The 2-chlorotrityl chloride resin (Cl-Trt-resin) preloaded with amino acid (1.1 mmol) was swollen in DCM (5 mL). The 9-fluorenylmethoxycarbonyl (Fmoc) amino acid (3.3 mmol) was dissolved in DMF (5 mL), and 1-hydroxybenzotriazole (HOBt) (3.3 mmol) and N,N′-dicyclohexylcarbodiimide (DCC) (3.3 mmol) were added as coupling reagents. The mixture was stirred for 3 h. The resin was washed three times with the following solvents: DCM (5 mL), MeOH (5 mL), and DMF (5 mL). The peptide bond formation was evaluated using the Kaiser test. The Fmoc protective group was removed by a solution of piperidine (20%) in DMF (5 mL) for 45 min at room temperature. After filtering and washing with DCM, MeOH, and DMF, the complete Fmoc deprotection was evaluated using the Kaiser test. The steps of coupling and deprotection were repeated for each amino acid.

The half peptidyl resin SS22 was treated with a solution of trifluoroacetic acid (TFA) and triisopropylsilane/water/phenol (TIPS/H₂O/PhOH (9:0.5:0.5 and v/v 20 mL)) and left to be stirred for 2.5 h at room temperature. After that, the suspension was filtered and washed with ether (Et₂O)/TFA (95:5 and v/v 10 mL), Et₂O, and methanol. The combined filtrates were evaporated under vacuum. The residual oil was treated with ice-cold ether to yield the product SS24 (325 mg, 0.40 mmol, 36.4%) as a white precipitate. The solid was recovered by centrifugation. Purities were determined to be >95% by analytical RP HPLC and elemental analysis (for the conditions, see General Methods). The identity of the compounds was confirmed by ¹H NMR, 2D NMR, and ESI MS analyses.

¹H NMR (400 MHz, [D6] DMSO) δ: 0.86 (d, J = 6.6 Hz, 6H, IleCH₃ + ValCH₃), 0.88 (d, J = 6.6 Hz, 3H, IleCH₃), 0.90 (d, J = 6.6 Hz, 3H, ValCH₃), 1.20 (d, J = 3.5 Hz, 3H, AlaCH₃), 1.35–1.40 (m, 4H, LysHγ + IleCH₂), 1.45–1.53 (m, 4H LysHβ + LysHδ), 1.70–1.74 (m, 4H, ValHβ + ProHβ + IleΗβ), 2.71–2.76 (m, 4H, ProHγ + LysHε), 3.30–3.33 (m, 2H, ProHδ), 3.76 (dd, J = 6.0. 15.0 Hz, 2H, GlyHα), 4.11 (t, J = 7.36 Hz, 2H, AlaHα + ValHα), 4.27–4.30 (m, 2H, LysHα + IleHα), 4.40–4.50 (m, 4H, ProHα+ FmocCH₂,CH), 7.34 (t, J = 7.16 Hz, 2H, ArFmoc), 7.42 (t, J = 7.56 Hz, 2H, ArFmoc), 7.51 (d, J = 8.52 Hz, 1H, AlaNH), 7.86 (d, J = 7.36 Hz, 1H, ValNH), 7.90–8.10 (m, 6H, ArFmoc + IleNH + LysNH), 8.14 ( t, J = 5.60 Hz, 1H, GlyNH). ES-MS m/z [M+2H]²⁺/2 and [M+H]⁺found: 403.7 and 806.2, calculated: 806.4 calculated for C₄₂H₅₉N₇O₉. Elementary analysis for C₄₂H₅₉N₇O₉ calculated: C 62.51, H 7.49, N 12.15, found: C 62.32, H 7.62, N 12.02.

Peptide cleavage Fmoc Ala-Val-Pro-Ile-Gly-Lys(Boc)-OH and Arg(Pbf)-Gly-Asp(OtBu)-phe-Lys(Cbz)OH (SS23 and SS16): the peptidyl resin was treated with a solution of trifluoroethanol/acetic acid/dichlomethane (TFE/CH₃COOH/DCM (1:1:4 and v/v 20 mL)) and allowed to stir for 90 min at room temperature. After that, the suspension was filtered and washed with Et₂O, DCM, and methanol. The solvents were collected and evaporated under vacuum. The residual oil was treated with ice-cold ether to yield products SS23 (325 mg, 0.40 mmol, and 32.7%) and SS16 (650 mg, 0.61 mmol, and 55.5%) as white precipitates. The solids were recovered by centrifugation.

¹ H NMR FmocAla-Val-Pro-Gly-Lys(Boc)OH (SS23) (400 MHz, [D6]DMSO) δ: 0.86 (d, J = 6.6 Hz, 6H, IleCH₃ + ValCH₃), 0.88 (d, J = 6.6 Hz, 3H, IleCH₃) 0.90 (d, J = 6.6 Hz, 3H, ValCH₃), 1.20 (d, J = 3.5 Hz, 3H, AlaCH₃), 1.35–1.40 (m, 3H, LysHγ + IleCH₂), 1.43 (s, 9H, Boc), 1.45–1.53 (m, 4H LysHβ + LysHδ), 1.70–1.74 (m, 4H, ValHβ + ProHβ + IleΗβ), 2.71–2.76 (m, 4H, ProHγ + LysHε), 3.30–3.33 (m, 2H, ProHδ), 3.76 (dd, J = 6.0. 15.0 Hz, 2H, GlyHα), 4.11 (t, J = 7.36 Hz, 2H, AlaHα + ValHα), 4.27–4.30 (m, 2H, LysHα + IleHα), 4.40–4.50 (m, 4H, ProHα+ FmocCH₂,CH), 5.60–5.65 (m, 1H, LysNHBoc), 7.34 (t, J = 7.16 Hz, 2H, ArFmoc), 7.42 (t, J = 7.56 Hz, 2H, ArFmoc), 7.51 (d, J = 8.52 Hz, 1H, AlaNH), 7.86 (d, J = 7.36 Hz, 1H, ValNH), 7.90–8.10 (m, 6H, ArFmoc + IleNH + LysNH), 8.14 ( t, J = 5.60 Hz, 1H, GlyNH). ES-MS m/z found: 805.5 [M-100] and 928.5 [M+Na], calculated: 906.5 calculated for C₄₇H₆₇N₇O₁₁. Elementary analysis for C₄₇H₆₇N₇O₁₁ calculated: C 62.30, H 7.45, N 10.82, found: C 62.49, H 7.32, N 10.94.

The linear peptide SS16 (0.6 mmol) was solubilized in DMF (5 mL) and added drop by drop using a syringe pump to a mixture formed by diphenyl phosphoryl azide (DPPA) (1.8 mmol) and NaHCO₃ (12 mmol) in DMF (10 mL) for 12 h at room temperature. After the reaction time, the solvent was removed under high vacuum. The solid was dissolved in 30 mL of ethyl acetate and washed with a solution of HCl 1 M (5 mL), a saturated solution of NaHCO₃ (5 mL), and brine (5 mL). The solvent was removed under vacuum, and the crude product was purified by column chromatography (EtOAc- > EtOAc/MeOH (95:5, v/v)) to yield the final product SS21 (550 mg, 0.53 mmol, and 88.3%). ES-MS m/z found for C₅₂H₇₁N₉O₁₂S 1046.2 [M+H]⁺ calculated: 1046.5.

Peptide SS21 was solubilized in iPrOH in a flask, and HCOONH₄ (4 equiv.) and Pd/C 10% (5% in weight with respect to the peptide) were added. The solution was irradiated in a microwave at 600 W for three cycles of 30 s each. The reaction was monitored by TLC under the disappearance of the starting material.

The Fmoc linear peptide SS23 (0.14 mmol) was dissolved in DCM/DMF (3:1), HOBt (0.16 mmol) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) (0.16 mmol) were added to it. After 10 min, the cyclic peptide SS25 (0.15 mmol) and N,N-diisopropylethylamine (DIPEA) were added (0.30 mmol) and stirred at room temperature. After 3 h, the solvent was removed under vacuum. The residual oil was solubilized with 30 mL of ethyl acetate and washed with HCl 1 M (5 mL) and a saturated solution of NaHCO₃ (5 mL). The solvent was evaporated under vacuum, and the crude product was purified by column chromatography (EtOAc- > EtOAc/MeOH (80:20, v/v)) to yield the final product SS30 (150 mg, 0.087 mmol, 62%) as colorless oil. ES-MS m/z found for C₉₁H₁₃₀N₁₆O₂₀S 1698.9 [M-100] and 1821.9 [M+Na]⁺calculated: 1799.9.

Peptides SS25 (0.50 mmol) and SS30 (0.087 mmol) were dissolved in a mixture formed by TFA, triisopropylsilane, and water (90:5:5 v/v/v and 20 mL). The final solution was stirred for 1 h. After removing the solvent, the products were triturated in ether and recovered by centrifugation to yield the final products SS27 and SS33 as TFA salt and used without further purification. Purities were determined to be >95% by analytical RP-HPLC and elemental analysis (for the conditions, see General Methods). The identity of the compounds was confirmed by ¹H NMR, 2D gCOSY, and ESI MS analyses.

Hexadecyltrimethylammonium bromide (CTAB) (1.33 mol) was dissolved in H₂O/ethanol (90 mL and 33 mL) and a 32 wt% ammonia (1.2 mL) solution. The reaction mixture was stirred at room temperature for 1 h before the addition of 1,2-bis(trimethoxysilyl)ethane (BTME) (0.005 mol). The aforementioned reaction mixture was continuously stirred for an additional time of 48 h at room temperature. The CTAB mesoporous template was removed by stirring the sample in ethanol (50 mL) with a 32 wt% aqueous solution of HCl (1.5 gr) at 50°C for 6 h. The resulting solid was recovered by centrifugation (6,000 rpm, 20 min), washed with ethanol three times, and dried at 60°C in vacuum (Motealleh et al., 2019).

IR-780 (10 mg) was dissolved in dimethylsulfoxide (DMSO) (5 mL) and sonicated to complete dissolution of the dye. Then, the mixture was added drop by drop to 10 mL of water containing PMO (100 mg). The solution was stirred, in the dark, overnight and at room temperature. After this, the PMO was washed thrice with water, and the nanoparticles were recovered by centrifugation and left to dry at room temperature (Ma et al., 2020).

To functionalize the ^IR-780PMO-OH, we coated them with 3-isocyanatopropyltrymethoxy silane (ICPTES) and then with peptides. For ICPTES coating, ^IR-780PMO-OH (100 mg) was suspended in toluene (5 mL) and triethylamine (TEA, 0.4 mL) and sonicated for 15 min. After that, ICPTES was added (1.6 mL). The reaction mixture was stirred overnight at room temperature. The nanoparticles were recovered by centrifugation and washed thrice with toluene.

Peptides (20 mg) were dissolved in DMSO (1 mL) and TEA (120 μL) was added. The reaction mixtures were left sonicated for 10 min and then added to the PMO (20 mg). After stirring overnight at room temperature, the nanoparticles were washed twice with water and ethanol and recovered by centrifugation. PMO was left to dry under vacuum at 60°C.

Fmoc-peptides@PMO were dissolved in a solution of 20% piperidine in DMSO. The reaction mixtures were sonicated for 10 min and stirred for 40 min. After that, the nanoparticles were washed thrice with ethanol and recovered by centrifugation. The step of deprotection was repeated for 25 min. The PMOs were recovered by centrifugation, then washed thrice with ethanol, and dried at room temperature.

Analytical RP HPLC was carried out on a C18 column (100 mm × 3 mm, particle size 3 μm, pore size 110 Å); DAD 210/246 nm, mobile phase from 9:1 H₂O/CH₃CN/0.1% TFA to 2:8 H₂O/CH₃CN/0.1% TFA in 20 min, flow rate 0.5 mL min⁻¹. The exact mass was determined using a QTOF apparatus. The products were purified by flash chromatography, which was performed with silica gel (particle size: 40–60 µm) obtained from Merck. The purity was determined to be ≥ 95% by analytical RP HPLC, exact mass, and elemental analysis. Elemental analysis was performed with an EACE 1110 CHNS/O analyzer (Thermo Fisher, United States of America). NMR spectra were recorded by Bruker at 400 MHz in 8:2 DMSO–d₆/H₂O (water presaturation) and ¹³C NMR spectra at 100 MHz. Two-dimensional methods (HSQC; COSY) were used to support and confirm the assignment. The chemical shift δ is reported in ppm relative to the residual proton signal of the solvent: DMSO-d6 2.50 ppm (¹H), 39.52 ppm (¹³C).

The nanoparticles were analyzed through dynamic light scattering (DLS) measurements performed with a Zetasizer Nano ZS (Malvern), He–Ne laser 633 nm, Max 4 mW, using polystyrene cuvettes (optical path length 1 cm). PMOs were dispersed in distilled water and sonicated for 20 min. The measurements were conducted at 20°C. Scanning electron microscopy, specifically the JSM-7610 FPLUS model by JEOL in Udine, Italy, was used to acquire high-resolution images of PMO nanoparticles. All images were collected using different magnifications.

To quantify the amounts of peptides on the PMO, a Varian Cary 50 bio benchtop spectrophotometer in the range of 200–800 nm with a 1-nm step was used (Faura et al. 2021). The calibration curves were determined by preparing five solutions at known concentrations of Phenylalanine and Fmoc-Alanine used as standards. Briefly, a stock solution of 5 mM Phe in absolute ethanol was diluted to the final concentrations of 0.2 mM, 0.5 mM, 1 m, 2 mM, and 5 mM. A standard solution of 52.7 μM Fmoc-Alanine (Ala) in absolute ethanol was diluted to the concentrations of 20 μM, 10 μM, 5 μM, and 2 μM. The fluorescence signals were recorded for Phenylalanine (Phe: λ_ex 240 nm and λ_em 282 nm) (Lakowicz, 2006) and Fmoc-Ala (λ_ex 397 nm and λ_em 280 nm) (Pappas et al., 2014) and plotted against their respective sample concentrations. The resulting data were subjected to linear regression analysis, which provided the slope and the R-square (COD) for the chromophores of each peptide.

IR spectra were measured within the wavenumber range of 4,000 to 650 cm^-1 using a Cary 630 spectrometer by Agilent. Background and sample spectra were acquired before each acquisition at a spectral resolution of 1 cm^-1, with 128 scans per measurement.

BJ, HeLa S3, SW480, and Ht-29 were purchased from ATCC (Manassas, United States of America), and HCT-116 was purchased from Ubigene (Austin, United States of America). MCF10A and 293 Ampho-Phoenix cells were previously described and characterized (Di Giorgio et al, 2021). Cancer cells were cultured in 10% FBS DMEM (Euroclone, Milan, Italy). BJ cells were grown in EMEM (Euroclone, Milan, Italy). Media were supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM), penicillin (100 U ml⁻¹), and streptomycin (100 μg ml⁻¹) (Lonza, Basel, Switzerland). MCF10A was cultured in Ham’s F12: Dulbecco’s modified Eagle’s medium (DMEM) 1:1 medium (Merck, Milan, Italy) supplemented with 5% horse serum (Gibco), penicillin (100 U/ml), streptomycin (100 μg/ml), L-glutamine (2 mM) (Lonza), insulin (0.01 mg/ml), hydrocortisone (500 ng/ml), cholera toxin (100 ng/ml) (Sigma-Aldrich, Milan, Italy), and epithelial growth factor (20 ng/ml) (Peprotech, United Kingdom). Oxaliplatin was purchased from Tocris (Bristol, United Kingdom) and dissolved in DMSO (Sigma-Aldrich, Darmstadt, Germany).

For the resazurin-to-resorufin reduction assays, cells were grown in a 96-well plate for 120 min at 37 °C with resazurin solution (0.15 mg/ml) (Sigma-Aldrich, Milan, Italy). The fluorescence of resorufin was quantified on a PerkinElmer EnSpire 2,300 Multilabel Reader (ex 550 nm/em 590 nm). For the Apo-ONE assay, 25,000 cells were seeded in a 96-well white polystyrene microplate (Corning, United States of America). After 24 h, cells were treated for 36 h with oxaliplatin and oxaliplatin+SS35. Cells were then lysed and subjected to caspase-3/7 (DEVDase) activity assays by using the Apo-ONE Homogeneous Caspase-3/7 Assay (Promega, Madison, United States of America) according to the manufacturer’s instructions. After 30 min of incubation, fluorescence was recorded in the linear phase of the kinetics with a Synergy H1 plate reader (BioTek, Winooski, United States of America). For the trypan blue viability assay, cells were harvested, pelleted at 1500 rcf, and incubated for 5 min with a PBS/0.4% Trypan Blue solution (Gibco). At least 300 cells were counted for each condition.

A total of 25,000 HeLa S-3 cells were seeded in a 96-well plate (Sarstedt, Nümbrecht, Germany). After 24 h, they were treated with 20 µM oxaliplatin or SS35 (0.1 μg/ml), or both, for 48 h. Then, living cells were harvested and counted. A total of 1000 living cells were seeded in 60-mm adhesion plates (Sarstedt, Nümbrecht, Germany), cultured for 14 days, and stained for 20 min with methylene blue (5 g/L of 50% (v/v) ethanol/water, 1 mL/plate). Colonies were counted with clone.JAR

Ht-29 and HeLa S-3 cells were treated with scaling doses of SS7, SS35, SS28, and SS36 for 3 h; cells were then harvested and resuspended in phosphate-buffered saline (PBS). Single-cell suspensions were prepared as previously described (Di Giorgio et al, 2021), and forward scatter (FSC), side scatter (SSC), and FL-3 were acquired on a BD FACSCalibur cytometer. Data were analyzed with FlowJO software.

Cell lysates were obtained by collecting living and dead cells after centrifugation at 1500 rcf for 5 min. Cells were lysed with Laemmli sample buffer 2x. After SDS-PAGE and immunoblotting on nitrocellulose (Whatman, United Kingdom), membranes were incubated overnight with the following primary antibodies: anti-caspase 3 (1:1000 Abcam, ab214430), anti-HA (HA-7, Sigma-Aldrich), anti-actin (MAB1501, Sigma-Aldrich), and anti-histone H3 (D1H2, Cell Signaling Technology). HRP-conjugated secondary antibodies were obtained from Cell Signaling Technology (Danvers, United States of America), and blots were developed with Super Signal West Dura (Pierce, United States of America). For fluorescence-based detection, AF660 or AF760 secondary antibodies were used (Merck, Milan, Italy), and images were acquired at Odyssey M Imaging System (LI-COR Biosciences, United States of America).

pWZL-Hygro Hemagglutinine-XIAP (HA-XIAP) was obtained by subcloning through a restriction-ligation-based approach the ORF of XIAP (BamHI/EcoRI) from pEBB-XIAP (Addgene Plasmid #11558) in pWZL-Hygro HA. Transfections of 293 Ampho-Phoenix cells were carried out with polyethylenimine (PEI, 1 μg/ml) using a 2:1 ratio of PEI (µl): DNA (µg). A pWZL-Hygro empty plasmid was used as a control. Retroviral infection of HeLa S-3 cells was performed with an M.O.I of 0.1–0.3 at 32°C by using Ampho cells as packaging cells. Two consecutive infections were performed, the second 48 h after the first. Cells were selected with 250 μg/ml hygromycin B (Euroclone, Milan, Italy). Polyclonal cultures were then obtained and started to be used 2 weeks after the end of selection. For Integrin Subunit Beta 3 (ITGB3) silencing, 300,000 HeLa cells were seeded in 35-mm plates and transfected with 40 ng endoribonuclease-prepared short interfering RNAs (esiRNAs) (EHU051661, Merck, Milan, Italy) and 5.5 µl Lipofectamine 3,000 (Life Technologies, Carlsbad, United States).

HeLa cells were lysed using TRIzol (Invitrogen, United States of America). Totally 1.0 μg of total RNA was DNAse I-treated (Ambion, United States of America) and retro-transcribed by using 100 units of M-MLV reverse transcriptase (Life Technologies, United States of America) in the presence of 1.6 μM oligo (dT) and 4 μM random hexamers (Euroclone, Milan, Italy). Real-time quantitative reverse transcriptions (qRT-PCRs) were performed using SYBR Green technology (KAPA Biosystems). Data were analyzed by the comparative threshold cycle (delta delta Ct ΔΔCt) using HPRT and ACTB as normalizers. The primers used for ITGB3 amplification in qRT-PCR are FW: GTG​ACC​TGA​AGG​AGA​ATC​TGC and RV: CCG​GAG​TGC​AAT​CCT​CTG​G.

For experimental data, Student’s t-test was employed. The Mann–Whitney test was applied when normality could not be assumed. For comparisons between more than two samples, the ANOVA test was applied coupled with Kruskal–Wallis and Dunn’s multiple comparison tests. Excel and GraphPad Prism were used for statistical analysis. We denoted significance as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Unless otherwise indicated, all the data in the figures were represented as arithmetic mean ± the standard deviation from at least three independent experiments.

To prepare the linear peptides SS23, SS24, and SS16, we used a Cl-Trt resin in the presence of HOBt/DCC under the previously optimized conditions (De Marco et al., 2020). The steps of protective group removal and coupling were repeated until the linear peptides were obtained and recovered by cleavage. SS24 was cleaved with TFA and scavengers (Figure 1A). For peptides SS23 and SS16, we used TFE and acetic acid in dichloromethane to maintain the protective group in the side chain (Figures 2A,B). The peptides are recognized by analytical RP-HPLC (see general method). The cyclization of SS16 was performed under high-dilution conditions in the presence of DPPA/NaHCO₃/DMF, yielding c [Arg (pbf)-Gly-Asp(OtBu)-phe-Lys (Cbz)] (Figure 2B). After hydrogenation (Daga et al. 2001) and treatment with TFA, we obtain the cyclic [Arg-Gly-Asp-phe-Lys]. To prepare the dual peptide Fmoc-AVPI@c [RGDfK], the peptides SS23 and SS25 were coupled in the presence of HOBt/TBTU/DIPEA. The deprotection under acid conditions with TFA led to obtaining the final product SS33 (Figure 2C). The peptides were purified by flash chromatography (see General Procedures) and characterized by reversed-phase HPLC and 1H NMR. The purities were determined to be >95% by analytical RP-HPLC (see General Procedures).

To prepare the nanoparticles, the surfactant CTAB was used to form the micellar solution in the presence of ammonia. BTME was used to form the walls of the nanoparticles, and the excess surfactant was removed by an acid solution of ethanol and HCl (Motealleh et al., 2019). The internal core was loaded with IR-780 according to the literature (Ma et al., 2020) (Figure 3A). Briefly, the dye was dissolved in DMSO and then added to the nanoparticles dispersed in H₂O. The reaction was stirred for 12 h in the dark, then washed with water, and recovered by centrifugation. To functionalize PMO with peptides, the outer shell of the nanoparticle was reacted previously with ICPTES and TEA, with a procedure optimized in our laboratory (Greco et al., 2015), and then with peptides in basic conditions (Figures 3A,B). The last step is the deprotection of the Fmoc protective group from peptides@PMO to obtain SS35 and SS36 with an amine group free of the alanine, which is the pharmacophore responsible for the inhibition of Xiap (Bourguet et al., 2014). PMOs were characterized by DLS, SEM, and IR to analyze the size, shape, and structure after each step.

Scanning electron microscopy shows a PMO-OH having a diameter of 556 nm measured at 50 k magnification, while the hydrodynamic light scattering distribution (dH) measured has a diameter of 495 nm with a PDI of 0.12 (Figure 4). After conjugation with cytotoxic AVPI, cyclicRGD and DTP with ICPTES, which was used to functionalize the outer shell of the nanoparticles, the reactions gave AVPI@PMO, c [RGDfK]@PMO, and DTP@PMO. The increase in size distribution and diameter measured by DLS and SEM confirms the successful functionalization, as the dH after conjugation increased to 856 nm compared with that of native nanoparticles (see Supplementary Table S1 and Supplementary Figures S1–S3).

The FT-IR was used to analyze the structure of PMOs. The stretching vibrations at around 693.28 cm^-1 were assigned to Si-C. The presence of a broad absorption band at 3384.42 cm^-1 was assigned to Si-OH. The presence of a urea bond is confirmed by stretching vibrations at 1647.48 and at 1546.84 cm^-1 in SS28, at 1651.21 and at 1558.02 cm^-1 in SS35, and at 1654.93 and at 1558.02 cm^-1 in SS36 spectrums (see Supplementary Material S4–S8) between the amino group of the peptides and PMOs.

Once calibration curves were established, solutions of SS28, SS35, and SS36 (ca.1 mg) in ethanol (2 mL) were prepared, and the absorption spectra were measured. The absorbance values of peptides linked to PMOs were adjusted to account for potential interference caused by PMOs, which can scatter or absorb light in the visible and UV ranges, leading to inaccuracies in absorbance measurements of chromophores. The absorbance spectrum of PMO was subtracted from the absorbance spectrum of the sample containing the peptide@PMO. In the region of absorption of the chromophore, we observed a linear and well-defined relationship between the absorbance of PMO and wavelength. To generate the PMO absorbance spectrum under the chromophore’s absorption band, we performed linear regression on the absorbance values outside the chromophore’s band. This allowed us to establish a baseline for the absorption of the PMO. The amount of peptide functionalized on the PMO was determined using the formula obtained from the calibration curves. The absorbance is at λ_max = 257 nm for Phe and λ_max = 267 nm for Fmoc-Ala. The quantification values are reported in Table 1 (for calibration curves, see Supplementary Material S9, S10).

To prove the successful uptake of the three molecules (SS28, SS35, and SS36), two cancer cell lines were selected: Ht-29, a human colorectal adenocarcinoma cell line expressing low levels of the heterodimeric αvβ3 integrin, and HeLa, a human cervical epithelioid carcinoma cell line expressing high amounts of αvβ3 (De Marco et al., 2020). Equal amounts of Ht-29 and HeLa cells, which were in the exponential growth phase, were loaded with 0.01 μg/ml and 0.1 μg/ml of the three functionalized nanoparticles SS28, SS35, and SS36 for 3 hours. Cellular uptake was monitored by flow cytometry by detecting the fluorescence of IR-780 loaded into the internal core of the nanoparticles. Background autofluorescence was quantified by treating the cells with empty, non-fluorescent nanoparticles SS4. As shown in Figure 5A, the uptake of SS36 (nanoparticles functionalized with AVPI) was equivalent between Ht-29 and HeLa cells. A more efficient uptake by HeLa cells was observed for SS28 (nanoparticles functionalized with RGD peptide) and SS35 (nanoparticles functionalized with RGD and AVPI) (Figure 5A, B). This evidence supports the efficacy of RGD-conjugates as targeting ligands for αvβ3 positive cancer cells, as previously shown by others (Danhier et al., 2012).

To prove that the differential uptake of SS28 and SS35 between Ht-29 and HeLa cells was indeed due to differential expression levels of αvβ3, we silenced ITGB3 expression in HeLa cells for 48 h and monitored the uptake of SS28, SS35, and SS36 for 3 hours after administration of the compounds. In HeLa cells successfully deprived of the integrin β3 (Figure 5C), the uptake of SS28 and SS35, but not of SS36, was severely impaired and comparable to the uptake of SS36 (Figure 5D, E). These data indicate that integrin β3 binding promotes the uptake of SS28- and SS35 nanoparticle-based compounds.

After establishing the differential uptake of the described compounds in Ht-29 and HeLa cells, we performed standard biological assays to evaluate the induction of apoptosis. First, Ht-29 and HeLa cells were treated with increasing doses (0.02, 0.04, and 0.06 μg/ml) of SS28, SS35, and SS36 for 48 h, and cell viability was assessed by resazurin reduction assays (Figure 6A). HeLa cells were found to be more sensitive to SS35 than to Ht-29 (Figure 6A), which is in excellent agreement with the uptake data (Figures 5A, B). Both Ht-29 and HeLa cells were moderately sensitive to SS36, whereas SS28 resulted in some toxicity only in HeLa cells (Figure 6A). Interestingly, increasing doses of SS28 did not correlate with an increase in toxicity, suggesting that a) the engagement of αvβ3 decreases HeLa fitness and b) the lower dose is sufficient to block αvβ3 and achieve efficacy.

To evaluate the superior efficacy of our peptide-to-nanoparticle conjugation system, we decided to evaluate the cytotoxicity of the free peptides. For this purpose, Ht-29 and HeLa cells were treated with 1, 5, and 25 µM of the free RGD peptide (referred to as p28 by analogy with the nanoparticle-conjugated peptide SS28) and the chimeric peptide RGD-AVPI (referred to as p35 by analogy with the nanoparticle-conjugated peptide SS35) (Figure 6B). Although the two experiments cannot be directly compared as the conjugation to the nanoparticles may affect other aspects than the bioavailability of the peptides, like their stability and activity, by comparing Figures 6A,B, it is clear that the effect of the unconjugated molecules is significantly attenuated. One of the reasons for the higher functionality of the peptides conjugated with nanoparticles compared to the unconjugated peptides could be their higher stability than that of the naked peptides, in addition to their higher uptake, as reported by others and reviewed in Lucana et al (2021). To indirectly test the stability of the nanoparticle-conjugated SS35 peptide, we exposed the SS35 molecule, and the empty nanoparticle SS7 as a control, to the action of extracellular proteases for 0, 1, 3, and 12 h, and then we tested their residual activity by evaluating their cytotoxicity on HeLa cells. SS35 was found to retain significant activity even after 12 h in an extracellular environment at 37°C (Figures 6C,D).

In molecular oncology, apoptosis inducers must not only be functional but also have selectivity toward cancer cells. As the expression level of αv integrin is heterogeneous in healthy tissues, we selected MCF10A and BJ to test the cytotoxicity of SS28 and SS35, as these normal cell lines were characterized by the highest mRNA levels of ITGAV on the basis of what emerges from the analysis of the transcriptome available on GENT2 (Park et al, 2019) (Figure 6E). MCF10A and BJ cells were loaded with increasing doses (0.02, 0.05, and 0.10 μg/ml) of SS28 and SS35, and no significant alteration of their viability was observed after 48 h of treatment (Figure 6F).

Finally, to prove that the high cytotoxicity of SS35 was due to the inhibition of IAP through AVPI, a retroviral plasmid was used to stably overexpress HA-XIAP in HeLa cells (Figure 6G). A retroviral empty plasmid encoding for hygromycin B resistance was used as a control. Polyclonal HeLa cultures overexpressing HA-XIAP and hygromycin B resistance or hygromycin B resistance alone, as a control, were established. As shown in Figure 6G, HA-XIAP overexpressing cells were much less sensitive to SS35 than hygro-expressing control cells (Figure 6G). This was correlated to a defective cleavage of caspase 3 in HA-XIAP overexpressing cells (Figure 6H).

Overall, these data demonstrate that the selective targeting of αvβ3-positive cells with the SS35 molecule leads to the induction of apoptosis, which can be overcome by increasing XIAP levels. Importantly, SS35 does not negatively impact the viability of the healthy cells evaluated, thus confirming its potential specific anti-neoplastic effect.

Inherent or acquired resistance to platinum compounds negatively affects their efficacy against cervical cancer (Bhattacharjee et al., 2022). Overexpression of anti-apoptotic Bcl2 family members is one of the most common resistance mechanisms. We therefore hypothesized that treatment with SS35 would enhance the toxicity of oxaliplatin (OxPt). To prove this, we treated HeLa cells with 10 µM OxPt as a single agent or in combination with 0.6 μg/ml SS35. As shown in Figure 7A, SS35 increases the anti-cancer activity of OxPt, and this effect correlates with increased activity of caspases 3 and 7 (Figure 7B) and increased proteolysis of caspase 3 (Figure 7B). Importantly, SS35 and OxPt showed synergistic anti-cancer effects in a 14-day colony formation assay, achieving a nearly complete response (Figure 7C). These data suggest that SS35 sensitizes αvβ3-positive HeLa cells to the genotoxic compound oxaliplatin.

To demonstrate that the cytotoxic effect of SS35 was not restricted to HeLa cells but can be expanded to other αvβ3-positive cancer cells, we adopted a panel of three colorectal cancer (CRC) cell lines, Ht-29, HCT-116, and SW480, that were previously described for having increasing expression levels of αvβ3: Ht-29 << HCT-116 < SW480 (Chin et al. 2019; Lei et al., 2011; Figure 7D). FOLFOX (folinic acid, 5-fluorouracil, and oxaliplatin) is the most commonly used neoadjuvant and adjuvant chemotherapy regimen for advanced colorectal cancers (CRC), and resistance to OxPt was observed in non-responsive patients (Hoang et al. 2022). Targeting αvβ integrins proved to be effective in potentiating OxPt effects in CRC cells (Dia and de Meija 2011). Engagement of αvβ3 was successfully exploited as a safe and precise exosome-based-therapy targeting strategy in CRC murine models to overcome resistance (Lin et al., 2021). For these reasons, we treated our selected panel of CRC cells with 6 µM OxPt and 0.6 μg/ml SS35, separately and in combination. Resazurin reduction assays were performed 96 h after the treatment to quantify cell survival (Figure 7E). SS35 proved to have limited efficacy in potentiating the OxPt effect in Ht-29, while being successful in strongly decreasing the viability of HCT-116 and SW480 cells in combined treatments.

Overall, our data suggest that the adoption of nanoparticles loaded with Smac mimetics and the ability to engage αvβ3 could be a good strategy to enhance the effects of oxaliplatin in αvβ3-positive cancers.