Deuterium oxide

Silver oxide, anhydrous sodium sulfate, iodomethane, iodomethane-d₃, l-phenylalanine methyl ester hydrochloride, mass spectrometric grade formic acid (~98%), and HPLC grade trifluoroacetic acid (99.0%) were obtained from Sigma-Aldrich; HPLC grade methanol, acetonitrile, ethyl acetate, and water from Fisher Scientific; Deuterium oxide (99.8%) and HPLC grade dichloromethane from Acros; l-alanyl-l-proline, N-(tert-butoxycarbonyl)-d-alanine, l-proline tert-butyl ester hydrochloride, d-proline tert-butyl ester hydrochloride, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) hydrochloride from Tokyo Chemical Industry. A Fisher Scientific vortex mixer was used for mixing and a Sorvall Legend Micro 21R microcentrifuge used for centrifugation of 1.5 mL Eppendorf tubes. A Corning Laboratory stirrer was used for mixing chemical reactions. Human plasma (Na₂-EDTA) was obtained from Bioreclamation and stored at −80 °C. An Argonaut SPE DRY™ 96 DUAL evaporator was used for solvent evaporation.

The degree of substitution (DS) was analyzed from ¹H-NMR spectra of gelatin and GelMA to quantify the methacrylate groups in GelMA using a Bruker DPX 500 spectrometer. Briefly, 30 mg of GelMA and gelatin were each dissolved in 1 mL of deuterium oxide (Acros Organics, Morris Plain, NJ, USA) at 40 °C and H-NMR spectra were collected at 40 °C. After phase and baseline corrections, the chemical shift scale was adjusted to the phenylalanine signal (6.9–7.5 ppm). The NMR spectra was also normalized to the phenylalanine signal, which was proportional with the concentration of the gelatin. DS was calculated by integrating the areas of lysine methylene signals (2.8–2.95 ppm) of GelMA and gelatin according to the following equation: $$\mathrm{DS}(\%)=\left(1-\frac{\mathrm{Peak}\mathrm{area}\left(\mathrm{lysine}\mathrm{methylene}\mathrm{of}\mathrm{GelMA}\right)}{\mathrm{Peak}\mathrm{area}\left(\mathrm{lysine}\mathrm{methylene}\mathrm{of}\mathrm{non}-\mathrm{modified}\mathrm{gelatin}\right)}\right)\times 100$$

The amphiphilic monomer AGC₁₂ was prepared and purified according to our previous work, and their synthetic routes are shown in Scheme 2 [25 (link)]. The detailed synthetic procedure of AGC₁₂ was given in Supplementary Materials. Deuterium oxide (99.9%) and chloroform-d (99.9%) were purchased from Acros (Geel, Belgium. Acryloyl chloride, 1-bromine octane and solvents were purchased from Beijing Chemical Co. (analytical reagent (AR) grade, Beijing, China). The used halloysite clay nanotubes (HNTs) were obtained from Nanyang Hongfa Bentonite Company in Nanyang, China. Acrylamide (AM) was obtained from Acros and purified via recrystallization before use. 3-aminopropyltriethoxysilane (APTES, 98%), serving as a silica source was supplied by Sigma-Aldrich (St Louis, MI, USA). and used as received. Methenamine, used for guest loading, was purchased from Alfa Aesar (Ward Hill, MA, USA).

Cuttlefish (Sepia pharaonis) bones were collected from a local cuttlefish processer (Hong Yu Foods Co., Ltd., Kaohsiung, Taiwan). The average size of cuttlefish caught in the winter season is 53–59 cm in length and 20–31 cm in width, the wet weight when received is ~5 kg, and the dry weight of the cuttlefish bone is ~80 g. The cuttlefish bones were buried in crushed ice during transportation and stored at −80°C until use. 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium bromide, ascorbic acid, potassium peroxydisulfate, trichloroacetic acid, and hydrogen chloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deuterium oxide and acetic acid-d4 were obtained from ACROS (Morris Plains, NJ, USA). 1,1-diphenyl-2-picrylhydrazyl (DPPH), sodium hydrogen phosphate, sodium dihydrogen phosphate, Ferrous chloride and hydrogen peroxide were obtained from Merck Co. (Darmstadt, Germany). Other chemicals used were of analytical grade.

Pluronic P-105 was purchased from BASF (Florham Park, New Jersey, USA). Pluronic P-123 was purchased from Millipore Sigma (St. Louis, Missouri). Perfluorooctyl bromide and perfluoro-15-crown-5-ether were purchased from Exfluor Research Corp (Round Rock, Texas, USA). Miglyol 812 (capric acid/caprylic acid triglycerides, known as medium chain triglycerides, MCT) was obtained from CREMER Oleo (Hamburg, Germany). Transcutol was purchased from Spectrum Chemical Mfg Corp (New Brunswick, New Jersey, USA). Trifluoroacetic acid and deuterium oxide, 99.8% were obtained from Acros Organics (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.). Dulbecco’s Modified Eagle’s Medium was supplied by Corning (Corning, New York, U.S.), and fetal bovine serum was supplied by ATCC (Manassas, Virginia). All chemicals and reagents were used without modification.

At the end of the MR studies, tumors were excised, snap-frozen in liquid nitrogen and stored at −80 °C until further investigation. For complementary ¹H MRS studies pieces of tumor tissue weighting 9 to 24 mg (wet mass) were homogenized in 500 μl ice cold phosphate buffer (PBS) with 1 μl/ml protease inhibitor cocktail set III (Calbiochem) using beads (TissueLyser LT, QIAGEN). The dual-phase extraction method described above for the cell extracts was then applied and the aqueous phase was reconstituted in 400 μl 100 mM phosphate buffer in deuterium oxide (Acros Organics).