MOF-based nanoagent for self-illuminating photodynamic therapy

Abstract

Photodynamic therapy (PDT) is a minimally invasive and local light-activated treatment for annihilating tumor tissues. However, it is often limited by the tissue penetration depth of common external light sources. To address this issue, we herein report a self-illuminating ZIF-90 MOF-based composite photosensitive system prepared using a combined one-pot self-assembly and post-synthetic modification (PSM) methodology. The obtained PME@Zn/Fe-ZIF-90-Lum consists of the Fe(II)-doped and luminol-decorated MOF host as well as the protoporphyrin IX dimethyl ester (PME) guest. Under the given conditions, the light, which is generated from the Fe(II)-catalysed luminol oxidation by endogenous H₂O₂, can in situ activate the encapsulated photosensitizer PME to produce reactive oxygen species (ROS) via a chemiluminescence resonance energy transfer (CRET) process. In the absence of an external light source, antitumor PDT is successfully realized and high phototoxicity is fully evidenced by in vitro and in vivo experiments.

---

1. Introduction

Photodynamic therapy (PDT) is a promising clinical cancer treatment method in which a photosensitizer interacts with light and oxygen to generate cytotoxic oxygen species (ROS), such as singlet oxygen (¹O₂), to cause cancer cell death.^1–3 Compared with chemotherapy and radiotherapy, PDT produces less collateral damage to normal tissues, because ¹O₂ is produced only in the illuminated area where the photosensitizer accumulates. However, PDT usually requires an external light source, and the penetration depth of external light is severely limited due to strong optical attenuation along the light propagation path from the outside in.

Although several types of nanomaterials, including two-photon excitation nanoparticles (NPs),⁴ upconversion NPs,⁵ and near-infrared photosensitizers,^6–8 have been used to improve tissue penetration for deep-seated treatment, deep-tissue PDT has not been satisfactorily achieved. It is conceivable that if PDT could be performed in the absence of external light sources, this bottleneck would be broken through.

Besides conventional light sources, light can also be generated from chemical reactions in principle. This chemiluminescence is a heaven-sent chance to fabricate a self-illuminating system which is supposed to activate the coexisting photosensitizers via chemiluminescence resonance energy transfer (CRET) to produce ROS for PDT.

Hitherto, several interesting self-illuminating organic transformations, including lophine autoxidation,⁹ metal-assisted luminol¹⁰ and lucigenin^11,12 oxidation, and the peroxyoxalate reaction,^13–17 have been found to exhibit chemiluminescence. However, only a few self-illuminating systems have been used for tumor therapy thus far.^18–25

Nanoscale metal–organic frameworks (NMOFs) have recently been explored as a promising class of nanoplatforms for tumor therapy due to their low toxicity and good biocompatibility.^26–28 Therein, on the one hand, organic photosensitizers can be nanocrystallized by loading them onto the NMOF-carriers via either a one-pot reaction or PSM for PDT. For example, ZIF-MOFs are capable of encapsulating rhodamine B,²⁹ BODIPY³⁰ and phthalocyanine^31,32 to afford photosensitizer-loaded host–guest photosensitive systems. On the other hand, the pre-embedded organic groups in NMOFs, such as the –CHO in Zn-ZIF-90, can be further functionalized by PSM,^33,34 consequently endowing NMOFs with more functionality.

In this contribution, we report a self-illuminating ZIF-90-based composite photosensitive system for tumor photodynamic therapy. As illustrated in Scheme 1, the synthesized PME@Zn/Fe-ZIF-90-Lum composite consists of an Fe(II)-doped, luminol chemiluminescent emitter-modified ZIF-90 host and a PME photosensitizer guest. Under specific conditions, the light generated by Fe(II)-catalyzed luminol oxidation via endogenous H₂O₂ can in situ activate the coexisting PME photosensitizer through a CRET process, thereby inducing the production of ¹O₂. Notably, antitumor PDT is successfully achieved without relying on an external light source, and its high phototoxicity is fully validated by both in vitro assays and in vivo experiments. Compared with the aforementioned studies,^24,25 our work has made significant breakthroughs in the synthesis strategy for composite nanomaterials. Firstly, we precisely grafted luminol onto the surface of the MOF via a covalent bonding strategy. This design not only significantly enhances the structural stability of PME@Zn/Fe-ZIF-90-Lum but also effectively reduces the risk of luminol being recognized and cleared by the immune system—owing to the covalent modification, luminol forms a tight chemical bond with the MOF framework, constructing an integrated and stable structure. These innovative designs offer a feasible approach for the development of efficient and stable self-illuminating photosensitive systems, which is critical for improving the therapeutic efficacy of PDT in deep-seated tumors.

Scheme 1 Schematic illustration of the fabrication of PME@Zn/Fe-ZIF-90-Lum and its working mechanism for self-illuminating PDT.

Specifically, the Fe(II)-doped PME@Zn/Fe-ZIF-90 host–guest system was prepared as purple-dark crystalline solids by one-pot in situ self-assembly of imidazole-2-carboxaldehyde (IcaH), FeSO₄/Zn(NO₃)₂ (molar ratio 0.42 : 1), and PME with trioctylamine (TOA) in DMF at room temperature.^30–35 The molar ratio of Fe/Zn in the resulting PME@Zn/Fe-ZIF-90 was found to be ca. 0.27 : 1 based on inductively coupled plasma optical emission (ICP-OES) analysis (Table S1). By mixing PME@Zn/Fe-ZIF-90 and luminol in methanol with the aid of diethylamine (DEA) at room temperature for 4 h, the self-illuminating nanoagent PME@Zn/Fe-ZIF-90-Lum was readily generated as a deep brown crystalline solid (Scheme 1), which was further supported by the model reaction between molecular IcaH and luminol to IcaH-Lum under the same reaction conditions (Fig. S1).

2. Experimental

2.1 Materials and instrumentation

All reactants were reagent grade and were used as purchased without further purification. Imidazole-2-carboxaldehyde (IcaH) was purchased from Bidepharm Co., Ltd, and trioctylamine (TOA) was purchased from Tokyo Chemical Industry Co., Ltd. Protoporphyrin IX dimethyl ester (PME) was obtained from Aladdin Reagent Co., Ltd. Luminol (Lum) was purchased from Energy Chemical Co., Ltd. Zinc nitrate hexahydrate (ZnNO₃·6H₂O) and ethylenediamine (EDA) were purchased from Sinopharm Chemical Reagent Co., Ltd. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (Shanghai) Trading Co. Ltd. Phosphate-buffered saline (PBS) and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Biological Industries USA, Inc. RPMI Medium 1640 basic (1×) (RPMI 1640), fetal bovine serum (FBS), penicillin–streptomycin mixtures (Pen–Strep) and trypsin–EDTA solution were purchased from HyClone Laboratories, Inc. Singlet Oxygen Sensor Green (SOSG) and Calcein Acetoxymethyl Ester (calcein-AM) were purchased from Shanghai Macklin Biochemical Co., Ltd. All organic solvents were purchased from Sinopharm Chemical Reagent Co., Ltd. MCF-7 cell lines were provided by the Institute of Basic Medicine, Shandong Academy of Medical Sciences (Jinan, China). MCF-7 cell lines were cultured in RPMI 1640 supplemented with 10% (v/v) FBS and 1% antibiotics (penicillin/streptomycin, 100 U mL⁻¹) at 37 °C under a 5% CO₂ atmosphere. Deionized water prepared with an AquaPro System (18 MΩ) was employed in all experiments.

Powder X-ray diffraction (PXRD) experiments were obtained on a D8 ADVANCE X-ray powder diffractometer with CuKα radiation (λ = 1.5405 Å) from 2θ = 5° to 50° with 0.01° increments. Ultraviolet-visible (UV-vis) absorption spectra were recorded with a UV-2600 Jingdao spectrophotometer. Fourier transform infrared (FT-IR) samples were prepared as KBr pellets, and FT-IR spectra were obtained in the 4000–400 cm⁻¹ range using a Thermo Scientific Nicolet iS50 FT-IR Spectrometer. Fluorescence spectra were obtained with an FLS-920 Edinburgh Fluorescence Spectrometer with or without a xenon lamp. Chemiluminescence (CL) spectra were obtained with a GloMax^TM 96 Microplate Luminometer. Scanning electron microscopy (SEM) micrographs were recorded on a SIGMA300 scanning electron microscope equipped with an energy-dispersive X-ray detector (X-MAX-20 EDX). X-ray photoelectron spectroscopy (XPS) spectra were obtained from a PHI VersaProbe II. Inductively coupled plasma (ICP) measurements were obtained on a Varian720 ICP-OES. Dynamic light scattering (DLS) and zeta potentials were obtained on a Malvern Zetasizer Nano ZS90 with water as the solvent at 25 °C. Confocal fluorescence imaging studies were performed with a TCS SP8 confocal laser scanning microscope (Leica Co., Ltd. Germany) with an objective lens (×20).

2.2 Model reaction of IcaH with luminol

A mixture of luminol (500 mg, 2.82 mmol), IcaH (271 mg, 2.82 mmol) and diethylamine (DEA) (600 μL) in MeOH (60 mL) was stirred at room temperature for 4 h. After addition of ether, the obtained crude product IcaH-Lum was collected by filtration, and purified by recrystallization in MeOH/ether. Yield: 94%. IR (KBr pellet cm⁻¹): 2985(w), 2486(w), 1632(w), 1574(m), 1507(s), 1413(w), 1362(w), 1319(m), 1271(w), 1090(m), 819(w), 769(m), 733(w). ¹HNMR (400 MHz, DMSO-d6, 25 °C, TMS, ppm): 11.01 (s, 2H, –CONH–), 8.05 (s, 1H, –CH=N–, Schiff base), 7.58–7.42 (m, 3H, –C₆H₃–), 7.32 (s, 1H, –NH–, IcaH), 6.96–9.94 (d, 1H, –CH–, IcaH), 6.88–6.86 (d, 1H, –CH–, IcaH). ESI-MS: m/z calcd for C₁₂H₉N₅O₂ [M⁻] 254.1; found 254.1. Anal. calcd for C₁₂H₉N₅O₂ (%): C, 56.47; H, 3.55; N, 27.44; found C, 56.13; H, 4.01; N, 27.21.

2.3 Synthesis of PME@Zn/Fe-ZIF-90

PME-encapsulating Fe-doped ZIF-90 (PME@Zn/Fe-ZIF-90) was synthesized via a one-pot in situ preparation method. ZnNO₃·6H₂O (31.5 mg, 0.106 mmol), FeSO₄·7H₂O (12.5 mg, 0.045 mmol) and protoporphyrin IX dimethyl ester (PME) (43.0 mg, 0.0728 mmol) were dissolved in 10 mL of DMF. In a separate vial, 40 mg (0.416 mmol) of IcaH was added to 20 mL of DMF and was heated at 50 °C with stirring until fully dissolved and then cooled to room temperature. Another 10 mL of DMF containing 172 μL (0.394 mmol) of TOA was prepared. All these three solutions were kept at about 4 °C. Then, the IcaH solution was added to the prepared Zn/Fe-containing solution under vigorous stirring before the addition of another 10 mL of DMF containing of TOA at room temperature. After 1 min, the reaction was quenched by addition of 20 mL of ethanol. Particles were then isolated by centrifugation and then washed with DMF until no color was observed for the DMF extract. After it was separately and completely washed with ethanol and ether, the product was dried at 40 °C to generate nanoparticles as dark purple crystalline solids. Yield: 28.8 mg (66.3%). FT-IR (ATR, cm⁻¹): 3446(m), 2975(w), 2850(w), 1673(s), 1456(s), 1416(s), 1363(s), 1325(m), 1200(w), 1169(s), 1130(w), 1047(w), 956(m), 879(w), 792(s), 699(w), 537(w).

2.4 Synthesis of PME@Zn/Fe-ZIF-90-Lum

PME@Zn/Fe-ZIF-90-Lum was synthesized via a post-synthetic method. PME@Zn/Fe-ZIF-90 (10 mg, 0.031 mmol for (Ica)₂ content in a host–guest system) and luminol (10 mg, 0.056 mmol) were stirred in 3 mL of methanol at room temperature. Then, 100 μL of diethylamine (DEA) was added to the mixture solution and stirred for 4 h. The resulting solids were collected by centrifugation, soaked and washed with DMF to completely remove the unreacted luminol molecule (monitored by UV-vis). After this it was separately and completely washed with ethanol and ether, and then dried at 40 °C to generate nanoparticles as yellowish-brown solids. Yield: 4.3 mg (37%). FT-IR (ATR, cm⁻¹): 3420(m), 2854(w), 1671(s), 1574(s), 1506(s), 1456(s), 1417(s), 1365(s), 1327(m), 1201(w), 1169(s), 1123(w), 1089(w), 956(m), 793(s), 721(w), 699(w), 541(w).

2.5 Synthesis of Zn/Fe-ZIF-90

Light-yellow Fe-doped Zn/Fe-ZIF-90 was prepared by the same synthetic procedure as that for PME@Zn/Fe-ZIF-90 in the absence of PME. Yield: 14 mg (41%). FT-IR (ATR, cm⁻¹): 3446(m), 2975(w), 2850(w), 1673(s), 1456(s), 1416(s), 1363(s), 1325(m), 1200(w), 1169(s), 1130(w), 1047(w), 956(m), 879(w), 792(s), 699(w), 537(w).

2.6 Singlet oxygen (¹O₂) generation

In order to verify the singlet oxygen producing ability of PME@ZnFe-ZIF-90-Lum with H₂O₂ in aqueous solution, Singlet Oxygen Sensor Green (SOSG) was used as an ¹O₂ detection agent by monitoring the fluorescence spectra (λ_ex = 480 nm, monitored at 510–580 nm) in the presence of H₂O₂. The samples were prepared by respectively mixing Zn/Fe-ZIF-90 (0.7 μg mL⁻¹), PME@Zn/Fe-ZIF-90 (0.9 μg mL⁻¹) and PME@Zn/Fe-ZIF-90-Lum (1.1 μg mL⁻¹) with SOSG (0.08 μM) in the presence or absence of H₂O₂ (20 μM) and allowing them to stand at −18 °C for 3 h.

2.7 Intracellular detection of H₂O₂

The H₂O₂ content of MCF-7 cells was detected using a hydrogen peroxide assay kit. This kit oxidizes divalent iron ions with hydrogen peroxide to produce trivalent iron ions, which are then combined with xylenol orange to form a purple product in a specific solution, detecting the hydrogen peroxide concentration. The lysis buffer was added in a ratio of 0.2 mL per 10⁶ cells. Then, the sample was thoroughly homogenized to lyse and disrupt the cells, and the supernatant was collected. Next, the absorption intensity at 560 nm of a mixed solution of the supernatant (50 μL) and H₂O₂ detection reagent (100 μL) was recorded using a Microplate reader.

2.8 Cell uptake

MCF-7 cells were seeded into glass-bottomed dishes in 2 mL of RPMI 1640 medium. The cells were treated with the sample PME@Zn/Fe-ZIF-90-Lum (40 μg mL⁻¹) at 37 °C. The cell culture medium was removed and cells were washed with ice-cold PBS buffer (pH = 7.4) twice before being fixed with fresh 4.0% paraformaldehyde (1 mL) for 10 min at room temperature. The fixed cells were again washed with PBS three times before observation using a confocal laser scanning microscope. To confirm the localization of photosensitizers in cells, the above fixed cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and then again washed with PBS three times before observation using a confocal laser scanning microscope excited at 405 nm and monitored at 425–490 nm.

2.9 Biocompatibility

To evaluate the biocompatibility of PME@Zn/Fe-ZIF-90-Lum, MCF-10A cells were separately seeded into 96-well plates and incubated in a humidified incubator in 5% CO₂/95% air at 37 °C for 24 h. The cells that were incubated with PME@Zn/Fe-ZIF-90-Lum (100 μL per well) served as the treatment group, and those treated with free RPMI 1640 (100 μL per well) served as the control group.

2.10 In vitro ¹O₂ generation

MCF-7 cells were incubated with samples of luminol, PME, Zn/Fe-ZIF-90, PME@Zn/Fe-ZIF-90 and PME@Zn/Fe-ZIF-90-Lum (60 μg mL⁻¹) with SOSG (5 μM) for 4 h, respectively, and then washed with RPMI 1640. Cell images were acquired using a confocal laser scanning microscope. The ¹O₂ probe was excited at 488 nm and monitored at 500–550 nm.

2.11 Cytotoxicity assay

The cytotoxicity levels of luminol, PME, Zn/Fe-ZIF-90, PME@Zn/Fe-ZIF-90 and PME@Zn/Fe-ZIF-90-Lum nanocrystals were evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MCF-7 cells were plated at a density of 1 × 10⁴ cells per well in a 96-well cell culture plate at 37 °C under a 5% CO₂ atmosphere overnight. The cells that were incubated with luminol, PME, Zn/Fe-ZIF-90, PME@Zn/Fe-ZIF-90 and PME@Zn/Fe-ZIF-90-Lum (100 μL per well) served as treatment groups, and those that were treated with free RPMI 1640 (100 μL per well) served as the control group for 24 h at 37 °C under a 5% CO₂ atmosphere and incubated for 24 h.

2.12 Calcein-AM staining

MCF-7 cells were incubated with samples luminol, PME, Zn/Fe-ZIF-90, PME@Zn/Fe-ZIF-90 and PME@Zn/Fe-ZIF-90-Lum (80 μg mL⁻¹) at 37 °C for 24 h before calcein-AM (calcein acetoxymethyl ester) was used to stain the cells and they were imaged using a laser scanning confocal microscope using a green channel for live cells (λ_ex = 488 nm, monitored at 500–550 nm).

2.13 In vivo antitumor therapy

Nude mice (BALB/c-JGpt-Foxn1^nu/Gpt, male, aged 5 weeks, ∼20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animal experiments were reviewed and approved by the Ethics Committee of Shandong Normal University (Jinan, P. R. China). All the animal experiments complied with relevant guidelines of the Chinese government and regulations for the care and use of experimental animals (approval number AEECSDNU 2019029).

MCF-7 cancer cells (10⁶ cells) suspended in DPBS (100 μL) were subcutaneously injected into the flanks of each mouse to establish an MCF-7 xenograft model. The length (L) and width (W) of the tumor were determined using digital calipers. The tumor volume (v) was calculated by the formula V = 1/2 × L × W². When the tumor size reached ∼100 mm³, the nude mice bearing MCF-7 tumors (n = 12) were randomly distributed into 3 groups, as follows: (i) control, (ii) PME@Zn/Fe-ZIF-90, and (iii) PME@Zn/Fe-ZIF-90-Lum. After intratumoral injection (1 mg mL⁻¹, 50 μL), the nude mice were fed for 3 days, and for the treatment group, injection of samples (1 mg mL⁻¹, 50 μL) was performed on the tumor site another three times every 3 days. The tumor volume and nude mouse body weight were recorded every two days during the experimental period. The major organs (e.g., heart, liver, spleen, lung, kidney, and tumor) were collected and sliced for H&E staining.

2.14 In vivo distribution analysis

50 μL of DPBS solution containing PME@Zn/Fe-ZIF-90-Lum (1 mg mL⁻¹) was injected into the tumor site. Subsequently, the main organs and tumor were collected at different time points (3 or 6 h) to systematically investigate the distribution of PME@Zn/Fe-ZIF-90-Lum.

3. Results and discussion

As shown in Fig. 1a, the powder X-ray diffraction (PXRD) patterns of Zn/Fe-ZIF-90, PME@Zn/Fe-ZIF-90, and PME@Zn/Fe-ZIF-90-Lum are identical to that of their pristine Zn-ZIF-90, indicating that the one-pot in situ Fe doping and PME encapsulation as well as the subsequent luminol decoration did not affect the Zn-ZIF-90 structural integrity and crystallinity. In addition, no PXRD peaks related to PME were detected in the PXRD patterns of PME@Zn/Fe-ZIF-90 and PME@Zn/Fe-ZIF-90-Lum, suggesting that PME herein is located in ZIF-90 pores instead of on the surfaces of the MOF NPs (Fig. S2), which is in good agreement with previous reports.^28,29 The scanning electron microscopy (SEM) images showed that the Zn/Fe-ZIF-90, PME@Zn/Fe-ZIF-90, and PME@Zn/Fe-ZIF-90-Lum NPs were uniformly distributed, and they featured the same morphology and particle size (ca. 72 nm in diameter) (Fig. 1b and S3), which is further supported by the dynamic light scattering (DLS) measurement (Fig. S3).

Fig. 1 (a) PXRD patterns. (b) SEM image of PME@Fe-ZIF-90-Lum NPs. (c) Normalized UV-vis spectra of luminol (80 μM), PME (5 μM), Fe-ZIF-90 (67 μg mL⁻¹), and PME@Fe-ZIF-90-Lum (100 μg mL⁻¹) in DMF. (d) Chemiluminescence spectrum of IcaH-Lum (40 mM) with FeSO₄ (1 mM) and H₂O₂ (100 mM) in DMF, and absorption spectrum of PME (5.0 μM) in DMF. (e) Fluorescence spectrum of PME (1.0 μM) in DMF (λ_ex = 405 nm). (f) Luminescence spectrum of IcaH-Lum (40 mM), PME (0.1 mM), and FeSO₄ (1 mM) in the presence of H₂O₂ (100 mM) in DMF without an external light source.

Different from Zn/Fe-ZIF-90, PME@Zn/Fe-ZIF-90-Lum exhibited one strong peak (463 nm) and four weak peaks (537, 560, 590, and 641 nm) in its UV-vis spectrum, which are associated with the typical porphyrin Soret and Q-band absorptions, respectively. In comparison with free PME, the corresponding absorption bands of the encapsulated PME in PME@Zn/Fe-ZIF-90-Lum were significantly broadened and red-shifted (Fig. 1c), further implying the formation of a host–guest complex rather than the surface adsorption or a simply physical blend.³⁶ The standard curve method revealed that the PME loading amount in PME@Zn/Fe-ZIF-90-Lum is up to ca. 19.0 wt% (Fig. S4). Of note, no absorptions for Zn- and Fe-porphyrin complexes have been found in the UV-vis spectrum,^37,38 indicating that the involved metal ions are only located in the MOF framework. In addition, the UV-vis absorption of the covalently grafted luminol was blue-shifted from 358 to 352 nm compared with that of the free luminol molecule (Fig. 1c).

The covalent decoration of luminol on the MOF framework was further confirmed by the Fourier-transform infrared (FTIR) spectra (Fig. S5). The stretching band of C=N in PME@Zn/Fe-ZIF-90-Lum at 1634 cm⁻¹ evidenced the formation of the imine linkage.^33,34 Meanwhile, the vestigial peak at 1673 cm⁻¹ associated with the aldehyde group suggested that the PSM herein was not quantitative. The amount of luminol decorated in PME@Zn/Fe-ZIF-90-Lum was determined to be ca. 14.4 wt% based on the standard curve method (Fig. S4). As is known, Zn-ZIF-90 possesses a pore size of 11.2 Å in diameter, which is perfectly size-matched to the porphyrin core size (ca. 9.8 Å × 9.8 Å).³³ On the other hand, its open pore diameter of 3.5 Å cannot allow luminol (ca. 6 × 7 Å) to get through the ZIF pores to form a host–guest system during the PSM process. Importantly, covalent decoration enables the close chemical binding of luminol to the MOF framework, forming an overall stable structure. This effectively reduces the risk of the luminol being recognized and eliminated by the immune system.³⁹

For tumor therapy, the stability of PME@Zn/Fe-ZIF-90-Lum NPs in aqueous media was examined. The measured PXRD indicated that its crystallinity and structural integrity were well maintained after soaking in PBS solution (pH = 7.4) for 24 h (Fig. S6). Furthermore, no obvious aggregation in PBS or cell culture media (RPMI 1640) was observed after five days (Fig. S7), which is well in accordance with the previous observation for ZIF-based nanoagents.^30,39–41 Different from BodipyPhNO₂@ZIF-90,³⁰ the ζ potential of the luminol-grafted PME@Zn/Fe-ZIF-90-Lum herein is negative. For example, the measured surface ζ potentials of PME@Zn/Fe-ZIF-90-Lum in aqueous solution with different pH values are ca. −15.6 (pH = 8.0), −13.4 (pH = 7.4), −12.2 (pH = 7.0), −11.6 (pH = 6.5), −9.0 (pH = 6.0), and −8.35 mV (pH = 5.9), respectively (Fig. S8). The negatively charged surfaces of PME@Zn/Fe-ZIF-90-Lum NPs enable the NPs to accumulate in the tumor sites through the enhanced permeability and retention (EPR) effect.^42,43

As shown in Fig. 1d, the chemiluminescence emission of IcaH-Lum (380–650 nm, emission maximum at ca. 429 nm) and the electronic absorption of PME (λ_max = 405 nm for the Soret band and λ = 504, 539, 575, 631 for four Q bonds) met the spectral overlap requirement for CRET as the donor–acceptor pair.⁴⁴ So we envisioned that the chemiluminescence energy from IcaH-Lum oxidation could trigger PME emission (λ_em = 633 nm, Fig. 1e) within the MOF platform. To further prove this CRET process, the fluorescence spectrum of IcaH-Lum/FeSO₄/PME/H₂O₂ was measured in DMF without external light irradiation. As shown in Fig. 1f, two emission maxima at 457 and 634 nm, which are, respectively, assigned to IcaH-Lum and PME, were observed. This result unequivocally indicated that the luminescent acceptor of PME could be activated by the in situ generated light from IcaH-Lum oxidation to emit fluorescence through a CRET process (Scheme 1).

Further study revealed that the PME@Zn/Fe-ZIF-90-Lum fluorescence was H₂O₂-dependent. To simulate the internal environment of cancer cells, the fluorescence was measured in PBS solution of H₂O₂ with a concentration range of 1–1000 nM.^45–47 As shown, PME@Zn/Fe-ZIF-90-Lum was luminous under the given conditions, and its emission intensity gradually improved with the increasing H₂O₂ concentration (Fig. S9). In addition, it can be clearly seen that the emission intensity of PME@Zn/Fe-ZIF-90-Lum also increased with elevated pH and incremental MOF concentrations (Fig. S10). All these observations demonstrate that the fluorescence performance of PME@Zn/Fe-ZIF-90-Lum herein is consistent with the molecular luminol emission behavior.¹⁰

Besides, Singlet Oxygen Sensor Green (SOSG), which is a specific intracellular green fluorescent probe for ¹O₂,⁴⁸ was also used to evaluate the singlet oxygen producing ability of PME@Zn/Fe-ZIF-90-Lum in cells. The fluorescence spectra of Zn/Fe-ZIF-90 (0.7 μg mL⁻¹), PME@Zn/Fe-ZIF-90 (0.9 μg mL⁻¹) and PME@Zn/Fe-ZIF-90-Lum (1.1 μg mL⁻¹) were measured with SOSG (0.08 μM) in the presence or absence of H₂O₂ (20 μM). Different from Zn/Fe-ZIF-90 and PME@Zn/Fe-ZIF-90, H₂O₂ can only effectively activate PME@Zn/Fe-ZIF-90-Lum to generate ¹O₂ under the given conditions (Fig. S11), indicating that the PME photosensitizer and luminol chemiluminescent emitter are essential for this ¹O₂ production via a CRET process. Then, we used the H₂O₂ detection kit to detect the H₂O₂ concentration in MCF-7 cells. The results showed that its concentration was approximately 1.2 nM, which was sufficient to drive the luminescent substrate to generate optical signals and effectively activate the PME photosensitizer, thereby achieving a self-illuminating photodynamic therapeutic effect (Fig. S12).⁴⁹ Furthermore, its intracellular ¹O₂ generation was also confirmed using a confocal laser scanning microscope (CLSM). When MCF-7 cells were incubated with PME@Zn/Fe-ZIF-90-Lum (60 μg mL⁻¹) with SOSG (5 μM) for 4 h, green fluorescence was clearly observed in MCF-7 cancer cells (Fig. 2), implying that the self-illuminating PME@Zn/Fe-ZIF-90-Lum can effectively induce ¹O₂ generation in living cells via the endogenous CRET process. In contrast, negligible changes were observed in a series of control experiments (blank, Luminol/SOSG, PME/SOSG, Zn/Fe-ZIF-90/SOSG, PME@Zn/Fe-ZIF-90/SOSG, PME@Zn/Fe-ZIF-90-Lum and SOSG only) under the same conditions (Fig. 2).

Fig. 2 Cell confocal fluorescence imaging for detecting intracellular ¹O₂. Cells were incubated with samples (60 μg mL⁻¹) and with or without SOSG (5 μM) for 4 h, and imaged with a laser scanning confocal microscope. Green images were obtained when the samples were excited with 488 nm light, and the emission wavelength range was 500–550 nm. The experiments for control groups were performed under the same conditions.

In addition, the localization of PME@Zn/Fe-ZIF-90-Lum NPs in cells was also examined based on the CLSM. As shown in Fig. 3a, the fixed MCF-7 cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and visualized using a blue channel for DAPI (λ_ex = 405 nm, monitored at 425–490 nm) and a red channel for porphyrin species (λ_ex = 514 nm, monitored at 640–750 nm), respectively.³⁶ We noticed that the red fluorescence was mainly located in the cytoplasm, indicating that PME@Zn/Fe-ZIF-90-Lum NPs were principally distributed in the cytoplasm. On the other hand, this test further demonstrated that the PME@Zn/Fe-ZIF-90-Lum nanoagent possesses good membrane permeability. Subsequently, the effect of PME@Zn/Fe-ZIF-90-Lum on the cell viability of normal breast epithelial cells (MCF-10A) was detected through MTT assay. The results showed that even at a concentration of up to 120 μg mL⁻¹, the cell survival rate remained above 90% (Fig. S13), indicating that PME@Zn/Fe-ZIF-90-Lum has no obvious toxicity against normal cells and possesses good biocompatibility.

Fig. 3 (a) In vitro CLSM images of MCF-7 cells treated with PME@Zn/Fe-ZIF-90-Lum (40 μg mL⁻¹) for 2 h and counterstained with DAPI, scale 25 μm. (b) In vitro cytotoxicity of PME@Zn/Fe-ZIF-90-Lum against MCF-7 for 24 h. For comparison, the cytotoxicity levels of PME, Zn/Fe-ZIF-90, PME@Zn/Fe-ZIF-90 and luminol with concentrations equivalent to 10.0, 20.0, 40.0, 60.0, 80.0, and 120.0 μg mL⁻¹ of PME@Zn/Fe-ZIF-90-Lum are also shown.

Encouraged by its novel self-illuminating feature, excellent stability, and prominent biocompatibility, the PDT efficacy of PME@Zn/Fe-ZIF-90-Lum was thus evaluated using MTT assays against MCF-7 cells at various concentrations (0–120 μg mL⁻¹). As shown in Fig. 3b, the in vitro phototoxicity of PME@Zn/Fe-ZIF-90-Lum against MCF-7 cells was rapidly enhanced with its increasing concentration. MCF-7 survival rates of ca. 62, 56, and 30% were observed in the presence of 40, 60 and 80 μg mL⁻¹, respectively. Meanwhile, nonlinear regression fitting was adopted to accurately calculate the half maximal inhibitory concentration (IC₅₀) value of each experimental treatment group. As shown in Fig. S14, PME@Zn/Fe-ZIF-90-Lum exhibited the strongest therapeutic efficacy, with the IC₅₀ value in MCF-7 cells being 68.89 μg mL⁻¹. This observation was further supported by Calcein-AM staining. For example, the proportion of dead MCF-7 cells was up to ca. 70% after incubation with PME@Zn/Fe-ZIF-90-Lum at 80 μg mL⁻¹ for 24 h, which is well in agreement with the MTT results (Fig. S15). Under the given conditions, PME@Zn/Fe-ZIF-90-Lum induced a minimum cell survival rate of ca. 3% at a concentration of 120 μg mL⁻¹, suggesting its excellent in vitro PDT efficiency in the absence of external light irradiation. In contrast, luminol (17.5 μg mL⁻¹, 120.0 μg mL⁻¹PME@Zn/Fe-ZIF-90-Lum equiv.) and PME (17.5 μg mL⁻¹, 120.0 μg mL⁻¹PME@Zn/Fe-ZIF-90-Lum equiv.) were basically nontoxic and they maintained high cell survival rates of ca. 86 and 90%, respectively. On the other hand, Zn/Fe-ZIF-90 (79.9 μg mL⁻¹, 120.0 μg mL⁻¹PME@Zn/Fe-ZIF-90-Lum equiv.) and PME@Zn/Fe-ZIF-90 (102.7 μg mL⁻¹, 120.0 μg mL⁻¹PME@Zn/Fe-ZIF-90-Lum equiv.) exhibited almost the same cytotoxicity towards MCF-7 cells with mortality rates of ca. 23 and 29%, respectively (Fig. 3b). This inconspicuous cytotoxicity might be caused by a tiny amount of hydroxyl radicals (·OH) which were generated from the Fenton reaction of doped ferric ions in the ZIF framework with the endogenous H₂O₂ under the given conditions.^50,51

Based on the above results, we can conclude that our self-illuminating nanoagent PME@Zn/Fe-ZIF-90-Lum enables PDT in the absence of an external light source via an endogenous CRET process to kill cancer cells.

We finally carried out in vivo antitumor experiments on PME@Zn/Fe-ZIF-90-Lum, which were performed on an MCF-7 xenograft model. Twelve nude mice bearing tumors were randomly divided into three groups. Group I was the control group without any treatment. Group II was given PME@Zn/Fe-ZIF-90 at a dose of 50 μL (1 mg mL⁻¹), with administration repeated four times at intervals of 3 days. Group III was given PME@Zn/Fe-ZIF-90-Lum at a dose of 50 μL (1 mg mL⁻¹), with administration repeated four times at intervals of 3 days. As shown in Fig. 4a and b, the tumor volume and weight increased rapidly in control group I. Compared to group I, a slightly slower tumor growth for group II was observed. In contrast, the tumor tissues were significantly eradicated in group III, and the tumor weight was only ca. 35 and 23% of those in groups I and II, respectively. The digital photographs of tumors also showed that the tumor sizes of group III after therapy were much smaller than those of the other groups (Fig. 4c), further confirming that this high self-illumination induced a PDT antitumor effect.

Fig. 4 Antitumor therapy in vivo. (a) Tumor volumes of the nude mice in various groups during the treatment. (b) Tumor weights of the nude mice in various groups during the treatment. (c) Photographs of the tumor tissues obtained after dissection. All data are presented as mean ± SD (n = 4).

Additionally, by monitoring the weight changes of nude mice for 11 days (Fig. S16), it was found that there was no significant difference between the experimental group and the control group. On further combinination with Hematoxylin & Eosin (H&E) staining to observe the main organs such as the heart, liver, spleen, lung, and kidney, no significant tissue damage or pathological changes were observed (Fig. S17). These results confirmed that PME@Zn/Fe-ZIF-90-Lum has good biocompatibility.

Then, the in vivo distribution of PME@Zn/Fe-ZIF-90-Lum was dynamically monitored using animal fluorescence imaging. The results showed that the fluorescence signal intensity of PME@Zn/Fe-ZIF-90-Lum at the tumor site exhibited a decaying trend over time, and fluorescence enrichment was observed in the liver site (Fig. S18). This indicates that PME@Zn/Fe-ZIF-90-Lum can be gradually cleared through liver metabolism, demonstrating good metabolic safety in the body.

4. Conclusion

In summary, a self-illuminating PME@Zn/Fe-ZIF-90-Lum nanoagent was successfully designed and fabricated. The photosensitizer PME and the chemiluminescent emitter luminol were logically integrated on the NMOF platform by combined one-pot in situ synthesis and PSM steps. Under the given conditions, the light that originated from the Fe(II)-catalyzed luminol oxidation by endogenous H₂O₂ can effectively activate the co-existing porphyrin photosensitizer via a CRET process to produce ¹O₂ that can efficiently kill cancer cells in the absence of an external light source. Its high PDT antitumor efficiency was fully supported by in vitro and in vivo experiments. The concept of NMOF-based multifunctional integration makes PDT feasible in the absence of an external light source, and it might provide an alternative way to realize deep-tissue photodynamic therapy. Study of the preparation of new MOF- and COF-based composite self-illuminating nanoagents for tumor treatment is underway.

Author contributions

^‡These authors contributed equally to this work. All authors contributed extensively to the work presented in this paper.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data presented in this study are available within the article and its SI: experimental section and supplementary figures. See DOI: https://doi.org/10.1039/d5bm00963d.

Acknowledgements

We are grateful for financial support from the National Natural Science Foundation of China (grant no. 22371172), the Taishan Scholars Climbing Program of Shandong Province, and the Shandong Provincial Natural Science Foundation (grant no. ZR2021QB035).

References

1. X. Li, S. Lee and J. Yoon, Chem. Soc. Rev., 2018, 47, 1174–1188.
2. M. Lismont, L. Dreesen and S. Wuttke, Adv. Funct. Mater., 2017, 27, 1606314.
3. W. Fan, B. Yung, P. Huang and X. Chen, Chem. Rev., 2017, 117, 13566–13638.
4. M. Pawlicki, H. A. Collins, R. G. Denning and H. L. Anderson, Angew. Chem., Int. Ed., 2009, 48, 3244–3266.
5. G. Chen, H. Qiu, P. N. Prasad and X. Chen, Chem. Rev., 2014, 114, 5161–5214.
6. T. Sun, J.-H. Dou, S. Liu, X. Wang, X. Zheng, Y. Wang, J. Pei and Z. Xie, ACS Appl. Mater. Interfaces, 2018, 10, 7919–7926.
7. H. Liu, Y. Yang, A. Wang, M. Han, W. Cui and J. Li, Adv. Funct. Mater., 2016, 26, 2561–2570.
8. L. Li, Y. Liu, P. Hao, Z. Wang, L. Fu, Z. Ma and J. Zhou, Biomaterials, 2015, 41, 132–140.
9. B. R. Radziszewski, Ber. Dtsch. Chem. Ges., 1877, 10, 70–75.
10. O. S. Fedorova and V. M. Berdnikov, Theor. Exp. Chem., 1983, 19, 307–312.
11. K. Gleu and W. Petsch, Angew. Chem., Int. Ed., 1935, 48, 57–59.
12. R. Maskiewicz, D. Sogah and T. C. Bruice, J. Am. Chem. Soc., 1979, 101, 5347–5354.
13. E. A. Chandross, Tetrahedron Lett., 1963, 4, 761–765.
14. M. M. Rauhut, Acc. Chem. Res., 1969, 2, 80–87.
15. U. Quaβ and D. Klockow, Int. J. Environ. Anal. Chem., 1995, 60, 361–375.
16. C. V. Stevani, D. F. Lima, V. G. Toscano and W. J. Baader, J. Chem. Soc., Perkin Trans. 2, 1996, 989–995.
17. A. V. Romanyuk, I. D. Grozdova, A. A. Ezhov and N. S. Melik-Nubarov, Sci. Rep., 2017, 7, 3410.
18. H. Yuan, H. Chong, B. Wang, C. Zhu, L. Liu, Q. Yang, F. Lv and S. Wang, J. Am. Chem. Soc., 2012, 134, 13184–13187.
19. J. Zhao, J. Fei, L. Gao, W. Cui, Y. Yang, A. Wang and J. Li, Chem. – Eur. J., 2013, 19, 4548–4555.
20. Y. Zhang, L. Pang, C. Ma, Q. Tu, R. Zhang, E. Saeed, A. E. Mahmoud and J. Wang, Anal. Chem., 2014, 86, 3092–3099.
21. Y. Yang, H. Liu, M. Han, B. Sun and J. Li, Angew. Chem., Int. Ed., 2016, 55, 13538–13543.
22. S. Kim, H. Jo, M. Jeon, M. G. Choi, S. K. Hahn and S. H. Yun, Chem. Commun., 2017, 53, 4569–4572.
23. H. Cao, Y. Yang, Y. Qi, Y. Li, B. Sun, Y. Li, W. Cui, J. Li and J. Li, Adv. Healthcare Mater., 2018, 7, 1701357.
24. L. Hu, C. Xiong, J.-J. Zou, J. Chen, H. Lin, S. J. Dalgarno, H.-C. Zhou and J. Tian, ACS Appl. Mater. Interfaces, 2023, 15, 25369–25381.
25. W.-X. Ren, F. Kong, Y.-Q. Shao, Q.-Y. Huang, L.-F. Sun, J. Feng and Y.-B. Dong, Chem. Commun., 2022, 58, 5245–5248.
26. C. He, D. Liu and W. Lin, Chem. Rev., 2015, 115, 11079–11108.
27. Q. Guan, Y.-A. Li, W.-Y. Li and Y.-B. Dong, Chem. – Asian J., 2018, 13, 3122–3149.
28. E. Ploetz, H. Engelke, U. Lächelt and S. Wuttke, Adv. Funct. Mater., 2020, 30, 1909062.
29. J. Deng, K. Wang, M. Wang, P. Yu and L. Mao, J. Am. Chem. Soc., 2017, 139, 5877–5882.
30. Q. Guan, L.-L. Zhou, Y.-A. Li and Y.-B. Dong, Inorg. Chem., 2018, 57, 10137–10145.
31. M.-R. Song, D.-Y. Li, F.-Y. Nian, J.-P. Xue and J.-J. Chen, J. Mater. Sci., 2017, 53, 2351–2361.
32. D. Xu, Y. You, F. Zeng, Y. Wang, C. Liang, H. Feng and X. Ma, ACS Appl. Mater. Interfaces, 2018, 10, 15517–15523.
33. W. Morris, C. J. Doonan, H. Furukawa, R. Banerjee and O. M. Yaghi, J. Am. Chem. Soc., 2008, 130, 12626–12627.
34. L. Garzón-Tovar, S. Rodríguez-Hermida, I. Imaz and D. Maspoch, J. Am. Chem. Soc., 2017, 139, 897–903.
35. P. Su, H. Xiao, J. Zhao, Y. Yao, Z. Shao, C. Li and Q. Yang, Chem. Sci., 2013, 4, 2941–2946.
36. J.-L. Kan, Y. Jiang, A. Xue, Y.-H. Yu, Q. Wang, Y. Zhou and Y.-B. Dong, Inorg. Chem., 2018, 57, 5420–5428.
37. H.-J. Son, S. Jin, S. Patwardhan, S. J. Wezenberg, N. C. Jeong, M. So, C. E. Wilmer, A. A. Sarjeant, G. C. Schatz, R. Q. Snurr, O. K. Farha, G. P. Wiederrecht and J. T. Hupp, J. Am. Chem. Soc., 2013, 135, 862–869.
38. K. Wang, D. Feng, T.-F. Liu, J. Su, S. Yuan, Y.-P. Chen, M. Bosch, X. Zou and H.-C. Zhou, J. Am. Chem. Soc., 2014, 136, 13983–13986.
39. R. Tenchov, R. Bird, A. E. Curtze and Q. Zhou, ACS Nano, 2021, 15, 16982–17015.
40. F.-K. Shieh, S.-C. Wang, C.-I. Yen, C.-C. Wu, S. Dutta, L.-Y. Chou, J. V. Morabito, P. Hu, M.-H. Hsu, K. C.-W. Wu and C.-K. Tsung, J. Am. Chem. Soc., 2015, 137, 4276–4279.
41. H. Zheng, Y. Zhang, L. Liu, W. Wan, P. Guo, A. M. Nyström and X. Zou, J. Am. Chem. Soc., 2016, 138, 962–968.
42. F.-F. An, W. Cao and X. J. Liang, Adv. Healthcare Mater., 2014, 3, 1162–1181.
43. Q. Jia, J. Ge, W. Liu, X. Zheng, M. Wang, H. Zhang and P. Wang, ACS Appl. Mater. Interfaces, 2017, 9, 21124–21132.
44. W. Fan, P. Huang and X. Chen, Chem. Soc. Rev., 2016, 45, 6488–6519.
45. Y. Kuang, K. Balakrishnan, V. Gandhi and X. Peng, J. Am. Chem. Soc., 2011, 133, 19278–19281.
46. C. de Gracia Lux, S. Joshi-Barr, T. Nguyen, E. Mahmoud, E. Schopf, N. Fomina and A. Almutairi, J. Am. Chem. Soc., 2012, 134, 15758–15764.
47. F. Liu, L. Lin, Y. Zhang, Y. Wang, S. Sheng, C. Xu, H. Tian and X. Chen, Adv. Mater., 2019, 31, 1902885.
48. Q. Guan, D.-D. Fu, Y.-A. Li, X.-M. Kong, Z.-Y. Wei, W.-Y. Li, S.-J. Zhang and Y.-B. Dong, iScience, 2019, 14, 180–198.
49. Y.-D. Lee, C.-K. Lim, A. Singh, J. Koh, J. Kim, I. C. Kwon and S. Kim, ACS Nano, 2012, 6, 6759–6766.
50. R. Jin, Z. Liu, Y. Bai, Y. Zhou, J. J. Gooding and X. Chen, Adv. Funct. Mater., 2018, 28, 1801961.
51. G. Lan, K. Ni, Z. Xu, S. S. Veroneau, Y. Song and W. Lin, J. Am. Chem. Soc., 2018, 140, 5670–5673.

---

Footnote

† These authors contributed equally to this work.

---