An intelligent and self-assembled nanoscale metal organic framework ( 99m TC-DOX loaded Fe 3 O 4 @Fe III -tannic acid) for tumor targeted chemo/chemodynamic theranostics

Background: Recent advances in clinical transformation research have focused on chemodynamic theranostics as an emerging strategy for tackling cancer. Neverthe-less, its effectiveness is hampered by the tumor’s glutathione antioxidant effect, poor acidic tumor microenvironment (TME) and inadequate endogenous H 2 O 2 . Hence, we designed an activatable theranostics ( 99m Tc-DOX loaded AA-Fe 3 O 4 @Fe III -TA) that effectively boost


Background
Worldwide, the prevalence and death rate of cancer are on the rise and the disease represents one of the leading causes of death.Chemotherapy (CT) is an essential therapeutic practice used to treat cancer.Doxorubicin (DOX) exhibits good clinical effectiveness as an anthracycline anticancer drug, effectively treating a wide range of tumors that acts on cancer cells via intercalating into DNA and interfering with topoisomerase-II-mediated DNA repair.Moreover, it may increase the intracellular hydrogen peroxide (H 2 O 2 ) levels by activating poly-ADP ribose polymerase (PARP) and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase posing enhanced oxidative stress (Thorn et al. 2011;Mizutani et al. 2005;Deng et al. 2007).Despite being widely used in medicine, DOX has been demonstrated to have a variety of dangerous side effects including hepatotoxicity, cardiotoxicity, neurotoxicity and nephrotoxicity (Ajaykumar 2020).
Recently, chemodynamic theranostic (CDT) has been perceived as an emerging and effective approach to cancer management.It is based on the fact that in the tumor microenvironment (TME), Fenton or Fenton-like agents catalyze the in-situ conversion of H 2 O 2 to very dangerous reactive oxygen species (ROS) (Tang et al. 2019;Wang et al. 2020a;Lin et al. 2019).CDT has numerous distinct advantages over traditional cancer treatment strategies, among them the capacity to regulate the TME, significant tumor specificity, regression and penetration, endogenous stimulus activation and reduced off-target concerns (Lin et al. 2018;Liu et al. 2020a;An et al. 2020;Ding et al. 2019).Additionally, it is more suited for treating deep tumors as it doesn't require external stimulation as sound or light, unlike sonodynamic therapy and photodynamic therapy (Cui et al. 2021;Shi et al. 2021).Unfortunately, CDT is encountering certain hurdles due to inadequate concentration of endogenous H 2 O 2 , insufficient acidity and a glutathione (GSH)-rich antioxidant system in TME (Liu et al. 2018;Gu et al. 2020;Han et al. 2019).Despite substantial advances via employing inorganic nano-catalytic processes including metal ions-induced Fenton-like reactions (Ce 3+ , Cu 2+ , Co 2+ and Mn 2+ ), these CDT agents are bound to confront biosafety concerns due to their high toxicity (Wang et al. 2019;Ma et al. 2018;Gao et al. 2019;Nie et al. 2020).In addition, the adoption of H 2 O 2 generators such as glucose oxidase (GOX) and calcium peroxide (CaO 2 ) nanoparticles encountered a major obstacle related to the quick burst release of H 2 O 2 during its generation process, which prevented it from being completely utilized in practice (Cui et al. 2021;Huang et al. 2021;Liu et al. 2020b).In recent times, GOX has been incorporated into nano-sized metal-organic framework (nano-MOF) or coupled with Fe 3 O 4 NPs, which are iron-based nanomaterials with good biosafety profile (Zheng et al. 2023;Feng et al. 2019).Through this process, GOX can convert glucose into gluconic acid, improving the acidic levels of tumors and enhancing Fenton-like reactions by increasing the generation of hydroxyl radicals (•OH).Furthermore, additional tactics like carbonic anhydride IX inhibition or siRNA silencing may also quicken the Fenton reaction of Fe 3 O 4 by raising the tumor's acidity level (Liu et al. 2018;Chen et al. 2020).However, throughout the blood circulation process, the unregulated reaction between GOX and glucose may result in "off-target" adverse effects for healthy tissues (Ding et al. 2020).Consequently, a great deal of work has been conducted to develop a variety of highly effective CDT strategies for treating cancer.The combined approach of CT with CDT may significantly increase the efficacy of cancer treatment because CDT increases the tumor suppressor effect of CT by upsetting both TME and tumor physiology (Wang et al. 2021a;Lei et al. 2021).MIL-101(Fe)-NH 2 nanoscale MOF demonstrated high DOX loading efficiency, pH-responsive release property, enhanced tumor cell uptake and efficient chemodynamic activity (Wang et al. 2015).In addition, Honghui Li et al. successfully prepared a pH-responsive iron-based MOF (MTD) that showed an effective concentration of DOX, elevated intracellular H 2 O 2 concentration at the tumor site and high therapeutic efficacy based Fenton reaction (Li et al. 2022).The majority of synergistic therapy-based drug delivery systems (DDSs) are sophisticated since they incorporate numerous diverse features.It is therefore crucial to determine the best strategy for integrating CT and CDT to produce multifunctional DDSs.
With the aforementioned issues in consideration, herein, we designed an activatable nanotheranostics ( 99m Tc-DOX loaded AA-Fe 3 O 4 @Fe III -tannic acid) affording pH-dependent spatiotemporal DOX release, H 2 O 2 self-supply and GSH depletion for effective tumor growth suppression.The platform was constructed in a core-shell MOFstructure where a cross-linked matrix of Fe III -TA served as a pH-responsive shell on the surface of AA-Fe 3 O 4 NPs, followed by loading of technetium-99m [ 99m Tc]-labeled DOX.Interestingly, the biocompatible and biodegradable platform had the ability to accumulate passively inside tumors with little payload leakage in the systemic circulation because of the MOF shell's protective function.Following the platform's entry into tumor cells, the acidic properties of TME may cause the MOF shell to break down, releasing free Fe III , TA and sustained release of 99m Tc-DOX.In addition to its chemotherapeutic and imaging properties, 99m Tc-DOX could increase the intracellular H 2 O 2 concentration.The liberated TA might be able to increase the acidification level of cancer cells overcoming the tumor's modest acidity.Crucially, both TA and GSH might quickly convert the liberated Fe III to ferrous ions (Fe II ), increasing GSH depletion.The generated Fe II ions along with the elevated level of intracellular H 2 O 2 might be effectively transformed into extremely reactive • OH via the Fenton reaction.Lastly, the ready platform may offer a different approach to combine CT and CDT to generate effective theranostics.

Chemicals and materials
All chemicals, unless noted differently, were delivered in analytical grade form and used right away without additional purification.

Equipment and characterizations
Transmission electron microscopy (TEM; JEM-1230; JEOL, Tokyo, Japan) was conducted with 200 kV accelerating voltage for the determination of the particles size.Dynamic light scattering (DLS; Zetasizer Nano-system Nano-ZS90, Malvern, UK) was utilized for hydrodynamic size and zeta potential determination.Fourier transform infrared (FTIR; VERTEX 70; Bruker, Bremen, Germany) was performed to investigate the composition of the as-prepared nanoparticles.UV-vis spectrophotometer (U-4100, Japan) was applied to investigate the UV-vis absorption spectra.A NaI (Tl) γ-ray scintillation counter (Scaler Ratemeter SR7 model, UK) was applied for radioactivity determination.

Preparation of ascorbic acid-coated iron oxide nanoparticles (AA-Fe 3 O 4 NPs)
The synthesis of AA-Fe 3 O 4 NPs was performed according to previous literatures with some modifications (Shagholani and Ghoreishi 2017;Swidan et al. 2022).Briefly, ferrous and ferric salts in molar ratio 1:2 were dissolved in 40 ml oxygen-free bi-distilled water before the temperature was gradually raised to 50 C in a nitrogen environment with steady stirring for 0.5 h.Then, the ammonia solution was added dropwise till pH 10 which was then maintained at that pH and temperature for an additional 0.5 h.The aforesaid reaction mixture was then supplied with a 20 mL addition of 10% AA (aqueous solution).The reaction took place for 1 h with constant stirring at 50 °C to functionalize the particles with AA.The obtained black precipitate (AA-Fe 3 O 4 NPs) was extracted from the separated solution by magnetic decantation and repeatedly washed with bidistilled water to bring a neutral pH for further study.
Preparation of nanoscale metal-organic framework (AA-Fe 3 O 4 @Fe III -Tannic acid) Tannic acid (TA) solution (40 mg/mL) was slowly added to the prepared solution of AA-Fe 3 O 4 NPs (10 mg/mL) in which the mixture was mechanically stirred for 2 h.Then, 10 mL solution of FeCl 3 .6H 2 O (10 mg/mL) was added while the pH ~ 7.4 was adjusted using HEPES buffer and the mechanical stirring was employed for another 2 h to facilitate the complexation reaction between TA and the metal ions (Fe III ) (Ejima et al. 2013).Finally, the obtained nanoscale MOF (AA-Fe 3 O 4 @Fe III -TA) was separated utilizing magnet, washed many times and dried under vacuum.

Drug loading and release studies
A definite volume of an aqueous drug solution (DOX.HCl, 1 mg/mL) was ultrasonically dispersed in an aqueous solution (5 mg/mL) of the nano-MOF in which the final mixture was mechanically stirred at ambient temperature for 6 h.Thereafter, DOX-loaded nano-MOF (DOX-loaded Fe 3 O 4 @Fe III -TA) was separated from the solution at different time intervals by a magnet in which the supernatant was utilized to determine the unloaded DOX via spectrophotometric analysis at 480 nm.The drug loading efficiency and capacity were estimated by the following formulas: The pH-triggered release study of the prepared DOX-loaded nano-MOF was conducted in phosphate-buffered saline (PBS) compromising pH of 7.4 and 5. Typically, 1 ml of DOX-loaded nano-MOF (1 mg/mL) was wrapped in the dialysis bag which placed within 19 mL PBS and exposed for continuous shaking at room temperature.At the scheduled intervals (0.5, 1, 2, 4, 8 h), 1 mL dialysis buffered-solution was withdrawn and retrieved for analysis using UV/vis absorption at 480 nm, before being replaced with new PBS of the same volume and pH value.

pH-dependent degradation of the nanoscale MOF (AA-Fe
The release of Fe II at various time intervals was evaluated using the 1,10-Phenanthroline colorimetric technique (Zhang et al. 2018).First, 200 µL of different concentrations of FeCl 3 solutions were incubated with 200 µL of ascorbic acid (1 mM) at ambient temperature for 3 min.After that, 200 µL of 1,10-Phenanthroline (1 mg/mL) was added, and after 10 min., the mixture's absorption at 510 nm was measured using a UV-vis spectrophotometer demonstrating the implementation of the calibration curve.The sample; (1) Loading efficiency % = Total DOX − unloaded DOX Total DOX × 100 (2) AA-Fe 3 O 4 @Fe III -TA (1 mL, 5 mg/mL) was put into dialysis bags, which were then submerged in buffer solutions (19 mL) with various pH levels (pH 7.4 and 5).A sample of the dialysis buffer (200 µL) was taken and new buffer (200 µL) was introduced at various intervals (0-8 h).After 10 min reaction with 1,10-phenanthroline, the released Fe II content was determined by measuring the absorbance at 510 nm of the withdrawn solution using a UV-vis spectrometer.

In-vitro Fenton-like reaction of the nanoscale MOF
The chemodynamic activity was investigated as follow (Guo et al. 2019;Guo et al. 2020): nano-MOF was incubated with 400 µl PBS (pH 5) containing methylene blue (MB; 0.1 M) and hydrogen peroxide (8 mM).After 1 h of incubation at 37 °C, the change in the absorbance value at 665 nm was used to track the hydroxyl radical ( • OH)-induced MB degradation.For control experiment, the exact concentrations of MB and a mixed solution of H 2 O 2 /MB as those of the aforementioned sample were also analyzed similarly.

Cell culture
The human breast cancer cells (MCF-7) were acquired from the VACSERA tissue culture unit (Giza, Egypt).They were cultivated in Dulbecco's modified Eagle's medium (DMEM) that contained gentamycin (1%), 10% fetal bovine serum (FBS) and L-glutamine.The cultures were maintained for 7 days at 37 C in a humid environment with 5% CO 2 .

Cell viability assay
The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examine the cell viability.In a 96-well plate, MCF-7 cells were sown and grown for 24 h at 37 °C in 100 µL of DMEM medium.The medium was then swapped out for 200 µL of new media that included various concentrations of DOX, nano-MOF or DOXloaded nano-MOF.The control cell line was assessed without using any of the tested samples.MTT (20 µL, 5 mg/mL) was then added and incubated for a further 24 h to create purple formazan.After centrifugation, an optical density microplate reader was used to track the results by contrasting the absorbance at 570 nm of treated and untreated cells.

Intracellular reactive oxygen species (ROS) determination
A 12-well plate supplemented with MCF-7 cells (6 × 10 4 cells/well) was left incubating for 24 h after which fresh culture medium containing DOX, nano-MOF or DOX loaded nano-MOF (IC 50 concentration for each one) was used to replace the original medium.
The culture medium was pulled out and wiped with PBS after 4 h of incubation.Each well was then filled with fresh culture medium (1 mL) containing 2,7-dichlorofluorescin diacetate (DCFH-DA; 10 µg/mL), and after 30 min of incubation, the culture medium was removed and cleaned with PBS.The cells were then centrifuged after being lysed in an alkaline solution.A microplate reader with 96-well filled with 200 µL of supernatant was used to monitor the fluorescence at 485 nm excitation and 520 nm emission.The results were displayed as a mean of the fluorescence intensity (Fukumura et al. 2012).

Intracellular glutathione (GSH) levels determination
A set of MCF-7 cells were exposed to DOX, nano-MOF or DOX-loaded nano-MOF for 2 h.A different group received catalase (CAT) before the treatment with the prepared formulations.The cells were taken out, cleaned, and lysed in 40 µL of Triton X-100 lysis buffer on ice.Twenty minutes later, lysates centrifugation was performed in which 10 µL of the supernatant was combined with 50 µL of Ellman's reagent (0.5 mM DTNB).Using a microplate reader, the absorbance at 405 nm was measured to determine the GSH concentration (Wang et al. 2021b).

Radiolabeling of DOX with 99m Tc
The radiolabeling of DOX with Tc-99m was accomplished using a simple reductive methodology (El-Ghareb et al. 2020).The radiolabeling technique was launched in which the reaction parameters including the concentration of DOX (0.25-1.5 mg/ mL), stannous chloride (25-200 µg/mL), reaction time (15-60 min) and pH (3-9) were tuned to get the highest radiochemical yield % (RCY).The radiolabeling efficiency was determined by means of ascending paper chromatographic analysis (Whatman paper no. 1 strips; 13 cm length and 1 cm width), whereby acetone and saline were used as mobile phases for the detection of free pertechnetate and other radioactive impurities, respectively. 99m

Tc-DOX loading study
As previously stated in the non-radioactive DOX loading fashion, a set volume of 99m Tc-DOX solution (1 mg/mL) was immediately dispersed with a definite volume of nano-MOF (1 mg/mL) followed by magnetic stirring for 3 h.At each predetermined time-step following magnetic decantation, the radioactivity in the precipitate ( 99m Tc-DOX loaded nano-MOF) and the supernatant ( 99m Tc-DOX) were measured and applied to compute the loading efficiency percent using the formula given:

In-vitro serum stability analysis
It is noteworthy that exploring the radiochemical procedure's tolerability in physiological settings should be investigated.In brief, a 1 mL reaction volume containing 99m Tc-DOX loaded nano-MOF product (0.1 mL) and Ringer's solution (0.9 mL, pH = 7.4) or mice serum (0.9 mL) was incubated at 37 °C for 24 h.Through using paper chromatographic procedures outlined before, the radiochemical yields were recorded at various intervals of time. ( Loading efficiency% = Radioactivity in the precipitate Radioactivity in the precipitate + Radioactivity in the supernatant × 100

Animal models and tumor inoculation
The National Cancer Institute (Cairo, Egypt) provided the primary tumor-induced mouse (Ehrlich Ascites Carcinoma) and all healthy Swiss albino male mice with usual weights of 25-35 g (n = 60).After the tumor in the primary mouse had formed for 5-6 days, the ascites fluids were collected and re-dispersed in 5 mL saline.For tumor inoculation, the healthy mice were subcutaneously injected in the right flank with the required volume (0.2 mL) of ascites fluids.The mice were monitored for 5-7 days until the tumor mass had fully formed and had an average volume of 1 ± 0.1 cm 3 (Korany et al. 2020).
All mice, both healthy and those had developed tumors, were kept in comfortable housing with consistent dietary and environmental parameters under the supervision of a veterinarian.

Bio-distribution studies
The mice were categorized into two groups, each with 30 animals.Group (A) had included normal mice, whereas Groups (B) had included mice that had undergone tumor induction.All of the two groups' members intravenously (i.v.) received treatment with 99m Tc-DOX loaded nano-MOF in the tail vein.Using a well-equipped NaI gamma counter, it was possible to quantify the accumulation of 99m Tc-DOX loaded nano-MOF in various organs and tumor tissues at 0.25, 0.5, 1, 2 and 4 h after injection.Percentage Injected Dose per Gram Organ (% ID/g ± SD) was used to examine the findings of 5 mice at every interval.Moreover, a time-dependent evaluation of the target/non-target (T/NT) ratios post-injection was also performed.

Statistical analysis
The data, with respect to the control group, were averaged over three replicates unless explicitly stated.A p-value of less than 0.05 from the One-Way ANOVA test indicated a statistically significant result.

Results and discussion
Synthesis and characterizations of the nanoscale AA-Fe 3 O 4 @Fe III -TA framework The synthesis of AA-Fe 3 O 4 NPs was made via a co-precipitation approach in which a stoichiometric mixture of ferrous and ferric salts in an aqueous environment was employed to produce Fe 3 O 4 NPs.While ascorbic acid was utilized as a functionalizing (coating) agent to provide Fe 3 O 4 NPs with the required stability because it is widely used as a biocompatible surfactant for superparamagnetic iron oxide nanoparticles (Özel et al. 2019;Sreeja et al. 2015;Ozel et al. 2018;Asefi et al. 2021).Subsequently, the nano-MOF (AA-Fe 3 O 4 @Fe III -TA) with core-shell structure was fabricated by simply mixing AA-Fe 3 O 4 NPs with the self-assembly TA and Fe III matrix.Throughout the assembly process, TA serves as an organic ligand whereas Fe III operates as an inorganic cross-linker, leading to the formation of a cross-linked Fe III -TA shell on the surface of the AA-Fe 3 O 4 NPs (Ejima et al. 2013).
According to TEM investigation, the produced AA-Fe 3 O 4 NPs proclaimed discrete and quasi-sphered morphology, uniform particles size ~ 34.4 ± 6.1 and narrow size distribution, as shown in Fig. 1a, b.The synthesized nano-MOF (AA-Fe 3 O 4 @Fe III -TA) was further examined by TEM, which revealed that the morphology was mostly still quasi-spherical but showed somewhat agglomeration, increasing the particle size to ~ 69.8 ± 11.8 as depicted in Fig. 1c, d.Zeta potential (ζ-potential) measurements were used to exhibit the core-shell structure after proper surface engineering.Fe III -TA shell caused a considerable change in the surface charge compared to the as-produced AA-Fe 3 O 4 NPs (ζ-potential: from -16 mV to -33 mV; Fig. 1e), which was attributed by the availability of many hydroxyl groups in TA represented in the construction of the nano-MOF (Chen et al. 2022a).The FTIR analysis (Fig. 1f ) was carried out to confirm the proper synthesis of AA-Fe 3 O 4 NPs and the efficient crosslinking of Fe III -TA shell onto AA-Fe 3 O 4 NPs.In contrast to pure AA, the spectra of AA-Fe 3 O 4 NPs showed the loss of the lactone ring's C = O band (1750 cm −1 ), a shift in the C = C band (1665 to 1635 cm-1), and the emergence of a new stretching band for Fe-O (589 cm −1 ).These are reliable signs that AA-Fe 3 O 4 NPs successfully formed.The spectrum of AA-Fe 3 O 4 @Fe III -TA displayed a broadband (3600-2700 cm −1 ) indicating the numerous hydroxyl groups of polyphenols, absorption peaks (1400-1600 cm −1 ) attributed to stretching and vibration of substituted benzene rings, as well as the distinctive band of Fe-O (589 cm −1 ).This FTIR investigation revealed further evidence of Fe III -TA coating on AA-Fe 3 O 4 NPs.

Doxorubicin loading and pH triggering release studies
The nano-MOF have been widely recognized as effective nano-carriers for loading diverse compounds due to their enormous surface area (Chen et al. 2022b).The goal of this study was to load DOX onto the synthesized nano-MOF (AA-Fe 3 O 4 @Fe III -TA).The UV/vis absorption of DOX at 480 nm was employed to track the DOX loading The dialysis technique was used to examine the drug-pH-triggered release profile.Under pH 7.4 which mimics the physiological environment, the loaded DOX exhibited low release behavior with an accumulative amount ~ 10 ± 0.93% after 8 h (Fig. 2d).So, it was possible to deduce that the system could be injected steadily during blood circulation with little to no pre-leakage.On the other hand, reducing the pH to 5 (simulating the tumor environment) resulted in reasonable acceleration of DOX release in a sustained manner with an accumulative amount ~ 98.3 ± 0.97% after 8 h (Fig. 2d).This is likely due to the dissociation of Fe III -TA matrix under an acidic condition (Chen et al. 2022a;Liu et al. 2019).

pH-dependent degradation inducing Fe II release
The traditional 1,10-phenanthrene colorimetric technique was used to quantify the generation of Fe II .In phosphate buffer solutions with pH values 7.4 and 5.0, the release of Fe II ions from AA-Fe 3 O 4 @Fe III -TA was monitored over time.Under neutral conditions, as shown in Fig. 3a, the released Fe II concentration was extremely low.On the other hand, under mild acidic circumstances, it displayed a time-dependent release pattern that revealed the release of Fe II ions with percentages of 5 ± 0.02 and 57 ± 0.03 of the initial concentration of Fe III ions at 1 and 8 h after the reaction, respectively (Fig. 3a).This might be the result of the Fe III -TA matrix dissociation in an acidic environment, releasing both Fe III and TA (Chen et al. 2022a;Liu et al. 2019).It is important to note that naturally occurring polyphenols, such as TA, have been shown to function as reducing agents for facilitating Fe II generation by accelerating the Fe III /Fe II conversion (Li et al. 2022;Guo et al. 2020).The foregoing findings showed that AA-Fe 3 O 4 @Fe III -TA exhibited good acidity responsiveness and effective production of Fe II to function as a Fenton reaction catalyst for producing hazardous • OH.

In-vitro Fenton-like reaction of the nanoscale MOF
The demonstration of the Fenton-like reaction ability (chemodynamic activity) of the nano-MOF (AA-Fe 3 O 4 @Fe III -TA) in the presence of H 2 O 2 under acidic conditions (simulating TME) was conducted using the MB colorimetric method (Guo et al. 2019;Guo et al. 2020).MB has a markedly UV-vis adsorption peak at 665 nm, despite that it is susceptible to being promptly degraded by • OH, triggering the disappearance of UV-vis adsorption (Guo et al. 2019;Guo et al. 2020).As seen in Fig. 3b, the absorbance of MB drastically dropped after incubation with AA-Fe 3 O 4 @Fe III -TA and H 2 O 2 , whereas H 2 O 2 treated alone with MB did not appear to experience any obvious drop.These results may be attributed to the ability of AA-Fe 3 O 4 NPs and Fe III -TA matrix to catalyze H 2 O 2 in a weak acidic environment to produce • OH efficiently (Cui et al. 2021;Chen et al. 2021;Dai et al. 2018).The acidic environment induces the dissociation of Fe III -TA shell to Fe III Fig. 3 In-vitro Fenton-like reaction of AA-Fe 3 O 4 @Fe III -TA: A time-dependent generation of Fe II ions in PBS with different pH values using 1,10-phenanthrene colorimetric method.B Determination of • OH using MB colorimetric method in the presence of H 2 O 2 under acidic conditions (pH 5).Data was represented as mean ± SD (n = 3).Means were compared using ANOVA, followed by the Tukey test for multiple comparisons, two-to-two.( * p ≤ 0.05, ( a p > 0.05) and TA which has the ability to reduce the generated Fe III to Fe II inducing the degradation of H 2 O 2 into highly cytotoxic • OH via Fe II -mediated Fenton-like reaction as declared by the following reaction (Liu et al. 2020a;Chen et al. 2022b):

Cell viability assay
The MTT assay was used to assess the cytotoxic activity of DOX, nano-MOF and DOXloaded nano-MOF against the MCF-7 cell line (Fig. 4).The outcomes supported the existence of a positive correlation between the examined concentrations per each model and the cytotoxic behavior.In contrast to DOX and nano-MOF, which had IC 50 values of 0.55 and 6.8 µg/ml, respectively, DOX-loaded nano-MOF was found to have an IC 50 value of just 0.08 µg/ml.With regard to ~ 7 times-fold reduction in the DOX inhibitory concentration necessary to kill 50% of MCF-7 cells, the beneficial contribution of the cytotoxic carrier (DOX loaded AA-Fe 3 O 4 @Fe III -TA) should be viewed as a synergistic effect of chemotherapy and chemodynamic therapy.The chemotherapeutic effect may be attributable to the (4) sustained release behavior of DOX inside the tumor cells while the chemodynamic activity may be due to the cytotoxic • OH via Fe II -mediated Fenton-like reaction (Chen et al. 2022a;Chen et al. 2022b;Liu et al. 2019).These findings concurred with those who investigated how nanoparticles and their associated Fenton reaction might have the potential of an efficient synergistic approach for CT and CDT (Cui et al. 2021;Nie et al. 2020;Chen et al. 2021;Fu et al. 2021;Liang et al. 2019).

Intracellular reactive oxygen species (ROS) determination
The ROS generating ability of the prepared nano-MOF in MCF-7 cells was initially studied utilizing DCFH-DA assay kit (Fukumura et al. 2012;Li et al. 2021).As seen in Fig. 5A, the Fe III -TA complex fluorescence intensity was remarkably low and similar to that of the control group.When cells were treated simply with either DOX or AA-Fe 3 O 4 @Fe III -TA, the fluorescence intensity increased in comparison to the control (~ 5.2 or 13.5 times, respectively) indicating ROS production which may be attributed to DOX-induced oxidative stress (Hernandes et al. 2023) or the enhanced Fenton reaction of AA-Fe 3 O 4 @Fe III -TA (Chen et al. 2021;Fu et al. 2021).While the treatment of MCF-7 cells with DOX-loaded AA-Fe 3 O 4 @Fe III -TA augmented the fluorescence intensity by ~ 19 times compared to the control, demonstrating the synergistic effect of CDT and CT.As an added benefit, DOX could produce more H 2 O 2 inside cells, which could mediate DOX cytotoxicity in speciesmediated ways (Wagner et al. 2005;Ubezio and Civoli 1994).This attribution stems from the metabolic reductive activation of DOX to a semiquinone that promotes the generation of superoxide and is then dis-mutated by superoxide dismutase enzymes, resulting in the synthesis of H 2 O 2 as represented in the subsequent equations: (5) The GSH depletion activity for 0.5 h incubation.Data was represented as mean ± SD (n = 3).Means were compared using ANOVA, followed by the Tukey test for multiple comparisons, two-to-two.* p ≤ 0.05 is deemed a significant difference

Intracellular glutathione (GSH) levels determination
Due to the availability of intracellular antioxidants, such as GSH, tumor cells have an increased capacity to scavenge RO;8S, which typically restricts the therapeutic efficacy of CDT.Therefore, a number of techniques have been devised to improve the CDT by decreasing intracellular GSH (Guo et al. 2019;Chen et al. 2021;Fu et al. 2021).This objective can be met in our system (DOX loaded AA-Fe 3 O 4 @Fe III -TA) by evaluation of the GSH depletion activity as illustrated in Fig. 5B.It showed that both nano-MOF and DOX-loaded nano-MOF-treated cells had a significantly low GSH level (47 and 32%, respectively), whereas DOX-treated cells only marginally consumed the cellular GSH in an approximately similar manner of the control group.Because of AA-Fe 3 O 4 @Fe III -TA's propensity to induce the Fenton reaction, DOX-loaded nano-MOF-treated cells have lower GSH levels as a result of the increased GSH depletion during the redox reaction with the released Fe III (Fu et al. 2021;Liang et al. 2019).To sum up, after DOX-loaded nano-MOF treatment, DOX could induce to some extent elevated levels of ROS production but produce less GSH depletion, while nano-MOF not only substantially elevated the ROS production but also facilitated the GSH depletion.So, DOX-loaded nano-MOF could effectively reinforce CDT and CT efficacy.These in-vitro biological findings encourage the pursuit of future in-vivo pharmacodynamic investigation in laboratory animals.

Synthesis of 99m
Tc-DOX loaded AA-Fe 3 O 4 @Fe III -TA The radiolabeling of DOX with Tc-99m was accomplished adequately via a straightforward reductive process in which stannous chloride was incorporated to reduce the pertechnetate ( 99m Tc +7 ) to a highly reactive form ( 99m Tc +5 ) (El-Ghareb et al. 2020;Ibrahim et al. 2014;Motaleb et al. 2018).According to Fig. 6A, the radio chromatogram of 99m Tc-DOX declaring the R f values of the two mobile phases used, the radiolabeling capacity of 99m Tc-DOX was estimated as ~ 96%.At room temperature, the following parameters yielded the best radiolabeling capacity: stannous chloride concentration (100 µg/mL; Fig. 6B), reaction pH (7; Fig. 6C), DOX concentration (1 mg/mL; Fig. 6D) and reaction time (30 min; Fig. 6E).The loading process of 99m Tc-DOX on nano-MOF shortly after its formation exhibited a time-dependent rhythm identical to that of the non-radioactive DOX loading outlined previously.The highest loading efficiency (98%) was attained after 4 h of stirring, which is close to the recommended time for non-radioactive DOX loading.
The in-vitro stability experiment of 99m Tc-DOX loaded nano-MOF in mice serum was evaluated since the serum protein corona's dynamic activity may change its thermodynamic and kinetic stability.Chromatographic estimates of the radiolabeling yields were made at time intervals up to 24 h in advance (Fig. 6F).It demonstrates how 99m Tc-DOX loaded nano-MOF maintained an acceptable level of physiological stability for at least 8 h, 94.7%, before displaying deterioration after this point.

In-vivo bio-distribution studies
The in-vivo distribution investigations are at the forefront as a crucial strategy for figuring out the pharmacokinetic parameters associated with any newly developed nanotheranostics (Taha et al. 2023;Swidan et al. 2023).Therefore, the biodistribution pattern of 99m Tc-DOX loaded nano-MOF had been explored in two different experimental groups of mice.After administering 99m Tc-DOX loaded nano-MOF to normal mice intravenously, the in-vivo distribution profile demonstrated an ordinary biodistribution pattern for a nanomaterial (Fig. 7A).It displayed a satisfactory blood circulation pattern where the radioactivity was washed out and declined from 22.45 ± 1.76 to 3.2 ± 1.12% ID/g at 0.25 h and 4 h p.i., respectively.Although most organs displayed minor radioactivity buildup, the liver and spleen tissues (reticloendothelial organs) exhibited the greatest accumulation (12.17 ± 0.98 and 8.68 ± 0.47% ID/g, respectively) at 1 h p.i. Due to these organs' leaky vasculature, the considerable hepatic and splenic accumulation could potentially be addressed (Sakr et al. 2018;Swidan et al. 2019;Nie 2010).In terms of class B, the in-vivo distribution in tumor-induced mice highlighted an upward accumulation in the tumor tissues with an optimal uptake of 16.8 ± 0.88% ID/g at 1 h p.i. and declined gradually to display the lowest level of 5.8 ± 0.62% ID/g at 4 h p.i. (Fig. 7B).It's crucial to mention that this significant radioactivity level in the tumor lesion is an advancement over the naked 99m Tc-DOX (only exhibited a maximum ID/g of 1.5%) (Fernandes et al. 2016).This high tumor accumulation might mainly rely on the enhanced permeability and retention (EPR) effect, which was due to the defective blood vasculature of tumor tissues, poor lymphatic drainage, and increased vessel permeability Values are expressed as ''mean ± SD'' (n = 3).Means were compared using ANOVA, followed by the Tukey test for multiple comparisons, two-to-two.( * p ≤ 0.05, ( a p > 0.05) (El-Ghareb et al. 2020;Wang et al. 2020b;Kim et al. 2009).Therefore, the platform could either simultaneously target the tumor passively (small-sized nanoparticles induced EPR effect) or actively (DOX-receptor interaction) in a synergistic manner.The ability to demonstrate a considerable target accumulation (tumor lesion) in contrast to nontarget (uninfected muscle), T/NT, is one of the critical obstacles in fabricating optimal radiopharmaceuticals (Mahmoud et al. 2023;El-Safoury et al. 2021a;Essa et al. 2015;El-Safoury et al. 2021b).Interestingly, Fig. 7C discovered encouraging T/NT findings along the experimental time points with a magnitude value of 8 at 1 h p.i. Herein, the considered formulation, 99m Tc-Dox loaded nano-MOF, could potentially turn out as being a tumor-imaging guided SPECT tracer.

Conclusion
A unique in-situ activatable nano-MOF, 99m Tc-DOX loaded AA-Fe 3 O 4 @Fe III -TA, had been effectively engineered to operate as a tumor-selective multifunctional platform that offers improved chemo/chemodynamic theranostics.Optimized loading efficiency, pH-responsive characteristics, and nanoparticle size were all features of the biocompatible nano-MOF, which could remain stable in the bloodstream for an extended duration of time leading to enhanced tumor accumulation.The selective disintegration of Tc-DOX loaded AA-Fe 3 O 4 @Fe III -TA was aided by the acidosis of the TME, which resulted in the release of TA, Fe III , and the cargo 99m Tc-DOX.Together, the released entities had a synergistic impact that combined SPECT imaging capabilities with a tumorcell chemotherapeutic effect.Additionally, the burst cascade of • OH-dependent Fenton reaction increased the oxidative stress to a large degree, therefore killing tumor cells specifically.Our work offers an entirely new viewpoint on chemodynamic theranostics and shows significant promise for precise tumour treatment.

Fig. 1
Fig. 1 Physicochemical characterization of AA-Fe 3 O 4 NPs and AA-Fe 3 O 4 @Fe III -TA.a-d The representative TEM images a and c and size distribution b and d of AA-Fe 3 O 4 NPs and AA-Fe 3 O 4 @Fe III -TA, respectively.e Zeta potential measurements in water.f FT-IR spectroscopic analysis

Fig.
Fig. Loading and release studies of DOX. a The calibration curve of by UV/vis absorbance at 480 nm.b DOX loading efficiency and capacity at different stirring intervals.c Hydrodynamic size distribution of DOX loaded AA-Fe 3 O 4 @Fe III -TA in aqueous medium.d Drug release profiles of DOX-loaded AA-Fe 3 O 4 @Fe III -TA in PBS buffer with different pH values.Data was represented as mean ± SD (n = 3)

Fig. 4
Fig.4The cell viability of MCF-7 cells treated with different formulations: A DOX B AA-Fe 3 O 4 @Fe III -TA and C DOX loaded AA-Fe 3 O 4 @Fe III -TA at various concentrations using MTT assay.D IC 50 comparative diagram.Data was represented as mean ± SD (n = 3)

Fig. 5
Fig. 5Oxidative stress markers determination in MCF-7 cells treated with different formulations: A Quantitative analysis of the fluorescence intensity (λex/em = 485/520 nm) of DCF-induced ROS production B The GSH depletion activity for 0.5 h incubation.Data was represented as mean ± SD (n = 3).Means were compared using ANOVA, followed by the Tukey test for multiple comparisons, two-to-two.* p ≤ 0.05 is deemed a significant difference

Fig. 6
Fig. 6The tuning profile of the radiolabeling yield percent of 99m Tc-DOX.a Paper radio-chromatographic analysis declaring the R f values of 99m Tc-DOX in different mobile phases.b-e Factors affecting the optimization of the radiolabeling yield percent of 99m Tc-DOX.f In-vitro stability of 99m Tc-DOX over time for 24 h at 37° C in mice serum (n = 3).Values are expressed as ''mean ± SD'' (n = 3).Means were compared using ANOVA, followed by the Tukey test for multiple comparisons, two-to-two.( * p ≤ 0.05, ( a p > 0.05)