Acetone

HSP70B-NIS-MSCs were seeded directly on FBS-coated microscope slides and grown until 60% confluent. 6 h after hyperthermia, the slides were air-dried overnight at room temperature and monolayers fixed with 80% methanol (Carl Roth) for 5 min at 4 °C, followed by 100% acetone (Carl Roth) for 2 min at -20 °C. Following blocking with 12% bovine serum albumin (Sigma-Aldrich) in phosphate- buffered saline (PBS; Sigma Aldrich) for 30 min, cells were then incubated with a primary mouse monoclonal NIS-specific antibody (Merck Millipore; dilution 1:500) for 90 min. A secondary Cy3 AffiniPure donkey anti-rabbit IgG antibody (Jackson ImmunoResearch; dilution 1:400) and bisbenzimide (Hoechst; Sigma Aldrich; dilution 1:2000) to counterstain nuclei were added for 30 min. Pictures were taken using an Axiovert 135 TV fluorescence microscope in combination with an AxioCam MRm CCD camera and the AxioVision Rel. 4.8 software (Carl Zeiss Microscopy GmbH, Jena, Germany).

Tetrahydrofuran (THF) and diethyl ether were HPLC-grade and obtained from VWR International GmbH (Darmstadt, Germany). For polymer synthesis, THF was dried by refluxing over potassium and sodium and subsequently distilled. Maleic anhydride (MA) and aniline were purchased from Thermo Fisher Scientific and VWR International GmbH, respectively. Tetradecyl acrylate (TDA) and poly(ethylene glycol) methyl ether methacrylate (methoxy-PEG-monomethacrylate, mPEG-MAc) with an average Mn of 950 were obtained from TCI Deutschland GmbH (Eschborn, Germany) and used as received. 2,2′-Azobis(2-methylpropionitril) (AIBN), 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine (azido-TEG-amine) and triethylamine (TEA) were from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Acetone was purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany), and aqueous ammonia (25% m/V) was obtained from Grüssing GmbH (Filsum, Germany). Deuterated solvents, CDCl₃, DMSO-D6, both with tetramethyl silane, and D₂O were purchased from ARMAR GmbH (Leipzig, Germany). Float-A-Lyzer^® dialysis devices from Repligen Europe B.V. (Dreda, The Netherlands), with a cellulose ester membrane and a molecular weight cut off of 0.1–0.5 kDa were used.

YCl₃·6H₂O (99.99%), YbCl₃·6H₂O (99.99%), ErCl₃·6H₂O (99.99%), oleic acid (90% technical grade), and NaOH (98%) were purchased from Sigma-Aldrich. 1-octadecene (ODE, 90% technical grade) and NH₄F (99.99%) were obtained from Alfa Aesar. Chloroform, cyclohexane, acetone, and ethanol were purchased from Carl Roth GmbH. Low-price Ln-chlorides were puchased from XI’AN FUNCTION MATERIAL GROUP. All chemicals were used without further purification.

Cells were grown for 24 h either in SCM or in CDM on coated or noncoated six-well plates. We fixed the cells by removing the medium, gently washing with 2 mL PBS, and incubating with acetone (Carl Roth, Karlsruhe, Germany) for 10 min at −20°C. After two washes with PBS, the sample was incubated with 2 mL blocking solution (10 mg mL⁻¹ BSA in PBS) for 30 min at room temperature. The sample was again washed twice and then incubated with a 1 : 80 dilution of Alexa Fluor® 555 Phalloidin (Life Technologies, Darmstadt, Germany, A340555) or a 1 : 200 dilution of DyLight 488 integrin α₄ antibody MM0417-2L30 (R&D Systems GmbH, Wiesbaden, Germany, NBP2-11738G) in PBS for 2 h at room temperature in the dark. Finally, the nuclei were counterstained with DAPI (AppliChem, Darmstadt, Germany) and the sample was embedded in Mowiol (Carl Roth) according to the manufacturers' recommendations.

Chemicals for the nanoparticle synthesis were purchased and used without further purification: ethanol absolut (VWR), diethylene glycol (99%, Alfa Aesar), N-methyl-2,2‘-iminodiethanol (Merck), sodium hydroxide pellets (Merck), Hydrochloric acid solution (in water 1 M, Grüssing GmbH), Sodium hydroxide solution (in water 1 M, Grüssing GmbH), acetone (technical grade, Carl Roth GmbH), iron(II) chloride tetrahydrate (Glentham Life Sciences) and iron(III) chloride hexahydrate (puriss. p.a. reag., ≥99%, Sigma-Aldrich). Ligands for the nanoparticle modification were obtained from Sigma-Aldrich L-histidine monohydrochloride monohydrate (≥99%), L-lysine monohydrochloride (BioUltra, ≥99.5% (AT)); 4-mercaptobenzoic acid (99%); 4-aminobenzoic acid (Reagent Plus, ≥ 99%); DL-Lactic Acid (~90%), phosphacholine chloride calcium salt tetrahydrate (Sigma grade); cysteamin (> 98%); L-(+)-tartaric acid (99%), from Alfa Aesar L-Arginine (98%), L-(−)-malic acid (99%), 4-hydroxybenzoic acid (99%), (±) Mandelic Acid (99%) and from Grüssing GmbH trisodium citrate dihydrate.

Monolayers were fixed in ice-cold methanol, while pellet cultures were placed in Tissue-Tek® Cryomold® Standard (Sakura Fintek, Torrance, CA, USA) before being cryopreserved in liquid nitrogen. The frozen pellets were sectioned, placed on SuperFrost® cryosection slides (Thermo Fischer Scientific GmbH) and fixed with 3% ice-cold acetone (Carl Roth GmbH). Alizarin Red S, Oil redO and alcian blue stainings were performed as outlined previously [23 (link), 24 (link), 26 (link)]. In addition, immunohistochemical stainings were performed on monolayers and pellets using the following antibodies: Col I - monoclonal anti Col Iα1 (5 μg/mL; Abcam pls, Cambridge, Great Britain); Col II - polyclonal Col IIα1 antibodies (5 μg/mL; Acris Antibodies GmbH, Herford, Germany); Col X - polyclonal Col X antibodies (5 μg/mL; Abcam pls). The immunostainings were visualised with the Avidin-Biotion complex method using the protocols, biotinylated antibodies, blocking serum and peroxidase from the VECTASTAIN® Universal Elite® ABC Kit (Vector Laboratories, Burlingame, CA, USA) and the VECTOR® NovaRED™ peroxidase substrate kit (Vector Laboratories). The slides and wells were counterstained with haematoxylin (Sigma-Aldrich). For control stainings the primary antibodies were replaced with non-immune IgG antibodies (Sigma-Aldrich).