*2.2. Synthesis of Peptides*

Peptides were synthesized manually by solid-phase peptide synthesis on Wang resin (0.25 g, 0.52 mmol/g) using the standard protocol of Fmoc/ *<sup>t</sup>*Bu strategy. The first amino acid derivative (5 equivalent to the resin capacity) was attached to the resin with a DIC coupling agent in the presence of 0.5 equivalent DMAP in DMF. The Fmoc group was removed with 2% DBU and 2% piperidine in DMF (four times; 2, 2, 5, 10 min, respectively). For the coupling of the following amino acid derivatives, DIC-HOBt mixture (3 equivalent each) were applied in DMF for 60 min. The ε-amino group of lysine used for the development of a branch was protected with 4-methyltrityl (Mtt) group that could be removed selectively next to the *tert*-butyl type protecting groups with 2% TFA and 2% TIS in DCM (7 times; 1, 1, 3, 3, 5, 10, 30 min, respectively). The aminooxyacetic acid used for the development of an oxime linkage was incorporated in its isopropylidene protected form [25] either to the *N*-terminus or to both *N*-termini (backbone and branch) of the peptides. DIC and HOBt coupling agents were used for this purpose, similar to the coupling of amino acid derivatives. The peptides were removed from the resin by cleavage with 5 mL TFA, containing 0.125 mL distilled water and 0.125 mL TIS (as scavengers). The crude product was precipitated by dry diethyl ether, dissolved in 10% acetic acid, freeze-dried and purified by RP-HPLC (Gradient I.).

### *2.3. Synthesis of Daunomycin Conjugates*

In the first step, the isopropylidene protecting group was removed from the aminooxyacetyl moiety of the purified peptide derivatives by methoxyamine (in 1.5 M concentration) in 0.2 M NH4OAc buffer solution (pH = 5) at RT for 2 h. The reaction took place quantitatively. The unprotected products were isolated by RP-HPLC (Gradient I.). Prior to the conjugation, the solvent was evaporated and then the rest was re-dissolved in 0.2 M NH4OAc buffer solution (pH = 5) and 2 equivalent Dau to the peptide was added to the mixture. The reaction was carried out overnight. The reaction mixture was injected directly to the HPLC in all cases and the conjugates were separated from the excess of Dau by RP-HPLC (Gradient II.)

### *2.4. Reverse Phase High-Performance Liquid Chromatography (RP-HPLC)*

The purification of the crude products was carried out by RP-HPLC using KNAUER 2501 HPLC system (Bad Homburg, Germany) and Phenomenex Luna (Torrance, CA, USA) C18 column (250 × 21.2 mm I.D.) with 10 µm silica (100 Å pore size). Experiments were carried out at a flow rate of 14 mL/min at room temperature. Linear gradient elution was applied. Gradient I: 0 min 5% B, 10 min 5% B, 10.1 min 20% B, 50 min 80% B. Gradient II: 0 min 20% B, 5 min 20% B, 50 min 80% B. Eluent A was 0.1% TFA in distilled water and eluent B was 0.1% TFA in CH3CN-water (80:20, *v*/*v*). Peaks were detected at λ = 220 nm.

Analytical RP-HPLC was performed on a Waters Symmetry (WAT 045905) C18 column (150 × 4.6 mm I.D.) with 5 µm silica (100 Å pore size) as a stationary phase. A linear gradient elution was developed: 0 min 0% B; 2 min 0% B; 22 min 90% B with eluent A (0.1% TFA in water) and eluent B (0.1% TFA in acetonitrile-water (80: 20, *v*/*v*)). A flow rate of 1 mL/min was used at ambient temperature. Samples were applied dissolved in eluent A and 20 µL was injected. Peaks were detected at λ = 220 nm.

#### *2.5. Mass Spectrometry (MS)*

The identification of the peptide analogues and conjugates was achieved by electrospray ionization mass spectrometry (ESI-MS) on a Bruker Daltonics Esquire 3000 Plus (Bremen, Germany) ion trap mass spectrometer, operating in continuous sample injection at 4 µL/min flow rate. Samples were dissolved in ACN-water (50:50 *v*/*v*%) mixture containing 0.1 *v*/*v*% AcOH. Mass spectra were recorded in positive ion mode in the *m*/*z* 50–2000 range.

For the stability and metabolism studies of the conjugates, liquid chromatography–mass spectrometry (LC-MS) analyses were performed on a Q ExactiveTM Focus, high resolution and high mass accuracy, hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) using on-line UHPLC coupling. UHPLC separation was performed on a Dionex 3000 UHPLC system using a Supelco Ascentis C18 column (2.1 × 150 mm, 3 µm). Linear gradient elution (0 min 2% B, 1 min 2% B, 17 min 90% B) with eluent A (0.1% HCOOH in water, *v*/*v*) and eluent B (0.1% HCOOH in acetonitrile/water, 80:20, *v*/*v*) was used at a flow rate of 0.2 mL/min at 40 ◦C. High-resolution mass spectra were acquired in the 200–1600 *m*/*z* range. LC-MS data were analyzed by XcaliburTM software (Thermo Fisher Scientific) and with Origin Pro 8 (OriginLab Corp., Northampton, MA, USA).

#### *2.6. Measurement of Lysosomal Degradation of Conjugates by LC-MS*

Conjugates were dissolved in distilled water in 2.5 µg/µL concentration followed by dilution with 0.2 M NaOAc solution (pH = 5.03) to 0.025 µg/µL. The lysosome-homogenate was prepared from rat liver and contained proteins in 16.6 µg/µL concentration. An aliquot (20 µL) of this stock solution was further diluted with 190 µL 0.2 M NaOAc solution, therefore the final protein concentration was 0.83 µg/µL. To prepare the reaction mixture, 15 µL (0.83 µg/µL) lysosome homogenate was added to 500 µL (0.025 µg/µL) conjugate solution. Furthermore, a control reaction mixture was always prepared which contained 500 µL conjugate solution and 15 µL NaOAc solution only. The solutions were stirred on 600 rpm at 37 ◦C and samples (50 µL) were taken out at 0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 6 h, 24 h, and 72 h. The enzymatic activity was quenched by adding 5 µL formic acid to the samples. After this procedure, samples were frozen immediately at −25 ◦C. Control samples were taken at 0 min, 15 min, 1 h, 6 h, 24 h and 72 h. Composition of the samples was determined by HPLC-MS as described above.

#### *2.7. Cell Cultures*

For the in vitro characterization of conjugates four different tumor cell lines were used: PANC-1 (human pancreatic carcinoma of ductal origin), Colo-205 (human colorectal adenocarcinoma), A2058 (human metastatic melanoma) obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK) and EBC-1 (human lung squamous cell carcinoma) purchased from the Japanese Research Resources Bank (Tokyo, Japan). Normal Human Dermal Fibroblasts (NHDF; Promocell, Heidelberg, Germany) as non-tumorous control cells were also investigated in order to determine the tumor selectivity of the proposed conjugates.

Dulbecco's Modified Eagle Medium (DMEM, Lonza, Basel, Switzerland) was used for the culturing of the PANC-1, Colo-205 and EBC-1 cell lines, while the A2058 cell line was maintained in RPMI 1640 (Lonza). These basal media were supplemented with 10% fetal bovine serum (FBS, Gibco®/Invitrogen Corporation, New York, NY, USA), L-glutamine (2 mmol/L) (Lonza) and 100 µg/mL penicillin/streptomycin (Gibco®/Invitrogen Corporation). The medium of the Colo-205 cell line also contained 4500 mg/L D-glucose (Sigma-Aldrich, St. Louis, MO, USA), while, in case of EBC-1 cells, 1% non-essential amino acids (NEAA, Gibco®/Invitrogen Corporation) and 1 mM sodium pyruvate (Sigma-Aldrich) were also added to the culturing medium. For the cultivation of NHDF cells, Promocell Fibroblast Growth Medium (Promocell, Heidelberg, Germany) was used after adding SupplementMix (supplements necessary for the optimal growth of human fibroblasts, Promocell, Heidelberg, Germany) and the aforementioned antibiotics. All cell lines were grown in a T25 culture flask (Sigma-Aldrich or Eppendorf AG, Hamburg, Germany) in an incubator providing an atmosphere of 37 ◦C and 5% CO2.

#### *2.8. Measurement of the In Vitro Cytotoxicity of Conjugates*

The PANC-1 model cell line exhibits adherent properties under laboratory conditions; therefore, the potential effects of novel antitumor conjugates on cell viability were measured by impedimetry allowing the real-time detection of cell adhesion. This measurement is based on the registration of electrical resistance (impedance, *Z*) in alternating current (AC) field. The living cells transplanted to the gold measuring electrodes are physically insulated by phospholipid bilayer that covers them. This instrumentally measurable property changes (decreases) in response to cellular cytotoxic agents. Our measurements were performed on xCELLigence single plate (ACEA Biosciences, San Diego, CA, USA) dedicated for impedimetric analysis of cellular samples at 37 ◦C and 5% CO2.

During the initial phase of the experiments—the baseline recording—a special 96-well cell culture plate, E-plate (ACEA Biosciences), equipped with measuring electrodes, was pretreated with freshly prepared cell culture medium (for 60 min; sampling frequency: 1 min). Subsequently, PANC-1 cells were plated at a cell density of 10<sup>4</sup> cells/well. During the 24 h incubation, the cells evenly covered the electrodes at the bottom of the wells of the E-plate. The resulting confluent cell cultures were then treated with the test substances at the following final concentrations: 10−<sup>6</sup> , 10−<sup>5</sup> , 10−<sup>4</sup> M. Total treatment duration was 72 h and the sampling rate was 1 min (0–24 h); and then 15 min (48–72 h). In our measurements, three replicates were used, the control was the drug-free medium. The device displays the impedance change in the form of a cell index (CI), which is a relative (to the start of the experiment) and dimensionless index. The CI results were analyzed with xCELLigence RTCA 2.0 software and Origin Pro 8.0 software. Normalized CI values, expressed as a percentage of control, were used to characterize the cell viability and hence the effect of conjugates.

EBC-1 and Colo-205 model cells have weak/negligible adherent properties, and A2058 cells could not produce constant cell index values. Therefore, a colorimetric assay (alamarBlue-assay) was chosen instead of the xCELLigence system to investigate the viability of these model cells treated with the conjugates. Due to the growth characteristics of NHDF cells, alamarBlue-assay was performed also on this cell line.

The protocol for the alamarBlue-assay was similar to the method which was published earlier [26], with some minor modifications. Briefly, the cell seeding occurred on 96-well cell culture plates (Sarstedt AG, Nümbrecht, Germany) at 10<sup>4</sup> cells/well concentration. After a 24 h long culturing period, the treatment was carried out with the conjugates at 10−<sup>4</sup> , 10−<sup>5</sup> and 10−<sup>6</sup> M final concentrations for 24, 48 and 72 h. In the next steps, the alamarBlue reagent (0.15 mg/mL, Sigma-Aldrich) dissolved in phosphate-buffered saline (PBS; pH = 7.2), was added to the wells. After 6 h incubation with the reagent, the fluorescence intensity of the samples was obtained by an LS-50B Luminescence Spectrometer (Perkin Elmer Ltd., Buckinghamshire, UK) or a FluoroskanTM FL Microplate Fluorometer and Luminometer (Thermo Scientific, Waltham, MA, USA) by using the following settings: λex = 560 nm and λem = 590 nm.

Three parallels were performed per treatment group. In the case of controls, an equivalent volume of cell culture media was added to the cell. Fluorescence intensities of the samples treated with various concentrations of conjugates were expressed as a percentage of the fluorescence of control.

#### *2.9. Flow Cytometric Measurement of Cell Surface Binding and Internalization*

Cell surface binding and internalization of the conjugates were performed by flow cytometry (FACS-Calibur, Becton Dickinson, San Jose, CA, USA) based on the detection of the fluorescence activity of Dau (λex = 488 nm, λem = 585 nm) linked to the peptides. Studies of binding and uptake were performed on PANC-1 cells.

Cells were seeded (2.5 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL, 900 µL/well) on 12-well plates, 24 h prior to the treatment with conjugates and free Dau. To distinguish the cell surface binding and internalization of conjugates, the cells were treated with the conjugate solutions at a final concentration of 10−<sup>5</sup> M at two temperatures (37 ◦C and 4 ◦C) in parallel. After the incubation period of 30 min, cells were washed with PBS and were removed from the plate using TrypLE (Thermo Fisher Scientific, Waltham, MA, USA) cell-dissociation reagent, thus avoiding cell surface protein degradation. To stop the enzymatic dissociation, 500 µL of fresh medium was added to the wells after 3–5 min and the cells were transferred to FACS-tubes. After the centrifugation of the cell suspension, the cell pellets were resuspended in PBS (400 µL/tube) and the samples were measured by a flow cytometer.

To determine the fluorescence intensity and to evaluate the results CellQuest Pro (Becton Dickinson) and Flowing2.5.1. (Turku Center of Biotechnology, Turku, Finland) software were used. The measurement was carried out twice with two parallels per treatment group. Samples containing cells treated with fresh cell culture medium at 37 ◦C and 4 ◦C were used as negative controls. The instrument determines the relative fluorescence intensity of Dau built in the conjugates as geometric mean channel (GeoMean) value.

GeoMean values of the 4 ◦C and 37 ◦C samples were corrected with GeoMean values representing the autofluorescence of negative control samples. The fluorescence intensity of cells treated at 4 ◦C is proportional to the amount of conjugates bound to the cell surface, whereas the fluorescence intensity of cells treated at 37 ◦C is composed of the signal of conjugates internalized by the cells and those bound to the cell surface, too. The fluorescence intensity specific for the amount of conjugates internalized by the cells was calculated by subtracting GeoMean values of the cells incubated at 4 ◦C from GeoMean values of the samples incubated at 37 ◦C.

#### *2.10. Experimental Animals*

The Balb/c mice and immunodeficient SCID mice used in these studies were kept as described previously [27] and 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. The permission license for breeding and performing experiments with laboratory animals: PEI/001/1738-3/2015 and PEI/001/2574-6/2015.
