Chemicals, Materials and Spectroscopy
Chemicals and solvents were purchased from Sigma-Aldrich, Fluka, Merck, KMF, LGC Promochem and Roth and were used without further purification. Doxorubicin hydrochloride was purchased from Yick-Vic Chemicals & Pharmaceuticals (HK) China. EMC-Arg-Ser-Ser-Tyr-Tyr-Ser-Arg-OH was custom-made by JPT Peptide Technologies GmbH, Berlin, Germany. Human serum albumin (5% solution) was purchased from Octapharma GmbH, Langenfeld, Germany that contained ∼60% free thiol groups as assessed with the Ellmann's test. The buffers used were vacuum-filtered through a 0.2 μm membrane (Sartorius AG, Göttingen, Germany) and thoroughly degassed with ultrasound prior to use. Enzymatically active PSA was purchased from Calbiochem (Bad Soden, Germany). Xenograft tumor tissues were gratefully received from Dr. I. Fichtner (EPO Exp. Pharmakol. & Onkol. GmbH, Berlin). Lyophilization was performed with a lyophilizer Alpha 2–4 (Christ, Osterode, Germany).
UV/vis-spectrophotometry was carried out with a double-beam spectrophotometer U-2000 from Hitachi. Mass spectra (ESI-MS) was performed with a Thermoelektron LCQ Advantage.
Column chromatographic separations were carried out as flash chromatography using reverse-phase Lichroprep® RP-C18 from Merck with a particle size of 0.040–0.063 mm. HPLC for the determination of purity of PSA5 was performed with a Waters System (pump: Waters 616, detector: Waters 996 Photodiode Array Detector; controller: Waters 600S; auto sampler: Waters 717; Empower PDA software); column: Waters Symmetry® C18 (300–5, 250 × 4.6 mm2) with pre-column; chromatographic conditions: flow: 1.2–1.8 mL/min; mobile phase A: 70% MeCN, 30% 20 mM potassium phosphate (pH 7.0), mobile phase B: 27.5% MeCN, 72.5% 20 mM potassium phosphate (pH 7.0), gradient: 0–26 min 100% mobile phase B; 26–41 min increase to 100% mobile phase A; 41–50 min 100% mobile phase A; 50–60 min decrease to 100% mobile phase B; injection volume: 50 μL.
HPLC for incubation and stability studies with PSA5 and the respective albumin-conjugate was performed with BioLogic Duo Flow System from Bio-Rad (Munich, Germany), which was connected with a Merck F-1050 Fluorescence Spectrophotometer (EX. 490 nm, EM. 540 nm) or a Lambda 1,000 visible detector from Bischoff (at λ = 495 nm); UV-detection at 280 nm; column: Waters, 300 Å, Symmetry C18 (4.6 × 250 mm2) with precolumn; chromatographic conditions: flow: 1.2 mL/min; mobile phase A: 27% MeCN, 73% 4 mM sodium phosphate buffer (pH 3.0); mobile phase B: 70% MeCN, 30% 4 mM sodium phosphate buffer (pH 3.0); gradient: 0–25 min 100% mobile phase A; 25–40 min increase to mobile phase B; 40–50 min 100% mobile phase B; 50–60 min decrease to initial mobile phase; injection volume: 50 μL.
Synthesis of H-Arg-DOXO
Doxorubicin hydrochloride (200 mg, 0.35 mmol), Fmoc-Arg-OH (305 mg, 0.77 mmol) and triethylamine (191 μL, 139.4 mg, 1.38 mmol) were dissolved in 25 mL of anhydrous DMF. After stirring at room temperature for 5 min, HATU (157 mg, 0.41 mmol) was added. After stirring at room temperature for 2 hr, the product was precipitated with 1,000 mL of diethyl ether, washed 3 times with diethyl ether and dried in vacuum to yield 400 mg of a red powder.
The protecting group was removed by dissolving 400 mg Fmoc-Arg-DOXO in 5 mL of 20% piperidine solution (DMF) and stirring for 5 min. The violet product was precipitated with 250 mL of diethyl ether, washed 3 times with diethyl ether and dried in vacuum. The product was purified on a diol column using chloroform/methanol 3:1 + 0.1% TFA. The remaining product on the column was eluted with chloroform/methanol 2:1 + 0.1% TFA to afford 323 mg of H-Arg-DOXO as a red powder after precipitating the combined fractions containing the product with diethyl ether. Mass [ESI-MS: 2.5 kV]: m/z = 700.2 [M + H]+, 722.2 [M + Na]+; HPLC (495 nm): >95%.
Synthesis of H-Ser-Arg-DOXO
H-Arg-DOXO (390 mg, 0.558 mmol), Fmoc-Ser-OH (391 mg, 1.192 mmol) and DIEA (50 mg, 426 μL, 2.512 mmol) were dissolved in 22 mL of anhydrous DMF. After stirring at room temperature for 5 min, HATU (318 mg, 0.837 mmol) was added. After stirring at room temperature for 2 hr, the product was precipitated with 200 mL of diethyl ether, washed 3 times with diethylether and dried in vacuum.
The product was purified on a diol column using chloroform/methanol 5:1 + 0.1% TFA to afford 410 mg Fmoc-Ser-Arg-DOXO as a red powder after precipitating the combined fractions containing the product with diethyl ether.
The protecting group was removed by dissolving 400 mg Fmoc-Ser-Arg-DOXO in 8 mL of 20% piperidine solution (DMF) and stirring for 5 min. The product was precipitated with 400 mL of diethyl ether, washed 3 times with ether and dried in vacuum to afford 312 mg of H-Ser-Arg-DOXO. Mass (ESI-MS: 3 kV): m/z 788.2 [M+H]+; HPLC (495 nm): >95%.
Synthesis of EMC-Arg-Ser-Ser-Tyr-Tyr-Ser-Arg-DOXO (2TFA) (PSA5)
H-Ser-Arg-DOXO (128 mg, 0.163 mmol), EMC-Arg-Ser-Ser-Tyr-Tyr-OH (159.3 mg, 0.184 mmol), HOBt (65.9 mg, 0.487 mmol) and 4-methylmorpholine (71 μL, 65 mg, 0.643 mmol) were dissolved in 10 mL of anhydrous DMF; after stirring at +5°C for 15 min, DIPC (151 μL, 123 mg, 0.975 mmol) was added. After stirring at +5°C for 72 hr, the product was precipitated with diethyl ether, washed 3 times with diethylether and dried in high vacuum. The product was dissolved in methanol/water 3:1 and purified first through size-exclusion chromatography on Sephadex™ LH-20 (Amersham Pharmacia Biotech AB) using methanol and in a second purification step on a reverse-phase column using acetonitrile/water + 0.1% TFA 30:70 to afford 152 mg of PSA5 as a red powder after lyophilizing the combined fractions containing the product. Mass (LC-MS-pos. ESI. 1.5 kV): m/z 1,636.5 [M]+, 1,749.7 [M++CF3COO−], 1,750.7 [M++CF3COOH]; HPLC (495 nm): >98%.
Synthesis of the albumin conjugate of PSA5
1.9 mg of PSA5 were dissolved in 2 mL human serum albumin (5% solution from Octapharma) and the solution was incubated at 37°C for 1 hr. The albumin conjugate was obtained after subsequent size-exclusion chromatography (Sephacryl® S-100; Tris-buffer: 50 mM Tris-HCl - pH 7.4). The content of anthracycline in the sample was determined using the ε-value for doxorubicin [ϵ495 (pH 7.4) = 10,650 M−1 cm1]. The concentration of PSA5 in the conjugate was adjusted to 400 ± 50 μM by concentrating the sample with CENTRIPREP®-10-concentrators from Amicon, FRG (4°C and 4,500 rpm). Samples were kept frozen at −20°C and thawed prior to use.
Incubation studies with human blood plasma
Human blood plasma (EDTA stabilized) was taken from healthy volunteers. 1.8 mg of PSA5 were dissolved in 200 μL of 10 mM sodium phosphate/5% D-glucose buffer pH 6.4 to give a 5 mM stock solution of PSA5. 30 μL was added to plasma preincubated at 37°C at a final concentration of 250 μM and the samples were incubated for 5 min, 4, 20 hr and for 24 hr at 37°C. A 50 μL sample for each time point was analyzed by HPLC.
In a further experiment 500 μL of human blood plasma was preincubated with 0.4 mg of EMC for 1 hr. Subsequently, 10 μL of PSA5 and 190 μL of the preincubated plasma was mixed and incubated for 10 min. 50 μL samples were analyzed with HPLC.
Incubation studies of the albumin conjugate of PSA5 with PSA at pH 7.4
100 μL of the albumin conjugate stock solution (420 μM) of PSA5 were mixed with 50 μL PSA (200 μg/mL) and 50 μL of buffer (Tris-buffer pH 7.4). The samples were incubated at 37°C. After 5 min, 1.5, 3 and 24 hr samples were collected and 50 μL of each sample were diluted 1:1 to a final concentration of 110 μM and analyzed by HPLC.
Preparation of LNCaP tumor tissue homogenates at pH 7.4
For obtaining LNCaP carcinoma tissue homogenates, all steps were carried out on ice where possible. Tissue of LNCaP xenograft tumors were cut into small pieces, and 200 mg samples were transferred in a 2 mL Eppendorf tube to which was added 800 μL of homogenate buffer (50 mM Tris-HCl buffer, pH 7.4 containing 1 mM monothioglycerol). Homogenization was carried out with a micro-dissmemberator at 3,000 rpm for 3 min with the aid of glass balls, and the samples were then centrifuged at 5,000 rpm for 10 min and kept frozen at −78°C prior to use.
Incubation studies of the albumin conjugate of PSA5 with LNCaP tissue homogenates
The albumin conjugate of PSA5 was incubated with LNCaP tissue homogenate at 37°C at a final concentration of 100 μM and chromatograms recorded at 495 nm using reverse phase HPLC at the time points stated in the figures.
In vitro cellular experiments
For the PSA/luciferase measurement, cells were plated at 200,000 cells/well into a 6-well plate. After 24, 48, 72 and 96 hr, medium supernatant was removed and stored at −80°C. Cells were then trypsinized, counted and cells frozen as a cell pellet. Once all samples were collected, the cell pellets were lysed in 90 μL of luciferase assay buffer (25 mM TRIS-phosphate pH 7.8; 2 mM EDTA; 2 mM DTT; 0.1% Triton X-100), and 10 μL of the undiluted, as well as 1/10 and 1/100 dilutions were measured in a Luminometer (BMG Lumistar) using the luciferase substrate from Promega (Promega E4550), according to the manufacturer's instructions. PSA values were determined nephometrically with a microparticale immunoassay from Abbot.
For IC50 measurements, 0.2 × 104 cells were plated per well in a 96-well plate. 24 hr later, serial dilutions of the drugs were added in triplicates. alamar blue was added to the medium after 69 hr, and the cells were incubated for another 3 hr, before the fluorescence was measured at 590 nm. Cells were then lysed in 100 μL of luciferase assay buffer, and an aliquot of the lysate was assayed for luciferase activity.
All animal experiments were performed in accordance to German Animal License Regulations (Tierschutzgesetz) identical to UKCCCR Guidelines for the welfare of animals in experimental neoplasia.18 Male SCID (C.B-17/IcrHanHsd-Prkdc-scid) mice were obtained from Harlan Winkelmann GmbH, Germany.
Mice were anesthetized with isofluorane, positioned and fixed on the back. The coat of the abdominal area was shaved and the skin was disinfected with alcohol (70%). A 3–4-mm incision was made alongside the linea alba in the lower abdomen using the cranial edge of the bladder for orientation. Seminal vesicles and prostate were pulled out partially and exposed carefully. 2 × 106 tumor cells, suspended in 20 μL PBS, were injected into the anterior part of the prostate using a 29-gauge needle syringe. The exteriorized organs were reinserted into the abdomen, the abdominal wall was closed using 5-0 Dexon sutures (DEXON®, B.Braun-Dexon, Spangenberg, Germany), and the skin was again disinfected using a Dibromol tincture (Trommsdorf GmbH, Germany).
Measurement of in vivo bioluminescence
The animals were injected with 100 μL of D-luciferin (Synchem OHG, Germany), and anesthesized using isofluorane. 10 min. after the injection, the mice were transferred into a NIGHTOWL camera (Berthold Technologies) equipped with isofluorane adaptors. An overview picture of the mice as well as 2 exposures, 5 min at 10 × 10 binning and 1 min at 2 × 2 binning were taken.
Raw, unmodified images from the camera were imported into Adobe Photoshop 7.0® and transformed to 8-bit indexed color format. To help visualizing the primary tumor, levels were set to 0-25, and the color table “spectrum” was applied (the latter 2 steps did not change the information content of the files, only helped to localize the signal for the graphical quantification in ImageJ). The files were then opened in imageJ, and the mean pixel intensity of 150-pixel squares drawn at the site of the primary tumor was measured; the results were used to generate growth curves.
Necropsy and luciferase assays of mouse organs
Mice were sacrificed, the abdominal cavity was opened and a picture of the tumor in situ was taken. The primary tumor was resected, measured and its volume was calculated using the formula V = a2 × A/2 (a - small diameter, A - large diameter). Tumors were weighed and cut in 2 pieces. One was shock frozen in liquid nitrogen for histology, the other homogenized in 2 mL of luciferase lysis buffer. To obtain a quantitative analyze of the metastatic spread into potential target organs, the kidney, liver, spleen and lungs as well as a sample of the femur, were resected from the animals taking great care to prevent cross-contamination of the tissues. Whereas a part of these organs were also cryo-preserved, samples from the intestine and the inguinal lymph nodes were only analyzed in luciferase assays. After homogenization, insoluble material was spun 10 min at 3,000 rpm in a Heraeus Megafuge 2.0. 5 μL were checked for protein concentration using a Bradford assay (Sigma B6916) with BSA serving as a standard protein, and 10 μL were measured in a luciferase assay (Promega E4550).
7 μm cryo-sections were prepared, and either processed for immunohistochemistry or assayed for luciferase activity, followed by H&E staining. For the former, the sections were fixed using 4% paraformaldehyde and stained for human cytokeratin using the biotinylated mouse monoclonal pan cytokeratin 1–8 antibody from Progen (Cat # 61506) according to the instructions from the manufacturer. In the latter case, the sections were overlaid with a luciferase substrate mix (Promega E4550), exposed under a Nightowl camera (Berthold) for 2 min. at 1 × 1 binning, washed in water and stained with H&E to reveal the morphology of the sections.