A water-soluble cationic [2]biphenyl-extended pillar[6]arene: synthesis, host–guest interaction with hemin and application in chemodynamic/photodynamic cancer therapy

Abstract

A water-soluble cationic [2]biphenyl-extended pillar[6]arene (CBpExP₆) was designed and synthesized successfully. It could form a stable 1 : 1 complex with hemin, thereby enhancing the stability of hemin in water, and can be further applied in cancer CDT and PDT.

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Supramolecular macrocycles possess the unique ability to selectively bind guest molecules through non-covalent interactions (including hydrogen bonding, π–π stacking, electrostatic interactions, van der Waals forces, etc.).^1–4 This highly specific selective recognition mechanism is just like a precisely matched key (i.e., the guest molecule) opening the corresponding lock (that is, the macrocyclic host compound), thus laying a solid foundation for the construction of intricate and complex supramolecular systems.⁵ Taking advantage of molecular recognition ability, many important applications such as the precise separation and sensitive detection of specific molecules can be achieved.⁶ In the context of biological systems, the recognition process of enzymes for substrates is also essentially a form of molecular recognition. Therefore, the molecular recognition characteristics exhibited by host macrocycles help us to gain a more in-depth and thorough understanding of the intricate interaction principles among biological molecules.^7–12

Traditionally macrocycles, such as crown ethers,¹³ cyclodextrins,^14,15 calixarenes,^16,17 cucurbiturils,^18,19 and pillar[n]arenes,^20–24 although they possess molecular recognition abilities to a certain extent, have relatively obvious limitations in terms of selectivity and recognition range.²⁵ With the rapid development and continuous deepening of supramolecular chemistry in cutting-edge fields such as materials science and nanotechnology, there is an urgent need to construct more intricate and diverse supramolecular structures.^26–28 Currently, the structures formed by the self-assembly of existing macrocycles can hardly fully meet the requirements under specific situations and application needs.^29,30

Therefore, it is particularly crucial to design and synthesize new macrocycles. By skilfully introducing new recognition sites and various functional groups into the macrocycles, the breadth and depth of their molecular recognition can be effectively expanded.^31–34 By precisely introducing nitrogen-containing heterocycles with specific coordination abilities onto the macrocyclic host compounds, the recognition efficiency for transition metal ions can be significantly enhanced.³⁵ For example, Prof. Yang designed and prepared an [2]biphenyl-extended pillar[6]arene with larger cavity, which can associate with more guest molecules.^36–40 Meanwhile, new macrocycles can also achieve the precise recognition of a wider range of guest molecules (including various organic molecules, biological molecules, etc.) by flexibly adjusting and optimizing key factors such as the size, shape, and flexibility of the ring, which undoubtedly has irreplaceable critical significance and far-reaching value for carrying out efficient molecular recognition and separation work in complex systems and will open up new paths and broad prospects for the further expansion and innovative application of supramolecular chemistry.^41–45

Hemin is a kind of porphyrin derivative containing two carboxyl groups. It can be directly absorbed by the human body and is the biological iron with the highest absorption rate currently known. Studies have found that hemin decomposes under light irradiation. Herein, we have designed and synthesized a cationic [2]biphenyl-extended pillar[6]arene (CBpExP₆), which has a larger cavity than that of pillar[6]arene and contains eight water-soluble quaternary ammonium cations. This enables it to form a stable 1 : 1 complex with hemin in water, thereby enhancing the stability of hemin. Further in vitro experiments have demonstrated that after the formation of CBpExP₆ ⊃ hemin, both its ability to generate reactive oxygen species (ROS) and kill tumor cells have been significantly improved.

As described in Scheme 1, CBpExP₆ was synthesized from 4,4′-bis(chloromethyl)-1,1′-biphenyl through three steps with a total yield about 70%. As expected, CBpExP₆ exhibited excellent water solubility due to it contains 8 quaternary ammonium cations. The structure of CBpExP₆ was confirmed through ¹H, ¹³C NMR, and electrospray ionisation mass spectrometry (ESI-MS) characterisations (Fig. S1–S5, ESI†). As CBpExP₆ presents ammonium cations on its macrocyclic framework, it can form a complex with hemin which contains anionic group efficiently. Firstly, the host–guest properties of CBpExP₆ with model guest (G_M: n-octanoic acid) and hemin were investigated in detail by ¹H NMR. All protons on G_M and hemin shifted upfield after complexation (Fig. S6 and S15, ESI†), suggested that linear guest G_M and hemin were threaded through the cavity of CBpExP₆ to form a [2]pseudorotaxane.⁴⁶ The formation of the complex might be mainly driven by hydrophobic and electrostatic interactions, because the hydrophobic cavity of CBpExP₆ could hold the hydrophobic alkyl chain of G_M and the cationic trimethylammonium groups of CBpExP₆ could bind the anionic carboxylate group of G_Mvia electrostatic interaction. Then, according to the isothermal titration calorimetry (ITC) results, CBpExP₆ and hemin form a 1 : 1 complex, and the association constant (K_a) between CBpExP₆ and hemin was calculated to be (2.46 ± 0.18) × 10⁴ M⁻¹, indicted CBpExP₆ ⊃ hemin was stable in water (Fig. 1). Further evidence for the formation of the desired 1 : 1 complex was obtained by LRESIMS, revealing peaks at m/z 1239.87 and 799.95, corresponding to [CBpExP₆ ⊃ hemin − 2H⁺ − 4Br⁻]²⁺ and [CBpExP₆ ⊃ hemin − 2H⁺ − 5Br⁻]³⁺, respectively (Fig. S8, ESI†).

Scheme 1 The synthetic route of cationic [2]biphenyl-extended pillar[6]arene (CBpExP₆) and chemical structures of model guest and hemin.

Fig. 1 (a) Isothermal titration calorimetry (ITC) studies between CBpExP₆ and hemin. (b) Schematic illustration of the formation of CBpExP₆ ⊃ hemin.

After confirming the host–guest interaction between CBpExP₆ and hemin, we probed the capacity of CBpExP₆ to improve the stability of hemin in water under irradiation. From the UV-vis spectrum, we found that under non-illumination conditions, the maximum absorption peak of the aqueous solution of hemin decreased from 2.3 to 1.6 within 60 minutes (Fig. S9, black line, ESI†). However, in the case of CBpExP₆ ⊃ hemin under the same conditions, only a slight decrease occurred in the maximum absorption peak (Fig. S9, blue line, ESI†). Even under the irradiation of a 660-nm laser, CBpExP₆ ⊃ hemin remained basically unchanged (Fig. S9, red line, ESI†), which demonstrates that CBpExP₆ can significantly enhance the stability of hemin in water.

Hemin could generate ROS under 660 nm laser irradiation as it contained the porphyrin core. Firstly, 1,3-diphenyl isobenzofuran (DPBF) was used as a ROS indicator to investigate the ROS generation ability of hemin. Upon irradiated with 660 nm laser, the UV absorbance decreased tremendously within 360s in the presence of CBpExP₆ (Fig. 2a), and the degree of decrease is much higher than that in the absence of CBpExP₆ (Fig. S14, ESI†), indicating the high efficiency of ROS generation of CBpExP₆ ⊃ hemin.⁴⁷ On the other hand, the structural feature of hemin contains ferrocene units, which can catalyze H₂O₂ to generate ˙OH via a Fenton-like reaction.⁴⁸ As shown in Fig. 2b, time-dependent UV-visible spectra for the solution containing CBpExP₆ ⊃ hemin, H₂O₂, and methylene blue (MB) further confirmed the increase in ˙OH generation and hence oxidation of MB. Afterward, the intracellular ROSs generation of CBpExP₆ ⊃ hemin in living cells under illumination with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as the probe was investigated by confocal laser scanning microscopy (CLSM). As shown in Fig. 2c, green fluorescence was observed when human cervical carcinoma (HeLa) cells were treated with both CBpExP₆ ⊃ hemin and 660 nm laser irradiation, this is because the nonfluorescent DCFH-DA was oxidized into green fluorescence 2′,7′-dichloroflorescein (DCF) by the generated ROSs. Furthermore, the generation of ˙OH, and ¹O₂ by CBpExP₆ ⊃ hemin was confirmed by aminophenyl fluorescein assay (APF), and singlet oxygen sensor green assay (SOSG), respectively (Fig. 2d and e). Electron paramagnetic resonance (EPR) spectra also confirmed the above results (Fig. S12, ESI†).

Fig. 2 (a) Time-dependent UV-vis spectra of CBpExP₆ ⊃ hemin with DPBF after irradiation (660 nm, 1.0 W cm⁻²). (b) Time-dependent UV-visible spectra for the solution containing CBpExP₆ ⊃ hemin (0.15 mg), H₂O₂ (10 mmol L⁻¹), MB (15 μg mL⁻¹). (c)–(e) CLSM images of various ROS species generated in HeLa cells after 660 nm light irradiation (1.0 W cm⁻²). (c) Total ROS was detected by DCFH, (d) ¹O₂ was detected by SOSG, and (e) ˙OH was detected by APF. Scale bar is 20 μm.

Since CBpExP₆ ⊃ hemin can generate ROSs under 660 nm-light irradiation and catalyze overexpressed H₂O₂ to produce ˙OH in tumor tissue, we then investigated its ability to kill cancer cells upon illumination. Through dynamic light scattering (DLS) and scanning electron microscopy (SEM) studies (Fig. S10, ESI†), it was found that CBpExP₆ ⊃ hemin can self-assemble in water to form nanoparticles with diameters around 200 nm. And the zeta potential of CBpExP₆ ⊃ hemin was 12.6 ± 0.5 mV (Fig. S13, ESI†). These enabled CBpExP₆ ⊃ hemin NPs to be effectively phagocytosed by cancer cells, achieving rapid enrichment of CBpExP₆ ⊃ hemin at the tumor site.

Then HeLa cells were also selected to investigate the phototherapy effect of CBpExP₆ ⊃ hemin in vitro, after incubating with different groups, its viability was investigated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. HeLa cells were cultivated with different concentrations (0–80 μg mL⁻¹) of hemin, CBpExP₆ ⊃ hemin, then irradiated with 10 min of 660 nm light (1 W cm⁻²). Fig. 3b showed that the viability of cells in control and near-infrared-radiation (NIR) groups all above 97%, indicating that there was no obvious cytotoxicity of these groups. Hemin exhibits relatively low toxicity in tumor cells. This is because although Fe(II) can catalyze the production of ˙OH from the overexpressed H₂O₂ in tumor cells, hemin is highly unstable and will quickly become ineffective.

Fig. 3 (a) Schematic illustration of hemin self-assembly into nanoparticles in the presence of CBpExP₆ and application in cancer therapy. (b) Cell viability of HeLa cells (4 h) incubated with control, NIR, hemin, hemin + NIR, CBpExP₆ ⊃ hemin and CBpExP₆ ⊃ hemin + NIR, respectively, at different concentrations.

In the hemin + NIR group, the cytotoxicity increases due to ¹O₂ is generated upon near-infrared light irradiation, which also could kill tumor cells. On the other hand, in the CBpExP₆ ⊃ hemin group, the cytotoxicity is also greater than that in the hemin group. This is because CBpExP₆ enhances the stability of hemin, enabling it to continuously catalyze the overexpressed H₂O₂ to produce ˙OH to kill tumor cells. Under the same conditions, the toxicity of hemin and CBpExP₆ ⊃ hemin to the normal cells HEK293 is significantly lower than that to HeLa cells (Fig. S11, ESI†). This is caused by the fact that the content of H₂O₂ in normal cells is lower than that in HeLa cells. Nevertheless, the CBpExP₆ ⊃ hemin + NIR group shows the greatest cytotoxicity. When the concentration is 80 μg mL⁻¹, the cell viability is only 8%. This is because it can not only continuously generate ˙OH but also produce ¹O₂ under NIR. The synergistic effect of the two significantly improves the ability to kill tumor cells.

At last, to check the phototherapy effect of CBpExP₆ ⊃ hemin more intuitively effect, live (green) and dead (red) cells was differentiated by calceinacetoxymethyl (calcein-AM) and propidium iodide (PI) staining. In control (Fig. 4a) and NIR (Fig. 4b) groups, the cells exhibited green fluorescence, indicating they are living well. On the other hand, when incubated with hemin, CBpExP₆ ⊃ hemin, or hemin + NIR, both red and green fluorescence were observed, indicating that partial cells were dead. However, when treated with CBpExP₆ ⊃ hemin then irradiated with NIR, almost the cells were dead and showed red fluorescence. These results clearly confirmed the satisfied therapeutic effect of CBpExP₆ ⊃ hemin upon irradiation.

Fig. 4 Fluorescent images of live HeLa cells after incubation for 4 h with (a) control, (b) NIR, (c) hemin, (d) CBpExP₆ ⊃ hemin, (e) hemin + NIR and (f) CBpExP₆ ⊃ hemin + NIR (concentration is 5.00 × 10⁻⁴ M). Scale bar is 50 μm.

In conclusion, we designed and synthesized a cationic [2]biphenyl-extended pillar[6]arene with eight quaternary ammonium salts (CBpExP₆). Isothermal titration calorimetry (ITC) studies have shown that CBpExP₆ can form a stable 1 : 1 complex with hemin in water, thus enhancing the stability of hemin in water. Moreover, CBpExP₆ ⊃ hemin can further self-assemble into nanoparticles with a diameter around 200 nm, enabling it to effectively be phagocytosed by tumor cells. Since the Fe(II) in CBpExP₆ ⊃ hemin can catalyze the H₂O₂, which is overexpressed in tumor cells, to generate ˙OH, and on the other hand, under light irradiation, the porphyrin core in CBpExP₆ ⊃ hemin can generate ¹O₂. The synergistic effect of these two processes can effectively kill tumor cells. This work provides a potential supramolecular strategy for enhancing the stability of photosensitizers and thus improving their anti-tumor therapeutic efficacy.

This work was supported by the National Natural Science Foundation of China (22007052), and College Students' Innovation and Entrepreneurship Project (202410304102Y). We also thank Nantong University Analysis & Testing Center for characterization.

Data availability

Data for this article are available at main article of this paper.

Conflicts of interest

There are no conflicts to declare.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Synthesis, additional ¹H NMR spectra and determination of the association constants. See DOI: https://doi.org/10.1039/d5cc00627a

‡ Y. Cai and Y. Zhang contributed equally to this article.

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