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1 1. 1 2. DDS 2 2.1. 2 2.2. 3 2.3. 3 2.4. 3 3. DDS 5 4. 7 5. 9 2 Solid-in-Oil 1. 11 1.1. 12 1.2. 14 1.3. 16 1.4. DDS 17 1.4.1. 18 1.4.1.1. 18 1.4.1.2. 18 1.4.1.3. 19 1.4.2. 19 1.4.2.1. 20 1.4.2.2. 21 1.5. DDS 23 1.5.1. Solid-in-Oil S/O 23 1.5.2. S/O 24 1.5.3. S/O 25 1.5.4. S/O 28 1.5.5. S/O 29 2. 31 2.1. 31 2.2. S/O 31 2.3. 32 2.4. 32 2.4.1. 32 2.4.2. ELISA 33 2.4.3. HRP 33 3. 34 3.1. S/O 34 3.2. FITC 36 3.2.1. YMP 36 3.2.2. FITC 37 3.2.3. FITC 39 3.3. EGFP HRP 41 3.4. 42 4. 45 5. 46

3 Solid-in-Oil 1. 51 1.1. 51 1.2. Gregory M. Glenn 52 1.3. 54 1.3.1. 54 1.3.2. 55 1.3.3. 57 1.3.3.1. 57 1.3.3.2. 59 1.3.3.3. 59 1.3.3.4. 60 1.3.3.5. Layer-by-layer-assembled films 60 1.3.3.6. 61 1.3.3.7. 61 1.4. Solid-in-Oil S/O 61 2. 62 2.1. 62 2.2. S/O 62 2.3. 63 2.4. OVA S/O 63 2.4.1. 63 2.4.2. 63 2.5. HEL S/O 64 2.5.1. 64 2.5.2. 64 2.6. Anti-OVA IgG from guinea pig, polyclonal ELISA 64 2.7. Anti-HEL IgG from rabbit, polyclonal ELISA 65 3. 66 3.1. OVA S/O 66 3.1.1. OVA S/O 66 3.1.2. Anti-OVA IgG from guinea pig, polyclonal ELISA 66 3.1.3. 68 3.2. HEL S/O 70 3.2.1. HEL S/O 70 3.2.2. 70 4. 72 5. 73

4 1. 76 1.1. 77 1.1.1. 77 1.1.2. 78 1.2. 81 1.2.1. lipoplex 82 1.2.2. polyplex 82 1.2.3. MEND 83 1.2.4. 84 1.3. 86 1.3.1. 86 1.3.2. 88 1.4. 92 1.5. 93 1.5.1. PLGA 93 1.5.2. ph 94 1.5.3. ph 94 2. 95 2.1. 95 2.2. 96 2.3. STR-R8 97 2.4. 100 2.4.1. pdna S/O 100 2.4.2. S/O 100 2.4.3. 101 2.5. 101 2.6. 101 2.7. 102 2.8. LIVE/DEAD assay 103 2.9. 104 2.10. WST assay 104 3. 105 3.1. 105 3.2. ER-290 : PC 106 3.3. 107 3.4. S/W 107 3.5. pdna 109 3.6. Carreir #1-2 110 3.7. 112 3.8. Carreir #4-5 113 3.9. 116 4. 117 5. 118 5 122 125

1 1. Drug Delivery System, DDS 4 DDS 1 DDS DDS DDS DDS 1 1 DDS Quality Of Life, QOL / 1000 % DDS 21 DDS 2 DDS DDS DDS DDS 1

1 2. DDS DDS DDS DDS DDS DDS DDS DDS [a] QOL 2.1. DDS 1 DDS 2 DDS 3 DDS 1 DDS DDS 2 3 2 4 DDS [a] intradermal i.d. /intracutaneous/ percutaneous injection subcutaneous s.c. /hypodermic injection intravenous i.v. injection intramuscular i.m. injection 5 1 ~ 2 2

1 3 DDS DDS 2.2. 2 2 PEG 3 [b] DDS PEG PLGA DDS 2.3. DDS DDS DDS 2.4. DDS 1 2 1 DDS [b] Polyethylene glycol PEG PEG PEG PEG DDS 3

1 PLGA DDS DDS ph 2 DDS DDS 4 ph enhanced permeation and retention EPR 5,6 [c] 3 2006 1 DDS 1986 EPR DDS EPR [c] EPR 200 nm 100 nm EPR 200 nm DDS EPR 4

1 3. DDS DDS DDS 1960 Alec Douglas Bangham DDS DDS 1 9 Fig.1.1 DDS DDS 7 [d] Fig.1.1 DDS 9 DDS 1990 Vadimir P. Torchilin Leaf Haung DDS 1 PEG 8 1980 immunoliposome PEG DDS PEG PEG DDS [d] 1964 Bangham TEM 5

1 PET MRI 4 in vitro 1990 2 3 1 DDS DDS 1 Table 1.1 DDS 1 1 DDS DDS multifunctional envelope type nano device, MEND DDS Table 1.1 DDS Core technologies Lipid-based carriers Liposomes, Lipid bilayers 9,10 DDS 2005, 2006 11 2009 12 MEND 2011 Oil-in-warer colloids, Solid-lipid particles; Water-in-oil colloids, Reverse micelles 13 Oil-in-water DDS 2004 14,15 Solid-lipid particles DDS 2002, 2007 16 2008 Micellar fibers, Supramolecular gels 17 2009 18 Samuel I. Stupp 2010 Inorganic carriers Carbon tubes 19 2010 20,21 PEG 2011 Quantum dots 22,23,24 2005, 2010 25 2006 Gold particles, Gold rods 26,27 DDS 2008, 2009 28 2009 Magnetic particles 29 DDS 2008 30,31 MRI 2008, 2009 Silica particles 32 2008 33 2008 6

1 Table 1.1 DDS Core technologies Polymeric carriers Linear chain, Macromolecular gels 34,35 2008, 2009 36 2011 37 2005 Polymeric micelles 38 Alexander V. Kabanov Pluronic 2008 39 Kazunari Kataoka 2009 Layer-by-layer (LbL) particles 40 LbL 2011 41 LbL MRI 2011 Spheres 42 PLGA 1997 43 PLGA 2011 Nanogels 44 2009 45,46 Kazunari Akiyoshi Kwangmeyung Kim 2010, 2009 Dendrimers 47,48,49 DDS 2004, 2005, 2011 50 2009 Naturally-derived carriers Viral vectors 51 Targeted viral vectors 2007 Virus like particles 52 Virus capsid 2006 4. DDS DDS 1990 Masahiro Goto Noriho Kamiya Eiichi Toorisaka Hongyu Piao Solid-in-Oil S/O 53,54,55,56 DDS DDS Fig.1.2 7

1 2 S/O DDS S/O DDS 3 2 QOL DDS 3 2 S/O DDS 4 DDS S/O 2 5 8

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2 Solid-in-Oil 1. mab Table 2.1 2009 10 2010 200 360 240 mab 6,000 2009 990 1 Products Campany Table 2.1 10 2009 Enbrel Amgen, Takeda Pharmaceuticals, (etanercept) Wyeth Centocor (Johnson & Johnson), Remicade Rheumatism Mitsubishi Tanabe Pharma, (infliximab) (mab) Schering-Plough Avastin Lung/bowel cancer Chugai, Genentech, Roche (bevacizumab) (mab) Rituxan/MabThera Lymphatic malignancy Biogen-IDEC, Genentech, Roche (rituximab) (mab) Humira Rheumatism Abbott, Eisai (adalimumab) (mab) Epogen/Procrit Amgen, Janssen-Cilag, (Renal) anemia /Eprex/ESPO Kyowa Hakko Kirin, Ortho (Glycoprotein hormone) (epoetin alfa) Herceptin Breast cancer Genentech, Chugai, Roche (trastuzumab) (mab) Lantus Diabetes Sanofi-aventis (insulin glargine) (Insulin derivative) Neulasta Cancer immunotherapy Amgen (pegfilgrastim) (PEG-cytokine) Aranesp/Nespo (Renal) anemia Amgen, Kyowa Hakko Kirin (darbepoetin alfa) (Glycoprotein hormone) s.c.: subcutaneous ; i.v.: intravenous. Indication Administration (Biopharmaceuticals) Methods Cycle and Periods Rheumatism Twice a week s.c. (TNF receptor-fc) for 3-6 months i.v. (3-4 hours) i.v. (90 min) i.v. (2 hours) s.c. s.c./i.v. i.v. (60 min) s.c. s.c. i.v. (60 min) Once a week for 6 months Once per 2 weeks for 2-6 months Once a week for 3-6 months Once per 2 weeks for 3-6 months Once a week for 6 months Once a week for 6 months Once a day Once a day for one month Once per 2 weeks for 6 months Table 2.1 Quality Of Life, QOL 11

2 1.1. DDS Fig.2.1 2 3 [a] 2 30 Alza Alejandro Zaffaroni Table 2.2 Zaffaroni 2 Advantages of the transdermal route for drug administation 1. Eliminates the vagaries of gastrointestinal absorption 2. Bypasses the portal circulation 3. Permits easy termination of therapy 4. Allows drug entry into the circulation when the gastrointestinal tract is not available or not suitable 5. Offers a means of painless systemic therapy for young children [a] Transdermal Transcutaneous Dermis Transdermal Epidermis Transepidermal Cutaneous Transcutaneous Dermatology Dermis Transdermal 12

2 Zaffaroni 1981 Table 2.2 5 2 ph 1 Table 2.3 FDA 3 Year Drug/Product name Indication Company 1979 Scopolamine/Transderm-Scop Motion sickness Novartis Consumer Health 1981 Nitroglycerin/Transderm-Nitro Angina pectoris Novartis 1984 Clonidine/Catapres-TTS Hypertension Boehringer Ingelheim 1986 Estradiol/Estraderm Menopausal symptoms Novartis 1990 Fentanyl/Duragesic Chronic pain Janssen Pharmaceutica 1991 Nicotine/ ProStep, Nicoderm, Habitrol Smoking cessation Elan, GlaxoSmithKline, Novartis Consumer Health 1993 Testosterone/Testoderm Testosterone deficiency Alza 1995 Lidocaine with epinephrine / Local dermal analgesia Iomed Iontocaine (iontophoresis) 1998 Estradiol with norethidrone/combipatch Menopausal symptoms Novartis 1999 Lidocaine/Lidoderm Post-herpetic neuralgia pain Endo Pharmaceuticals 2001 Ethinyl estradiol with norelgestromin/ortho Evra Contraception Ortho-McNeil Pharmaceutical 2003 Estradiol with levonorgestrel/ Climara Pro Menopausal symptoms Bayer Healthcare Pharmaceuticals 2003 Oxybutynin/Oxytrol Overactive bladder watson Pharma 2004 Lidocaine (ultrasound)/sonoprep Local dermal anesthesia Echo Therapeutics 2005 Lidocaine with tetracaine/synera Local dermal analgesia Endo Pharmaceuticals 2006 Fentanyl HCl/Ionsys (iontophoresis) Acute postoperative pain Alza 2006 Methylphenidate/Daytrana Attention deficit hyperactivity disorder Shire 2006 Selegiline/Emsam Major depressive disorder Bristol-Myers Squibb 2007 Rotigotine/Neupro Parkinson s disease Schwarz Pharma 2007 Rivastigmine/Exelon Dementia Novartis FDA Table 2.3 QOL 3 Zaffaroni 13

2 1.2. Fig.2.2 5,9! Fig.2.2 200 µm 2 mm 3 15 µm keratinocyte [b] melanocyte [c] [c] corneocyte, horny cell [b] 10 ~ 20 2 ~ 4 10 ~ 25 % 70 % ph 4.2 ~ 5.6 1 cm 2 10 100 4,5,6,7,8 2 [d] [b] [c] 4 [d] 1 1 M mg S cm 2 # L cm J = dm " 1 & % ( mg/ $ dt S ' cm 2 dc h x dx mg/cm3 /cm D! cm 2 /h J = D dc dx = D C 1 " C " 2 L! K = C 1 = C % 2 $ # C i C ' f & J = DK L C " ( i " C f ) C f = 0 P = DK % $ ' cm/h J = PC i # L & J! DDS C i DDS! P P 14!

2 1,000 1 1,000 1 9 Fig.2.3 a b c 5,9 Fig.2.3a Fig.2.3a Fig.2.3bc Fig.2.3b Fig.2.3c Fig.2.4 15

2 1.3. Fig.2.4 11 Jan D. Bos 2000 The 500 Dalton rule for the skin penetration of chemical compounds and drugs 500 10 The 500 Dalton rule 2003 Samir Mitragotri 11 Fig.2.4a Mitragotri P cm/h 10 10 1 Fig.2.4b Kenji Sugibayashi 2007 11 20 300 DFNa 4 1 4 100 1000 10000 1 3 LSE-High, TOYOBO 16

2 1.4. DDS Zaffaroni 30 DDS 3 1 2008 sirna 12 2 3 Table 2.1 3 DDS DDS 1 DDS DDS 2 3 Fig.2.5 DDS 13 Fig.2.5 occlusive dressing therapy, ODT Fig.2.5a transdermal 3 Fig.2.5b 14 15 Fig.2.5c Fig.2.5d Fig.2.5e DDS Fig.2.5f 17

2 1.4.1. 1.4.1.1. 20 k ~ 16 MHz 100 khz 16 Samir Mitragotri [e] Mitragotri 1995 17 Fig.2.6 18 18 19 Table 2.1 Mitragotri 20 1.4.1.2. 0.01 ~ 100 msec 21 1993 22 -MIT James C. Weaver [e] 23 1900 1980 24 1990 Yie W. Chien Fig.2.7 Fig.2.8 25 [e] Mark R. Prausnitz 1993 -MIT James C. Weaver James C. Weaver Robert Langer Samir Mitragotri 1995 Robert Langer 1990 18

2 Calcitonin LH-RH Somatorelin Vasopressin somatostatin 25 26 1.4.1.3. Fig.2.9 1970 1990 27 Mark R. Prausnitz [e] 1998 Fig.2.9 28,29 28 Prausnitz 2003 PLA PGA 29 2005 2008 PLGA 30 50 µm 200 ~ 800 µm 10 1.4.2. 1990 ~ 2000 QOL 30 Zaffaroni Table 2.2 1 19

2 3 1.4.2.1. Table 2.4 Table 2.4 Peptides Sequences References Oligo-arginine TAT YARA WLR Antennapedia homeodomain Haptide LMWP TD-1 R n (n = 7-9) (YG)RKKRRQRRR YARAAARQARA (WLRRIKA) n (n = 1-3) RQIKIWFQNRRMKWKK TRWYSMKKTTMKIIPFNRL VSRRRRRRGGRRRR ACSSSPSKHCG 31,32,33,40,41 32,33,34,35,36,40,41 37,38 38 42 43 44 45 2000 Paul A. Khavari Paul A. Wender R 7 A CsA 31 CsA 1200 1 Wender R 7 4 Khavari R 7 CsA ph 7.4 CsA 2001 2005 S.Y. Choi R 9 TAT 32,33,34,35,36 L.B. Lopes YARA WLR heat shock protein 20 37,38,39 Lee R 9 TAT GFP 40,41 SIINFEKL CpG 42 haptide 43 LMWP 44 20

2 Chen in vivo TD-1 45,46 10 2000 Khavari Khavari Lopes 38 Fig.2.10 47,48,49 Fig.2.10a 47,48 2003 Kazuo Ohtake Poly-L-Arg Fig.2.10b 49 50 51 1.4.2.2. 1990 Gregor Cevc 1998 Cevc edge activator Fig.2.11a Transfersome Cevc IDEA AG 52 Transfersome IDEA AG 53 21

2 1 54,55 2 56,57 1 Joke A. Bouwstra 2000 Fig.2.11e 58,59 Bouwstra Transdersome Fig.2.11e 60 Fig.2.11b 61 Fig.2.11c 62 1 2 15 63 Fig.2.11d Fig.2.11 22

2 1.5. DDS Solid-in-Oil Solid-in-Oil 1.5.1. Solid-in-Oil S/O Fig.2.12 S/O 1994 2000 Masahiro Goto Noriho Kamiya - 1990-20 % - Goto Kamiya - 64,65,66,67,68,69,70 1997 Solid-in-Oil S/O water-in-oil W/O 71,72,73,74,75,76,77,78 Fig.2.13a Fig.2.13b - 79 80,81,82,83,84,85,86 87,88 89 PEG 90,91,92,93,94,95 96,97,98 23

2 99,100,101,102 1990 - Eiichi Toorisaka 2003 W/O - DDS Toorisaka 2C 18 9 GE Toorisaka 103 S/O Toorisaka S/O oil-in-water Solid-in-Oil-inWater S/O/W 103 Hongyu Piao S/O 104 2003 2008 S/O 105,106,107,108,109,110,111,112,113,114 2011 S/O 115 Piao 2008 S/O 116 117,118 119 2 120 3 121 122,123,124,125,126,127,128, 129,130 Fig.2.13 S/O 81,71,103,104 1.5.2. S/O S/O W/O W/O W/O 24

2 - W/O Oil Water W/O Oil Solid Solid-in-Oil S/O Fig.2.14 S/O 1.5.3. S/O 1990 2000 Masahiro Goto S/O Solid-in-Oil S/O 131,132,133 PLGA S/O Fig.2.15a W/O/W Fig.2.15b 4 S/O PLGA S/O 20 % 1990 Goto PLGA Table 2.5 S/O 25

2 1.6 wt.% W/O 1 PEG PLGA S/O 2010 S/O PLGA 134 Fig.2.15 PLGA 133, 135 Table 2.5 http://macro.lsu.edu/howto/index.htm Solvent Solubility in water Solubility of water Dielectric constant Density g/ml Melting point C Boiling point C Vapor pressure Torr 20 C Non-polar n-hexane Cyclohexane Toluene 1,4-Dioxane Isopropyl Myristate Chloroform Diethyl ether 0.014 % 20 C 0.006 % 25 C 0.052 % 25 C! < 0.1 % 20 C 0.815 % 20 C 6.89 % 20 C 0.01 % 20 C 0.01 % 20 C 0.03 % 25 C! < 0.1 % 20 C 0.06 % 20 C 1.26 % 20 C 1.9 2.0 2.4 2.3 3.2 4.8 4.3 0.655 0.779 0.867 1.034 0.853 1.489 0.713 95.3 6.5 95 11.8 3 63.6 117.4 68.7 80.7 110.6 101.3 192.6 61.2 34.6 124 77.5 28.5 29 < 1.0 158.4 442 Polar aprotic Ethyl acetate Dichloromethane Tetrahydrofuran (THF) Acetone Dimethylformamide (DMF) Acetonitrile Dimethyl sulfoxide (DMSO) 8.7 % 20 C 1.60 % 20 C!!!!! 3.3 % 20 C 0.24 % 20 C!!!!! 6.0 8.9 7.6 20.7 36.7 37.5 46.7 0.901 1.326 0.888 0.790 0.949 0.782 1.100 84.0 95.1 108.5 94.7 60.4 43.8 18.5 77.1 39.8 66 56.3 153.0 81.6 189.0 73.0 350 142 184.5 2.7 73 0.4 Polar protic Acetic acid Trifluoroacetic Acid (TFA) Isopropanol (Isopropyl alcohol) Ethanol (Ethyl alcohol) Methanol (Methyl alcohol) Water!!!!!!!!!!!! 6.2 8.6 19.9 24.6 32.7 80.1 1.049 1.489 0.785 0.789 0.791 1.000 16.5 15.3 88.0 114.1 97.7 0.0 118.5 71.8 82.3 78.3 64.7 100.0 11.8 97.5 32.4 43.9 97 17.5 26

2 1 S/O Fig.2.16 Masato Kukizaki 55 C 60 C W/O S/O 136 shirasu porous glass SPG 137 60 C S/O/W solid lipid particle S/O 60 C 60 C Fig.2.16 S/O/W SPG 137,136 W/O Solid-in-Oil 1 nm ~ 1 µm emulsion µm nm Solid-in-Oil Solid-in-Oil Solid W/O Solid-in-Oil Solid Solid-in-Oil Solid 27

2 1.5.4. S/O S/O 138 W/O S/O 1 W/O 2-3 1 2 S/O 5.0 mg/ml 3 W/O W/O 5 wt% 20 wt% 4 S/O 0.2 % w/v S/O Fig.2.16 Fig.2.17 S/O 116,117,118 2008 Hongyu Piao S/O 116 Fig.2.17a 117 118 S/O Fig.2.17a ER-290 S/O 2012 S/O 2005 Masahiro Goto S/O ASPION 2009 SO 4349639 S/O VIVCO Fig.2.17c 28

2 1.5.5. S/O Table 2.6 S/O pdna DFNa NaCl L1695 W/O Milli-Q ER-290 L-195 W/O dinitrochlorobenzene IPM W/O Table 2.6 S/O S/O W/O Milli-Q W/O S/O ph W/O Milli-Q S/O Table 2.4 Fig.2.18 S/O W/O S/O ER-290 [f] L-195 [f] Fig.2.18 ER-290 L-195 S/O W/O Table 2.5 [f] ER-290 HLB 2 HLB 0 ~ 20 HLB ER-290 ER-290 ER HLB 2 90 L-195 ER-290 L-195 ER-290 L-195 Fig.2.18 29

2 S/O S/O W/O W/O W/O Table 2.5 6.5 C S/O W/O - Fig.2.19 IPM Isopropyl myristate, IPM Fig.2.19 IPM 167 C 3 C IPM 141 S/O Fig.2.18 S/O S/O S/O ER-290 drug ER-290 [g] 1 50 S/O W/O ER-290 2.0 mg/ml = 0.2 % w/v W/O 4 S/O W/O POLYTRON PT2500E POLYTRON!196 C FD5N; EYELA, Tokyo, Japan 20 C S/O W/O S/O [g] S/O S/O S/O 1 30

2 2. 2.1. fluorescein isothiocyanate FITC Sigma-Aldrich Tokyo, Japan horseradish peroxidase HRP Wako Pure Chemical Industries Osaka, Japan ER-290 L-195 Mitsubishi-Kagaku Foods Co. Tokyo, Japan isopropyl myristate IPM Tokyo Kasei Co. Tokyo, Japan Yucatan micropig YMP, 5-month-old female 139 [h] Charles River Japan, Inc. Tokyo, Japan metal enhanced diaminobendizine DAB substrate kit Pierce Rockford, IL, USA 4 % Rhodamine-labeled 1,2-dioleoyl-sn-glycero- 3-phosphoethanolamine DOPE Avanti Polar Lipids Inc. Alabaster, AL HistoPrep TM Fisher Scientific NJ, USA Bovine insulin Enzyme-Linked ImmunoSorbent Assay ELISA kit Mercodia Wild-type enhanced green fluorescent protein EGFP His-tag 2.2. S/O S/O [i] 1 1.0 mg/ml 5.0 ml [j] 25 mg/ml ER-290 10 ml W/O ER-290 2 1 IPM 5.0 ml S/O 1.0 mg/ml ER-290 50 mg/ml Fig.2.20 [h] in vitro 1997 Makiko Fujii in vitro Yucatan micropig YMP 139 YMP 25 kg 1 15 20 µm Yucatan micropig skin 40 ~ 200 µm 1.5 ~ 2 mm Wang 40 EGFP R9 YMP YMP 1000 YMP in vitro [i] OVA : ER-290 1 : 50 w:w 2006 ~ 2008 2012 ER-290 S/O 4 [j] FITC Milli-Q Milli-Q 2 HCl ph NaOH ph HCl ph PBS HCl NaOH PBS S/O 5.0 mg 0.01 M HCl 1.0 ml 0.01 M NaOH 0.50 ml Milli-Q 3.5 ml ph 3.5 1.0 mg/ml 5.0 ml Milli-Q 1.0 ml 1.0 M HCl 40 µl 50 mg 0.01 M NaOH 0.7 ml Milli-Q 3.26 ml ph 3.5 10 mg/ml 5.0 ml 31

2 2.3. Microviscometer AWVn; Anton Parr GmbH, Graz, Austria Zetasizer Nano-ZS Malvern, Worcestershire, UK [p] 2.4. in vitro 1 80 C YMP 2 mm 2! 2 cm YMP 2 0.785 cm 2 5.0 ml PBS ph 7.4 32.5 C 500 rpm 1 YMP 3 S/O 0.50 ml 2.4.1. 4 3 YMP [k] 4 % 5 5 HistoPrep TM YMP 80 C CM1510; Leica, Wetzlar, Germany 20 µm 20 C 6 20 C PBS HistoPrep TM Fig.2.21 [k] 32

2 2.4.2. ELISA 7 3 YMP 1 mm [l] 140 [m] 1.5 ml 3 FITC 8 2 % BSA in PBS [l] 100 ~ 200 Mercodia Bovine insulin ELISA kit [n] calibrator 25 µl/well 9 ELISA kit HRP-labeled anti-insulin IgG from mouse, monoclonal conjugation buffer 11 100 µl/well 10 2 ELISA kit wash buffer 5 times/well 11 Mercodia ELISA kit TMB 200 µl/well 12 37 C 15 0.5 M H 2 SO 4 50 µl/well 13 450 nm O.D. Fig.2.22 2.4.3. HRP HRP 14 6 DAB H 2 O 2 [o] 15 15 HRP [l] 2 % BSA PBS ELISA 3 titer BSA [m] 0.04 M H 3PO 4 0.04 M NaH 2PO 4 ph 2.3 A A 95 % B 3 : 7 A:B, v:v 140 [n] Mercodia bovine insulin ELISA kit ELISA conjugation buffer 1 1 HRP 1 2 ELISA ELISA kit calibrator FITC [o] HRP DAB HRP DAB HRP 2 33

2 3. 3.1. S/O Table 2.7 FITC EGFP HRP S/O 1.0 mg/ml Fig.2.23 FITC S/O IPM Fig.2.23a S/O Fig.2.23b IPM Fig.2.23a ER-290 EGFP HRP FITC EGFP HRP S/O dynamic light scattering, DLS Fig.2.24 Fig.2.26 Table 2.7 mean Fig.2.23 FITC a IPM b S/O particle size poly dispersion index PDI distribution width S/O PDI 0.2 1 distribution width IPM ER-290 100 ~ 400 nm 1 1 S/O W/O S/O FITC Fig.2.27 3.4 nm FITC 1 S/O ER-290-FITC 257 nm 1 40 FITC ER-290 FITC S/O 1 DLS Fig.2.28 Proteins FITC-labeld insulin EGFP HRP m.w. Table 2.7 S/O n = 6 [p] ca. 6 kda ca. 27 kda ca. 40 kda S/O nanodispersion Proteins ER-290 Weight ratio 5.0 mg 250 mg 1:50 Mean particle size [nm] 257 ± 14 236 ± 38 214 ± 22 PDI 0.16 ~ 0.37 0.13 ~ 0.40 0.23 ~ 0.48 Distribution width [nm] 115 ± 31 81 ± 16 59 ± 19 [p] DLS DLS 1 3 3 6 DLS 3 1 18 Fig.2.24a 1-1 1-2 1-3 DLS 3 3 S/O PDI PDI > 0.2 Z average mean particle size distribution width DLS 68 % mean particle size ± distribution width 34

2 Fig.2.24 FITC S/O n = 6 [p] Fig.2.25 EGFP S/O n = 6 [p] Fig.2.26 HRP S/O n = 6 [p] 35

2 Fig.2.27 FITC n = 1 Fig.2.28 1 FITC S/O n = 1 2008 Hongyu Piao S/O IPM 116 FITC EGFP HRP IPM 3.2. FITC FITC S/O S/O 116 3.2.1. YMP YMP YMP Fig.2.29 Fig.2.29a Fig.2.29b 15 µm Fig.2.29b 200 µm Fig.2.29a 2 mm Fig.2.29c IPM 72 72 Fig.2.29 YMP a 10 b 40 c 72 36

2 3.2.2. FITC Fig.2.30 FITC FITC S/O FITC PBS YMP 48 Control PBS Control 0.4 3 Fig.2.30 YMP FITC S/O IPM Fig.2.30A Fig.2.30D Fig.2.30 FITC 48 [q] YMP Fig.2.31 0 Fig.2.31A Fig.2.31A Fig.2.31E FITC Fig.2.31F FITC S/O IPM 12 24 FITC Fig.2.31E 48 Fig.2.31A 0 24 FITC Fig.2.31 [q] [q] 10 200 µm 37

2 Fig.2.32 48 [q] Fig.2.33 24 [q] 38

2 Proteins Table 2.8 FITC S/O FITC-labeld insulin S/O nanodispersion Proteins ER-290 L-195 Weight ratio 125 mg 1:25 5.0 mg 250 mg 1:50 250 mg 1:50 Fig.2.34 48 [q] Fig.2.32 Fig.2.31 48 S/O 24 FITC Fig.2.33 24 FITC 4 ~ 12 12 Table 2.8 Fig.2.34 W/O FITC S/O L-195 3.2.3. FITC FITC 6,000 Sintov 140 Sintov HPLC 210 nm ELISA Table 2.9 [r] FITC Fig.2.35 YMP FITC ELISA Fig.2.35 S/O nanodispersion *p<0.05 FITC S/O 39

2 S/O 7 Lopes 2,000 P20 37 5 ~ 7 Table 2.8 Fig.2.36 3 S/O 6,000 FITC S/O IPM IPM Table 2.9 1.0 ml [r] Samples FITC-labeled insulin ER-290 IPM Milli-Q water PBS Aqueous solution (PBS) IPM solution W/O emulsion Physical mixture S/O nanodispersion 1.0 mg 1.0 mg 1.0 mg 1.0 mg 1.0 mg 50 mg 50 mg 50 mg 1.0 ml 0.9 ml 1.0 ml 1.0 ml 0.1 ml 1.0 ml Fig.2.35 FITC 48 [s] [r] IPM FITC IPM W/O 10 mg/ml FITC A ER-290 IPM B 1:9 A:B, v:v FITC ER-290 IPM 3 1 [s] Bartlett one-way ANOVA Dunnett post-hoc test 5 % *p<0.05 vs 40

2 Fig.2.36 48 n = 1 3.3. EGFP HRP S/O 6,000 ELISA Enhanced green fluorescent prtotein, EGFP 27,000 Horseradish peroxidase, HRP 40,000 Table 2.7 Fig.2.25 Fig.2.26 S/O FITC Fig.2.37 EGFP HRP 24 [t] FITC EGFP HRP EGFP HRP HRP HRP Fig.2.37A-D Fig.2.37E-H EGFP HRP 6 kda EGFP [t] A,C,E,G 10 200 µm B,D,F,H 40 15 µm 41

2 27 kda HRP 40 kda S/O Fig.2.37D Fig.2.37H EGFP HRP 15 µm FITC 3.4. S/O EGFP HRP Piao DFNa YMP PBS 116 ER-290-DFNa PBS ER-290-DFNa ER-290 DFNa ER-290-DFNa EGFP HRP S/O - Table 2.10 S/O 1.0 ml FITC-labeld insulin Rhodamine-labeld DOPE ER-290 1.0 mg 1.0 mg 50 µg 50 µg 50 mg 50 mg 50 mg Fig.2.38 S/O ER-290 FITC S/O DOPE [u] DOPE ER-290 W/O 25 mg/ml ER-290 DOPE 25 µg/ml [u] DOPE 1302 IPM ER-290 L-195 IPM ER-290 DOPE ER-290 42

2 FITC DOPE Table 2.10 S/O Fig.2.38 S/O FITC DOPE DOPE S/O Fig.2.39 FITC DOPE 48 [v] Fig.2.39 Table 2.10 3 FITC DOPE 1,302 S/O 1,055 ER-290 ER-290 S/O Fig.2.40 Fig.2.40 [v] FITC DOPE 20 100 µm 43

2 S/O IPM Sato IPM 141 Bouwstra 58 Fig.2.3a IPM S/O Fig.2.3a YMP FITC EGFP PBS 6 kda FITC 27 kda EGFP FITC transdermal 6 kda FITC Fig.2.41 EGFP FITC 48 [w] S/O IPM ER-290 FITC Fig.2.30 Fig.2.34 EGFP HRP Fig.2.37 [w] 10 200 µm 44

2 4. Yoshiro Tahara, Shota Honda, Noriho Kamiya, Hongyu Piao, Akihiko Hirata, Eiji Hayakawa, Takeru Fujii, Masahiro Goto, A solid-in-oil nanodispersion for transcutaneous protein delivery, Journal of Controlled Release 131 (2008) 14 18. FITC 6,000 EGFP 27,000 HRP 40,000 Solid-in-Oil S/O IPM ER-290 L-195 FITC EGFP HRP S/O 200 nm IPM 1 Yucatan micropig YMP S/O in vitro S/O FITC YMP FITC ELISA ELISA IPM W/O S/O EGFP YMP EGFP HRP YMP HRP HRP 4 S/O FITC S/O DOPE YMP S/O IPM ER-290 S/O IPM DDS 45

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Goto, Preparation of surfactant-coated lipase for the esterification of geraniol and acetic acid in organic solvents, Enzyme and Microbial Technology 22 (1998) 552 557. 80 N. Kamiya, M. Goto, S. Furusaki, Surfactant-histidine-heme ternary complex as a simple artificial heme enzyme in organic media, Biotechnology and Bioengineering 64 (1999) 502 506. 81 N. Kamiya, M. Inoue, M. Goto, N. Nakamura, Y. Naruta, Catalytic and structural properties of surfactant-horseradish peroxidase complex in organic media, Biotechnology Progress 16 (2000) 52 58. 82 S. Okazaki, Y. Uchimura, M. Goto, S. Furusaki, Surfactant-lactoperoxidase complex catalytically active in organic media, Biochemical Engineering Journal 6 (2000) 103 107. 83 S. Okazaki, M. Goto, S. Furusaki, H. Wariishi, H. Tanaka, Preparation and catalytic performance of surfactant-manganese peroxidase-mnii ternary complex in organic media, Enzyme and Microbial Technology 28 (2001) 329 332. 84 N. Kamiya, T. Nagamune, Effect of water activity control on the catalytic performance of surfactant-arthromyces ramosus peroxidase complex in toluene, Biochemical Engineering Journal 10 (2002) 55 59. 85 J. Michizoe, Y. Uchimura, T. Maruyama, N. Kamiya, M. Goto, Control of water content by reverse micellar solutions for peroxidase catalysis in a water-immiscible organic solvent, Journal of Bioscience and Bioengineering 95 (2003) 425 427. 86 J. Michizoe, Y. Uchimura, H. Ichinose, T. Maruyama, N. Kamiya, H. Wariishi, S. Furusaki, M. Goto, Activation of manganese peroxidase in an organic medium using a mediator, Biochemical Engineering Journal 19 (2004) 43 46. 87 S. Okazaki, M. Goto, H. Wariishi, H. Tanaka, S. Furusaki, Characterization and catalytic property of surfactant-laccase complex in organic media, Biotechnology Progress 16 (2000) 583 588. 88 J. Michizoe, H. Ichinose, N. Kamiya, T. Maruyama, M. Goto, Biodegradation of phenolic environmental pollutants by a surfactant-laccase complex in organic media, Journal of Bioscience and Bioengineering 99 (2005) 642 647. 89 S. Okazaki, M. Goto, S. Furusaki, Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents, Enzyme and Microbial Technology 26 (2000) 159 164. 48

2 90 T. Maruyama, T. Kotani, H. Yamamura, N. Kamiya, M. Goto, Poly(ethylene glycol)-lipase complexes catalytically active in fluorous solvents, Organic and Biomolecular Chemistry 2 (2004) 524 527. 91 T. Maruyama, H. Yamamura, T. Kotani, N. Kamiya, M. Goto, Poly(ethylene glycol)-lipase complexes that are highly active and enantioselective in ionic liquids, Organic and Biomolecular Chemistry 2 (2004) 1239 1244. 92 M. Goto, T. Maruyama, N. Kamiya, Poly(ethylene glycol)-lipase complex highly active in ionic liquids, AIChE Annual Meeting, Conference Proceedings (2004) 8711 8715. 93 T. Maruyama, S. Nagasawa, M. Goto, Poly(ethylene glycol)-lipase complex that is catalytically active for alcoholysis reactions in ionic liquids, Biotechnology Letters 24 (2002) 1341 1345. 94 H. Sawae, A. Sakoguchi, F. Nakashio, M. Goto, Surfactant-lipase complexes immobilized in PEG microspheres, Journal of Chemical Engineering of Japan 35 (2002) 677 680. 95 H. Sawae, A. Sakoguchi, F. Nakashio, M. Goto, Important factors affecting enzymatic functions of PEG microspheres containing lipase complexes, Journal of Chemical Engineering of Japan 38 (2005) 54 59. 96 E. Miyako, T. Maruyama, N. Kamiya, M. Goto, Highly enantioselective separation using a supported liquid membrane encapsulating surfactant-enzyme complex, Journal of the American Chemical Society 126 (2004) 8622 8623. 97 E. Miyako, T. Maruyama, N. Kamiya, M. Goto, A supported liquid membrane encapsulating a surfactant-lipase complex for the selective separation of organic acids, Chemistry-A European Journal 11 (2004) 1163 1170. 98 E. Miyako, T. Maruyama, F. Kubota, N. Kamiya, M. Goto, Optical resolution of various amino acids using a supported liquid membrane encapsulating a surfactant-protease complex, Langmuir 21 (2005) 4674 4679. 99 S. Egusa, T. Kitaoka, M. Goto, H. Wariishi, Synthesis of cellulose in vitro by using a cellulase/surfactant complex in a nonaqueous medium, Angewandte Chemie-International Edition 46 (2007) 2063 2065. 100 S. Egusa, S. Yokota, K. Tanaka, K. Esaki, Y. Okutani, Y. Ogawa, T., Kitaoka, M. Goto, H. Wariishi, Surface modification of a solid-state cellulose matrix with lactose by a surfactant-enveloped enzyme in a nonaqueous medium, Journal of Materials Chemistry 19 (2009) 1836 1842. 101 K. Esaki, S. Yokota, S. Egusa, Y. Okutani, Y. Ogawa, T. Kitaoka, M. Goto, H. Wariishi, Preparation of lactose-modified cellulose films by a nonaqueous enzymatic reaction and their biofunctional characteristics as a scaffold for cell culture, Biomacromolecules 10 (2009) 1265 1269. 102 S. Egusa, T. Kitaoka, K. Igarashi, M. Samejima, M. Goto, H. Wariishi, Preparation and enzymatic behavior of surfactant-enveloped enzymes for glycosynthesis in nonaqueous aprotic media, Journal of Molecular Catalysis B: Enzymatic 67 (2010) 225 230. 103 E. Toorisaka, H. Ono, K. Arimori, N. Kamiya, M. Goto, Hypoglycemic effect of surfactant-coated insulin solubilized in a novel solid-in-oil-in-water (S/O/W) emulsion, International Journal of Pharmaceutics 252 (2003) 271 274. 104 H. Piao, N. Kamiya, J. Watanabe, H. Yokoyama, A. Hirata, T. Fujii, I. Shimizu, S. Ito, M. Goto, Oral delivery of diclofenac sodium using a novel solid-in-oil suspension, International Journal of Pharmaceutics 313 (2006) 159 162. 105 67 2003 696 698. 106 Membrane 29 2004 98 104. 107 2004 137 148. 108 E. Toorisaka, M. Hashida, N. Kamiya, H. Ono, Y. Kokazu, M. Goto, An enteric-coated dry emulsion formulation for oral insulin delivery, Journal of Controlled Release 107 (2005) 91 96. 109! 50 2005 924 928. 110 S/O 42 2006 823 828. 111 H. Piao, A. Hirata, H. Yokoyama, T. Fujii, I. Shimizu, S. Ito, N. Kamiya, M. Goto, Reduction of gastric ulcerogenicity during multiple administration of diclofenac sodium by a novel solid-in-oil suspension, Pharmaceutical Development and Technology 12 (2007) 321 325. 112 H. Yoshiura, M. Hashida, N. Kamiya, M. Goto, Factors affecting protein release behavior from surfactant-protein complexes under physiological conditions, International Journal of Pharmaceutics 338 (2007) 174 179. 113 43 2007 351. 114 H. Yoshiura, Y. Tahara, M. Hashida, N. Kamiya, A. Hirata, T. Fujii, M. Goto, Design and in vivo evaluation of solid-in-oil suspension for oral delivery of human growth hormone, Biochemical Engineering Journal 41 (2008) 106 110. 115 E. Toorisaka, K. Watanabe, H. Ono, M. Hirata, N. Kamiya, M. Goto, Intestinal patches with an immobilized solid-in-oil formulation for oral protein delivery, Acta Biomaterialia in press (2011), doi:10.1016/j.actbio.2011.09.023. 116 H. Piao, N. Kamiya, A. Hirata, T. Fujii, M. Goto, A novel solid-in-oil nanosuspension for transdermal delivery of diclofenac sodium, Pharmaceutical Research 25 (2008) 896 901. 117 Solid-in-Oil Membrane 34 2009 227 232. 118 H. Piao, N. Kamiya, F. Cui, M. Goto, Preparation of a solid-in-oil nanosuspension containing l-ascorbic acid as a novel long-term stable topical formulation, International Journal of Pharmaceutics 420 (2011) 156 160. 119 Y. Tahara, S. Honda, N. Kamiya, H. Piao, A. Hirata, E. Hayakawa, T. Fujii, M. Goto, A solid-in-oil nanodispersion for transcutaneous protein delivery, Journal of Controlled Release 131 (2008) 14 18. 120 Y. Tahara, K. Namatsu, N. Kamiya, M. Hagimori, S. Kamiya, M. Arakawa, M. Goto, Transcutaneous immunization by a 49