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10 protocols using pheophorbide a pha

1

Synthesis of Photosensitizer-Conjugated Nanocarriers

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mPEG-OH, anhydrous CH2Cl2, docosanedioic acid 6b, KI, dexamethasone and streptomycin were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Triethyl amine and anhydrous DMSO were purchased from Acros Organics. Mesyl chloride, I2, 11-mercaptoundecanoic acid 5a and N-hydroxysuccinimide (NHS) were purchased from Aldrich. NH3 25% solution, NaSO4, MeOH, Na2SO3 and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) were purchased from Carl Roth (Carl Roth GmbH + Co. KG, Karlsruhe, Germany). NaOH and HCl were bought from Fisher Scientific (Fisher Scientific GmbH, Schwerte, Germany). 2-Morpholinoethanesulfonic acid (MES), GSH, oxidized glutathione (GSSG), TCEP and fluorescein isothiocyanate (FITC) were purchased from TCI (TCI Deutschland GmbH, Eschborn, Germany). Hydroxyethyl cellulose (HEC) gel was purchased from Ashland (Düsseldorf, Germany). Diethyl ether, acetone and acetonitrile were purchased from VWR Chemicals (Avantor, Darmstadt, Germany). Meso-tetra (m-hydroxyphenyl) porphyrin (mTHPP) was purchased from Biolitec (Jena, Germany). Pheophorbide A (PhA) was purchased from Frontier scientific (Logan, UT, USA). Rapamycin was bought from LC Laboratories (Woburn, MA, USA). Aminated hyperbranched polyglycerol (hPG-NH2, 10 kDa, 70% functionalized, 20 mg/mL in MeOH) was synthesized according to Roller et al. [18 (link)].
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2

Photosensitizer and BCRP Inhibitor Preparation

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Hoechst 33342 was obtained from Invitrogen. Pheophorbide a (PhA), chlorin e6, and purpurin 18 were obtained from Frontier Scientific. PhA, chlorin e6, and purpurin 18 were dissolved in DMSO and aliquots protected from light at −20 °C. [14C]-erlotinib was obtained from Moravek Biochemicals. Ko143 was obtained from Enzo Life Sciences. Lapatinib and tariquidar were obtained from Selleckchem. Ko143, Lapatinib, tariquidar, and other compounds were stored at −20 °C at 10 mM in DMSO.
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3

Synthesis and Characterization of Photosensitive Conjugates

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Penicillin/Streptomycin, Trypsin-EDTA, Methylene tetrazolium (MTT), dihydroethidium (DHE), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 4-dimethylaminopyridine (DMAP), N-hydroxysuccinimide (NHS), poly-l-Lysine (PLL, 25 kDa), and other fine chemicals were obtained from Sigma (Buchs, Switzerland). Antibodies including CD90, CD44, CD29, and E-64d were purchased from Abcam (Cambridge, UK). Cathepsin B polyclonal antibody (USCPAC964Mu01) was purchased from Chemie Brunschwig (Basel, Switzerland). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from vWR International (Nyon, Switzerland). Pheophorbide a (Pha) was purchased from Frontier Scientific (Logan, UT, USA). GAGRRAAG peptide was obtained from Caslo Laboratory (Lyngby, Denmark). mPEG-NHS 20 kDa was purchased from NANOCS (New York, NY, USA). O-(7-azabenzotriazole-1-yl)-N,N,N′N′-tetramethyluronium hexafluorophosphate (HATU) was obtained from GenScript Corporation (Piscataway, NJ, USA). Trifluoroacetic acid (TFA, 99%), N,N′-diisopropylethylamine (DIPEA), N,N′-dimethylformamide (DMF), and dimethylsulfoxide (DMSO) were obtained from Acros Organics (Thermo Fisher Scientific, Wohlen, Switzerland). N-succinimidyl(1-methyl-3pyridinio)formate iodide was synthesized according to the literature [26 (link)]. Pha-NHS was prepared as described previously [27 (link)].
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4

Photosensitizer-Conjugated Nanoparticles

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Yttrium(III) chloride hexahydrate (YCl3·6H2O), erbium(III) chloride hexahydrate (ErCl3·6H2O), ytterbium(III) chloride hexahydrate (YbCl3·6H2O), ammonium fluoride (NH4F), 4-(dimethylamino) pyridine (DMAP), and triphosgene oleic acid were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Sodium borohydride, N,N’-dicyclohexylarbodiimide (DCC), and N-hydroxysuccinimide (NHS) were obtained from Fluka (Buchs, Switzerland). 4-Hydroxy-2-butanone was purchased from TCI (Tokyo, Japan). α-Lipoic acid (LA), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), folic acid (FA), β-benzyl-L-aspartate (BLA), triethylamine (TEA), hydrazine monohydrate, PEG-bis(amine) (molecular weight: 3.350 kDa), sodium bicarbonate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Tetrahydrofuran (THF), n-hexane, benzene, N,N-dimethylformamide (DMF), methanol, chloroform, diethyl ether, dimethyl sulfoxide (DMSO), 1, 4-dioxane, acetic acid, and dichloromethane (DCM) were obtained from Samchun Pure Chemical Co., Ltd. (Gyeonggi-do, Korea). Pheophorbide a (Pha) was obtained from Frontier Scientific, Inc. (Logan, UT, USA).
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5

Quantification of ABC Transporter Inhibition

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Crenolanib was purchased from Selleck Chemicals, Houston, TX, and was stored at −80 °C as a 100 mM stock solution in dimethyl sulfoxide. Cell Proliferation Reagent WST-1 was purchased from Roche Diagnostics (Indianapolis, IN). The fluorescent ABCB1 substrate 3,3′-diethyloxacarbocyanine iodide [DiOC2(3)] was purchased from Sigma-Aldrich (St Louis, MO), the fluorescent ABCG2 substrate pheophorbide A (PhA) from Frontier Scientific (Logan, VT) [40 (link)], and the ABCC1 protein substrate rhodamine 123 (RH 123) from Sigma-Aldrich [40 (link)]. The ABCB1 inhibitor PSC-833 was obtained from Novartis Pharmaceutical Corporation (East Hanover, NJ), the ABCG2 inhibitor fumitremorgin C (FTC) was purchased from Sigma-Aldrich and the ABCC1 inhibitor p-[dipropylsulfamoyl] benzoic acid (probenecid) from Sigma-Aldrich [40 (link)]. [125I]-Iodoarylazidoprazosin ([125I]-IAAP) (2200 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA).
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6

Compound Preparation and Storage

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Fumitremorgin C (FTC) and Ko143 were obtained from Enzo Life Sciences. Hoechst 33342 was obtained from Life Technologies. Pheophorbide a (PhA) was obtained from Frontier Scientific Inc PhA was dissolved in DMSO and aliquots protected from light at −20°C. Other compounds were stored at −20°C at 10 mM in DMSO.
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7

Preparation of Drug Solutions for Cell Studies

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5-ALA hydrochloride from Frontier Scientific Inc. (Logan, Utah) and rhodamine 123 from Life Technologies (Grand Island, New York) were dissolved in phosphate-buffered saline (PBS). Lapatinib, gefitinib, and sunitinib malate salt (all from LC Laboratories, Woburn, Massachusetts), PD169316 (from Millipore, Burlington, Massachusetts), pheophorbide a (Pha, from Frontier Scientific Inc.), Ko143 (from Santa Cruz Biotechnology, Santa Cruz, California) were all dissolved in DMSO. All chemicals were sterilized through 0.22-μm filters and stored in a 20°C freezer.
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8

Photodynamic Therapy Reagent Preparation

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Delta-aminolevulinic acid hydrochloride (ALA) from Frontier Scientific Inc. (Logan, UT) and rhodamine 123 from Life Technologies (Grand Island, NY) were dissolved in phosphate buffered saline (PBS) solution. Pheophorbide a (Pha) from Frontier Scientific Inc. and ABCG2 inhibitor Ko143 from Santa Cruz Biotechnology (Santa Cruz, CA) were dissolved in dimethyl sulfoxide (DMSO). All chemicals were sterilized through filters and stored in a −20 °C freezer.
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9

Photosensitizer Preparation and Characterization

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ALA hydrochloride, PpIX and pheophorbide a (Pha) were obtained from Frontier Scientific Inc. (Logan, UT) and dissolved in PBS (for ALA) or DMSO (for PpIX and Pha). Lapatinib was obtained from LC Laboratories (Woburn, MA) and dissolved in DMSO. Ko143, DFO and palmitic acid were purchased from Sigma (St. Louis, MO) and dissolved in DMSO. Ethylenediaminetetraacetic acid (EDTA), zinc acetate, and Triton X-100 were from Sigma and dissolved in deionized water. Protease inhibitor cocktail (1× working solution) includes aprotinin (2 μg/mL), leupeptin (2 μg/mL), pepstatin A (1 μg/mL) and phenylmethanesulfonyl fluoride (PMSF, 1 mM), all from Sigma. All chemicals were sterilized through filtration using 0.22-μm pore size filters and stored in a −20°C freezer.
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10

Characterization of Multidrug Transporter Inhibitors

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Bosutinib (SKI-606), and C-14 radiolabeled bosutinib (C-14 bosutinib) were obtained from Pfizer Inc, New York, NY. Both drugs were dissolved in DMSO to obtain a 10-mM stock solution. Imatinib (STI-571, Gleevec) was synthesized by Enrico Rosso, PhD, University of Venice, Italy and was dissolved in water to obtain a 10-mM stock solution.
Doxorubicin (Sigma-Aldrich, Saint Louis, MO) was dissolved in DMSO to obtain a final concentration of 10 mM. Geneticin and puromycin, (Euroclone Milan, Italy) were both dissolved in water to obtain stock solutions of 400 mg/mL and 25 mg/mL, respectively.
Verapamil hydrochloride (Sigma-Aldrich, Saint Louis, MO) was dissolved in ethanol to obtain a final concentration of 100 mM and used as a selective ABCB1 inhibitor. Amantadine hydrochloride (Sigma-Aldrich, Saint Louis, MO) was dissolved in ethanol to obtain a final concentration of 1 M and used as a selective SLC22A1 inhibitor. Fumitremorgin C (FTC) (Sigma-Aldrich, Saint Louis, MO) was dissolved in DMSO to obtain a final concentration of 1 mM and used as a selective ABCG2 inhibitor.
Rhodamine 123 (Rho-123) (Sigma-Aldrich, Saint Louis, MO), 4-Di-2-ASP (4-(4-Diethylaminostyryl)-1-methylpyridinium iodide, ASP) (Invitrogen, Carlsbad, CA), and Pheophorbide A (PhA) (Frontier Scientific, Logan, UT) were dissolved in DMSO to obtain a final concentration of 2 mM, 10 mM, and 1 mM, respectively.
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