Fmoc-Rink-Amide MBHA resin, 1-hydroxybenzotriazole hydrate (HOBt), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), N,N′-diisopropylcarbodiimide (DIC), triisopropylsilane (TIS), piperidine, trifluoroacetic acid (TFA), diisopropylethylamine (DIPEA), and ninhydrin were purchased from Sigma-Aldrich Kft (Budapest, Hungary). Daunorubicin hydrochloride was provided from IVAX (Budapest, Hungary). N,N-dimethylformamide (DMF), dichloromethane (DCM) and diethyl ether (Et2O) were delivered by Molar Chemicals Kft (Budapest, Hungary). All the amino acid derivatives used for the preparation of the conjugates were obtained from Merck KGaA (Darmstadt, Germany) or Iris Biotech GmbH (Marktredwitz, Germany) with the highest available purity.
Preparation of Cyclic NGR Peptide-Daunorubicin Conjugates
The cyclic NGR peptide-daunorubicin conjugates were prepared by a combination of solid phase peptide synthesis and chemoselective ligation (oxime bond formation) in solution as described in Tripodi et al. . The crude peptides and conjugates were purified on a KNAUER 2501 HPLC system (KNAUER, Bad Homburg, Germany) was applied with a semi-preparative Phenomenex Luna C18 column (250 mm × 21.2 mm) with 10 μm silica (100 Å pore size) (Torrance CA). Linear gradient elution (0 min 5% B; 60 min 90% B) with eluent A (0.1% TFA in water) and eluent B (0.1% TFA in MeCN-H2O (80:20, v/v)) was used at a flow rate of 4 mL/min. The resulting fractions were lyophilized. Electrospray Ionization (ESI)-mass spectrometric analyses were carried out on an Esquire 3000+ ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany). The freeze-dried bioconjugates were directly used for the in vitro and in vivo studies.
Stability in Murine Plasma
NGR-Dau conjugates were dissolved in ddH2O, murine plasma was added, the obtained final concentration of the conjugates was 10 μM. Samples were incubated at 37 °C, and aliquots were taken at 0.5, 1, 2, 4, and 8 h. The experiment was concluded by addition of 10 μL pure acetic acid. The low molecular weight samples were analyzed by LC-MS, while the high molecular weight murine plasma proteins were removed via ultracentrifuge filters with a cut-off of 10 kDa. The same measurements were performed in ddH2O as a control (data not shown).
Cell Lines and Culture Conditions
KS (Kaposi’s sarcoma) derived from human Kaposi sarcoma  and HT-29 (human colorectal adenocarcinoma) cell line obtained from ATCC were cultured in RPMI 1640 medium with glutamine (Roswell Park Memorial Institute Medium, Lonza, Basel, Switzerland), and MRC-5 (normal fibroblast) cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium, Lonza). All media were supplemented with 10% heat-inactivated FBS (Fetal Bovine Serum, Euroclone, Milan, Italy), and with 1% penicillin/streptomycin (Sigma-Aldrich). Cells were cultured in sterile T25 or T75 flasks with ventilation cap (Sarstedt, Nümbrecht, Germany) at 37 °C in a humidified atmosphere with 5% CO2.
In Vitro Antiproliferative Activity of NGR-Dau Conjugates and Free Dau
For evaluation of in vitro antiproliferative activity of NGR-Dau conjugates and free Dau, cell viability was determined by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (Sigma Aldrich)). After standard harvesting of the cells by trypsin-EDTA (Lonza), 5 × 103 till 10 × 103 cells per well depending on cell line, were seeded in serum containing growth medium to 96-well plates and incubated. After 24 h, cells were treated with various concentrations of conjugate 1 and 2 (32 nM-100 μM) or free Dau (0.1-10 μM), dissolved in serum containing medium, and incubated under standard conditions. Control wells were treated with serum containing medium. Two treatment regimens were used. According to the first type, after 24 h of treatment, cells were washed with serum free medium, and then cultured in serum containing medium for an additional 48 h. In case of the second type, cells were treated for 72 h continuously. Afterward, MTT assay was performed in order to determine cell viability, by adding 20 μL of MTT solution (5 mg/mL in PBS) to each well and after 2 h of incubation at 37 °C, the supernatant was removed. The formazan crystals were dissolved in 100 μL of a 1:1 solution of DMSO (Sigma-Aldrich):EtOH (Molar Chemicals) and the absorbance was measured after 15 min at λ = 570 nm by using a microplate reader (Bio-Rad, model 550, Hercules, CA, USA). The IC50 values of the conjugates and free drug were calculated using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). The experiments were done in triplicate, and each experiment was repeated two times.
CD13 Cell Surface Expression Level Determination by Immunocytochemistry
CD-13 expression was detected by confocal microscopy. KS and HT-29 cells were seeded (105 cells/well) to coverslip-containing (Assistant, Karl Hecht GmbH&Co KG, Sondheim/Rhön Germany) 24-well plates (Sarstedt) one day before immunostaining. Nuclei were stained with Hoechst 33342 solution (0.2 μg/mL, Thermo Fisher Scientific, Rockford, IL, USA, diluted in serum-free medium) for 10 min at 37 °C. After washing, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 min at 37 °C that was followed by blocking with 3% Bovine Serum Albumine (BSA, Sigma-Aldrich, dissolved in PBS) for 1 h at room temperature. Anti-CD-13 antibody (clone: WM-15, FITC-conjugated, eBioscience, 1:100, diluted in 1% BSA containing PBS) was added to the wells overnight at 4 °C. After washing three times with PBS, coverslips were mounted to cover glasses using Mowiol 4-88 (Sigma-Aldrich). Imaging was carried out using a ZEISS LSM-710 system (Carl Zeiss microscopy GmbH) with a 40x/1.4 Plan-Apochromat oil immersion objective. Images were processed with ZEN (Carl Zeiss microscopy GmbH).
Cellular Uptake Determination by Flow Cytometry
The cellular uptake of the bioconjugates was studied on KS and HT-29 cells. Cells were seeded (1.5 × 105 cells/well) to 24-well plates (Sarstedt), incubated for 24 h at 37 °C, and treated with conjugates at concentrations 2, 10, 50 and 100 μM for 6 h. After harvesting, cells were washed with PBS. Fluorescence intensity was detected using the PE-A channel of Attune NxT Flow Cytometer (Thermo Fisher Scientific). Number and proportion of the cells with intracellular fluorescence were evaluated and calculated using Attune NxT 2.6. software (Thermo Fisher Scientific).
Adult inbred BALB/c mice from a specified pathogen free (SPF) breeding of the National Institute of Oncology (Budapest, Hungary) were used in acute and chronic toxicity studies. Mice were kept in a sterile environment in Makrolon® cages at 22-24 °C (40-50% humidity), with a lighting regulation of 12/12 h light/dark. The animals had free access to tap water and were fed with a sterilized standard diet (VRF1, autoclavable, Akronom Kft., Budapest, Hungary) ad libitum.
The immunodeficient SCID mice on a C.B.-17 background were bred in specific opportunistic and pathogen free isolator breeding rooms. The breeding isolator was supplied with corn-cob bedding and standard VRF1 rodent chow and with acidified (pH = 3) sterilized distilled water. The mice from the breeding rooms were used for the subcutaneous model of KS and orthotopic model of human colon cancer. They were held in filter-top boxes in the experimental barrier rooms, and every box-opening was performed under a Class 100 laminar-flow hood. The animal housing density was in accordance with the international recommendations. The cage components, corn-cob bedding and food (VRF1 from Special Diet Services) were steam-sterilized in an autoclave (121 °C, 20 min). The animals used in these studies were cared for according to the “Guiding Principles for the Care and Use of Animals” based upon the Helsinki declaration, and they were approved by the local ethical committee. Permission license for breeding and performing experiments with laboratory animals: PEI/001/1738-3/2015 and PEI/001/2574–6/2015.
Acute and Chronic Toxicity Studies of NGR Conjugates
In order to determine toxicity of conjugates on healthy animals in vivo, acute and chronic toxicity studies were performed. In acute toxicity study, adult BALB/c male mice (26-32 g) were treated by a single intraperitoneal (i.p.) injection of both conjugates at the start of the experiment, administrating 4 different doses: 25, 12.5, 6.25 and 3.125 mg/kg Dau content (3 mice per group). In chronic toxicity studies, adult male BALB/c mice (25-31 g) were treated with both conjugates at dose of 10 mg/kg Dau content on day 1, 3, 7, 9 and 11 (5 treatments, 3 mice per group). The toxicity was evaluated on the basis of life span, behavior and appearance of the mice, as well as body weight. Parameters were followed for 14 days.
Mouse Model of Subcutaneous Kaposi’s Sarcoma
Kaposi’s sarcoma (KS) cells were injected into SCID female mice (19-24 g) (s.c.), 3 × 106 cells per animal in volume of 200 μL M199 medium per animal. Treatment started 35 days after cell inoculation when average tumor volume was 66 mm3, by i.p. administration. Four groups by 5 animals were established and treated with the following doses and schedule: control group was treated with sterile water (Pharmamagist Kft., Budapest, Hungary) as solvent due to better solubility of Dau and conjugates than in saline solution; free Dau group was treated with a dose of 1 mg/kg on days 35, 42, 49, 56 and 63 after cell inoculation, while the groups treated with conjugates 1 and 2 were administered with a dose of 10 mg/kg Dau content, i.e. 33.8 and 33.5 mg/kg of each conjugate respectively, on days 35, 37, 39, 42, 45, 49, 52, 56, 59, 63 and 66 after cell inoculation. Treatment volume was 0.2 mL/animal. Animal weight and tumor volumes were measured initially when the treatment started and at periodic intervals according to the treatment schedule. A digital caliper was used to measure the longest (a) and the shortest diameter (b) of a given tumor. The tumor volume was calculated using the formula V = ab2 × π/6, whereby a and b represent the measured parameters (length and width). The experiment was terminated on day 70 after tumor transplantation (day 36 of treatment). The mice from all groups were sacrificed by cervical dislocation. Their primary tumors and livers were harvested and weighed.
Mouse Model of Orthotopic Human Colon Adenocarcinoma
HT-29 human colon cancer cells were injected into SCID female mice subcutaneously (s.c.), 3 × 106 cells per animal in volume of 200 μL M199 medium per animal, in order to establish tumor for transplantation. After 2 weeks, the mice with palpable tumors were sacrificed by cervical dislocation, and the subcutaneous tumor was dissected out aseptically. Tumor pieces of 2 mm3 were transplanted orthotopically, under aseptic conditions into anesthetized (narcotic mixture: tiletamine, zolazepam, xylazine, butorphanol) SCID female mice (19-25 g). A small midline incision (0.5 cm) was made and the colorectal part of the intestine was exteriorized. Serosa of the site where the tumor pieces were to be implanted were removed. Tumor tissue fragments of HT-29 human colon tumor were implanted on the top of the animal intestine; an 8/0 surgical (polypropylene) suture was used to suture it on the wall of the intestine. The intestine was returned to the abdominal cavity, and the abdominal wall was closed with 4/0 surgical (polyglycolic acid) sutures. The wound was sterilized and the animals were kept in a sterile environment. On the next day, no sign of pain and/or stress of the mice was observed. The treatments started 6 days after tumor transplantation by i.p. administration of the compounds dissolved in distilled water for injection. 8 mice per group were used.
One group of mice were treated with free Dau (1 mg/kg body weight) on days 6, 13 and 20 after tumor transplantation. Animal groups treated with compounds 1 and 2 were administered with a dose of 10 mg/kg Dau content (33.8 and 33.5 mg/kg of each conjugate, respectively) on days 6, 8, 10, 13, 17, 20 and 24 after tumor transplantation. Control group was treated with sterile water as solvent in 0.2 mL volume per animal. The experiment was terminated on day 27 after tumor transplantation (day 22 of treatment). Daunorubicin treated group was terminated on day 24 after tumor transplantation (day 19 of treatment) due to significant weight loss of the animals. The mice from all groups were sacrificed by cervical dislocation. Their primary tumors and livers were harvested and weighed, while metastases were counted in other organs.
Determination of the Proliferative Index and Vascularization in Tumor Tissues
The routinely formalin-fixed tumors were dehydrated in a graded series of ethanol, infiltrated with xylene and embedded into paraffin at a temperature not exceeding 60 °C. Two microns thick sections were mounted on Superfrost slides (Thermo Shandon, Runcorn, UK) and manually deparaffinized. To block endogenous peroxidase activity, slides were treated for 20 min at RT with 3% H2O2 in methanol. Slides were immersed in 6% citrate buffer (pH = 6) and exposed to 98 °C water bath for 40 min. Afterwards, slides were primarily treated with antibody against human KI-67 (DAKO, Glostrup, Denmark, 1:40) and endothelial marker CD31 (Dianova, Hamburg, Germany, 1:20) incubated for 1 h at RT. After washing, Biotinylated Link (Dako) secondary antibody was applied for KI-67 samples for 10 min at RT, while rabbit anti-rat IgG (Novus Biologicals, Centennial, CO, USA) was applied for CD31 samples for 1 h at RT. For visualization of KI-67 samples, supersensitive one step polymer HRP (Biogenex, Fremont, CA, USA) was used with 3-amino-9-ethylcarbazole (AEC) as chromogen, while for visualization of CD31 samples match 2 rabbit-HRP polymer (Biocare Medical, Concord, CA, USA) with AEC (Vectorlabs, Burlingame, CA, USA) were used. Staining without the primary antibody served as negative control. The KI-67-positive tumor cells were counted manually per fields of vision under light microscope (400-fold magnification), and 3 fields of view per tumor were evaluated. Proliferation index was calculated as percentage of KI-67 positive cells from all cells in the field of view. The CD31-positive blood vessels were counted manually using light microscope (200-fold magnification), whereby 3 fields of view per tumor were evaluated, and blood vessel density was calculated as number of blood vessels per mm2.
The statistical analyses were performed by GraphPad Prism 6 (GraphPad Software) using the non-parametric Mann-Whitney (independent samples) test. The experimental data were filtered by Gaussian statistics where P-values lower than 0.05 were considered statistically significant.