Recent trends in the design and delivery strategies of ruthenium complexes for breast cancer therapy

Utpal Das ^a, Uttara Basu ^b and Priyankar Paira *^a
aDepartment of Chemistry, School of Advanced Sciences, Vellore Institute of Technology, Vellore-632014, Tamilnadu, India. E-mail: priyankar.paira@vit.ac.in
^bDepartment of Chemistry, Birla Institute of Technology & Science (BITS) Pilani, K K Birla Goa Campus, NH 17B Bypass Road, Goa - 403726, India

First published on 14th August 2024

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Utpal Das

Utpal Das received his Bachelor of Science degree from West Bengal State University, India, in 2017 and his Master of Science degree from West Bengal State University, India, in 2019 with a specialization in Organic Chemistry. He is pursuing his PhD (from 2020) at the Vellore Institute of Technology, Vellore, under the supervision of Dr Priyankar Paira. His research interests include the synthesis of Ru(II), Ir(III), and Re(I) metal complexes for cancer therapy.

Uttara Basu

Dr Uttara Basu obtained her Bachelor of Science with Chemistry Honors from Presidency College, the University of Calcutta, in 2008. She obtained her MS and PhD from the Indian Institute of Science in 2015 under the supervision of Prof. Akhil R. Chakravarty, where she designed and synthesized iron-based anticancer agents. She then joined the group of Prof. Shanta Dhar (FRSC) at the Miller School of Medicine, the University of Miami, USA, for her postdoctoral research from 2016 to 2017. Later, she worked as a postdoctoral fellow in the group of Prof. Gilles Gasser (FRSC), Chimie ParisTech, France (2017–2018), and with Prof. Ingo Ott, at the Technical University of Braunschweig, Germany (2019–2022). She joined the Department of Chemistry at BITS Pilani K K Birla Goa Campus in 2022, as an Assistant Professor. Her research interests include the development of metal complexes and nanomaterials for various therapeutic and diagnostic applications.

Priyankar Paira

A/P Priyankar Paira obtained his BSc from the University of Calcutta and MSc from the University of Delhi. He pursued his PhD degree at the Indian Institute of Chemical Biology. His postdoctoral training was at the National University of Singapore in the Department of Pharmacy under Prof. Giorgia Pastorin and the Department of Chemistry under Prof. Ang Wee Han. His postdoctoral training was on the development of target-specific organoruthenium complexes for cancer therapy and the application of bionanomaterials in drug delivery. He joined the Department of Chemistry at the Vellore Institute of Technology in 2013 as Assistant Professor, and he is currently an Associate Professor. His research interests lie in the area of transition metal complexes in cancer therapy and diagnosis. Currently, he is involved in many independent and collaborative projects including A3 receptor-targeting phototoxic transition metal complexes for cancer diagnosis and therapy, heterodimetallic biotin conjugates and their encapsulated MWCNT for target delivery, and organelle-specific cyclometaleted complexes for phototheranostic application. He has over 100 scientific publications in internationally acclaimed journals including Inorganic Chemistry, Dalton Transactions, Journal of Inorganic Biochemistry, etc. Research conducted in his laboratory has been featured as editor's choice and cover page articles. Under his mentorship, several UG students completed their final year thesis project, and four scholars received their PhD degrees. Dr Paira has secured 3 patents in Medicinal Chemistry and has completed the DST-SERB-YS (YSS/2014/000842) project, the DST-SERB project (EMR/2017/000816), and two ICMR projects. At present, he is working on another DST-SERB-CRG project (CRG/2021/002267). In particular, he has always been fascinated by research involving a combination of Engineering, Medicine, and Science, as demonstrated by his publications and collaborations with several people from different Institutes having diverse research expertise. He is associated as a reviewer with several RSC, ACS, Wiley, and Elsevier journals. Currently, he is serving as Executive Guest Editor in Current Organic Chemistry. He is also an Editorial Board Member of Current Electrocatalysis, Bentham Science, and Frontiers in Chemistry journals. He is associated with professional membership in the RSC, ACS, and SBIC.

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1. Introduction

In affluent countries, the prevention, diagnosis, and treatment of neoplastic diseases present significant challenges. Breast cancer ranks as the most common cancer among females, with 7.8 million cases reported in the last five years. Particularly in less developed countries, it has emerged as the most prevalent cancer type and the second leading cause of cancer-related deaths worldwide. According to the World Health Organization (WHO), around 2.3 million females were diagnosed with breast cancer in 2020, globally, which resulted in nearly 685000 deaths. The lifelong risk of invasive breast cancer, estimated at 12.6% for American females, means that approximately 1 in 8 females will face a diagnosis of breast cancer at some point in their lives.^1–3 Although less than 1% of breast cancer cases are male, male breast cancer also deserves attention and requires appropriate treatment. Furthermore, there is a notable worldwide increase in cases of breast cancer in young women between the ages of 35 and 40, with the tumors developing more aggressively than in older women. Evidence from recent research indicates that young women's breast cancer subtypes are more aggressive and invasive, and it is unquestionably the leading cause of cancer-related mortality among women under the age of 45.^4,5

Breast tissue, specifically the innermost layer of the ducts or the lobules that supply milk to the milk ducts, is where breast cancer usually begins to grow. The unrestrained growth and division of cells that start in tissues of the breast is referred to as “breast cancer” (Fig. 1).⁶ The two main types of breast tissues are the stromal (supporting) tissues and glandular tissues. The glandular tissues contain the lobules and ducts that create milk, whereas the stromal tissues are composed of the fatty and fibrous connective tissues of the breast. There are various places in the breast where breast cancer may begin. The glands called lobules produce breast milk and “lobular cancer” originates here. Ducts are tiny channels that come out of the lobules and carry milk to the nipple; this is where ductal cancers develop. One less common type of breast cancer that can start in the nipple is called Paget's disease. Fatty tissue and connective tissue envelop the ducts and lobules, helping to keep them in place. The stroma may be the starting point for phyllodes tumor, a less common type of breast cancer. Additionally, the individual breast contains lymphatic and blood arteries.⁷ An uncommon kind of breast cancer called angiosarcoma can start in the lining of these veins.⁸

Although the mortality rate for breast cancer decreased by 40% from 1989 to 2016, some forms are still incurable.^9,10 Based on the available data, it appears that multiple subtypes of breast cancer exhibit varying rates of growth as well as dissemination and therapy responses. Specifically, at least four primary molecular subtypes of invasive breast cancer have been identified as a result of genome and transcriptome deep sequencing (luminal A and luminal B hormone receptor-positive, HER-2-positive, and basal-like). Eighty percent of luminal breast cancers are ER+ (estrogen receptor positive), and sixty-five percent of them are PR+ (progesterone receptor positive). The best prognosis is typically associated with Luminal A tumors that are ER/PR-positive and HER-2 negative because they grow slowly in response to hormone stimulation and may therefore respond to hormone therapy. Compared with luminal A tumors, luminal B cancers grow more quickly and have a marginally worse prognosis. They exhibit high levels of Ki-67 and are hormone-receptor positive (estrogen and/or progesterone receptor positive), as well as either HER-2 positive or HER-2 negative. Tumors that overexpress (HER-2+) respond to targeted therapy, such as trastuzumab (Herceptin) which targets the HER-2 protein, and are typically more aggressive than conventional malignancies.^11–19 Finally, because basal-type tumors are devoid of the estrogen receptor (ER), progesterone receptor (PR), and HER-2, they are often referred to as triple-negative breast cancer (TNBC). Despite being present in just 15% of cases of breast cancer, research has demonstrated that the basal subtype is the most aggressive phenotype, resistant to therapy, and typically associated with a poor prognosis. Breast cancer patients with ER+ or PR+ status may benefit from endocrine therapy; nevertheless, other therapeutic options, such as chemotherapy, are often explored despite the unfavorable side effects and elevated risk of recurrence after treatment. It should be highlighted that patients with ER+ breast cancer have a much higher survival rate when receiving both chemotherapy and endocrine therapy.^20–24

2. Types of breast cancer

2.1. Non-invasive breast cancer

Cells that fail to spread to the connective and fatty tissues of the breast, but stay in the ducts, are known as non-invasive breast cancer cells. The most frequent form of non-invasive breast cancer is called ductal carcinoma in situ (DCIS).

2.2. Invasive breast cancer

Invasive breast cancer cells penetrate the duct and lobular wall to target the connective and fatty tissues around the breast.

2.3. Lobular carcinoma in situ (LCIS)

“In situ” refers to cancer that is still contained within its original site. In LCIS, there is a marked increase in the cell number in the breast's lobules, or milk glands.

2.4. Infiltrating lobular carcinoma (ILC)

ILC typically begins in the breast milk glands, or lobules, though it typically spreads to other areas of the body.

2.5. Ductal carcinoma in situ (DCIS)

DCIS, the most common type of non-invasive breast cancer, is only found in the breast ducts.

2.6. Infiltrating ductal carcinoma (IDC)

IDC begins in the breast's milk ducts, penetrates the duct wall, and spreads to the breast's fatty tissue and possibly other body areas.

2.7. Mucinous carcinoma

An uncommon kind of breast cancer called mucinous carcinoma is created by cancer cells that produce mucus.

2.8. Medullary carcinoma

A distinct separation is made between the tumor and healthy tissue in an invasive breast cancer known as medullary carcinoma.

2.9. Tubular carcinoma

This type of breast cancer is invasive. Women with this carcinoma usually have a better prognosis than those with more common invasive carcinoma types.

2.10. How does breast cancer spread?

When cancer cells are transported to different body parts by the blood or lymphatic system, breast cancer can spread. The lymphatic system is the immune system of the body. Lymph fluid is transported away from the breast by lymph vessels. Breast cancer cells can penetrate these lymphatic channels and proliferate in lymph nodes. There are several different types of lymph nodes (Fig. 2), including internal mammary lymph nodes, which are located in the chest, close to the breastbone, axillary lymph nodes located under the arm, supraclavicular lymph nodes, located both above and below the collar bone, and infraclavicular lymph nodes which are located around the collar bone. If cancer cells have already entered the lymph nodes through the lymphatic system, the chances are higher that they have migrated to other parts of the body.⁸

Metal ions have a wide range of coordination numbers and geometric shapes, achievable redox states, thermodynamic and kinetic characteristics, and coordinating ligand natures, and inorganic compounds can benefit from these unique features to generate novel anticancer therapeutics.^25,26 A widely recognized chemotherapeutic drug, cisplatin, has been one of the most frequently administered drugs for the treatment of breast cancer. It is undeniable that cisplatin and its derivatives, which do not specifically target malignant sites, usually cause significant adverse consequences despite helping treat breast cancer.^27–30 Since many ruthenium-based compounds were claimed to have fewer negative consequences because of their distinct mechanisms of action, most research endeavors have been directed at developing ruthenium-based compounds as a replacement for platinum-based chemotherapeutic therapies.^31–35 Several times, it was discovered that ruthenium compounds exhibited strong cytotoxicity against cancer cell lines resistant to platinum, which indicated their potential for additional study.^36,37 The first one to enter a clinical trial was the complex with a Ru(III) centre called NAMI-A. Under physiological environments, this compound was able to attach to some bio-molecules like DNA and RNA, as well as serum albumin. The next-generation anticancer complex, KP1019, was created by the Keppler group and completed Phase-I clinical trials. The sodium salt of KP1019, called KP1339, was eventually developed and underwent clinical testing because of better water solubility.^38–40

There has been a lot of focus in the past couple of decades towards the discovery of anticancer therapeutics by the coordination of biologically active compounds to metals, including ruthenium. Recently, Sadique et al. have summarized the findings on ruthenium complexes for breast cancer therapy.^24b In another article, Golbaghi and Castonguay have highlighted the design strategies adopted to improve the anticancer properties of ruthenium complexes specifically for breast cancer therapy.^24c Considering that this is an extremely relevant topic in the current scenario, in this article, we have tried to provide an overview of the latest ruthenium-based compounds that have been reported and have the potential to be developed for the treatment of breast cancer, highlighting the design aspects. In addition, we have also focused on the drug delivery strategies for targeted drug delivery using nanoformulation for greater therapeutic efficacy and reduced side effects, which holds the promise of personalized medicine in the future.

3. Ruthenium complexes used for triple-negative breast cancer (TNBC)

Since TNBC does not respond to hormonal therapy, standard anticancer drugs for HR+ breast cancers, such as P450 enzyme suppressants or estrogen receptor-directed molecules, do not function as ligands in the structure of ruthenium complexes. This section discusses several ruthenium compounds with outstanding anticancer properties in TNBC.

Based on the inhibitor of PARP-1, Zhu et al. reported a ruthenium(II) anticancer complex (1). In Hcc1937 TNBC cells, the complex exhibited 1.5 times more cytotoxicity with an IC₅₀ value of 93.3 ± 11.4 μM compared with an IC₅₀ value of 143.0 ± 6.3 μM in non-cancerous MRC-5 cells. The authors concluded that the complex of Ru–PARP inhibitor showed marginally greater PARP inhibitory characteristics than the corresponding free inhibitor.⁴¹ Another arene-based ruthenium complex of dichloroacetate (DCA) (2) with significant cytotoxicity and antimetastatic effects in MDA-MB-231 cells was described by Brabec et al. When MDA-MB-231 cells were treated with the compound, the IC₅₀ value was found to be 0.86 ± 0.01 μM in comparison with immortalized human embryonic kidney cells, HEK-293 (IC₅₀ 9.4 ± 0.5 μM). The authors also noted that complex 2 may have antimetastatic effects by reducing the re-adhesion, migration, and invasion of TNBC cells. Additionally, whereas cisplatin had little to no effect on the glycolysis of MDA-MB-231 cells, complex 2 could modestly reduce it.⁴² A ruthenium complex of lapachol (3) was reported and investigated by Batista et al. for its cytotoxic properties against MDA-MB-231 cell lines. Lapachol is 1,4-naphthoquinone, which occurs naturally and has cytotoxic and antimetastatic properties. After complex formation, lapachol exhibited anticancer properties with an IC₅₀ value 0.20 ± 0.01 μM against the TNBC cell line.⁴³ A ruthenium(II) complex (4) was discovered by Amici et al. and showed in vivo anticancer activity. The authors observed that 4 significantly inhibited the development of the TNBC cells A17 (Fig. 3) implanted into FVB syngeneic mice when given in a 4-dose course, reiterating a single dosage every 3 days (52.4 mg kg⁻¹). Due to its higher solubility in aqueous medium, 4 was quickly removed from the kidneys, liver, and circulation, according to the pharmacokinetic studies, and exhibited great therapeutic impact with few adverse effects. From the immunohistological investigations, the authors revealed that the efficacy of complex 4 mainly stemmed from its capacity to counteract the immune suppression linked with tumors by significantly reducing the quantity of regulatory T cells that infiltrate tumors.⁴⁴ Chen et al. synthesized ruthenium polypyridyl complex RuPOP (5) and investigated its antitumor activity in MDA-MB-231 cell lines. It successfully prevented the growth and metastasis of MDA-MB-231 cells. The complex exhibited a highly cytotoxic effect against human breast cancer cell lines (Fig. 3) when they were exposed to high concentrations of 5. Remarkably, 5 was found to be an effective antimetastatic drug and metal-based chemosensitizer of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) toward MDA-MB-231 cells, as demonstrated in Fig. 3. 5 was able to synergistically augment TRAIL-induced apoptosis and successfully reduce the metastatic potential of the MDA-MB-231 cells even at minimal concentrations (1–2 μM). Further research into the molecular mechanisms showed that 5 might amplify TRAIL-induced apoptosis in MDA-MB-231 cells by inhibiting FAK-mediated ERK and Akt activation, blocking the secretion of vascular endothelial growth factor (VEGF), and altering the expression levels of metastatic regulatory proteins (Fig. 4).⁴⁵

An iminophosphorane ligand containing water-soluble ruthenium(II) complex (6) was synthesized by Contel et al., compared with cisplatin, 6 was more cytotoxic against human cancer cell lines. The authors investigated in vivo experiments on MDA-MB-231 xenografts in NOD.CB17-Prkdc SCID/J mice (Fig. 5A) and after twenty-eight days of the treatment (14 doses of 5 mg kg⁻¹ every other day), they noticed a remarkable tumor shrinkage of fifty-six percent with no adjacent effects. It is noteworthy that in contrast to kidney and liver tissues, complex 6 accumulated mostly in the tissues of breast tumors, which may help to explain its great in vivo efficacy.⁴⁶ Mei et al. synthesized the polypyridine Ru–DPPZ complex Ru(bpy)₂BEDPPZ (7), and it demonstrated admirable inhibitory efficacy against the triple-negative MDA-MB-231 breast cancer cells. Complex 7 exhibited an IC₅₀ value of 17.2 ± 0.9 μM against MDA-MB-231, which was superior to cisplatin (20.9 ± 1.9 μM). The authors performed a wound-healing assay to determine the anti-metastatic activity of the complex and to gauge cell migration and repair capacity. In the untreated cells, there was significant wound closure in 72 h, indicated in Fig. 5B. However, when cells were treated with 7, the wound closure was repressed. The authors also observed that the wound-healing rate was <½ of the control in the presence of 2 μM of 7. The rate of wound healing further diminished when the dose of 7 was increased to 4 μM, suggesting that 7 prevented migration in a dose-dependent fashion. Again FITC-gelatin assay was used to investigate how 7 inhibited the invasion of MDA-MB-231 cells. As shown in Fig. 5D, it was observed that the invasive potential of MDA-MB-231 cells was significantly lowered by 5 μM treatments of 7, and the amount of black holes in FITC–gelatin was suppressed. The authors deduced from these data that this complex efficiently blocked the MDA-MB-231 cells’ invasion and migration.⁴⁷ Gallic acid-containing ruthenium complex, Ru(GA) (8), was developed by Cominetti et al. and cytotoxicity was tested against TNBC. Boyden chamber assay and wound healing were used to assess the effects of 8 on migration and invasion, respectively. It reduced the cell migration and invasion ability and triggered apoptosis. The authors performed the MTT assay and found the IC₅₀ values 4.50 ± 0.29 μM, 1.62 ± 0.20 μM, and 12.04 ± 2.33 μM for (TN basal A) MDA-MB-231, (TN basal B) MDA-MB-468, and MCF-10A cells, respectively, with selectivity indexes (SI) 2.68 for MDA-MB-231 and 7.43 for MDA-MB-468, respectively. When the authors compared it with untreated cells, the complex significantly reduced wound closure to around ten percent at 0.195 μM, twenty percent at 0.78 μM, and fifty percent at 1.56 μM and 3.12 μM after 24 hours of treatment. It was demonstrated by the invasion assay results that the therapy dramatically reduced TNBC cell invasion at 1.56 and 3.12 μM. To evaluate the apoptosis-inducing capacity of the complex against MDA-MB-231 cells, the authors performed DAPI and PE–annexin-V staining. It was clear that in a concentration-dependent way, apoptosis was induced in tumor cells. It was also evident from the DAPI staining that after 8 hours’ treatment, greater chromatin condensation, damage to nuclei, and the presence of apoptotic bodies were found than in untreated MDA-MB-231 cells.⁴⁸ According to reports, ruthenium-based complexes may be effective anticancer agents for TNBC and BRCA1-mutant breast cancer. Additionally, BRCA1 controls chemotherapy-induced DNA damage.^49,50 In this aspect Ratanaphan et al. synthesized two ruthenium complexes (9, 10) which exhibited an anticancer effect on the BRCA1-defective (HCC1937) TNBC cell line. The authors observed that 10 exhibited more cytotoxicity against BRCA1-defective HCC1937 cells with an IC₅₀ value of 1.8 ± 0.1 μM compared with the cell lines MDA-MB-231 with an IC₅₀ value of 13.2 ± 0.3 and MCF-7 with 8.2 ± 0.1 μM. The interesting part was after 12–48 hours of incubation, complex 10 entered and was retained to the greatest extent possible in the nuclear fraction of HCC1937 cells, whereas complex 9 accumulated in the nuclear fraction at 48 hours of the incubation. This result indicated that HCC1937 cells predominantly internalize complex 10 compared with complex 9. The DNA was damaged as a result of the high ruthenium concentration in the nuclear component, which led to apoptosis. In breast cancer cells treated with 10, the authors also observed an excessive regulation of p53 mRNA and a deregulation of BRCA1 mRNA.⁵¹ A new sequence of ruthenium(II) complexes was synthesized by Doriguetto et al. Complex 11 exhibited a highly cytotoxic nature with an IC₅₀ value of 9.18 ± 0.30 μM. The compound exhibited anti-clonogenic properties on MDA-MB-231 because it caused morphological alterations and reduced the size and quantity of colonies.⁵² A sequence of biphosphine bipyridine complexes of ruthenium(II) for anticancer treatment against TNBC cells was developed by Cominetti et al. Complex 12 demonstrated the greatest anticancer effects. The complex exhibited IC₅₀ values of 31.16 ± 0.04 for MDA-MB-231, >200 for MCF7, and 48.89 ± 0.09 μM for MCF-10A cell lines. That means the complex exhibited its potency on the MDA-MB-231 cell line rather than the MCF7 cell line. The authors investigated tumor cell migration on MDA-MB-231 cell lines with the help of wound healing and Boyden chamber migration assays. Complex 12 inhibited MDA-MB-231 cell migration, as shown by Boyden Chamber and wound-healing experiments, in a concentration-dependent manner. Complex 12 was capable of preventing the invasion of MDA-MB-231 cells by about 80% when compared with control at a dose of 20 μM. At doses of 2.5, 5, and 10 μM, 12 did not prevent MMP-9 from being expressed in MDA-MB-231 cells. However, when compared with untreated control, greater concentrations (20 μM) significantly reduced the expression of MMP-9 by 60%.⁵³

The RAPTA complexes were introduced by the Dyson group, and had a significant impact on the subject. The group described RAPTA-C (13), RAPTA-T (14), and RAPTA-B (15), and looked into how breast cancer metastasized. The authors also examined the resistance of three breast cell lines (MDA-MB-231, MCF-7, and HBL-100) with varying degrees of malignancy to detachment, re-adhesion, migration, invasion, and MMP activity. They discovered that RAPTA-T selectively inhibits certain stages of the metastatic process, including detachment, migration, invasion, and re-adhesion, particularly in the highly invasive MDA-MB-231 cells.⁵⁴ Following that, the T. Nhukeaw group also investigated the significance of the RAPTA-T complex. They looked at how RAPTA-T, which has encouraging antimetastatic qualities, affected BRCA1-defective HCC1937 breast cancer cells and contrasted them with MCF-7 breast cancer cells that are BRCA1-competent. A variety of techniques, including the MTT assay, flow cytometry, QPCR, and western blot, were employed by the authors to assess the BRCA1 expression, cellular accumulation, apoptosis, cell cycle, and cytotoxicity of RAPTA-treated cells. According to their findings, RAPTA-T causes both cell lines to undergo apoptosis and G2/M cell cycle arrest while having very little cytotoxicity. Furthermore, compared with MCF-7 cells, RAPTA-T damages and downregulates BRCA1 in HCC1937 cells more severely.⁵⁵ Recently K. Hongthong et al. investigated the anticancer activity of RAPTA-EA1 (16) on triple-negative BRCA1 wild-type breast cancer MDA-MB-231 cells, and compared it with sporadic BRCA1 wild-type MCF-7 cells. They also explored the combination treatment of RAPTA-EA1 with olaparib, a PARP inhibitor. They reported that RAPTA-EA1 is more effective than cisplatin (IC₅₀ 10.5 ± 0.5 in MDA-MB-231 and 20.0 ± 2.2 in MCF-7) in inhibiting the growth of both cell lines, and accumulates mainly in the nuclear fraction of MDA-MB-231 cells. RAPTA-EA1 induced G2/M arrest, nuclear condensation, and cell death in MDA-MB-231 cells. RAPTA-EA1 also damaged and inhibited the replication and expression of BRCA1 in MDA-MB-231 cells. The combination of RAPTA-EA1 and olaparib shows a synergistic effect in MCF-7 cells and an additive effect in MDA-MB-231 cells, and reduced the expression of BRCA1 protein in both cells.⁵⁶ Very recently S. A. P. Pereira et al. explored the activity of a series of ruthenium(II) arene compounds, similar to RAPTA compounds, against breast cancer cell lines. They demonstrated that the cytotoxicity and selectivity of the compounds can be fine-tuned by changing the phosphine ligands and the counter ions. Among all the complexes, complex 17 (Fig. 6) exhibited remarkable stability, high cytotoxicity, and selectivity to breast cancer cells over non-tumorigenic cells. The authors also suggested that the minor groove of DNA is a relevant target for this complex.⁵⁷ Dyson's group reported novel derivatives of complex 18, ruthenium(II)–arene with a ligand modified with perfluoroalkyl, that demonstrated noteworthy selectivity against cancer cells. Without influencing noncancerous cells (HEK-293) or endothelial cells (ECRF24 and HUVEC), the novel combination suppressed the migration of breast cancer cells (MDA-MB-231). The novel complex demonstrated more potent antivascular properties in vivo when tested against the well-established antiangiogenic ruthenium(II)–arene complex RAPTA-C, utilizing the chicken chorioallantoic membrane (CAM) model. The authors also concluded that structural alterations could improve the efficacy of the complex and adaptability even more, and that the perfluoroalkyl chain is necessary for its specific anticancer action.⁵⁸

The use of poly(ADP-ribose) polymerase (PARP) inhibitors in conjunction with ruthenium(II) polypyridyl complex (RPC) metallo-intercalator [Ru(dppz)₂(PIP)]²⁺ (complex-19) has been investigated by N. A. Yusoh et al. in a recent study to treat triple-negative breast cancer (TNBC) in BRCA wild-type. The researchers used two BRCA wild-type breast cancer cell lines, MDA-MB-231 (TNBC) and MCF-7, and compared the results with normal human fibroblasts to examine the anti-proliferative, clonogenic, cell cycle, apoptotic, DNA damage, and cell migration effects of the RPC-PARP inhibitor combination. Additionally, the authors suggested that, with little impact on normal fibroblasts, the combination of RPC and PARP inhibitors synergistically inhibited cell survival and proliferation, induced G2/M arrest and apoptosis, enhanced DNA double-strand break (DSB) damage, abrogated pChk1 signaling, and impeded cell migration in breast cancer cells. They also concluded that the RPC renders the breast cancer cells hypersensitive to the PARP inhibitor olaparib, resulting in a 300-fold increase in olaparib potency in TNBC cells.⁵⁹

Gurgul et al. explored the impact of three polypyridyl Ru(II) complexes, designated as complexes 20–22, on the metastatic behavior of cancer cells, including their adhesion, migration, invasion, and transmigration capabilities. The researchers observed that these complexes demonstrated considerable cytotoxic effects on cancer cells while showing reduced toxicity toward non-tumor cells. Additionally, the complexes were found to enhance the adhesion strength of cancer cells, hinder their detachment, migration, invasion, and transmigration processes, and enhance their elasticity. Furthermore, the study suggested that these complexes influenced the activity and expression levels of integrins and focal adhesion components, such as vinculin and paxillin, resulting in an increased number of focal adhesion contacts.⁶⁰ Using biotin as a targeting agent for cancer cells, L. Côrte-Real et al. synthesized a series of novel ruthenium(II)–cyclopentadienyl complexes 23–26. The compounds showed good cytotoxicity against MCF7 and MDA-MB-231 breast cancer cell lines, with lower IC₅₀ values than cisplatin, and the authors showed that the compounds were stable in physiological conditions. Additionally, they demonstrated that the biotinylated compounds exhibited greater cellular absorption in comparison with their non-biotinylated counterparts, indicating a potential internalization process controlled by receptors. In zebrafish embryos, the substances caused apoptosis and prevented cancer cells from forming colonies. The authors concluded that the new ruthenium(II)–cyclopentadienyl compounds with biotin are promising candidates for anticancer therapy, as they combine the advantages of organometallic complexes with the specificity of biotin targeting. They also suggested that further studies are needed to elucidate the mechanism of action and the biodistribution of the compounds in vivo.⁶¹

The group of A. A. Batista synthesized six novel ruthenium complexes (27–32) with distinct acyl thiourea and diphenylphosphine ligands. By employing the MTT assay to assess the cytotoxic potential of the complexes on breast and non-tumor cell lines, the authors discovered that complex 32 had the lowest IC₅₀ value (0.112 ± 0.018) when it came to killing MDA-MB-231 cell lines. The findings demonstrated the complexes’ strong anti-proliferative properties, particularly against the MDA-MB-231 breast cancer cell line, while posing little risk to healthy cells. Additionally, they used flow cytometry, wound-healing assay, and microscopy to examine the effects of the complexes on the MDA-MB-231 cell line's cell morphology, migration, and apoptosis. The results indicated that the complexes caused alterations in the cell shape and size, inhibited cell migration, induced cell cycle arrest in the Sub-G1 phase, and triggered cell death by apoptosis.⁶² The Contel group has made significant advancements in the development of compounds targeting triple-negative breast cancer. Their previous work on complex 6, as discussed in their earlier paper, demonstrated its potency against TNBC. Building upon this, they have recently developed two luminescent analogs, complexes 33 and 34, based on a ruthenium(II) compound (complex 6), which exhibited high anticancer activity against TNBC cells and tumors. Importantly, they found that these new compounds were stable and luminescent in aqueous media. Furthermore, the authors demonstrated that both complexes 33 and 34 exhibited similar cytotoxicity to complex 6 in TNBC cells. However, they observed lower toxicity of these compounds in nonmalignant cells, indicating a potentially favorable therapeutic profile. A key finding of their study was the differential subcellular localization of complex 34 compared with complex 6 in TNBC cells. 34 was shown to accumulate in the endoplasmic reticulum, mitochondria, and lysosomes, whereas 6 primarily accumulated in the mitochondria and cytoplasm. This differential localization suggested distinct mechanisms of action for these compounds. Moreover, 34 was found to induce the generation of reactive oxygen species and apoptosis specifically in TNBC cells, further highlighting its potential as a targeted therapeutic agent for this aggressive cancer subtype. Overall, the development of luminescent analogs of complex 6 by the Contel group represents a promising advancement in the field of TNBC treatment, offering compounds with potent anticancer activity and improved selectivity for malignant cells.⁶³ Q. Wu et al. have synthesised a series of arene Ru(II) complexes, 35–39. These complexes were designed to target and stabilize c-myc G-quadruplex DNA, which is recognized as a potential target for anticancer agents due to its involvement in cancer progression. In their study, the authors investigated the inhibitory activity of these complexes against the proliferation, migration, and invasion of MDA-MB-231 breast cancer cells. Additionally, they assessed the in vivo toxicity of these complexes using zebrafish embryos. The arene Ru(II) complexes possessed a typical “piano stool” structure and exhibited the ability to bind to the groove of c-myc G-quadruplex DNA. The binding affinities of these complexes to the DNA structure varied depending on the substituent in the main ligand. Among the complexes tested, complex 37 demonstrated particularly promising results. It effectively inhibited the growth, migration, and invasion of MDA-MB-231 cells. The mechanism of action involved inducing S-phase arrest and apoptosis in the cancer cells. Furthermore, the complexes exhibited low toxicity in vivo, as evidenced by their minimal adverse effects on the development of zebrafish embryos. Overall, the research conducted by Q. Wu et al. highlights the potential of arene Ru(II) complexes, especially 37, as anticancer agents targeting c-myc G-quadruplex DNA. These findings contribute to the development of novel strategies for breast cancer treatment with reduced toxicity to normal cells.⁶⁴ The study conducted by A. A. Batista et al. focused on synthesizing and characterizing three new ruthenium complexes (40–42) with alizarin, a natural dye derived from plants, as potential anticancer agents. Alizarin is known for its various biological activities, including antioxidant, anti-inflammatory, and antimicrobial effects. The researchers evaluated the anticancer activity of these complexes both in vitro and in vivo, using breast cancer cell lines and zebrafish embryos as models. Among the complexes tested, complex 41, which contains a phosphine ligand, exhibited the most promising results for drug development. It displayed potent and selective cytotoxicity against triple-negative breast cancer cells, which are typically resistant to many conventional therapies. The IC₅₀ values were determined to be 42.2 ± 3.6 μM for complex 40, 6.5 ± 0.1 μM for 41, and 45.4 ± 1.4 μM for complex 42. Furthermore, 41 demonstrated additional beneficial effects, including inducing cell cycle arrest, inhibiting colony formation, and impairing cell migration, all of which are crucial in preventing cancer metastasis. Importantly, complex 41 also exhibited low toxicity in zebrafish embryos, indicating a favorable safety profile. The researchers suggested that the anticancer activity of these complexes might be attributed to their interaction with DNA and their ability to generate reactive oxygen species, which can cause damage to cancer cells. Additionally, the phosphine ligand in complex 41 likely played a role in enhancing the stability and selectivity of the complexes. In conclusion, the study proposed that these new ruthenium complexes with alizarin hold promise as potential anticancer agents, particularly for the treatment of triple-negative breast cancer. Further investigations are warranted to fully understand their mechanisms of action and to advance their development as therapeutic agents.⁶⁵

The study conducted by D. P. Dorairaj et al. involved the synthesis and characterization of twelve Ru(II)–arene complexes with furoylthiourea ligands bearing various substituents (43–54). The interaction of these complexes with calf thymus DNA and bovine serum albumin (BSA) was investigated using absorption, emission, and viscosity measurements. The findings suggested that the complexes bind to DNA via intercalation and to BSA via static quenching. Notably, the binding affinities varied depending on the presence of PPh₃ and the nature of substituents on the ligands. Furthermore, the cytotoxicity of these complexes was assessed against three breast cancer cell lines: MCF-7, T47-D, and MDA-MB-2314. The complexes exhibited higher activity compared with their ligands and displayed selectivity for cancer cells over normal cells. Particularly, complexes containing PPh₃ and electron-donating substituents exhibited the highest cytotoxicity. Among the complexes tested, complexes 48 and 54 demonstrated the most significant activity, with remarkable IC₅₀ values against the MDA-MB-231 cancer cell line (8.5 and 0.1 μM, respectively). Moreover, 48 and 54 induced apoptosis in the MDA-MB-231 cancer cell line, as evidenced by AO/EB staining, DAPI staining, western blot, and flow cytometry analyses. Finally, in vivo experiments revealed that these two complexes did not cause noticeable damage to the organs of mice, suggesting their potential as candidates for further investigation as anticancer agents.⁶⁶ K. M. Oliveira et al. synthesized six new ruthenium(II) complexes (55(a, b), 56(a, b), 57(a, b)) with lapachol and lawsone, two natural naphthoquinones with anticancer activity. They aimed to explore the potential of these complexes as chemotherapeutic agents, especially for triple-negative breast cancer (TNBC). They evaluated the cytotoxicity of the complexes against four cancer cell lines (MCF-7, MDA-MB-231, DU-145, and PC-3) and one normal cell line (MCF-10A), and compared them with cisplatin, lapachol and lawsone. They found that one of the complexes, (55a) exhibited significant selectivity for TNBC (IC₅₀ = 0.15 ± 0.01 μM for MDA-MB-231, and 2.76 ± 0.30 μM for MCF-10A) cells (MDA-MB-231) over normal cells (MCF-10A), and induced apoptosis through mitochondrial dysfunction and ROS generation. The authors concluded that the ruthenium complexes containing lapachol and lawsone as ligands are promising candidates as chemotherapeutic agents, especially for TNBC. The selectivity of complex (55a) for TNBC cells may be related to its preferential cellular uptake and its ability to interact with DNA (Fig. 7).⁶⁷

Three cyclometalated complexes (58–60) with anti-metastasis and anti-proliferation activity containing ruthenium complexes were synthesized by L. Xie and co-workers. These complexes selectively accumulated in mitochondria, causing oxidative stress and energy depletion, leading to cancer cell death. The complexes disrupted redox balance by increasing iron content and reactive oxygen species while reducing glutathione and ATP levels. The authors investigated the anti-metastasis and anti-angiogenesis properties of the complexes (Fig. 8A). Fig. 8A represents experimental data showing the effects of Ru(II) complexes on cell migration. In their experiment, the researchers treated cells with Ru(II) complexes and compared them with a control group that did not receive treatment. After 24 hours, they observed that the gap between cells in the treated group was not healing as rapidly as in the control group. This slower healing suggests that the Ru(II) complexes inhibited the migration of cells, indicating an anti-migration property of these complexes. Furthermore, in Fig. 8B, a transwell invasion assay was performed to assess the ability of cancer cells to invade through a membrane barrier, mimicking the process of cancer cell invasion into surrounding tissues. The results showed a significant decrease in invasive cells after treatment with the Ru(II) complexes at a concentration of 0.5× IC₅₀ value. This indicates that the complexes effectively suppressed the invasion of MDA-MB-231 cells, further supporting their anti-metastasis properties. In Fig. 8C, the expression level of vascular endothelial growth factor (VEGF), a protein crucial for angiogenesis, was evaluated after treatment with the Ru(II) complexes. A down-regulated expression of VEGF was observed in MDA-MB-231 cells treated with the complexes at IC₅₀ value. This suggests that the complexes inhibited the angiogenesis capability of cancer cells, which is essential for supplying oxygen and nutrients to support tumor growth and metastasis. Additionally, a tube formation assay was conducted on human umbilical vein endothelial cells (HUVECs), Fig. 8E. This assay assesses the ability of endothelial cells to form capillary-like structures, which is indicative of angiogenesis. The results showed that fewer tubes and networks were formed after treatment with the Ru(II) complexes compared with the control cells, demonstrating their potential as anti-angiogenesis agents.⁶⁸ A. Mukherjee's group synthesized eight ruthenium(II) complexes with pyrazolyl benzimidazole ligands and examined their anticancer property against MDA-MB-231 cell lines. They found the IC₅₀ values of the complexes were >75, >50, 13.0 ± 1.6, 9.7 ± 0.5, 11.9 ± 0.7, 9.5 ± 0.2, 10.1 ± 2.2, and 9.0 ± 1.8 μM for complexes 61–68. The ligands and complexes inhibited VEGFR2 phosphorylation, a key factor in angiogenesis, which is crucial for cancer progression. Some compounds also affected other kinases like ERK1/2 and Src. The study in in vivo models also showed effectiveness in inhibiting angiogenesis.⁶⁹

Six new N,O-donor ligands with or without pendant morpholine units were synthesized and their ruthenium(II) complexes (69–74) were also characterized by the same group (Fig. 9). The complexes exhibited significant in vitro antiproliferative activity against TNBC cancer cell lines, with IC₅₀ values 1.9 ± 0.4, 1.6 ± 0.3, 4.4 ± 0.4, 3.0 ± 0.8, 1.5 ± 0.3, and 1.2 ± 0.3 μM for 69–74. Treatment with the complexes affected the acidic vesicles in cells and caused the translocation of cathepsin D to the nucleus, indicating a potential necrotic pathway of cell killing rather than apoptosis.⁷⁰ Another interesting study was also conducted by Mukherjee's group, which involved the synthesis of nine Ru (II) complexes, designated as 75–83 (Fig. 9), utilizing N,N, and N,O coordination with 3-aminobenzoate Schiff bases. These complexes were then evaluated for their activity against triple-negative breast cancer cell lines. The results indicated that the synthesized complexes exhibited significant antiproliferative activity against metastatic triple-negative breast carcinoma (MDA-MB-231) cells. Notably, 79 and 80 demonstrated high activity, with IC₅₀ values of 2.5 ± 0.3 and 2.3 ± 0.2 μM, respectively. In addition to assessing cytotoxicity, the authors delved into the solution stability and elucidated the cell-killing pathways of these complexes. They highlighted the differences between N,N and N,O coordination in terms of their biological effects. Interestingly, the study revealed that different complexes exerted their cytotoxic effects through distinct pathways. Specifically, N,N coordinated complexes induced cell cycle arrest at the G2/M phase, whereas the N,O coordinated complex arrested the cell cycle at the G0/G1 phase. These findings underscore the importance of coordination chemistry in dictating the biological activity of metal complexes and provide valuable insights into the mechanisms underlying the cytotoxicity of these Ru(II) complexes against triple-negative breast cancer cells.⁷¹ Very recently, the same group discussed the role played by nuclear factor kappa beta (NF-κB) in breast cancer, particularly triple-negative breast cancer, and how its upregulation promotes inflammation and angiogenesis. The Ru(II) complexes (84–95) were studied as inhibitors of NF-κB, potentially reducing inflammation and angiogenesis. The authors introduced Ru(II) complexes with methyl- and dimethyl pyrazolyl-benzimidazole ligands that inhibit phosphorylation of p65 and VEGFR2, disrupting proinflammatory signaling and angiogenesis. The complexes showed non-toxicity to zebrafish embryos at certain concentrations and demonstrated strong antiangiogenic activity, suggesting potential for therapeutic use. The findings highlighted the significance of NF-κB as a therapeutic target in TNBCs and the promise of small-molecule drugs, like the Ru(II) complexes, in targeting molecular characteristics of TNBCs. They suggested these complexes could improve current chemotherapy treatments for TNBCs by inhibiting NF-κB and angiogenesis.⁷² The study conducted by A. Arunachalam et al. focused on synthesizing and evaluating the cytotoxicity of six new arene ruthenium(II) complexes, specifically complexes 96–101 (Fig. 9), which incorporated biphenyl benzhydrazone ligands. These complexes were assessed for their cytotoxic effects on both MDA-MB-231 cancerous and noncancerous cell lines. Among the synthesized complexes, 100 demonstrated notable cytotoxicity against cancer cells, with an IC₅₀ value of 9.53 ± 0.71. To further understand the mechanism of action, staining studies were conducted using acridine orange and ethidium bromide (AO–EB) as well as Hoechst 33342. These studies confirmed that the complexes induced apoptosis in cancer cells. Furthermore, additional investigations revealed that the complexes triggered the generation of reactive oxygen species (ROS) and affected the mitochondrial membrane potential (MMP). These findings suggest that apoptosis induced by the complexes proceeded via the mitochondrial pathway.⁷³

S. Gupta et al. were involved in the synthesis of seven Ru(II) polypyridyl complexes, denoted as complexes 102–108 (Fig. 9), containing acetylacetonate (acac) ligands. These complexes were then evaluated for their anticancer activity against breast cancer cells. Among the synthesized complexes, 102 exhibited remarkable selectivity for cancer cells compared with normal cells, with up to 2.5 times higher selectivity. Additionally, the authors observed that 102 induced apoptosis specifically in MDA-MB-231 cell lines. While cytotoxicity was not directly correlated with the electrochemical potentials of the complexes, the authors noted a moderate linear correlation between lipophilicity and toxicities. This suggests that the lipophilicity of the complexes may play a role in their cytotoxic effects. Overall, the study highlights the potential of 102 as a selective anticancer agent against breast cancer cells. The observed induction of apoptosis and the correlation between lipophilicity and cytotoxicity provide valuable insights for further research into the development of Ru(II) polypyridyl complexes for cancer therapy.⁷⁴ M. Pavlović et al. focused on synthesizing four benzamide derivatives containing ruthenium complexes, designated as complexes 109–112 (Fig. 9), for their anticancer activity against triple-negative breast cancer. Among these complexes, 109 demonstrated the highest antiproliferative activity against breast cancer cells. Furthermore, the complexes were observed to enter cells within 24 hours and exhibited nuclear-targeting properties, suggesting potential for intracellular action. 110 was found to display the highest inhibition of PARP-1 enzymatic activity in vitro, indicating its potential as a PARP inhibitor. Importantly, 109 exhibited the highest intracellular accumulation and DNA binding properties among the complexes. This resulted in cell cycle arrest in the S phase, suggesting a mechanism of action involving interference with DNA replication. Overall, the study highlighted the potential of these benzamide derivatives containing ruthenium complexes as promising candidates for the treatment of triple-negative breast cancer. 109, in particular, demonstrated significant antiproliferative activity, intracellular accumulation, and DNA-binding properties, suggesting its potential for further development as a targeted anticancer agent (Table 1).⁷⁵

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The work by L. J. Stephens et al. presented a notable advancement in the field of ruthenium complexes (113–119) for potential anticancer applications. They synthesized new ruthenium complexes, particularly with α-mercapto carboxylic acids and β-mercapto carboxylic acids, offering a diverse set of compounds for investigation. Among these, complexes 114-a, b, and 117 exhibited significant antiproliferative effects against MDA-MB-231 cell lines, indicating potential efficacy against invasive breast cancer. Complex 117 stood out for its selective cytotoxicity towards MDA-MB-231 cells, demonstrating an IC₅₀ value of 39 ± 4 μM. This selectivity, coupled with low systemic toxicity, positions it as a promising candidate for further development as a therapeutic agent against breast cancer. The proposed mechanism of action involved cellular uptake of the complexes, followed by catalytic oxidation of cellular thiols by oxygen. This process ultimately leads to cell death through the generation of reactive oxygen species (ROS). This mechanism aligned with the growing interest in exploiting ROS-mediated pathways for cancer therapy, showcasing the potential of ruthenium complexes as ROS-inducing agents.⁷⁶ The work by A. E. Graminha and colleagues presented an intriguing exploration of gallic acids and their derivatives containing ruthenium complexes (120–122) as potential agents for cancer treatment, particularly targeting triple-negative breast cancer (TNBC). The observed higher cytotoxicity against MDA-MB-231 cells compared with hormone-dependent MCF-7 cells highlights the potential selectivity of these complexes towards aggressive cancer phenotypes, such as TNBC. Of particular interest is the Ru(GAC) (complex 121), featuring a polyphenolic acid. This complex demonstrated interaction with the apo-transferrin (apo-Tf) structure and function in a manner dependent on transferrin, a protein crucial for iron transport. Ru(GAC) also exhibited the ability to inhibit the formation of reactive oxygen species (ROS), suggesting a potential mechanism for mitigating oxidative stress in cancer cells. Furthermore, Ru(GAC) was found to disrupt the cellular cytoskeleton, leading to the inhibition of critical cellular processes in TNBC cells, including invasion, migration, and adhesion (Fig. 10A–C).⁷⁷

A. A. Batista and group reported cinnamic acid or its derivatives containing five ruthenium complexes (123–127). Among all the complexes, 126 and 127 exhibited remarkable cytotoxicity against MDA-MB-231 cancer cell lines, with IC₅₀ values of 1.90 ± 0.05 and 2.14 ± 0.44 μM, respectively. The authors observed at the concentration of 1.56 μM, 126 inhibited colony formation, as well as reduced colony size and efficiency of the plate, whereas 127 exhibited the similar activities but at a higher concentration of 2.5 μM. They also arrested cell invasion, migration, and adhesion.⁷⁸

C. Yuan et al. developed novel chiral Ru(II) complexes (Δ/Λ128–Δ/Λ130) as potential stabilizers of c-myc G-quadruplex DNA, which could help suppress the progression of TNBC (Fig. 11). Among all the complexes, Δ130 exhibited efficient cytotoxicity results, with an IC₅₀ value of 25.51 ± 1.42 μM. Their finding also established that the DPPZ-based Ru(II) complex containing a 3-aminophenyl ethynyl group (dextro-isomer) entered the nucleus more efficiently and induced better inhibition activity than the levo-isomer. Through intermolecular H bonding and π–π stacking interactions, Δ130 stabilized the c-myc G-quadruplex, consequential in the suppression of transcription and expression of c-myc. The authors also revealed that complex Δ130 exhibited proliferation, migration, and invasion inhibitory effects against the TNBC cell line. Finally, they also found that the complex could reduce the growth of TNBC cells in the zebrafish xenograft model.⁷⁹ A. A. Batista and group reported amino acids containing four ruthenium complexes (131–134, Fig. 12) and investigated the cytotoxicity against MDA-MB-231 cell lines. All the complexes exhibited significant cytotoxicity values with IC₅₀ values of 33.6 ± 3.9, 25.9 ± 5.8, 12.1 ± 0.7, and 23.9 ± 1.2 μM, respectively. In their study, they also found that all the complexes reduced the percentage of viable cells and induced apoptosis. Additionally, they also proved that after 48 h with low concentration the complexes inhibited the cell migration of MDA-MB-231.⁸⁰

G. Gasser's group reported four ruthenium polypyridyl complexes (135–138) with flavonoid ligands and investigated their activity against TNBC cell lines. Complex 136 exhibited a remarkable cytotoxicity value among all the complexes, with an IC₅₀ value of 2.64 ± 0.43 μM. Metabolic studies also specified that the complex significantly diminished mitochondrial respiration and glycolysis in cancer cells.⁸¹ Eight new water-soluble ruthenium cyclopentadienyl complexes (139–146) and their anticancer activity against the TNBC cell line were reported by M. H. Garcia et al. They found all the complexes exhibited their cytotoxic activity against MDA-MB-231 cell lines. The quenching study also revealed that complex 144 interacted remarkably with HSA and fatty-acid-free HSA.⁸²

A new family of ruthenium(II) organometallic complexes (147–150) was designed by A. Valente et al. for targeting TNBC. The main focus of the synthesized complexes was both active and passive targets using biotin and polylactide (PLA), respectively. All the complexes exhibited remarkable cytotoxicity against MDA-MB-231 cell lines with IC₅₀ values 2.3 ± 0.1, 14.6 ± 0.4, 6.5 ± 0.3, and 3.4 ± 0.1 μM for complexes 147–150, respectively. The authors included biotin for cellular uptake through SMVT and the complexes induced apoptosis and proliferation, as well as cytoskeleton disruption. The in vivo study also suggested that the complex tends to control the growth of tumors.⁸³ W. L. Kwong and colleagues discovered that complex 151 effectively inhibited angiogenesis and reduced VEGFR2 expression, leading to the inhibition of tumor growth. Remarkably, it demonstrated no toxicity towards endothelial or cancer cells while specifically targeting angiogenic processes. In vivo experiments confirmed its efficacy, as it suppressed tumor growth in mice and inhibited angiogenesis in zebrafish models.⁸⁴

The use of biologically active peptides as drug delivery methods for tumors has grown in popularity because it allows for more selectivity and, as a result, less severe side effects. In triple-negative breast cancer cells, there is an overexpression of the fibroblast growth factor receptor (FGFR), which is a sign of poor prognosis, early relapse, and metastasis. The four subtypes of this receptor from FGFR1 to FGFR4 have various manifestation arrangements in the cells of breast cancer. FGFR-targeting peptides are therefore potentially useful delivery systems for targeted tumor damage.^85–88 In this regard, Marques et al. first developed a ruthenium complex named TM34 and investigated the activity against the MDA-MB-231 cell line.⁸⁹ In MDA-MB-231 cell lines, TM34 exhibited greater cellular accumulation. Even so, in these cell lines, the complex was less cytotoxic. The TM34 was later changed to TM281 to conjugate a peptide that might be used as an arsenal to attack tumors (Fig. 13).

The three ruthenium(II)–cyclopentadienyl conjugates of peptide (152–154) were then reported, along with a preliminary biological study of their effectiveness towards FGFR (+) and FGFR (−) human breast cancer cells.⁹⁰ The conjugates were more damaging to SKBR cells that excessively express FGFR receptors, as shown in Fig. 13. The upregulation of the hormone steroid's receptor and its capacity to bind in human carcinoma cells provide a useful strategy for cancer therapy.^91,92 Mei et al. used the Sonogashira coupling process and microwave irradiation to create two chiral ruthenium complexes (155A and 155B). Using the MTT assay, the authors investigated the activity of 155A towards the growth of different cells, namely HaCaT and MDA-MB-231. Even with 80 μM of [Ru] followed by 24 h treatment with 155A, the survival rate of MDA-MB-231 cells was still around 70%, and that of HaCaT cells was 95%. When treated with 40 μM, there were only 6.2% of cells in the late stage of apoptosis and 21.8% of cells in the early stage of apoptosis, meaning that around 70% of the cells were surviving, according to a subsequent study that utilized flow cytometry analysis. Blue staining of the nucleus with Hoechst-33528 was first noticed in MDA-MB-231 cells, with hardly any red phosphorescence of 155A throughout the entire cell population. After 30 minutes of incubation with 155A, the nucleus displayed a little red phosphorescence. The red phosphorescence reached its peak intensity after 90 minutes of incubation, after which it remained nearly unchanged. The red phosphorescence's intensity peaked after 120 minutes, and cellular uptake had reached about 100%. This discovery demonstrated the effectiveness of 155A as a nucleus-targeting probe for potential clinical uses (Table 2).⁹³

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4. Ruthenium complexes used for hormone receptor-positive breast cancer (HR+)

Candidates for ligands include anticancer drugs that are known to deprive cancer cells of the hormones necessary for their proliferation, like progesterone and estrogen, to create multi-targeting ruthenium complexes to facilitate the therapy of HR+ breast cancer. Examples of anticancer drugs covered in this section include steroid hormone receptor-targeting moieties and P450 enzyme inhibitors. This section also covers several successful instances of ruthenium complexes that contain other bioactive ligands, such as NSAIDs, EGFR inhibitors, and GST inhibitors. P450 enzymes catalyze processes that are involved in the metabolism and production of several important substances. As an illustration, the P450 enzyme aromatase (CYP19A1) is essential for steroid synthesis and, consequently, for the development of ER+ breast cancer.^94–96 Third-generation aromatase inhibitors are thought to impede the enzyme's ability to catalyze reactions by interacting with the iron in the cofactor through the N-position of their triazole rings. In this aspect, ruthenium(II) complexes containing anastrozole, a slightly different third-generation aromatase inhibitor, were reported by Castonguay et al. Complex 156 showed the highest in vitro cytotoxicity response. The authors found that the IC₅₀ value of the complex was 4 μM against both MCF7 and T47D breast cancer cell lines, which was better than cisplatin (>150 and 37.0 ± 2.4 μM for T47D and MCF7 cell lines, respectively). They also found that 156 exhibited a decrease in aromatase activity in H295R cells. Complex 156 (12.5 μM) is a promising choice for additional in vivo studies because it did not cause observable toxicity symptoms in zebrafish embryos over 96 hours.⁹⁷ A variety of third-generation ruthenium(II) arene complexes aromatase inhibitors that are therapeutically used were investigated for their preliminary in vitro anticancer potential in breast cancer cells by Maysinger et al. The cytotoxicity of complex 158 in the ER+ MCF7 was the most encouraging of the ruthenium complexes described in the investigation. This compound was shown to have much more cytotoxicity (IC₅₀ = 36 ± 6 μM, 24 h) than the control complex 159 (without an aromatase inhibitor), pointing to a role for the letrozole ligand in the drug's action.⁹⁸ Nonsteroidal anti-inflammatory medications (NSAIDs), which have shown encouraging anticancer effects in several studies, are good ligands for the creation of versatile ruthenium anticancer complexes. It is believed that the key method of action of NSAIDs in breast cancer involves suppressing the COX enzyme. The clearest effect of the overproduction of the COX (precisely COX-2) is the creation of inflammatory (PG) prostaglandins, which could exacerbate cancer, hasten the growth of cancer cells, and impair immunity.^99–102 In this aspect a series of ruthenium complexes (160–163) carrying several NSAID ligands, including naproxen, diclofenac, ibuprofen, and aspirin, was described by Mukhopadhyay et al. It was found that in the MCF7 cell line, all complexes had a lower (<0.1 μM) IC₅₀ value than their corresponding (>80 μM) free NSAID, except the Ru–Asp complex. It was demonstrated that all of the complexes had superior in vitro inhibition of COX compared with the equivalent free NSAID. Most importantly, 162 was more effective at inhibiting the cyclooxygenase enzyme than either 160 or 161, suggesting a potential link between COX inhibition and anticancer properties.¹⁰³ Another highly sought-after target for cancer therapies is the epidermal growth factor receptor (EGFR). It might be a good place to start when developing cutting-edge anticancer medications to treat breast cancer.^104,105 According to Wang et al. several 4-anilinoquinazoline derivative ruthenium(II) complexes (164) displayed abilities, including a considerable EGFR inhibition and a high tendency for DNA binding through the groove (minor) binding mode in various breast cancers. The authors also evaluated the cytotoxicity of 164 against human MCF7 cancer cells, in the presence or absence of EGF (100 ng mL⁻¹), to ascertain how the inhibitory effectiveness of the investigated complexes will be impacted by blocking the EGF's signal transduction. They found that when exogenous EGF was introduced, 164 exhibited inactiveness with >100 μM IC₅₀, despite exhibiting moderate cytotoxicity toward MCF7 cells in the absence of EGF, with 54 ± 4 μM IC₅₀ value (Fig. 14).¹⁰⁶

Zhao and colleagues presented an innovative strategy for targeted chemotherapy employing a hybrid of a ruthenium catalyst and antibody (Fig. 15). This hybrid, dubbed Ru-HER2, was tailored to specifically target cancer cells expressing the HER2 receptor. By binding selectively to HER2-positive cancer cells, Ru-HER2 facilitated the activation of a gemcitabine prodrug, thereby improving drug selectivity and minimizing adverse effects. Their findings demonstrated that Ru-HER2 effectively bound to the HER2 receptor, catalyzing the conversion of the inactive prodrug into the active chemotherapy agent gemcitabine. This process induced DNA damage and obstructed the HER2 signaling pathway in cancer cells. The hybrid exhibited significant anticancer activity against HER2-positive cancer cells in both laboratory (in vitro) and animal (in vivo) models, including multicellular tumor spheroids and a zebrafish xenograft model. Notably, the study revealed that the combination of Ru-HER2 and the prodrug elicited a synergistic effect, simultaneously inducing DNA damage and blocking the HER2 signaling pathway. This combined action inhibited cancer cell proliferation more effectively than either treatment alone, underscoring the promise of this approach in targeted chemotherapy.¹⁰⁷

Lin et al. combined carbene complex (NHC-Ru) of ruthenium N-heterocyclic with 17-ethynyl testosterone using a disulfide linkage to create a novel compound known as Te-S-S-NHC-Ru (complex 165). Research indicates that the pairing of testosterone bestows comparatively high efficacy on metal-based anticancer medications. After being exposed to Te-S-S-NHC-Ru at increasing concentrations (5, 10, and 15 M) for 48 hours, both MCF7 and MBA-MD-231 cells displayed a concentration-dependent increase in the percentage of the apoptotic population. The anticancer effect of Te-S-S-NHC-Ru towards MCF-7 cells was suggested by the greater apoptotic effect of MCF7 cells at concentrations of 5 and 10 μM compared with MBA-MD-231 cells, as indicated by the cytotoxicity results. The authors concluded that targeting group–drug conjugates effectively improved the targeting ability of metallodrugs.¹⁰⁸ Hannon et al. found that a steroid-conjugated (levonorgestrel) ruthenium(II) complex (166) was 8 times more effective than cisplatin in T47D human breast cancer cells. Additionally, it was revealed that the antiproliferative effects of free levonorgestrel and a control complex (167) were substantially less potent than those of the ruthenium bioconjugate complex. When a levonorgestrel group was added to a ruthenium(II) complex, the metallic center and the steroidal ligand worked in concert to produce very powerful ruthenium(II) complexes from the inactive parts.¹⁰⁹ Due to their aptitude for binding estrogen receptors, flavonoids, of which flavones are a member, are known to exhibit a variety of biological roles, including some antiestrogenic action.¹¹⁰ In this aspect, Arshad et al. reported ruthenium complexes (168–171) with substituted flavones as ligands. Among all the complexes, complex-168 was the most potent, with an IC₅₀ value of 16 μM against MCF7 cells. To investigate the anti-proliferative mechanism of the most active complex 168 in a single dose (16 μM), the authors performed cell-cycle phase distribution analysis. They demonstrated that 168 interfered with the G1 checkpoint.¹¹¹ Chakraborty et al. investigated the possible routes of action of a combination of ruthenium(III)–flavone (chrysin) (complex 172). The effects of the 172 on the MCF7 cell line were the subject of their next investigation. The ruthenium–chrysin combination inhibited cell growth and triggered apoptosis, according to the results of DAPI staining for oligonucleosomal fragmentation and cell viability. According to the findings, 172 produced a higher percentage of early apoptosis in MCF-7 cells which culminated in death. The authors concluded that complex 172 functions as a chemotherapeutic agent in the treatment of breast cancer because it causes cell cycle arrest and induces apoptosis, which included the overexpression of p53 and Bax and the consequent downregulation of Bcl2, VEGF, and mTOR (mammalian target of rapamycin).¹¹²

Very recently Potapov et al. reported six arene-based ruthenium complexes (173–178) with oxime chelating ligand. The authors studied their cytotoxicity against MCF7, MCF7CR, and MCF10A. With IC₅₀ values of 9.0 ± 4.4 against MCF7 and 8.9 ± 1.5 μM (175) against MCF7CR, it was discovered that hexamethylbenzene complexes were significantly more cytotoxic than p-cymene and benzene derivatives. This was likely caused by their increased lipophilicity, which allowed their efficient transport through the cell membranes.¹¹³ M. Khater et al. investigated the impact of ruthenium (Ru) complexation on the pharmacological activities of two bioactive flavones, designated as 179 and 180, which are being explored as potential anticancer agents. The results indicated that while the Ru complexes derived from these flavones (179Ru and 180Ru) showed alterations in their biological activities compared with the parent molecules, the effects varied depending on the specific flavone and the type of activity being assessed. The Ru complexes (179Ru and 180Ru) led to a loss of antiangiogenic activity when tested using an endothelial cell tube formation assay. This suggested that the Ru chelation affected the ability of the flavones to inhibit the formation of blood vessels from endothelial cells, which is a crucial process in tumor growth and metastasis. For compound 179, complexation with Ru (179Ru) resulted in enhanced antiproliferative and anti-migratory activities against MCF-7 breast cancer cells. Specifically, 179Ru exhibited a lower IC₅₀ value (66.15 ± 5 μM) compared with the parent molecule, indicating increased potency in inhibiting cell proliferation. Additionally, 179Ru significantly inhibited cell migration at a concentration of 1 μM (p < 0.01), further underscoring its improved efficacy in preventing cancer cell spread. In contrast, compound 180 showed a different pattern of activity upon Ru complexation. While the cytotoxic activity of 180Ru against MCF7 and MDA-MB-231 breast cancer cells was diminished compared with the parent molecule, it significantly enhanced the migration inhibition activity of compound 180, particularly in the MDA-MB-231 cell line (p < 0.05). This suggests that 180Ru may be more effective in impeding cancer cell migration, despite its reduced cytotoxicity. Overall, the study highlighted the complex interplay between ruthenium complexation and the pharmacological activities of bioactive flavones. While some activities, such as antiangiogenic effects, were lost upon Ru chelation, others, such as antiproliferative and antimigratory activities, were either enhanced or altered. These findings underscored the importance of understanding the structure–activity relationships of metal–flavonoid complexes for the rational design of novel anticancer agents.¹¹⁴ L. G. Julius and colleagues synthesized three ruthenium complexes, designated as complexes 181–183, and evaluated their anticancer efficacy against MCF-7 cell lines. Among these complexes, 182 demonstrated the most potent cytotoxicity, with an IC₅₀ value of 45.23 ± 6.96 μM. Docking studies further indicated the compounds’ ability to bind to estrogen receptor alpha (ER-α), with 182 exhibiting the most favorable binding energy, suggesting stable interactions.¹¹⁵ P. J. Sadler and colleagues unveiled a fascinating attribute of four ruthenium complexes (184–187), each featuring two distinct ancillary ligands: chloride (Cl) and iodide (I). These complexes showcased remarkable potency surpassing that of conventional platinum drugs while demonstrating a notable preference for cancer cells over normal cells. Notably, the iodide complexes exhibited superior efficacy compared with their chloride counterparts. Crucially, they displayed no cross-resistance with platinum drugs, exhibiting heightened accumulation in cell membranes and inducing late-stage apoptosis. Moreover, these complexes elicited cell growth arrest in the G1 phase, a point of difference from S-phase arrest mechanism showed by cisplatin. Among the complexes, 187 emerged as particularly cytotoxic, boasting an IC₅₀ value of 0.8 ± 0.1 μM against the MCF7 cell line. This discovery heralds promising implications for the development of potent and selective anticancer agents.¹¹⁶

F. Althobaiti and colleagues unveiled the anticancer potential of a uracil-containing ruthenium complex, denoted as 188, against the MCF7 cell line. Their findings showcased the complex's formidable anticancer efficacy, specifically its ability to trigger apoptosis in breast cancer cells. This apoptotic process was mediated by the augmentation of caspase-9 protein levels, a key regulator of apoptosis, and the inhibition of PCNA (proliferating cell nuclear antigen) activity, crucial for cancer cell proliferation. Thus, 188 emerges as a promising candidate for combating breast cancer, offering insights into novel mechanisms for inducing cell death in cancer cells.¹¹⁷ S. Sirasani and colleagues unveiled the potent anticancer properties of three polypyridyl ruthenium complexes, (189–191), against the MCF7 cell line. These complexes displayed remarkable cytotoxicity, with IC₅₀ values of 0.41 ± 0.014 μM, 2.3 ± 0.021 μM, and 1.5 ± 0.019 μM for complexes 189, 190, and 191, respectively. Notably, they exhibited the capacity to induce apoptosis in MCF-7 breast cancer cells by disrupting cell cycle checkpoints and upregulating caspase-8, a pivotal initiator of apoptosis. Moreover, these complexes demonstrated the ability to induce DNA cleavage via the production of singlet oxygen, further contributing to their anticancer efficacy. This groundbreaking research underscores the potential of polypyridyl ruthenium complexes as promising candidates for combating breast cancer through multiple mechanisms of action.¹¹⁸ X. He and colleagues presented their research on the synthesis of four half-sandwich ruthenium complexes featuring quinoline derivative ligands (192–195). These complexes demonstrated promising antitumor activity both in laboratory experiments and animal studies. Mechanistically, they induced lysosomal damage, leading to the release of cathepsin B and the initiation of apoptotic signals. Furthermore, the complexes heightened the production of reactive oxygen species (ROS) by compromising mitochondrial membrane potential, thereby exacerbating lysosomal damage. Importantly, in mouse models, these complexes exhibited significant antitumor effects without causing substantial changes in body weight, indicating favorable biological efficacy and highlighting their potential as therapeutic agents against cancer (Fig. 16).¹¹⁹

A groundbreaking series of studies by various research groups has illuminated the potent anticancer properties of ruthenium complexes, opening new avenues in cancer therapy. In particular, the utilization of 2-aminomethyl pyridine and its derivatives has emerged as a promising strategy, as evidenced by the remarkable findings. Led by S. Mukhopadhyay and colleagues, the investigation into 2-aminomethyl pyridine-containing ruthenium complexes (196–199) uncovered their exceptional efficacy against the MCF-7 cell line, a representative model of breast cancer. Notably, these complexes exhibited profound cytotoxicity, underscoring their potential as potent anticancer agents. Mechanistic insights revealed that the complexes catalyzed hydrogen transfer from NADH to NAD⁺ within cancer cells, thereby inducing intracellular accumulation of reactive oxygen species (ROS) and triggering apoptosis—a pivotal process in halting cancer progression.¹²⁰ Further enhancing the therapeutic arsenal against breast cancer, J. Qian and collaborators explored the role played by arene Ru(II) complexes (200–202) as stabilizers of KRAS G-quadruplex DNA—a novel target implicated in cancer pathogenesis. Their findings unveiled the selective binding of these complexes to KRAS G-quadruplex DNA, with 200 exhibiting the highest affinity. Importantly, 200 demonstrated potent induction of apoptosis in MCF-7 cells through DNA damage and cell cycle arrest, thereby underscoring its promise as a targeted chemotherapeutic agent.¹²¹ In a complementary endeavor, T. K. Mondal and team reported on two novel cyclometallated ruthenium(II) carbonyl complexes (203, 204) boasting a strong affinity for DNA and BSA protein—an attribute vital for therapeutic efficacy. Notably, these complexes exhibited compelling in vitro cytotoxicity against cancer cell lines, surpassing the potency of cisplatin—a cornerstone chemotherapy drug. This observation suggests their potential as efficacious anticancer agents, with the added advantage of potentially lower toxicity.¹²² Expanding the repertoire of ruthenium-based anticancer agents, DMSO-containing 2-aminophenyl benzimidazole-based ruthenium complexes (205, 206) emerged as potent contenders against breast cancer (Fig. 17). In vitro studies showcased their remarkable cytotoxicity against cancer cell lines, coupled with low toxicity towards normal cells—a crucial hallmark of effective chemotherapy. Encouragingly, these complexes demonstrated enhanced anticancer activity in a preclinical mouse model of Ehrlich Ascites Carcinoma, positioning them as promising candidates for further clinical exploration.¹²³

Collectively, these findings underscore the versatility and promise of ruthenium complexes in combating breast cancer, offering novel avenues for targeted therapy with enhanced efficacy and reduced toxicity—a beacon of hope in the fight against this devastating disease (Table 3).

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5. Nanoparticle-based formulations of ruthenium compounds for the management of breast cancers

The discovery of ruthenium complexes as potent chemotherapeutic agents against breast cancer cells has been the subject of our discussion. One of the main obstacles to their clinical application, however, lies in their effective biodistribution and selective accumulation in malignant cells. This has been addressed, to a certain extent, by the development and optimization of nanoparticle formulations, that due to an enhanced penetrability and retention (EPR) outcome can effectively improve drug accumulation and control dosage with reduced side effects. In the following section, we will highlight the advances made towards the development of various nano-formulations of therapeutic ruthenium complexes either by themselves or in collaboration with other therapeutic agents for breast cancer therapy.

The limitations and side effects of conventional therapeutic strategies have resulted in the growth of nanocarrier-mediated approaches for cancer therapy. Different biodegradable formulations of nanoparticles (NPs), such as liposomes, polymeric NPs, hydrogels, solid–lipid NPs, micelles, dendrimers, etc., have been studied for the roles they can play in drug delivery systems.¹²⁴ The short half-life of conventional chemotherapeutic drugs makes their direct administration difficult and in turn leads to an increasing demand for synthesizing nanocarriers that may have a characteristic controlled release pattern of the payload. Hence, understanding the interaction between the biological components of an organism and nanomedicine has led to the promotion of innovative cancer nanotherapeutics. Nanomedicine approaches typically lie at the interface between medicine and nanotechnology, covering the various pharmaceutical and biomedical sciences. Currently, numerous investigational NP delivery systems have been approved for various cancer treatments. The limitations of anticancer therapy are specific targeting and delivery of the conventional drug to the tumor cells to eradicate them, keeping the normal, healthy cells unaffected. Classical chemotherapy, which has been widely used for different types of cancers, is unfortunately accompanied by major off-target effects to the extent that the cure may become worse than the disease. In practice, free-form chemotherapeutic drugs have very little specificity for the tumor cells, which is why there is a very low level of drug accumulation inside the tumor. This leads to either moderate or severe side effects depending on the lethality of the cancer type. Another major bottleneck of classical chemotherapy is drug resistance which develops in the tumor cells either due to genetic factors, enzymatic deactivation of drugs, or due to the DNA repair machinery of the cells. The potential of drug delivery systems using nanocarriers is a very relevant subject that has been evolving over the last few decades. The nanocarriers provide stability to the drugs by enhancing the blood circulation time, preventing their premature degradation, and eliminating phagocytic cells. These particles can be designed in a way to regulate the percentage of drug accumulation in the tumor tissues and eventually in the tumor cells. The active and passive targeting ability of the NPs can be modulated using ligands designed to either recognize tumor markers or take advantage of the enhanced permeability and retention (EPR) effect, respectively. A liposome-based theranostic nanodelivery system for a dipyrodophenazine/ruthenium complex, [Ru(phen)₂dppz](ClO₄)₂ (207) (Lipo-Ru, Fig. 18) was investigated for its therapeutic efficacy in the MDA-MB-231 cancer model. Incubation of MDA-MB-231 cancer cells with Lipo-Ru induced double-strand DNA disruptions and initiated apoptosis. Treatment with Lipo-Ru in a mouse model notably reduced tumor progression. These results indicate that Lipo-Ru could be a promising theranostic system for cancer.¹²⁵

Lu and co-workers reported a self-assembled, biodegradable, dual drug nanocarrier system with Ru-based compound RAPTA-C covalently conjugated to the polymer backbone and chemotherapeutic drug Paclitaxel encapsulated in the micellar core. The water-soluble polymeric backbone was derived from fructose for effective targeting of GLUT-5, which is overexpressed in breast cancer cells. The dual-action therapeutic particles were about 226 nm in size and had an IC₅₀ value of 1223 nM in MDA-MB-231 cells. RAPTA-C alone exhibited negligible cytotoxicity, which has been previously reported in literature. The particles were found to inhibit cell invasion and migration; the cytotoxicity of the nanotherapeutic system in MDA-MB-231 was attributed to paclitaxel, whereas cell migration and cell incursion inhibition (an indicator of antimetastatic properties) were due to the effect of RAPTA-C. Overall, this is a rational approach for evaluating the efficacy and anticancer activity of two individual drugs that operate by completely different mechanisms. Paclitaxel inhibits cell splitting, thereby inducing apoptosis, while RAPTA-C affects the cytoskeleton to stop migration (Fig. 19).¹²⁶

A decade ago, a study conducted by Stoddart's group showed the photoinduced release of a surface-grafted ruthenium(II)–dipyridophenazine (dppz) complex from the surface of a mesoporous silica nanoparticles (MSNPs) hybrid (Fig. 20). The ruthenium complex was attached to the surface of the MSNPs using a photocleavable covalent bond, while paclitaxel could be loaded inside the pores of the nanoparticles. The reformed MSNPs underwent rapid cellular uptake, and after visible light photoactivation, expulsion of an aqua Ru(II) complex was detected using confocal microscopy. The cytotoxicity of the paclitaxel-loaded Ru-MSNP in MDA-MB-468 cells was drastically enhanced upon irradiation with visible light, whereas no such effect was observed with the free drug. It was noteworthy that ruthenium complexes may boost the cytotoxicity of emitted paclitaxel by prompting DNA damage. This was one of the first rationally designed hybrid material-based theranostic platforms reported for the combination therapy of breast cancer.¹²⁷

Ultra-small iridium/ruthenium alloy nanoparticles (IrRu NPs) with dual enzyme activities were synthesized loaded with glucose oxidase (GOx) and surface functionalized with polyethylene glycol to produce a biocompatible, multi-enzyme nanoreactor (IrRu-GOx@PEG NPs) for catalytic anticancer therapy application in 4T1 cells. In the first step of the cascading catalytic cycle, GOx in IrRu-GOx@PEG NPs reduced tumor tissue-sensitive glucose to hydrogen peroxide, and deprived the tumor cells of their nutrient source, thus inhibiting tumor growth by starvation therapy. In the following catalytic stage, IrRu NPs in IrRu-GOx@PEG NPs catalyzed the upstream endogenous H₂O₂ to highly toxic singlet oxygen ¹O₂ and O₂. Singlet oxygen, an extremely powerful oxidant, acted by oxidative therapy and directly induced cell apoptosis. The oxygen produced allowed for the continuation of the catalytic cycle and rectified the hypoxia issue that had caused the starvation therapy response to end in the tumor microenvironment. In the 3D cell clusters model of 4T1 cells, NPs had efficient tumor penetration (85 μM), due to their tiny size and efficient biocompatibility. The particles exhibited strong, pH-dependent cytotoxicity in 4T1 mouse breast cancer cells. Additionally, they prevented hypoxia-induced tumors from spreading. Experimental evidence was found to conclude that the cytotoxicity arose from their ability to produce reactive oxygen species. Biosafety studies in BALB-C mice revealed no apparent disturbance to the animals’ metabolic parameters. To evaluate in vivo efficacy, a 4T1 mouse model was developed by the group by subcutaneous transplant of the tumor cells. The vehicle or the control nanoparticle systems did not alter the tumor growth pattern, whereas NPs considerably inhibited tumor growth.¹²⁸ In our previous discussion on ruthenium-based small molecules for breast cancer therapy, we discussed the advantages of using photodynamic therapy (PDT). In this regard, a study on a hybrid nanotherapeutic particle was reported where ultraminiature palladium ruthenium alloy (sPdRu) and Ru(II) were combined with thermally responsive phase change materials (PCMs) [PdRu-RCE@PCM] NPs. Thermally responsive nanoparticles (PdRu-RCE@PCMNPs) were obtained by co-encapsulation of polypyridyl ruthenium complex (RCE), and hyaluronic acid was used for effective tumor aggregation of the particles. After entry of the NPs into the tumor, PdRu produced heat via photothermal action, which melted PCM, a heat-sensitive material, and released the Pd/Ru particles. The nanoparticles could impair the 4T1 mouse breast tumor cells through both PTE and PDT effects, the latter by catalyzing hydrogen peroxide to oxygen, with soaring ROS levels. It is to be noted that the NPs could successfully inhibit the primary growth of the tumor as well as inhibit metastasis of the tumor. Additionally, the strong fluorescence of the NPs could be engaged for the photothermal imaging of tumors (Fig. 21).¹²⁹

An effective way to deliver the antiproliferative ruthenium(III) complex AziRu for invasive neoplasms such as TNBC was investigated by Santamaria, Piccolo, and colleagues using a biocompatible cationic liposomal nanoformulation called HoThyRu/DOTAP. The group studied the cytotoxicity of their therapeutic particles, naked Ru-compound, and cisplatin as a reference compound in various breast cancer cells. The IC₅₀ value of the HoThyRu/DOTAP in MDA-MB-231 cells was found to be similar (9 μM) to that of cisplatin (around 7 μM). According to expectations, in the identical experimental setup, the naked AziRu did not affect the cell viability. To further substantiate this observation, clonogenic analysis in an investigational model of TNBC was performed which showed that the cells had a reduced ability to survive, reproduce, and grow upon treatment with HoThyRu/DOTAP. The cell migratory capacity of HoThyRu/DOTAP-treated MDA-MB-231 cells was reduced by nearly 50% from the beginning to the endpoint (96 h) of the experiment. To study the in vivo anticancer effectiveness of the therapeutic particles, a TNBC xenograft model was established in nude mice. The mice were injected with the HoThyRu/DOTAP formulation and tumor growth was monitored for 5 weeks. There was a considerable reduction in the tumor volumes in comparison with the control group of animals. Anticancer ruthenium complexes are often less toxic compared with platinum chemotherapeutics, and additionally they have excellent antimetastatic properties. Thus, they can act by a “multi-targeted” approach, ensuring both enhanced antitumor efficacy and reduced chemoresistance. Management of heterogeneous TNBC phenotypes could benefit from effective curative agents acting simultaneously on multiple targets.¹³⁰ M. Piccolo et al. focused on evaluating the efficacy and safety of a cationic nucleolipid nanosystem incorporating a ruthenium(III) complex, known as HoThyRu/DOTAP, as a potential treatment for human breast cancer. The researchers utilized a mouse xenograft model derived from MCF-7 cells to assess the in vivo effects of HoThyRu/DOTAP. The researchers measured tumor volume and weight to evaluate the impact of HoThyRu/DOTAP on tumor progression. The results indicated a significant reduction in both tumor volume and weight, suggesting the potential efficacy of the nanosystem in inhibiting tumor growth. The study monitored the survival rates of the mice following treatment with HoThyRu/DOTAP to assess its impact on overall survival. Changes in body weight were monitored as an indicator of potential toxicity or adverse effects associated with the treatment. The absence of significant alterations in body weight suggested that the nanosystem was well tolerated by the mice. The researchers analyzed various blood parameters, such as hematological and biochemical markers, to assess the systemic effects of HoThyRu/DOTAP. The absence of alterations in blood function suggested minimal systemic toxicity associated with the treatment. The distribution of ruthenium in different organs and tissues was examined to evaluate the biodistribution and pharmacokinetics of HoThyRu/DOTAP. The results indicated that ruthenium content was primarily localized in tumor lesions, suggesting selective delivery and activation of the complex within the tumor microenvironment.¹³¹

The study conducted by C. Riccardi et al. presents the synthesis and characterization of a novel amphiphilic amino acid-based Ru(III) complex, referred to as 208 (Fig. 22), which holds promise as a potential anticancer agent. The Ru(III) 208 was synthesized by coupling a glutamic acid derivative with a NAMI-A-like complex (AziRu). This process involved chemical reactions to form the desired complex with specific structural and physicochemical properties. The complex was thoroughly characterized to understand its molecular structure and properties. The study observed that complex 208 exhibited a slower hydrolysis rate compared with other Ru(III) complexes, namely AziRu and ToThyRu. This slower hydrolysis rate could contribute to its improved stability and potentially prolonged activity in biological systems. Complex 208 was aggregated with a cationic lipid (DOTAP) to form stable nano aggregates in water. This formulation strategy aimed to enhance the solubility, stability, and bioavailability of the complex, facilitating its delivery to target cells or tissues. Various techniques, namely UV–vis spectroscopy, dynamic light scattering (DLS), small-angle neutron scattering (SANS), and MTT assay, were employed to characterize the hydrolysis, aggregation, and antiproliferative properties of complex-208 and its DOTAP/208 formulation. These techniques provided valuable insights into the behavior and efficacy of the synthesized complex and its formulation. The DOTAP/208 formulation demonstrated significant antiproliferative activity against cancer cells (MCF-7, HeLa, and C6), with low IC₅₀ values, particularly notable against MCF-7 cells (104 ± 9 μM). This suggests the potential of 208 as an effective agent for inhibiting cancer cell growth. Importantly, the DOTAP/100 formulation exhibited low toxicity on normal cells (3T3-L1 and HaCaT). This selective cytotoxicity toward cancer cells while sparing normal cells is a desirable characteristic for anticancer therapeutics, as it minimizes adverse effects. The amino acid-based Ru(III) complex 208, particularly when delivered via liposomal formulations, is emerging as a promising candidate for further development as a new metal-based anticancer drug. Future research should investigate the effectiveness and safety of 208 in both lab and clinical settings, with emphasis on improving its formulation and delivery for medical use. This study highlights the promise held by 208 as a new metal-based anticancer treatment, particularly when administered through liposomal formulations to boost its effectiveness and safety.¹³²

D. de Oliveira Silva et al. presented the synthesis and characterization of two novel compounds, RuIbp and RuNpx, which involve coordination of the anti-inflammatory drugs ibuprofen and naproxen to a di-ruthenium(II,III) core. The authors developed a method to encapsulate the Ru₂(NSAID) metallo drugs (RuIbp and RuNpx) in SPLNs (Solid Polymer Lipid Nanoparticles), which are hybrid nanocarriers consisting of both polymer and lipids. This encapsulation strategy aimed to improve the drug delivery properties of the compounds, including drug loading efficiency, colloidal stability, and suitability for intravenous injection. The cytotoxicity of the Ru₂(NSAID)-SPLNs was evaluated against breast and prostate cancer cells. The results indicated that the SPLN formulations enhanced the anticancer activity of the metallodrugs compared with free metallodrugs, parent NSAIDs, and a non-drug compound (RuAc). This enhancement suggested that the SPLNs may facilitate cellular uptake and stability of the metallodrugs, leading to improved anticancer efficacy. The authors utilized fluorescence-labeled SPLNs to investigate their biodistribution and tumor accumulation in an orthotopic breast tumor model in mice. The results demonstrated that the SPLNs effectively delivered the metallodrugs to the tumor site via the enhanced permeability and retention (EPR) effect, a phenomenon where nanoparticles preferentially accumulate in tumors due to leaky blood vessels and impaired lymphatic drainage. Additionally, the SPLNs exhibited longer tumor retention time compared with other organs, indicating their potential as effective drug delivery vehicles for targeted cancer therapy. Overall, this study highlighted the potential of RuIbp and RuNpx encapsulated in SPLNs as promising candidates for cancer therapy. The enhanced anticancer activity facilitated cellular uptake, and favorable biodistribution and tumor accumulation properties suggest that these formulations could be further explored for clinical applications in cancer treatment.¹³³

Very recently, S. Michlewska et al. reported the synthesis and characterization of a carbosilane ruthenium metallodendrimer (209, Fig. 22) and its complexes with three anti-cancer drugs: doxorubicin (DOX), 5-fluorouracil (5-Fu), and methotrexate (MTX). The authors investigated the cytotoxicity of the 209/drug complexes against human breast cancer cells (MDA-MB-231) and normal human fibroblasts (BJ cell line) using the MTT assay. An in vivo experiment was performed by using a mouse model of triple-negative breast cancer (TNBC) to assess the tumor weight reduction and the ruthenium biodistribution in different tissues after treatment with 209, DOX, or 209/DOX. They found that 209 formed stable nano complexes with all three drugs, enhancing their effectiveness against cancer cells. 209 alone or in combination with DOX caused a decrease in tumor weight in mice with TNBC, and ruthenium accumulated mostly in the tumor tissue. They also suggested that 209 is a promising anti-cancer drug carrier with selective delivery and pro-apoptotic properties.¹³⁴

6. Light-activated ruthenium complex for breast cancer

Ruthenium complexes have emerged as a pivotal element in advancing light-activated chemotherapeutic methodologies, notably in photodynamic therapy (PDT) and photoactivated chemotherapy (PACT). These techniques harness the unique properties of ruthenium compounds to selectively target and destroy malignant cells upon exposure to light. Through intricate molecular design, ruthenium-based agents exhibit remarkable photochemical properties, enabling precise activation within targeted tissues while minimizing collateral damage to healthy cells. The utilization of ruthenium(II) underscores a promising avenue in the development of innovative cancer treatment strategies, offering enhanced efficacy and reduced side effects compared with conventional therapies. PDT, or photodynamic therapy, relies on a photosensitizer that absorbs light to generate reactive oxygen species (ROS) or singlet oxygen (¹O₂), impacting cancer cells through oxidative stress or electron transfer to biomolecules like proteins or lipids. However, PDT's effectiveness hinges on oxygen levels within tumors, limiting its efficacy in hypoxic environments. In contrast, PACT, or photoactivated chemotherapy, operates independently of oxygen availability. Upon light activation, PACT complexes disassemble into toxic ligands, enhancing treatment selectivity. This approach ensures the systemic inactivity of compounds, with effectiveness confined to sites of localized light activation.^135–139 So, in this section we discuss some light-activating ruthenium complexes which have exhibited good anticancer activity against breast cancer cell lines.

A new method for selectively tagging biomolecules in the living cells called bioorthogonal labeling was spearheaded by Carolyn Bertozzi. The CuAAC reaction, which is copper-catalyzed azide–alkyne cycloaddition, is a frequently used biorthogonal reaction for in vitro labeling. CuAAC focuses on the distinctions in highly expressed proteins between tumor cells and normal cells, making tumor cell identification easier. The azide-modified sugar N-azidoacetylmannosamine-tetraacylated (Ac4Man-NAz) is an important metabolic precursors. Azide groups can be accurately inserted into tumor cells’ plasma membranes by incubation. Tumor selectivity was effectively demonstrated by photosensitizers using the CuAAC reaction.^140–144 Lin et al. created Ru(II) complexes with an alkyne group using these ideas (210, 211). The MDA-MB-231 cells were then used to validate the in vitro bioorthogonal labeling of 211. The researchers used the MTT assay to look into phototoxicity. In the dark, complex 211 demonstrated negligible cytotoxicity toward MDA-MB-231 cells (Fig. 23a and b). They found that when exposed to a low dose of two-photon radiation, an increasing concentration of the Ru(II) complex decreased cell viability. The absence of bioorthogonal labeling was found to cause moderate photocytotoxicity. However, under identical circumstances, the complex showed no noteworthy behavior toward the non-cancerous human cell line MCF-10A. A large number of red spots were seen after Ac4ManNAz pretreatment and irradiation, which were caused by EthD-1 and suggested cell death, as seen in the illustration (Fig. 23c). On the other hand, Ac4ManNAz pretreatment without irradiation led to an intense greenish fluorescence and afterward the disappearance of red color fluorescence. When no Ac4ManNAz pretreatment was applied, only green fluorescence could be seen in either the presence or absence of light. This finding was supported by a ROS experiment using (DCFH-DA). Finally, the authors concluded that the bioorthogonal labeling effect had a significant impact on photosensitizer tumor selectivity.¹⁴⁵

Specific receptors regulate the biological activities of steroid hormones on the tissues of interest, and they are crucial to the regulation of specific gene networks’ expression. Targeting hormone receptors in breast tumors has been demonstrated in several studies to inhibit their interaction with hormones, and result in cancer cell death.^146–148 In this regard, Zhao et al. synthesized Ru-tmxf (212) by combining tamoxifen ligand with a Ru(II) polypyridyl moiety, which effectively targeted estrogen receptors to ER+ breast cancer cells. MCF-7 cell viability persisted at about ninety percent even after the concentrations of Ru-OMe (213) and Ru-tmxf (212) were increased, indicating minimal cytotoxicity in the dark (Fig. 24a, black and blue bar). On the other hand, complex 212 upon radiation for 2.5 hours considerably diminished the viability of cancer cells in culture (Fig. 24a, green bar). Only 17% of the cells under the same conditions died as a result of treatment with 213 (Fig. 24a, red bar). Overall, complex 212 demonstrated greater phototoxicity as compared with complex 213.¹⁰³ In the presence of complex 212 and light irradiation, the authors also compared the phototoxicity against COS-7, MDA-MB-231, HL-7702, and MCF-7 cells. They discovered that MCF-7 exhibited large red fluorescence that was PI-attributed and had no green fluorescence after PDT treatment (Fig. 24c). They investigated potential action patterns and an experiment on organelle colocalization by using several organelle-specific trackers. Lyso Tracker green and complex 212 were found to have the same distribution in MCF-7 cell lines. This outcome demonstrated the lysosomal localization of the complex 212. MCF-7 cells showed red fluorescence in lysosomes when exposed to irradiation or simply upon treatment with complex 212. When the cell was exposed to light or dark conditions while being treated with complex 213 (a control molecule), the same situation was also noted. Therefore, under this circumstance, the lysosomes of the MCF-7 cell were found to be in an intact state. Red fluorescence, however, was eliminated when the cell was treated with 212 followed by light irradiation (12 J cm⁻²). They deduced from this experiment that complex 212-mediated PDT could cause lysosome damage (Fig. 24d–f). Green fluorescence was seen in live MCF-7 cells treated with complex 212 after TP irradiation. No cell death was detected when only light or 212 alone was present, but following PDT, a faint green or evident red fluorescence was seen. From all of these experiments it was concluded that complex 212 upon irradiation could destroy ER+ breast cancer cells.¹⁴⁹

The study by F. Qu et al. involved the synthesis of five ruthenium complexes, designated as complexes 214–218, each with different ligands. The primary aim was to investigate their cytotoxicity and selectivity, particularly focusing on their potential as anticancer agents. Among these complexes, 216 exhibited the highest cytotoxicity, displaying IC₅₀ values as low as 3.7 μM against MDA-MB-231 breast cancer cell lines when activated by blue light. Notably, the authors explored dual activation mechanisms involving low pH and blue light, which further enhanced the activity of complex 216. The results of this study underscore the potential of pH-activated ruthenium complexes for selectively targeting cancer cells. By exploiting both low pH conditions typically found in tumor microenvironments and light activation, these complexes demonstrate a promising approach for cancer therapy. This dual activation strategy represents a novel proof of concept for achieving selective cancer cell targeting, which could have significant implications for the development of more effective and targeted cancer treatments.¹⁵⁰ In their study, H. Chan et al. proposed an innovative approach for chemotherapy by employing photolabile ruthenium(II)–purine complexes (219–221). These complexes released a purine ligand upon exposure to visible light, offering a targeted treatment for cancer by converting a non-toxic prodrug into its active form specifically within diseased tissue. This research marked a significant milestone as it introduced the first instance of a ruthenium prodrug that liberates the anticancer agent 6-mercaptopurine upon irradiation. Notably, the prodrug exhibited stability in darkness but manifested toxicity toward breast cancer cells post-light exposure. The investigation elucidated how the properties of the complexes, such as absorbance maxima, photostability, and the binding mode of 6-mercaptopurine, were influenced by the nature of the polypyridyl ligand. This exploration not only demonstrated the feasibility of utilizing photolabile ruthenium complexes for delivering purine-based anticancer agents, but also hinted at the potential for enhanced bioavailability and reduced side effects. In essence, this research opens doors to a safer and more effective avenue in chemotherapy, offering promise for improved cancer treatment outcomes.¹⁵¹ In their groundbreaking research, A. M. Mansour et al. delved into the synthesis and evaluation of Ru(II) complexes (222–226) featuring benzimidazole ligands, aiming for light-activated cytotoxicity against breast cancer cells. Their study marked a significant advancement by introducing dark-stable photoactivatable carbon monoxide (CO) prodrugs, adorned with diverse substituents on the phenyl ring. Upon exposure to 365 nm light, these complexes elegantly released CO molecules. The evaluation of the cytotoxic effects against breast cancer (MCF-7) cells yielded fascinating results. Remarkably, except for the 4-COOCH₃ group, all complexes remained inert in the absence of light, yet swiftly transformed into potent cytotoxic agents upon illumination. This light-induced cytotoxicity was not solely attributed to the liberated CO; instead, it involved the synergistic action of both the liberated CO and the CO-depleted metal fragments, including the benzimidazole ligands. The implications of this study are profound, hinting at promising clinical applications for precise CO delivery in cancer treatment. By harnessing light as a trigger, these complexes offer a controlled and targeted approach for combatting breast cancer, holding immense potential for advancing the landscape of cancer therapeutics.¹⁵² C. Fayad and their team directed their research efforts towards investigating the potential of four ruthenium(II) complexes (227–230) as promising chemotherapeutic agents, with a unique characteristic of releasing a ligand upon activation by blue light. Among these, complex 229 emerged as noteworthy, exhibiting remarkable cytotoxicity against triple-negative breast cancer (MDA-MB-231) cells. In their study, complex 229 displayed an impressive ability to induce cytotoxic effects, with IC₅₀ values of 4.72 ± 0.33 in the absence of light and a substantially enhanced potency of 0.74 ± 0.20 upon exposure to light. This heightened efficacy was accompanied by a notable increase in reactive oxygen species (ROS) production and the induction of apoptosis, underscoring the potential of complex 229 as a potent anticancer agent. Furthermore, the study revealed that complex 229 possessed superior cellular uptake properties compared with the other complexes investigated. This characteristic is of paramount importance, as it ensures efficient delivery of the therapeutic agent to its target site within cancer cells, thus maximizing its effectiveness as a photoactivated chemotherapy agent. Overall, the findings of this study shed light on the promising role of complex 229 and its counterparts as innovative candidates for photoactivated chemotherapy, offering new avenues for the development of targeted and potent treatments against aggressive forms of breast cancer.¹⁵³

In a recent breakthrough, Y. Sun and their research group unveiled a series of nine ruthenium CNC pincer complexes (231–239) and explored their potential as anticancer agents under both light and dark conditions. Their study uncovered intriguing insights into the anticancer activity of these complexes, particularly highlighting the most lipophilic ones (236–239) as exhibiting exceptional efficacy against MCF-7 cancer cells. Among these lipophilic complexes, complexes 236 and 239 emerged as cytotoxic entities, while complexes 237 and 238 demonstrated a fascinating phenomenon of light-activated photocytotoxicity. Complex 237, for instance, displayed EC₅₀ values of 3.9(6) and 1.2(1) μM under dark and light conditions against the MCF-7 cell line, and 9.5(7) and 2.7(1) μM under dark and light conditions against the MDA-MB-231 cell line, respectively. Similarly, complex 238 showcased noteworthy EC₅₀ values of 5.3(1) and 2.2(1) μM under dark and light conditions against the MCF-7 cell line, and 81(1) and 3.9(5) μM under dark and light conditions against the MDA-MB-231 cell line, respectively. These findings not only underscore the significant anticancer potential of ruthenium CNC pincer complexes, but also shed light on the nuanced effects of light activation on their cytotoxicity (Fig. 25). The identification of these lipophilic complexes as potent agents against cancer cells opens up promising avenues for further exploration in the development of targeted and effective cancer therapeutics.¹⁵⁴

N. Mansour et al. have unveiled a compelling discovery in the realm of cancer therapeutics, introducing a photoactive ruthenium complex, [Ru(bipy)₂(dpphen)]Cl₂ (complex 240), as a potent chemotherapeutic agent targeting triple-negative breast adenocarcinoma cells (MDA-MB-231). This complex, comprising bipyridine (bipy) and diphenylphenanthroline (dpphen) ligands, demonstrated remarkable efficacy upon photoactivation. Upon illumination, complex 240 exhibited profound cytotoxic activity, eliciting a cascade of cellular responses including cell rounding, detachment, accumulation of reactive oxygen species (ROS), DNA damage, and ultimately, apoptosis. Notably, its mechanism of action involved induction of cell death through both intrinsic and extrinsic pathways, offering multiple avenues for therapeutic intervention. Moreover, the complex exerted inhibitory effects on crucial signaling pathways implicated in cancer progression, namely the MAPK and PI3K pathways, further augmenting its therapeutic potential. The cytotoxicity profile of complex 240 revealed compelling results, with cytotoxicity values of 8.30 ± 0.11, 3.98 ± 0.13, and 1.00 ± 0.78 μM observed after 24, 48, and 72 hours of treatment in the presence of light, respectively. In stark contrast, negligible cytotoxicity was observed in the dark (>100 μM), underscoring the pivotal role played by light activation in unleashing the therapeutic efficacy of this complex. This groundbreaking study not only highlights the remarkable cytotoxic potential of complex 240 against triple-negative breast adenocarcinoma cells, but also underscores its multifaceted mechanism of action and specificity toward light activation. These findings pave the way for the development of novel photoactivated chemotherapeutic strategies with enhanced efficacy and selectivity for combating aggressive forms of cancer.¹⁵⁵ In a pioneering endeavor, a novel near-infrared-II (NIR-II) Ru(II) polypyridyl complex, designated as complex 241, has been ingeniously crafted for the purpose of chemophotothermal therapy against 4T1 breast cancer. This complex, adorned with unique properties, heralds a new era in cancer treatment strategies. The introduction of complex 241 dots brings forth a paradigm shift in therapeutic efficacy and precision in tracking intracellular delivery and biodistribution in real time. These dots exhibit a remarkable ability to precisely navigate through cellular landscapes, providing invaluable insights into their journey within the body. In vivo studies have unequivocally demonstrated the exceptional targeting prowess of complex 241 toward tumors, ensuring their accumulation at the desired site of action. Furthermore, the synergy between chemophotothermal effects orchestrated by complex 241 dots has been revealed to exert a formidable onslaught against cancer cells, all while maintaining minimal side effects.¹⁵⁶

A uridine-modified Ru(II) complex (242) was synthesized by Q. Wu et al. This complex was selectively localized in lysosomes and enhanced ROS production. It induced ferroptosis in triple-negative breast cancer (TNBC) cells under light irradiation and also bound to lysosomal integral membrane protein 2 (LIMP-2) with high affinity. The complex exhibited IC₅₀ values of 1.09 ± 0.22 and 0.36 ± 1.61 under normoxic and hypoxic conditions in the presence of light. The authors also evaluated that in tumor-bearing mice, the complex showed promising photodynamic therapeutic effects on TNBC tumors.¹⁵⁷ New ruthenium complexes with bathophenanthroline and dihydroxy bipyridine ligands (243, 244) exhibited remarkable anticancer activity against breast cancer cell line. These complexes demonstrated photocytotoxicity, upon exposure to light, which is beneficial for targeted cancer therapy. The complexes introduced ROS upon light activation and contributed a cytotoxic effect on a cancer cell line.¹⁵⁸ In their groundbreaking research, F. Qu et al. delved into the realm of ruthenium complexes (245–249, Fig. 26) with protic diimine ligands, shedding light on their fascinating potential as cytotoxic agents when illuminated with blue light. Their study presented compelling cellular evidence, showcasing the formation of reactive oxygen species (ROS) and apoptosis indicators following treatment with blue light, hinting at the intricate mechanisms underlying their cytotoxicity. A particularly intriguing revelation from their investigation was the stark contrast in quantum yields for singlet oxygen formation between the basic forms of the complexes (bearing O⁻ groups) and the acidic forms (bearing OH groups). This intriguing finding suggests that the process of deprotonation amplified the photocytotoxicity of these complexes under physiological conditions, unveiling a crucial aspect of their mode of action. Furthermore, the authors of the study ventured into uncharted territory by exploring the potential of these complexes to exert their cytotoxic effects under hypoxic conditions. This line of inquiry holds immense promise for addressing the challenges posed by tumors that are less responsive to traditional therapies due to their low oxygen levels, offering a glimmer of hope for patients facing daunting treatment obstacles. In essence, the research conducted by F. Qu et al. not only unveiled the photodynamic potential of ruthenium complexes with protic diimine ligands, but also highlighted their versatility and adaptability in the face of challenging tumor microenvironments. These findings pave the way for the development of innovative therapeutic strategies aimed at tackling cancer with precision and efficacy, ultimately offering renewed hope in the fight against this formidable disease.¹⁵⁹ G. He and colleagues presented an exciting advancement in cancer therapy, unveiling a groundbreaking ruthenium complex-based photocage activated by near-infrared (NIR) light at 760 nm. This innovation holds immense significance for in vivo phototherapy, boasting deep tissue penetration capabilities and minimal phototoxicity, crucial for effective treatment. At the heart of this development lies a cleverly designed photocage housing the potent anticancer agent tetrahydrocurcumin (THC). Upon exposure to NIR light, the cage efficiently releases THC, unleashing its therapeutic potential. In compelling in vivo experiments, this strategy demonstrated remarkable efficacy in inhibiting tumor growth. To further enhance the delivery of this promising therapy, the authors engineered a sophisticated self-assembled nanoparticle system using amphiphilic block copolymers. This delivery mechanism offered targeted cancer therapy, precisely delivering the photocage to tumor sites, thereby minimizing off-target effects and maximizing therapeutic impact. The study's findings represent a significant leap forward in cancer treatment paradigms, offering compelling proof-of-concept for the photocage's ability to combat tumor proliferation in vivo effectively. With its remarkable therapeutic potential and targeted delivery system, this novel approach holds immense promise as a future cornerstone in the fight against cancer.¹⁶⁰ Very recently, A. Dao et al. developed a novel dinuclear Ru(II) complex (250) that was activated by 700 nm NIR light for targeted anticancer therapy. Complex-250 was synthesized using a donor–acceptor–donor (D–A–D) linker, enhancing its NIR absorption and extending its triplet excited state lifetime. This complex exhibited unique slowed photodissociation kinetics, facilitating synergistic photosensitization and photocatalytic activity.

When activated by 700 nm NIR light, 250 showed nanomolar photocytotoxicity toward 4T1 cancer cells by inducing calcium overload and endoplasmic reticulum (ER) stress. This led to intracellular redox imbalance and tubulin polymerization inhibition. In vivo and in vitro experiments demonstrated that 250 effectively destroyed tumor cells both in vitro and in vivo, providing a robust foundation for the development of NIR-activated Ru(II) PACT complexes for phototherapeutic applications.¹⁶¹

7. Other ruthenium complexes against breast cancer

In this section, we discuss some other ruthenium complexes that have a great impact on breast cancer cells. In this aspect, A. A. Batista et al. focused on developing and evaluating three ruthenium complexes (251–253) for their anticancer potential against MDA-MB-231 cell lines. The findings indicated that all three complexes demonstrated cytotoxicity against the studied cell lines, with IC₅₀ values lower than those observed for cisplatin, a commonly used chemotherapeutic agent. Among the complexes tested, complex 252 exhibited the most promising selectivity against MDA-MB-231 cancer cell lines, showing an IC₅₀ value 12 times lower than that observed on MCF-10A cells, indicating a higher potency against cancer cells while sparing normal cells to some extent. Furthermore, complex 252 induced notable changes in the morphology of MDA-MB-231 cells, including loss of cellular adhesion and inhibited colony formation. It also caused an accumulation of cells at the sub-G1 phase, suggesting induction of apoptosis, along with alterations in the cell cycle dynamics, with an increase in cells at the S-phase and a decrease in cells at the G2 phase. Additionally, the study explored the interaction of the complexes with the human serum albumin (HSA) protein, revealing a static mechanism of interaction. The negative values for changes in enthalpy (ΔH) and entropy (ΔS) suggested the involvement of van der Waals forces and hydrogen bonding in the interaction between the complexes and HSA. Overall, these findings indicated that the investigated ruthenium complexes hold promise as potential anticancer candidates, warranting further detailed studies to elucidate their mechanisms of action and therapeutic potential.¹⁶² Another ten ruthenium, neutral, and ionic, complexes (254(a–e)–255(a–e)) were synthesized by activating C–H bonds of 2-phenylindole ligands. These complexes exhibited significant cytotoxicity against MCF7 and MDA-MB-231 breast cancer cell lines. The research group also highlighted the promise of these Ru(II) complexes as antitumoral drugs with greater activity and potentially fewer side effects than current platinum-based treatments.¹⁶³ Other research explored the use of heteroscorpionate ligands as scaffolds for creating ruthenium(II) metallodrugs (256–260) for breast cancer therapy. Complex 257 exhibited remarkable cytotoxic activity, with IC₅₀ values of 3.19 ± 0.76 and 5.10 ± 0.20 μM against MDA-MB-231 and MCF-7 cell lines. Uptake studies also indicated distribution in the cytoplasm, and stability studies proposed structure–activity relationships.¹⁶⁴ N. Balakrishnan et al. designed two ruthenium complexes (261, 262) for breast cancer treatment. The complexes exhibited better stability as well as a good cytotoxic profile with IC₅₀ values. Between the two complexes, complex 261 exhibited IC₅₀ values of 23.69 ± 0.02 and 27.62 ± 0.15 μM against MDA-MB-231 and MCF-7 cell lines. The complexes induced cell death through apoptosis.¹⁶⁵ Six new ruthenium complexes (263–268) with salicylic acid derivatives were synthesized for induced cell death in triple-negative breast cancer cells. The authors investigated the cytotoxic effects of these complexes on various human breast tumor cells and non-tumor cell lines, and found complex 267 exhibited the best result with an IC₅₀ value of 0.15 ± 0.00 μM against the MDA-MB-231 cell line (Table 4). The selected complex induced apoptosis through the intrinsic pathway, which was confirmed by increased levels of proteins involved in apoptotic cell death (Fig. 27).¹⁶⁶

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8. Conclusion

Breast cancer remains a formidable challenge in the realm of oncology, necessitating innovative approaches for more efficacious treatment. In our review, we have meticulously explored the burgeoning landscape of ruthenium complexes as potential therapeutic agents against the formidable adversary of breast cancer. Through an exhaustive analysis of the existing literature, we unveil a rich tapestry of compounds, each holding promise in confronting this challenging disease. Our examination traverses a diverse spectrum of ligands, ranging from the elegant simplicity of organic molecules like dichloroacetate and polypyridine to sophisticated bioactive ligands such as biotin and lapachol. Each of these compounds contributes its unique arsenal to the fight against breast cancer, offering distinct avenues for therapeutic intervention. The compelling evidence of these complexes’ activities against breast cancer cell lines is central to our discussion. We have dissected their mechanisms of action, revealing how the interplay between ligands and complexes orchestrates a symphony of molecular interactions. From the precise inhibition of key enzymes to the strategic targeting of hormone receptors and growth factors, these complexes exhibit a multifaceted approach to combating breast cancer. Furthermore, our review sheds light on the transformative potential of these compounds in reshaping the treatment landscape for breast cancer. By elucidating how ligands can modulate the mechanisms of action of ruthenium complexes, we unveil new avenues for innovation and precision in cancer therapeutics. Our review serves as a beacon of hope in the ongoing battle against breast cancer. It underscores the remarkable diversity and efficacy of ruthenium complexes, offering a glimpse into a future where targeted interventions promise to transform the lives of those affected by this challenging disease. The combination of the central metal with different ligand systems has shown various mechanisms of cytotoxic effect, often amounting to synergism in their mode of action. Metal-based drug discovery research faces certain bottlenecks like lack of knowledge of biodistribution, intracellular targets, pharmacokinetic and pharmacodynamic behavior, systemic toxicity, etc., and, except for a few, the reported ruthenium complexes are no exceptions. Extensive studies on the in vivo behavior of the complexes under similar experimental conditions (animal species, tumor xenograft model, dosage, duration of treatment) can truly substantiate the efficacy of the complexes in more realistic cases of breast cancer. Furthermore, analysis of the effects of the ruthenium complexes on patient tissue samples may provide further information on their effects on cancer cell metabolism. In addition, to mitigate the side effects of chemotherapy, it is also necessary to target and activate the molecules specifically to the tumor sites, especially with spatiotemporal control of drug release that may be achieved by suitable nano-formulations of the appropriate ruthenium complexes and in the presence of light activation not in dark condition. We hope this review serves not only as a compendium of current knowledge, but also as a catalyst for future research directions in the development of novel ruthenium-based complexes for the treatment of breast cancer, the most prevalent malignancy affecting women worldwide. By shedding light on promising avenues and encouraging further exploration, it holds the promise of revolutionizing breast cancer therapy and improving outcomes for countless patients globally.

Data availability

All the data provided in the manuscript are genuine and authentic.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

PP are grateful to the Department of Science and Technology, Government of India, for supporting the work through the DST-SERB CRG project grant (CRG/2021/002267). UD and PP thank the Vellore Institute of Technology for arranging VIT SEED funding. UD and PP acknowledge DST, New Delhi, India, for the DST-FIST project. UB would like to thank BITS Pilani for the New Faculty Seed Grant (NFSG/GOA/2023/G0900).

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