The peptides P6c [6, 8], P6c-PbK and P6c-BN consist of a pentameric coiled-coil domain derived from cartilage oligomeric matrix protein (COMP) , linked to a trimeric coiled-coil domain that was de novo designed [28, 29]. The P6c peptide has a His-tag sequence (22 amino acids, black) at the N-terminus, followed by a pentameric domain (36 amino acids, green) and a trimeric domain (46 amino acids). The P6cPbK peptide was created based on the P6c peptide, but with a lysine residue at its C-terminus. In the peptide P6c-BN, the sequence was extended at its C-terminal end with a flexible linker (GSGSGSGSGS) and nine residues (NQWAVGHLM) from the peptide bombesin. The sequences of P6c, P6c-PbK and P6c-BN are shown in Figure 2.
Plasmids carrying the genes for P6c and P6c-BN were transformed into competent BL21(DE3) pLysS E. Coli cells (Novagen, Madison, WI) by adding 5 μL plasmid to 100 μL cells. After allowing the mixture to cool on ice for 30 minutes, the cells were treated with a heat shock at 42°C for 90 seconds. After cooling the cells on ice for 2 minutes, 800 μL SOC medium were added and the cultures grown at 37°C, under shaking at 300 RPM, for 45 minutes. 100 μL of the cultures were spread on LB-agar plates containing 100 μg/mL ampicillin and 30 μg/mL chloramphenicol and left overnight at 37°C.
Peptide expression and purification
Peptides were expressed by inoculating 50 mL Luria broth containing 100 μg/mL ampicillin and 30 μg/mL chloramphenicol with a single, isolated colony from the transformation plate. The culture was grown overnight at 37°C. The following day, 30 mL were used to inoculate 3 L of Luria broth containing 100 μg/mL ampicillin and 30 μg/mL chloramphenicol. The cells were allowed to grow at 37°C, under shaking at 180 RPM, until the OD600 = ~0.5. Expression was induced by addition of isopropyl β-D-thiogalactopyranoside. The cells were grown for an additional 4 hours and harvested by centrifugation at 4,000 RPM for 15 minutes. Cell pellets were stored at -20 °C until purification.
Cells were resuspended in lysis buffer (9 M urea, 10 mM Tris pH 8.0, 100 mM NaH2PO4) and lysed by sonication. The lysates were clarified by centrifugation at 30,500xg for 45 minutes. Cleared lysate was then passed through a nickel affinity column (GE Healthcare, Waukesha, WI), which was washed first with lysis buffer then with high phosphate buffer (9 M urea, 10 mM Tris pH 8.0, 500 mM NaH2PO4, 20 mM imidazole). The column was then washed with 9 M urea, 20 mM citrate, 100 mM NaH2PO4, 20 mM imidazole, at three different pH values: 6.3, 5.9, and 4.3. Finally, the column was washed with lysis buffer containing increasing concentrations of imidazole. Purity of the collected fractions was determined by SDS PAGE.
The purified peptide was then dialyzed into imidazole-free buffer: 8 M urea, 20 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA.
PEGylation of P6c and P6c-PbK
The PEGylation agent mPEG-NHS (1 kDa) was purchased from Nanocs, Inc., New York, USA. The peptides were concentrated to 1–5 mg/ml in a buffer containing 50 mM sodium phosphate, pH7.5, 0.05% SDS. Then the mPEG-NHS powder was added to the concentrated peptide solution in 50 mM sodium phosphate, pH 7.5, 0.05% SDS and incubated with 1000 rpm shaking at room temperature. In the reaction mixture, the molar ratio of mPEG-NHS to peptide was about 100:1. After the PEGylation reaction, the excess of mPEG-NHS was removed using a dialysis membrane with MWCO of 6000–8000 kDa (Spectrum Laboratories, Inc., CA, USA). The product mixture was analyzed using SDS-PAGE. They were used without further purification.
For the un-PEGylated particles, the two peptides P6c and P6c-BN were first dialyzed into a denaturing buffer (8 M urea, 20 mM Hepes pH 7.5, 150 mM NaCl, 5% glycerol) then filtered with a 0.1 μm filter (Millipore, Billerica, MA). They were then mixed so that the ratio of moles P6c to moles P6c-BN was 50:10. The mixed peptide solution was then diluted with filtered denaturing buffer such that the final peptide concentration was approximately 0.1 mg/ml. Refolding was done by stepwise dialysis into buffers with 20 mM Hepes pH 7.5, 150 mM NaCl, 5% glycerol and successively lower urea concentration. The steps were: 6, 4, 2, 1, 0, 0 M urea. 0 M urea was used twice to ensure removal of any residual urea.
For the PEGylated samples the PEGylated products from the previous steps were directly used for co-assembling with unPEGylated P6c-BN without further purification. The PEGylated products were filtered and then denatured in the same denaturing buffer as is described above. They were then mixed to the appropriate molar ratios with unPEGylated P6c-BN (Table 1) and diluted to approximately 0.1 mg/ml. Refolding was performed by direct dialysis into 0 M urea buffer. The 0 M urea step was performed twice to remove residual urea.
Dynamic light scattering
Dynamic light scattering experiments were carried out on a Zetasizer Nano S Instrument (Malvern, Worcestershire, UK), with a 633 nm He-Ne laser. All measurements were carried out at 25°C in 20 mM Hepes pH 7.5, 150 mM NaCl, 5% glycerol.
Transmission electron microscopy
Samples were negatively stained with 1% uranyl acetate (SPI Supplies, Westchester, PA, USA) and observed with a FEI Tecnai T12 S/TEM at an accelerating voltage of 80 kV (FEI, Hillsboro, Oregon).
Labeling with 99mTc
Labeling with 99mTc was done in two steps. First, 1 ml of [99mTc]pertechnetate, obtained from a Mo/Tc-Generator (Ultratechnecow, Mallinckrodt), was added to a mixture of 4.5 mg sodium boranocarbonate, 2.9 mg borax and 9 mg sodium potassium tartrate and heated at around 95°C for 20 min. The obtained solution with [Tc(CO)3(H2O)3]+ was cooled to room temperature, neutralized with hydrochloric acid and buffered with phosphate buffer at pH 6–7. In a second step 10 – 50 μl of this solution (depending on the activity concentration of the generator eluate) was mixed with 0.1 ml of a suspension of 0.8 mg/ml of the respective SAPN and kept at 40–50°C for 1 h. After cooling, the final product was purified over a PD10 column since the labeling yield was only up to 80%. Two flow-through cells, one equipped with an UV detector, the other with a radioactivity detector, were used to analyze the eluate.
Cell line for pharmacological tests
The human prostate adenocarcinoma cell line PC-3 was obtained from the European Collection of Cell Culture (CRL-1687, ECACC; Salisbury, England). The cells were maintained in DMEM GLUTAMAX-I supplemented with 10% FCS, 100 IU/mL penicillin G sodium, 100 μg/mL streptomycin sulfate and 0.25 μg/mL amphotericin B. The cells were incubated at 37°C in an atmosphere containing 5% CO2 and twice weekly subcultured after detaching with trypsin/EDTA (0.25%).
For internalization, PC-3 cells at confluence were placed in 6-well plates and left to attach overnight. Cells were incubated with the labeled analogues (4 kBq) in culture medium for 0.5, 1, 2, 4 and 24 h at 37°C (final volume 1 mL/well). Nonspecific binding was determined with 10 μM unlabeled BN (residues 1–14) or unlabeled particles. After the different incubation times, cells were washed twice with cold PBS to discard unbound peptide. Surface-bound activity was removed by a 5-min acid wash (50 mM glycin-HCl, 100 mM NaCl, pH 2.8), which was twice applied at room temperature. Afterwards, the plates were washed with cold PBS and the cells were lysed with 1 M NaOH twice. Surface-bound and internalized radioactivity was measured in the gamma counter. Additional experiments were carried out with internalization inhibitors, such as sucrose 0.4 mM, Phenylarsinoxid 0.01 mM, Methyl-β-cyclodextrin 10 mM, Genistin 0.1 mM, Chloroquine 0.1 mM, Cytocalsin B 0.05 mM.
PC-3 cells were placed at different concentrations (0.0625 – 2 million cells/well) in 12-well plates. They were incubated for 2 h at 37°C with 20 kBq 99mTc labeled SAPN-BN (250 μl). Nonspecific binding was determined in presence of 1 μmol/l unlabeled bombesin (1–14). After incubation the supernatant was discarded and the cells washed twice with PBS. To detach the cells the wells were washed 2 times with 500 μl 1 N NaOH and the activity measured in a -counter.
All animal experiments were conducted in compliance with the Swiss animal protection laws and with the ethical principles and guidelines for scientific animal experimentation established by the Swiss Academy of Medical and Natural Sciences. Biodistribution studies were performed with 6- to 8-week-old female CD-1 nu/nu mice (20–25 g) purchased from Charles River Laboratories (Sulzfeld, Germany). For the induction of tumor xenografts, each mouse received subcutaneously 8x106 PC-3 cells in 150 μL culture medium without supplements. The tumors were allowed to grow for at least three weeks. On the day of the experiment, the mice (3 per group) received the labeled nanoparticles intravenously. At 1, 4 and 24 h post injection (p.i.), the animals were euthanized and dissected. Blood, tumors and various healthy tissues and organs were collected and weighed. The amount of radioactivity in each tissue was determined by measuring the samples with the gamma counter and decay corrected (reference time = time of injection). Results are expressed as percentage of injected dose per gram of tissue (%ID/g).