2 iminothiolane hydrochloride

Cetuximab was thiolated using Traut’s reagent (2-Iminothiolane hydrochloride, Sigma-Aldrich) for 1 h at room temperature in HEPES buffer (25 mM HEPES, 140 mM NaCl, 2 mM EDTA, pH 8.0). Unreacted Traut’s reagent was removed by passing through the PD-10 column (Merck KGaA) with HEPES buffer (25 mM HEPES, 140 mM NaCl, pH 7.4). The thiolated antibodies were conjugated to Mal-PEG2000-DSPE/mPEG2000-DSPE (1:4 molar ratio) micelles at the molar ratio of 0.2:1 (Antibody:Mal-PEG2000-DSPE) by incubation for 16 h at 4 °C with continuous stirring. After the incubation of the antibody with the micelles, the conjugate of cetuximab and lipid was purified using chromatography on sepharose CL-4B columns in HEPES buffer (25 mM HEPES, 140 mM NaCl, pH 7.4). The antibody conjugation yield and purity were measured by Lowry protein assay using a bovine gamma globulin (BGG) standard set (Bio-Rad, Hercules, CA, USA).

DH was provided from Jai Radhe Sales (Gujarat, India). Chitosan (103.2 g/mol, degree of deacetylation 76.6%), 2-imino thiolane hydrochloride, 2-mercaptoethanol, triethanolamin reagent grade, formic acid (ultra-performance liquid chromatography MS/MS (UPLC MS/MS) grade), clopidogrel (as internal standard (IS)), ammonium formate, and acetonitrile were purchased from Sigma Aldrich Company (St. Luis, MO, USA). Cholesterol (CHOL) and dipalmitoylphosphocholine (DPPC) were purchased from Fisher Scientific Company (Fair Lawn, NJ, USA) and Avanti Polar Lipid Inc., (Birmingham, AL, USA), respectively. Polyvinyl pyrolidone (PVP), known as Luviskol® K-9, was purchased from BASF (Aktien gesellschaft Ludwig Shafen, Germany). Polyethylene glycol 600 (PEG600) was obtained from Merck Schuchardt (Hohenbrunn, Germany). Lactic acid glutaraldehyde 50% solution and chloroform were purchased from BDHL laboratory Supplies (PooleBH151TD, England). Mono basic potassium dihydrogen phosphate and cellophane membrane (Spectra/Por® Dialysis Tubing from regenerated cellulose) with cut-off 12–14000 Da were purchased from panreac Quimica, SA, (Barcelona, Espana) and Spectrum Laboratories (Houston, TX), respectively. All chemicals used were of analytical grade and were used as received.

Soybean phosphatidylcholine (S100-PC) was purchased from Lipoid GmbH (Ludwigshafen, Germany). DC-chol, 2-Iminothiolane hydrochloride (2-IT, Traut’s reagent), CTX and sulforhodamine B (SRB) were provided by Sigma-Aldrich (Saint Louis, MO, USA). Mal-PEG2000-DSPE was obtained from Avanti Polar Lipids (Alabaster, AL, USA). Anti-Transferrin Receptor antibody (OX-26) and anti-Telomerase reverse transcriptase antibody were purchased from Abcam (Cambridge, UK). Sephadex G-50 and Sepharose CL-4B were provided by Science and Technology Development Co., Ltd. (Beijing, China). Centriprep-10 concentrators were purchased from Millipore Amicon (Bedford, MA, USA). NanoOrange® Protein Quantitation Kit was purchased from Invitrogen (California, USA). YOYO-1 and LysoTracker Red were purchased from Molecular Probes (Eugene, OR, USA). pIRES2-EGFP (pEGFP) and pIRES2-EGFP/hTERTC27 (pC27) were purified using a E.Z.N.A.TM Fastfilter Plasmid Maxi Kit (Omega, Norcross, GA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Tokyo, Japan). Lipofectamine™ 2000 Reagent (Lipo2000) was purchased from Invitrogen (California, USA). pGL3 Luciferase Reporter Vectors (pGL3-luc) and Luciferase Reporter Assay Kit were purchased from Promega (Madison, WI, USA).

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1’,3’-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol (18:1 cardiolipin), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)−2000] (DSPE-PEG₂₀₀₀), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)−2000] (DSPE-PEG₂₀₀₀-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5.5) (18:0 Cy5.5 PE), and L-α-Phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhod-PE) were all purchased from Avanti Polar Lipids. Cholesterol and 2-iminothiolane hydrochloride were purchased from Sigma Aldrich. diABZI STING agonist (Compound 3) was purchased from Selleckchem. Temozolomide was purchased from Cayman Chemical. InVivoMAb antibodies including anti-mouse PD-L1 (B7-H1, clone 10F.9G2), anti-mouse CD47 (clone MIAP301), anti-human PD-L1 (B7-H1, clone 29E.2A3), and anti-human CD47 (clone B6.H12) were all purchased from BioXCell.

Dipalmitoylphosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycerol-3 phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (mPEG–DSPE) and 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (Mal–PEG–DSPE) were obtained from NOF Co. (Tokyo, Japan). Cholesterol (Chol) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Thiazolyl blue tetrazolium bromide (MTT), 2-iminothiolane hydrochloride, human IgG, RPMI 1640 medium, and DMEM were from Sigma-Aldrich (St Louis, MO, USA). Doxorubicin hydrochloride was purchased from Wako Pure Chemical (Osaka, Japan). Amicon Ultra filters were from Merck Millipore Ltd. (Billerica, MA, USA). PD-10 columns were from GE Healthcare (Carlsbad, CA, USA).

Alpha-mangostin, Resomer® RG 503 H (acid-terminated poly (lactide-co-glycolide) (PLGA-COOH), lactide: glycolide 50 : 50, MW 24000–38000 Da, viscosity 0.32–0.44 dL/g), human holo-transferrin, 2-iminothiolane hydrochloride (Traut's reagent), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT), and all other chemicals that are not specifically mentioned below were purchased from Sigma Aldrich, Germany. DSPE-PEG2K-MAL was obtained from Jenkem Technology, USA. Acetone and dimethyl sulfoxide (DMSO) were purchased from RCI Labscan, Thailand. Dulbecco's Modified Eagle Medium (DMEM), minimal essential medium (MEM), Roswell Park Memorial Institute 1640 (RPMI-1640), fetal bovine serum (FBS), and penicillin-streptomycin were obtained from Gibco, USA.