As previously described by Pleiko et al. [52], peptide-encoding region of bacteriophage genome was amplified by PCR using Phusion Green Hot Start II High-Fidelity DNA Polymerase (#F537L, Thermo Scientific) in 25 μL reaction volume. Cycling conditions: denaturation at 98 °C for 30 s, followed by 25 amplification cycles (10 s at 98 °C, 21 s at 72 °C), and final elongation (72 °C for 5 min). Polymerase chain reaction (PCR) products were purified using AMPure XP Bead Based Next-Generation Sequencing Cleanup system (Beckmann Coulter, A63881) using 1.8 μL of beads per 1 μL of PCR products. Purified PCR products were quantified using Agilent Bioanalyzer 2100 Instrument using the High sensitivity DNA Kit (#5067–4626, Agilent). Ion Torrent Emulsion PCR and enrichment steps were performed using Ion PGM HiQ View OT2 kit (#A29900, Life Technologies). High throughput sequencing (HTS) was performed using Ion Torrent™ Personal Genome Machine™ (ION-PGM) using Ion PGM HiQ View sequencing kit (#A30044, Life Technologies) and Ion 316v2 chips (#448,149, Life Technologies). The FASTQ sequence files were converted to text files and translated using in-house developed python scripts.

Enzyme/uPA pretreatment

Per 50 µL of peptide-AgNPs (5.6 nM) or 50 µL of phage (1 × 109 pfu/mL) 2–32 µL of uPA (10,000 U/mL; #672,112, Sigma-Aldrich) was added and incubated at 37 °C for 1 h.

In enzymatic cleavage specificity experiments, AgNPs were treated with 2 mg/mL of cathepsin B (#SRP0289, Sigma-Aldrich), 1.3 mg/mL cathepsin K (in-house), 1 mg/mL hyaluronidase (#H3884, Sigma-Aldrich) or 1X TrypLE™ Express (#12,604–019, Gibco) in addition to 32 µL of uPA (10,000 U/mL; #672,112, Sigma-Aldrich) in the same experimental conditions.

Cell-free AgNP binding experiments

Cell-free binding studies were done as described in chapter 4.4 “Cell-free phage display”, except 2 mg/mL of hexahistidine-tagged recombinant b1 domain of NRP-1 (in-house) or C-domain of TNC (TNC-C; in-house) was used for coating. For NRP-1 b1, Tris buffer (50 mM, pH 7.0) containing 5 mM imidazole, 1 M NaCl and 0.05% Igepal CA-630 (all from Thermo Scientific Inc.), or for TNC-C and negative control NRP-1 b1 mutant (mut), PBS containing 0.05% Igepal CA-630 (Thermo Scientific Inc.), was used for the protein binding step. For incubations, 0.5 nM peptide-AgNPs were used, and eluted proteins with bound peptide-AgNPs were quantified by UV–vis spectrometry at 415 nm using a Nanodrop 2000c spectrophotometer (Thermo Scientific Inc.).

Peptides

All synthetic peptides used herein were ordered as powder with a purity of > 95%, and reconstituted in PBS. Sequences of peptides are shown in Table 2 (all from TAG Copenhagen, Denmark).

Table 2 Synthetic peptidesSynthesis and functionalization of AgNPs

Silver nanoparticles were synthesized according to the Lee and Meisel citrate method [53]. Surface functionalization of AgNPs was done as previously published [42, 54]. Briefly, AgNO3 (360 mg; #209,139, Sigma-Aldrich Co., LLC, Darmstadt, Germany) or 107AgNO3 or 109AgNO3 (Isoflex USA, San Fransisco, CA, USA) was added to ultrapure Milli-Q (MQ) water (2 L; resistivity 18 MΩ cm−1) in a flask cleaned with a piranha solution (H2SO4/H2O2). Next, trisodium citrate hydrate (400 mg; #25,114, Sigma-Aldrich Co., LLC) was dissolved in MQ water (40 mL) and added to the vessel. The solution was boiled for 30 min in the dark. The resulting Ag-citrate was used directly in the next step.

Next, NeutrAvidin (NA; #31,055, Thermo Scientific Inc., Washington, USA) was modified with a OPSS-PEG(5 K)-SCM linker (OPSS; JenKem Technology USA Inc., Texas, USA) according to the procedure described by Braun et al. [42] Subsequently, NeutrAvidin-OPSS (3.9 mL, 2.9 mg/mL) was added to Ag-citrate (500 mL). After 2 min, 4-morpholineethanesulfonic acid hemisodium salt (5 mL, 0.5 M in MQ water; #M0164, Sigma-Aldrich Co., LLC) was added. The pH of the solution was adjusted to 6.0, and the solution was kept at 37 °C for 24 h. The solution was brought to room temperature (RT) and 10X phosphate buffered saline (50 mL; in-house) was added, followed by Tween® 20 (250 µL; #P9416, Sigma-Aldrich Co., LLC). The solution was centrifuged at 12,200 × g for 1 h at 4 °C, the supernatant was removed, and the particles were resuspended in PBST (0.005% Tween® 20 in PBS). Next, tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP; #646,547, Sigma-Aldrich Co., LLC) was added to a final concentration of 1 mM, followed by a 30-min incubation at RT. Then, lipoic acid-PEG(1 k)-NH2 (#PG2-AMLA-1k, Nanocs Inc., New York, USA) was added to a final concentration of 5 µM, and the mixture was incubated at RT for 2.5 h. The solution was centrifuged at 17,200 × g for 20 min at 4 °C, the supernatant was removed, and the particles were resuspended in PBST to half of the initial volume. The AgNP solution was filtered through a 0.45 µm filter and stored at 4 °C.

NHS-functionalized CF555 dye (#92,130, Biotium Inc., California, USA) was coupled to the NH2 groups on the AgNPs. For this, NHS-CF555 (5 µL, 2 mM) in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co., LLC) was added to AgNA (500 µL), followed by an overnight incubation at 4 °C. The particles were washed 3 times by centrifugation at 3,500 × g for 10 min at 4 °C, followed by resuspension of the particles in PBST. Next, biotinylated peptides were coupled to the particles by adding peptide (10 µL, 2 mM in MQ water) to AgNPs (500 µL), followed by incubation at RT for 30 min. The AgNPs were washed, 0.2 µm filtered and stored at 4 °C in the dark.

Etching of AgNPs

To eliminate extracellular AgNPs, we exposed the live cells to a cell membrane-impermeable biocompatible etching solution (10 mM), 1: 1 solution of tripotassium hexacyanoferrate(III) (K3Fe(CN)6; CAS# 13,746–66-2, Sigma-Aldrich, Co., LLC) and sodium thiosulfate pentahydrate (Na2S2O3; CAS# 10,102–17-7, Sigma-Aldrich, Co., LLC) in PBS. After incubation with AgNPs, the cells were treated with the etching solution (400 µL, 10 mM) for 3 min at RT, followed by 2 washes with PBS. During etching, the AgNPs are dissolved by oxidation with hexacyanoferrate, and the Ag+ ions form a complex with the thiosulfate component. After the treatment only membrane-protected intracellular AgNPs remain intact and detectable.

Cell culture

Prostate carcinoma cells (PPC1; ATCC) and melanoma cells (M21; ATCC) were grown as attached cultures in tissue culture-treated flasks (Corning Inc., USA) in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Lonza Ltd, Basel, Switzerland) with added 10% fetal bovine serum (FBS; Thermo Scientific Inc.), penicillin (Thermo Scientific Inc.) and streptomycin (Thermo Scientific Inc.).

Glioblastoma cells (U87-MG; ATCC) were grown as attached cultures in tissue culture-treated flasks (Corning Inc., USA) in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Lonza Ltd, Basel, Switzerland) containing 10% FBS (Thermo Scientific Inc.), 100 IU/mL penicillin (Thermo Scientific Inc.), 100 IU/mL streptomycin (Thermo Scientific Inc.), 1% sodium pyruvate (Sigma-Aldrich) and 1% non-essential amino acids (Sigma-Aldrich).

WT-GBM and VEGF-KO-GBM cell lines [55], which were a gift from Gabriele Bergers (Leuven, Belgium), were cultured in MEM with Earl's salts (Capricorn Scientific, Germany) supplemented with 100 IU/mL penicillin (Thermo Scientific Inc.), 100 IU/mL streptomycin (Thermo Scientific Inc.), 1% sodium pyruvate (Sigma-Aldrich), 0.01 M HEPES, 0.6% glucose (Applichem, USA) and 5% of heat-inactivated fetal bovine serum (GE Healthcare, UK). NCH421k cells [56], also a gift from G. Bergers, were cultivated in neurobasal medium complemented with B27 neurobasal supplement (Thermo Fisher Scientific, USA), 100 IU/mL of penicillin/streptomycin (Thermo Scientific Inc.), 0.4 mM Glutamax, 20 ng/ml basic FGF, 20 ng/ml EGF and 40 μg/ml of heparin (all Sigma-Aldrich, USA).

All cell lines were cultured at 37 °C in humidified atmosphere with 5% CO2. Before use, cells were rinsed with DPBS, dissociated with Cellstripper (Thermo Scientific Inc.), counted with a TC20™ Automated Cell Counter (Bio-Rad Laboratories AB, Sundyberg, Sweden), and diluted with supplemented medium or PBS as needed.

In vitro experiments with AgNPs

Semiconfluent cells cultured on coverslips (d = 12 mm; Paul Marienfeld GmbH & Co. KG) were incubated with CF555-labeled AgNPs (1.5 nM final concentration) at 37 °C for 1 h. Optionally, prior to adding the AgNPs, the AgNPs were preincubated with 2 µL of uPA (#672,112, Sigma-Aldrich) reconstituted at 10,000 U/mL in MQ water per sample for 1 h at 37 °C in 1.5 mL Eppendorf tubes (Sigma-Aldrich). After incubation with the AgNPs and optional etching (see above), the cells were washed 3 times with 500 µL of DPBS, fixed with − 20 °C methanol (MeOH; Sigma-Aldrich) for 1 min, washed 2 times with DPBS, and counterstained with DAPI (500 µL, 1 µg/mL; Thermo Scientific Inc.). For confocal microscopy imaging, microscopy slides (76 × 26 mm, Glaswarenfabrik Karl Hecht GmbH & Co. KG) were coverslipped using an aqueous mounting medium Fluoromount-G (Electron Microscopy Sciences).

Animal work

Athymic nude mice were purchased from the Tartu University Laboratory Animal Centre (Estonia). Female mice were used in all in vivo experiments if not stated otherwise. For induction of orthotopic GBM xenografts in nude mice, 7 × 105 (WT-GBM, VEGF-KO-GBM) or 3 × 105 (NCH421k, U87-MG) cells were implanted intracranially in the right striatum of the brain (coordinates: 2 mm laterally and 2 mm posteriorly from bregma and at 2.5 mm depth). Intracranial tumors were allowed to develop for 6–7 (WT-GBM), 12–14 (VEGF-KO-GBM, U87-MG), or ~ 30 (NCH421k) days before performing experiments, and mice were monitored for signs of tumor burden.

For AgNP and FAM-peptide biodistribution studies, tumor-bearing nude mice were i.v. injected with 100 μL of AgNPs (11.3 nM) or FAM-peptide (200 μg; TAG Copenhagen, Denmark). After 3 h (AgNP) or 1 h (FAM-peptide) of circulation, the mice were anesthetized with intraperitoneally (i.p.) injected 350 μL of 0.1 mg/kg dexmedetomidine and 75 mg/kg ketamine dissolved in saline, and perfused with PBS. The brain, tumor, lungs and liver were collected, frozen in OCT (Leica Camera AG, Germany), sectioned with a Leica CM1520 (Leica Camera AG, Germany) at 15 μm (for immunostaining) onto Superfrost Plus slides (Thermo Fisher, USA), and dried in a vacuum desiccator (Novus DN 200, Buch & Holm, Germany).

For LA-ICP-MS analysis, the 107AgNPs (functionalized with PL3, SKLG or PL3uCendR peptide) and control 109AgNPs (blocked with biotin) were mixed in equimolar ratio and i.v. injected into orthotopic WT-GBM-bearing nude mice (Envigo). In total, 100 μL of AgNPs (11.3 nM) was injected per mouse. After 3 h of circulation, the mice were anesthetized with intraperitoneally (i.p.) injected 350 μL of 0.1 mg/kg dexmedetomidine and 75 mg/kg ketamine dissolved in saline, and perfused with PBS. The brain, tumor, lungs and liver were collected, frozen in OCT (Leica Camera AG, Germany), sectioned with a Leica CM1520 (Leica Camera AG, Germany) at 30 μm onto Superfrost Plus slides (Thermo Fisher, USA), and dried in a vacuum desiccator (Novus DN 200, Buch & Holm, Germany).

Immunohistochemistry and immunofluorescence analyses

For uPA expression staining on 2D cultured cells, the cells were fixed in − 20 °C MeOH for 1 min, washed with PBS and blocked with 300 μL of 5% blocking solution containing 0.05% Tween-20 (Sigma-Aldrich, USA), 5% fetal bovine serum (FBS), 5% bovine serum albumin (BSA), and 5% goat serum (all from GE Healthcare, USA) in PBS for 1 h. The sections were incubated for 1 h at RT with 300 μL 0.9 mg/mL polyclonal rabbit anti-uPA IgG (#17,968–1-AP, Proteintech Group, Inc., USA) as the primary antibody which was diluted 1: 100 with 1% blocking solution. After washing with PBST, sections were incubated for 30 min at RT with 250 μL of 2 mg/mL Alexa 647-conjugated goat anti-rabbit IgG (#A21245, Invitrogen, USA) as the secondary antibody, which was diluted 1: 1000 in 1% blocking solution. After washing sequentially with PBST and PBS, nuclei were counterstained with 1 μg/mL DAPI (Sigma-Aldrich, USA) in PBS.

For immunofluorescence analysis, cryosections (15 μm) on Superfrost Plus slides were fixed in − 20 °C MeOH for 2 min, washed with PBS, and blocked with 300 μL of 5% blocking solution containing 0.05% Tween-20 (Sigma-Aldrich, USA), 5% FBS, 5% BSA, and 5% goat serum (all from GE Healthcare, USA) in PBS for 1 h. The sections were incubated for 1 h at RT with 250 μL of 0,5 mg/mL polyclonal rat anti-mouse CD31 (#553,370, BD Biosciences, USA), 250 μL of 1.2 mg/mL polyclonal rabbit anti-NRP-1 (in-house), 1.4 mg/mL polyclonal rabbit anti-TNC (in-house) or 0.9 mg/mL polyclonal rabbit anti-uPA IgG (#17,968–1-AP, Proteintech Group, Inc., USA) as primary antibodies, which were diluted 1: 200 with 1% blocking solution. After washing with PBST, sections were incubated for 30 min at RT with 250 μL of 2 mg/mL Alexa 647-conjugated goat anti-rabbit IgG (#A21245, Invitrogen) or Alexa 546-conjugated goat anti-rat (#A11081, Invitrogen, USA) as the secondary antibody, which was diluted 1: 1000 in 1% blocking solution. After washing sequentially with PBST and PBS, nuclei were counterstained with 1 mg/mL DAPI (Sigma-Aldrich, USA) in PBS.

For hematoxylin & eosin (H&E) staining, cryosections (15 μm) on Superfrost Plus slides were air-dried at RT, fixed in − 20 °C MeOH for 2 min, and rinsed in MQ. Slides were stained with Mayer’s hematoxylin (#180,210, Reagena, Finland) for 5 min, rinsed with running cold tap water for 5 min, stained with 0,5% eosin (#230,251, Sigma-Aldrich, USA) in 95% ethanol (EtOH, Sigma-Aldrich, USA) with 0,5% of acetic acid (Sigma-Aldrich, USA) for 5 min, and rinsed again with cold tap water and MQ for 5 min. After air-drying the sections, they were mounted with 130 μL of Depex (Merck Millipore, USA), and imaged with the Aperio VERSA 10 (Leica Biosystems, Germany).

For 3,3’-diaminobenzidine (DAB) staining, cryosections (15 μm) on Superfrost Plus slides were air-dried at RT, fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich, USA) for 20 min, permeabilized with 0.2% Triton X-100 (AppliChem PanReac, USA) for 15 min. Sections were washed with 0.1% Tween 20 (Sigma-Aldrich, USA) in PBS and blocked with 3% H2O2 (Sigma-Aldrich, USA) in PBS on a rocking platform (SIA BioSan, Latvia) for 30 min at RT. After washing with PBS, sections were blocked with 5% heat-inactivated normal donkey serum (GE Healthcare) and 5% BSA (GE Healthcare), 0.3 M glycine in PBS for 60 min at RT. Sections were incubated overnight at + 4 °C with 250 µL of 1: 200 diluted 1 mg/mL rabbit anti-FAM IgG (#A889, Invitrogen, USA) in blocking buffer as the primary antibody. After washing with PBS, sections were incubated at RT for 1 h with 250 µL of 1: 200 diluted donkey-anti-rabbit HRP (#406,401, Biolegend, USA) as the secondary antibody. After washing with PBS, sections were incubated with 500 µL of ImmPACT DAB (#SK-4105, Vector Laboratories) for 1 min at RT. After washing with MQ for 30 min, sections were air-dried, mounted with 130 μL Depex (Merck Millipore, USA), and imaged with the Aperio VERSA 10 (Leica Biosystems, Germany).

Microscopy

For H&E and DAB staining imaging, parallel tissue sections were immunostained with H&E and DAB as described above. Sections were imaged with a 10 × objective (HC PL FLUOTAR 10 × /0.32; Leica Microsystems, Germany) mounted to an Aperio VERSA 10 Brightfield, Fluorescence FISH Digital Pathology Scanner (Leica Biosystems, Germany). Images were analyzed with Aperio ImageScope v. 12.4.3.5008 software (Leica Biosystems Pathology Imaging, Germany).

Confocal imaging was performed using an Olympus FV1200MPE confocal microscope (Olympus Europa SE & Co. KG), and images were analyzed with FluoView FV10-ASW 4.0 software (Olympus Europa SE & Co. KG).

LA-ICP-MS analysis

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) analysis of the tissue samples was performed using Agilent 8800 ICP-MS/MS coupled to a Cetac LSX-213 G2 + laser ablation unit equipped with HelEx II ablation cell and connected using ARIS (Aerosol Rapid Introduction System) sample introduction system. The system was tuned using NIST 610 glass. Ablation was performed as line scans using 65 μm square spot, scan speed of 260 μm/s, 20 Hz and fluence of 12 J/cm2. All tissue samples were fully ablated using 65 μm spacing of ablation lines. ICP-MS was operated in single quad mode. Data collection was performed in TRA mode with dwell times of 9.5 ms on mass 13C and 14 ms on mass 107Ag and 109Ag corresponding to a total duty cycle of 50 ms. Data reduction of LA-ICP-MS raw data was performed using Iolite v3.62 and Microsoft Excel. Median with MAD error 2 SD outlier reject was used for data selection in Iolite. To remove non-tissue areas in the analytical data, signal masking based on 13C signal was employed. 2D 109Ag/107Ag signal ratio maps were then constructed using Iolite v3.62 and exported as numeric maps for statistical analysis.

Statistical analysis

To assess statistical significance, one- or two-way analysis of variance (ANOVA) with Tukey or Dunnett post-hoc test, as appropriate, was performed using GraphPad Prism Software v. 10.1.2 (GraphPad Software, LLC, CA, USA).

Artwork and illustrations

The graphical abstract as well as Figs. 1a and 4a were created with BioRender.com software in accordance with the BioRender’s Academic License to Allan Tobi. Microsoft Office PowerPoint was used to create the figure panels.