Lighting up cancer: latest discoveries in ruthenium(ii) polypyridyl complexes for anticancer photodynamic therapy

Abstract

Photodynamic therapy (PDT), which utilises light-sensitive compounds to specifically target cancer cells while preventing harm to neighbouring healthy tissues, presents a potentially viable alternative treatment option. Ruthenium (Ru) polypyridyl complexes have garnered considerable attention as prospective PDT agents owing to their capability to generate stable and selective molecules by structural modification through ligands replacement. Additionally, their intricate geometries and oxidation states contribute to their therapeutic potential. This review provides the evolution and application of Ru(ii) polypyridyl complexes in PDT as a cancer treatment modality. It analyses the efficacy, mechanisms of action, and advancements of these complexes in comparison to conventional methods. We present the design and optimisation of these complexes, emphasising their critical importance in combination therapies to circumvent the shortcomings of existing PDT methods and achieve enhanced therapeutic outcomes. Our review subsequently examines the practical implications of the results, highlighting the need for further in vivo experiments to validate Ru(ii) polypyridyl complexes as viable therapeutic agents for cancer. Future studies aiming to enhance the effectiveness of PDT could significantly benefit from the incorporation of nanotechnology and advanced delivery systems.

Rationally engineered Ru(ii) polypyridyl complexes enable precision photodynamic therapy through tunable photophysics and targeted cancer cell eradication.

1. Introduction

Cancer remains a major global health threat with projections from the International Agency for Research on Cancer (IARC) suggesting a dramatic increase in cancer incidence and cancer-related mortality. Estimations suggested approximately 20% of the population will experience cancer during their lifetime, with a mortality rate around one in nine men and one in 12 women.¹ In 2025, the American Cancer Society (ACS) predicted 2.04 million new cancer cases in the United States, averagely 5600 new cases each day, with prominent diagnosis from female breast, lung, colorectal, prostate, and stomach cancers. However, globalisation and urbanisation-induced lifestyle changes, such as smoking, poor diet, and physical inactivity, led to the rise of infection-related cancers in developing countries.² By 2040, global cancer rates are anticipated to rise to 28.0 million incident cases and 16.2 million deaths due to population growth and aging. In nations classified as having low to medium Human Development Index levels, where medical resources are scarce, new cases are projected to increase by 95% and 64%, respectively.

Despite the frequent practice of conventional treatments like surgery, radiation therapy, and chemotherapy to enhance the survival rates and quality of life of cancer patients, the limitations of high invasiveness, unwanted side effects, increased recurrence rates, and metastasis have not been fully addressed. Therefore, innovative strategies are urgently required in developing and evaluating anticancer treatments.³ In response to these challenges, photodynamic therapy (PDT) and photosensitisers (PSs) medications have emerged as promising areas of cancer research.

2. Photodynamic therapy

The concept of PDT originated during the early 1900s when it was observed that acridine dye irradiated with sunlight eradicated protozoan paramecia compared to cells treated with acridine dye in the absence of sunlight.⁴ PDT represents a groundbreaking approach to cancer treatment, using PSs that are activated by specific wavelengths of light in the presence of oxygen to generate reactive oxygen species (ROS). Upon activation, the absorption of energy excites the PSs to an excited singlet state, followed by intersystem crossing to excited triplet state. The PSs then react with biomolecules and kill cancer cells through vascular structure destruction, or by stimulating an immune response like apoptosis and necrosis.⁵ Ultimately, PDT should be designed with high precision and selectivity to eradicate cancerous cells while simultaneously protecting neighbouring healthy tissues.^5,6

As depicted in Fig. 1, PDT harnesses two distinct pathways to damage cancer cells. Type I mechanism relies on photoinduced electron transfer to generate superoxide (˙O₂⁻), hydroxyl radicals (˙OH), and hydroperoxyl radicals (˙O₂H). On the contrary, type II transfers energy from the PSs to ground-state triplet oxygen (³O₂), which produces singlet oxygen (¹O₂), inducing instantaneous localised responses.^6–8

Fig. 1. Mechanism of PDT.

One of the major challenges in PDT for conventional PSs is the substantial reliance of oxygen for ROS generation. In hypoxic tumour environments, the scarcity of oxygen hinders the oxygen-dependent type II pathway. In an effort to address this, Li et al. designed a series of photodegradable squarine-π systems modified with dicyanovinyl groups, which acted as multimodal PDT agents.⁹ Even under hypoxic environments, the efficient generation of ˙O₂⁻ and ¹O₂ induced cytotoxicity among cancer cells, concurrently ˙OH aided in the self-degradation upon endogenous oxidation.⁹ This highlights the possibilities in the fabrication of next-generation PSs, whereby multimodal photoreactivity becomes the primary focus instead of maximisation of ¹O₂ quantum yield. Ultimately, the hybridisation of ROS generation and exploitation of endogenous substrates for metabolic clearance in PDT boosts safety and clinical translational potential.⁹

2.1. Different types of PSs

In the 1970s, the potential therapeutic value of hematoporphyrin derivative (HpD) as a PS, a water-soluble mixture of porphyrins and their esters (e.g. hematoporphyrin, protoporphyrin, and deuteroporphyrin) was examined by Dougherty and his colleagues.^6,10–12 This marked the beginning of the commercial application of PSs in PDT. Since then, many PSs, both natural and synthetic, have been discovered as shown in Table 1.¹³

Table 1. Example of different types of PSs.

PSs	Absorption range	Applications	Classification	Ref.
	∼630 nm	Treatment for bladder cancer, early-stage lung cancer	Synthetic	12, 14 and 15
	410–630 nm	Treatment for vulvar lichen sclerosis	Natural	12 and 16
	∼630 nm	Treatment for basal cell carcinoma	Natural	17 and 18
	∼689 nm	Treatment for gastric cancer, basal cell carcinoma	Synthetic	3 and 19

2.2. Development of the porphyrin PSs

Porphyrin is a group of heterocyclic organic compounds characterised by a large ring containing four linked nitrogen atoms. The exceptional capacity to harness light energy is contributed by light absorbing chromophores of porphine, making them exceptional PSs for PDT.^4,6–8

Photofrin was the first PSs to receive clinical licence for use in PDT. However, the subpar tissue selectivity and insignificant red-light absorption (Absorption coefficient ≤2500 M⁻¹ cm⁻¹ at 630 nm) are exacerbated with the limited penetration of visible light with shorter wavelengths into deep tissue layers.¹⁰

Subsequently, second-generation PSs with refined purity were designed to increase tumour selectivity, minimise the dosage and unfavourable side effects of systemically delivered PSs.^4,12 Different derivatives of porphyrins, chlorins, and others were included to promote light absorption at longer wavelengths, which increases their tissue penetration for treating solid tumours in more inaccessible anatomical locations.

Several PSs have either been clinically approved for therapeutical application or are currently undergoing clinical trials as shown in Table 2.^12,17 Second-generation PSs utilise porphyrinoid chemicals that provide increased tumour selectivity and deeper tissue penetration, owing to the immense spectral absorption between 650 and 800 nm. The side effects are minimised coupled with shortened durations (<2 weeks) for the patients to be located in a dark room, attributed to the rapid clearance of these substances from the body.^12,17 Nevertheless, the poor aqueous solubility causes agglomeration in physiological environments, limiting the production of ROS hence therapeutic potential.^4,12,13 Thus, second-generation PSs are restricted by the struggle to reconcile the trade-off between high tumour affinity and optimal photophysical and photochemical properties.

Table 2. List of second-generation PSs currently in clinical use or undergoing clinical trials.

Second-generation PSs	Trade names/Manufactures/Country
	Foscan®, Biolitec, Germany
	Tookad® soluble, Negma-Lerads in Elancourt, France
	Purlytin®, Miravant, Santa Barbara, California

As a resolution, third-generation PSs equipped with active or passive targeting mechanisms that enable superior selectivity for cancerous tissues were developed.^4,20,21 Through conjugation or encapsulation of PSs within biodegradable/biocompatible nanoparticles (NPs), the PSs stability, hydrophilicity and in vivo functions were enhanced.^20–22 Concomitantly, these nanoformulations possess superior pharmacokinetics, pharmacodynamics, and biodistribution profiles, with mitigated undesired side-effects and dark toxicity.²² Such revolutionary approaches herald a new era of enhanced efficacy and safety for PDT.

Despite notable strides in the field, third-generation PSs remain a work in progress. The current limitations of parenteral administration of PSs pose a significant hurdle in the clinical applicability of PDT, underscoring the pressing need for innovative drug delivery systems that can enhance the PDT bioavailability.^4,22 As promising as the advancements have been, much work remains to be done before the full potential of PSs can be realised in the fight against cancer.

3. Combination therapy

PDT is a minimally invasive technology ideal for treating end-stage cancer patients, as it poses a minimal risk of complications. Nonetheless, it's in vivo and clinical application remains restrained by low PSs solubility, aggregation, limited light penetration through biological tissues, and photosensitivity issues. Additionally, the hypoxic environment of tumours impedes type II PDT pathway.^23,24

To overcome these challenges, PDT is progressively integrated with distinct techniques to enhance the targeting effect on cancer cells, vascular system, immune pathways, and compositions of the tumour microenvironment. The combination therapies include radiotherapy, chemotherapy, immunotherapy, surgery, photothermal therapy (PTT), and sono-photodynamic therapy.^25–27

PTT can complement PDT by generating ROS without oxygen dependency, concurrently promote blood flow, enhance drug delivery efficiency, and alleviate tumour tissue hypoxia by increasing local temperature. A promising clinical approach, near-infrared photo-immunotherapy (NIR-PIT), uses monoclonal antibodies conjugated to near-infrared (NIR) dye to treat metastases by triggering the release of tumour-associated antigens and promoting localised immunogenic cell death.^26–28 Another approach is photodynamic priming (PDP), which uses sub-lethal photodynamic doses to sensitise the tumour microenvironment including tumour cells, stroma, and the vasculature. In addition, the utilisation of PDT in conjunction with chemotherapy demonstrates promise in the treatment of oncological conditions. Moreover, X-ray induced PDT permits deeper penetration and higher efficacy against tumour types with poor efficacy and limited standard protocols. Additionally, nanocarriers-aided PDT improves targeting, cellular uptake, and tumour accumulation.²⁹ Lastly, combining multiple PSs for instance Photofrin II with anionic porphyrin, TPPS4, assessed on BALB/c mice bearing EMT-6 mammary tumours resulted in a complete treatment success when compared to a 50% and 60% of cure rate for Photofrin and TPPS4 individually, respectively.^26–28

Overall, combination therapy improves therapeutic efficacy through lowering PSs dosage, preventing drug resistance, reducing treatment frequency, and reducing dark toxicity by targeting different pathways, which can prevent skin photosensitivity.^26–28,30

4. Transition metal complexes as anticancer drugs

The serendipitous discovery of cisplatin and success in testicular cancer treatment since 1970s has led to the extensive clinical application of metal complexes in oncology therapies.³¹ Platinum-class anticancer drugs particularly cisplatin, carboplatin, and oxaliplatin are predominantly used for chemotherapy in modern settings.³² This success naturally pivoted anticancer research towards the exploration of other metal-based drugs. However, in spite of considerable efforts, none have surpassed the three primary Pt(ii) drugs.³³

Transition metal complexes possess inherently beneficial chemical and physical characteristics for biomedical purposes, including their ability to catalyse redox reactions, bind to biologically relevant molecules, and maintain high stability and selectivity. The rational design achieved by fine-tuning the ligands, metal core, or oxidation state can further enhance these intrinsic properties.³⁰

To date, several d- and f-block metals have been investigated for their electronic properties as possible PSs with enhanced light-induced cytotoxicity for PDT in cancer treatment. The promising in vitro anticancer activity of these complexes is tempered by concerns over toxicity profiles and tumour treatment resistance. Nonetheless, PDT has demonstrated potential in reducing the harmful effects of metal-based medicines on human organs compared to traditional chemotherapy.³⁰ Encouragingly, new photocytotoxic metal complexes with favourable safety and efficacy profiles continue to be discovered.^30,34 In particular, Ru(ii) complexes have been the most extensively investigated for PDT applications, as elaborated in the following sections. Table 3 presents selected examples of metal-based complexes and their applications.

Table 3. Examples of metal-based complexes and their applications.

Metal based complexes	Structure	Applications	References
Platinum-based complexes		Treating cancer, by inducing damage to their deoxyribonucleic acid (DNA) through the creation of gaps in single strands, along with the production of ROS	35
		FDA-approved first-generation Pt(II) drug, widely applied for malignant breast, colorectal, ovarian, and testicular cancer due to the strong DNA damaging activity	36
		FDA-approved second-generation Pt(II) drug for ovarian cancers, with decreased systemic toxicity	36
		FDA-approved third-generation Pt(II) drug predominantly used against colorectal and gastrointestinal cancers, possess broad spectrum of action against cisplatin and carboplatin-resistant lines	36
Aluminium-based complexes		Treatment for melanoma skin cancer	37 and 38
Iron-based complexes		Antitumour activity without harming normal cells	39

5. Ru complexes

As a dynamic transition metal, Ru showcases an array of oxidation states and intricate geometries, spurring curiosity in its potential for applications in PDT and the emerging domain of photoactivated chemotherapy (PACT).^40–42 The distinct properties of Ru metal, attributed to its Lewis acidic and decreased oxophilic nature, present promising opportunities for developing innovative therapeutic agents.⁴⁰

The oxidation states of Ru(ii) and Ru(iii) exhibit stability within a biological context, with the more reduced state demonstrating enhanced reactivity.⁴⁰ Every oxidation state in both Ru(ii) and Ru(iii) can assume a six-coordinated octahedral geometry, which enables it to engage with a diverse array of ligands and facilitate numerous redox reactions within living organisms.^40,41 Through meticulous adjustment of the steric and electronic characteristics of a complex via the control of ancillary ligands, a wide variety of chiral Ru complexes can be created. These complexes are frequently produced by incorporating rigid bidentate auxiliary ligands, such as 2,2′-bipyridine (bpy) and 1,10-phenanthroline (phen), or tridentate auxiliary ligand of 2,2′,6′,2″-terpyridine (tpy) as depicted in the Fig. 2. These cations establish non-covalent, reversible associations with DNA.^44,45

Fig. 2. Examples of rigid bidentate and tridentate auxiliary ligands (a) general structures of octahedral Ru(ii) complexes containing bidentate ligands (b) bidentate auxiliary ligands (bpy and phen) (c) general structure of octahedral Ru(ii) complexes containing tridentate ligands (d) tridentate auxiliary ligand (tpy), followed by chemical structures of biologically active Ru polypyridyl complexes developed by Dwyer et al. (e)–(g).

Remarkably, Ru complexes display an extended luminescence lifetime, substantial Stokes shifts, and second-order optical properties when excited to the triplet metal-to-ligand charge transfer (MLCT) form. Prolonged emission lifetimes enable Ru(ii) complexes to be susceptible towards the intracellular microenvironment, thereby functioning as potential ‘see and cure’ agents. As they are stable under prolonged one- and two-photon illumination, the PSs can be reused, and thus lower dose of PSs is required. These features collectively enhance their potential as PDT agents.⁴⁵ Since Dwyer et al. introduced a range of bioactive Ru polypyridyl compounds in 1952 as depicted in Fig. 2, the investigation into Ru polypyridyl complexes has been comprehensive and widespread.^25,45 Multiple mechanisms contribute to the anticancer properties of Ru complexes, such as their capacity to impede topoisomerase activity, diminish metastasis, effect of ROS, interact with proteins and DNA, and induce apoptosis.⁴⁶

At present, a selected group of Ru-derived anticancer agents are subjects for clinical investigation, including NAMI-A, KP1019, NKP-1339, and TLD1433 as shown in Table 4.⁴⁷ Despite significant research and development efforts, the advancement of Ru complex-based therapies has encountered obstacles such as suboptimal antitumour efficacy (NAMI-A) and inadequate solubility (KP1019).^47,48 To date, no Ru-based compound has entered widespread clinical application. However, in 2017, phase II clinical trial with TLD1433 showcased therapeutic potential for human muscle-invasive bladder cancer.⁴⁷ Additionally, RAPTA-C is another promising anticancer drug candidate which is progressing toward clinical trials, while NKP-1339 also known as BOLD-100 (IT-139) has just entered Phase I clinical trial.^47,49–52 Each metal complex has demonstrated distinct in vitro and in vivo activities regardless of their structural similarity. KP1019 is more prone to targeting primary tumours while NAMI-A displays antiangiogenic and antimetastatic effects in secondary tumours.^47–52 NKP-1339 which was first discovered as a precursor molecule for KP1019, underwent modifications in the clinical setting due to its high aqueous solubility.^47–52

Table 4. Details of Ru complexes in clinical trials.

Compound	Clinical trial phase and number	Structure	Clinical findings	References
NAMI-A	Phase II (N/A)		Monotherapy was less effective than standard drug gemcitabine against advanced non-small cell lung cancer (NSCLC), hence studies are discontinued	48
KP1019	Phase I (N/A)		Antitumour activity exhibited by stabilised conditions of NSCLC, colon cancer, and neuroendocrine tumour. Dose escalation study revealed low solubility	48
NKP-1339/IT-139/BOLD-100	Phase I (NCT01415297)		Targets GR78 stress-response protein. Moderate antitumour activity at maximum tolerated dose (MTD), without notable toxicity	49 and 50
	Phase I/II (NCT04421820)		Combination with FOLFOX (folinic acid, fluorouracil, and oxaliplatin) improved treatment efficacy against pre-treated advanced biliary tract and gastric cancer	51 and 52
TLD1433	Phase II (NCT03945162)		PDT treatment was safe and effective against BCG-unresponsive NMIBC	53–55

Intriguingly, TLD1433 demonstrated low dark toxicity with exceptional phototoxicity ranging between 0.0039–9.9 µM in various cancer cells using green and red light at 525 and 625 nm respectively.⁵³ The immense photodynamic activity of TLD1433 compared to alternative Ru(ii) complexes highlights the potential for clinical translation as a PDT agent. Notably, the success of PDT treatment amongst Bacillus Calmette–Guérin (BCG)-unresponsive non-muscle invasive bladder cancer (NMIBC) was a milestone which marked the inaugural clinical trial for a Ru-based PSs in PDT.^43,53–55

The progression of Ru anticancer agents, encompassing NAMI-A, KP1019, NKP-1339, and TLD1433, reflects an ongoing transition from immunotherapeutic strategies to combination therapy approaches.⁵⁶ It has been discovered that the cancer-fighting capacities of Ru complexes hinge significantly on the specific ligands present, the complexes' intrinsic properties, and the existence of unbound sites in the metal centre's coordination sphere.⁵⁶ Thus, it is theorised that the introduction of diverse ligands for structural adjustments could enhance and diversify their antiproliferative prowess.

6. Bpy-based Ru complexes

6.1. Fundamental structural alterations influencing the phototoxic impact of Ru polypyridyl complexes

A research endeavour led by Han et al. examined a Ru polypyridyl complex furnished with bpy ligands and an altered phen ligand (1) (Fig. 3). Within the human estrogen positive breast adenocarcinoma cells (MCF-7), this compound exhibited low dark toxicity (half-maximal inhibitory concentration, IC₅₀ = 21.33 µM) and significant photoactivity (IC₅₀ = 1.75 µM) upon exposure to 450 nm light of 30 J cm⁻² for 5 minutes. Consequently, a phototherapeutic index (PI = IC₅₀ (dark)/IC₅₀ (light)) greater than 11.5 was achieved. Contrarily, the control (2) revealed no notable disparity in antitumour cytotoxicity between the dark and illuminated conditions. Importantly, the heightened photodynamic performance of complex 1 was linked to its unique ¹O₂ production and increased cellular uptake efficiency, both of which were ascribed to the modified, expanded ligand.⁵⁷

Fig. 3. Examples of bpy-based Ru complexes 1–13.

At the same time, Karges and coworkers investigated many Ru polypyridyl complexes generated from bpy (Fig. 3). When tested against human cervical adenocarcinoma cells (HeLa), complexes 3 and 4 showed poor dark toxicity and even less impressive phototoxicity when irradiated for at 480 nm (10 minutes, 3.1 J cm⁻²) and 540 nm (40 minutes, 9.5 J cm⁻²). The phototoxicity of complex 3 was mild (IC_50,480nm = 152.4 ± 3.8 µM; IC_50,540nm = 146.3 ± 4.2 µM), while that of complex 4 was undetectable (IC₅₀ > 200 µM). A possible explanation for the decreased phototoxicity of complex 3 is its weak capacity for ¹O₂ production and its lack of stability in human plasma. In addition, complex 3 showed a striking red shift of 65 nm in its absorption property. Complex 3 demonstrates enhanced absorption as a result of an extended π-system and the incorporation of dimethylamine groups at the distal end of the parent complex 5 and 6.¹⁵

Solís-Ruiz and colleagues conducted in vitro tests on cisplatin-resistant gastric adenocarinoma (AGS) and carcinoma (KATO III) cells using a series of cyclometalated Ru polypyridyl complexes that featured increased π-conjugation (Fig. 3). One of the investigated complexes, complex 6 displayed outstanding phototoxicity when irradiated (590 nm, 60 minutes, 20.3 J cm⁻²) against KATO III, achieving an IC₅₀ value of 0.27 ± 0.06 µM, while complex 7 demonstrated a significantly higher IC₅₀ value of 4.89 ± 0.15 µM. This disparity could be caused by the enhanced lipophilicity of complex 6, which could impact cellular uptake. Furthermore, synthesised complex 8 exhibited better cytotoxicity against AGS, with an IC₅₀ value of 0.75 ± 0.08 µM, when compared to its coordination counterpart 9. The substantial variation in cytotoxic effects underscores the significance of the carbon–metal bond in altering the biological efficacy of Ru complexes.⁵⁸

In a study conducted by Welsh et al., Ru(ii) polypyridyl complexes bearing 2-pyridylbenzimidazole or 2-quinolylbenzimidazole ligands and s-triazine or trisamine core were synthesised (Fig. 3). Complex 10, a mononuclear Ru complex possessed the highest quantum yield for ¹O₂ emission (Φ_Δ = 0.79) compared to trinuclear Ru complexes 11, 12, and 13 (Φ_Δ = 0.26, 0.31, and 0.13 respectively). This suggested that the excitation could be partially quenched through energy dissipation by vibrational relaxation of the more flexible trinuclear complex structures. During the cytotoxicity evaluation against HeLa cells upon exposure to 455 nm light of 1188 J cm⁻² for 60 minutes, complex 10 exhibited low phototoxicity (IC₅₀ > 45 µM). On the contrary, the trinuclear Ru complexes 11, 12 and 13 displayed moderate phototoxicity, with IC₅₀ value of 42.53 ± 1.16 µM, 34.05 ± 1.09 µM and 34.63 ± 1.11 µM respectively. All complexes 10 to 13, exhibited very poor dark toxicity (IC₅₀ > 300 µM). Despite the moderate phototoxicity of complex 11, 12, and 13, it is noteworthy that the PI ranges between 7.05 to 8.81 was achieved, which were relatively higher than clinical drugs of 5-aminolevulinic acid (PI > 1) and Photofrin (PI = 4.4). This demonstrates that low ¹O₂ quantum yield does not diminish the phototoxicity effect, indicating that Ru complexes participate in both Type I and II photodynamic therapy mechanisms. Noticeably, the nuclearity and overall metal complex charge affects the phototoxicity, whereby higher nuclearity and higher overall charge increases the uptake of metal complexes by cells.⁵⁹

6.2. Impact of conjugation on the phototoxic effect of Ru complexes

A Ru complex with two boron dipyrromethene (BODIPY) ligands were synthesised by Aksakal and coworkers (complex 14) (Fig. 4). Following the substitution of BODIPY ligands, the ¹O₂ quantum yield of complex 14 increased significantly. The heavy atom effect could be accountable for the enhanced spin–orbit coupling (SOC) and the accelerated rate of intersystem crossover (ISC), both factors promoting the rapid generation of ¹O₂. Furthermore, compound 14 exhibits strong cytotoxic effects, with an IC₅₀ value of 5 µM against both HeLa and human myelogenous leukemia cells (K562) when subjected to red light (632 nm, 5 hours, 45 J cm⁻²), but not in darkness. The enhanced ability of the conjugated BODIPY ligand to generate ¹O₂ is associated with its potent cytotoxic activity.⁶⁰

Fig. 4. Examples of bpy-based Ru complexes 14–31.

Jakubaszek and her team devised an approach to enhance the delivery of Ru(ii) complexes to cancer cells by incorporating two conjugated cobalamin derivative Ru(ii) polypyridyl complexes (Fig. 4). Nonetheless, both complex 15 (IC₅₀, dark and light condition >100 µM) and complex 16 (IC₅₀, dark and light condition >50 µM) demonstrated minimal cytotoxicity when evaluated against human retinal epithelial cells (RPE-1) or HeLa cells, with photoirradiation at 480 nm for 10 minutes of 3.21 J cm⁻². The diminished cytotoxic activity of the Ru complexes could be contributed by the substantial size. As a result, the current investigation suggests that the conjugation of cobalamin might not substantially influence the transport efficiency of Ru complexes as PSs, ultimately affecting the enhancement of the anticancer efficacy of PDT in the context of cancer management.⁶¹

In an innovative endeavour, Liu's team crafted a pair of groundbreaking Ru(ii) polypyridyl structures, fusing imidazo-phenanthroline with hydroxybenzoic acid elements (Fig. 4). Intriguingly, both compounds (17 and 18) demonstrate a remarkable affinity for cancer cells, owed to the presence of carboxyl and hydroxyl groups that amplify the amphiphilic nature of Ru structures and enable seamless transmembrane navigation in the tumour's acidic milieu. Although minimal cytotoxicity was observed under dark conditions (IC₅₀ > 100 µM), these complexes displayed a cytotoxic potential upon exposure to light (460 nm, 30 minutes, 18 J cm⁻²), effectively targeting the cancer cells under investigation (Table 5).⁶²

Table 5. IC₅₀ values for complexes in varying cell lines in both dark and light conditions (460 nm, 30 minutes, 18 J cm⁻²)⁶².

IC50a (µM)	Conditions	Complex 17	Complex 18	Cisplatin
Human lung carcinoma cells (A549)	Dark	>100	>100	8.4 ± 0.8
	460 nm	40.7 ± 1.6	35.1 ± 1.5
Human gastric epithelial cells (GES-1)	Dark	>100	>100	9.5 ± 0.6
	460 nm	83.2 ± 4.8	61.4 ± 3.9
Human hepatocellular carcinoma cells (HepG2)	Dark	>100	>100	10.2 ± 1.3
	460 nm	9.2 ± 0.6	7.2 ± 0.4
Human umbilical endothelial cells (Huvec)	Dark	>100	>100	10.1 ± 0.8
	460 nm	94.6 ± 8.2	80.1 ± 7.8
MCF-7	Dark	>100	>100	22.1 ± 3.7
	460 nm	25.7 ± 1.2	29.6 ± 2.1
Human triple negative breast adenocarcinoma cells (MDA-MB-231)	Dark	>100	>100	11.2 ± 0.8
	460 nm	24.8 ± 1.1	19.9 ± 1.0

^aIC₅₀ = half-maximal inhibitory concentration.

In 2020, Chen's group created a Ru(ii) polypyridyl compound, complex 19, that targets lysosomes by combining it with a traditional Chinese medicine herb, rhein. Complex 20, which is also known as Ru(bpy)₃ consist of Ru as metal ion in the centre, coordinated by three bpy ligands which allows it to be a stable structure (Fig. 4). These properties also make it highly versatile in PDT. In contrast, through in vitro testing, it was observed that complex 19 had low cytotoxic effects in the dark on various cancer cells and normal liver cells, with IC₅₀ values ranging from 35.1 ± 6.2 to 250.6 ± 0.2 µM as presented in Table 6. However, when exposed to light (450 nm, 15 minutes, 3.15 J cm⁻²), the cytotoxicity of complex 19 significantly increased, and the IC₅₀ values dropped to 2.4 ± 0.02–8.1 ± 0.3 µM, demonstrating the compound's strong photocytotoxic properties and its ability to target different types of cancer cells. Notably, complex 19 showed higher cytotoxicity than complex 20 in both dark and light conditions, which was due to the increased cellular uptake efficiency caused by the addition of Rhein. Furthermore, complex 19 showed promise in overcoming cisplatin resistance during cancer therapy.⁶³

Table 6. IC₅₀ values for complexes in varying cell lines in both dark and light conditions (450 nm, 15 minutes, 3.15 J cm⁻²)⁶³.

Complexes	Complex 19			Rhein		Complex 20		Cisplatin
IC50a (µM)	Dark	450 nm	PIb	Dark	450 nm	Dark	450 nm	IC50a (µM)
Human ovarian endometroid adenocarcinoma cells (A2780)	49.3 ± 1.1	5.2 ± 1.1	9.5	126.9 ± 4.7	>100	>200	>200	3.2 ± 0.0
Human ovarian endometroid adenocarcinoma resistant cells (A2780R)	91.3 ± 1.1	3.2 ± 0.1	28.5	116.5 ± 6.3	129.9 ± 6.7	>200	>200	18.0 ± 0.4
A549	64.7 ± 0.3	2.4 ± 0.02	26.9	130.6 ± 6.2	144.8 ± 3.1	>200	>200	13.9 ± 0.2
MCF-7	250.6 ± 0.2	8.1 ± 0.3	20.9	>100	>100	>200	>200	14.4 ± 0.1
Human foetal hepatocellular cells (LO2)	35.1 ± 6.2	8.7 ± 0.2	4.0	123.8 ± 6.1	144.7 ± 2.0	>200	>200	10.1 ± 0.1

^aIC₅₀ = half-maximal inhibitory concentration.

^bPI represents the ratio between IC₅₀ values of complex 19 in dark and light conditions.

Furthermore, Zhang's research team achieved red light-induced anticancer activity utilising a Ru(ii) biq (2,2′-biquinoline) complex, in conjunction with a nitroanthraquinone group (complex 21) (Fig. 4). This compound demonstrated efficacy against A549 and human drug-resistant lung adenocarcinoma cells (A549R), in addition to the human ovarian adenocarcinoma cells (SKOV-3). Due to the presence of Ru(iii) and anthraquinone anion centres that produce ˙O²⁻ radicals upon exposure to light, this compound has potential applications in PDT and photoredox catalysis. Light can also be employed to convert a non-toxic pro-drug into an anticancer agent through a process known as PACT, as demonstrated by the complex within tumours cells. In contrast to PDT and photoredox, PACT does not require oxygen. Phototherapy is most effective between 600 and 900 nm. Subjecting cells to 600 nm irradiation for 30 minutes at 40.5 J cm⁻² yielded an IC₅₀ value of 1 µM for A549 and SKOV-3, and 0.96 µM for A549R. Under low-oxygen conditions in A549 cells, the IC₅₀ value was 6.5 µM, attributed to PACT's contribution.⁶⁴

Recently, Wu's team synthesised a lysosome-targeting PSs through conjugation of uridine to Ru(ii) polypyridyl complex via click chemistry (complex 22) (Fig. 4). The covalent linkage on uridine recognised the lysosomal integral membrane protein 2 (LIMP-2) abundant in triple negative breast cancer (TNBC) cells, thus improved the targeting ability and sensitivity for PDT. In comparison to reference complex 23, complex 22 exhibited higher efficiency in ROS generation attributed to the triazole conjugation of uridine. Upon photoirradiation at 650 nm for 10 minutes at 180 J cm⁻², complex 22 showed excellent phototoxicity against breast cancer cells of MDA-MB-231 in normoxic conditions (IC₅₀ = 1.09 ± 0.22 µM) and hypoxic conditions (IC₅₀ = 0.36 ± 1.61 µM). On the contrary, complex 22 displayed moderate dark toxicity in both normoxic conditions (IC₅₀ = 36.41 ± 3.97 µM) and hypoxic conditions (IC₅₀ = 32.06 ± 5.87 µM), ultimately yielding a PI of 33.4 and 89.1 respectively. On the other hand, the PI for cancerous MCF-7 cells and normal human breast epithelial cells (MCF-10A) under normoxic conditions were 3.39 and 2.01 respectively, indicating low toxicity against both cell lines. Moreover, in vivo tests of complex 22 on nude mice transplanted with MDA-MB-231 cells revealed complex accumulation in tumour, biocompatibility, targeting of lysosomes for ferroptosis induction and ROS generation.⁶⁵

Moreover, Fennes et al. synthesised complex 24, a Ru polypyridyl complex bearing an (E,E′)-4,4′-bis[p-(N,N-phenylamino)styryl]-2,2′-bipyridine group for red-light activation (Fig. 4). The presence of highly conjugated groups extended the absorption wavelength until 630 nm. Additionally, complex 24 displayed enhanced aqueous stability, membrane permeability, and production of ROS compared to parent compound of complex 23. As shown in Table 7, the cytotoxicity evaluation against cancerous mouse large intestinal carcinoma cells (CT-26), MCF-7, human pancreatic carcinoma cells (PT-45), including healthy human skin fibroblast cells (GM-5657) demonstrated poor dark toxicity (IC₅₀ > 100 µM), however photoirradiation at 450 nm (10 minutes, 1.2 J cm⁻²) provided low IC₅₀ values ranging between 0.8 ± 0.1 to 3.3 ± 0.4 µM, whereas 630 nm (15 minutes, 0.9 J cm⁻²) exhibited IC₅₀ values ranging from 1.3 ± 0.2 to 6.4 ± 0.3 µM. Comparatively, clinical PSs of photofrin and drug of cisplatin, both were less therapeutically effective due to the stronger dark toxicity but weaker phototoxicity at 450 nm and 630 nm in all tested cell lines.⁶⁶

Table 7. IC₅₀ values for complex 24, photofrin and cisplatin in varying cell lines in dark, 450 nm (10 minutes, 1.2 J cm⁻²), and 630 nm (15 minutes, 0.9 J cm⁻²) conditions⁶⁶.

IC50a (µM)	Complex 24			Photofrin			Cisplatin
	Dark	450 nm	630 nm	Dark	450 nm	630 nm	Dark
CT-26	>100	0.8 ± 0.1	1.3 ± 0.2	>50	4.1 ± 0.6	2.4 ± 0.5	6.3 ± 0.5
GM-5657	>100	3.3 ± 0.4	5.4 ± 0.3	>50	4.8 ± 0.5	3.9 ± 0.4	9.3 ± 0.6
MCF-7	>100	2.4 ± 0.2	6.4 ± 0.3	>50	3.6 ± 0.3	2.9 ± 0.3	8.1 ± 0.6
PT-45	>100	1.6 ± 0.1	2.7 ± 0.3	>50	9.7 ± 1.1	7.4 ± 0.6	11.3 ± 0.4

^aIC₅₀ = half-maximal inhibitory concentration.

In a subsequent study, Abad-Montero and collaborators designed three Ru(ii) polypyridyl complexes bearing coumarins fluorophores (complexes 25–27) which efficiently induced phototoxicity within hypoxic tumours (Fig. 4). All complexes demonstrated bathochromic shift in absorption maxima (λ_max), which permitted the application of NIR wavelength light for photoirradiation to achieve deeper tissue penetration. According to the photochemical characterisations, the alterations in fluorescent probe signals confirmed the production of ¹O₂, ˙O₂⁻, and ˙OH by complexes 25–27 upon photoirradiation, evidently triggering oxidative damage under hypoxic conditions. As listed in Table 8, under normoxic conditions, complexes 25–27 and reference of protoporphyrin IX (PpIX) were inactive at dark conditions (IC₅₀ > 100 µM) when tested against CT-26 cancer cells but displayed significant phototoxicity at six different wavelengths ranging between 540 nm to 740 nm, for 60 minutes at 3.4 to 13.5 J cm⁻². On the contrary, as shown in Table 9, only complex 25 and 27 showed significant cytotoxicity upon photoirradiation under hypoxia. Ultimately, the PI profiles for complex 27 at NIR light was excellent, supporting their application for hypoxic tumour eradication. Subsequently, complex 27 was selected for in vivo assay against BALB/c mice carrying CT-26 cells, in which an effective dose of 3 mg kg⁻¹ coupled with photoirradiation at 660 nm for 15 or 20 minutes at 90 or 120 J cm⁻² successfully inhibited tumour growth by 69–83%.⁶⁷

Table 8. IC₅₀ values and PI for complex 25–27 and PpIX and in CT-26 cells in dark and six photoirradiation wavelengths (540–740 nm, 60 minutes, 3.4–13.5 J cm⁻²) under normoxic (21% O₂) conditions⁶⁷.

Complexes	25		26		27		PpIX
Normoxia (21% O2)	IC50a (µM)	PIb	IC50a (µM)	PIb	IC50a (µM)	PIb	IC50a (µM)	PIb
Dark	>250	—	>250	—	>250	—	>100	—
540 nm, 9.0 J cm−2	0.0082 ± 0.0006	>30 487	0.033 ± 0.004	>7575	0.025 ± 0.002	>10 000	0.320 ± 0.09	>312
595 nm, 3.4 J cm−2	0.013 ± 0.003	>19 230	0.076 ± 0.007	>3289	0.025 ± 0.004	>10 000	0.400 ± 0.01	>250
620 nm, 6.7 J cm−2	0.042 ± 0.006	>5952	0.118 ± 0.027	>2118	0.017 ± 0.003	>14 705	0.660 ± 0.210	>151
645 nm, 9.0 J cm−2	0.048 ± 0.003	>5208	0.117 ± 0.002	>2136	0.0074 ± 0.0006	>33 783	0.170 ± 0.210	>588
670 nm, 13.5 J cm−2	1.460 ± 0.450	>171	1.11 ± 0.32	>225	0.036 ± 0.003	>6944	0.770 ± 0.200	>129
740 nm, 12.6 J cm−2	31.3 ± 6.1	>8	32.8 ± 4.6	>7.6	0.76 ± 0.06	>329	2.100 ± 0.200	>50

^aIC₅₀ = half-maximal inhibitory concentration.

^bPI represents the ratio between IC₅₀ values of complexes in dark and light conditions.

Table 9. IC₅₀ values and PI for complex 25 and 27 and in CT-26 cells in dark and four photoirradiation wavelengths (540–740 nm, 60 minutes, 9.0–13.5 J cm⁻²) under hypoxic (2% O₂) conditions⁶⁷.

Complexes	25		27
Hypoxia (2% O2)	IC50a (µM)	PIb	IC50a (µM)	PIb
Dark	>250	—	>250	—
540 nm, 9.0 J cm−2	0.035 ± 0.005	>7143	0.086 ± 0.011	>2907
645 nm, 9.0 J cm−2	0.920 ± 0.09	>272	0.076 ± 0.008	>3290
670 nm, 13.5 J cm−2	13.24 ± 3.64	>19	0.074 ± 0.005	>3378
740 nm, 12.6 J cm−2	>100	—	9.56 ± 2.15	>26

^aIC₅₀ = half-maximal inhibitory concentration.

^bPI represents the ratio between IC₅₀ values of complexes in dark and light conditions.

One of the latest enhancements of PSs were made by Ng and her team through conjugation of peptides to Ru(ii) polypyridyl complexes for complementary membrane-targeting effects in PDT (Fig. 4). The parent compound of complex 28 possessed 4-methyl-4′-carboxy-2,2′-bipyridine, which permitted the conjugation with arginine–tryptophan peptides (Arg–Trp) of varying lengths via amide linkage formation, ultimately producing modified Ru(ii) complexes 29, 30 and 31. The presence of the positively charged Arg–Trp imparted high binding affinity towards the negative surface of the cells membranes. However, it was noteworthy that the inclusion of Arg–Trp limited the yield of ¹O₂ in aqueous phosphate buffered saline (PBS) solution to 7–8%, which restricted the PDT action via Type II pathway. Among the breast cancer cell lines tested, only conjugated complexes (29–31) display selectivity against human breast metastatic adenocarcinoma cells (MDA-MB-468) cells (Table 10). Moreover, the process of photoirradiation (365 nm, 2.5 minutes, 0.258 J cm⁻²) predominantly reduces the IC₅₀ values for the tested cells. Evidently, the inclusion of Arg–Trp imparted the targeting effects of PSs on cancer cells, whereby the overall cytotoxic activity improves proportionally according to the peptide length.⁶⁸

Table 10. IC₅₀ and SI values for compounds 28–31 and cisplatin and in varying breast cancer cells in dark and light conditions (365 nm, 2.5 minutes, 0.258 J cm⁻²)⁶⁸.

Compounds	Condition	Cell lines
		MCF-7		MDA-MB-231		MDA-MB-468		MCF-10A
		IC50a (µM)	SIb	IC50a (µM)	SIb	IC50a (µM)	SIb	IC50a (µM)
28	Dark	>100	—	>100	—	>100	—	>100
	365 nm	>100	—	>100	—	>100	—	>100
29	Dark	93.08 ± 3.74	0.9	71.48 ± 6.61	1.2	31.61 ± 0.24	2.6	82.33 ± 3.91
	365 nm	75.73 ± 13.96	1.0	60.66 ± 1.84	1.3	11.57 ± 1.37	6.7	77.69 ± 1.99
30	Dark	19.24 ± 1.93	3.4	29.75 ± 3.43	2.2	15.11 ± 1.26	4.3	64.72 ± 1.38
	365 nm	17.35 ± 1.69	3.6	25.56 ± 2.29	2.4	7.28 ± 1.44	8.5	61.60 ± 5.08
31	Dark	15.34 ± 1.08	2.6	23.62 ± 2.92	1.7	7.92 ± 0.54	5.1	40.46 ± 3.18
	365 nm	15.51 ± 1.96	2.3	25.00 ± 0.04	1.5	5.44 ± 0.36	6.7	36.26 ± 3.99
Cisplatin	Dark	20.87 ± 1.17	1.7	36.59 ± 1.77	1.7	6.97 ± 0.09	5.0	34.56 ± 2.73
	365 nm	23.40 ± 0.58	1.5	33.68 ± 0.23	1.5	6.82 ± 0.23	5.2	35.55 ± 2.16

^aIC₅₀ = half-maximal inhibitory concentration.

^bSelectivity index (SI) represents the ratio between IC₅₀ of MCF-10A and IC₅₀ of MCF-7, MDA-MB-231, and MDA-MB-468 respectively.

7. Phen-based Ru complexes

7.1. Fundamental structural alterations influencing the phototoxic impact of Ru polypyridyl complexes

Roy and collaborators investigated a collection of halogenated Ru(ii) polypyridyl complexes to determine their phototoxicity as shown in Fig. 5. All the complexes were non-toxic when tested in the dark, indicating their viability as PDT agents. After being exposed to light irradiation (420 nm, 20 minutes, 9.27 J cm⁻²), HeLa cancer cells were more sensitive to the lethal effects of complex 32 (IC₅₀ = 5.45 ± 0.35 µM) than those of complex 33 (IC₅₀ = 55.4 ± 8.4 µM), despite both complexes containing the dipyridophenazine (dppz) ligand.⁶⁹ The dppz ligand is a phen derivative that features modified phenyl groups at the 4 and 7 positions, altering the core structure of the phen. This alteration enables the phen structure to enhance their lipophilicity. In addition, an increase in the atomic radius of the halogen group (from Cl to F) can significantly reduce the phototoxicity of the complex. The underlying reason is that an enlarged halogen group enhances the complex's bulkiness, which consequently impacts its binding capacity to DNA, a critical factor for anticancer activity.⁶⁹

Fig. 5. Examples of phen-based Ru complexes 32–39.

In a separate investigation, Fayad and her team developed a series of light-sensitive Ru(ii) polypyridyl compounds as potential candidates for PACT. Complex 34 was shown to be the most effective, with IC₅₀ values of 1.25 ± 0.17 µM and 0.74 ± 0.20 µM against A549 and MDA-MB-231 cells, respectively (Fig. 5). Complex 35 was less effective, with an IC₅₀ value of 2.9 ± 0.83 µM for A549 and 18.22 ± 4.3 µM for MDA-MB-231. The key factor contributing to the enhanced efficacy of complex 34 was attributed to the presence of the hydrophobic 4,7-diphenyl-1,10-phenanthroline ligands. The hydrophobic nature of these ligands facilitated superior cellular uptake and DNA intercalation, ultimately resulting in increased phototoxicity (460 nm, 30 minutes, 180 J cm⁻²). Impressively, complex 34 not only exhibited cancer-targeting properties but also demonstrated a remarkable ability to generate ROS.⁷⁰

Subsequently, Wang et al. investigated Ru(ii) polypyridyl complexes bearing β-carboline for mitochondrial adenosine triphosphate synthase (ATPase) targeted PDT (365 nm, 20 minutes) containing varying bidentate ligands (Fig. 5). As summarised in Table 11, in comparison to clinical drug of cisplatin, the cytotoxicity evaluation demonstrated that complex 36 possessed stronger anticancer effect and high selectivity against cancerous cell lines including HeLa, A549, A549R, human prostate adenocarcinoma cells (PC-3), MDA-MB231, and human glioblastoma cells (U87) (IC₅₀ = 2.1 ± 0.8–14.7 ± 0.7 µM), with moderate toxicity against healthy human primary lung fibroblast cells (HLF) (IC₅₀ = 33.9 ± 0.2 µM). In contrast, complex 37 had a weaker cytotoxicity effect against cancer cell lines (IC₅₀ = 2.5 ± 0.4–13.4 ± 0.4 µM), but higher toxicity against HLF cells (IC₅₀ = 23.2 ± 0.3 µM), thus reducing the selectivity. Overall, complex 37 modified with a photoaffinity tag revealed that complex 36 targets ATPase for inhibition of angiogenesis and epithelial-to-mesenchymal transition, subsequently eradicating cancer cells via ferroptosis and mitophagy induction.⁷¹

Table 11. IC₅₀ values for complex 36, complex 37 and cisplatin in multiple cancer cell lines⁷¹.

IC50a (µM)	Complex 36	Complex 37	Cisplatin
A549	2.1 ± 0.8	2.5 ± 0.4	5.9 ± 1.0
A549R	3.3 ± 0.7	4.2 ± 0.6	28.1 ± 4.2
HeLa	3.1 ± 0.7	3.8 ± 0.1	10.3 ± 1.3
HLF	33.9 ± 0.2	23.2 ± 0.3	15.4 ± 1.1
MDA-MB-231	7.7 ± 0.2	8.4 ± 0.6	9.0 ± 0.2
PC-3	2.7 ± 0.4	3.4 ± 0.6	13.1 ± 2.7
U87	14.7 ± 0.7	13.4 ± 0.4	14.8 ± 1.2
SIb	16.1	9.3	2.6

^aIC₅₀ = half-maximal inhibitory concentration.

^bSI represents the ratio between IC₅₀ of HLF and IC₅₀ of A549.

In a study conducted by Mansour et al., Ru(ii) polypyridyl complex (38) bearing a 2,9-diphenyl-1,10-phenanthroline and bpy groups were synthesised and assessed against A549 lung cancer cells and human mesenchymal stem cells (MSCs) as depicted in Fig. 5. In dark conditions, complex 38 did not demonstrate any cytotoxicity (IC₅₀ > 100 µM) against both A549 and MSCs cells. On the contrary, light activation resulted in phototoxicity effect against A549 cells (IC₅₀ = 2.6 ± 1.7 µM) and remained inactive towards MSCs cells (IC₅₀ > 100 µM). Photoirradiation (460 nm, 40 minutes, 240 J cm⁻²) of complex 38 formed complex 39 bearing positive charge, which possess high affinity towards the cancer cell membranes with negative charges. Evidently, the high selectivity of complex 38 against A549 cells was supported by the high PI (PI > 39). Upon endocytosis, complex 39 eradicated A549 cells by disrupting the cellular morphology and integrity, damaging DNA, inhibiting cell attachment, and inducing apoptosis.⁷²

Exploring the potential of dppz-containing Ru(ii) polypyridyl complexes, Gandosio et al. investigated a compound featuring a 7-methoxy substitution, which demonstrated a promising PI of 42 against HeLa cells under 420 nm light exposure for 20 minutes at 9.27 J cm⁻². Subsequently, two versions of the [Ru(phen)₂dppz]²⁺ complexes with different dppz ligands functional groups (dppz-7,8-(OMe)₂] (40) and [dppz-7,8-(OH)₂] (41) were synthesised to investigate the effects of these small modifications on the photophysical properties and phototoxicity of the complexes, as shown in Fig. 6. Compounds 40 and 41 displayed similar absorption spectra in the 400–500 nm visible light range, characteristic of MLCT. The lipophilic compound 40 was absorbed by HeLa cancer cells, subsequently distributed uniformly throughout the cytoplasmic and nucleic regions. Alternatively, the complex 41 bearing hydroxyl groups predominantly accumulated in the cell membranes, exhibiting approximately three times lower uptake efficiency. Complex 40 showed toxicity in the dark against HeLa cells (IC₅₀ = 36.5 µM), while complex 41 was non-toxic. Interestingly, upon exposure to 420 nm light, both complexes demonstrated increased cytotoxicity, with respective PI values of 12 and 6. Moreover, HeLa cell spheroids were sensitive to complex 40, presenting an IC₅₀ value of 9.5 µM and a PI of 11 when subjected to 800 nm light, a consequence of two-photon absorption.⁷³

Fig. 6. Ru(ii) complexes 40 and 41 with detailed chemical structures for use in PDT.

7.2. Impact of conjugation on the phototoxic effect of Ru complexes

A research team led by Lu Y. has investigated the effects of altering the electron-donating properties of 1,10-phenanthroline ligands on enhancing the performance of Ru(ii) polypyridyl complexes. They created a series of novel Ru(ii) complexes and assessed their cytotoxicity against various cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The chemical structures are illustrated in Fig. 7, and the corresponding IC₅₀ values are presented in Table 12. Among these compounds, complex 45 demonstrated the highest potency as an inhibitor. The IC₅₀ value for compound 42 was larger than 50 µM, rendering it ineffective, while the IC₅₀ value for complex 43 was only 31.51 ± 3.21 µM, indicating promising cytotoxicity against human lymphoblastic leukemia cells (CEM). The cytotoxicity of Complex 44 was low when tested on two different cell lines, human colorectal carcinoma cells (HCT-116) and CEM. The research shows that optimising the physicochemical profiles of the complexes can be achieved by altering the ligands' substituent structures and regulating the electron distribution among Ru(ii) ligands to achieve the required biological activity. Complex 45 presents a favourable basis for the creation of Ru(ii)-based cancer treatments, owing to its capacity for specific interactions with proteins and DNA.⁷⁴

Fig. 7. Chemical structures of target compounds (42–45).

Table 12. IC₅₀ values for complexes in comparison with 5 different cell lines from MTT assay⁷⁴.

IC50a (µM)	Complex 42	Complex 43	Complex 44	Complex 45
HCT-116	>50	>50	43.17 ± 3.84	3.19 ± 0.46
A549	>50	>50	>50	7.87 ± 1.04
HepG2	>50	>50	>50	9.86 ± 0.67
Human promyelocytic leukemic cells (HL60)	>50	>50	>50	3.19 ± 0.62
CEM	>50	31.51 ± 3.21	46.20 ± 3.40	4.60 ± 0.93

^aIC₅₀ = half-maximal inhibitory concentration.

Mascheroni and colleagues presented a study involving two distinct polyamidoamines ligands and their corresponding Ru(phen)₃²⁺ complexes as displayed in Fig. 8. Interestingly, the Ru complex combined with cationic polyamidoamine ligands (46) showed improved cellular uptake, while the Ru complex containing zwitterionic polyamidoamine ligands (47) demonstrated reduced uptake efficiency. The superior cellular uptake and nuclear accumulation observed in complex 46 contributed to its enhanced PDT efficacy. When tested against HeLa cells, complex 46 exhibited a high phototoxic effect (half-maximal effective concentration, EC₅₀ = 0.7 µΜ) and low dark toxicity (EC₅₀ > 5 µM), whereas complex 47 displayed minimal toxicity (EC₅₀ > 50 µM) in the absence of light but no significant change in cytotoxicity upon LED light irradiation with 400–800 nm for 40 minutes at 56.88 J cm⁻².⁷⁵

Fig. 8. Chemical structures of target compounds (46–63).

In a separate study by Silva-Calderia and colleagues, they investigated two distinct phenanthroline-hydrazone ligands and their corresponding Ru complexes (Fig. 8).⁷⁶ The findings revealed that incorporating a hydrazone moiety with different atom substitutions in the heterocyclic compound within the Ru complex considerably decreased cancer cell proliferation, particularly in K562 cells. Heterocycles are significant in drug design development, as they are found in most drugs available on the market. Nitrogen, oxygen, and sulfur-based heterocyclic structures have been recognised for their role in enhancing the pharmacokinetics and pharmacodynamics of anticancer drugs by improving properties such as lipophilicity, polarity, and other physicochemical features.^76,77 In low light conditions, complex 48 displayed IC₅₀ values of 55.06 ± 5.5 µM, while complex 49 exhibited IC₅₀ values of 51.81 ± 5.2 µM.⁷⁶ Notably, both complexes demonstrated a threefold decrease in IC₅₀ values of 18.82 ± 1.9 µM for complex 48 and 17.26 ± 1.8 µM for complex 49 when subjected to photoirradiation (365 nm, 5 minutes, 0.183 J cm⁻²).^76,77 Importantly, the cytotoxic effects of these complexes were found to be concentration-dependent.

Karges and his team introduced a Ru polypyridyl compound with dual aldehyde groups, designated as complex 50, for potential use as a PDT PSs (Fig. 8). The experiments demonstrated that this complex exhibited strong luminescence properties and an elevated ¹O₂ quantum yield. Following this, complex 50 was combined with benzylamine units to create conjugate 51. This new conjugate did not possess dark toxicity (IC₅₀ > 100 µM) but showed significant cytotoxic effects when exposed to light (IC₅₀ = 18.2 ± 0.3 µM) in tests against HeLa cells. On the other hand, complex 50 had reduced cytotoxicity when exposed to light (IC₅₀ = 81.3 ± 8.2 µM) and no cytotoxicity in dark conditions (IC₅₀ > 100 µM). Additionally, conjugate 51 phototoxic impacts spanned a range of 450 nm to 540 nm for 10 to 40 minutes with 3.1 to 9.5 J cm⁻², suggesting an enhanced penetration depth potential during PDT applications.⁷⁸

In an investigation conducted by Roque III and collaborators, varying Ru(ii) complexes containing 2,9-dimethyl-1,10-phenanthroline and imidazo[4,5-f][1,10]phenanthroline (complexes 52–57) were synthesised with up to four conjugated thiophene rings (Fig. 8). The inclusion of additional thiophene groups resulted in the bathochromic shift of λ_max with complex 57 having the longest λ_max of 488 nm due to the reduced HOMO–LUMO energy gap. Additionally, the transient absorption during excited state of complex 57 was extended NIR region of 675–700 nm. The oligothiophene also attributed to the enhanced production of ¹O₂, however the presence of steric methyl groups on phen limited the ligand dissociation and quantum yield of ¹O₂. Under normoxic conditions, complex 57 displayed low cytotoxicity (EC₅₀ = 67.6 µM) without light irradiation against human malignant melanoma cells (SK-MEL-28). Impressively, exposure of 400–700 nm visible light (75–92.5 minutes, 100 J cm⁻²) on complex 57 resulted in intense cytotoxicity of EC₅₀ = 82.4 pM, whereas photoirradiation at 523 nm and 633 nm (75–92.5 minutes, 100 J cm⁻²) also demonstrated high cytotoxicity (EC₅₀ = 1.79 nM and 1.80 µM respectively). Under hypoxic conditions, light activation of complex 57 at visible light and 523 nm were EC₅₀ = 2.09 nM and 7.20 nM respectively. Overall, the PI for complex 57 was significantly higher than the other active complex 56 as shown in Table 13. Despite having reduced photosubstitution reaction due to the increased the intersystem crossing pathways caused by oligothiophene structure, complex 57 remained the most potent, which proved that the complex 57 acts as excellent PDT agent.⁷⁹

Table 13. EC₅₀ values and PI for active Ru(ii) complexes in different conditions against SK-MEL-28 melanoma cell line (400–700 nm, 75–92.5 minutes, 100 J cm⁻²).⁷⁹.

Complexes	Oxygen %	Cell viability assay (SK-MEL-28)
		EC50a ± SEMb (µM)				PIc
		Dark	400–700 nm	523 nm	633 nm	400–700 nm	523 nm	633 nm
56	18.5	50.7 ± n.dd	0.0294 ± 0.004	0.0771 ± 0.0046	12.5 ± 0.3	1724	658	4
	1	50.4 ± n.dd	0.320 ± 0.070	0.370 ± 0.035	50.6 ± n.dd	158	136	1
57	18.5	67.6 ± 0.8	(8.24 ± 3.38) × 10−5	(1.79 ± 0.18) × 10−3	1.80 ± 0.04	820 388	37 765	38
	1	69.5 ± 1.1	(2.09 ± 0.25) × 10−3	(7.20 ± 0.70) × 10−3	67.9 ± 1.9	33 254	9653	1

^aEC₅₀ = half-maximal effective concentration.

^bSEM = standard error of the mean.

^cPI represents the ratio between IC₅₀ values of complexes in dark and light conditions.

^dn.d. = not determined.

In the latest research initiated by de la Torre-Rubio et al., a series of photoactive Ru(ii) phen complexes (58–63) were synthesised which included N-glycopiranosylamines in aim to achieve enhanced cancer targeting effects via glycoconjugation (Fig. 8). The presence of carbohydrate groups improved the photostability, aqueous solubility, and cellular uptake of complexes. In comparison to the standard PSs of rose Bengal (Φ_Δ = 0.53), complexes 59–63 produced lower ¹O₂ quantum yields (Φ_Δ = 0.11–0.37) except for complex 58 (Φ_Δ = 0.55). Intriguingly, the ¹O₂ quantum yields did not correlate to the phototoxicity of complexes in MCF-7, PC-3, and non-cancerous human foreskin fibroblast cells (HFF-1). As shown in Table 14, the complexes did not exhibit dark toxicity within all three cell lines (IC₅₀ > 500 µM) but displayed moderate phototoxicity with high PI ranging from >9 to >44. Based on the trypsin resistance assay, cells treated with complexes and 30 minutes photoirradiation (433–480 nm) exhibited improved resistance to detachment by trypsin compared to control (36–84% remain attached). Further supported by wound healing assays, PC-3 cells treated and photoirradiated with complexes 60, 62, and 63 demonstrated wound closure between 53 to 65% after 48 hours recovery, but controls were fully healed. Evidently, the glycoconjugated Ru(ii) complexes influence metastatic cascade processes upon photoirradiation, especially on the detachment and migration abilities of cancer cells.⁸⁰

Table 14. IC₅₀ values, PI and SI for complexes 58–63 and reference drug of cisplatin against MCF-7, PC-3, and HFF-1 cells with and without photoirradiation (433–480 nm, 30 minutes).⁸⁰.

Cancer cell line	Conditions	Compounds, IC50a (µM)
		58	59	60	61	62	63	Cisplatin
MCF-7	Dark	>2000	>2000	>2000	>1000	>2000	>500	9.8 ± 1.05
	433–480 nm	104.2 ± 1.05	113.1 ± 1.04	108.1 ± 1.05	68.82 ± 1.04	67.47 ± 1.07	56.75 ± 1.22	NAd
	PIb	>19	>18	>18	>14	>30	>9
	SIlightc	1.0	0.9	0.9	0.6	0.7	0.7
PC-3	Dark	>1000	>1000	>2000	>500	>1000	>500	16.52 ± 1.05
	433–480 nm	101.6 ± 1.04	104.1 ± 1.03	47.61 ± 1.09	33.31 ± 1.11	22.47 ± 1.11	58.06 ± 1.07	NAd
	PIb	>10	>10	>42	>15	>44	>9
	SIlightc	1.4	1.0	1.8	1.1	2.1	0.7
HFF-1	Dark	>1000	>2000	>2000	>2000	>1000	>1000	NAd
	433–480 nm	143.6 ± 1.07	105.6 ± 1.05	86.87 ± 1.03	39.74 ± 1.09	47.82 ± 1.08	41.13 ± 1.04

^aIC₅₀ = half-maximal inhibitory concentration.

^bPI represents the ratio between IC₅₀ values of complexes in dark and light conditions.

^cSI^light represents the ratio between IC₅₀ of HFF-1 and IC₅₀ of PC-3 and MCF-7 in light conditions respectively.

^dNA = not available.

7.3. Mixed-ligand Ru complexes

The research team led by Li examined various mixed ligand Ru derivatives as possible PDT candidates and their chemical structures are depicted in Fig. 9. Compound 64 exhibited substantially higher phototoxic effects (IC₅₀ of A549 = 56 nM, IC₅₀ of HepG2 = 63 nM) compared to compound 65 (IC₅₀ of A549 = 53.1 µM, IC₅₀ of HepG2 = 49.1 µM) when irradiated with 650 ± 10 nm red light at 144 J cm⁻². The observed phototoxic impact could be linked to the inclusion of the 3,6-bis(2′-pyridyl)pyridazine (dppn) ligand, which has the ability to intercalate with DNA due to its flat conjugated structure.⁸¹ Complex 64 demonstrates significant phototoxicity as a result of its ability to cleave DNA by photodamage. Complex 66 has an elevated production of ¹O₂, which is linked to a potent cytotoxic impact. The only plausible explanation for this impact is the presence of BODIPY moieties.⁷⁷ Complex 66 exhibits a heightened anticancer effect due to its amphiphilic polyethylene glycol (PEG) groups, which facilitate cellular uptake. Studies on PDT applications have demonstrated that utilising a mixed ligand conjugation method might significantly enhance their advancement.⁸²

Fig. 9. Structure of mixed-ligand Ru complexes (64–66).

8. Tpy-based Ru complexes

8.1. Fundamental structural alterations influencing the phototoxic impact of Ru polypyridyl complexes

A research initiated by Ryan et al. involved the synthesis of Ru(ii) complexes bearing tridentate ligands of tpy as shown in Fig. 10. In comparison to the λ_max of reference compound [Ru(bpy)₃]²⁺ (λ_max = 450 nm), complex 67 and 68 displayed an extended λ_max of 475 and 495 nm respectively, contributed by the extended π-system of polypyridyl rings. However, the cytotoxicity evaluation against HL60 cells demonstrated that complex 67 had low dark toxicity (IC₅₀ ∼ 65 µM), low phototoxicity at 405 nm for 0.5 minutes at 17.4 J cm⁻² (IC₅₀ ∼ 50 µM) and 450 nm for 1 minute at 29.1 J cm⁻² (IC₅₀ ∼ 50 µM). Complex 68 was inactive in both conditions (IC₅₀ > 100 µM). On the contrary, [Ru(bpy)₃]²⁺ was inactive in dark conditions (IC₅₀ > 100 µM), but highly potent at 405 nm (IC₅₀ = 5.2 ± 1.0 µM) and 450 nm (IC₅₀ = 0.3 ± 0.03 µM). Generally, tpy are weak field ligands which contribute to small orbital splitting, thus forming destabilised excited states. Consequently, fast quenching of triplet excitation states hinders ROS generation of tpy complexes, limiting the phototherapeutic action.⁸³

Fig. 10. Examples of tpy-based Ru complexes 67–78.

Similarly, Karges and collaborators investigated the antiproliferative activity of Ru(ii) tpy complexes with varying substituents (complexes 69–75) against RPE-1 and HeLa cancer cell lines (Fig. 10). All complexes except for complex 74 were stable in human plasma. During cytotoxicity evaluation, complex 69–74 were inactive in both dark and light conditions for RPE-1 and HeLa cells. Conversely, complex 75 had moderate toxicity in the dark (IC₅₀ = 39.7 ± 3.6 µM) and upon irradiation at 480 nm for 10 minutes of 3.1 J cm⁻² (IC₅₀ = 27.5 ± 1.1 µM) for RPE-1 cells. For HeLa cells, complex 75 also exhibited similar range of dark toxicity (IC₅₀ = 35.1 ± 0.6 µM) and phototoxicity (IC₅₀ = 24.5 ± 2.6 µM). Nevertheless, the individual tpy ligands of complex 69–72 and 75 displayed a better range of cytotoxicity in dark (IC₅₀ = 8.7 ± 0.8 to 40.9 ± 2.1 µM) and light conditions (IC₅₀ = 6.9 ± 0.1 to 40.2 ± 0.3 µM). The insignificant effect of Ru(ii) tpy complexes could be explained by the short-lived excited triplet states, which limited the PSs effect. Overall, the activity of Ru(ii) complexes was significantly impacted by changes of tpy substituents.⁸⁴

Recently, the team led by Mandal designed several tpy-based Ru(ii) complexes (76–78), which functioned dually as PSs and photocatalysts (Fig. 10). Upon exposure to green light at 525 nm (30 minutes, 5.0 J cm⁻²), complexes 78 exhibited the most significant increase in nicotinamide adenine dinucleotide (NAD⁺) and decrease in reduced NAD⁺ (NADH) absorbance band, indicating the stimulation of oxidation by photoirradiation. Complex 78 possessed the smallest energy gap (ΔE_g) which allowed the generation of H₂O₂ from O₂ and enhanced photocatalysis process. Likewise, the excellent intracellular ROS generation capacity of complex 78 was indicated by the intense fluorescence of ROS marker upon green light-activation. The presence of additional conjugated hydrophobic groups enhanced the lipophilicity of complex 78, thus yielding the highest cellular uptake. Complex 78 altered membrane potential (ΔΨ_m) which resulted in disruption and damage of mitochondria, leading to apoptosis. The overall photocatalytic and photosensitising nature of complex 78 attributed to the excellent phototoxicity compared to other tested compounds as shown in Table 15.⁸⁵

Table 15. IC₅₀ values and PI for complexes upon photoirradiation (400–700 nm; 525 nm, 30 minutes, 5.0 J cm⁻²) against splenocytes and HeLa cell line⁸⁵.

Cell viability assay	Splenocytes	HeLa
	IC50a ± SEMb (µM)				PIc
Complexes	Dark		400–700 nm	525 nm	400–700 nm	525 nm
76	55.6 ± 1.3	94.2 ± 2.8	6.8 ± 0.2	8.5 ± 5.0	13.77	11.11
77	79.0 ± 3.0	95.5 ± 2.0	> 10	> 10	NAd	NAd
78	75.7 ± 2.2	66.2 ± 2.0	0.75 ± 0.12	1.9 ± 0.2	88.37	34.52
Cisplatin	NAd	16.5	NAc	25.3	NAd	NAd
ALA	NAd	> 10 000	NAc	151.1	NAd	6.6

^aIC₅₀ = half-maximal inhibitory concentration.

^bSEM = standard error of the mean.

^cPI represents the ratio between IC₅₀ values of complexes in dark and light conditions.

^dNA = not available.

8.2. Impact of conjugation on the phototoxic effect of Ru complexes

In efforts to resolve the photoreactivity and geometrical issues of Ru(ii) tpy complexes, Liu et al. proposed a series of Ru(ii) complexes (79–81) bearing varying conjugated groups as displayed in Fig. 11. The introduction of oligoether promoted the aqueous solubility, whereas BODIPY and pyrene moiety improved the photophysical properties. Comparatively, complex 80 exhibited the most prolonged excited triplet state lifetime and enhanced ROS production followed by complex 81, then only 79. Evidently, the phototoxicity assessment in A549 lung cancer cells demonstrated that complex 80 was significantly more active (EC₅₀ in dark = 53.0 µM, EC₅₀ at 500 nm, 1 minute, 0.048 J cm⁻² = 23.9 µM, EC₅₀ at 500 nm, 10 minutes, 0.48 J cm⁻² = 1.50 µM) compared to complex 81 (EC₅₀ in dark = 36.3 µM, EC₅₀ at 500 nm, 1 minute, 0.048 J cm⁻² = 11.1 µM, EC₅₀ at 500 nm, 10 minutes, 0.48 J cm⁻² = 7.41 µM). Intriguingly, complex 81 was mainly localised within lysosomes, which upon photoirradiation triggered lysosome damage and mitochondrial membrane potential alteration.⁸⁶

Fig. 11. Examples of tpy-based Ru complexes 79–88.

Moura and his team devised a strategy to refine the photophysical properties of Ru(ii) tpy complexes through conjugation of natural PSs of porphyrin to complex 82, forming complex 83 and 84 (Fig. 11). The quantum yield of ¹O₂ produced was notable for all complexes 82–84 (ϕ_Δ = 0.67, 0.89 and 0.65 respectively). Impressively, complex 84 exhibited the longest λ_max at 684 nm. The inclusion of porphyrin augmented the intersystem crossing process via prolonging the excited triplet state of complex 84. The dark cytotoxicity of all complexes was insignificant against mouse melanoma cells (B16F10) (IC₅₀ > 10 µM). Intriguingly, the photodynamic activity was dependent on the dosage of light applied for instance 5 µM of complex 84 eradicated 15% more cells with an increase in light power from 5 to 10 J cm⁻² for 3.42 to 6.83 minutes at 405 nm. At 405 nm light of 10 J cm⁻², 10 µM of complex 83 and 84 exhibited strong anticancer effect (IC₅₀ < 10 µM) with complex 84 inducing 89 ± 2.7% of cell death.⁸⁷

Similarly, a research team led by Paul investigated the function of biotin and BODIPY in boosting the PSs effect of Ru(ii) tpy complexes (85 and 86) as shown in Fig. 11. Comparatively, the presence of BODIPY chromophore enhanced the molar absorption coefficient of complex 86 three-fold higher than complex 85. Complex 86 characterised with dyad system formed by BODIPY core and biotin had augmented ¹O₂ generation capacity (ϕ_Δ = 0.63), which promoted photocleavage of DNA. Moreover, the conjugated biotin group also improved targeting ability and cellular uptake of complex 85via biotin-mediated receptor endocytosis, subsequently accumulating in cellular lysosomes. For cytotoxicity evaluation against HeLa, human lung large carcinoma cells (H1299), and human embryonic kidney cells (HEK293), both complex 85 and 86 did not exhibit dark cytotoxicity (IC₅₀ > 100 µM). Upon visible light activation (400–700 nm, 30 minutes, 5 J cm⁻²), complex 85 remained inactive against all cells (IC₅₀ > 100 µM). Contrarily, complex 86 demonstrated remarkable phototoxicity against HeLa, H1299, and HEK293 (IC₅₀ = 0.17 ± 0.015 µM, 0.57 ± 0.062 µM, 3.20 ± 0.41 µM respectively) with PI ranging between > 18 to 625. Fascinatingly, complex 86 displayed the capability to allocate into multicellular tumour spheroids, indicating the penetration across extracellular matrix into solid tumours.⁸⁸

Recently, Wang and collaborators modified the structure of complex 87 through conjugation of donor–acceptor-donor (DAD) moiety (Fig. 11). Complex 88 possessed two Ru(tpy)₂ cores linked with the chromophore group, resulting in a bathochromic shift with an intense absorption peak at 521 nm. Consequently, the extended conjugation system enhanced ROS generating ability through electron transfer via intramolecular route upon photoirradiation. Complex 88 also displayed remarkable phototoxicity at 595 nm for 30 minutes with 28.26 J cm⁻² against non-functioning pituitary adenomas (NFPAs) and human pituitary tumour derived folliculostellate (PDFS) with IC₅₀ values of 3.36 µM and 0.90 µM respectively. In comparison, clinically applied drug of temozolomide was inactive against PDFS with IC₅₀ value of 591.0 µM. Additionally, complex 88 possessed excellent antiproliferative activity against PDFS in light conditions. Intriguingly, mechanistic studies revealed that binuclear complex 88 attenuated the activity of nicotinamide adenine dinucleotide phosphate (NADPH), thus disrupting redox balance and causing oxidative damage, eventually triggering cellular apoptosis.⁸⁹

9. Recent advances in PDT applications for cancer treatment

PDT is a cutting-edge, non-invasive treatment approach that has captured the attention of the medical community for its diverse applications. Combining light-sensitive compounds called PSs, specific wavelengths of light, and oxygen, PDT triggers a series of photochemical reactions that can selectively target and destroy diseased cells.^17,90 This innovative therapy has been successfully applied in various medical fields, such as oncology for targeted cancer treatment, dermatology for skin conditions, urology for bladder and prostate issues, ophthalmology for age-related macular degeneration, and dentistry for oral infections.^91,92 One of the primary strengths of PDT lies in its precision in targeting cancerous cells while minimising harm to healthy tissue. In 1976, individuals with bladder cancer participated in the first PDT human studies.^12,92 Promising results were seen in multiple trials for patients with early-stage lung, oesophagus, and gastric cancers. Cancers of the brain, eyes, breast, head, and neck, pancreas, and genitourinary system have all been successfully treated with PDT.^92,93

The fundamental tactic for improving PDT action of PSs is through tailoring the structure of PSs to include additional targeting or therapeutic functionality. In a notable advance, Dong's team synthesised two Ru(ii) complexes of bpy and phen respectively, each containing a specific ligand of 4-nitro-3-trifluoromethylaniline-1H-imidazo[4,5-f][1,10]phenanthroline (NFIP).⁹⁴ The terminal nitro group functioned as a nitric oxide (NO) donor upon photoirradiation. Ultimately, combined ROS and NO production effectively induced HeLa cancer cell death via caspase-mediated pathway and mitochondrial damage.⁹⁴ This highlights the potential of including combinational photoresponsive functional groups to be released upon light induction for therapeutic applications.

Nanotechnology-based PDT has emerged as a novel approach to treat cancer. By modifying PSs with nanotechnology or delivering them via nanocarriers like NPs, nanoliposomes (NLPs), and nanohydrogel particles, the targeting, specificity, and efficacy of PDT against tumours can be improved.^95,96 Examples of NPs are gold NPs (AuNPs), quantum dots (QDs), superparamagnetic iron oxide NPs (SPIONs), and upconverting nanomaterials.^95,96 Tumourous angiogenesis has a different structure and appearance than normal blood vessels, with wider gaps between endothelial cells. As tumours do not possess lymphatics, there is no lymphatic reflux or enhanced permeability and retention effect. As a result, small particles like NPs can selectively accumulate in tumour sites for an extended period, increasing the effectiveness of PDT while minimising side effects in normal tissues surrounding the tumour.⁹⁵ Due to their minimal toxicity and chemical inertness, AuNPs have been investigated for several biological uses.⁹⁵ Li et al. utilised hexadecyl dimethylammonium bromide-coated gold nanorods (AuNRs) carrying positive groups and negatively charged sulfonated aluminium phthalocyanines (AlPcS) to electrostatically bond and create AuNRs-AlPcS.⁹⁵ The products generated a five-fold higher fluence of fluorescence and triggered light dose-dependent PDT-induced cytotoxicity in liver cancer cells (QGY). Simultaneous enhancement in photodynamic detection and treatment efficacy was achieved due to a synergistic reaction.⁹⁵

PDP is also a new treatment strategy that combines subtherapeutic photochemistry with biological inhibitors to treat advanced cancer.⁹⁷ This method is gaining attention due to its therapeutic benefits and potential for approval as a combination regimen. The process works by using light to trigger a PSs molecule, which then produces ROS capable of oxidising biomolecules. PDT is known to cause direct cell death through this method, but sub-lethal effects are also achievable with PDP.⁹⁷ These effects have a wide range, including vascular modulation, chemo-sensitisation, and antitumour immune activation, and are currently being actively studied. A recent study has shown that PDP can boost the effectiveness of an anti-migratory agent that targets the prostaglandin E receptor 4 in ovarian cancer cells. This promising development offers hope for the treatment of ovarian cancer.⁹⁷

The clinical field has widely used X-rays for imaging and radiation therapy because of their ability to penetrate tissues deeply.⁹⁸ A new derivative of PDT, known as X-ray-induced PDT (X-PDT), has been developed to overcome the limitations of shallow penetration by utilising X-rays as an energy source. The combination of PDT and radiation therapy work together to attack both the cell membranes and DNA, resulting in lethal damage that cannot be repaired by cells.⁹⁸ This synergy is what makes X-PDT more effective than radiation therapy alone, even for radioresistant cells. Studies have shown that X-PDT can effectively kill H1299 cells, which are known to be radioresistant NSCLC cells.⁹⁸ The improved efficacy has been noticed both in vitro and in vivo with a subcutaneous tumour model or when H1299 cells were implanted into the lung percutaneously. Besides, it was also demonstrated that mesoporous silica NPs co-loaded with SrAl₂O₄:Eu²⁺ NPs (SAO:Eu) and MC540 (a PSs) can convert X-ray photons to visible photons and produce ¹O₂ under X-ray radiation.⁹⁸ This leads to efficient cancer cell death, even when the cells are located beneath thick tissues. These findings have significant implications for the development of X-PDT as a viable treatment option for cancer patients.⁹⁸

In addition to its use in oncology, PDT has demonstrated its effectiveness in various other areas, including the treatment of bacterial, dental infections and ensuring food safety. This highlights the wide range of applications for PDT's principles.^12,99–102 Nevertheless, the focus on cancer treatment using advanced PDT strategies continues to be a crucial area of interest, in line with current research trends and the aim of maximising the therapeutic potential of this state-of-the-art technology. With the ongoing advancements in research and clinical applications, PDT is proving to be a promising avenue for more targeted and effective cancer treatments, offering hope to patients worldwide.

10. Future perspective

Ru(ii) complexes have illustrated their great potential as an anticancer agent in PDT. Nevertheless, current efforts to address PDT limitations are immensely complicated which results in the slow progression of this sector. A few areas should be taken into consideration for future improvement of PDT therapy. First is the design of highly selective PSs and PSs-vehicle conjugates for cancer over healthy tissues (yet sufficiently general for application against varying types of cancer cells), which is safe and effective for systemic administration.^40,103 Next would be the application of X-ray-activatable PSs which involve the concept of radiotherapy and PDT for aggressive tumours.^40,103 Lastly, the advancement of PDT/PACT regimens which can provoke an antitumour immune response enables the transition of local PDT treatment to targeted therapy for metastatic tumours.⁴⁰ These perspectives highlight the significant progress achieved in this field, along with the opportunities and challenges encountered, thus making them well worth exploring.

Additionally, the PDT evaluation within in vitro cell culture models is insufficient to provide accurate predictive values of PDT as the tissues layers and tumour complexity is poorly represented by monolayer cells.¹⁰⁴ Growing interest has emerged in applying spheroids and organoids to mimic three-dimensional spatial arrangement of organs. This simulates a physical barrier against light penetration, which provides better insights into wavelength selection for PDT. The assessment for PDT in vivo should also be emphasised to determine the safety, selectivity, biodistribution, and PI of novel PDT agents.¹⁰⁴

By delving into strategies emphasised by researchers, one can gain valuable insights into shaping the future design of molecules. Using two-photon PDT, an advanced technique that enhances the effectiveness of Ru complexes in PDT, is one such approach.^3,105 Two-photon absorption (TPA) is a fascinating nonlinear optical phenomenon that enables the activation of PSs.^3,105 These PSs have the unique ability to absorb light of short wavelengths by simultaneously absorbing two photons with lower energies. Hence, this will effectively double the absorbed energy without the need for higher energy single photons. In contrast to single-photon absorption techniques, TPA has the ability to penetrate deeper into tissues, allowing for potential treatment of tumours in difficult-to-access areas. Furthermore, the utilisation of TPA allows for precise targeting by delivering laser photons directly to the tumour site, resulting in improved spatial control over the activation of PSs and minimising damage to surrounding healthy tissues.^3,105,106

11. Conclusion

In conclusion, this review highlights the significant potential of Ru polypyridyl complexes as anticancer PDT agents. By exploring the extensive research conducted on these complexes, it becomes evident that the thoughtful selection of ligands and modifications to the coordination environment can lead to improved anticancer efficacy. Moreover, advancements in NPs formulations and combination therapies show promise in further enhancing their therapeutic potential. Although in vitro results have been promising, more in vivo studies are needed to fully assess the clinical applicability of these compounds. This review underscores the importance of continued research to unlock the full potential of Ru polypyridyl complexes in PDT applications, paving the path for innovative and efficient cancer therapies in the future.

Author contributions

The authors have confirmed their contribution to the paper as follows: P. Y. T, K. H. L., K. T. T., and M. L. L. were responsible for study conception and design, while P. Y. T, K. H. L., K. T. T., E. W. J. K. and X. Q. C. handled literature search and data analysis. P. Y. T, K. H. L. and K. T. T. also drafted the manuscript, and W. M. L., C.-W. M., L. C. W. and M. L. L. critically revised the work. Finally, all authors reviewed the paper and approved the final version of the manuscript.

Conflicts of interest

The authors have no competing interests to declare that are relevant to the content of this article.

Biographies

Biography

Lai Chun Wong.

ChM. Dr Lai Chun Wong is a Senior Lecturer in Pharmaceutical Chemistry at IMU University, Malaysia. A Chartered Chemist and Secretary of the Royal Society of Chemistry Malaysia Local Section, her research integrates synthetic medicinal chemistry, analytical method development, and nanomedicine for ocular drug delivery. Her work focuses on the design and evaluation of bioactive molecules with anticancer and antimicrobial potential, as well as formulation strategies to enhance therapeutic efficacy. Dr Wong has secured national and international research grants and actively contributes to peer review, accreditation, and advancing chemical sciences education and professional standards.

Biography

May Lee Low.

ChM. Dr May Lee Low is an Assistant Professor of Pharmaceutical Chemistry and Head of Postgraduate Studies at the Faculty of Pharmaceutical Sciences, UCSI University Malaysia. Her research interests encompass metal-based compounds for therapeutic, imaging, and sensing applications, as well as photodynamic therapy, nanomedicine, analytical and green chemistry, and chemical education. She has published extensively in international peer-reviewed journals and has secured competitive national and international research grants. Dr Low currently serves as the Immediate Past Chair of the American Chemical Society Malaysia Chapter and as a committee member of the Analytical Chemistry Section of the Malaysian Institute of Chemistry.

Data availability

There were no new data generated or analysed in this article.

Supplementary information (SI): photoirradiation conditions of Ru(ii) complexes discussed throughout section 6.1 to section 8.2. See DOI: https://doi.org/10.1039/d6ra01698g.