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8 protocols using monomethoxy peg

1

Synthesizing Biodegradable Polymers

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HPMC (meets USP testing specifications), N,N-diisopropylethylamine (Hunig’s
base), hexanes, diethyl ether, N-methyl-2-pyrrolidone
(NMP), dichloromethane (DCM), lactide (LA), 1-dodecylisocynate, and
diazobicylcoundecene (DBU) were purchased from Sigma-Aldrich and used
as received. Monomethoxy-PEG (5 kDa) was purchased from Sigma-Aldrich
and was purified by azeotropic distillation with toluene prior to
use.
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2

Synthesis of PEG-Dextran Copolymers

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L-Lactide and D-lactide were obtained from Musashino Chemical Laboratory, Ltd. (Tokyo, Japan). PEG (number-averaged molecular weight (Mn) = 4600) and monomethoxy-PEG (Mn = 1000) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Carbonyl diimidazole (CDI), tin-2-ethylhexanoate, and dextran (Dex) (Mn = 150,000) were obtained from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). All other chemicals were of reagent grade and were used without further purification.
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3

Multifunctional Nanoparticle Synthesis for Immunotherapy

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4-Cyano-4-(dodecylsulfanylthiocarbonyl) sulfanyl pentanoic acid (DCT), N,N′-dicyclohexylcarbodiimide (DCC), acetone oxime, monomethoxy PEG (Mw = 5000 Da), and 2,2′-azobis(2-methylpropionitrile) (AIBN) were purchased from Sigma–Aldrich. 2-Hydroxyethyl disulfide was purchased from Macklin, and 4-(N,N-dimethylamino) pyridine (DMAP) was purchased from Heowns. Triphosgene, methacryl chloride, N-ethylethanolamine, propyl bromide, and camptothecin were obtained from Energy Chemical. 5(6)-TAMRA cadaverine (Rho) was purchased from Haoran Biological Technology Co., Ltd. Cyanine 5.5 amine was purchased from Lumiprobe. 4′,6-Diamidino-2-phenylindole (DAPI) was purchased from Beyotime Biotechnology Co., Ltd. Cell counting kit-8 (CCK-8) was purchased from Beijing Solarbio Technology Co., Ltd. An Annexin V-FITC/PI apoptosis detection kit was purchased from Meilun Biological Technology Co., Ltd. (Dalian, China). 1-(4-(Aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c] quinoline-4-amine (IMDQ) was provided by Nanjing Aikon Chemical Ltd. Anti-CD11c, anti-CD86, anti-CD80, anti-CD40, anti-GrB, anti-TNF-α, anti-CD3e, anti-IFN-γ, anti-MHC-I, anti-MHC-II, anti-CD103, anti-F4/80, anti-CD206, anti-iNOS, anti-CD4, anti-CD8, anti-Foxp3, anti-CD44, anti-CD62L were purchased from Biolegeng (American).
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4

Synthesis of PEGylated Polymer Conjugates

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HPMC (meets USP testing specifications), N,N‐diisopropylethylamine (Hunig's base), hexanes, diethyl ether, N‐methyl‐2‐pyrrolidone (NMP), dichloromethane (DCM), lactide (LA), 1‐dodecylisocynate, and diazobicylcoundecene (DBU) were purchased from Sigma‐Aldrich and used as received. Monomethoxy‐PEG (5 kDa) was purchased from Sigma‐Aldrich and was purified by azeotropic distillation with toluene prior to use.
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5

Lipid-based Nanomedicine Preparation

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GRVRRSC (G: glycine; R: arginine; V: valine; S: serine; C: cysteine) was provided by GL Biochem Co., Ltd. (Shanghai, China). MonomethoxyPEG (mPEG) was provided by Sigma-Aldrich (St. Louis, MO). 4-Dimethylaminopyridine (DMAP), 1, 1-dioctadecyl-3, 3, 3, 3-tetramethylindodicarbo cyanine iodide (DiD) and Triethylamine (TEA) were all provided by Aladdin Co., Ltd. (Shanghai, China). Octadecanol, succinic anhydride, maleic anhydride, and tripterine (purity >98.0%) were all purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Coix seed oil (extracted by carbon dioxide supercritical technology, purity >85%). RH 40 and PEG 400 were purchased from BASF Co., Ltd. (Ludwigshafen, Germany). Dipalmitoylphosphatidylcholine (DPPC), stearoyllysophosphocholine (S-lysoPC), and Distearoylphosphoethanolamine-PEG2000 (DSPE-MPEG2k) were provided by A.V.T Co., Ltd. (purity > 98.0%, China). Double-distilled water was purified via Milli Q purification system (Merck Millipore). All other reagents used were of analytical grade.
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6

Synthesis of PEG-Lactide Copolymer

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HPMC (meets USP testing specifications), N,N-Diisopropylethylamine (Hunig's base), hexanes, diethyl ether, N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), lactide (LA), 1dodecylisocynate, and diazobicylcoundecene (DBU) were purchased from Sigma Aldrich and used as received. Monomethoxy-PEG was purchased from Sigma Aldrich and was purified by azeotropic distillation with toluene prior to use.
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7

Expi293F and HEK293T Cell Culture Protocol

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Mammalian Cell culture: Expi293F cells (ThermoFisher) were maintained in a 2:1 mix of FreeStyle293:Expi293 Expression medium (ThermoFisher) and grown at 37 °C and 8% CO2 while shaking at 120 rpm. HEK293T cells (ATCC) were maintained in DMEM (Cellgro) supplemented with 10% FBS, L-glutamate, 1% penicillin-streptomycin, and 10 mM HEPES. ACE2/HeLa cells were generously provided by Dennis Burton and were maintained in DMEM (Cellgro) supplemented with 10% FBS, L-glutamate, 1% penicillin-streptomycin, and 10 mM HEPES. [47] Chemicals: HPMC (meets USP testing specifications), N,N-diisopropylethylamine (Hunig's base), hexanes, diethyl ether, N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), lactide (LA), 1-dodecylisocynate, and diazobicylcoundecene (DBU) were purchased from Sigma-Aldrich and used as received. Monomethoxy-PEG (5 kDa) was purchased from Sigma-Aldrich and was purified by azeotropic distillation with toluene prior to use. For in vitro studies, 2'3'cyclic-GMP-AMP (cGAMP) was synthesized and purified in-house as previously described. [27] For in vivo studies, vaccine-grade 2'3'-cyclic-GMP-AMP (cGAMP) was purchased from Invivogen.
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8

Synthesis of PLGA-COOR and PEG-PLGA Copolymers

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The synthesis of PLGA-COOR was adapted from the ring-opening polymerization method described by Yoo and Park [37] . Briefly, a mixture of DL-lactide (Purasorb® DL) and glycolide (Purasorb® G) in the molar ratio of 3:1 was heated with the initiator benzyl alcohol to 140 °C under nitrogen atmosphere for complete melting. The use of this initiator would result in a benzyl group being the R-group in the PLGA-COOR product. Then, 0.04% (w/w) stannous octoate was added, and the reaction mixture was further heated to 180 °C. The temperature was maintained for 3 hours for polymerization to take place under static vacuum. The polymer was then recovered by dissolution in dichloromethane before precipitation in heptane. The precipitate was subsequently filtered and dried at 25 °C for 24 hours under vacuum. For the synthesis of PEG-PLGA co-polymer, the same procedure was adopted except that monomethoxy-PEG of number average molecular weight (Mn) of 5 kDa (Sigma-Aldrich) was used as an initiator instead of benzyl alcohol, and the precipitation of PEG-PLGA was carried out in diethyl ether chilled to -20 °C.
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