Abstract

   Iron is an essential element for various cellular metabolism. Cancer
   cells also have high requirement of iron in their proliferation,
   invasion, and metastasis processes. Alendronate (ALN), a kind of
   FDA‐approved bisphosphonates with metal‐chelating capability, is
   initially certified to selectively bind to intracellular Fe^3+
   theoretically and experimentally in this study. Hence, CaALN iron
   nanochelator is rationally designed to kill cancer cells by synergism
   of Fe‐depletion and calcium accumulation. In vitro experiments and RNA
   sequencing analysis indicate that CaALN nanomedicine inhibits the
   proliferation of cancer cells by depleting Fe, interfering with DNA
   replication, and triggering intracellular reactive oxygen species
   (ROS). Meanwhile, released Ca^2+ and ROS mutually promote and induce
   damage of cellular macromolecules, which leads to mitochondrial
   apoptosis of cancer cells. In an intraperitoneal disseminated mouse
   model with the human ovarian cancer cells SKOV3, CaALN nanoparticles
   selectively accumulate in tumor tissues and result in significant
   retardation of tumor growth and ascites formation. The mean survival
   time of SKOV3‐bearing mice in treatment group is prolonged from 33 to
   90 d. These results indicate that the alendronate‐originated iron
   chelator can serve as an efficient strategy for the treatment of
   peritoneal carcinomatosis.

   Keywords: calcium overload, chemotherapy, infinite coordination
   polymers, iron chelator, ovarian cancer
     __________________________________________________________________

   An alendronate‐composed iron nanochelator is designed for peritoneal
   carcinomatosis treatment. Satisfactory antitumor effect is achieved by
   synergism of Fe‐depletion and calcium accumulation, which promote
   mutually and eventually leads to the apoptosis of cancer cells.

   graphic file with name ADVS-9-2203031-g003.jpg

1. Introduction

   Iron is an essential requirement for both human normal cells and cancer
   cells. It plays crucial roles in many biological processes, such as
   cellular metabolism, DNA synthesis and repair, oxygen transport, and
   energy production.^[ [44]^1 ^] It has been confirmed that several
   cancer cells have higher transferrin receptor 1 (TfR1) expression than
   normal cells because they have high requirements of iron to support
   their rapid growth, proliferation, and metastasis.^[ [45]^2 ^] Thus,
   targeting iron metabolism in cancer cells (Fe‐deficiency or
   ferroptosis) represents a new treatment strategy for cancers.^[ [46]^3
   ^] Several small molecule iron chelators, such as deferoxamine (DFO),
   deferasirox (DFX), and some polyphenols were reported to preferentially
   reduce cancer cell growth and reduce tumor burden in vitro and in
   vivo.^[ [47]^4 ^] In addition, iron depletion, mostly using of DFO, was
   proved as a potential strategy for clinical cancer treatment.^[ [48]^5
   ^]

   Ovarian cancer is one of the most lethal gynecological cancers and
   characterized by diffuse peritoneal metastasis.^[ [49]^6 ^]
   Intraperitoneal chemotherapy has been demonstrated to be very effective
   for the treatment of ovarian cancer with peritoneal metastasis, which
   can prominently increase the drug concentration in the abdominal cavity
   with reduced systematic toxicity.^[ [50]^7 ^] The current first‐line
   drugs for intraperitoneal perfusion are mainly antineoplastic drugs
   which are designed for intravenous injection, such as platinum‐based
   drugs, 5‐fluorouracil, paclitaxel, etc.^[ [51]^8 ^] However, due to the
   lack of tumor specificity and low bioavailability, the efficacy of
   small molecule based intraperitoneal chemotherapy is severely
   compromised.^[ [52]^9 ^] It has been reported that not only the level
   of Fe in serum and tumor tissues in advanced‐stage increased as
   compared with early‐staged ovarian cancer patients, but also proteins
   related to Fe uptake, such as transferrin receptor 1 (TFR1), ferritin,
   divalent metal ion transporter 1 (DMT1) increased in ovarian cancer
   tissues.^[ [53]^10 ^] Thus, it is plausible to believe that targeting
   Fe metabolism may be an effective therapeutic strategy for ovarian
   cancer.

   Bisphosphonates, with the P‐C‐P bond in their structure, have been
   extensively used in the treatment of osteoporosis and skeletal
   metastases due to their low toxicity, high thermostability and
   inhibition of bone resorption.^[ [54]^11 ^] In the last two decades,
   nitrogen‐containing bisphosphonates have been used in clinical for the
   adjuvant treatment of breast cancer and prostatic cancer to reduce the
   incidence of skeletal‐related events.^[ [55]^12 ^] Alendronate, one of
   the nitrogen‐containing bisphosphonates, has been reported to exert an
   antiangiogenic effect and inhibit the invasiveness of human ovarian
   cancer cells. Current reports of alendronate were focused mainly on the
   antiangiogenic effect by inhibiting enzymes of the mevalonate
   pathway.^[ [56]^13 ^] In fact, bisphosphonates have also been proposed
   as potent chelators for metallic ions. The coordination of alendronate
   with metallic ions, especially Ca (II), has been exclusively studied.^[
   [57]^14 ^] ALN‐functionalized polymers and nanoparticles have been
   reported as carriers for bone‐targeting drug delivery.^[ [58]^15 ^] But
   few works studied alendronate on its homeostasis of intracellular
   essential metallic ions yet. On the other hand, alendronate small
   molecule is not a desirable anticancer medicine due to its unfavorable
   pharmacokinetics. After intravenous administration of alendronate
   salts, the majority of alendronate binds to the mineralized bone
   matrix, and the remainder is excreted by the kidney.^[ [59]^11 ,
   [60]^16 ^]

   In recent years, nanoformulations have been demonstrated as a viable
   strategy to solve the problems associated with conventional
   chemotherapeutic agents by the composition of nanotechnology,
   biomedicine, and pharmaceutical science, thus achieving reduced system
   toxicity, increased tissue selectivity, and enhanced therapeutic
   efficacy.^[ [61]^17 ^] Nevertheless, clinical applications of
   nanomedicine are limited by the low drug‐loading efficiency and
   immunogenicity of carriers.^[ [62]^18 ^] Infinite coordination polymers
   (ICPs) are amorphous compounds bearing polydentate ligands and metal
   ions, exhibiting higher tailorability and degradation than the regular
   crystalline coordination polymers.^[ [63]^19 ^] Therapeutic agents,
   such as methotrexate, oxaliplatin, gallic acid, and doxorubicin have
   been reported to coordinate with metal ions forming ICPs, showing
   superior antitumor efficacy and slight side effects.^[ [64]^20 ^]

   In this work, we reported for the first time that alendronate inhibited
   cancer cell proliferation by interference with intracellular iron
   metabolism. Alendronate‐based nanomedicine (CaALN) was rationally
   designed as iron nanochelator to deplete intracellular iron. In vitro
   study revealed that CaALN nanoparticles not only chelated intracellular
   iron and perturbed DNA replication, but also disturbed Ca^2+
   homeostasis, which induced intracellular ROS elevation and
   mitochondrial apoptosis of cancer cells. RNA sequencing has indicated
   that Fe deficiency and apoptosis related pathways were upregulated,
   while DNA replication and cell‐migration related pathways were
   downregulated. In an intraperitoneal xenograft model with SKOV3 cells,
   CaALN prominently suppressed the tumor progression and ascites
   formation. Generally, alendronate‐based iron nanochelator features
   considerable potential in clinical peritoneal carcinomatosis treatment.

2. Results and Discussion

2.1. Specific Binding of Alendronate with Fe and Synthesis of ICPs

   Alendronate, a clinically approved drug for the treatment of osteolytic
   metastases,^[ [65]^21 ^] has been reported to have high efficiency in
   binding with metal ions. However, the interference of alendronate on
   intracellular ions metabolism was rarely investigated. In this study,
   chemical binding energies of alendronate and several intracellular
   metallic ions were obtained by density functional theory (DFT)
   calculation. As show in Figure [66] 1A and Figure [67]S1 (Supporting
   Information), alendronate could chelate intracellular metal ions
   through the coordination between O atoms of phosphate and metals. Among
   the six types of common intracellular metal ions, Fe displayed the
   strongest binding with alendronate (Figure [68]1B), reminding us that
   alendronate may be a potential Fe chelator to disrupt Fe metabolism of
   cancer cells. To verify the combination of alendronate and metal ions,
   alendronate‐based ICPs were achieved by the quick reaction of
   alendronate and metal ions at 4 °C for 30 min. The obtained CaALN,
   MnALN, and FeALN ICPs exhibited rodlike morphology with amorphous
   states (Figure [69]1C–F). Energy dispersive X‐ray (EDX) spectrometer
   and elemental mapping recognized the existence of metal elements and
   phosphonate in ICPs (Figures [70]S2–S5, Supporting Information). The
   nitrogen–adsorption–desorption isotherms and low specific surface areas
   also confirmed their disordered interior structures (Figure [71]S6,
   Supporting Information). The broad absorption band centered at 1100
   cm^−1 (metal—O—P stretching vibration, blue arrow) and disappeared peak
   centered at 1235 cm^−1 (P=O stretching vibration, black arrow) in
   Fourier transform infrared (FTIR) spectra confirmed the covalent
   binding of metal ions to phosphate (Figure [72]1J).^[ [73]^21 , [74]^22
   ^] The positive zeta potentials (19.1 ± 0.83 mV for CaALN, 12.06 ±
   0.10 mV for MnALN, and 24 ± 1.38 mV for FeALN) could be attributed to
   the amino group in alendronate, which further verified the successful
   coordination of alendronate and metal ions. Figure [75]1G–I showed the
   pH‐triggered degradation behaviors of CaALN and MnALN, while the
   degradation rate of FeALN was less than 5% at both pH 7.0 and 5.0
   (Figure [76]1K–M). These results together demonstrated that alendronate
   had much higher affinity to Fe rather than other metallic ions.
   However, the high affinity to bone tissue limits its clinical
   application as a versatile antitumor drug.

Figure 1.

   Figure 1
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   Characterization of alendronate‐assembled ICPs. A) Schematic
   illustration of coordination between alendronate and Ca^2+, Mn^2+, and
   Fe^3+ in normal saline (pH 7.0). B) Calculated binding energy of
   alendronate and metal ions in normal saline (pH 7.0). TEM images of C)
   CaALN, D) MnALN, and E) FeALN NPs. F) XRD patterns of CaALN, MnALN, and
   FeALN. G) Degradation of CaALN in PBS with different pH values (7.0 or
   5.0). H) Degradation of MnALN in PBS with different pH values (7.0 or
   5.0). I) Degradation of FeALN in PBS with different pH values (7.0 or
   5.0). J) FTIR spectra of NaALN, CaALN, MnALN, and FeALN. The
   photographs of K) CaALN, L) MnALN, and M) FeALN after 24 h incubation
   in PBS with different pH values (7.0 or 5.0).

2.2. Intracellular Fe‐Depletion of NaALN and CaALN in Cancer Cells

   Herein, we report a nanoformulation strategy of alendronate to improve
   its tumor targeting and anticancer potency. Figure [78] 2A showed that
   at equipotent concentration, CaALN nanoparticles revealed the strongest
   cytotoxicity on SKOV3 cells, followed by the commercial Fe‐chelator
   DFX. This result might be attributed to the synergistic effect of
   alendronate and Ca^2+. Moreover, it is noteworthy that CaALN was
   ≈150 nm in size and positive charged (19.1 ± 0.83 mV), which makes
   CaALN well adapted for cancer cell uptake (Figure [79]2B and Figure
   [80]S7, Supporting Information). Likewise, MnALN also exhibited potent
   anticancer ability, while FeALN featured no cytotoxicity due to its
   undegradability in physiological environments (Figure [81]2C).
   Figure [82]2D showed that CaALN revealed relatively lower cytotoxicity
   than MnALN and DFX in normal human epithelial cell (HK2). The serum
   calcium concentration of normal human is 2.25–2.75 mmol L^−1, which is
   higher than the serum manganese concentration (0.073–0.255 mmol L^−1),
   demonstrating Ca ‐baesd material is more tolerable for human body.
   Considering the excellent biocompatibility and clinical availability of
   Ca‐based biomaterials, CaLAN was eventually optimized for human ovarian
   cancer treatment. Next, the chelation exchange behavior of CaALN in
   Fe^3+ solution was studied. As shown in Figure [83]S8 (Supporting
   Information), white CaALN sample turned into yellow after immersing in
   FeCl[3] solution for 24 h. X‐ray photoelectron spectroscopy (XPS) test
   was performed to study the chemical composition of these powders. As
   shown in Figure [84]2E, disappearance of Ca2p peak at 359 eV and
   emergence of Fe2p peak at 739 eV revealed that most Ca has been
   replaced by Fe. The intracellular Fe‐chelation ability of NaALN and
   CaALN was further investigated by an established calcein method.^[
   [85]^23 ^] In brief, the free iron in cytosol and labile iron pool
   (LIP) can combine with carboxyl group of calcein AM and quench the
   fluorescence of calcein AM. After co‐incubation with powerful Fe
   chelating agents, the fluorescence intensity of calcein AM could be
   recovered. As shown in Figure [86]2F,G, in comparison with DFX,
   treatment with NaALN and CaALN revealed analogous Fe‐depletion ability
   in MGC803 and SKOV3 cells. As Fe is critical for DNA synthesis, Fe
   deficiency would inhibit the activity of ribonucleotide reductase,^[
   [87]^24 ^] the cell cycle distributions of cancer cells were detected
   by flow cytometry (FCM). Figure [88]2H showed obvious G1/S cell cycle
   arrest after NaALN and CaALN treatment in MGC803 and SKOV3 cells, which
   was similar with DFX treatment. Moreover, NaALN and CaALN treatment
   increased the expression of TfR1 protein and Fe^2+ supplement could
   promote the recovery of cancer cells after treatment of CaALN, implying
   that Fe‐deficiency was one of the action modes of CaALN in cancer cell
   (Figure [89]2I–K). In addition, CaALN exhibited broad‐spectrum
   antitumor activity, the cytotoxicity of CaALN against human pancreatic
   tumor cell PSN1, human osteosarcoma cell line MG63, human breast cancer
   cell SKBR3 and human bladder cancer cell MGH‐U3 were shown in
   Figure [90]2L.

Figure 2.

   Figure 2
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   In vitro Fe‐depletion abilities of NaALN and CaALN in SKOV3 and MGC803
   cells. A) Relative viabilities of SKOV3 cells after treatment with
   various concentrations of NaALN, CaALN, and DFX for 48 h. B) Abberior
   STEDYCON super‐resolution images of SKOV3 cells incubated with CaALN
   NPs at different time points, bar = 20 µm. C) Relative viabilities of
   SKOV3 cells after treatment with various concentrations of FeALN,
   CaALN, and MnALN for 48 h. D) Relative viabilities of human renal
   tubular epithelial cell HK2 after treatment with various concentrations
   of DFX, CaALN, and MnALN for 48 h. E) XPS analysis of CaALN powder
   before and after immersing in FeCl[3] solution for 24 h. Detection of
   intracellular Fe concentration using calcein‐AM in F) MGC 803 and G)
   SKOV3 cells after treatment with NaALN, CaALN, and DFX at 25 mg L^−1
   for 24 h by FCM analysis (mean ± SD, n = 3, ***P < 0.001 vs control
   group). H) Cell cycle distributions of G1/S phase in MGC803 and SKOV3
   cells treated with NaALN (25 mg L^−1), CaALN (25 mg L^−1), and DFX
   (25 mg L^−1) for 24 h and analyzed by FCM. I) Western blot analysis of
   MGC 803 and SKOV3 cells treatment with NaALN, CaALN, and DFX at
   concentration of 25 mg L^−1 for 24 h. Treatment of J) MGC803 and K)
   SKOV3 cells with CaALN in the presence or absence of FeSO[4] for 48 h.
   L) Relative viabilities of PSN1, MG‐63, MGH‐U3, and SKBR3 cells after
   treatment with various concentrations of CaALN for 48 h.

2.3. Synergetic Anticancer Effect of Alendronate and Ca^2+

   The underlying anticancer mechanism of CaALN on SKOV3 cells was further
   studied. Ca^2+, as an important second messenger, plays essential roles
   in a range of cellular processes such as gene expression, cell
   proliferation, and apoptosis. Intracellular Ca^2+ level is precisely
   regulated by an effective Ca^2+ homeostatic system, which is 10 000
   times lower than that of the extracellular fluid, thus both excess and
   deficiency of Ca^2+ in cells may result in undesirable cytotoxicity.
   Co‐incubation of CaALN could cause calcium accumulation in SKOV3 cells,
   as detected by intracellular calcium fluorescent probe (Figure [92] 3A
   and Figure [93]S9, Supporting Information). Thus, the Ca^2+ retention
   synergized with alendronate to induce high ROS generation in cancer
   cells (Figure [94]3B and Figure [95]S10, Supporting Information), which
   leads to the oxidative damage to membrane systems, including
   mitochondria. On the other hand, it has been reported that iron
   deficiency decreased mitochondrial function.^[ [96]^25 ^] Herein, the
   mitochondrial membrane potential (MMP) of SKOV3 cells after being
   treated with NaALN or CaALN was assessed using JC‐1 fluorescence probe
   (Figure [97]3C). The remarkably enhanced green/red fluorescence
   intensity ratio stated the significant mitochondrial membrane potential
   loss and mitochondrial injury caused by NaALN or CaALN. Finally, Ca^2+
   accumulation, ROS generation, and mitochondrial dysfunction jointly
   resulted in the apoptosis of SKOV3 cells (Figure [98]S11, Supporting
   Information). In addition, alendronate indicated to prevent cancer cell
   migration in vitro and in vivo by inhibiting the mevalonate pathway.^[
   [99]^13 , [100]^26 ^] The wound healing assay showed that both NaALN
   and CaALN could effectively inhibit in vitro migration of SKOV3 cells
   in a time‐ and dose‐dependent manner (Figure [101]3D–F), suggesting
   that CaALN nanoparticles would inhibit cancer growth via multiple
   mechanisms.

Figure 3.

   Figure 3
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   In vitro anticancer evaluation of alendronate‐assembled ICPs.
   Fluorescence microscope images of SKOV3 cells treated with CaALN for
   24 h and stained with A) Fluo‐3, AM, B)DCFH‐DA, and C) JC‐1 , scale bar
   = 100 µm. D) Microscope images and E,F) corresponding quantitative
   analyses of wound healing in SKOV3 cells treated with NaALN and CaALN
   at different concentrations. The wound gap was calculated as
   dehiscence/initial dehiscence × 100%.

2.4. RNA Sequencing Analysis of CaALN against SKOV3 Cells

   We further conducted RNA sequencing analysis to assess the regulation
   of gene expression and analyze differentially expressed genes.
   Statistically significant genes with a log[2] (fold change) expression
   of ≥1.0 were considered to be upregulated and ≤−1.0 were considered to
   be downregulated (treated group vs control group). RNA sequencing
   analysis showed that compared with the cells in the control group,
   there were significant changes in mRNA abundance of 1243 upregulated
   and 1517 downregulated genes in cells treated with NaALN (Figure [103]
   4A), while 1938 upregulated and 3683 downregulated genes in cells
   treated with CaALN (Figure [104]4B). Moreover, CaALN and NaALN treated
   cells shared 973 upregulated and 1338 downregulated genes (Figure
   [105]S12, Supporting Information). Fe‐deficiency was reported its
   significant effects on cell cycle and cell metabolism.^[ [106]^27 ^] As
   shown in Figure [107]4C, cell cycle‐associated genes, including cyclin
   A2 (CCNA2), cyclin B1 (CCNB1), and cyclin‐dependent kinases 2 (CDK2)
   were downregulated after treatment with CaALN. Meanwhile, TP53 tumor
   suppressor gene and its downstream genes, growth arrest‐ and DNA
   damage‐inducible genes (GADD45A and GADD45G) were upregulated. We
   subsequently applied a bioinformatics approach to explore the upstream
   regulators and functional significance of such alterations according to
   the identified differential expression genes. Kyoto Encyclopedia of
   Genes and Genomes (KEGG) enrichment analysis revealed the top 10
   upregulated (Figure [108]4D) and downregulated pathway
   (Figure [109]4E). Therein, p53 signaling pathway and Ras signaling
   pathway, which were involved in cell proliferation and apoptosis, were
   prominently upregulated. The upregulation expression of P53 protein and
   key genes on TNF signaling pathway were shown in Figures [110]S13 and
   [111]S14 (Supporting Information). The activation of RAS pathway might
   be attributed to the acquired and intrinsic resistance of cancer cells
   in chemotherapy.^[ [112]^28 ^] Meanwhile, the upregulated mitophagy
   confirmed the mitochondria damage, consistent with the result of the
   JC‐1 fluorescence probe in Figure [113]2A. In addition, Gene Set
   Enrichment Analysis (GSEA) of the downregulated genes in the DNA
   replication and cell cycle was further confirmed, which indicated that
   the treatment of CaALN induced SKOV3 cell apoptosis by removing iron,
   interfering with the cell cycle and activating TNF‐mediated
   caspase‐dependent pathway (Figure [114]4F–H). Upregulation of key genes
   enriched in the TNF signaling pathway were further confirmed by qRT‐PCR
   analysis, including upregulation of the Tumor Necrosis Factor Receptor
   Type 1 Associated Death Domain Protein (TRADD), Apoptosis‐Related
   Cysteine Peptidase (Caspase8), Receptor Interacting Serine/Threonine
   Kinase 1(RIPK1), and Receptor Interacting Serine/Threonine Kinase 3
   (RIPK3) gene (Figure [115]S15, Supporting Information). Based on those
   results, a possible antitumor mechanism of CaALN against SKOV3 cells
   was shown in Figure [116]4I. After being internalized by tumor cells,
   CaALN could induce cell cycle arrest, ROS elevation, and mitochondrial
   damage, which eventually leads to caspase‐dependent apoptosis of SKOV3
   cells.

Figure 4.

   Figure 4
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   RNA sequencing analysis of SKOV3 cells treated with NaALN or CaALN.
   Volcano plots for all the expressed genes of control versus A) NaALN
   and B) CaALN treatments. Red (upregulated) and green (downregulated)
   dots mean that the genes have significant differences (FDR ≤ 0.001, and
   at least with twofold differences). C) Heat map of differentially
   expressed genes related to intracellular Fe‐deficiency. D,E) The top 10
   remarkably enriched pathway of upregulated and downregulated of
   differentially expressed genes by KEGG analysis. GSEA enrichment plots
   of differentially expressed genes centralized in F) regulation of DNA
   replication, G) cell cycle, and H) TNF signaling pathway. I) Schematic
   illustration of anticancer mechanism of CaALN in SKOV3 cells.

2.5. In Vivo Biodistribution of CaALN Nanomedicine

   Although alendronate exhibited strong antineoplastic ability in vitro,
   the bone affinity hindered its clinical applications as an
   antineoplastic drug. In this study, alendronate coordinated with Ca^2+
   to improve its tumor targeting efficiency. To evaluate the in vivo
   distribution of CaLAN, an intraperitoneal disseminated tumor xenograft
   model was established by intraperitoneal injection of 5 × 10^6 SKOV3
   cells into NOD/SCID mice. It is noted that SKOV3 cells were stably
   transfected with lentivirus carrying luciferase (Luc) gene, which could
   exhibit strong bioluminescent signal and be captured by the In Vivo
   Imaging System(IVIS, AniView 100). To enable in vivo real‐time
   monitoring, CaALN nanoparticles were labeled by DiR fluorescence. As
   shown in the first row of Figure [118] 5A, luciferase signals in the
   abdominal cavity could be detected, implying successful establishment
   of intraperitoneal xenograft mice model. After 14 d of tumor
   establishment, the in vivo distribution of CaALN nanoparticles was
   evaluated by i.p. injection of CaALN/DiR or free DiR into mice. The
   images and quantitative fluorescence intensities of tumors and organs
   confirmed that i.p. injection of CaALN nanoparticles could target tumor
   tissue post 4 h injection (Figure [119]5A,B). In the free DiR group,
   fluorescence distributed not only in the tumor sites, but also in the
   liver and spleen (Figure [120]5C,D). The tumor targeting advantage of
   CaALN could be attributed to the existence of the peritoneal‐plasma
   barrier, which maintained a high regional concentration of CaALN
   nanoparticles in the peritoneal cavity and was eventually uptaken by
   cancer cells (Figure [121]5E). In comparison, small molecules could
   enter subperitoneal capillaries via the peritoneal–plasma barrier and
   metabolize by liver and spleen (Figure [122]5F).

Figure 5.

   Figure 5
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   In vivo biodistribution of CaALN. In vivo time‐dependent
   bioluminescence and fluorescence images of SKOV3 tumor‐bearing mice
   after i.p. injection of A) CaALN/DiR and C) free DiR i.p. treatment.
   B,D) In vitro bioluminescence and fluorescence images of tumors (Tu),
   livers (Li), hearts (He), spleens (Sp), lungs (Lu), and kidneys (Ki)
   dissected from SKOV3 tumor‐bearing mice post 72 h i.p. injection. E)
   Illustration of in vivo distribution of CaALN nanoparticles and small
   molecules. F) Fluorescence intensity of in vitro tissues from mice
   treated with CaALN/DiR and DiR (mean ± SD, n = 3, ***P < 0.001, **P <0
   .01).

2.6. In Vivo Anticancer Evaluation of CaALN Nanomedicine

   Encouraged by the in vitro antitumor activity and in vivo tumor
   targeting capability, the in vivo tumor inhibition activity of CaLAN
   nanoparticle was further evaluated. Five days after tumor cell
   implantation, SKOV3‐bearing mice were treated with PBS and two
   different doses of CaALN (10 and 25 mg kg^−1, n = 8). Five of the mice
   were periodically imaged and monitored for collecting data of body
   weight, abdominal girth, and survival day. The other three mice of each
   group were sacrificed at day 31 for real efficacy and biocompatibility
   evaluation (Figure [124] 6A). As shown in Figure [125]6B,C, both doses
   of CaALN‐treated groups maintained the bioluminescence by day 15,
   implying the complete suppression of tumor progression. The tumor
   inhibition effect of the high dose (25 mg kg^−1) group was better than
   that of the low dose (10 mg kg^−1) group. In comparison, treated mice
   began to exhibit abdominal swelling (Figure [126]6E) with ascites at 25
   d post SKOV3 cell inoculation, the average survival day was 34 d
   (Figure [127]6F). At day 31, three mice of each group were sacrificed,
   the tumor burden and ascites were quantified, and the main organs were
   extracted for hematoxylin and eosin (H&E) dyeing. As shown in Figure
   [128]S16 (Supporting Information), the mean tumor weight was 650.1 ±
   135.4 mg in the PBS group, which was significantly reduced in CaALN
   treatment groups (238.8 ± 22.8 mg in the low CaALN dose group and 174.6
   ± 24.1 mg in the high CaALN dose group). The ascites formation of mice
   was also inhibited by treatment of CaALN, where the mean weight of
   ascites in the PBS group was 3163 ± 450 mg, only 502 ± 154 mg in the
   low CaALN dose group, and 57.2 ± 8.5 mg in the high CaALN dose group
   (Figure [129]S17, Supporting Information). Meanwhile, H&E and TUNEL
   staining also identified the severe cancer cell apoptosis and less
   tumor cell invasion (black arrow) in the spleen in both CaALN treatment
   groups (Figure [130]6G,H). Finally, the mean survival time in the CaALN
   treatment groups was significantly longer than the control group
   (Figure [131]6F).

Figure 6.

   Figure 6
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   In vivo antitumor effect of CaALN on SKOV3 bearing mice. A) Scheme
   illustration of the in vivo antitumor experiment using SKOV3
   intraperitoneal xenograft model. B) Bioluminescence images, C)
   bioluminescence intensity, D) body weight, E) abdominal girth, and F)
   survival curves of SKOV3 tumor‐bearing mice in each treatment group. G)
   H&E staining images and tunnel histochemical images of representative
   tumor sections at day 31. H) H&E staining images of organs from
   experimental mice.

   After six cycles of treatment, the mice in all the CaALN‐treated groups
   showed stable body weights (Figure [133]6D). The body weight of the PBS
   group rose dramatically at day 38 due to the formation of massive
   ascites. However, H&E staining of organs revealed that in the high dose
   of CaALN group, the liver of mice had bile canaliculus proliferation
   (black arrows), suggesting of hepatic fibrosis. By contrast, the low
   dose CaALN group showed normal cellular morphology of organs.
   Therefore, from bioluminescence intensity, tumor and ascites weight
   results, high dose of CaALN induces the higher
   antiproliferation/antimigration activity of ovarian cancer in vivo.
   However, tumor‐bearing mice treatment with a low dose of CaALN exhibits
   longer survival due to its better biocompatibility. These results
   suggested that CaALN nanoparticles have considerable potential in
   treating peritoneal disseminated ovarian cancer at moderate
   concentration.

3. Conclusions

   In summary, we have reported the selective Fe‐chelation ability of
   alendronate, based on which, an alendronate‐composed CaALN has been
   rationally designed for efficient peritoneal carcinomatosis treatment.
   In vitro experiments demonstrated that CaALN with pH sensitive
   degradation induced strong antiproliferation/antimigration activities
   in human cancer cell lines by causing Fe‐deficiency and cell cycle
   arrest. Importantly, exogenous Ca^2+ aggravated mitochondrial
   dysfunction and intracellular ROS elevation, which involved in the
   apoptosis of SKOV3 cancer cells. Based on RNA sequencing analysis, we
   further confirmed that apoptosis related pathways were upregulated, and
   DNA replication, cell cycle and migration related pathways were
   downregulated. In an intraperitoneal xenograft model with SKOV3, it has
   been demonstrated that CaALN nanoparticles after i.p. administration
   accumulated mainly in tumor tissues. In addition, both doses of CaALN
   nanoparticles treatments (10 and 25 mg kg^−1) not only decreased tumor
   burden, but also inhibited the formation of ascites, which presents a
   considerable clinical application for the management of ovarian cancer.
   The mice treated with a low dose of CaALN (10 mg kg^−1) have longer
   lifetime with no obvious toxic effects observed. Generally, by using
   alendronate formed iron nanochelator, this study has demonstrated a
   distinct strategy to enhance the therapeutic potency and reduce the
   side effects of small molecule for efficient peritoneal carcinomatosis
   treatment.

4. Experimental Section

Materials

   CaCl[2], MnCl[4]•4H[2]O, FeCl[3]•6H[2]O, alendronate sodium trihydrate,
   deferasirox (DFX), ethanol, and NaOH were purchased from Sigma‐Aldrich.
   6‐FAM SE (Ex/Em = 495/519 nm) was bought from Fanbo Biochemicals Co.,
   Ltd. DiR Iodide (DiIC18(7), Ex/Em = 748/780 nm) was bought from Yeasen
   Biological Technology Co., Ltd. All chemical reagents were analytically
   pure and used without further purification. Cell Counting Kit‐8 (CCK8
   kit) and calcein acetoxymethyl ester (Calcein‐AM) were bought from MCE
   (MedChemExpress, Shanghai). Fluo‐3 AM ester was bought from US
   Everbright Inc. AO/EB assay kit and Mitochondrial Membrane Potential
   Detection Kit (JC‐1) were bought from LEAGENE. Antibodies against TFR1
   and p53 were purchased from Abclonal. Human ovarian cancer cell line
   SKOV3 and Human Gastric Carcinoma Cell Line MGC803, human pancreatic
   tumor cell PSN1, human osteosarcoma cell line MG63, human breast cancer
   cell SKBR3, and human bladder cancer cell MGH‐U3 were purchased from
   the American Type Culture Collection (ATCC). Female nude mice (5–6
   weeks) were bought from Guangdong Province Animal Center, China.

Synthesis of ICPs

   For preparing CaALN, 243 mg of alendronate sodium trihydrate was
   dissolved in 40 mL of deionized water. CaCl[2] solution (0.1 m, 10 mL)
   was added to the above alendronate sodium solution. Then, the pH value
   of the mixture was adjusted to 7.0 by 0.05 m NaOH and stirred for
   30 min at 4 °C in the water bath. CaALN nanoparticles were obtained by
   centrifugation and washing by ethanol. FeALN and MnALN were synthesized
   in a similar way. For in vitro imaging, 1 mg of CaALN and 0.1 mg of
   6‐FAM SE reacted in ethanol in dark for 12 h. After dialysis,
   6‐FAM‐CaALN nanoparticles were obtained. For in vivo real‐time imaging,
   CaALN/DiR was prepared by mixing CaALN and DiR for 12 h and dialysis
   for 24 h.

Characterizations

   The morphologies of as‐synthesized ICPs were examined with transmission
   electron microscope (TEM, JEM‐2100F) and element analysis was carried
   out with an energy‐dispersive X‐ray spectroscopy (XPS, Thermo
   Scientific K‐Alpha). X‐ray diffraction (XRD) was obtained by Rigaku
   Ultima IV X‐ray diffractometer. Inductively coupled plasma‐optical
   emission spectrometry (ICP‐OES, Agilent 700 Series, Agilent
   Technologies, USA) was used for the quantitative elemental analysis.
   FTIR spectra were conducted on a Nicolet 7000‐C spectrometer in the
   range of 400–4000 cm^−1. Brunauer–Emmett–Teller (BET) specific surface
   areas were measured with a surface area and porosity analyzer
   (Micromeritics ASAP 2460). The particle size and zeta potential were
   detected by Zetasizer Nano Series (Malvern, USA)

Calculations

   The binding energy of metal ions and alendronate was carried out with
   the Gaussian 16 software. The B3LYP functional and 6‐311G(d) basis set
   were adopted for all calculations.^[ [134]^29 ^] The IEFPCM implicit
   solvation model was applied to account for the solvation effect of
   water.^[ [135]^30 ^] The DFT‐D3 dispersion correction with BJ‐damping
   was applied to correct the weak interaction to improve the calculation
   accuracy.^[ [136]^31 ^] Then, the interaction energies between metal
   ion and alendronate were calculated by the following formula: ΔG = G
   [complex] − (G [metal] + G [alendronate]).

In Vitro Degradation

   10 mL of ICP was dispersed in 10 mL of Ca^2+ and Mg^2+ free PBS. After
   different incubation time at 37 °C, 100 µL of supernatant was collected
   by centrifugation and analyzed by ICP. The degradation rate was
   calculated according to the metal concentrations in the supernatant.

Chelation Exchange of CaALN in Fe^3+ Solutions

   20 mg of CaALN were dispersed in 20 mL of deionized water. Then, 20 mL
   of FeCl[3]•6H[2]O solution (1 × 10^−3 m) was added into CaALN and
   stirred for 24 h at 37 °C. After centrifugation and drying, the
   obtained yellow product was collected for XPS analysis.

In Vitro Cytotoxicity Assays

   SKOV3 and MGC803 cells were seeded in 96‐well plates and cultured at
   37 °C under 5% CO[2] for 24 h. Cells were incubated with treatment
   agents at series of concentrations. Fe^2+ supplemental experiment was
   conducted after 2 h treatment of CaALN. After 48 h of incubation, the
   culture media was washed with PBS and replaced with CCK8 solution, and
   the OD values were recorded at the wavelength of 450 nm. AO/EB
   staining, mitochondrial membrane potential, intracellular calcium ions,
   and ROS generation were carried out following the instructions. The
   mean fluorescence intensities of fluorescence microscope images were
   calculated by ImageJ software.

Measurement of the Cellular Labile Iron Pool (LIP)

   The measurement of LIPs was performed as a described calcein‐AM
   method.^[ [137]^32 ^] Briefly, SKOV3 and MGC803 were treated with DFX,
   NaALN, or CaALN at 50 mg L^−1 for 48 h. Then these cells were washed
   with PBS and incubated with calcein‐AM (0.5 × 10^−6 m) for 15 min at 37
   °C in the dark. Finally, cells were collected and analyzed by flow
   cytometry.

Cell Cycle Analysis

   SKOV3 and MGC803 cells were seeded in six‐well plates and treated with
   NaALN, CaALN, and DFX at concentration of 25 mg L^−1 for 24 h. After
   rinsing with PBS, cells were harvested and fixed in 75% ethanol for
   24 h at 4 °C. The fixed cells were centrifuged and washed twice with
   PBS, then incubated with propidium iodide (PI) solution containing
   RNase A at 37 °C for 30 min in the dark. After washing and
   centrifugation, cells were analyzed by flow cytometry.

Wound Healing Assay

   SKOV3 cells were seeded in six‐well plates at a density of 6 × 10^5
   cells/well. When the cells grew to 90% confluence, one wound per well
   was made using a tip and the floating cells were removed by PBS
   washing. Then, serum‐free media with different concentrations of NaALN
   or CaALN were added. After 2, 4, and 8 h of incubation, photographs
   were taken by microscope and the migration distance was measured.

Cellular Uptake of CaALN by SKOV3 Cells

   SKOV3 cells were seeded into confocal dishes and incubated with
   6‐FAM‐CaALN at concentration of 20 mg L^−1 for 1, 2, 4, and 24 h. Then,
   the cells were washed twice with PBS and stained by DAPI and DiR. The
   cells were observed by an Abberior STED Superresolution System
   (STEDYCON, Abberior Instruments, Germany).

Western Blotting (WB)

   Cells were incubated with NaALN, CaALN, and DFX for 48 h at
   concentration of 50 mg L^−1 and proteins were collected after lysis and
   quantified by BCA assay. The retrieved proteins were separated by using
   SDS‐PAGE and analyzed by immunoblotting. Protein bands were quantified
   by ImageJ software.

Quantitative Real‐Time PCR (qPCR)

   The cells of each well were harvested separately after coincubation
   with CaALN for 24 and 48 h. Total RNA of cells was extracted by the
   RNAex Pro Reagent Kit (AG21102, Accurate Biotechnology, China).
   First‐strand cDNA was synthesized using Evo M‐MLV RT Premix for the
   qPCR kit (AG11706, Accurate Biotechnology, China). qRT‐PCR was
   performed using SYBR Green Premix Pro Taq HS qPCR Kit (AG11701,
   Accurate Biotechnology, China) on CFX96 Real‐Time System (BIO‐RAD,
   Singapore). mRNA expression levels were analyzed by the 2^−ΔΔCt method
   and compared to the control. The specific primers were purchased from
   Sangon Biotech Co., Ltd. (Shanghai, China) and the primers sequences
   are listed in Figure [138]S18 (Supporting Information).

RNA Extraction and Sequencing

   SKOV3 cells were treated with NaALN (0.1 mg mL^−1) or CaALN (0.1 mg
   mL^−1) for 24 h. Three biological replicates were carried out for each
   group and total RNA was extracted by Trizol reagent kit (Invitrogen,
   USA) following the manufacturer's protocol.^[ [139]^33 ^] Agilent 2100
   Bioanalyzer (Agilent Technologies, USA) was used to assess total RNA
   quality.^[ [140]^34 ^] RNA library construction and sequencing were
   performed on the Illumina HiseqTM 2500/4000 by Gene Denovo
   Biotechnology Co., Ltd. (Guangzhou, China). The differential expression
   between given pairwise comparison groups was evaluate by DESeq2
   software.^[ [141]^35 ^] Bioinformatic analysis such as GO enrichment
   analysis, pathway enrichment analysis, and GESA were performed using
   Omicsmart, a real‐time interactive online platform for data analysis.

Ovarian Peritoneal Disseminated Tumor Model Establishment

   The ovarian peritoneal disseminated tumor model using SKOV3 cells was
   established as previously.^[ [142]^36 ^] Briefly, SKOV3 cells (2 ×
   10^6) were intraperitoneally injected as a cell suspension into
   NOD/SCID mice. Five days post inoculation, animals were imaged using
   the IVIS spectrum system to monitor the tumor progression.

In Vivo Imaging Study

   To study the biodistribution of CaALN nanoparticles in mice,
   tumor‐bearing mice (n = 3) were intraperitoneally injected with
   CaALN/DiR and free DiR at an equivalent DiR dose of 0.5 mg kg^−1. The
   IVIS imaging system was used to acquire real‐time bioluminescence and
   fluorescence images at 0, 1, 4, 24, and 72 h. Finally, the mice were
   sacrificed, and major organs and tumor tissue were collected for
   imaging.

In Vivo Therapeutic Effect of CaALN

   SKOV3 tumor‐bearing mice were randomly divided into three groups (n =
   8), as follows: PBS, low dose CaALN (10 mg kg^−1), and high dose CaALN
   (25 mg kg^−1). Mice were intraperitoneally injected with drugs for
   seven weeks. All the mice were imaged using the IVIS Spectrum to
   monitor the extent of tumor progression. On day 31, three mice of each
   group were sacrificed. The weight of ascites and tumor weight of each
   mouse were measured, and the main organs and tumor tissues were
   extracted for H&E and TUNEL staining. Other mice were treated for seven
   cycles and fed until death. Animal procedures were reviewed and
   authorized by the Animal Ethics Committee of Sun Yat‐sen University
   (SYSU‐IACUC‐2021‐B0815).

Statistical Analysis

   Statistical analysis was performed using SPSS software. Data were
   presented as the mean ± SD (n = 3). The differences between two
   independent groups were analyzed using T‐test. Statistical significance
   was indicated by P‐values as follows. ***P < 0.001, **P <0 .01, *P <
   0.05.

Conflict of Interest

   The authors declare no conflict of interest.

Author Contributions

   The manuscript was written through contributions of all authors. All
   authors have given approval to the final version of the manuscript.
   J.Z., X.H., and P.L. contributed equally to this work. J.Z., M.Y., and
   X.H. performed investigation, formal analysis, data curation, software,
   and writing original draft; P.L. carried out formal analysis, data
   curation, resources, and validation; B.L., S.L., and M.W. performed
   investigation, writing – review and editing; M.Q. and Q.W. provided
   resources and software; Y.C. and Y.P. performed conceptualization,
   methodology, funding acquisition, project administration, and
   supervised the study.

Supporting information

   Supporting Information
   [143]Click here for additional data file.^ (846.8KB, pdf)

Acknowledgements