177lucl3

For the purpose of radiolabeling the liposomes, 4 mCi of ¹⁷⁷Lu was typically used. A total of 20 μL (0.05 N HCl) of ¹⁷⁷LuCl₃ (Perkin Elmer Inc.,) was taken in a tube and 9 μL of 0.5 M sodium acetate was added to adjust the pH to ~5. Then, 100 μL (200 µg) of ILs or non-targeted CLs was added to the tubes and incubated for 1 h at RT (~20 µCi/µg ILs). Subsequently, the ¹⁷⁷Lu-labeled CLs (¹⁷⁷Lu-CL) or ILs (¹⁷⁷Lu-IL) were purified by using a PD-10 column with PBS as a mobile phase. Labeling efficiency of ¹⁷⁷Lu-CL or ¹⁷⁷Lu-IL was calculated from the ratio of radioactivity associated with the liposome fraction compared to the total added radioactivity. The radioactivity was measured with the CRC-55tW Dose Calibrator/Well Counter (Capintec, Inc., Ramsey, NJ, USA).

Dara was prepared for radiolabeling via the conjugation with SCN‐Bn‐deferoxamine (Df) or p‐SCN‐Bn‐CHX‐A″‐diethylenetriaminepentaacetic acid (DTPA, Macrocyclics) at the mole ratio of mAb:chelator at 1:5–1:10 following a standard protocol.^[19, ^35] In brief, for ⁸⁹Zr labeling, ⁸⁹Zr‐oxalate in 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) buffer (0.5 m) was added into the Na₂CO₃ solution containing Df–dara (pH = 7.0) and mixed for 1 h at 37 °C. For ¹⁷⁷Lu labeling, lutetium chloride (¹⁷⁷LuCl₃) in sodium acetate buffer (pH 6.5) was mixed gently with DTPA–dara (0.15 µg µCi⁻¹) at 37 °C.^{[35] 89}Zr‐oxalate was produced using a PETrace cyclotron (GE Healthcare). ¹⁷⁷LuCl₃ (t_1/2 = 6.65 d) was purchased from PerkinElmer. Radiolabeled products were purified using PD‐10 columns (GE Healthcare) with a phosphate‐buffered saline (PBS) mobile phase. A radiolabeled nonspecific isotype control immunoglobulin G (IgG, Invitrogen) was prepared as a control group.

All the lipids and their derivatives, including 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (POPC), 1,2dimyristoyl-sn-glycero-3-phospho-ethanolamine-N-diethylene-triamine-pentaaceticacid (DMPE-DTPA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-mPEG₂₀₀₀), 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG₂₀₀₀-MAL), and cholesterol, as well as the mini extruder were purchased from Avanti Polar Lipids (Alabaster, AL, USA). 2-mercaptoethanolamine (2-MEA) was purchased from Thermo Scientific (Wilmington, DE). Dimethyl sulfoxide (DMSO, anhydrous), chloroform (anhydrous), and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). PD-10 columns (Sephadex G-25) were purchased from GE healthcare (Chicago, IL, USA). ¹⁷⁷LuCl₃ was purchased from Perkin Elmer (Boston, MA, USA).

Buffers used for labeling were prepared from high-quality Milli-Q water and purified from metal contamination using Chelex 100 resin (Bio-Rad Laboratories, USA). ¹¹¹InCl₃ was purchased from Mallinckrodt Sweden AB (Stockholm, Sweden). Carrier-free ¹⁷⁷LuCl₃ was purchased from PerkinElmer (Waltham, MA, USA). Radioactivity was measured using an automated gamma-spectrometer with a NaI(TI) detector (1480 Wizard, Wallac, Finland). Her2-expressing SKOV3, BT474 and DU145 cells were purchased from the American Type Culture Collection (ATCC) and were cultured in complete RPMI-medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin in a humidified incubator with 5% CO₂ at 37 °C, unless stated otherwise.
Production and purification of PNA-based probes have been described previously in Westerlund et al.^{13 (link)} and Altai et al.^{12 (link)}. Briefly, the PNA-based probes HP1 and HP2 were synthesized manually using solid phase synthesis with commercially available building blocks. The primary agent, Z_HER2:342-SR-HP1, was produced by site-specifically attaching HP1 to the anti-HER2 affibody using a sortase A mediated ligation strategy. Z_HER2:342-SR-HP1 and the secondary agent HP2 were both purified using reversed phase HPLC to a final purity of ≥95% and kept lyophilized at −20 °C until use.

¹¹¹InCl₃ or ¹⁷⁷LuCl₃ (Perkin Elmer) were buffered with 1/10th (v/v) of 1 M ammonium acetate solution pH 7.1 and then added to DOTAGA-F(ab′)₂ in 0.1 M ammonium acetate buffer pH 5.9 (600 MBq mg^− 1 and 1 GBq.mg^− 1, respectively). See details in Supplemental methods.