Multi-functional nanocarriers for drug delivery The combination of photodynamic therapy and chemotherapy with anti-angiogenesis in the treatment of cancers)

近年來奈米載體作為藥物傳輸的研究與臨床應用極為熱門且具前瞻性。而合併不同療法，如光動力治療、化學療法、與抗血管新生療法等，也成為腫瘤治療上的新趨勢。為藉由奈米藥物載體整合不同療法，故本論文的架構先由小分子光感藥物有機合成推向高分子奈米載體的製備；由單一治療推向多療法的合併；由細胞實驗推向動物實驗的評估多功能奈米載體在藥物傳輸上與合併抗腫瘤治療上的應用。 本論文分為三的部分，第一部分是光感藥物的合成與鍵結在光動力治療上的評估。此部分有兩個研究主題，一是非對稱卟啉類光感藥物的合成與抗癌評估。二是鍵結光感藥物與樹枝狀高分子應用於光動力治療與基因轉植的載體的評估。本研究合成一系列非對稱卟啉類光感藥物，並針對其在腫瘤細胞所表現的特殊胞器標的特性與光動力效果家以評估。研究中所得知的卟啉結構與生物交互作用的特性關係，可作為未來光感藥物設計的借鏡。進一步利用所合成出的卟啉與樹枝狀高分子鍵結，提升其在藥物傳輸與基因治療的應用性。 第二部分是製備雙效的奈米微胞作為合併光動力療法與化學療法的藥物載體。此部分有兩個研究主題。首先是合成以光感藥物為中心的放射狀雙性高分子，其可在水相環境中自組形成奈米微胞，包覆輸水性化療藥物。以紫杉醇為標的藥物作為乳癌合併療法的評估。進一步包覆喜樹鹼類藥物SN-38於大腸直腸癌症動物模式評估本載體合併治療的效果。結果顯示，有六成的老鼠腫瘤經過重覆的合併治療後完全消失。而治療對於腫瘤細胞的複製與血管密度均有抑制的效果。 第三部分是評估合併抗血管新生對於光動力治療與奈米藥物在腫瘤治療上的影響。在合併光動力治療部分，發現抗血管新生藥物-癌思停(抗血管內皮生長因子抗體, Bevacizumab)可增加光動力治療的的效果，尤其是以先光動力後再給與抗血管內皮生長因子抗體的效果較具有統計上的加成效果。所給予的抗體可有效降低光動力治療後所產生的血管內皮生長因子含量，抑制血管再生、降低腫瘤復發。抗血管內皮生長因子抗體對於奈米藥物在腫瘤上的累積量與治療效果的評估。以喜樹鹼微胞為測試奈米藥物，以大腸直腸癌的動物模型進行測試。初步結果顯示，抗血管內皮生長因子抗體對於較大尺寸的喜樹鹼微胞的腫瘤累積量與抗癌效果並不影響，對於一百奈米左右的顆粒較具有統計意義的累積量抑制。In recent years, the nanomaterials, such as polymer-drug conjugates, nanoparticles, and polymeric micelles, have been considered as potential carriers for hydrophobic drug delivery that may resolve the mentioned problems. The combination of photodynamic therapy, chemotherapy, or anti-angiogenesis had been a development tendencies for the cancer treatments.his dissertation was divided into three parts. The first part is synthesis of photosensitizers and their conjugates for photodynamic therapy. There are two topics of research in this part. The first topic is to synthesis and evaluate the asymmetric porphyrins on intracellular uptake, subcellular localization and phototoxicity in cancer cells. The second topic is synthesis and evaluate of PAMAM- porphyrin conjugates for photodynamic therapy and gene transfection. In first study, we prepared a series of asymmetric porphyrins with varying proportion of substituents, such as 4-hydroxyphenyl, 4-aminophenyl, and 4-pyridine, with a varied degree of hydrophobic/hydrophilic substitution as model compounds for localization studies of photosensitizers and its photodynamic activity in tumor cells. In addition, we conjugated photosensitizers (TAMCPP, P35) with PAMAM dendriers to increase the hydrophilicity for clinical applicaion. And G4-TAMCPP became less aggregation, more photocytotoxitic efficacy, and lysosomal targeting that could be applied on delivery system for the photochemical internalization.he second part is to prepare the dual functionalized micellar delivery system for the combination of photodynamic therapy and chemotherapy. This part has two topics of research, the first is to synthesis and evaluate the self-assembled chlorin-cored poly(ε-caprolactone)-poly(ethylene glycol) diblock copolymer (CSBC) micelles for paclitaxel-based chemotherapy combinated with photodynamic therapy in MCF-7 breast cancer cells. The second topics is to use CSBC micelles for SN-38 delivery and evaluate its combination efficacy effects in a HT-29 human colon cancer xenograft model. Combined SN-38/CSBC-mediated PDT synergistically inhibited tumor growth, resulting in up to 60% complete regression of well-established tumors after 3 treatments. These treatments also decreased the microvessel density (MVD) and cell proliferation within the subcutaneous tumors. he third part is Anti-angiogenesis therapy combinated with photodynamic therapy or nanomedicines. The first topic is anti-angiogenic treatment (Bevacizumab) enhances the responsiveness of photodynamic therapy in a HT-29 human colon cancer xenograft mode. Our results demonstrated that combination therapy protocol of PDT then bevacizumab exhibited greater tumor response in comparison with other treatment protocols. The last topic is to investigate the relationship between angiogenesis and nanomedicines on the therapeutic efficacy in colon cancer. he vascular-targeting PDT induce the loss of vascular barrier function and increase in vascular permeability. In this study, we demonstrated that alteration in tumor vascular barrier by vascular-targeting PDT, which enhances the delivery of macromolecules. It also demonstrated whether pretreatment with bevacizumab (anti-angiogenesis) or vascular-targeting PDT (pro-angiogenesis) influence the efficacy of systemically administered nanomedicines in mice bearing human colon HT-29 adenocarcinoma, and to investigate the optimal conditions (time window, particle size) of anti-angiogenesis and pro-angiogenesis and their effects on the delivery and efficacy of nanomedicines. Bevacizumab had been shown to decrease microvessel density (MVD) and vascular perfusion in solid tumours. However, the effects of bevacizumab on the delivery of micellar SN-38 formulations therapeutic efficacy are not effected, because of the larger size of SN-38/micelles.CONTENS文摘要..................................................1BSTRACT..................................................2HAPTER 1ENERAL INTRODUCTION......................................4eferences................................................6HAPTER 2YNTHESIS OF PHOTOSENSITIZERSOPIC1. INFLUENCE OF SUBSTITUTIONS IN ASYMMETRIC ORPHYRINS ON INTRACELLULAR UPTAKE, UBCELLULAR LOCALIZATION AND PHOTOTOXICITY IN HELA CELLSBSTRACT .................................................8. INTRODUCTION.....................................9. MATERIALS AND METHODS...........................11SYNTHESIS OF MESO-SUBSTITUTED PORPHYRINS 1-6....11PARTITION COEFFICIENTS..........................12SINGLET OXYGEN QUANTUM YIELDS...................13CELL CULTURE AND INCUBATION CONDITIONS..........13CELLULAR UPTAKE.................................14CYTOTOXICITY AND PHOTOTOXICITY OF PORPHYRINS....14INTRACELLULAR LOCALIZATION OF PORPHYRINS........15. RESULTS AND DISCUSSION..........................16CHEMISTRY.......................................16SINGLET OXYGEN QUANTUM YIELDS (Φ△) ...........16PARTITION COEFFICIENT...........................16ABSORBANCE AND EMISSION SPECTRA.................16CELL CULTURE STUDIES............................16CELLULAR UPTAKE OF PORPHYRINS...............................................16CYTOTOXICITY AND PHOTOTOXICITY OF PORPHYRINS....17INTRACELLULAR LOCALIZATION OF PORPHYRINS........17. Discussion......................................19. References......................................31ABLESABLE 1. PHOTOPHYSICAL PROPERTIES OF MESO-SUBSTITUTED PORPHYRINS...............................................23ABLE 2. LIGHT DOSE (J/cm2) FOR LD50.....................24IGURESIG. 1 (A) UV–VIS ABSORBANCE SPECTRA OF PORPHYRIN DERIVATIVES (1-6, 1ΜM) IN THF; (B) EMISSION SPECTRA OF CATIONIC PORPHYRIN DERIVATIVES (1-6, 1μm) IN THF........25IG. 2 DOSE-DEPENDENCE OF DRUG UPTAKE BY HELA CELLS INCUBATED IN THE MEDIUM WITH DIFFERENT PORPHYRIN DERIVATIVES FOR 24H. CELLULAR UPTAKE WAS DETERMINED USING A 96 WELLS FLUORESCENCE READER.............................26IG. 3 COMPARISON OF HELA CELL PHOTOTOXICITY ( LD50 IN J /CM2) AND CELLULAR UPTAKE (IN RFI / 3X103 CELLS) OF A SERIES OF PORPHYRIN DERIVATIVES (2μM) AS A FUNCTION OF THEIR PARTITION COEFFICIENT (LOG P), AFTER 24 H INCUBATION...............................................27IG. 4 DRUG DOSE-RESPONSE CURVES OBTAINED IN HELA CELLSOLLOWING EXPOSURE TO DIFFERENT PORPHYRIN DERIVATIVES FOR 24H (A); AND IRRADIATION WITH BLUE LIGHT (ΛMAX = 435nm) FOR 40S ( 0.28 J/ cm2)(B). THE COLORIMETRIC MTT TEST WAS USED FOR CELL DEATH ESTIMATION...........................28IG. 5 (A) CELLULAR UPTAKE OF PORPHYRIN DERIVATIVES AT LOWER DRUG DOSE (2μM). (B) LIGHT DOSE-RESPONSE CURVES OBTAINED IN HELA CELLS FOLLOWING EXPOSURE TO THE DIFFERENT PORPHYRIN DERIVATIVES AT LOWER DRUG DOSE (2μM) FOR 24H AND IRRADIATION WITH BLUE LIGHT (ΛMAX = 435Nnm) FOR 0~200S ( 0~1.4 J/ cm2). THE COLORIMETRIC MTT TEST WAS USED FOR CELL DEATH ESTIMATION. LIGHT SOURCE :LUMISOURCE® (PEAK WAVELENGTH 435 nm, 13.5mW/ cm2)..........................29IG. 6. COMPARATIVE INTRACELLULAR LOCALIZATION OF PORPHYRINS WITH MITOTRACKER GREEN AND LYSOTRACKER GREEN: CONFOCAL LASER SCANNING MICROSCOPY STUDIES (CLSM)........30OPIC2. SYNTHESIS AND EVALUATE OF PAMAM-PORPHYRIN CONJUGATES FOR PHOTODYNAMIC THERAPY AND GENE TRANSFECTIONBSTRACT ................................................35. INTRODUCTION....................................36. MATERIALS AND METHODS...........................39.1 SYTHESIS OF MESO-SUBSTITUTED PORPHYRINS.............39.2 THIN LAYER CHROMATOGRAPHY ANALYSIS..................40.3 PARTITION COEFFICIENTS..............................41.4 DETERMINATION OF PARTICLE MORPHOLOGY AND SIZ........41.5 CELL CULTURE AND INCUBATION CONDITIONS..............41.6 INTRACELLULAR LOCALIZATION BY CONFOCAL MICROSCOPY....42.7 LYSOSOME MEMBRANE STABILITY ASSAY...................42.8 CELLULAR UPTAKE.....................................43.9 CYTOTOXICITY AND PHOTOTOXICITY EXPERIMENTS..........43.10 DNA RETARDATION TEST - AGAROSE GEL ELECTROPHORESIS..44.11 TRANSFECTION EFFICACY – FLOW CYTOMETRY AND FLUORESCENCE MICROSCOPY..................................44. RESULTS AND DISCUSSION..........................46.1 ABSORPTION SPECTRUM..................................46.2 THIN LAYER CHROMATOGRAPHY ANALYSIS...................46.3 PARTITION COEFFICIENT................................46.4PARTICLE MORPHOLOGY AND SIZE OF PAMAM-PORPHYRIN CONJUGATES...............................................46.5 INTRACELLULAR LOCALIZATION....................... ...47.6 LYSOSOME MEMBRANE STABILITY ASSAY………………………..47.7 Cellular Uptake……………………………………………..........................47.8 Cytotoxicity and Phototoxicity………………………………………..........48.9.SINGLET OXYGEN GENERATION OF G4-TAMPP PLUS IRRADIATION. …………………………………………..…………………48 .10 Evaluation of Complex Formation with Plasmid DNA……….....................49.11 Transfection Efficacy of G4-TAMCPP Conjugates.......................................50. Conclusion…………………………………………………………..50. References…………………………………………………………..66ABLESABLE 1 PARTITION COEFFICIENTS...................................................................55ABLE 2 SUMMERY OF THE RESULTS OF FIG. 20. EFFP PLASMID TRANSFECTION EFFICACY…...............................................................65IGURESIG. 1 ABSORPTION SPECTRUM………………………………………………..52IG. 2 THIN LAYER CHROMATOGRAPHY ANALYSIS………………………..54IG. 3 PARTITION COEFFICIENTS MEASUREMENT OF TAMCPP AND G4-TAMCPP………………………………………………………………...55IG. 4 MORPHOLOGY AND SIZE DISTRIBUTION OF G4-TAMCPP CONJUGATES………………………………………………………………56IG. 5 INTRACELLULAR LOCALIZATION……………………………………..57IG. 6 ESTIMATION OF LYSOSOME DISRUPTION CAPABILITY FOR G4-TAMCPP CONJUGATES……………………………………………….58IG. 7 TIME-DEPENDENCE OF TAMCPP AND G4-TAMCPP UPTAKE BY HELA CELLS………………………………………………………………………..59IG. 8 CYTOTOXICITY AND PHOTOCYTOTOXITY OF TAMCPP ND G4-TAMCPP CONJUGATES………………………………………………60IG. 9.SINGLET OXYGEN GENERATION OF TAMCPP (P35) AND G4-TAMCPP (GP) IN DIFFERENT PH 7.5 TRIS BUFFER BY SINGLET OXYGEN SENSOR GREEN REAGENT. ALL SAMPLES WERE EXPOSED TO LED IRRADIATION, RESULTING IN GENERATION OF 1O2…………..…....61IG. 10 EVALUATION OF COMPLEX FORMATION WITH PLASMID DNA…62IG. 11 LIGHT-INDUCED DNA TRANSFECTION EFFICACY…………………63HAPTER 3UAL FUNCTIONALIZED MICELLAR DELIVERY SYSTEMOPIC 1: SELF-ASSEMBLED CHLORIN-CORED POLY(ε-CAPROLACTONE) -POLY(ETHYLENE GLYCOL) DIBLOCK COPOLYMER MICELLES FOR DUAL CHEMO-PHOTODYNAMIC THERAPIES BSTRACT .……………………………………...………………...…...69. INTRODUCTION………………………………………………….70. MATERIALS AND METHODS……………………………..71.1 MATERIALS…………………………………….………………..……..71.2 SYNTHESIS OF 5, 10, 15, 20-TETRAKIS (4-AMINOPHENYL)-21H, 23H-CHLORIN……………………………..………..…………723 SYNTHESIS OF ACTIVATED AMPHIPHILIC BLOCK COPOLYMER………………………………………………….…………72.4 SYNTHESIS OF CHLORIN-CORE STAR BLOCK COPOLYMER (CSBC)……………………………………………………………….….….73.5 PREPARATION OF CSBC MICELLES WITHOUT OR WITH PACLITAXEL LOADING……...........…………………...74.6 SINGLET OXYGEN PRODUCTION OF CSBC-M-MEDIATED PDT……………………………………………………………….…74.7 RELEASE PROFILES OF PACLITAXEL FROM PCSBC-M……….....…75.8 TREATMENT OF MICELLES WITHOUT OR WITH PACLITAXEL BY PDT...............................................................................................76.9 CYTOTOXICITY ASSAY S…………………………………….77.10 IMMUNOFLUORESCENCE……………………………………………..77.11 MEDIAN EFFECT ANALYSIS …………………………………………77. RESULTS AND DISCUSSION……………………………………78.1 SYNTHESIS AND CHARACTERIZATION OF CSBC….................……...78.2 MICELLAR PROPERTIES OF CSBC-M AND PCSBC-M...........79.3 SINGLET OXYGEN GENERATION OF CSBC PLUS IRRADIATION......81.4 IN VITRO PACLITAXEL-RELEASE STUDY …………....................……..82.5 EFFECTS OF PACLITAXEL ON MICROTUBULES IN MCF-7 CELLS………………………………………………………….....82.6 CYTOTOXICITY AND PHOTOTOXICITY OF PACLITAXEL-LOADED CSBC-MICELLES……………………………….…………………..…..83. CONCLUSION………………………………………………….….84. REFERENCES……………………………………………………..95ABLESABLE 1 CHARACTERISTICS OF MPEG-PCL COPOLYMERS..........................87ABLE 2 CHARACTERISTICS OF THE CHLORIN-CORE COPOLYMERS……87ABLE 3 COMBINATION INDEXES OF PCSBC-58M WITH IRRADIATION IN MCF-7 CELLS…………………………………………………………...88IGURESIG. 1 H NMR SPECTRA OF (A) MPEG-PCL(M58) COPOLYMER AND (B) M58C COPOLYMER IN CDCL3………………………………………..89IG. 2 PLOT OF THE INTENSITY RATIO (I1/I3) VERSUS CONCENTRATION OF THE CSBC COPOLYMERS: (A) CSBC-52 AND (B) CSBC-58………………………………………………………………..89IG. 3 FIG. 3. MORPHOLOGY OF THE PREPARED PCSBC-58M BY (A) AFM AND (B) TEM……………………………………….….…………...90IG. 4 SINGLET OXYGEN GENERATION OF CHLORIN AND CSBC IN (A) MEOH BY 1,3-DIPHENYLISOBENZOFURAN AND (B) PH 7.5 TRIS BUFFER BY SINGLET OXYGEN SENSOR GREEN REAGENT. ALL SAMPLES WERE EXPOSED TO LED IRRADIATION (660+/-10nm, 19.5 mW), RESULTING IN GENERATION Of 1O2…………………..…………90IG. 5 THE IN VITRO RELEASE PROFILE OF PCSBC-58M AT PH 5.0 AND PH7.4……………………………………………………………………….91IG. 6 INDIRECT IMMUNOFLUORESCENCE OF MICROTUBULES IN MCF-7 CELLS. CELLS WERE TREATED WITH DISTINCT PACLITAXEL FORMULATIONS FOR 24 h. (A) Control; (B) paclitaxel; (C) CSBC-58M; and (D) PCSBC-58M…………………………………………..…….92IG.7 THE CYTOTOXICITY OF CSBC-58M UNDER DIFFERENT IRRADIATION (A) AND PCSBC-58M IN MCF-7 CELLS WITH 7 J/cm2 IRRADIATION (B)………………………………………………………….93OPIC 2: DUAL CHEMOTHERAPY AND PHOTODYNAMIC THERAPY IN A HT-29 HUMAN COLON CANCER XENOGRAFT MODEL USING SN-38-LOADED CHLORIN-CORE STAR BLOCK COPOLYMER MICELLESBSTRACT .……………………………………...………………...…...99. INTRODUCTION………………………………………………100. MATERIALS AND METHODS……………………………..71.1 MATERIALS………………………………...…….………………..…101.2 PREPARATION OF SN-38-LOADED CSBC MICELLES…….………102.3. CHARACTERIZATION OF SN-38 LOADED CSBC MICELLES…103.4 RELEASE PROFILES OF SN-38 FROM THE CSBC MICELLE...…103.5 IN VITRO CYTOTOXICITY…………………………………………...104.6. ANTI-TUMOR EFFICACY OF THE SN-38-LOADED CSBC MICELLES…………………………………………………………..105.7. BIODISTRIBUTION OF THE SN-38-LOADED CSBC MICELLES…..106.8. PHARMACOKINETIC AND STATISTICAL ANALYSES………….....106.9 NECROPSY AND IMMUNOHISTOCHEMICAL ANALYSI……………107.10 STATISTICAL ANALYSIS…………………….………………………..108. RESULTS AND DISCUSSION……………………………..…108.1. SYNTHESIS AND CHARACTERIZATION OF CSBCS…………….......108.2 CSBC AND SN-38-LOADED CBSC MICELLAR PROPERTIES……......108.3. IN VITRO SN-38 RELEASE STUDY ……………….…………………..109.4CYTOTOXICITY AND PHOTOTOXICITY OF SN-38/CSBC MICELLES...................................................................................................110.5. PHARMACOKINETICS OF CPT-11 AND SN-38/CSBC MICELLES….111.6 THE BIODISTRIBUTION OF SN-38/CBSC MICELLES IN HT-29 BEARING MICE…………………………………………………………..113.7.IN VIVO ANTITUMOR EFFECTS OF SN-38/CBSC MICELLES WITHOUT OR WITH IRRADIATION IN HT-29 TUMOR-BEARING MICE……………………………………………………………………….114.8. THE EFFECT OF SN-38/CSBC MICELLES-MEDIATED PDT/CHEMOTHERAPY ON CELLULAR PROLIFERATION AND MICROVESSEL DENSITY…………………………………….………….117. CONCLUSION…………………………………………….….119. REFERENCES……………………………………………………..95ABLESABLE 1 CHARACTERISTICS OF SN-38/CSBC MICELLES ……………....…120ABLE 2. PHARMACOKINETIC ANALYSIS OF SN-38 PLASMA AND TUMOR CONCENTRATIONS AFTER INTRAVENOUS ADMINISTRATION OF SN-38/CSBC MICELLES (10 mg/kg) OR CPT-11 (10 mg/kg) TO NUDE MICE BEARING HT-29 TUMORS…………………..……….120ABLE 3. EFFECT OF CPT-11, SN-38/CSBC MICELLES, AND COMBINATION THERAPIES ON TUMOR GROWTH IN A HT-29 HUMAN COLON CANCER XENOGRAFT MODEL…………………………………....121IGURESIG. 1 CHEMICAL STRUCTURE OF CHLORIN-CORE STAR BLOCK COPOLYMERS (CSBC) AND SCHEMATIC DRAWING OF SELF-ASSEMBLED SN-38/CSBC MICELLES……………………….…122IG. 2.SIZE AND SHAPE OF SN-38-LOADED CSBC MICELLES. THE MORPHOLOGY OF THE PREPARED CSBC MICELLES (A) AND SN-38/CSBC-58 MICELLES (B) WERE DETERMINED BY AFM. (C) SIZE DISTRIBUTION OF SN-38/CSBC-58 MICELLES WITH VARIOUS D/P RATIOS WAS ANALYZED BY DLS………………………..…………..123IG. 4. THE EFFECT OF SN-38 CONCENTRATION, PDT, MICELLAR DELIVERY ON CELLULAR CYTOTOXICITY. THE CYTOTOXICITY OF SN-38, SN-38-LOADED MICELLES, AND CPT-11 IN HT-29 CELLS WITHOUT OR WITH 7 J/CM2 IRRADIATION WAS DETERMINED BY THE MTT ASSAY. ………………………………………………………..124IG. 5. PHARMACOKINETIC ANALYSIS OF SN-38 AFTER INTRAVENOUS ADMINISTRATION OF FREE CPT-11 OR SN-38/CBSC MICELLES. (A) PLASMA SN-38 CONCENTRATION–TIME PROFILES AFTER INTRAVENOUS ADMINISTRATION OF CPT-11 OR SN-38/CBSC MICELLES IN MICE BEARING SUBCUTANEOUS HT-29 TUMORS. (B) TUMOR CONCENTRATION OF SN-38 AFTER INTRAVENOUS ADMINISTRATION OF SN-38/CBSC MICELLES OR CPT-11 (10 MG/KG)…………………………………………………………………….125IG. 6. BIODISTRIBUTION OF SN-38 IN MICE BEARING HT-29 SOLID TUMORS. AFTER INTRAVENOUS ADMINISTRATION OF SN-38/CSBC MICELLES AT A DOSE OF 10 MG/KG. EACH COLUMN REPRESENTS THE MEAN ±S.D……………………………………………….…………126IG. 7. THE ANTITUMOR EFFECTS OF CPT-11 VERSUS SN-38/CBSC MICELLES. (A) ANTITUMOR EFFICACY OF FREE CPT-11 OR SN-38/CBSC MICELLES WERE DETERMINED IN AN HT-29 HUMAN COLON CANCER XENOGRAFT MODEL. DRUG EFFICACY WAS ASSESSED BY MEASURING TUMOR VOLUME AS A FUNCTION OF TIME. (B) CHANGES IN THE RELATIVE BODY WEIGHT (%) AS A MEASURE OF TOXICITY WERE ALSO DETERMINED. (C AND D) AFTER THE COMBINATION THERAPY (SN-38/CSBC MICELLES WITH PDT) OF 1 OR 3 DOSES, CHANGES IN TUMOR VOLUME (C) AND RELATIVE BODY WEIGHT (D) WERE DETERMINED. POINTS, MEAN; BARS, SD.*, P<0.05 AND **, P < 0.01, AS COMPARED WITH CPT-11 OR CONTROL……………………………………………………127IG. 8. THE EFFECTS OF COMBINATION THERAPY ON PDT-MEDIATED PHOTOXICITY IN NUDE MICE BEARING HT-29 HUMAN COLON CANCER XENOGRAFTS. (A)THE MICE WERE TREATED WITH EACH FORMULATION WITH OR WITHOUT PDT, AND THE TUMOR SIZE WAS MEASURED DURING THE 30-DAY EVALUATION PERIOD. (B) MACROSCOPIC EVALUATION OF SKIN PHOTOTOXICITY IN MICE TREATED WITH FREE M-THPC (META-TETRA(HYDROXYPHENYL) CHLORIN, 0.3MG/KG), CSBC MICELLES (EQUIVALENT CHLORIN UNIT 2.03MG/KG), AND SN-38/CSBC MICELLES (EQUIVALENT CHLORIN UNIT 2.03MG/KG) AT 4 DAYS AFTER LIGHT IRRADIATION IN DORSAL SKIN USING A DIODE LASER (FLUENCE: 30 J/CM2). ARROWS, IRRADIATION SITE IN DORSAL SKIN OR TUMOR…………………………………………………………………….128IG. 9. IMMUNOHISTOCHEMICAL ANALYSIS IN HT-29 XENOGRAFTS IN TUMORS TREATED WITH CPT-11 OR MICELLULAR SN-38. (A) TUMORS WERE ANALYZED BY H&E STAINING. (B) CELLULAR PROLIFERATION WAS QUANTIFIED BY ASSESSING THE NUMBER OF PCNA-POSITIVE CELLS (MAGNIFICATION ×200). (C) QUANTIFICATION OF MICROVESSEL DENSITY (MVD) WAS DETERMINED BY CD31 EXPRESSION; VESSELS IN 10-20 DISTINCT REGIONS WERE COUNTED AT 100X MAGNIFICATION. RESULT REPRESENT THE MEAN ± S.D. IN 10-20 DISTINCT REGIONS FROM 5 TUMORS EXAMINED PER GROUP. *, P<0.05 AND **, P < 0.01…129HAPTER 4NTIANGIOGENESIS IN EXPERIMENTAL ONCOLOGYOPIC 1: ANTI-ANGIOGENIC TREATMENT (BEVACIZUMAB) ENHANCES THE RESPONSIVENESS OF PHOTODYNAMIC THERAPY IN A HT-29 HUMAN COLON CANCER XENOGRAFT MODEBSTRACT .……………………………………...………………...….134. INTRODUCTION………………………………………………105. MATERIALS AND METHODS…………………………….137.1 CHEMICALS………………………………......…….…………..……..…137.2 ANIMALS AND TUMOR MODEL…………………………………..…137.3 IN VIVO TREATMENT PROTOCOLS………………………………….137.4 HISTOLOGIC AND IMMUNOHISTOCHEMICAL STUDIES……...…138.5 MEASUREMENT OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) LEVELS IN TUMOR…………………………….…...138.6. CONCENTRATION OF MTHPC IN BEVACIZUMAB-TREATED TUMOR……………………………………………………………...…….139.7. LOCALIZATION OF MTHPC IN TUMOR……………………..………..139. RESULTS AND DISCUSSION……………………………..…141.1. EFFECTS OF BEVACIZUMAB ON ANTI-TUMOR EFFICACY OF PDT………………………………………………………………………...141.2 THE EFFECT OF PDT COMBINED WITH BEVACIZUMAB ON LEVEL OF VEGF AND MICROVESSEL DENSITY (MVD)………...…………..142.3. EXPRESSION OF PROANGIOGENIC MOLECULES AND CYTOKINES IN TUMORS ……………………………………………………………....143.4 THE EFFECT OF BEVACIZUMAB ON LOCALIZATION AND ACCUMULATION OF MTHPC IN TUMOR..............................................143. CONCLUSION…………………………………………….….144. REFERENCES……………………………….………………….152ABLESABLE 1. EFFECT OF PDT, BEVACIZUMAB, AND COMBINED THERAPIES ON TUMOR GROWTH IN THE HT-29 HUMAN COLON CANCER XENOGRAFT MODEL……………………..………………………..146ABLE 2. P VALUE OF THE DIFFERENT GROUPS IN THE KAPLAN-MELER CURVES…………………………………………………………….147IGURESIG.1. TIME LINE SKETCH ILLUSTRATING DIFFERENT TREATMENT PROTOCOLS…………………………………………………..……….145IG. 2. TUMOR VOLUME OF HT-29 TUMORS AFTER TREATMENTS. FOR PDT, MTHPC (0.3 MG/KG) WAS ADMINISTERED 24 HOURS BEFORE LASER ILLUMINATION (652 NM, FLUENCE RATE: 100 MW/CM2，FLUENCE: 10 J/CM2). FOR ANTIANGIOGENIC TREATMENT, BEVACIZUMAB (BEV) WAS ADMINISTERED I.P. EVERY OTHER DAY FOR TOTAL SIX DOSES AFTER OR BEFORE PDT LIGHT TREATMENT. THE TREATMENT PROTOCOLS AS SHOWN AS FIG. 1. EACH GROUP REPRESENTS THE MEAN (BARS, SE) OF 10 ANIMALS..........................................................................................……146IG. 3. KAPLAN-MEIER CURVES SHOWING SURVIVAL OF MICE TREATED WITH EACH TREATMENT. TUMOR GROWTH WAS MONITORED FOR 36 DAYS OR UNTIL TUMORS REACHED A VOLUME OF 400 MM3……………………………………………………………………….147IG. 4. IMMUNOHISTOCHEMICAL STAINING OF VEGF AND CD-31 IN HT-29 TUMORS. THE HT-29 BEARING NUDE MICE WERE TREATED SALINE(CONTROL), PDT ALONE, BEVACIZUMAB ALONE; PDT THEN BEVACIZUMAB;