lung cancer targeted photodynamic therapy and imaging

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1 99m Tc-Hematoporphyrin linked albumin nanoparticles for lung cancer targeted photodynamic therapy and imaging Su-Geun Yang, Ji-Eun Chang, Byungchul Shin, Sanghyun Park, Kun Na and Chang-Koo Shim* *Corresponding author. Materials Bovine serum albumin (BSA, fraction V), fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA), 2,4,6-trinitrobenzenesulfonic acid (TNBS) and n-hydroxysuccinimide were purchased from Sigma-Aldrich Co. (St. Louis, MO). Hematoporphyrin HCl and dicyclohexylcarbodiimide (DCC) was obtained from TCI (Tokyo, Japan). A549 (human alveolar epithelial cancer cell line) and CT-26 (murine metastasis colon cancer cell line) were from Korea cell line bank (KCLB, Seoul, Korea). Sodium 99m Tc-pertechnetate solution was supplied by Korea Atomic Energy Research Institute (KAERI, Daejeon, Korea). All other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO) in analytical grade. Preparation of N-hydroxysuccinimide ester of HP 10 g of hematoporphyrin HCl was dissolved in 200 ml of dry dimethyl sulfoxide and 5.0 ml triethylamine was added to this solution. In the presence of dicyclohexylcarbodiimide (9.4 g), hematoporphyrin HCl was reacted with N-hydroxysuccinimide (5.2 g) overnight at room temperature. The by-product, dicyclohexylurea, was removed by filtration. The dimethyl sulfoxide solution was then concentrated under high vaccumm pressure. The resulting paste was dissolved with methanol and mixed again with cold diethyl ether to get precipitation of HP-

2 NHS. The acquired HP-NHS was washed several times with anhydrous ether, dried in vacuum, and yielded as red-brown powder. Figure S1. TEM images of ANP and HP-ANP after negative staining. Quantification of amino groups of ANP and HP-ANP The content of amino groups at ANP and HP-ANP was determined following method which consisted of the incubation of ANP or HP-ANP with an excess of the 2, 4, 6- trinitrobenzenesulfonic acid (TNBS) and the back titration of the unreacted amount of the reagent. ANP was purified three times with distilled water by centrifugation at 20,000 g for 20 min, followed by redispersion with 4% sodium bicarbonate solution (ph 8.5). Two milliliters of the ANP dispersion and 4.0 ml of TNBS solution (4.0 μmol/ml in 4% sodium bicarbonate solution) were added. The reaction mixture was shaken at 400 rpm for 1 h at 40 C. The samples were centrifuged (30,000 g; 1 h) for separating the nanoparticles from the supernatant. In order to measure an unreacted TNBS, 0.9 ml of the supernatant was added to 0.1 ml of valine solution (40 μmol/ml) and incubated at 40 in the dark for 1 h. Then, 5ml of HCl (0.5 mmol/l) were added and the absorbance of solution was measured at 410 nm against a blank sample prepared as described above, but containing 0.1 ml of trichloroacetic acid (1%) instead of valine solution. The content of amino groups on the particle surface was calculated relative to a TNBS reference

3 that was treated in the same manner as described above, using water instead of the ANP dispersion. Figure S2. Residual amino group (%) after treatment of ANP with aldehyde (A) and HP- NHS (B) (n=3~4, mean ± s.d.). Applied amount of aldehyde and HP-NHS was expressed based on the 1 mg of albumin X-ray diffractometry of HP-ANP Crystallinity of HP powder, ANP and HP-ANP was observed on X-ray diffractometry (D-5005, Siements, Karlsruhe, Germany). Dried powder of each sample was mounted on a goniometer and the diffraction angle (2θ) was recorded from 0º to 64º with a scanning speed of 3º/min. Figure S3. Residual amino group (%) after treatment of ANP with aldehyde (A) and HP- NHS (B) (n=3~4, mean ± s.d.). Applied amount of aldehyde and HP-NHS was

4 expressed based on the 1 mg of albumin 99m Tc labeling of HP-ANP Ten milligrams of HP-ANP was thoroughly dispersed with 1 ml water. ph of the solution was adjusted to be 8.0 using 0.1 N NaOH under nitrogen gas purging. Then, 50 μg of stannous chloride dihydrate (Sigma Chemical Co., St. Louis, MO) in 50 μl of 0.1N HCl was added. One milliliters of sterile 99m Tc-pertechnetate was dropped to the vial over a period of 30s. The reaction mixture was incubated at room temperature for 30 min under continuous nitrogen gas purging and purified with three-cycles of centrifugation. The radiochemical purity of 99m Tc-HP-ANP was determined by an instant thin layer chromatography (ITLC) using silica gel coated fiber glass sheets (Gelman Sciences Inc., Ann Arbor, MI USA) and dual solvent systems, namely physiologic saline (0.9% NaCl) and a solvent mixture of acetone, ethyl acetate, water, and ammonium hydroxide (7:3:3:0.3, v/v) as mobile phase. Figure S4. The radiochemical purity of 99m Tc-pertechnetate (A) 99m Tc-HP-ANP (B), which was determined by an instant thin layer chromatography (ITLC).

5 Figure S5. The whole-body scintiimages of rabbit after IV injection of 99m Tc-HP (A) 99m Tc-HP-ANP (B) at a dose of 0.7 mci. Images were captured over 30 minutes after injection. Figure S6. Histology of normal lung (A), and metastatic lung cancer (B, C and D) which were recovered after 1, 2 and 3 weeks of CT-26 cell injection via tail vein, D and F are representative lung images for normal and metastatic lung cancer, respectively.

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