[FeIIICl(TMPPH2)][FeIIICl4]2: A Stand-Alone Molecular Nanomedicine That Induces High Cytotoxicity by Ferroptosis

Developing clinically meaningful nanomedicines for cancer therapy requires the drugs to be effective, safe, simple, cheap, and easy to store. In the present work, we report that a simple cationic Fe(III)-rich salt of [FeIIICl(TMPPH2)][FeIIICl4]2 (Fe-TMPP) exhibits a superior anticancer performance on a broad spectrum of cancer cell lines, including breast, colorectal cancer, liver, pancreatic, prostate, and gastric cancers, with half maximal inhibitory concentration (IC50) values in the range of 0.098–3.97 μM (0.066–2.68 μg mL−1), comparable to the best-reported medicines. Fe-TMPP can form stand-alone nanoparticles in water without the need for extra surface modification or organic-solvent-assisted antisolvent precipitation. Critically, Fe-TMPP is TME-responsive (TME = tumor microenvironment), and can only elicit its function in the TME with overexpressed H2O2, converting H2O2 to the cytotoxic •OH to oxidize the phospholipid of the cancer cell membrane, causing ferroptosis, a programmed cell death process of cancer cells.


Introduction
Materials designed to target the unique features of the tumor microenvironment (TME), such as the over-expression of H 2 O 2 and glutathione (GSH), weak acidity, hypoxia, and immunosuppression, are attracting intensive interest in the field of nanomedicine [1][2][3][4][5][6].Materials that are responsive exclusively to the TME imply low systemic toxicity and thus fewer side effects.Disturbing the complex homeostasis of the TME can further result in effective cancer therapy by novel cell death modalities (e.g., ferroptosis, pyroptosis, and immunogenic cell death) [7][8][9], and the resensitization of cells to conventional therapies [2,9,10].Molecular complexes of Pt [11], Au [12], Fe [13], Os [14], Re [15], Ru [16], and Ir [17] have been reported to induce ferroptosis; their molecular structures are shown in Scheme 1.Nevertheless, most of the current formulations are far from satisfactory for practical use due to their formidable structural complexity, high cost, and low cellular uptake that subsequently leads to low anticancer efficacy.
GSH may further facilitate the ferroptosis process [6,22,23].Fe-TMPP, reported herein, offers some advantages, such as iron-based chemotherapy, including inexpensive and facile synthesis, and direct nanoparticle formation without the need for complicated and costly surface modification.This instant nanoparticle formation by adding Fe-TMPP to water also permits convenient storage and simplifies the nanomedicine preparation process.
Scheme 1.The structures of some reported ferroptosis inducers.Some useful strategies have been proposed to promote cellular uptake and the accumulation of the drugs in organelles (particularly mitochondria) [18,19] and the synergistic operation between different therapeutic modalities, such as chemotherapy, chemodynamic therapy (CDT), and photodynamic therapy (PDT), by taking advantage of the chemical properties of the individual building blocks of nanomedicines.For example, Ke et al. designed a cyclometallated Ir(III) complex featuring a −SH terminal as the cationic and photoactive building block [20].Upon polymerization by disulfide bond formation, followed by DSPE-PEG-Biotin coating to endow aqueous solubility and cell specificity, the particle (denoted as IrS NPs) could be used for TME-responsive cancer therapy by harnessing both apoptosis and ferroptosis pathways.When IrS NPs were taken up by the cells, the disulfide bond was cleavaged by cross-coupling with the intracellular GSH to release the cationic subcomponent, which in turn, selectively accumulated in mitochondria.Notably, the use of Ir(III)-based photosensitizers elicited type I PDT, which circumvented the need for molecular oxygen.The oxidative stress imposed by the generated reactive oxygen species (ROS) from PDT resulted in the apoptosis of the lung cancer A549 cell line, while the depletion of GSH by IrS NPs weakened the reductive defense of the cells, leading to the effective peroxidation of the phospholipid, causing ferroptosis.In a similar effort, Yuan et al. designed a small molecular drug, MitoIrL2, featuring a cyclometallated Ir(III) skeleton for the hybrid treatment of breast and pancreatic cancer via a shared mechanism (apoptosis from type I PDT and ferroptosis) [21].In MitoIrL2, the presence of an additional cationic phosphonium center resulted in more efficient mitochondrial accumulation and, therefore, better treatment outcomes relative to its phosphonium-free counterpart.
Iron(II)-/Iron(III)-based nanoformulations have been widely reported for anticancer application via catalytic Fenton chemistry to convert the overexpressed H 2 O 2 in TME into cytotoxic hydroxyl radicals (•OH) [6,22,23].In addition, Fe 3+ can be further reduced by the excess intracellular GSH into Fe 2+ to initiate the Fenton process, and the exhaustion of GSH may further facilitate the ferroptosis process [6,22,23].Fe-TMPP, reported herein, offers some advantages, such as iron-based chemotherapy, including inexpensive and facile synthesis, and direct nanoparticle formation without the need for complicated and costly surface modification.This instant nanoparticle formation by adding Fe-TMPP to water also permits convenient storage and simplifies the nanomedicine preparation process.

Material Synthesis and Structure Descriptions
The assembly of FeCl 3 •6H 2 O and H 2 TMPP under solvothermal conditions in CHCl 3 / EtOH using HAc as the modulating reagent resulted in the formation of Fe-TMPP.The successful synthesis was authenticated by single-crystal X-ray crystallographic analysis.Fe-TMPP crystallizes in the triclinic space group P−1 (Table 1), featuring a cation of [Fe III Cl(TMPPH 2 )] 2+ and a pair of [FeCl 4 ] − counterions (Figure 1a).The [Fe III Cl(TMPPH 2 )] 2+ cation features a 'sit-atop' Fe 3+ which is chelated by four of the N atoms from the H 2 TMPP ligand and one Cl − to give a five-coordinate square pyramidal configuration (Figure S1).Notably, of the four methylpyridyl peripheries of the H 2 TMPP ligand, the two N at the para-positions are protonated, as suggested by the presence of two [FeCl 4 ] − ions for each [Fe III Cl(TMPPH 2 )] 2+ cation, in addition to the relative dispositions of these methylpyridyl moieties in the molecular packing diagram (Figure S2).The structure, refined with full protonation of the methylpyridyl N sites or misplaced protons, inevitably led to short H•••H contacts.
where n = number of reflections and p = total number of parameters refined.
Notably, of the four methylpyridyl peripheries of the H2TMPP ligand, the two N at the para-positions are protonated, as suggested by the presence of two [FeCl4] − ions for each [Fe III Cl(TMPPH2)] 2+ cation, in addition to the relative dispositions of these methylpyridyl moieties in the molecular packing diagram (Figure S2).The structure, refined with full protonation of the methylpyridyl N sites or misplaced protons, inevitably led to short H•••H contacts.

Spectroscopic Characterizations of Fe-TMPP
Fe-TMPP was spectroscopically characterized and compared with its H 2 TMPP precursor.The in-plane N-H vibration at 964 cm −1 in the Fourier transform infrared (FT-IR) spectrum of the ligand disappeared (Figure S3), and a new peak at 997 cm −1 appeared in Fe-TMPP, indicating the metalation of the porphyrin center.Fe 2p X-ray photoelectron spectroscopy (XPS) of Fe-TMPP revealed two peaks at binding energies of 711.3 eV and 724.8 eV, which are assignable by the spin-orbit splitting of the Fe 2p 3/2 and Fe 2p 1/2 of Fe 3+ (Figure 1c), while a pair of satellite peaks at around 717.6 eV and 730.7 eV were also present, presumably due to the photoreduction of Fe 3+ during the measurement [24,25].
In the ultraviolet-visible (UV-Vis) spectra (Figure 1d), the Soret band of the free H 2 TMPP at 419 nm underwent a significant blueshift to 398 nm upon the chelation of Fe 3+ .In addition, the Q bands coalesced into two, due to the change of symmetry from D 2h of H 2 TMPP to C 4v of Fe-TMPP [26,27].Notably, an additional absorption band at around 650 nm also appeared, presumably due to the charge transfer between Fe 3+ and the ligand [28].Notably, the UV-Vis spectrum of Fe-TMPP exhibited an undulating baseline, indicating lighting scattering by nanoparticle formation in an aqueous solution [29].The formation of a stand-alone nanoparticle of Fe-TMPP could be directly observed with the Tyndall effect (Figure S4).Energy dispersive X-ray spectroscopy (EDS) showed an Fe:Cl:N atomic ratio of 1.0:2.6:3.0 in Fe-TMPP (Figure S5a), which is close to the value of 1.0:3.0:2.7 found in the single crystal data.

Characterizations of Fe-TMPP Nanoparticles
The direct nanoparticle formation of Fe-TMPP in water was observed in the transmission electron microscopic (TEM) images and also by dynamic light scattering (DLS) analysis.TEM showed that Fe-TMPP formed particles of variable sizes (Figure 1b).DLS gave different results from batch to batch but all contained considerable numbers of particles with sizes less than 100 nm (Figure S6).We argue that the accuracy of the TEM results may be compromised, as the particles of Fe-TMPP, without any surface modification, may undergo an Ostwald ripening process upon solvent evaporation during sample preparation [30].The zeta potential of Fe-TMPP was 23.4 mV (Figure S7), which is ideal for ensuring good circulation stability with favorable adhesion to cell membranes for enhanced intracellular uptake [31].
Nanoparticles can be assembled and stored in an aqueous solution for short periods, or lyophilized for longer-term preservation.However, lyophilized particles can change their morphology, aggregate, or slowly degrade [32,33].Antisolvent precipitation is an often-used alternative to prepare nanoparticles of organic drugs.A risk with this approach, however, is the encapsulation of toxic organic solvents, and sometimes the need for a supercritical antisolvent (e.g., CO 2 ) [34,35].The simple chemical formulation and good water-dispersing ability of Fe-TMPP is thus an advantage for storage and facile particle preparation.
Fresh samples of Fe-TMPP in aqueous solutions maintained their color and homogeneity under ambient conditions for three days (Figure S8), as also evidenced by their UV-Vis spectra (Figure S9).In addition, Fe-TMPP was stable in PBS (0.1×) for 0, 12, and 24 h, as indicated by only insignificant changes in particle size (Figure S10) and zeta potential (Figure S11).To further confirm the stability of Fe-TMPP in water, we sonicated the aqueous suspensions of Fe-TMPP and then lyophilized.The powder X-ray diffraction (PXRD) analysis revealed that the diffraction patterns of the water-treated samples resembled that of the as-synthesized crystalline samples (Figure S12).Moreover, the EDS spectra of the lyophilized sample of Fe-TMPP showed an Fe:Cl:N atomic ratio of 1.0:2.4:3.2 (Figure S5b), which was close to that of the as-synthesized crystalline sample (1.0:2.6:3.0),indicating that there was no obvious compositional change upon suspending Fe-TMPP in water.We postulate that the four methyl groups at the ortho position of the pyridyl N sites significantly boost the hydrophobicity of the molecule to facilitate the aggregation of the polar Fe-TMPP featuring multiple hydrophilic moieties (i.e., [FeCl 4 ] − , protonated pyridyl ligands, the Fe-Cl, and free pyridyl N site) and thereby protecting the salt from hydrolyzation.

Detection of Fe-TMPP-induced Generation of ROS in Solution
High valent transition metal ions Fe 3+ and Cu 2+ exhibit peroxidase-like activity to generate reactive •OH, by utilizing the overexpressed H 2 O 2 in the TME, thereby eliciting nonspecific damage inside cancer cells [6][7][8]26].The TME also reduces Fe 3+ and Cu 2+ to Fe 2+ and Cu + through a reaction with the overexpressed GSH to initiate Fenton and Fentonlike reactions, which also generate •OH from H 2 O 2 .The peroxidase-like activity of the Fe 3+ -rich Fe-TMPP was therefore assayed using 3,3 ′ ,5,5 ′ -tetramethylbenzidine (TMB) [36].Different concentrations of Fe-TMPP were added to TMB solutions containing 100 µM H 2 O 2 , the approximate concentration in tumors, and five times higher than in normal cells [1,2].It can be seen from Figure 1e that Fe-TMPP produced considerable •OH in a concentration-dependent manner upon incubating with H 2 O 2 over 3 h.The generation of •OH was confirmed by electron paramagnetic resonance (EPR) spectroscopy, using DMPO as the spin trap.As shown in Figure 1f, the EPR spectrum of Fe-TMPP gave rise to a strong four-line signal with a 1:2:2:1 peak-to-peak intensity pattern, characteristic of the generation of •OH from H 2 O 2 [4,5].
Fe-TMPP is supported by a porphyrin-based ligand, which is often used for type II PDT of cancer [3,7,37].We employed diphenylisobenzofuran (DPBF) as a probe to compare the singlet oxygen's ( 1 O 2 ) generating capacity with that of TMPP-F127 (H 2 TMPP encapsuled by Pluronic F-127, Figure S13) [38], using blank DPBF as a control [39,40].DPBF was added to aqueous solutions of TMPP-F127 and Fe-TMPP, followed by laser irradiation (650 nm, 25 mW cm −2 ).As shown in Figure S14, at equivalent H 2 TMPP concentrations, neither species could generate sufficient 1 O 2 to degrade DPBF under laser irradiation.The 1 O 2 generated by Fe-TMPP was negligible, which might be due to the presence of redoxactive Fe 3+ that can quench the excited state of the molecule by electron transfer [41,42].

In Vitro ROS Detection and Cytotoxicity Assay
Given the efficient •OH production in water induced by Fe-TMPP, we next investigated in vitro ROS generation by Fe-TMPP in the representative 4T1 cell line using 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescence probe [4].As shown in Figure 2a and Figure S15, green fluorescence was detected in the cells treated with Fe-TMPP, which indicated the production of ROS in the cells from the peroxidase-like activity of Fe 3+ .By comparison, TMPP-F127 produced few ROS, which may be due to the aggregation of the H 2 TMPP ligand within the hydrophobic core of the TMPP-F127 micelles.
Molecules 2024, 29, x FOR PEER REVIEW 6 of 14 to a strong four-line signal with a 1:2:2:1 peak-to-peak intensity pattern, characteristic of the generation of •OH from H2O2 [4,5].Fe-TMPP is supported by a porphyrin-based ligand, which is often used for type II PDT of cancer [3,7,37].We employed diphenylisobenzofuran (DPBF) as a probe to compare the singlet oxygenʹs ( 1 O2) generating capacity with that of TMPP-F127 (H2TMPP encapsuled by Pluronic F-127, Figure S13) [38], using blank DPBF as a control [39,40].DPBF was added to aqueous solutions of TMPP-F127 and Fe-TMPP, followed by laser irradiation (650 nm, 25 mW cm −2 ).As shown in Figure S14, at equivalent H2TMPP concentrations, neither species could generate sufficient 1 O2 to degrade DPBF under laser irradiation.The 1 O2 generated by Fe-TMPP was negligible, which might be due to the presence of redoxactive Fe 3+ that can quench the excited state of the molecule by electron transfer [41,42].

In Vitro ROS Detection and Cytotoxicity Assay
Given the efficient •OH production in water induced by Fe-TMPP, we next investigated in vitro ROS generation by Fe-TMPP in the representative 4T1 cell line using 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescence probe [4].As shown in Figures 2a and S15, green fluorescence was detected in the cells treated with Fe-TMPP, which indicated the production of ROS in the cells from the peroxidase-like activity of Fe 3+ .By comparison, TMPP-F127 produced few ROS, which may be due to the aggregation of the H2TMPP ligand within the hydrophobic core of the TMPP-F127 micelles.The cytotoxicity of Fe-TMPP against diverse cell lines including 4T1 (breast cancer), HCT-116 (colorectal cancer), DLD-1 (colorectal cancer), HuH-7 (liver cancer), BXPC3 The cytotoxicity of Fe-TMPP against diverse cell lines including 4T1 (breast cancer), HCT-116 (colorectal cancer), DLD-1 (colorectal cancer), HuH-7 (liver cancer), BXPC3 (pancreatic cancer), PC3 (prostate cancer), and AGS (gastric cancer), were evaluated using either 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or cell counting kit-8 (CCK-8) assays.As shown in Figure 2b and Figure S16, Fe-TMPP exhibited concentration-dependent cytotoxicity against all tested cell lines.The half maximal inhibitory concentration (IC 50 ) values for Fe-TMPP were relatively low, down to 0.0975 µM (0.0658 µg mL −1 ) for AGS and up to 3.97 µM (2.68 µg mL −1 ) for HCT-116 (Table 2).These IC 50 values are comparable to cisplatin and to complex formulations that integrate multiple therapeutic modalities.The above results indicate that Fe-TMPP exhibits significant cytotoxicity against a broad spectrum of cancer cell lines.

Ferroptosis Assay
As noted above, when Fe-TMPP is internalized by cancer cells, Fe 3+ can oxidize GSH to give glutathione disulfide (GSSG) and is concomitantly reduced to Fe 2+ .The exhaustion of GSH in these cells can downregulate the expression of glutathione peroxidase 4 (GPX4), making the cell membrane phospholipid more susceptible to peroxidation [49,50].GSH depletion and phospholipid peroxidation are two characteristic features of ferroptosis, a novel form of iron-dependent programmed cell death [49,50].We therefore examined the possibility of ferroptosis induced by Fe-TMPP using 4T1 cell line as a representative.Phospholipid peroxidation levels of 4T1 cells were measured using C11-BODIPY (BODIPY™ 581/591 C11) as a fluorescent probe, as observed under confocal laser scanning microscopy (Figure 3) [51].We also used DAPI to stain the nucleus of the cells.The cells treated with Fe-TMPP showed significantly stronger lipid peroxidation than those treated with TMPP-F127 or PBS.In addition, lipid peroxidation can be inhibited by the presence of ROS scavengers, such as ferrostatin-1 (Fer-1), liproxstatin-1, and vitamin E [21,52].In the present experiments, lipid peroxidation was completely inhibited by the introduction of Fer-1, as indicated by the restoration of the red emission of C11-BODIPY.These results indicated that Fe-TMPP can induce ferroptosis for cancer treatment.Flow cytometry using Annexin-V/PI double staining further confirmed that 4T1 cells treated with Fe-TMPP showed obviously late apoptosis relative to TMPP-F127 and PBS-treated cells, supporting this alternative cell death mechanism (Figure 2c) [53,54].
HCT-116, HuH-7, BXPC3, DLD-1, PC3, AGS, and 4T1 cell lines were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.Phosphate buffer solution (PBS) and fetal bovine serum (FBS) supplemented with penicillin and streptomycin were bought from Elabscience Crop (Guangzhou, China).Fourier transform infrared (FT-IR) spectra were recorded on a Bruker VERTEX 70+HY-PERION 2000 spectrometer.Ultraviolet-visible spectroscopy (UV-Vis) absorption spectra were obtained on a Varian Cary-50 UV-Visible spectrophotometer (Varian, Inc., Palo Alto, CA, USA).X-ray photoelectron spectroscopy (XPS) was conducted on an EXCALAB 250 XI X-ray photoelectron spectrometer (Thermo Scientific, Waltham, MA, USA).The EPR measurement was conducted by a Bruker EMXnano spectrometer, with DMPO as the spintrapping agent.The optical density (OD) values were collected using a TECAN M1000PRO microplate reader (Tecan, Zürich, Switzerland).The flow cytometry was performed using a BD FACS Caliber instrument (BD Biosciences, San Jose, CA, USA).The transmission electron microscopic (TEM) images were obtained by dropping the sample in water onto a copper net under a HITACHI HT7700 transmission electron microscope.Zeta potentials were measured on an LA-95052 laser particle size analyzer using dynamic light scattering (DLS).The cytotoxicity assay was conducted on a multifunction microplate detector by recording the absorption at 570 nm using a TECAN M1000PRO microplate reader.The cell staining results were observed with a confocal laser scanning microscope (CLSM, AxioObserver A1, Germany).The EDS and elemental mapping diagrams were obtained on the EVO18 scanning electron microscope.

Synthesis and Characterization of TMPP-F127
Pluronic F-127 (10.6 mg) was introduced to 1 mL of DMSO solution containing H 2 TMPP (4.5 mg), and the homogenous mixture formed was then added dropwise into 3.5 mL of deionized water with tip sonication.The micelle formed was dialyzed using a dialysis bag (molecular weight cut-off of 2000 Da) for 24 h to obtain TMPP-F127.TEM indicated that the particle sizes for TMPP-F127 were 170 ± 25 nm (Figure S13), which is conducive to cellular uptake.

Single Crystal X-ray Crystallography
Data collections were performed on a Bruker APEX II CCD X-ray diffractometer using Mo Kα (λ = 0.71073 Å) irradiation.Refinement and reduction of the collected data were achieved using the program Bruker SAINT and absorption corrections were performed using a multi-scan method [55].Crystal structures were solved by direct methods and refined on F 2 using full-matrix least-squares techniques with SHELXTL-2016 [56].
For Fe-TMPP, the [Fe III Cl 4 ] − anion displays a conformational disorder with a relative ratio of 0.73:0.27refined for the disordered domains.The [Fe III Cl(TMPPH 2 )] 2+ cation also has two conformations due to the presence of 'sit-atop' Fe 3+ , and the disorder is at about the center of the porphyrin ligand.Thus, the occupancy factor of the coordinated FeCl moiety was fixed at 0.5.For the porphyrin ligand, only two pyridyl groups were protonated to balance the charge of the anion.The protonation of half of the four pyridyls can also be judged from the packing of the molecules.In the packing diagram, two pyridyls were brought to proximity by an N-H•••N bond, thereby excluding the full protonation of the pyridyls and corroborating the charge counting.A small amount of spatially delocalized electron density in the lattice was found but acceptable refinement results could not be obtained for this electron density.The solvent contribution was then modeled using SQUEEZE in the Platon program suite [57].
Crystallographic data for Fe-TMPP have been deposited in the Cambridge Crystallographic Data Center (CCDC) as supplementary publication number 2330690.These data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk/data_request/cif accessed on 3 February 2024) or from the Supplementary Materials.A summary of the key crystallographic data for Fe-TMPP is listed in Table 1.

•OH Detection
Different concentrations of Fe-TMPP were added to a TMB solution containing 100 µM H 2 O 2 .After reacting for 3 h at room temperature, the solution was centrifuged to remove the influence of nanoparticles on absorbance.The ultraviolet absorption curve of the supernatant at 652 nm was determined.

1 O 2 Detection
Identical concentrations of DPBF were added to aqueous solutions of TMPP-F127 and Fe-TMPP relative to the concentration of TMPP, to give uniform mixtures, with the concentrations of DPBF and TMPP being 33 µg mL −1 and 7 µg mL −1 , respectively.The solutions were then irradiated by a laser (650 nm, 25 mW cm −2 ).After each irradiation for 30 s, the absorption of DPBF was immediately measured.By monitoring the decreasing rate of the ultraviolet absorption peak at 416 nm, the 1 O 2 generation capacities of these materials could be evaluated.

MTT or CCK-8 Cytotoxicity Assay
The MTT assay of 4T1 cells is detailed as an example.The 4T1 cell line was cultured in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% FBS and 1% penicillin-streptomycin at 37 • C in 5% CO 2 .Cells grew as a monolayer and were detached upon confluence using trypsin (0.5% w/v in PBS).The cells were harvested from the cell culture medium by incubating in trypsin solution for 2 min, and then centrifuged with the supernatant subsequently discarded.A 3 mL portion of serum-supplemented cell culture medium was added to neutralize any residual trypsin.The cells were re-suspended in serum-supplemented RPMI 1640 at a concentration of 5 × 10 4 cells/mL.Cells were cultured at 37 • C and 5% CO 2 for the MTT studies.
4T1 cells were inoculated into 96-well plates (5 × 10 3 cells per well) and incubated for 24 h.After 24 h of incubation, the cells were treated with different concentrations of drugs for 24 h, and, upon completion of the treatment, the MTT solution was added and incubated for 4 h, and DMSO was added and shocked for 15 min.The cell viability was determined using an enzyme marker.
The relative cell viability (%) related to control cells was calculated using the following equation: where V% is the percentage of cell viability, [A] experimental is the absorbance of the wells culturing the treated cells, [A] blank is the absorbance of the blank, and [A] control is the absorbance of the wells culturing untreated cells.The cell viability data for HCT-116 (CCK-8), DLD-1 (CCK-8), HuH-7 (CCK-8), BXPC3 (CCK-8), PC3 (CCK-8), and AGS (CCK-8) were obtained in a similar manner using protocols listed in Table S1.

Intracellular ROS Detection
4T1 cells were inoculated in 12-well plates (1 × 10 5 cells per well), incubated for 24 h and then treated with different drugs for 24 h.After completion of the treatment, the cells were treated with DCFH-DA, and the cellular reactive oxygen species results were determined using an inverted fluorescence microscopy.

Flow Cytometric Apoptosis Assay
4T1 cells were inoculated in 6-well plates (3 × 10 5 cells per well) and incubated for 24 h.After 24 h of incubation, the cells were treated with different drugs for 24 h.After the completion of the treatment, the cells were processed using an Annexin V-FITC/PI apoptosis kit, and the results of apoptosis were determined using flow cytometry.

Ferroptosis Assay
4T1 cells were inoculated in confocal glass dishes (1 × 10 5 cells per dish) and incubated for 24 h.Fer-1 (10 µM), an inhibitor of ferroptosis, was added and incubated for 12 h.The cells were then treated with different drugs for 24 h.After the completion of the treatment, the cells were incubated for 30 min with the ferroptosis probe C11-BODIPY 581/591 (10 µM) and the results of the ferroptosis were observed using CLSM.

Conclusions
In this work, a simple ferroptosis inducer of Fe-TMPP was readily synthesized from earth-abundant metal and common organic materials.Fe-TMPP can directly form nanoparticles without requiring extra surface modification or organic-solvent-assisted antisolvent precipitation.Compared to the reported ferroptosis inducers based on noble metals such as oxaliplatin [11] and other coordination complexes derived from Au [12], Os [14], Ru [16], and Ir [17], the advantages of using Fe-TMPP include inexpensive fabrication, in addition to its cancer-specific activity and promising cytotoxicity, as described herein.The instant nanoparticle formation by adding Fe-TMPP to water also ensures convenient storage and simplifies the potential nanomedicine preparation process.All these features are important for potential clinical use.However, it should also be noted that iron overloading can potentially lead to cardiotoxicity, also caused by the ferroptosis process [58,59].Nevertheless, the unexpectedly high cytotoxicity originating from ROS alone inspires us to investigate the possibility of other structure analogs based on metal ions, such as Cu 2+ , and to further explore their mechanisms of action by examining the sub-organelle locations of these metallodrugs.

Figure 1 .Figure 1 .
Figure 1.The crystal structure of Fe-TMPP with the disordered domain omitted for clarity ((a); Fe dark magenta, Cl green, N blue, C black, H light pink).TEM image (b) and the Fe 2p XPS spectra (c) of Fe-TMPP.A comparison of the Ultraviolet-visible (UV-Vis) spectra of H2TMPP (in N,N-dimethylformamide) and Fe-TMPP (in water) ((d); inset: the absorption in the range of 475−675 nm as amplified 10 times).Change of UV-Vis absorption intensity at 652 nm (oxTMB) upon treatment with different concentrations of Fe-TMPP in the presence of 100 µM H2O2 (e).Electron paramagnetic Figure 1.The crystal structure of Fe-TMPP with the disordered domain omitted for clarity ((a); Fe dark magenta, Cl green, N blue, C black, H light pink).TEM image (b) and the Fe 2p XPS spectra (c) of Fe-TMPP.A comparison of the Ultraviolet-visible (UV-Vis) spectra of H 2 TMPP (in N,Ndimethylformamide) and Fe-TMPP (in water) ((d); inset: the absorption in the range of 475−675 nm as amplified 10 times).Change of UV-Vis absorption intensity at 652 nm (oxTMB) upon treatment with different concentrations of Fe-TMPP in the presence of 100 µM H 2 O 2 (e).Electron paramagnetic resonance (EPR) spectrum showing •OH production from H 2 O 2 as induced by Fe-TMPP with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin trap (f).

Figure 2 .
Figure 2. The generation of intracellular ROS by TMPP-F127, Fe-TMPP, and PBS control, as measured by DCF fluorescence (a).A comparison of the 4T1 cell viability when treated with gradient concentrations of TMPP-F127 and Fe-TMPP (b).Flow cytometric evaluation of 4T1 cell apoptosis by Fe-TMPP, TMPP-F127, and a PBS using the Annexin V-PI double staining method (c).

Figure 2 .
Figure 2. The generation of intracellular ROS by TMPP-F127, Fe-TMPP, and PBS control, as measured by DCF fluorescence (a).A comparison of the 4T1 cell viability when treated with gradient concentrations of TMPP-F127 and Fe-TMPP (b).Flow cytometric evaluation of 4T1 cell apoptosis by Fe-TMPP, TMPP-F127, and a PBS using the Annexin V-PI double staining method (c).

Figure 3 .
Figure 3.The confocal laser scanning microscopic images of the accumulation of lipid peroxidation in 4T1 cells when treated with PBS control, TMPP-F127, Fe-TMPP, and Fe-TMPP in the presence of Fer-1 as the ferroptosis inhibitor using C11-BODIPY as the fluorescent probe.

Figure 3 .
Figure 3.The confocal laser scanning microscopic images of the accumulation of lipid peroxidation in 4T1 cells when treated with PBS control, TMPP-F127, Fe-TMPP, and Fe-TMPP in the presence of Fer-1 as the ferroptosis inhibitor using C11-BODIPY as the fluorescent probe.

Table 1 .
Crystal data and structure refinement parameters for

Table 2 .
A comparison of the IC 50 values of cell lines induced by the reported drugs and Fe-TMPP.