Barnstead nanopure system

Poly (D,L-lactide-co-glycolide) (PLG) (50:50, ester terminated, M_W 7,000–17,000), Poly (vinyl alcohol) (PVA) (M_W 13,000–23,000, 87–89% hydrolyzed), dichloromethane (DCM), coumarin 6, fluorescein isothiocyanate isomer 1 (FITC) and mannan from Saccharomyces cerevisiae were purchased from Sigma (St. Louis, MO). α-D-mannopyranosyl 4-phenylisothiocyanate (MITC) was purchased from Synthose (Concord, ON, Canada). Ethanol was purchased from Decon Laboratories (King of Prussia, PA). Dimethyl sulfoxide (DMSO), annexin v binding buffer, fetal bovine serum (FBS), trypsin with 0.25% EDTA and pen/strep were purchased from Fisher (Hampton, NH). Ultrapure water was obtained from a Thermo Scientific Barnstead Nanopure system. Dimethyl sulfoxide d6 was purchased from Cambridge Isotope Labs (Tewksbury, MA). Concanavalin A rhodamine (ConA) was purchased from Vector Labs (Burlingame, CA). RAW 264.7 macrophages were obtained from ATCC (TIB-71). Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose and L-glutamine and DMEM/F12 was purchased from Corning Cellgro (Corning, NY). Recombinant macrophage colony stimulating factor (MCSF) was purchased from Cell Guidance Systems (Cambridge, UK). Antibodies for flow cytometry, Trustain fcX (anti-CD16/32) and APC anti-mouse F4/80 antibody (clone BM8) were purchased from BioLegend (San Diego, CA, USA).

Chemicals were acquired from Sigma-Aldrich Chemical, St. Louis, MO: A deoxynucleotide set, 10 mM (deoxyadenosine triphosphate: Product#: D6920, MW: 535.15; deoxycytidine triphosphate: Product#: D7045, MW:511.12; deoxyguanosine triphosphate: Product#: D7170, MW: 573.13; and thymidine triphosphate: Product#: T7791, MW: 504.15). Internal standards were purchased from Cambridge Isotope Laboratories, Inc. Andover, MA including 2′-deoxyadenosine-¹⁵N₅ (dA-IS, Cat#: PR-15211, MW: 256.21), 2′-deoxycytidine-¹⁵N₃ (dC-IS, Cat#: NLM-3897-0, MW: 230.19), 2′-deoxyguanosine:H₂O-¹⁵N₅ (dG-IS, Cat#: NLM-3899-CA-0, MW: 290.22), and Thymidine-¹³C₁₀, ¹⁵N₂ (T-IS, Cat#: CNLM-3902-0, MW: 254.14). Analytical grade reagents were obtained from: Fisher Scientific, Fairlawn, NJ: methanol, 2-propanol, acetic acid, and potassium chloride; Sigma-Aldrich Chemical: alkaline phosphatase; and JT Baker, Phillipsburg, NJ: acetonitrile. Ultrapure (UP) water was prepared in house from deionized water with a Barnstead Nanopure System (Thermo Fisher Scientific, Waltham, MA). Consumables included Waters Sep-Pak Accell Plus QMA Cartridge, 3cc/500 mg (Water Corporation, Milford, MA); Phenomenex Strata-X-CW 33 μm Polymeric Weak Cation Mixed Mode Cartridge 3cc/200mg (Phenomenex, Inc., Torrance, CA); and blood products for the lysed cellular matrix (Bonfils, Denver, CO).

Except as noted, all reagents are analytical grade and purchased from Sigma-Aldrich. Solutions are prepared from deionized-distilled water (ddH₂O) from a Barnstead Nanopure System (Thermo-Fisher Scientific, Waltham, MA USA). Prototype injectors were machined from polycarbonate plastic (#63404230, MSC Supply Co, Melville, NY USA) at the University of Notre Dame Chemistry Machine Shop. For production runs, a 3-D desktop printer was used to manufacture devices at Notre Dame’s Fabrication Lab using a Stratasys Objet30 Prime 3-D printer supplied with RGD-720 transparent print material (Eden Prairie, MN USA). Modification of the device was done in the local machine shop.

DNA oligonucleotides consisting of polythymidylic acids (dT₅, dT₁₀, dT₁₅, dT₂₀, dT₃₀ and dT₅₀) were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). RNA oligonucleotides were purchased from Dharmacon, Inc. (Lafayette, CO) and de-protected, as needed, according to the manufacturer’s protocol. The phosphorothioate oligonucleotide sample and corresponding shortmer failure sequences (n-1, n-2, and n-3) were purchased from Biosearch Technologies (Petaluma, CA). All oligonucleotides used in this study are summarized in Table 1. Oligonucleotide concentrations were measured on an IMPLEN Pearl nanophotometer (Westlake Village, CA).
LC-MS grade ammonium acetate and ammonium formate, 1,1,13,3,3-hexafluoro-2-propanol (HFIP, >99%), and triethylamine (TEA, HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO). LC-MS grade formic acid, methanol, and acetonitrile were purchased from Fisher Scientific (Waltham, MA). Acetic acid was purchased from VRW International (Radnor, PA). High-purity water (18.2 MΩ.cm) was produced in-house with Barnstead Nanopure system (Thermo Scientific, Waltham, MA).

75:25 poly (D,L-lactide-co-glycolide) (PLG) with a lauryl ester end group and an inherent viscosity of 0.79 dL/g was purchased from Evonik (Birmingham, AL). Rosiglitazone was purchased from Cayman Chemical (Ann Arbor, MI). Dichloromethane (DCM) and polyvinyl alcohol (PVA, Mw 13,000–23,000, 87–89% hydrolyzed) were purchased from Sigma (St. Louis, MO). Ethyl acetate was purchased from Macron Fine Chemicals (Center Valley, PA). 200 proof ethanol was purchased from Decon Laboratories (King of Prussia, PA). Dimethyl sulfoxide (DMSO) was purchased from Fisher (Hampton, NH). Ultrapure water was obtained from a Thermo Scientific Barnstead Nanopure system. Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose and L-glutamine was purchased from Corning Cellgro (Corning, NY). Trypsin with 0.25% EDTA, Fetal Bovine Serum (FBS) and Pen/Strep were purchased from Fisher (Hampton, NH). Super Calf Serum was purchased from GemCell (Sacramento, CA). Insulin (bovine), dexamethasone, and methylisobutylxanthine (IBMX) were purchased from Sigma (St. Louis, MO). 10% Neutral Buffered Formalin was purchased from Sigma (St. Louis, MO). Nile Red was purchased from Molecular Probes (Eugene, OR). Adiponectin ELISA kit was purchased from R&D systems (Minneapolis, MN).