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. 2025 Aug 23;15:31008. doi: 10.1038/s41598-025-15892-y

Porphyrinic MOF-derived novel nanocomposite for gastric anticancer and Helicobacter pylori photoantibacterial effect assay

Sajedeh Tehrani Nejad 1, Rahmatollah Rahimi 1,✉, Saeideh Eslaminejad 1
PMCID: PMC12375092  PMID: 40849528

Abstract

Porphyrinic MOF-based nanocomposites provide an applied attraction that overcomes the harmful effects of existing anticancer drugs. In this work, a nanorod porphyrinic metal-organic framework, PCN-222, was synthesized to serve as a high-surface-area drug carrier with photochemical properties. This material was then used for the production of Au nanoparticles. The PCN-222/Au/DOX NCs were provided with a low dosage of doxorubicin (DOX) as a novel nanocomposite with multifunctional anti-cancer properties on gastric cancer cells. The cytotoxicity effect was surveyed with an MTT assay on the MKN-45 cell line. The Annexin-V/propidium iodide technique and the colorimetric methods were utilized for apoptosis detection and caspase 8 and 9 pathway determination, respectively. The produced intracellular reactive oxygen species (ROS) were assigned using flow cytometry. Besides, the antibacterial activity of the nanocomposite was examined by conducting minimum inhibitory concentration (MIC) and Minimum Bactericidal Concentration (MBC) tests on Helicobacter pylori under LED light and dark conditions. Achieving a 91% ratio of the apoptotic cells versus the total dead cells rate, the high ROS production with the main Florence intensity (MIF) value of about 247.5, and an approximately two-fold increase in caspase 9 compared to caspase 8, with a MIC value of 375 µg/mL result under LED light for antibacterial activity, make the PCN-222/Au/DOX NCs an applicable promising agent with anticancer properties for gastric cancer cure.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-15892-y.

Keywords: Metal-organic frameworks, Porphyrin, PCN-222/Au/DOX NCs, Gastric cancer, ROS

Subject terms: Biotechnology, Cancer, Medical research, Chemistry

Introduction

Discovering Multipurpose materials for cancer cure remains a significant challenge despite the development and advancement of current medical therapies1–4. Metal-organic framework (MOFs) nanocomposites can be drug carriers, in addition to the possibility of combining several anticancer species in one structure3–12. This integration can be created by further shrinking the size of the host MOFs and the guest functional elements to the nanoscale regime, more suited for bio-circulation. MOF nanocomposites have gained remarkable attention in curative and energy-based applications13–15. MOFs have spawned a new point of view in combination with anti-cancer drugs10,16,17. Porphyrin-based MOFs are emerging as cancer therapeutic platforms, with the advantage of focusing on the possibility of photo-synthesis and becoming nanocomposites with thermodynamic stability, which can be utilized in biological systems. Among variable MOF nanocomposites, Zr-based porphyrinic MOFs share several characteristics. Because both the MOFs and their porphyrinic ligands function as outstanding drug carriers and photosensitizers (PSs), respectively. They provide the structural design adjustability of MOFs, alongside the enhancement of photochemical, catalytic, and biological properties of porphyrins. These materials robustly enhance the ability to make multimodal stabilized anticancer biocompatible structures versus common anticancer drugs. Presently, most of their uses include in vivo imaging and anticancer medication treatment7–9. Moreover, Porphyrins can be targeted in the structure of metal-organic frameworks due to their specific anti-cancer function, which is praised for various optical applications such as photosynthesis, photo-antibacterial, and photodynamic therapy due to their band gap in the visible region18,19.

The generated reactive oxygen species (ROS) in the porphyrinic MOF nanocomposites are the most important anti-cancer mechanism, including singlet oxygen, hydroxyl, and superoxide radicals, which penetrate cancer cells and increase the percentage of apoptosis. Moreover, certain chemotherapy drugs have anti-cancer activity with an identical mechanism, including the production of ROS as well20. Doxorubicin (DOX) is a chemotherapy drug utilized that treats several types of cancers, which causes an intense cardiotoxicity effect21–25. Combination therapy of DOX with PDT displayed synergic and enhancement effects of the cancer therapy26. Availing of Low-dose DOX with other anticancer agents provides the possibility of profiting from the benefits of its properties while minimizing its toxic effects27–29.

Since utilizing nanotechnology has created significant efficiency in anti-cancer agent drug delivery applications, exploiting the efficient nanomaterials with potential anticancer properties, has progressed the drug function30. Metallic nanoparticles have recently attracted a lot of attention due to the unique properties of these nanomaterials, which include high surface area and tunable optical, electrical, and magnetic properties31–33. Au nanoparticles, due to their special chemical and physical qualities (such as increased catalytic activity and plasmonic resonance) are useful for drug delivery and targeting, which recently attracted enormous interest34–36.

Our previous study corroborated the anticancer effect of porphyrinic MOF nanocomposite on colorectal cancer cells32.

It is known that the prevalence of gastrointestinal cancer continues to rise. The incidence of gastric cancer (GC) is rising, making it one of the most common cancers associated with mortality worldwide37–40. Besides, discovering the applicable integrated anticancer agents remains a fundamental challenge apart from surgery41–43. Herein, we targeted gastric cancer cells with an improved multifunctional PCN-222/Au/DOX nanocomposite. Initially, a nanorod porphyrinic metal-organic framework (MOF) was synthesized as a biocompatible, high surface area agent for potential cancer treatment applications. Furthermore, the synthesized nanorod Zr-based porphyrinic MOF, PCN-222, was applied as a photochemical agent to produce sustainable Au nanoparticles in a fast and facile green system. Afterward, the DOX chemotherapy drug was loaded on the nanocomposite at a low dose. Some previous studies demonstrated the curative application of MOFs and ZIFs in gastric cancer. MOF materials are generally utilized as a chemotherapeutic drug delivery system and benefit from their high surface area and structural stability, while the MOF part of our synthesized nanocomposite has anticancer properties with the capability of generating ROS that performs more than a drug delivery system44–48.

The cytotoxic function of the nanocomposite was investigated through the 3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay on the MKN-45 gastric cancer cell line. The apoptotic induction and the caspase activity were clarified utilizing the Annexin- V/propidium iodide (PI) staining and colorimetric caspase activity, respectively. The determination of the intracellular generated ROS of the PCN-222/Au NCs occurred via the flow cytometry method for the mechanism’sf investigation of the anticancer performance. Besides, for more applied performance determination of the synthesized final agent, the photo-antibacterial activity was investigated with minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) values measurement in the dark and under LED light as for gram-negative Helicobacter pylori as well. Eventually, utilizing high surface and biocompatible nanorod porphyrinic MOF with the capability of sustainable Au nanoparticle photochemical green synthesis, the facilitated structure which reduced the dosage of a DOX drug, the favorable apoptosis induction results alongside the photo-antibacterial study for Helicobacter H pylori as a check on the preventive effect against gastric cancer, all in all, provide the current research as an applicable and promising work in cancer remedy.

Experimental

Materials and methods

The certified analytical grade of pyrrole (C4H4NCH3), propionic acid (CH3CH2CO2), 4-formylbenzenecarboxylic acid (OCHC6H4CO2H), dimethylformamide (DMF), zirconyl chloride (ZrOCl2.8H2O), tetrachloroaurate (III) trihydrate (HAuCl4⋅3H2O), trifluoroacetic acid (TFA, CF3CO2H), benzoic acid (C6H5CO2H), tetrachloroauric(III) acid trihydrate (HAuCl4⋅3H2O), L-ascorbic acid (AA, HC6H7O6), hydrochloric acid, acetone and ethanol, were acquired from Sigma-Aldrich for this study. All of these chemicals were utilized in their original state, except for pyrrole, which was subjected to distillation before its usage. Doxorubicin (DOX) was also procured from Pfizer USA.

The Shimadzu FTIR-8400 S spectrophotometer was applied to accurately register the Fourier transform infrared spectra in the range of 400–4000 cm−1. An XRD diffractometer (Bruker D8 ADVANCE, Germany) equipped with Cu Kα radiation (λ = 1.5406 Å) in a 2θ range of 5° ≤ 2θ ≤ 80° was utilized to capture the X-ray diffraction (XRD) patterns. The specific surface area and pore width were studied using the Brunauer–Emmett–Teller (BET) method through N2 adsorption/desorption isotherms, utilizing a Micromeritics ASAP2020 system at 77 K. The ultraviolet-visible (UV-vis) spectra were taken using a Shimadzu UV-1700 spectrophotometer to illustrate the porphyrin compound. The morphology of the nanocomposites was persuaded by employing field emission scanning electron microscopy (FE-SEM) with an MRIA3 instrument (TESCAN-XMU), armed with energy-dispersive X-ray (EDX) equipment. Philips EM208S 100 KV instrument (Netherlands) was employed to conduct Transmission Electron Microscopy (TEM) imaging. Moreover, photochemical synthesis was carried out using a halogen incandescent light (82 V, 360 W, DONAR DN-30059). The MOF thermal stability surveying occurred utilizing a TGA (Thermal Gravimetric Analyzer, TA-Q600, USA).

Synthesis of Tetrakis (4-carboxyphenyl) porphyrin (H2TCPP)

In a 2-neck round bottom flask, 180 cc of propionic acid was initially heated until it reached 140 °C. Following that, 4-formylbenzenecarboxylic acid (10 mmol) was increased to refluxed propionic acid. Then 0.5 mL of pyrrole was diluted with 10 mL of propionic acid and cautiously added to the refluxing mixture dropwise. The refluxing process was sustained for 1 h. The flask was stayed to cool down to room temperature. the final mixture was securely kept in a dark environment for three days. Subsequently, the purple precipitate was recrystallized from the absolute ethanol. The obtained H2TCPP was then dried at 60 °C.

Synthesis of PCN-222 and PCN-222/Au NCs

PCN-222 was synthesized by adapting to the procedure reported by Nazari et al.49 with slight changes. The process was as follows: ZrOCl2.8H2O (0.2 g) and benzoic acid (3.0 g) were added to 20.0 mL of DMF and sonicated for 5 min. The solution was then incubated at 100 °C for 1 h. After cooling to room temperature, H2TCPP (0.1 g) and TFA (1 mL) were added to the solution and sonicated for 30 min. The resulting solution was refluxed at 120 °C for 48 h. The brown powder was obtained, separated, and then soaked in 40 mL of fresh DMF and 1 mL of 8 M HCl. It was placed in a digital oven at 120 °C for 18 h for pre-activation treatments. The solid was washed with DMF and acetone several times. Finally, the brown product was activated at 100 °C for 24 h to give PCN-222. Production of the Au nanoparticles on PCN-222 with a photochemical reaction was synthesized according to the reported procedure50. At first, 0.2 g of synthesized PCN-222 was added to 50 mL of deionized water and stirred for 5 min to disperse it. Afterward, 1 mL of HAuCl4·3H2O and 1 mL of 0.1 M L-Ascorbic acid solution were added and stirred for 10 min under 20 cm distance from the halogen lamp. The resulting mixture was double-washed and dried after centrifuging.

Cytotoxicity trial

MKN-45 cell line was prepared from the Pasture Institute national cell bank (Tehran, Iran). For the color intensity determination, the DNM-9602G microplate reader was utilized.

Cell viability test

The prepared final nanocomposite cytotoxic effect on the MKN-45 cancer cells was determined by applying the MTT method. For 24 h, 1 × 104 cells were seeded to the wells of the 96-well plate. PE38 and the synthesized final product were separately added to the plates in 15.625, 31.25, 62.5, 125, 250, and 500 µg/mL of concentration (eight replicates). The incubation of the plates was done for 48 h. Afterward, 100 µL of MTT solution (0.05 mg/well) was increased. Thereupon, the solubilized formed formazan resulting crystals appeared in cells after the 100 µL/well of dimethyl sulfoxide utilization. By a microplate reader, the absorption at 570 nm was registered. The surface of the wells was eliminated with a sampler. After adding 100 µl of DMSO, the wells were shaken for 20 min.

Apoptosis test

The differential counting was accomplished to determine the percentage of the viable, necrotic, and apoptotic cells by operating the Annexin-V dye fluorescein isothiocyanate (FITC), propidium iodide (PI), C27H34I2N4, with an apoptosis detection kit (Hoffman-La Roch Ltd, Basel, Switzerland). The viable cell detection was performed by a flow cytometry machine.

Annexin-V/PI iodide apoptosis test

The PCN-222/Au/DOX NCs treated population of gastric cancer cells was investigated to determine the apoptotic percentage in a comparison with the negative control cell population. Briefly, the Annexin-V and propidium iodide (PI) staining of the MKN-45 cells was done by Apoptosis Detection Kit, (Roch, Switzerland). Next, in a double IC50 concentration (µg/mL) of PCN-222/Au/DOX NCs, the cells were treated and incubated for 24 hours, then trypsinized and washed with sterile phosphate-buffered saline (PBS). Then, the solution of binding buffer (100 mL) was added in a 1.5 ml microtube to the achieved centrifuged sediment of cells. Afterward, the propidium iodide (10 mL) with Annexin-V (5 mL) dyes was increased and mixed slowly by shaking with a hand to dissolve the condensate cells. Thereupon, the sample was incubated for 10 minutes in the dark situation at room temperature (298 K). The viable, apoptotic, and necrotic cell’ rate was analyzed by a flow cytometry device51.

Caspase-8, and − 9 activity assays

The in vitro caspase activity was determined utilizing a colorimetric caspase activity assay Kit (Abnova). The cells were treated with 15.35 µg/L of PCN-222/Au/DOX NCs after 48 h incubation and lysed. After centrifugation of lysate, the 50 µl of the obtained supernatant was added to a fresh tube with 50 µl of reaction buffer containing 10 mM dithiothreitol (DTT) transferred to it. Thereupon, the 200 µM concentration of specified pNA-conjugated caspase-related substrates were surcharged into each tube individually and the incubation was performed at 37 °C for 2 h. Eventually, the absorbance was measured at 400 nm.

Intracellular ROS flow cytometry

The general oxidative stress indicator CM-H2DCFDA was utilized to measure the intracellular ROS. The aggravation and incubation of the DCFDA (10 mL; Sigma, Life Technologies C6827) to the cells were done at 37 °C for 25 min respectively. The intracellular H2O2 reacted with the DCFDA fluorescent probe to reproduce fluorescence emission for indication by the flow cytometry machine. Measurement of the generated intracellular H2O2 in SACS, before and after culture, was performed by flow cytometry utilizing DCFDA. The twice washing of the cells was done with PBS and afterward, for 5 min centrifuged at 2500 g. The emission of green fluorescence within 500 to 530 nm was measured utilizing a flow cytometry system.

Antibacterial test

Microorganism Preparation

For antibacterial activity surveying, the Helicobacter pylori (ATCC43504) was prepared from the microorganism bank of the Scientific Industrial Research Organization of Iran.

MIC and MBC method

The antibacterial activity was determined via the MIC and MBC assay in the presence of PCN-222/Au/DOX NCs below LED and in darkness as well. In this way, a fresh culture was prepared from the Helicobacter pylori bacteria in the brain-heart infusion (BHI) culture medium under microaerophilic conditions. Then, a quantity of bacteria was dissolved in sterile physiological serum to obtain a turbidity equal to OD = 0.1, which is equal to half McFarland. On the other hand, different concentrations of the sample (from 1 mg/ml dilution to lower concentrations) were prepared and sterilized using a BHI broth culture medium in a volume of 1 ml. Then the sample was placed under the LED light for 2 h. At the end, a volume of each of the substances in serum was added to a sample which was increased by one hundred thousand bacteria in one milliliter and placed in an anaerobic jar at 37 o C. The culture medium contains bacteria with and without the test substance utilizing the negative and positive control. After 72 h, the turbidity of the bacteria was checked and the samples in which the turbidity of bacterial growth was not observed were recorded to determine the MIC. To determine MBC, the samples where turbidity was not observed were cultured in BHI culture medium, and if there was no growth of bacteria, MBC was considered.

Statistical analysis

The Microsoft Excel software and the Statistical SPSS v16.0 Package are used for descriptive analysis and data analytical surveying, respectively.

Results and discussion

Nanorod Zr-based Porphyrinic metal-organic frameworks, PCN-222 were synthesized as the result of the reaction of H2TCPP with ZrOCl2 in the presence of benzoic acid, and trifluoroacetic acid as modulators in the two levels of heating respectively. The result of the photochemical reaction of HAuCl4, PCN-222, and L-ascorbic acid mixture stirring for 10 min under an incandescent halogen lamp created the growth of gold nanoparticles on PCN-222. The PCN-222/Au/DOX NCs were prepared by stirring the PCN-222/Au NCs and DOX in darkness for the doxorubicin loading 24 h (Fig. 1).

Fig. 1.

Fig. 1

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The PCN-222/Au/DOX NCs schematic synthesis.

All of our generated data is placed in the submitted manuscript and supporting file. The demonstrated FT-IR spectra in Fig. 2a were related to the H2TCPP, PCN-222, and PCN-222/Au/DOX NCs structures. The FT-IR spectra of H2TCPP represented the climax at 963 cm−1,which was assigned to the vibration of the pyrrole N-H band. Therewith, the observed bands at 1410 cm−1and 1606 cm−1in the PCN-222 spectra were ascribed to the –(O − C−O) − dicarboxylate groups in the MOF structure52. In addition, the observed peaks presented at 1264 cm−1and 1684 cm−1were attributed to the C-OH and C = O bonds of the carboxylic acid group, respectively. In the synthesized PCN-222, the mentioned bond vibration was decreased since the symmetric vibration of the O-Zr-O begets at 724 cm−153,54. Eventually, a remarkable diversity in the FT-IR spectra of the PCN-222/Au/DOX NCs and PCN-222 MOF 58 was not detected. The XRD patterns of the prepared PCN-222, PCN-222/Au NCs, and DOX/PCN-222/Au NCs have been depicted in Fig. 2b. The prominent peaks observed at 2θ = 4.9, 6.7, 7.1, and 9.8° correspond to reflections belonging to planes of (200), (2–11), (201), (400), and (4–21), respectively. Furthermore, the diffraction peaks observed at 38.01° and 44.10° in PCN-222/Au NCs and likewise 37.80 and 44.90 in PCN-222/Au/DOX NCs ascribed to the crystalline facets indexed as (111) and (222) of Au nanoparticles. Figure S1a demonstrates the UV–Vis spectrum of the porphyrin compound, H2TPP, in DMF. The typically sharp Soret band at 419 nm and Q bands at 516, 550, 592, and 650 nm were seen as well. The stability and structural integrity of PCN-222 were assessed at high temperatures through Thermal Gravimetric Analysis (TGA) after the material underwent vacuum drying to eliminate residual solvent. TGA results showed a weight loss of approximately 43.24% as the temperature increased from ambient to 1073 K. Weight loss below 373 K was attributed to acetone trapped in the material’s pores. The next stage of the weight loss occurred between 673 and 873 K, which was related to the dissociation of organic ligands (Figure S1b)55,56.

Fig. 2.

Fig. 2

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FT-IR spectra of TCPP, PCN-222, and PCN-222/Au/DOX NCs (a), PXRD patterns of the prepared PCN-222, PCN-222/Au NCs, and PCN-222/Au/DOX NCs in the range of 30–80 degrees (b).

Nitrogen adsorption-desorption tests were conducted at 77 K to assess the porosity of PCN-222. The obtained results, depicted in Fig. 3a, demonstrated a type-IV behavior of the adsorption-desorption isotherm, indicating the existence of mesopores (a sharp rise at about P/P0 = 0.3). Utilizing the Brunauer-Emmett-Teller (BET) method, the BET surface area of PCN-222 was achieved at about 2282.7 m2 g−1, and the total pore volume at P/P0 = 0.989 was established as 1.46 cm3.g−1. Moreover, for the calculation of the average pore diameter, the Barrett-Joyner-Halenda (BJH) method was used, resulting in a value of 2.55 nm (Fig. 3b)57,58. In addition, the BET surface area of the PCN-222/Au/DOX NCs was about 21.291 m2. g −1 and the pores’ total volume was about 0.051106 cm3.g−1 at P/P0 = 0.990 according to Fig. 3c, d. The specified reduction of the surface area attributed to the loading of the DOX confirmed that the PCN-222 performed as a drug carrier in the nanocomposite structure. Figure 4 panels a to i show field emission scanning electron microscopy (FE-SEM) images of the morphology of PCN-222, PCN-222/Au NCs, and PCN-222/Au/DOX NCs. As illustrated in the images of the synthesized PCN-222 (Fig. 4a-c), the rod-like structure exhibited conformity with previously documented research findings, displaying bars characterized by polygonal cross-Sects59–61.. Furthermore, the achieved images from FE-SEM of the prepared PCN-222/Au NCs and PCN-222/Au/DOX NCs were displayed in Fig. 4d–i, indicating the homogeneous distribution of Au nanoparticles on the surface of PCN-222 without any noticeable aggregation.

Fig. 3.

Fig. 3

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The BET and BJH analysis of the synthesized PCN-222 (a, b) and PCN-222/Au/DOX NCs (c, d).

Fig. 4.

Fig. 4

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The FE-SEM images of PCN-222 (a-c), PCN-222/Au NCs (d-f), and PCN-222/Au/DOX NCs (g-i).

In the SEM images, the nanorod synthesized PCN-222 was observed with one dimension under 200 nm. After gold nanoparticles undergo photosynthesis, several nanorods appear to have accumulated together, maintaining their shape while exhibiting some increase in size.

The three components of the prepared PCN-222/Au NCs were obtained from transmission electron microscopy (TEM) images (Fig. 5a-d). The nanorod morphology of the MOF fuselage, with spherical Au nanoparticles, was observed. The aggregated circular particles were attributed to the loaded DOX drug. The set of TEM and SEM images indicated the presence of the Au nanoparticles with a size of less than 10 to 20 nm within the nanocomposite construction, which confirmed each other.

Fig. 5.

Fig. 5

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The TEM images of PCN-222/Au/DOX NCs in the scale of 50 nm (a-d).

To examine the structural elemental distribution, the SEM images, related mapping, and EDS analyses were carried out for PCN-222, PCN-222/Au NCs, and PCN-222/Au/DOX NCs represented in Figs. 6a-f and 7a-g and a-g, and 8a-g, respectively.

Fig. 6.

Fig. 6

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The FE- SEM image (a), and the elemental mapping spectra of synthesized PCN-222 (b-f).

Fig. 7.

Fig. 7

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The FE- SEM image (a), and the elemental mapping spectra of synthesized PCN-222/Au NCs (a-g).

The presence of elements with weight% of C (61.28%), N (7.53%), O (20.53%), and Zr (10.67%) indicate the PCN-222 synthesis which were changed to C (55.08%), N (9.78%), O (19.98%), Zr (12.11%), and Au (3.06%) after the entry of Au nanoparticles with a monotonous distribution in PCN-222/Au NCs according to the image map (Fig. 7b, f).

The mapping analyses of PCN-222/Au/DOX NCs reveal the presence of all five materials within the sample, with the weight% value of C (51.35%), N (9.79%), O (24.24%), Zr (10.76%), and Au (3.86%) indicating the dispersion of these metals, especially Au, across the area of the crystalline particles (Fig. 8a-g). It seems that the final nanocomposite contains more carbon content distribution due to the DOX being added.

Fig. 8.

Fig. 8

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The FE- SEM image (a), and the elemental mapping spectra of PCN-222/Au/DOX NCs (a-g).

As depicted in Fig. 9a-d, general and pointwise EDS analyses with the FE-SEM images of synthesized PCN-222/Au/DOX NCs were performed on specific points containing individual Au nanoparticles.

Fig. 9.

Fig. 9

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The EDS spectra of PCN-222/Au/DOX NCs in the general and pointwise form (a, b), and the FE-SEM images of PCN-222/Au/DOX NCs (c, d).

Cell viability assay outcome

The MTT results represented that the exposure of the MKN-45 cell line to different increasing.

Concentrations of PCN-222/Au/DOX NCs up to 250 µg/mL in the plate, after 24 h, have been found half-maximal inhibitory concentration (IC50) value equal to 271 µg/mL for PCN-222/Au/DOX NCs treatment (Fig. 10a).

Fig. 10.

Fig. 10

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The MKN-45 cancer cells viability after 24 h of treatment with PCN-222/Au/DOX NCs (a), column chart representing caspase activity (b), the Annexin-V/PI staining dot plots of untreated MNK-45 cells (c), and the Annexin-V/PI staining dot plots MNK-45 cells treated with PCN-222/Au/DOX NCs in 24 h incubation (d).

Cell death assessments and the caspase activity

The numerating of the apoptotic, necrotic, and viable cells occurred by the fluorescein isothiocyanate (FITC) conjugated with Annexin-V. The changes in the activation of caspases 8 and 9 activity were determined to realize the mechanism of cell death in the treated cells. Figure 10b demonstrates that caspase activity changes in the MNK-45 cell treated with PCN-222/Au/DOX NCs represent that the caspase-8 and 9 activity was enhanced in comparison with untreated cells by 1.88 and 2.90 times, respectively. Furthermore, for the treated MKN-45 cells and the control group, Annexin-V/PI staining dot plots are displayed in Fig. 10c, d, respectively. For 24 h incubation, the results indicated that 11.04% of the treated cells were in early-stage apoptosis, 33.75% were in late-stage apoptosis, and 4.11% of the cells were necrosed. The results ascertain that the ratio percentage of the apoptotic cells to the total death cells was 91.59% (Fig. 8d).

Intracellular ROS generation results

The high obtained amount of fluorescence from the flow cytometry utilized method for ROS determination after treating the MKN-45 cells with the 222/Au/DOX NCs indicates that synthesized nanocomposite particularly expands the intracellular ROS generation with the main fluorescence intensity (MIF) reported value of about 247.5 which was demonstrated in comparison with control in Fig. 11a, b.

Fig. 11.

Fig. 11

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The ROS production histograms for the (a) untreated MKN-45 cells (control group) and (b) the MNK-45 after treatment with PCN-222/Au/DOX NCs.

The presence of gold nanoparticles has been confirmed by the peaks seen in XRD analysis. In addition, their dimensions below 5 nm are visible in SEM analysis, as confirmed in TEM images, which also causes the enhancement in permeability of the drug. Moreover, the performance of porphyrinic MOF as a DOX drug carrier is consistent with the final structure observed in TEM and SEM images. TEM images confirm the ternary structure of the designed and synthesized nanocomposite for enhanced functional anticancer. The presence of porphyrin in the structure, which is stable even after the formation of the final nanocomposite, is evident from FT-IR analysis and leads to the production of reactive oxygen species in the final structure as a main key that determinant the apoptosis pathway mechanism.

The porphyrinic nanocomposites’ particular localization within the cell can affect the pathway of apoptosis and the cell death mechanism. Caspase-9 is a key initiator caspase in the intrinsic apoptotic pathway, activated by the apoptosome. Singlet oxygen is an electronically excited state of molecular oxygen where all electrons are spin-paired, while the superoxide radical has an unpaired electron. In the Type I mechanism of the Porphyrinic nanocomposite photosensitizing, for creating radicals, the excited state transfers an electron to a substrate molecule or oxygen. Type II depends on energy transfer from the photosensitizer to oxygen and occurs with the conversion of triplet oxygen to singlet by intersystemic passage producing singlet oxygen, which then causes cellular damage. Porphyrins primarily induce apoptosis through the type II mechanism. Both singlet oxygen and superoxide are ROS that can influence caspase activity and apoptosis, The singlet oxygen has been considered the main ROS through which the photosensitizer exert their photodynamic action the main key that could advance PDT in the presence of porphyrinic anticancer drugs is the robust singlet oxygen, which directly affects mitochondria and the intrinsic caspase 9 cellular pathway, leading to apoptotic cell death. In this study, the synthesized porphyrinic nanocomposite activated caspase 9, 1.5 times more than caspase 8. It demonstrates that the mitochondrial intrinsic pathway was more activated according to the achieved increase in measured intracellular ROS. This concept strengthens and determines the Type II mechanism, while causing less caspase 8 activation, demonstrating less Type I progress62–65.

Antibacterial result

The under LED light sample containing Helicobacter pylori treated with PCN-222/Au/DOX NCs exposure has more antibacterial properties with a MIC value of 375 µg/mL indicating the photo-antibacterial performance of the synthesized nanocomposite (Table S1).

Conclusion

In this study, PCN-222/Au/DOX NCs were designed and synthesized with a green method for the photo-synthesis of gold nanoparticles by the PCN-222 visible light performance and using the low dose rate of DOX as a nanocomposite containing three anticancer components. The porphyrinic MOF nanocomposite ascertained the anticancer properties against MKN-45 gastric cancer cells with apoptotic induction as the major cell death pathway, with more than a 91% ratio of apoptotic cells versus total dead cells. The changes in the caspase activation demonstrate that the treated MKN-45 cells have mostly increased the caspase 9 activity and some in the caspase 8 pathway as well. The intracellular ROS was significantly enhanced after the cancer cells were treated with PCN-222/Au/DOX NCs with an MIF value of 247.5. Besides, the synthesized anticancer nanocomposite demonstrated the photo-antibacterial activity against Helicobacter pylori under LED light with the MIC value of 375 µg/mL. The PCN-222/Au/DOX nanocomposites, designed and fabricated as a porphyrinic MOF-derived structure, contain a low dose of DOX, which can be proposed as a novel nanocomposite with anticancer properties, including some photo-antibacterial functions.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (103KB, docx)

Acknowledgements

This work was partially supported by the Iran National Science Foundation: INSF (Project No. 4023314).

Author contributions

Sajedeh Teheraninejad and Saeideh Eslaminejad wrote the main manuscript text and prepared figures. All authors reviewed the manuscript. Rahmatollah Rahimi was also the supervisor of the research.

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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