Glycine hcl

Herceptin (trastuzumab) was from Roche (Basel, Switzerland) and Kadcyla (ado-trastuzumab emtansine (T-DM1)) from Genentech Inc. (South San Francisco, CA, USA). Human HER2/ErbB2s protein (His tagged) was obtained from Sino Biologicals (North Wales, USA). Ethylenediaminetetraacetic acid (EDTA), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), Tween 20, disodium hydrogen phosphate dihydrate, phosphate-buffered saline (PBS) tablets, ethanolamine hydrochloride, N-hydroxy succinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), hydrochloric acid 37% (HCl), acetic acid (≥ 99%), glycine-HCl, ammonium acetate, sodium hydroxide (NaOH), sodium chloride (NaCl), and sodium acetate were purchased from Sigma-Aldrich (Steinheim, Germany). Sodium azide (toxic, should be discarded in hazardous waste) was obtained from Mallinckrodt Baker B.V. (Deventer, the Netherlands). β-mercaptoethanol was obtained from Merck (Darmstadt, Germany). Deionized water was produced by a Milli-Q purification system from Millipore (Amsterdam, the Netherlands).

Two elution buffers were tested, including glycine-HCl (Sigma) and Pierce IgG elution buffer (Thermofisher). For glycine-HCl, 200 mM and 300 mM were tested. SEVs collected from the outlets of the NanoEPIC device were treated with an equal volume of elution buffer and immediately incubated at 37 °C for 30 min. The sEV samples were then separated from eluted MNPs through sucrose gradient centrifugation (0.5 M and 1.5 M sucrose concentration) running at 120,000 g for 1.5 h. sEVs were collected from the interface between the 0.5 M and 1.5 M sucrose while the released MNPs were pelleted at the bottom of the tube. To remove the excessive sucrose, the collected sEVs were washed with 5 mL of PBS and pelleted through centrifugation at 100,000 g for 2 h.

Carnauba wax, toluene, and ethyl acetate
were purchased from Acros Organics. Tween 20 (poly(ethylene glycol)
sorbitan monolaurate), squalene, and the chemicals used for buffer
preparation (glycine–HCl, MES hydrate, HEPES, NaHCO₃, NaOH) were purchased from Sigma-Aldrich. Rice bran oil was purchased
from TEA Natura (TEA Prodotti Naturali di Manzotti P., Italy). TDI
dye (a drug mimetic, λ_exc = 630 nm, λ_em = 700 nm; the structure is given in Figure 1) was synthesized as reported previously.^{34 (link)} Mouse IgG1 (clone: MOPC-21) and antihuman CD340
(erbB2/HER2; clone: 24D2) were purchased from Biozol Diognostica Vertrieb
GmbH and used as received. Sterile water (Braun Melsungen AG) was
used for the experiments unless otherwise stated.

Fusion proteins were dialysed into 25 mM Na Acetate solution, pH 8 (Sigma Aldrich) using Maxi GeBAflex dialysis tubes with a 3.5 kDa molecular weight cut off (Gene Bio-Application Ltd.). NHS-DOTA (Macrocyclics Inc.) was then added in a 4-fold molar excess and reacted overnight at room temperature. Conjugation solutions were then applied to Protein A columns (GE Healthcare) equilibrated in Chelex 100 (Bio-Rad Ltd.) treated PBS, pH 7.4 (PAA Laboratories GmbH), washed with Chelex treated PBS and eluted in 0.5 ml fractions of 0.1 M glycine/HCl, pH 2 (Sigma Aldrich), into tubes containing ammonium acetate (final concentration/pH of fractions was 0.46 M ammonium acetate, pH 5). Conjugation efficiency and activity were determined as described previously [26 (link)].

ACTH_, human chorionic gonadotropin hormone (hCG), and Glycine-HCl were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Isoflurane was purchased from Patterson Veterinary (Saint Paul, MN, USA). Heparin and saline were purchased from Hospira (Lake Forest, IL, USA). EDTA-containing vacutainer tubes were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). Etomidate was purchased from Bachem (Torrance, CA, USA). Enzyme-linked immunosorbent assay kits for corticosterone were purchased from IDS (Gaithersburg, MD, USA), whereas those for aldosterone, dehydroepiandrosterone (DHEA), and testosterone were purchased from Enzo Life Sciences (Farmingdale, NY, USA), and those for androstenedione and dihydrotestosterone were purchased from MyBioSource (Cambridge, MA, USA). Dimethoxy-Etomidate, (R)-ethyl 1-(1-(3-isopropoxyphenyl)ethyl)-1H-imidazole-5-carboxylate (isopropoxy-Etomidate), (R)-ethyl 1-(1-(naphthalen-2-yl)ethyl)-1H-imidazole-5-carboxylate (naphthalene-Etomidate), and (R)-ethyl 1-(1-(naphthalen-1-yl)ethyl)-1H-imidazole-5-carboxylate (naphthalene (2)-Etomidate) were custom synthesized by Aberjona Laboratories (Beverly, MA, USA) and their structures confirmed by nuclear magnetic resonance spectroscopy.