Traut s reagent

The thiolated gelatin was synthesized based on a previously published protocol [40 ]. Gelatin type B (Nitta Gelatin, Inc. Naniwa-ku, Osaka, Japan) was dissolved in MilliQ water at 37°C at 1% w/v. Traut’s reagent (Thermofisher Scientific, Waltham, MA) was added to the dissolved gelatin at 20 mg or 80 mg Traut’s reagent per 1 g of gelatin (Gel-SH 20 and 80) and allowed to stir overnight while protected from light at 37°C. The thiolated gelatin was then collected and purified via dialysis in 5 and 1 mM HCl (ThermoFisher Scientific, Waltham, MA) in MilliQ water for 24 h each at 37°C. Following dialysis, the solutions were flash frozen and freeze-dried to isolate the thiolated gelatin groups. Once dry, all thiolated gelatin was stored at –20°C.

Hydrogen tetrachloroaurate trihydrate (HAuCl₄·3H₂O; 99%), cetyltrimethylammonium bromide (CTAB), sodium borohydride (NaBH₄; 99%), silver nitrate (AgNO₃; 99%), L-ascorbic acid (AA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC), sodium oleate (NaOL; >97%), hydrochloric acid (HCl, 37%), (3-mercaptopropyl)trimethoxysilane (MPTMS), ethylenediaminetetraacetic acid (EDTA), poly(ethylene glycol) methyl ether thiol (PEG-SH, M_w ≈ 5000), goat anti-human IgG, dithiotreitol (DTT), thiol-poly(ethylene glycol)amine (SH-PEG-NH₂), and human serum IgG were provided by Sigma–Aldrich (St. Louis, MO). Traut’s reagent, Zeba^TM spin desalting columns (7K MWCO), and Ellman’s reagent were purchased from Thermo Scientific (Rockford, IL). PEG6-NHNH2 was obtained from SensoPath Technologies (Bozeman, MT). Glass slides (7 mm × 50 mm × 0.7 mm) were from Delta Technologies (Loveland, CO).

The cationized antibodies were obtained according to Ma’s report [17 (link)]. Briefly, the anti-CD63 antibody (Cosmobio, Tokyo, Japan) was thiolated using Traut’s reagent (Thermo Scientific, Waltham, MA, USA) in phosphate buffer (pH 8.0) containing 2 mM EDTA and then conjugated with Cys(Npys)-(D-Arg)₉ (AnaSpec, Fremont, CA, USA). The anti-CD63 IgG-9r was purified by size-exclusion chromatography (Figure S2) and concentrated using Amicon-Ultra 40K (Merck Millipore, Burlington, MA, USA). The stoichiometry of thiol modification on the antibody was calculated based on the signals resulting from reaction with Ellman’s reagent (Thermo Scientific) (Figure S3). The introduction number of arginine moieties to IgG was estimated to be 2.8 molecules.
The anti-CD63 IgG-9r/anti-miR complex was obtained by mixing anti-CD63 IgG-9r and anti-miR in PBS at the indicated molar ratios and incubating the mixtures at room temperature for 20 min. The sequence of anti-miR21 [18 (link)] was as follows: 5′- U ^mC A A ^mC A U ^mC A G T C U G A U A A G ^mC U A -3′. The control sequence [18 (link)] was as follows: 5′- U T C U ^mC C G A A C G U G T C A ^mC G U T A U -3′ (plain: 2′-O-methly RNA, underlined: locked-nucleic acid (LNA)). The chemical modification patterns of those oligonucleotides are different from those of antagomirs [10 (link)].

Tetraethyl orthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), 3-(trihydroxysilyl) propyl methylphosphonate, and aminopropyltriethoxy silane (APTS) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Branched-polyethylenimine (PEI, 10 kDa) was purchased from Alfa Aesar (Ward Hill, MA, USA). Maleimide-PEG (5 kDa)-NHS was purchased from JenKem Technology USA (Plano, TX, USA). Trastuzumab (Herceptin®, Genentech) and cetuximab (Erbitux®, Eli Lilly) were obtained from the OHSU pharmacy (Portland, OR, USA). Phosphate-buffered saline (PBS) (pH 7.2) was obtained from Life Technologies (Carlsbad, CA, USA). Zeba spin desalting columns (MW 40 kDa), RNase-free water, Traut’s reagent, ethanol, HCl, NHS-rhodamine, and sodium hydroxide were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All reagents were of the highest purity grade available. Cell lines (MDAMB468, BT549, MDAMB231, KPL4 and MCF7) were obtained from American Type Culture Collection and maintained following the supplier’s recommendation.