fi gure 4 6 ). Results showed that the fluorescence was observed in Caco 2 cells treated by FITC labeled BEN ( Figure 4 6B ), which indicates that FITC labeled BEN were taken up by cells after 60 min incubation time. Similar observations on BSA nanoparticle cell ular uptake were reported

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72 in the previous studies ( 202 204 ) Compared with FITC labeled BEN, FITC labeled PBEN and CBEN sh owed more intense fluorescence suggesting that poly lysine and chitosan coatings enhanced the cellular uptake of the nanoparticles. The enhancement of cellular uptake of nanoparticles coated with poly lysine or chitosan were also reported in earlier studies ( 20 5 207 ) Discussion After fabrication of BSA nanoparticles with a desolvation method, EGCG was loaded onto these nanoparticles using the polyphenol protein affinity. EGCG binds with BSA via hydrogen bonds and hydrophobic interactions ( 170, 208 ) Poly lysine and chitosan were coated on the surface of BSA EGCG nanoparticles via electrostatic interactions. Both poly lysine and chitosan had a large number of NH 2 groups. These groups ionized into NH 3 + in water. BSA nanoparticles had negative charges i n water (pH 5.5) because the isoelectric point of BSA is 4.7. These positively charged poly lysine or chitosan interacted with negatively charged BSA nanoparticles by electrostatic interactions. Moreover, the affinity between poly lysine or chitosan an d BSA can also be attributed to hydrogen bonds and hydrophobic interactions ( 209 210 ) EGCG was loaded on BSA nanoparticles before the coating step, therefore loading efficiency or loading capacity of EGCG was not c hanged by the addition of the coating. The scanning electron microscope revealed that BEN, PBEN, and CBEN had the spherical morphology. Dynamic light scattering measures the sizes of nanospheres with higher accuracy than nano materials of other morphology ( 77 ) The pH environment in the human gastrointestinal tract varies from acidic to neutral. Therefore it is critical to understand the change of particle sizes and zeta potentials under different pH conditi ons. Protein nanoparticles in suspension are

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73 stabilized primarily by inter particle electrostatic repulsion, which is measured by zeta potential ( 75 ) Protein nanoparticles tend to a ggregate at pH near its isoelectric point due to reduced zeta potential ( 75 ) The isoelectric point of BSA protein is around 4.7 and inter particle repulsions were significantly reduc ed in pH between 4.5 and 5.0, causing the aggregation of BEN at these pH values ( 211 ) Poly lysine and chitosan coatings altered both the size and zeta potential of BEN at the pH range 1.5 7.0. Both PBEN and CBEN prevented particle aggregation at and below pH 6.0. However, when pH was 6.5 and 7.0, PBEN dramatically increased the particle size to above 5000 nm due to particle aggregation. CBEN had a particle size 1300 nm and 2600 nm at pH 6.5 and 7.0. These aggregations were also accompanied with the decrease of zeta potentials. The gastrointestinal tract consists of different pH areas, starting from 6.5 7.0 in the mouth, to 1.5 to 5.0 in the stomach, and then to 5.0 to 7.0 in the intestine. Poly lysine or chitosan coatings were expected to stabilize BSA EGCG nanoparticles in the stomach and the upper portion of the small intestine (duodenum) where the pH changes from 1.5 to 6.0. However, particle aggregation is speculated to occur in the jejunum and ileum where the pH is about 7.0. Zeta potentials of the nanoparticles under different pH conditions also impact the interaction of particles with mucus and epithelial cells in intestine. Goblet cells secrete mucus to form barrier layers in intestinal epithelium. Negatively charged nanoparticles are repulsed by mucus layers ( 212 ) Positively charged nanoparticles exhibited absorption rates by adhering to the negatively charged mucus layer of small intestine ( 212 )

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74 There are two factors that may affect the releases of loaded EGCG from protein nanoparticles. First, EGCG can be released from the nanoparticles by diffusion. In the present study, about 25% of EGCG was released from all the nanoparticles in SGF without pepsin. BEN released about 40% of EGCG in SIF without p ancreatin, whereas less than 30% of EGCG was released from PBEN and CBEN ( Figure 4 3 ). Protein nanoparticles may also be digested by proteinases in the gastrointestinal tract. Digestion causes protein nanoparticles to lose structural integrity and the subs equent release of loaded EGCG. The SGF with pepsin and the SIF with pancreatin caused BEN to release about 50% and 70% of loaded EGCG, respectively. Higher percentage releases of EGCG were also observed in the coated nanoparticles with the presence of enzy mes in simulated digestive fluids. The additional release of EGCG was primarily attributed to the digestion of BSA protein by proteinases. However, coatings on the surface of the nanoparticles delayed EGCG release. Coating provided an additional barrier fo r diffusion. It may also decrease accessibility or digestibility of BSA by proteinases. Less than 40% or 55% of EGCG was released from the coated nanoparticles in SGF and SIF with proteinases compared to about 50% and 70% of EGCG released from BEN. Thus, c oatings appeared to protect the nanoparticles against release of EGCG in the gastrointestinal tract, which might improve the stability of EGCG in the gastrointestinal tract and potentially enhance the absorption of EGCG ( 213 214 ) Because nanoparticles have very high surface area/mass ratio, compounds loaded on nanoparticles are more susceptible to oxidation. Indeed, degradation of EGCG on nanoparticle at 60 o C was accelerated compared with pure EGCG ( Figure 4

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75 4 ). Coa tings created an additional barrier on the nanoparticles and protected EGCG against degradation at the early stage of the storage period Caco 2 monolayers were widely used to evaluate the intestinal absorption of EGCG and other flavonoids ( 215 216 ) The P app of EGCG in the present study was between 2.86 10 7 cm/s and 4.27 10 7 cm/s ( Figure 4 5 A ), which was consistent with previous studies ( 68, 195 ) The slight decrease on TEER of Caco 2 monolayer treated by EGCG during the incubation period suggested that EGCG concentration had no adverse effect on Caco 2 monolayers ( Figure 4 5 B ). N anoparticles have been proposed to enhance absorption of bioactive compounds through two mechanisms. Tight junctions connect cells in the Caco 2 monolayers. Nanoparticles with the average size below 20 nm are known to reversibly disrupt tight junctions. Th is will facilitate paracellular absorption of bioactive compounds released from nanoparticles. The decrease of TEER reflected the disruption of tight junctions on Caco 2 monolayers. Furthermore, Caco 2 cells may take up nanoparticles through clathrin media ted or caveolae mediated endocytosis. Nanoparticles internalized into Caco 2 cells via endocytosis can be transported into endosome and lysosome. Acid and enzyme s in lysosome degrade nanoparticle and release the loaded bioactive compounds ( 118, 217 ) In the study, TEER percentage of the Caco 2 monolayer treated by BEN did not show significant differences compared to that of EGCG solution, suggesting that the BEN did not disrupt the tight junctions among the Caco 2 cells during the incubation period. The P app of EGCG in BEN showed a decreasing trend from 9.22 10 7 cm/s to 3.73 10 7 cm/s during the incubation period. Confocal microscopic results suggested that BEN were taken up by Caco 2 cells ( Figure 4 6B ). Both poly lysine an d chitosan

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76 have been used as absorption enhancers to increase the absorption of drugs ( 193, 196, 218 ) Poly lysine has a structure similar to cell penetrating peptides that may improve the transport of bioactive c ompounds by enhancing endocytosis ( 196 ) The highest P app value of EGCG in PBEN was 9.41 10 7 cm/s at 30 min. After 120 min, the P app of EGCG was 5.43 10 7 cm/s in PBEN, which was higher than that in EGCG solution. In the meantime, TEER percentage of Caco 2 monolayer treated by PBEN was similar to th ose by EGCG solution at 90 min. Thus, we speculate that poly lysine enhanced the cellular uptake of nanoparticles in Caco 2 cells and increased the absorption of EGCG on Caco 2 monolayers The confocal microscopy indicated that poly lysine coating enhanced the cellular uptake of the FITC labeled nanoparticles by Caco 2 cells ( Figure 4 6C ). Chitosan coating significantly increased the P app of EGCG (11.17 10 7 cm/s) on Caco 2 monolayers at 30 min compared to EGCG solution. Furthermore, the P app of EGCG remained 6.27 10 7 cm/s at 120 min. CBEN also caused a grea ter drop of TEER compared with that of EGCG solution during the incubation period. Two mechanisms were proposed to explain the enhancement of EGCG absorption by CBEN. First, chitosan had the capacity to open up tight junction among Caco 2 cells. Therefore, EGCG released from nanoparticles may transport across monolayers through paracellular way ( 190, 193, 218 ) Second, chitosan coating may enhance cellular uptake of nanoparticles, which was suggested by the cellular uptake of FITC labeled CBEN by Caco 2 cells ( Figure 4 6D ). Summary BEN with poly lysine and chitosan coatings had the spherical morphology, and the loading efficiency and capacity of EGCG were 35.4% and 32.7%, and 17.0% and 16.0%, respectively. These coatings significantly stabilized the characterization of BEN

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77 in the pH range from 1 .5 to 6.0. Higher release of EGCG was observed in all the nanoparticles incubated in simulated gastric and intestinal fluids with proteinases. However, the coatings delayed the release of EGCG from the nanoparticles. The storage stability study showed fast er degradation of EGCG in all the nanoparticles compared to free EGCG. CBEN significantly enhanced EGCG absorption on Caco 2 monolayer, which was attributed to the enhancement of cellular uptake of CBEN by Caco 2 cells.

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78 Table 4 1. Loading efficiency and loading capacity of EGCG in BSA EGCG nanoparticles (BEN), poly lysine coated BSA EGCG nanoparticles (PBEN), and chitosan coated BSA EGCG nanoparticles (CBEN) Nanoparticles L oading efficiency L oading capacity (%) (w/w %) BSA EGCG nanoparticles (BEN) 32 .3 1.0 b 18.9 0.9 a Poly lysine coated BSA EGCG nanoparticles (PBEN) 35.4 1.4 a 17.0 1.0 a Chitosan coated BSA EGCG nanoparticles (CBEN) 32.7 0.9 ab 16.0 1.3 a Data are mean s tandard deviation of triplicate tests Different letters in t he column represent significant difference at p 0.05

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79 Figure 4 1 Scanning electron microscopy images. A) BSA EGCG nanoparticles (BEN). B) P oly lysine coated BSA EGCG nanoparticles (PBEN). C) C hitosan coated BSA EGCG nanoparticles (CBEN)

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80 Figu re 4 2. Particles size and zeta potential of BSA EGCG nanoparticles (BEN), poly lysine coated BSA EGCG nanoparticles (PBEN), and chitosan coated BSA EGCG nanoparticles (CBEN) in the pH range from 1.5 to 7.0. A) Particles size. B) Zeta potential. Data ar e mean standard deviatio n of duplicate tests

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81 Figure 4 3. Cumulative release of EGCG from nanoparticles in simulated fluids with and without digestive enzymes at 37 o C A) Simulated gastric fluid (SGF) with and without pepsin for 0.5 hour. B) Simulat ed intestinal fluid (SIF) with and without pancreatin for 2 hours. Data are mean standard deviation of duplicate tests Different low case letters indicate significant differences in SGF or SIF without digestive enzyme. Different upper case letters indic ate significant differences in SGF or SIF with digestive enzyme. ind icates significant differences of two groups using t test (with digestive enzyme versus without digestive enzyme).

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82 Figure 4 4. Stability of EGCG, EGCG in dry BSA EGCG nanoparticles (BEN), poly lysine coated BSA EGCG nanoparticles (PBEN), and chitosan coated BSA EGCG nanoparticles (CBEN) at 60 o C fo r 0, 3, 6, 9, 12, and 24 hours. Data are mean standard deviation of duplicate tests Different letters at each incubation time period represent sig nificant differences.

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83 Figure 4 5 Apparent permeability coefficient ( P app ) and transepithelial electric resistance (TEER) of EGCG in solution, BSA EGCG nanoparticles (BEN), poly lysine coated BSA EGCG nanoparticles (PBEN), and chitosan coated BSA EGC G nanoparticles (CBEN) at 37 o C for 30, 60, 90, and 120 min. A) P app B) TEER. Data are mean s tandard deviation of triplicate tests Different letters at each incubation period represent significant difference s

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84 Figure 4 6 FITC labeled BSA EGCG nano particles (FITC labeled BEN), FITC labeled poly lysine coated BSA EGCG nanoparticles (FITC labeled PBEN), and FITC labeled chitosan coated BSA EGCG nanoparticles (FITC labeled CBEN) cellular upta ke by Caco 2 cells for 60 min at 37 o C. A ) Control. B ) FITC labeled BEN. C) FITC labeled PBEN. D) FITC label ed CBEN. Red: cell membrane stained with Alexa TM 594 concanavalin A; Green: FITC labeled nanoparticles

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85 CHAPTER 5 FABRICATION OF SELF ASSEMBLED ( ) EPIGALLOCATECHIN GALLATE (EGCG) OVALBUMIN DEXTRAN CONJUGATE NANOPARTICLES AND THEIR TRANSPORT ACROSS MONOLAY ERS OF HUMAN INTESTINAL EPITHELIAL CACO 2 CELLS Background ( ) Epigallocatechin gallate (EGCG) has been of particular interest in both food and pharmaceutical sciences since it was reported to possess anticancer properties against brain, prostate, cervica l, and bladder tumors ( 180 184 ) However, its low absorption rate limits its bioactivity in vivo because EGCG is absorbe d via passive diffusion coupled with active efflux ( 185 188 ) Nanoparticle delivery system s have been widely used to enhance absorption of bioactive compounds becau se nanoparticles can penetrate small intestinal epithelium by the paracellular and/or endocytotic pathway ( 13, 118, 121 ) Protein is a major material that has been widely used for nano carrier formation ( 82, 170, 202 ) However, protein based nanoparticles have several drawbacks. First, proteins are sensitive to pH cond itions and tend to precipitate at a pH around their isoelectric points ( 74 75 ) Second, ionic effect on proteins can also cause the aggregation of proteins ( 219 ) More importantly, digestive enzymes in the gastrointestinal tract can readily hydrolyze protein to polypeptide s and amino acids, which causes burst release of bioactive compounds and subsequent drug degradation and poor absorption ( 201 ) Ovalbumin is a major protein in egg white. It can be easily extracted and pu rified ( 220 ) Ovalbumin has been reported to resist pepsin digestion ( 221 ) Dextran consists of a complex branched polysaccharide and has been reported to decrease vas cular thrombosis, prolong antithrombitic and colloidal effect, and provide anticoagulation

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86 benefits ( 222 224 ) Dextran is not digestible by amylase in the small intestine and can only be degraded by some microbial enzymes in the large intestine ( 225 ) The Maillard reaction c onjuga tes reducing sugars and amines ( 226 228 ) In the present study, we applied the Maillard reaction to conjugate ovalbumin and dextran to form the ovalbumin dextran conjugates. Then, EGCG and ovalbumin dextran co njugates were self assembled using a heating process to form EGCG ovalbumin dextran conjugate nanoparticles. Glutar aldehyde was used to crosslink EGCG ovalbumin dextran conjugate nanoparticles. The aim of this study was to fabricate protein polysaccharide nanoparticles with improved stability in the gastrointestinal tract and to enhance the absorption of loaded EGCG. Materials and M ethods Chemicals ( ) Epigallocatechin gallate (EGCG) was purchased from Quality Phytochemicals LLC (Edison, NJ) Ovalbumin ( 98% purity) was obtained from Sigma Adrich (St. Louis, MO). Dextran (low fraction, molecular weight of 60 to 90 kDa), simulated gastric fluid (SGF, pH adjusted to 4.0), simulated intestinal fluid (SIF, pH adjusted to 6.0) with pancreatin, and pepsin were p urchased from Fisher Scientific (Pittsburg, PA). Caco 2 cells were obtained from American Type Culture Collection (Manassas, VA). Preparations of Ovalbumin dextran C onjugates Ovalbumin (250 mg) and an equal amount of dextran were mixed and dissolved in 1 00 mL of water. The pH of the solutio n was adjusted to pH 8.0 using 0.1 N NaOH. Afterwards, this solution was lyophilized to form a fine powder. The dry powder was heated at 60 o C with 79% relative humidity in a desiccator containing saturated KBr

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87 solution for 2 days to form the ovalbumin dextran conjugates. The conjugates were stored at 20 o C and used without any further purification. SDS PAGE Analysis of Ovalbumin dextran C onjugates The formation of ovalbumin dextran conjugates was confirmed using SDS PA GE on a gel electrophoresis apparatus to detect the molecular weight distributions of ovalbumin and the ovalbumin dextran conjugates. Separating gels made of 10% poly acrylamide were used to separate proteins. Sta c king gel made of 5% poly acrylamide and 0.1% SDS was used to load the samples Samples were treated with 100 mM dithiothreitol and boiled for 5 min before loading. After electrophoresis, the gel was stained for protein and carbohydrate using Coomassie Blue R 250 and Fuchsin solution, respectively. T he protein and carbohydrate stain was destained with 10% acetic acid (v/v) containing 10% methanol (v/v) and 7.5% acetic acid (v/v) water solution, respectively The ovalbumin content in each lane was analyzed using UN SCAN IT gel analysis software (Versi on 6.1, Silk Scientific, Inc., Orem, Utah). Preparations of EGCG Ovalbumin dextran Conjugate Nanoparticles and Crosslinked EGCG Ovalbumin dextran Conjugate N anoparticles Briefly, 50 mg of ovalbumin dextran conjugate and 25 mg of EGCG were dissolved in 10 mL of de ionized water (mass ratio of ov a l bumin to EGCG was 1:1). After adjusting pH to 5.2 the solution was incubated at 80 o C for 60 min to form EGCG ovalbumin dextran conjugate nanoparticles. The resulting nanoparticle suspension was stored at 4 o C before analysis. Crosslinked EGCG ovalbumin dextran conjugate nanoparticles were prepared by adding 5 L of glutaraldehyde to 1 mL of nanoparticle suspension. The crosslinking

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88 process was carried out at room temperature for 24 hours under stirring speed of 750 r pm. Particle Size and Particle M orphology Particle size was measured in aqueous suspension. One mL of EGCG ovalbumin dextran conjugate nanoparticles or crosslinked EGCG ovalbumin dex tran conjugate nanoparticles were centrifuged at 13,300 rpm for 5 min and the sediments were re suspended in 5 mL of de ionized water M ean par ticle size ( n umber weighted distribution) was measured on Zetatrac (Microtrac Inc., Largo, FL). Samp les were measured in four replicates at 25 o C to calcul ate the average particle size T he morphology of dry EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles were measured using Field Emission Scanning Electron Microscope (Model JSM 633OF, JEOL Ltd, Tokyo, Japan). Loading Efficiency and Loading Capacity A ssessments The nanoparticle suspensions were centrifuged at 13,300 rpm for 5 min to collect the supernatant. The supernatant was analyzed using HPLC for EGCG content. The loading efficiency and the loading capacity were calculated u sing the equations below: Loading efficiency = 100%. Loading capacity = 100%. Nanoparticle Ch aracterization under Different p H C onditions Briefly, 1 mL of EGCG ovalbumin dextran conjugate nanoparticles or crosslinked EGCG o valbumin dextran conjugate nanoparticles were centrifuged at 13,300 rpm for 5

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89 min and the sediments were re suspended in 5 mL of deionized water. The pH of the suspension was adjusted to 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, and 7.5 using a diluted HCl or NaOH sol ution. M ean par ticle size ( n umber weighted distribution) and zeta potential were measured on Zetatrac (Microtrac Inc., Largo, FL). Samp les were measured in duplicate tests at 25 o C to calcul ate the average particle size and zeta potential. Nanoparticle Stab ility in Simulated Gastric or Intestinal F luid One mL of EGCG ovalbumin dextran conjugate or crosslinked EGCG ovalbumin dex tran conjugate nanoparticles were centrifuged at 13,300 rpm for 5 min and the sediments were re suspended in 5 mL of simulated gastr ic fluid (SGF, pH 4.0) or simulated intestinal fluid (SIF, pH 6.0) at 37 o C for 0.5, 1, 1.5, a nd 2 hour. At each time point, m ean p ar ticle size (number weighted distribution) was measured on Zetatrac (Microtrac Inc., Largo, FL). Samp les we re measured in dup licate tests to calcul ate the average particle size. Release of EGCG in Simulated Digestive F luids The cumulative release of EGCG in the nanoparticles was measured in simulated gastric fluid (SGF) with out or with pepsin and in simulated intestinal fluid ( SIF) without or with pancreatin ( 201 ) Briefly, nanoparticl es were suspended in 5 mL of SGF (pH 4.0) without or with 0.1% pepsin (w /v) and incubated at 37 o C for 0.5 h. Subsequently, 1 mL of the sample was centrifuged at 13,300 rpm for 5 min. Supernatant was measured by HPLC for EGCG content. The nanoparticles were suspended in 5 mL of SIF (pH 6.0) without or with 1.0% pancreatin (w/v ) and incubated at 37 o C for 2 hours. Then, 1 mL of sample was centrifuged at 13,300 rpm for 5 min. The supernatant obtained was measured by HPLC analysis. Samples were measured in four replicates An Agilent Zorbax SB C18 column (4.6 mm 25 0 m m) was used to

PAGE 90

90 analyze EGCG The binary mobile phase consisted of 0.04 % acetic acid: water (A) and acetonitrile (B). A 22 min gradient was used 30 % B l inear; 20 22 min, 30 10% B linear; followed by 2 min of re equilibration of the column before the next run. The detection wavelengt h on the diode array detector was 280 nm The injection volume was 5 L with the flow rate at 1 mL/min. The cumulative EGCG release was calculated using the following equation: Cumulative EGCG release (%) = 100% EGCG Absorption on Caco 2 Cell M onolayers Caco 2 cells were seeded at 6 well transwell plates with the internal diameter of 24 mm and membrane growth area of 4.67 cm 2 (Corning Inc, Lowell, MA). The cells serum, 1% penicillin streptomycin, and 1% non essential amino acids. The cells were grown to confluence and allowed to mature for 17 days at 37 o C and 5% CO 2 The cell culture media were changed every 1 2 days. The permeability stud y was carried out in triplicate tests after the transepithelial electrical resistance (TEER) was 500 600 cm 2 measur ed using an epithelial volt ohm meter (Millicell ERS 2, Millipore Corp, Billerica, MA). The permeability study followed a published paper with minor modifications ( 68 ) EGCG or EGCG ovalbumin dextran conjugate nanoparticles were added into balanced salt solution ( HBSS, pH adjust ed to 6.0) to control EGCG concentration at 0.025 mg/mL One and half milliliter s of each sample was transferred to the api cal chamber of the tra nswells and 2.6 mL of the fresh buffer was added to the basolateral chamber. After incubation at 37 o C for 30, 60, 90, and 120 min, the transepithelial electric resistance (TEER) was measured. Subsequently, 0.5 mL of the samples was

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91 ta ken from the basolateral chamber, acidified with ascorbic acid solution to adjust pH to 2.5 and stored in 80 o C until analyses. The fresh buffer (0.5 mL) was re filled to the basolateral chamber. The samples were analyzed by HPLC ESI MS n using a published method with minor modifications ( 68 ) An Agilent Zorbax SB C18 column (4.6 mm 25 0 m m) was used for compound separation The binary mobile phase consist ed of 0.04 % acetic acid: water (A) and acetonitrile (B). A 22 min gradient 10 30 % B l inear; 20 22 min, 30 10% B linear; followed by 2 min of re equilibration of the column before the next run. The injection volume was 20 L with a flow rate at 1 mL/min. Electrospray ionization in negative mode was perfo rmed using nebulizer 65 psi, drying gas 11 L/min, drying temperature 350 o C, and capillary 4000 V. The MRM was used to monitor the transition of deprotonated EGCG m/z 457 [M H] to the product ion m/z 169 EGCG was used as an external standard. Quantifica tion was conducted using QuantAnalysis ( Version 2.0 Bruker Daltonics Inc, Billerica, MA). The apparent permeability coefficient ( P app ) of EGCG was calculated using the following equation: P app = x W here dQ/dt is the permeabili ty rate ( g/s), C 0 is the in itial concentration in the apical chamber ( g/mL), and A is the surface area of filter (cm 2 ), which wa s 4.67 cm 2 in this study. Statistical Analyse s Data was expressed as mean compare mean values between two groups using JMP software (Version 8.0, SAS Institute Inc., Cary, NC). A difference with p 0.05 was considered significant.

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92 Results Synthesis of Ovalbumin dextran C onjugates After electrophoresis ovalbumin showed a single band whereas dextran did not show any bands ( Figure 5 1 A ). The o valbumin/dextran physical mixture only showed the same ban d as the ovalbumin sample, indicating that no reaction occurred between ovalbumin and dextran. After the ovalbumin and dextran mixture was incubated at 60 o C under relative humidity of 79% in a desiccator containing saturated KBr solution for 2 days, a new smear band appeared, and the ovalbumin single band became smaller. This suggested that ovalbumin dextran conjugate was generated In Figure 5 1 B the ovalbumin and the ovalbumin/dextran physical mixture showed the single band in carbohydrate staining gel b ecause ovalbumin is a glycoprotein. N o band appeared on the dextran sample because dextran did not enter the gel due to lack of any charge However, ovalbumin dextran conjugate samples showed the smear band and the ovalbumin band became lighter which furt her confirmed that dextran was covalently conjugated with ovalbumin. The content of ovalbumin conjugated with dextran was calculated using UN SCAN IT gel analysis software Results suggested that 27% of ovalbumin reacted with dextran to form the conjugates Particle S iz e, Morphology, EGCG Loading Efficiency and Loading C apacity After re suspending nanoparticles in the water, both EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles showed average pa rticle size s of 2 85 nm and 339 nm, respectively ( Figure 5 2 ) Both dry EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles showed the spherical morphology, visualized by SEM .The sizes measured by Ze tatrac were bigger than those

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93 measured by SEM because the dry particles were used in SEM assessment ( 170 ) The loading efficiency of EGCG in EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles was 23.4% and 30.0%, respectively ( Table 5 1 ). The loading capacity of EGCG in the nanoparticles was 19.6% and 20.9%, respectively. No significant differences were observed in either loading efficiency or loading capacity between EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles. Nanopart icle C haracterizat ion under Different p H C onditions EGCG ovalbumin dextran co njugate nanoparticles showed a particle size of 210 nm in the aqueous suspension at pH 2.5, and the size remained at 323 nm and 308 nm when pH was adjusted to 3.0 and 4.0, respectively ( Figure 5 3 A ). The particle size increased to 634 nm when the pH of EGCG ovalbumin dextran conjugate nanoparticle suspension was 5.0, and then dropped to between 168 nm and 211 nm in a pH range from 6.0 to 7.5. Crosslinked EGCG ovalbumin dextran conjugate nanoparticl es showed particle size s of 173 nm, 336 nm, and 347 nm at pH 2.5, 3.0, and 4.0, respectively. When the pH of the nanoparticle suspension was adjusted to 5.0, crosslinked EGCG ovalbumin dextran conjugate nanoparticles had a size of 325 nm, and the size rema ined between 244 nm and 340 nm in a pH range of 6.0 to 7.5. EGCG ovalbumin dextran conjugate nanopar ticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles had comparable zeta potentials in a pH range from 2.5 to 7.5 ( Figure 5 3B ). Both EGCG ovalbumin dextran conjugate nanopar ticles and crosslinked EGCG ovalbumin dextran co njugate nanoparticles showed a zeta potential of around 10 mV at pH of 2.5 and 3.0. Lower surface ch arges were observed in EGCG ovalbumin dextran conjugate nanoparticles (ze ta potential of

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94 between 6.71 mV and 1.37 mV) and crosslinked EGCG ovalbumin dextran conjugate nanopartic les (zeta potential of between 1.45 mV to 6.07 mV) in the pH range of 4.0 to 6 .0. This was because such pH ranges were close to the isoelectric point of ovalbumin (around 4.5 to 4.8). When pH was above 7, both nanoparticles had the zeta potential around 10 mV. Stability of Nanoparticles in Simulated Digestive F luids The initial particle size of EGCG ovalbumin dextran conjuagate nanoparticles and cros slinked EGCG ovalbumin dextran conjugate nanoparticles re suspended in simulated gastric fluid (SGF) was 261 nm and 380 nm, respectively ( Figure 5 4A ). Incubating these two nanoparticles in SGF at 37 o C for 2 hours did not cause a significant change in part icle sizes. The particle size of EGCG ovalbumin dextran conjugate nanoparticles was 183 nm, 349 nm, 294 nm, and 299 nm at 0.5, 1, 1.5, and 2 hours, respectively. Similarly, crosslinked EGCG ovalbumin dextran conjugate nanoparticles showed average particle size s of 478 nm, 294 nm, 527 nm, and 439 nm after 0.5, 1, 1.5, and 2 hour of incubation, respectively. Both EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles appeared stable in the simulated intes tinal fluid (SIF) at 37 o C for 2 hours ( Figure 5 4B ). After EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugated nanoparticles were re suspended in SIF, the ir particle size s were 232 nm and 206 nm, respectively. A fter a 0.5 hour incubation, the size s of these nanoparticles were 217 nm and 280 nm, and the size s remained at 291 nm and 265 nm at 2 hours.

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95 EGCG Release from Nanoparticles in Simulated Digestive F luids After incubation of the nanoparticles in simulated g astric fluid (SGF) for 0.5 hour, 12.3% and 8.3% of EGCG was released from EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles, respectively ( Figure 5 5 A ). Introducing pepsin into the SGF led to a si gnificantly faster release of EGCG from the nanoparticles in SGF. They released 17.2% and 14.9% of EGCG, respectively. Similarly, 27.1% and 21.9% of EGCG was release from EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles in simulated intestinal fluid (SIF) for 2 hours, respectively ( Figure 5 5 B ). In SIF with pancreatin for 2 hours, EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles released 37.9% and 32.5% of EGCG, respectively. Crosslinked EGCG ovalbumin dextran conjugate nanoparticles retarded the EGCG release compared to EGCG ovalbumin dextran conjugate nanoparticles in SIF without and with pancreatin. EGCG T ransport on Caco 2 M onolayers The transepithelial electric resistance (TEER) percentage of Caco 2 monolayer s treated by HBSS buffer (Blank) dropped to 100.6%, 95.6%, 94.1%, and 89.9% of initial value at 30, 60, 90, and 120 min, respectively ( Figure 5 6A ). No significant decreases on T EER of Caco 2 mo nolayer s treated by EGCG were observed compared to the blank. The TEER of Caco 2 monolayer s treated by EGCG was 98.1%, 95.1%, 91.7% and 85.0% of initial value respectively EGCG ovalbumin dextran conjugate nanoparticles caused the decrease of TEER to 95.2%, 90.9%, 89.3%, and 79.8% of initial value at 30, 60, 90, and 120 min, respectively. However, there were no significant

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96 differences on TEER of Caco 2 monolayer s treated by the nanoparticles, EGCG, or blank. The apparent permeability coeffi cient ( P app ) of EGCG from the apical to the basolateral side on the Caco 2 monolayer s was investigated after incubating EGCG solution or EGCG ovalbumin dextran conjugate nanoparticles with Caco 2 monolayer s for up to 120 min ( Figure 5 6B ). The P app of EGCG in EGCG solution was between 0.28 0.42 10 6 cm/s during the incubation period. However, the P app of EGCG in the nanoparticles was between 0.52 0.78 10 6 cm/s, and significantly higher than those of the EGCG solution. Discussion The Maillard reaction f orms covalent bonds between carbonyl groups and amino groups ( 226 ) The ovalbumi n dextran conjugates were generated by cont rolling the Maillard reaction at an early stage, the Amadori rearrangement ( 227 229 ) SDS PAGE showed that a new smear band appeared in both protein staining and carbohydra te staining gel after incubating the ovalbumin and dextran mixture at 60 o C under a relative humidity of 79% in a desiccator containing saturated KBr solution for 2 days. In the meantime, the ovalbumin band became smaller compared to unconjugated ovalbumin ( Figure 5 1 ). This confirmed the generat i o n of conjugates. The gel analysis revealed that about 27% of ovalbumin reacted with dextran to form the conjugates. After mixing the ovalbumin dextran conjugates with EGCG and adjusting the pH of the solution to 5 .2 heating the mixture at 80 o C for 60 min caused the formation of a white milky suspension. The heating step induced the gelation of ovalbumin, which led to the heat induced unfolding of ovalbumin ( 230 ) The protein protein interactions (hydrogen bonding electrostatic and hydrophobic interactions, disulfide sulphydryl

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97 interchange) and protein EGCG interactions (hydrog en bond ing and hydrophobic interactions) caused the self assembly of EGCG and ovalbumin dextran conjugates This resulted in encapsulation of EGCG in the nanoparticles ( 82, 170, 230 ) Conjugation of ovalbumin with d extran increased the hydrophilic property of ovalbumin dextran conjugates, which helped to stabilize the nanoparticles in the aqueous suspension. Glutaraldehyde was added to the nanoparticle suspension to crosslink the ovalbumin proteins, which further sta bilized the structure of EGCG ovalbumin dextran conjugate nanoparticles. The loading efficiency and the loading capacity of EGCG in EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles were not signi ficantly affected by additional crosslinking because glutaraldehyde was added after the heating step ( Table 5 1 ). The formation of EGCG ovalbumin dextran conjugate nanoparticles is illustrated in Figure 5 7 We hypothesized that the core of the nanoparticl es was formed by the hydrophobic peptides of ovalbumin and the hydrophilic dextran was on the surface of the nanoparticles as the shell. Our data showed that the heating process degraded about 2.4% EGCG. Therefore, the degradation of EGCG is not a concern. The pH in the human gastrointestinal tract varies drastically in different segments. For example, pH in mouth stomach, small intestine, and large intestine were 6.5 7.0, 1.5 (fast) 5.0 (fed) 5.0 7.0, and 7.0 7.4 respectively ( 13, 118 ) The s ize and surfa ce charge of protein based nanoparticles depends on pH conditions. Weakened repulsion among the nanoparticles caused particle precipitation when the pH was around the isoelectric point of the selected protein ( 75 ) Therefore, it is important to know the change of particle size and zeta potential under different pH conditions. In the

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98 present study, both EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextra n conj ugate nanoparticles remained at nano scale sizes in a pH range from 2.5 to 7.5 ( Figure 5 3 ) Dextran has been reported to enhance colloidal stability ( 223, 231 232 ) Thus, hydrophilic dextran on the surface of the nanoparticles may retard the aggregation of ovalbumin proteins. Zeta potential of the nanoparticles is primarily determined by the protein and zeta potential var ies according to pH conditions. Both EGCG ovalbumin dextran con jugate and crosslinked EGCG ovalbumin dextran conjugate nanoparticles showed positive zeta potentials in a pH range from 2.5 t o 3.0, whereas negative charges were obser ved when pH was above 7.0 It was noticed that both nanoparticles had weak er zeta potentials ( 6.07 mV to 6.76 mV) at pH 4.0 6. M ucus layers in the small intestine serve as a barrier between drugs and intestinal epithelia and they possess a negative charge ( 212 ) Thus, nanoparticles with strong negative or positive charge s may be repulsed or adsorbed by mucus layers to hinder the ir contact with epithelial cells, whereas weak ly charged nanoparticles may readily penetrate the mucus layers. EGCG ovalbumi n dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles had weak zeta potentials at pH 5.0 6.0, which may enhance the penetration of nanoparticles through the mucus layers to reach epithelial cells in the upper port ion of the small intestine. I t takes about 0.5 to 2 hours and 2 to 4 hours for ingested food to pass the stomach and small intestine, respectively ( 13, 118 ) Thus, the stability of nanoparticle s in the stomach and small intestine are critical. The m ajority o f ingested nanoparticles sh ould maintain nano size to enhance the absorption of loaded drugs. In simulated gastric fluid (SGF, pH 4.0, fed state) for 2 hours, EGCG ovalbumin dextran co njugate

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99 nanoparticles showed an average particle size between 183 nm and 349 nm, whereas the sizes of crosslinked EGCG ovalbumin dextran conjugate nanoparticles were between 294 nm and 527 nm. More importantly, both nanopartic les showed particle sizes below 300 nm in simulated intestinal fluid (SIF, pH 6.0, upper portion of sm all intestine) for up to 2 hours. This was explained by the capacity of dextran to stabiliz e the nanoparticles in the gastrointestinal tract with high salt concentration and different pH ( 232 ) N anoparticles with better stability in the gastrointestinal tract may increase p ermeation of EGCG in the small intestine. Release of bioactive compounds from nanoparticles in the gastrointestinal tract is attributed to the diffusion of bioactive compounds in the gastrointestinal fluid. More importantly, degradation of nanoparticles by digestive enzymes in the gastrointestinal tract may cause the burst release of bioactive compounds ( 201, 210 ) For example pepsin in the stomach, and trypsin, chymotrypsin, elastase, and carboxypeptidas A and B in the amylase can hydrolyz e 1,4 D glucosidic linkages of polysaccharides. Dextran consists of straight 1,3 linkage s Thi s structure makes dextran resistant to hydrolysis by amylase in the gastrointestinal tract ( 225 ) In the present study, the release of EG CG from both of the nanoparticles in simulated fluids with presence of digestive enzymes was more than that wi th absence of digestive enzymes. This suggests that ovalbumin was hydrolyzed by proteinases in both SGF and SIF. It should be noted that EGCG oval bumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles show ed similar EGCG release percentage in the simulated fluid wi th

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100 digestive enzymes. This may be because the dextran on the surface of the nanoparticles p r otected the protein from digestion by hindering the contact between ovalbumin and proteinase. Also, ovalbumin was reported to be resistan t against pepsin digestion in the stomach at fed state ( 221 ) C onformation change of ovalbumin after thermal process ing provided resistance against trypsin, chymotrypsin, and elastate in the small intestin e ( 220, 233 ) Furthermore, crosslinked EGCG ovalbu min dextran conju gate nanoparticles decreased the EGCG release compared to EGCG ovalbumin dextran conjugate nanoparticles in simulated fluids without and with digestive enzymes, which indicated the crosslinking process further improved the integrity of the nanoparticles in the presence of proteinases. Caco 2 cell monolayers have been widely used to estimate the permeability of phenolic compounds in vitro ( 215 216 ) M onolayers were formed after the TEER was between 1 50 and 1600 cm 2 ( 234 ) The TEER value of Caco 2 monolayer s in the present study was 500 600 cm 2 A significant decrease of TEER indicated that the tight junctions of the monolayer s were disrupted or monolayer integrity was compromised Nanoparticles with a size <20 nm have the capacity to reversibly open tight junctions among cells and facilita te the permeation of loaded drugs via the paracellular pathway ( 118 ) In this study, TEER values of Caco 2 monolayers were similar among the b lank, EGCG solution, and EGCG ovalbumin dextra n conjugate nanoparticles. This suggested that the concentration of EGCG had little adverse effect on the integrity of the monolayer, and the nanoparticles did not open tight junctions of the monolayer to facil itate the permeation of EGCG by paracellular pathway.

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101 The main mechanism for nanoparticles to enhance the permeability of bioactive compounds was the cellular uptake of the nanoparticles via endocytosis, including clathrin and/or caveolae mediated endocyt osis ( 118, 121 122 ) It has been proposed that clathrin mediated endocytosis al lowed the nanoparticles with an average size below 300 nm to penetrate the membrane of the cells whereas nanoparticles with a size belo w 500 nm could be internalized into cells by caveolae mediated endocytosis ( 127 ) Upon internalization, nanoparticles can either be delivered into the endo/lysosome for the degradation to release the bioactive compounds, or escape the endo/lysosome to arrive at the basolateral side by extocytosis ( 13, 118 ) In the present study, the P app of EGCG in EGCG solution was 0.28 0.42 10 6 cm/s, which was comparable with previous studies ( 68, 195 ) EGCG ovalbumin dextran conjugate nanoparticles significantly improved the P app of EGCG to 0.54 0.78 10 6 cm/s. We hypothesized that the internalization of the nanoparticles by Caco 2 cells was mediated by clathrin mediated endocytosis since EGCG ovalbumin dextran conjugate nanoparticles had a size below 300 nm in SIF (pH 6.0). Summary Ovalbumin and dextran were conjugated using the Maillard reaction. EGCG ovalbumin dextran conjugate nanoparti cles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles were formed by self assembly under a heating process. These particles showed a spherical morphology measured by SEM and the sizes were 285 nm and 339 nm in aqueous suspension. The loading efficiency of EGCG in these conjugate nanoparticles was 23.4% and 30.0%, whereas the loading capacity was 19.6% and 20.9%, respectively. EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conj ugate nanoparticles remained at nano scale sizes in the

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102 pH range from 2.5 to 7.5, and they showed stable particle sizes in SGF and SIF at 37 o C for 2 hours. The limited release of EGCG from these two nanoparticles in SGF and SIF with out and with digestive enzymes was observed. EGCG in EGCG ovalbumin dextran conjugate nanoparticles possessed the higher apparent permeability coefficient ( P app ) on Caco 2 monolayers compared to EGCG solution.

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103 Table 5 1. Loading efficiency and loading capacity of EGCG in EGCG ovalbumin dextran conjugate na noparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles Nanoparticles L oading efficiency L oading capacity (%) (w/w %) EGCG ovalbumin dextran conjugate nanoparticles 23.4 2.9 19.6 3.2 Crosslinked EGCG ovalbumin dextran conjugate n anoparticles 30.0 4.9 20.9 3.8 D ata are mean stand ard deviation of four replicate tests No significant differences are observed between the samples using student t test at p 0.05.

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104 Fig ure 5 1. SDS PAGE analysis of ovalbumin, dextran, ovalbumin/dextran physical mixture, and ovalbumin dextran conjugates. A ) Ovalbumin stain. B ) C arbohydrate stain. Lane M represents molecular weight marker. Lane 1, 2, and 3 represent ovalbu min, dextran, and ovalbumin/dextran physical mixture, respectively. Lane 4, 5, and 6 represent triplicates of ovalbumin dextran conjugates. The amount of ovalbumin in each lane was 22 g. The amount of dextran was 22 g

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105 Figure 5 2. Particle size of an d particle morphology of EGCG ovalbumin dextr an conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanopartic les A1) Particle size of EGCG ovalbumin dextran conjugate nanoparticles. A2) Particle size of crosslinked EGCG ovalbumin dex tran conjugate nanoparticles. B1) Particle morphology of EGCG ovalbumin dextran conjugate nanoparticles. B2) Particle morphology of EGCG ovalbumin dextran conjugate nanoparticles.

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106 Figure 5 3. Particle size and zeta potential of EGCG ovalbumin dextran co njugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles in the pH range from 2.5 to 7.5 A) Particle size. B) Zeta potential. Data are mean standard deviation of duplicate tests

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107 Figure 5 4. Particle size of EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dext ran conjugate nanoparticles in simulated fluid s at 37 o C for 0, 0.5, 1, 1.5, and 2 hour A) Simulated gastric fluid (SGF). B) Simulated intestinal fluid (SIF). Data are mean standard devi ation of duplicate tests

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108 Fig ure 5 5 Cumulative release of EGCG from EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dext ran conjugate nanoparticles in simulated fluid s without and with digestive enzymes at 37 o C. A) Simul ated gastric fluid (SGF) without and with pepsin for 0.5 hour. B) Simulated intestinal fluid (SIF) without and with pancreatin for 2 hours. Data are mean stand ard deviation of four replicate tests # denotes significant differences between EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles using t test at denotes significant difference s between simulated fluid without enzyme and fluid with enzyme using t

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109 Figure 5 6. Transepithelial electric resistanc e (TEER) and apparent permeability coefficient ( P app ) of EGCG in solution and EGCG ovalbumin dextran conjugate nanoparticles at 37 o C for 30, 60, 90, and 120 min. A) TEER. B) P app Data are mean s tandard deviation of triplicate tests at each incubation period represents significant difference s

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110 Fig ure 5 7 Illustration of formation of EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles.

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111 CHAPTER 6 CONCLUSIONS The PPE obtained from pomegranate pericarp contained punicalagin A and B ellagic acid hexoside, and ellagic acid. Punicalagin s had the capacity of binding gelatin to form nanoparticles. The nanoparticle fabricated using three PPE to gelatin mass ratios had the particle size < 250 nm, zeta potential around +18 mv sphe rical morphology, and good loading capacity. PPE gelatin nanoparticles had lower apoptotic effects in HL 60 cells compared to PPE solution. PPE to gelatin mass ratio was a major factor affecting characteristics and stability of nanoparticle suspension. The critical point for PPE gelatin nanoparticles was at a mass ratio of 8:5. Nanoparticle s prepared in a pH range from 4 to 5 had particle sizes and zeta potentials that may favor the absorption of loaded ellagitannins. Reaction temperature s from 25 to 50 o C h ad a positive influence on nanoparticle characteristics. Self assembly occurred rapidly between gelatin and ellagitannin and fabrication was done within 12 hours and remained stable for 4 days. BEN with poly lysine and chitosan coatings had a spherical morphology, and the loading efficiency and capacity of EGCG were 35.4% and 32.7%, and 17.0% and 16.0%, respectively. These coatings significantly stabilized BEN in the pH range from 1.5 to 6.0. Faster release of EGCG was observed in all the nanoparticles incubated in simulated gastric and intestinal fluids with proteinases. However, the coatings delayed the release of EGCG from the nanoparticles. The storage stability study showed faster degradation of EGCG in all the nanoparticles compared to free EGCG. CBEN significantly enhanced EGCG absorption on Caco 2 monolayer, which was attributed to the enhancement of cellular uptake of CBEN by Caco 2 cells.

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112 Ovalbumin and dextran were conjugated using the Maillard react ion. EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conjugate nanoparticles were formed by self assembly under a heating process. These particles showed a spherical morphology measured by SEM and the sizes were 285 nm and 339 nm in aqueous suspension. The loading efficiency of EGCG in these two conjugate d nanoparticles was 23.4% and 30.0%, whereas the loading capacity was 19.6% and 20.9%, respectively. EGCG ovalbumin dextran conjugate nanoparticles and crosslinked EGCG ovalbumin dextran conj ugate nanoparticles remained at nano scale sizes in the pH range from 2.5 to 7.5, and they showed stable particle sizes in SGF and SIF at 37 o C for 2 hours. Partial release of EGCG from these two nanoparticles in SGF and SIF with out a nd with digestive enzymes was observed. EGCG in EGCG ovalbumin dextran conjugate nanoparticles showed higher apparent permeability coefficient ( P app ) on Caco 2 monolayers compared to EGCG solution.

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113 LIST OF REFERENCES ( 1 ) Khknen, M. P .; Hopia, A. I.; Vuorela, H. J.; Rauha, J. P.; Pihlaja, K.; Kujala, T. S.; Heinonen, M., Antioxidant activity of plant e xt racts containing phenolic c ompounds. Journal of Agricultural and Food Chemistry 1999, 47 3954 3962. (2) Asen, S.; Stewart, R. N.; No rris, K. H., Co pigmentation of anthocyanins in plant tissues and its effect on color. Phytochemistry 1972, 11 1139 1144. (3) Jackman, R. L.; Yada, R. Y.; Tung, M. A.; Speers, R. A., Athocyanins as food colorants A review Journal of Food Biochemistry 1 987, 11 201 247. (4) Castaeda Ovando, A.; Pacheco Hernndez, M. d. L.; Pez Hernndez, M. E.; Rodrguez, J. A.; Galn Vidal, C. A., Chemical studies of anthocyanins: A review. Food Chemistry 2009, 113 859 871. (5) Ly nn, D. G.; Chang, M., Phenolic sign als in cohabitation: implications for plant d evelopment. Annual Review of Plant Physiology and Plant Molecular Biology 1990, 41 497 526. (6) Labrecque, L.; Lamy, S.; Chapus, A.; Mihoubi, S.; Durocher, Y.; Cass, B.; Bojanowski, M. W.; Gingras, D.; Blivea u, R., Combined inhibition of PDGF and VEGF receptors by ellagic acid, a dietary derived phenolic compound. Carcinogenesis 2005, 26 821 826. (7) Rice Evans, C.; Miller, N.; Paganga, G., Antioxidant properties of phenolic compounds. Trends in Plant Scienc e 1997, 2 152 159. (8) Cai, Y.; Luo, Q.; Sun, M.; Corke, H., Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sciences 2004, 74 2157 2184. (9) Frankel, E. N.; German, J. B.; Ki nsella, J. E.; Parks, E.; Kanner, J., Inhibition of oxidation of human low density lipoprotein by phenolic substances in red wine. The Lancet 1993, 341 454 457. (10) Hollman, P. C. H.; Katan, M. B., Absorption, metabolism and health effects of dietary fl avonoids in man. Biomedicine & Pharmacotherapy 1997, 51 305 310. (11) Kanaze, F. I.; Bounartzi, M. I.; Georgarakis, M.; Niopas, I., Pharmacokinetics of the citrus flavanone aglycones hesperetin and naringenin after single oral administration in human subjects. Eur J Clin Nutr 2006, 61 472 477. (12) Appeldoorn, M. M.; Vincken, J. P.; Gruppen, H.; Hollman, P. C. H., Procyanidin d imer s A1, A2, and B2 are absorbed without conjugation or methylation from the small intestine of r ats. The Journal of Nutrit ion 2009, 139 1469 1473.

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134 BIOGRAPHIC AL SKETCH Zheng Li was born in Beijing, China. He rec eived his B S. degree in food b ioengineering at China Agricultural University in 2007, and enrolled into program in food b iotechnology at the same u niversity. He had three publications as the f irst author from his master proj ect. Af ter his graduation with M. S. degree from China Agricultural University in 2009 he entered the food science doctoral program at the University of Florida under the supervision of Dr. Liwei Gu. During his doctoral p eriod, Zheng published three papers as the first author Upon his completion of the Ph D. degree in August 2013, Zheng plans to continue his research as a post doc at other university and look s forward to any challenge that life will bring