Selective delivery of G-quadruplex ligand in glioma cell lines: the power of cyclic-RGD peptide

doi:10.21203/rs.3.rs-4984467/v1

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Selective delivery of G-quadruplex ligand in glioma cell lines: the power of cyclic-RGD peptide

https://doi.org/10.21203/rs.3.rs-4984467/v1

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Compounds targeting non-canonical secondary structures of nucleic acids, known as G-quadruplexes, are highly cytotoxic, both for cancer and healthy cells, because of their action mechanism's lack of appropriate selectivity. The targeted delivery of cytotoxic molecules to cancer cells is a valuable strategy to expand the repertoire of potential drugs, especially for cancer types for which new therapeutic tools are urgently needed, like glioblastoma. In this work, we conjugated a cyclic arginyl-glycyl-aspartic acid peptide to a naphthalene diimide, previously described as a highly performing stabilizing ligand for DNA G-quadruplexes, to specifically target glioma cells overexpressing RGD-binding integrin receptors. Our results, which include confocal microscopy and cell toxicity assays, demonstrated improved efficacy and selective cellular absorption of the new conjugate.

The selective targeting by small molecules of non-canonical secondary structures of nucleic acids known as G-quadruplexes (G4s) is recognized as a cutting-edge and robust strategy against different pathologies.^1–5 G4s are highly structured conformations within key genomic regions, such as telomers, gene promoters, and untranslated regions.⁶ Because of their acknowledged involvement in essential biological processes along with the countless experimental data proving their existence in living cells, they gained significant importance in medicinal chemistry.^7,8 Among all, over the years, many efforts have been made to strategically manipulate the folding of G4s with the ambitious aim of pioneering groundbreaking anticancer therapies.^1,9 Nevertheless, among the numerous organic small molecules designed and synthesized to selectively recognize the G4s of interest, only some of these have reached the pre-clinical phase.¹⁰ Indeed, despite the promising biological outcomes as potential anticancer drugs, many of these compounds lack the specificity needed to differentiate between cancerous and normal cells. This lack of specificity leads to systemic distribution, increasing the risk of off-target interactions and undesirable side effects. Significant progress has been made in the selective delivery of G4-ligands to cancer cells,^11,12 however, ongoing research and innovative strategies are crucial to overcoming the remaining challenges and improving treatment outcomes. In this context, we have undertaken a proof-of-concept study to enhance the cellular uptake of a tetrasubstituted naphthalene diimide (NDI-1 in Fig. 1), widely recognized as a highly effective G4-stabilizing ligand,^13,14 by a covalent linkage with a cyclic arginyl-glycyl-aspartic acid peptide (1a-cRGD in Fig. 1).^15,16 NDI-1 previously demonstrated significant stabilization of telomeric G4 structure over duplex DNA, with an increase in melting temperature of about 30°C (calculated ΔTm = 32.2°C for G4 vs ΔTm = 4.5°C for duplex DNA by FRET-melting), and exhibited good telomerase inhibition, with an EC₅₀ value comparable to other well-known G4-ligands: Braco-19 and TMPyP4 (25 µM vs 7.9 µM and 16.4 µM respectively).¹⁴ However, it did not show a marked preference for breast and lung cancer cell lines over normal human fibroblast with selective indexes equal to 2.8 (IC₅₀WI38/IC₅₀MCF7) and 10.2 (IC₅₀WI38/IC₅₀A549) respectively.¹⁴ cRGD is a well-known integrin-binding sequence specifically recognized by ανβ3, ανβ5, and α5β1 integrins, which are overexpressed on the surface of different cancer cells and angiogenic blood vessels and minimally expressed in normal cells.¹⁷

In principle, conjugating a promising G4-ligand, as NDI-1, with the RGD-based cyclopentapeptides, such as 1a-cRGD sequence, is expected to lead to specific internalization processes by integrin-mediated endocytosis. This targeted delivery could enhance the cellular uptake of the drug, ensuring its accumulation within the target cells improving the drug efficacy, also reducing systemic toxicity and side effects on healthy cells.¹⁸ Therefore, we modified one of the substituents of the NDI-1 by introducing a propargylamine pendant via nucleophilic aromatic substitution on the naphthalene core (Scheme 1). This reactive moiety is essential for the covalent linkage of the cRGD moiety, previously functionalized with a terminal azide according to the synthesis procedure already reported,¹² through a copper-catalyzed click chemistry reaction (Scheme 1). This process resulted in the new conjugate NDI-cRGD (Fig. 1).

Chemistry

The synthetic pathway for the conjugate NDI-cRGD is illustrated in Scheme 1. Initially, the di-substituted naphthalene diimide (compound 1) was refluxed in acetonitrile for three hours to yield the bromo-substituted propargylated derivative (compound 3). Next, compound 3 was dissolved in neat N,N-dimethyl-1,3-diaminopropane and heated for 10 minutes under microwave-assisted conditions (120°C, 250 psi, 200 W), leading to the formation of compound 4. Finally, a click reaction with the azide derivative, cRGD-N₃, in the presence of CuSO₄·5H₂O and sodium ascorbate at room temperature resulted in the desired NDI-cRGD conjugate.

Cellular Activity

To investigate the interaction of NDI-cRGD with integrin receptors, we strategically selected three glioma cell lines as a suitable model for evaluating our approach. Specifically, the quantitative real-time PCR analysis (primers listed in Table S1) displayed abundant expression of the integrin subunits of interest in U251 and U373 cell lines, with U251 expressing particularly high levels of α and β subunits. In addition, D384 cells were used as a negative control as αv, β3, and β5 mRNA expression were undetectable, while α5 and β1 mRNA expression were considerably lower compared to U251 and U373 cell lines (Fig. 2).

In previous work, dose/response curves for NDI-1 on glioma cells indicated that the concentration of 20 µM was quite toxic to any cell line after 24 and 48 hours of exposure.¹² Conversely, the same concentration of the 1a-cRGD peptide did not exhibit any toxic effects under identical experimental conditions.¹⁹ Based on these findings, we selected this concentration to investigate whether NDI-1 toxicity could be targeted and modulated through conjugation with cRGD peptide.

Taking advantage of the intrinsic red-emission of tetrasubstituted NDIs, the cellular permeability of the reference NDI-1 and the new NDI-cRGD conjugate were evaluated by confocal fluorescence microscopy. D384, U251, and U373 cell lines were treated with 20 µM NDI-1 or NDI-cRGD, recording the emission signals at different times between 575 and 700 nm range (excitation wavelength at 561 nm). As expected, NDI-1 exhibited rapid and indiscriminate cellular uptake, without any cell line preference. This was evident from images recorded just 5 minutes after treatment and further confirmed by images taken 30 minutes post-treatment (Figure S1). In contrast, the introduction of the cRGD sequence enabled selective cellular uptake of the new NDI-cRGD conjugate, which became significantly more evident after 24-, 48-, and 72 hours of treatment (Fig. 3 and S2). Specifically, the emission of NDI-cRGD conjugated was highly detectable in the U251 cells and, to a slightly lesser extent, in the U373 cells, where the emission signal increased over time. On the other hand, a negligible signal was observed in D384 cells, which do not express the integrins of interest, even 72 hours after treatment (Fig. 3).

These results perfectly correlate with cell viability tests. NDI-1 and NDI-cRGD were evaluated on U373, U251, and D384 cells at a 20 µM concentration over increasing contact times (24, 48, 72 hours). Under these conditions, NDI-1 exhibited high toxicity across all cell lines, reducing cell viability by over 80% within 24 hours (Fig. 3). In contrast, NDI-cRGD decreased cell viability by approximately 60% in U251 cells after just 24 hours, maintaining this effect consistently for up to 72 hours. In U373 cells, NDI-cRGD achieved similar toxic effects after 72 hours in a time-dependent manner. Fascinating, NDI-cRGD remained completely ineffective on D384 cells, even after 72 hours of treatment. The RGD peptide alone, 1a-RGD, showed a significant effect on U373 and U251 cells only after 72 hours, consistent with previous observations.¹⁹ These data align well with PCR analysis and strongly suggest that the differential response of glioma cells is related to their varying integrin receptor expression patterns.

To detect cell death, the annexin V binding assay was used (Annexin V-FITC Apoptosis Detection Kit, eBioscience) for FACS analysis, as described in the methods section (Supporting Information).

Following 24-hour treatments with 20 µM NDI-1 and NDI-cRGD, striking different results were obtained. While the cells treated with NDI-1 were predominantly necrotic and early apoptotic, cells treated with the new conjugate NDI-cRGD exhibited a markedly lower percentage of necrotic/apoptotic cells (Fig. 4). Notably, U251 cells were more sensitive to the NDI-cRGD compound than U373 cells. These findings align perfectly with the qRT-PCR and cell viability experiments, suggesting a correlation between RGD-binding integrin expression levels and sensitivity to the RGD-conjugated NDI compound. Once again, these data strongly support that the differential response of glioma cells is related to the cRGD-driven selective targeting of the different expression patterns of integrin receptors.

Overall, this proof-of-concept study underscores the potential of covalently linking the cyclic RGD peptides to potent G4-stabilizing ligands, as tetrasubstituted naphthalene diimide NDI-1, to enhance the selective cellular uptake and improve their safety profile as promising anticancer agents. The targeted delivery mechanism mediated by the cRGD sequence ensures a higher accumulation of NDI-1 within cancer cells, enhancing its efficacy while reducing systemic toxicity and side effects on healthy cells. By leveraging the targeting capability of the RGD motif and the biological pathways it interacts with, this approach holds significant potential to improve patient outcomes and quality of life during treatment, particularly for cancers that urgently need new therapeutic strategies, such as glioblastoma.

Chemical synthesis of the new conjugate NDI-cRGD

All the solvents and reactants for the synthesis were purchased from Merck and Fluorochem and were used as supplied without further purification. HPLC analyses were performed using an Agilent system SERIES 1260 with XSelect® HSS C18 column (2.5 µm, 4.6 x 50 mm) and the following method was used: flow 1.4 mL/min, isocratic gradient over 2 min 95% of H₂O + 0.1% TFA (5% CH₃CN), gradually to 40% aqueous solvent over 6 min, then isocratic flow for 4 min (λ = 256 nm). HPLC purifications were carried out on an Agilent Technologies 1260 Infinity preparative HPLC provided with a diode array UV-vis detector, using a Waters XSelect® CSH Prep C18 OBD^™ column (5 µm, 100 x 30 mm) at a 30 mL/min flow rate. Using acidic water (0.1% of TFA) and acetonitrile as eluents, the following methods were employed for purifying the crude. Method A: 2 min 80% acidic water, followed by a gradient step of 18 min reaching 40% water phase (λ = 254nm, 500nm, 600 nm). Method B: 2 min 95% acidic water, followed by a gradient step of 14 min reaching 40% water phase (λ = 254nm, 500nm, 600 nm). UPLC-MS data were recorded, using a surveyor UPLC system (Thermo Finnigan, San Jose, CA, USA) equipped with a BEH Acquity UPLC column (1.7 µm) 2.1 x 50 mm, and an LCQ ADV MAX ion-trap mass spectrometer, with an ESI ion source. ¹H- and ¹³C-NMR spectra were recorded on Bruker Avance 300 MHz.

Synthesis of compound 4

350 mg of 1 (0.59 mmol), prepared using our previously published protocol,²⁰ was dissolved in 120 mL of acetonitrile and 3 eq. of propargylamine (83µL, 1.77 mmol) was added under stirring (Scheme 1, step a). The mixture was refluxed for 3 hours under an argon atmosphere, and the crude product was verified by analytic HPLC obtaining compounds 2 and 3 as a mixture. The solvent was removed under vacuum and the red crude was purified by preparative HPLC following method A. Compounds 2 and 3 were obtained as red (yield 75%) and fuchsia (yield 45%) solids, respectively. NMR spectra are consistent with those reported in the literature.²¹ Purified compound 3 (30 mg, 0.05 mmol) was dissolved in neat N,N-dimethyl-1,3-diaminopropane (1 mL), and the mixture was stirred at 120°C for 10 min (250 psi, 200 W) under microwave assistance (Scheme 1, step b). The crude was acidified and purified by preparative HPLC following method A, obtaining compound 4 with 88.5% yield.

Compound 4. Analytic HPLC retention time: 5.210 min (100.0% pure).

UHPLC-MS (positive mode): 590.33 [4 + H]⁺; 295.86 [4 + 2H]²⁺ m/z

¹H-NMR (300 MHz, D₂O, 60°C) δ ppm: 7.62–7.61 (m, 2H⁺), 4.20 (s, 2H⁺), 4.08–4.05 (m, 4H⁺), 3.51 (t, J = 6.72 Hz, 2H⁺), 3.31–3.26 (m, 3H⁺), 3.21–3.16 (m, 4H⁺), 2.88 (s, 6H⁺), 2.85 (s, 12H⁺), 2.25-2.00 (m, 6H⁺). ¹³C-NMR (75.4 MHz, D₂O) δ ppm: 165.2, 165.2,163.2, 163.0, 162.9, 162.4, 161.9, 148.4, 147.5, 124.5, 124.0, 122.0, 120.3, 119.9, 118.1, 117.5, 117.5, 114.2, 101.7, 100.8, 79.6, 73.3, 55.2, 42.7, 39.5, 37.2, 24.0, 22.8.

Synthesis of NDI-cRGD

8 mg of pure RGD-N₃ (8.24 µmol)¹² were dissolved in 400 µL of a 1:1 H₂O:tBuOH mixture. Purified compound 4 (4.85 mg, 8.24 µmol) was added after its dissolution in the same mixture (100 µL). Subsequently, 1 eq. of ascorbic acid (1.62 mg) and 1 eq. of CuSO₄·5H₂O (1.31 mg) were simultaneously added and the solution was left under stirring at room temperature for 2h (Scheme 1, step c). The crude was purified by preparative HPLC following method B, obtaining compound NDI-cRGD as a blue solid with a 53.2% yield.

NDI-cRGD compound. Analytic HPLC retention time: 5.996 min (95.7% pure).

UHPLC-MS (positive mode): 1446.73 [NDI-cRGD + H]⁺; 724.4 [NDI-cRGD + 2H]²⁺; 483.2 [NDI-cRGD + 3H]³⁺; 362.8 [NDI-cRGD + 4H]⁴⁺ m/z.

¹H-NMR (700 MHz, D₂O) δ ppm: 1.12 (bs, 4H), 1.42–1.50 (m, 8H), 1.88–1.95 (m, 4H), 2.00–2.07 (m, 8H), 2.27 (bs, 2H), 2.64–2.75 (m, 5H), 2.84-.85 (m, 2H), 2.86 (s, 12H), 2.88 (s, 6H), 3.03–3.06 (m, 1H), 3.08–3.12 (m, 2H), 3.19–3.22 (m, 6H), 3.32 (bs, 2H), 3.50 (d, J = 14 Hz, 1H), 3.96–4.00 (m, 6H), 4.03–4.04 (m, 1H), 4.17 (bs, 1H), 4.48 (t, J = 7 Hz, 2H), 4.78–4.81 (m, 2H), 6.58 (bs, 2H), 6.73 (bs, 2H), 6 7.19 (s, 3H), 7.76–7.77 (m, 1H), 7.96 (s, 1H), 8.04–8.03 (m, 1H), 8.19 (s, 1H).

Biological Experiments

Cell Culture

U373, U251, and D384 human glioblastoma cell lines were purchased from the ECACC collection (Merk Life Science, Milan, Italy) and maintained in culture under standard growth conditions.¹²

Real-time PCR

To assess integrin subunit expression in U373, U251, and D384 cells, the mRNA expression of αν, α5, β3, β5, and β1 subunits was evaluated by quantitative real-time RT-PCR as previously described.¹² The primers used were fully listed in Table S1. β-actin was used as a housekeeping gene. Experiments were performed on three different cell preparations, and each run was analyzed in duplicate. Data are expressed as DCq (difference between reference and housekeeping gene Cq).²²

Confocal microscopy analysis

Cells were plated in 35 mm cell culture plastic bottom dishes at a density of 50000 cells/well and 24 h after plating in a serum-free medium were used for confocal microscopy analysis using a Leica TCS SP8 confocal microscope equipped with a 25× water immersion objective (Fluotar VISIR 25x/0.95 WATER, Leica). During the acquisitions, cells were maintained in an environmental chamber at 37°C in an atmosphere of 5% CO₂. A 561 nm laser line was used to excite NDI-1 and NDI-cRGD conjugate fluorescence and the emission was collected between 575 and 700 nm. For each field of view, a z-stack was acquired using a 2x optical zoom. Image visualization and processing were performed with ImageJ.

Cytotoxic Activity Evaluation

Cell viability assays were performed using the MTS assay after 24, 48, and 72 h treatments. The cells were seeded in a 96-well plate (5000 cells/well) and cultured as described above. 24 h after plating, the medium was replaced by 100 µL of serum-free medium, and 24 h after this medium change, the medium was replaced again with serum-free culture medium containing samples. In previous work, dose/response curves of NDI-1 were performed on glioma cells, and 20 µM was found to be a quite toxic NDI-1 concentration for any cell line after 24 and 48 h contact.¹² Conversely, the same cRGD peptide concentration (20 µM) does not exert any toxic effect under the same experimental condition.¹⁹ Therefore, we decided to use this concentration to asses whether NDI-1 toxicity could be modulated and targeted by the conjugation with RGD peptide. In detail, NDI-1 was tested at 20 µM on U373, U251, and D384 cells for increasing time. After an incubation of 24, 48, and 72 h, supernatants were discarded, cells were rinsed by PBS, and 20 µL of MTS solution was added to 100 µL of fresh medium in each well. After a 1.5 h incubation, the plates were read at 490 nm by a Synergy HT plate reader (BioTek, Swindon). The experiments were performed three times in quadruplicate, and data are expressed as percent of living cells in control wells.

FACS analysis

U373 and U251 cells were subjected to FACS analysis by the Annexin/PI protocol, as previously described.¹⁹ Briefly, after treatment (20 µM NDI-1 or NDI-cRGD, 24 h) the cells were collected and rinsed by PBS twice. Subsequently, Fluorescein-conjugated annexin V was added and cells were incubated for 15 min at room temperature. After the addition of propidium iodide, samples were acquired using a Navios EX Flow Cytometer (Beckman Coulter) and analyzed by Navios EX Software (Beckman Coulter). At least 10,000 events per sample were recorded. Each experiment was repeated three times.

Statistical Analysis

The software STATGRAPHICS XVII (Statpoint Technologies, Inc., Warrenton, VA, USA) was used to elaborate raw data. As all data presented a normal distribution, a linear generalized analysis of variance model (ANOVA) was used, and the differences between the groups were evaluated with Fisher’s least significant difference (LSD) procedure. Statistical significance was set at p < 0.05.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the Supplementary Information.

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Scheme 1 is available in the Supplementary Files section.

No competing interests reported.

Scheme1.png
Scheme 1. Synthesis of compound 4 and NDI-cRGD conjugate: a) propargylamine (3 eq.), CH₃CN, reflux, 3h; b) neat N,N-dimethyl-1,3-diaminopropane, 120°C, 250 psi, 200 W, 10 min, microwave-assisted; c) ascorbic acid (1 eq.), CuSO₄·5H₂O (1 eq.), H₂O:tBuOH=1:1 mixture, rt, 2h.
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You are reading this latest preprint version

Selective delivery of G-quadruplex ligand in glioma cell lines: the power of cyclic-RGD peptide

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Version 1

Abstract

Figures

Introduction

Results and Discussion

Chemistry

Cellular Activity

Conclusion

Materials and methods

Chemical synthesis of the new conjugate NDI-cRGD

Synthesis of compound 4

Synthesis of NDI-cRGD

Biological Experiments

Cell Culture

Real-time PCR

Confocal microscopy analysis

Cytotoxic Activity Evaluation

FACS analysis

Statistical Analysis

Declarations

References

Scheme 1

Additional Declarations

Supplementary Files

Status:

Version 1