Kolliphor hs 15

The synthesis and radiolabeling of the dual-modality probes was reported previously [18 (link)]. A total of four probes were generated by introducing a linker (p-aminomethylanilinediglycolic acid (pADA) or PEG₄) and lysine between the binding domain of NeoB and DOTA chelator, in combination with or without a PEG₄ linker coupling the lysine and trans-cyclooctene (TCO) moiety (Figure 1). A short overview of the methodology can be found in the Supplementary Materials.
The radiolabeling of the four dual-modality probes with [¹¹¹In]InCl₃ (Curium, Petten, The Netherlands) was performed in water containing Kolliphor^® HS 15 (Merck, Amsterdam, The Netherlands) (0.5 mg/mL) in the presence of sodium acetate, gentisic and ascorbic acid, which acted as radiolysis quenchers [19 (link)]. The radiolabeling mixture was incubated at 90 °C for 20 min. Then, diethylenetriaminepentaacetic acid was added to complex free radionuclide. The radiochemical yield (>95%) and radiochemical purity (>95%) of the radiopeptides were determined using instant thin-layer chromatography and radio high-performance liquid chromatography, respectively. The radiolabeled dual-modality probes were prepared with a molar activity of 20 MBq/nmol for all in vitro experiments.

Soy phosphatidylcholine (PC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) were purchased from Avanti Polar Lipids Inc. (Cat #1069-79-0 and 810103, Alabaster, AL, USA). Kolliphor^® HS 15 and α-tocopherol acetate (α-TA) were purchased from Sigma-Aldrich, Inc. (Cat #42996 and T3001, St. Louis, MO, USA). The three CTPs (GWLSEAGPVVTVRALRGTGSWGGC, GVTVRALRGTGSWGGC, and GWLSEAGPVVTVRALRGTGGGC, respectively) were synthesized by GenScript USA Inc. (Piscataway, NJ, USA). DSPE-PEG2K-maleimide and DSPE-PEG2K (SUNBRIGHT) were purchased from NOF Inc. (Cat #DSPE-020MA and DSPE-020CN, Tokyo, Japan). 1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindotricarbocyanine Iodide (DiR) was purchased from Thermo Fisher Scientific Inc. (Cat #D12731, San Jose, CA, USA).

All animal protocols were approved by the Animal Care and Use Committee at the Second Hospital of Jilin University (Jilin, China, Permit No. 2018178). Male SD rats (8 weeks old, weighing 200 ± 20 g; n = 32) were purchased from the Animal Center at Jilin University. The rats were housed in a temperature-controlled room (21 ± 2 °C) at the Animal Center with a 12-h light:12-h dark cycle. After a one-week acclimatization period, the rats were randomly and equally divided into four groups: (1) control (normal rats), (2) sham-operated, (3) UUO-operated + vehicle, and (4) UUO-operated + FFNT25. UUO was performed under complete chloral hydrate anesthesia (200 mg/kg, intraperitoneally) by ligating the left lateral ureter. Two weeks after UUO, the rats were gavaged with either FFNT25 (20.6 mg/kg/day) in vehicle or vehicle only for two weeks. Vehicle contained 3% kolliphor@HS15 (Sigma-Aldrich, St. Louis, MO) and 3% glycerol (Beijing Chemical Works, Beijing, China) dissolved in physiological saline. The chemical property of FFNT25 (Sunshine Lake Pharma Co., Guangdong, China) is shown in Table 1.

Plasma extravasation was evaluated using the near-infrared fluorophore IR-676 (0.5 mg/kg, Spectrum-Info Ltd., Kyiv, Ukraine) dissolved in 5% (v/v) aqueous solution of Kolliphor HS 15 (polyethylene-glycol-15-hydroxystearate; Sigma-Aldrich, St. Louis, MO, USA), yielding a micellar contrast agent accumulating in inflamed areas due to increased vascular permeability [40 (link)]. The formula was injected intravenously (iv.) into anesthetized mice through the retroorbital veins with a 0.5 mL insulin needle, the bevel oriented downwards as described earlier [41 (link)] 5 h after the K/BxN serum transfer and on day 2. Short-term anesthesia of multiple subjects has been done by administration of 120/6 mg/kg ketamine-xylazine ip. Fluorescence imaging of both ankle joints was performed in pairs 20 min post-injection using the IVIS Lumina III (PerkinElmer, Waltham, MA, USA) in vivo imaging system with the following settings: auto acquisition time, Binning = 2, F/stop = 2, excitation/emission filter: 660/710 nm). Data were analyzed and regions of interests (ROI) were drawn around the ankle joints. Fluorescence was expressed as total radiant efficiency ([photons/s/cm²/sr]/[μW/cm²]).

Citric acid monohydrate, acetyl-β-methylcholine chloride, salbutamol sulfate, atropine, cinnamaldehyde, codeine phosphate, chlorpheniramine maleate (Sigma-Aldrich, Poole, UK), and levodropropizine, (Eurodrug, The Hague, Netherlands), were all dissolved in sterile saline (0.9%; Baxter Healthcare Ltd., Thetford, UK). Tiotropium bromide (Chiesi Farmaceutici, Parma, Italy) was prepared as a stock solution of 6 mM and diluted with sterile saline. All drugs were prepared fresh on the day of use. Roflumilast (CAS162401-32-3; Kemprotec, Carnforth, UK) is poorly soluble in saline and was therefore prepared in neat solutol (2%, Kolliphor HS15, 42966; Sigma-Aldrich) and diluted to the final concentration on the day of use. Capsaicin (Sigma-Aldrich) was prepared as a stock solution of 25 mg/ml (8:1:1 saline:ethanol:Tween 20; Fisher Scientific, Loughborough, UK). HC-030031 (2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide; Chiesi Farmaceutici) was prepared as a stock solution of 300 mg/ml in sterile saline and 1% solutol.

SK-N-AS (1.8 × 10⁶) or SK-N-BE(2) (1.5 × 10⁶) cells in 25% Matrigel™ (BD Biosciences) were injected into the right flank of 6-week-old female athymic nude mice (Fredricks, Charles River, Wilmington, MA, USA) (n = 5 for SK-N-AS vehicle treated, n = 7 for SK-N-AS 792 or 1154 treatment and all SK-N-BE(2) experimental groups). Calipers were used to measure tumors three times per week, and volumes were calculated by the formula (width² × length)/2, where width was the smaller measurement. After tumors reached a volume of 100 mm³, animals were randomized to three groups to receive 100 µL of either vehicle (N,N-dimethylacetamide (DMA, 271012, Sigma Aldrich) and Kolliphor^® HS 15 (Solutol, 42996, Sigma Aldrich)), 792 (50 mg/kg in DMA and Solutol), or 1154 (50 mg/kg in DMA and Solutol) twice daily by oral gavage; dosing based on previously published in vivo cancer models [22 (link)]. Animals were weighed weekly and were humanely euthanized in their home cages with CO₂ and cervical dislocation when control tumors reached 2000 mm³; either 9 (SK-N-AS tumors) or 19 days (SK-N-BE(2) tumors) following treatment initiation, or when IACUC parameters were met. Power analysis was completed prior to the study.

Kolliphor® HS 15 (Solutol® HS 15), a mixture of free polyethylene glycol 660 and polyethylene glycol 660 hydroxy stearate) was purchased from Sigma-Aldrich, Zwijndrecht, Netherlands. Labrafac lipophile WL1394 (triglyceride medium chain, caprylic/capric TG) and soybean phosphatidylcholine (Epikuron™170 (EP) were a kind gift from Gattefosse, Saint-Priest, France. Sodium chloride, methanol, and n-hexane were purchased from Adwic, El-Nasr Pharmaceutical Co., Cairo, Egypt. Hydroxyethyl cellulose (HEC) (2% solution has a viscosity of 640.9 cP) was kindly supplied by Memphis Co., Cairo, Egypt.