Encapsulation of Ru(II) Polypyridine Complexes for Tumor-Targeted Anticancer Therapy

Ru(II) polypyridine complexes have attracted much attention as anticancer agents because of their unique photophysical, photochemical, and biological properties. Despite their promising therapeutic profile, the vast majority of compounds are associated with poor water solubility and poor cancer selectivity. Among the different strategies employed to overcome these pharmacological limitations, many research efforts have been devoted to the physical or covalent encapsulation of the Ru(II) polypyridine complexes into nanoparticles. This article highlights recent developments in the design, preparation, and physicochemical properties of Ru(II) polypyridine complex-loaded nanoparticles for their potential application in anticancer therapy.


Introduction
In the developed world, cancer is one of the leading causes for death with about 19.3 million new cases and 10.0 million deaths recorded in 2020.These numbers are projected to surge in the upcoming decades, with an anticipated 28.4 million new cases annually by 2040 [1].The prevalence of cancer has created a pressing need for the development of effective treatment methods.Traditional treatment modalities involve a combination of techniques whereby the tumor is removed in a surgical procedure and the patient is further treated by immunotherapy, radiotherapy, or chemotherapy.Since the discovery of cisplatin in the late 1960s, metal-based drugs have been extensively studied as chemotherapeutic agents.The biomedical mechanism of cisplatin is thought to be related to DNA damage, inhibition of replication and transcription, or a combination of both processes.One of the main factors contributing to the cytotoxicity of cisplatin is the formation of covalent cross-links when it interacts with DNA.The cytotoxicity of cisplatin is further amplified by overwhelming the cellular ability to repair the platinum-DNA adducts.Despite being one of the most widely used and effective chemotherapeutic drugs thus far, cisplatin is associated with severe side effects, including kidney damage, peripheral nerve damage, severe nausea, vomiting, and bone marrow suppression.These side effects, along with the development of tumor resistance, have limited the clinical use of cisplatin [2][3][4][5][6][7].One promising alternative is the development of a new class of compounds such as ruthenium (Ru) complexes.
Over the last decades, the development of Ru complexes as chemotherapeutic agents has received increasing attention because of the generally non-toxic nature of these compounds and the presence of these metal complexes in variable oxidation states.Several Ru complexes have shown clinical potential as anticancer agents.Notably, the compounds imidazolium trans-[tetrachloro(dimethylsulfoxide) imidazole ruthenium(III)] (NAMI-A) and imidazolium trans-[tetrachloro(dimethylsulfoxide) imidazole ruthenium(III)] (KP1019), as well as its sodium salt KP1339, have advanced into clinical trials.NAMI-A, as the first Ru complex to undergo clinical trials, has demonstrated potent tumor growth inhibition effects on primary and secondary metastatic tumors in animal models.Its antitumor mechanism involves enhancing actin-dependent cell adhesion while reducing cell invasion and migration.This results in the disturbance of the communication of the cancer cells with the extracellular matrix.The phase 2 clinical study yielded unsatisfactory results in terms of drug activation against disease progression and adverse effects on patients.These outcomes limited its further clinical development [8][9][10].Subsequently, KP1019, a structural analog of NAMI-A with a different mechanism of action on cancer cells, was introduced into clinical trials.KP1019 disrupts the redox balance inside the cancer cells, leading the inhibition of DNA synthesis and G2/M cell cycle arrest.These responses ultimately induce cell death by apoptosis.Despite these promising biological effects, the poor water solubility poses a limitation for further clinical advancement [11,12].To overcome this limitation, the sodium salt of KP1019, known as KP1339, has been pursued as a drug candidate [13].
Besides chemotherapeutic agents, in recent years, considerable attention has been devoted to Ru(II) polypyridine complexes.This is largely due to their attractive biological properties and unique photophysical and photochemical properties, which can be fine-tuned by altering the number and nature of the polypyridyl ligands surrounding the Ru(II) metal center.On the basis of these properties, Ru(II) polypyridine complexes are extensively studied as photosensitizers for photodynamic therapy or photoactivated chemotherapy [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28].It is important to highlight that one of these compounds, namely, TLD-1433 is currently studied as a photosensitizer for photodynamic therapy in phase 2 clinical trials for the treatment of nonmuscle invasive bladder cancer [29,30].Despite their promising therapeutic profile, the vast majority of these complexes are associated with poor water solubility, non-specific biodistribution, lack of tumor-targeting properties, systemic toxicity, and, consequently, a low therapeutic index that limits their clinical application.Moreover, while the photosensitizer itself should be non-toxic in the absence of light, its exposure to light can result in cellular damage.Because of the strong light scattering of the skin and tissue during treatment and the challenge of precisely irradiating the tumor site, healthy surrounding tissue is also typically damaged in a photodynamic therapy treatment.To date, various types of delivery systems have been generated.In general, these systems can be categorized as active or passive pathways.In active tumor targeting, a particular molecule such as a signaling peptide [31][32][33][34][35][36], oligonucleotide [37][38][39], oligosaccharide [40,41], protein [42,43], receptor targeting moiety [44][45][46][47][48][49], or antibody [50,51] is employed to transport the therapeutic molecule.In passive tumor targeting, the leaky and highly permeable vasculature as well as poor lymphatic tissue characteristics of the tumor are targeted through selenium nanoparticles [52][53][54], silver nanoparticles [55], gold nanoparticles [56][57][58], silica nanoparticles [59][60][61][62][63][64], upconverting nanoparticles [65][66][67], carbon nanotubes [68][69][70], or metal-organic frameworks [71,72].Notably, polymeric nanoparticles have not been mentioned here as these are in-depth described below.Despite the endeavors made, most of the aforementioned transportation systems suffer from drawbacks such as low water solubility, complicated preparation methods, high cost, or reduced therapeutic efficacy.To address these limitations, there is a pressing need to develop a drug delivery system that can selectively transport Ru(II) polypyridine complexes to its intended target.

Advantages of the Encapsulation
Among other strategies, the encapsulation of metal complexes into polymeric materials, also referred to as nanomedicines, could present a viable solution to overcome the pharmacological limitations of the molecular therapeutic agents.Because of their unique features, such as small size, high surface area, surface chemistry, water solubility, and multifunctionality, polymeric materials are highly suitable for drug delivery purposes.By utilizing the abnormalities of the tumor vasculature, nanoparticles can accumulate in malignant tumors.This phenomenon is commonly referred to as the enhanced permeation and retention effect.Capitalizing on this, research efforts have been devoted to the incorporation of drugs into nanoparticles to overcome physical and pharmacokinetic limitations of molecular therapeutic agents [73][74][75][76][77][78][79].
Yu et al. [80] and Karges [81] have described a new research direction upon combination of material science with biosafety science termed as biosafety materials.The development of new materials that are able to influence biological or medicinal environments is expected to provide novel solutions for known and new medicinal problems and, therefore, actively shape research communities.Within this article, the encapsulation of anticancer agents into polymeric materials is systemically discussed.

Encapsulation-Dependent Parameters
The encapsulation efficiency as well as biological and pharmacological properties of the formed nanoparticles are dependent on various factors.Some of the most important parameters are highlighted here.
• Particle size: The efficiency of targeted delivery of encapsulated Ru(II) polypyridine complexes to cancerous tissues is directly influenced by the size of the nanocarrier.Small, molecular Ru(II) polypyridine complexes can diffuse into the interstitial fluid, causing undesired side effects.To overcome this, efficient macromolecular delivery systems have been designed by exploiting the structural differences between tumorous and healthy tissues.
For biological applications, the ideal diameter range for a carrier is 10 to 1,000 nm, but ideally, it should not exceed 300 nm to enable the enhanced permeation and retention effect and to ensure efficient passive targeting of tumor tissues [82,83].• Particle charge: The stability and targeting efficiency of nanocarriers are directly influenced by their surface charge.Nanoparticles with a positively charged surface can typically easily enter cancerous cells through endocytosis.In contrast, nanoparticles with a neutral or negatively charged surface rely on specific interactions, resulting in reduced levels of non-specific adsorption of proteins and non-specific phagocytosis [84,85].• Chemical structure of the polymer: The selection of a biocompatible polymer and the preparation method are crucial for ensuring the compatibility between the polymer and the metal complex.To achieve sufficient loading of the complex, a well-defined carrier structure is preferable to avoid phase separation and increase biological efficacy.Additionally, the integrity of the carrier nanostructure should be maintained post-encapsulation.Because of the charges of the Ru(II) polypyridine complexes, hydrophobic or electrostatic interactions between the polymer and the metal complex must be controlled [86,87].• Biodegradability of the polymer: To ensure optimal performance of the polymeric nanocarriers for the Ru(II) polypyridine complex delivery, the biodegradability and composition of the polymer must be carefully controlled.
For most applications, biodegradable polymers are required to regulate selective and specific release of the Ru complex.While nonbiodegradable polymers can improve properties such as stability and hydrophilicity, their elimination can occur without releasing the active compound.When biodegradable polymers are used, the surface-adsorbed Ru(II) polypyridine complex is released through initial hydrolysis of cleavable linkages, followed by slow polymer degradation over weeks to years to control payload release.Factors influencing the rate of Ru complex release include concentration gradient, mobility and diffusion within the nanocarrier, and polymer degradation rate.These properties are dependent on the composition, molecular weight, distribution, crystallinity, and chemical structure.Different degradation kinetics can be obtained depending on the regio-or stereoregularity of the polymer sequence in copolymers.Physical entrapment of the Ru(II) polypyridine complex is preferred over covalent conjugation as it maintains the integrity of the complex, but nanocarrier stability and preservation remain as challenges.A balance must be struck between too much stability, which can lead to poor release, and too little stability, which can result in premature disassembly or poor targeting efficiency [88,89].

Polymeric Nanoparticles
In the 1980s, polymeric nanoparticles were reported for the first time as carriers for drug delivery [90,91].These nanoscale particles are self-assembled usually in an aqueous solution from amphiphilic block copolymers.Spherical polymeric micelles typically have a diameter ranging from 10 to 100 nm [92].However, their size can increase when proteins are adsorbed, leading to the formation of particles that are too large for renal excretion [93].Apart from traditional spherical shapes, polymeric micelles can also self-assemble into flexible and cylindrical structures [94].
To achieve stable dispersion in aqueous environments, coreshell micelle architectures are typically obtained using diblock copolymers.The outer shell consists of hydrophilic blocks to protect the encapsulated Ru(II) polypyridine complex from adsorption of biomolecules during circulation or interaction with cellular membranes.The inner core, made up of the hydrophobic block, is stabilized by hydrophobic interactions and serves as a reservoir for the encapsulating hydrophobic Ru(II) polypyridine complexes.Amphiphilic diblock copolymers with longer hydrophilic segments are used to form spherical micelles.However, the limited kinetic stability can pose a challenge because they exist in a dynamic equilibrium between the selfassembled micelle and the bulk phase.To enhance the targeting specificity toward diseased tissues or organelles, the surface of a polymer can be functionalized with recognition motifs [95][96][97].
Polymer-based nanocarriers are widely employed as the preferred drug delivery system because of their facile synthesis, diverse composition, architecture, functionalization, and ability to degrade in physiological media.With a wide range of polymer architectures available, these are among the most promising drug delivery systems, including polymeric micelles, nanogels, vesicles, dendrimers, and nanoparticles.There are 2 strategies for encapsulating drugs within a polymer matrix: (a) physical encapsulation, which relies on noncovalent interactions between the drug and the polymer matrix, and (b) covalent encapsulation, which involves the covalent conjugation of the drug to the polymer [98][99][100].Subsequently, these types of encapsulations are separately discussed.

Physical encapsulation into polymeric nanoparticles
Polymeric nanoparticles are created by self-assembling amphiphilic polymers, which form a hydrophobic core and a hydrophilic shell to encapsulate therapeutic compounds and stabilize the interface between the core and the aqueous medium.As the predominant method, therapeutic compounds are physically encapsulated with amphiphilic polymers because of their facile synthesis and easy optimization into nanoparticles with tailored properties.The most widely used biocompatible and biodegradable polymers are aliphatic polyesters, such as the Food and Drug Administration-approved polylactide and poly(D,L-lactide-co-glycolide).Polylactide and poly(D,Llactide-co-glycolide) break down into non-toxic acidic products, specifically lactic acid and glycolic acid, which can be metabolized to produce harmless by-products such as carbon dioxide and water.Despite these promising properties, this method of encapsulation is associated with several limitations including (a) the burst release, which involves the sudden release of the drug; (b) difficulties in encapsulating drugs that are poorly miscible with the polymer matrix; and (c) poor drug loading, necessitating a high concentration of the nanoparticles to achieve a therapeutic effect [101][102][103].
Chan et al. [104] described the encapsulation of the anticancer agent [Ru(1,10-phenanthroline) 2 (2-(4-methoxyphenyl)imidazo [4,5-f][1,10]phenanthroline)] 2+ in poly(D,L-lactide-co-glycolide) nanoparticles using the nanoprecipitation technique.To improve their pharmacological properties, the nanoparticles were coated with polyethylenimine that was previously prepared from biotin and polyethylene glycol (Fig. 1).The resulting nanoparticle 1 had a diameter of 150 nm and showed tumor-targeting capabilities, particularly toward cancer cells that overexpressed sodium multivitamin transporter receptors.The researchers confirmed the spherical shape of the nanoparticles using transmission electron microscopy.The nanoparticles demonstrated high stability in cell media and human plasma.In comparison to the molecular metal complex or unmodified Ru(II) polypyridine complex nanoparticles, the loaded nanoparticle 1 exhibited 3-to 4-fold higher toxicity against human hepatocellular carcinoma cells (HepG2).In a xenograft mouse model, the biodistribution analysis revealed that the loaded nanoparticles accumulated primarily in the liver and tumor, while the molecular metal complex was distributed throughout the body, indicating the potential of the nanoparticle formulation to improve the biodistribution of the metal complex in the animal model.
Boeuf et al. [105] developed nanoparticles that encapsulate 5-substituted-1,10-phenanthroline functionalized Ru(II) polypyridine complexes using poly(D,L-lactide-co-glycolide).The nanoparticles were generated through nanoprecipitation in the presence of Poloxamer 188 and acid-terminated poly(D,Llactide-co-glycolide) (Fig. 2).The drug loading efficiency was approximately 1%.The researchers successfully obtained spherical nanoparticle 2 with a size of 100 nm and low polydispersity index.Upon irradiation, around 50% of the Ru(II) polypyridine complex payload was released within 2 days, while only 10% was released after 6 days of incubation in the dark.The nanoparticles showed minimal toxicity in the dark, but when exposed to white light irradiation (30 min, 17 mW/cm 2 ), the entire cell population of glioma (C6) cells was eliminated at a concentration of 0.1 μM.
Dickerson et al. [110] developed cross-linked nanoassemblies using polyethylene glycol-block-poly(L-aspartate) copolymers as a nanogel delivery platform for the Ru(II) polypyridine complex [Ru(4,7-diphenyl-1,10-phenanthroline) 3 ] 2+ (Fig. 5).The nanoassembly 6 had an average diameter of 19 nm and achieved a drug loading efficiency of up to 20%.The release rate of the metal complex was influenced by its hydrophobicity and the ionic strength of the solution, while pH changes had minimal impact.This suggests that the drug release can be controlled and tailored for specific applications.In terms of cytotoxicity, the nanoparticles and the unformulated Ru(II) polypyridine complex showed similar profiles in the absence of light (CC 50,dark,complex = 0.6 ± 1.1 μM, CC 50,dark,nanoparticle > 0.6 ± 1.2 μM) and when exposed to irradiation (>400 nm) (CC 50,light,complex = 0.1 ± 1.0 μM, CC 50,light,nanoparticle > 0.1 ± 1.1 μM) against A549 cells.

Covalent encapsulation into polymeric nanoparticles
To formulate polymer-drug conjugates into nanosized constructs, covalent encapsulation is utilized.This approach involves linking the drug covalently to a hydrophilic polymer [111].The connectivity and position of the modification of the Ru(II) polypyridine complex onto the polymer such as a terminal group or the polymeric backbone determines its biological properties and the specific synthetic strategies used for its preparation.
Maggioni et al. [116] have polymerized polyamidoamine chains to 1,10-phenanthroline that were further coordinated to Ru(II) polypyridine center 10 (Fig. 7).The resulting polymeric material self-assembled into nanoparticles with an average diameter of ~20 nm.The zwitterionic nature of the polymeric chain in an aqueous solution and stability studies in the presence of cysteine suggest its suitability for biological applications.The nanoparticles were readily internalized into HEK-293 cells through endocytosis or micropinocytosis, accumulating in vesicular compartments of the cytoplasm.Subsequently, Mascheroni et al. [117] incorporated the Ru(II) polypyridine complex [Ru(1,10-phenanthroline) 3 ] 2+ into a polyamidoamine polymer 11 (Fig. 7).The resulting polymeric material self-assembled into nanoparticles with an average diameter of ~10 nm.In an aqueous solution, the cationic nature of the polymer distinguished it from the analogous polymer 10.Both 10 and 11 efficiently generated singlet oxygen upon light exposure.The zwitterionic polymeric nanoparticle 10 was non-toxic in the dark and under light irradiation (CC 50,dark/light > 50 μM) against HeLa cells.In contrast, the cationic polymeric nanoparticle 11 was non-toxic in the dark (CC 50,dark > 5 μM) but exhibited low micromolar cytotoxicity (CC 50,light = 0.7 μM) against HeLa cells upon irradiation with visible light (400 to 800 nm, 40 min, 23.7 mW/cm 2 ).

Liposomes
Liposomes are small vesicles ranging from nanosized to microsized, which contain an aqueous core enveloped by a phospholipid bilayer.They have made a significant breakthrough as a nanomedicine delivery system, becoming the first to transition from theory to clinical application, thus establishing them as a well-established technological platform with extensive clinical acceptance [120,121].

Summary and Perspectives
Extensive research has been conducted on the application of Ru(II) polypyridyl complexes for anticancer therapy.However, their low cellular uptake and lack of specificity for cancer cells and tumors have initiated the necessity of the development of nanomaterials that incorporate Ru(II) polypyridine complexes.The encapsulation of these metal complexes through into nanoparticles is a promising strategy for overcoming the pharmacological limitations of molecular agents and provides cancer selectivity.The development of nanomaterials loaded with Ru(II) polypyridine complexes is still in its early stages, and much remains to be understood.Various nanoplatform constructs are available for incorporating these metal complexes depending on the intended application.Some potential areas of focus for future studies include: • Optimization of nanoparticle design: There is a need to optimize the design of nanoparticles to improve their efficiency and effectiveness in delivering Ru(II) polypyridine complexes to cancer cells.This could involve tailoring the size, shape, surface charge, and surface functionalization of nanoparticles to enhance their cellular uptake and targeting.• Evaluation of toxicity and biodistribution: It is essential to investigate the toxicity and biodistribution of Ru(II) polypyridine complex-loaded nanoparticles to ensure their safety and efficacy for clinical use.Preclinical studies can provide valuable insights into the pharmacokinetics, pharmacodynamics, and potential adverse effects of these nanoparticles.• Development of combination therapies: Combining Ru(II) polypyridine complex-loaded nanoparticles with other cancer therapies, such as chemotherapy, radiation and immunotherapy, could enhance their therapeutic efficacy and overcome resistance to treatment.This approach could also reduce the dosage of each therapy, minimizing side effects and improving patient outcomes.• Translation to clinical applications: Ultimately, the development of Ru(II) polypyridine complex-loaded nanoparticles needs to be translated into clinical applications to benefit patients with cancer.This involves rigorous testing in clinical trials to demonstrate their safety and efficacy, as well as regulatory approval for clinical use.
Overall, the development of Ru(II) polypyridine complexloaded nanoparticles has the potential to revolutionize cancer therapy by providing targeted and multimodal treatments with reduced side effects.Continued research and development in this area could lead to significant advances in the field of oncology.

Fig. 2 .
Fig. 2. Structure of the physical encapsulation of the Ru(II) polypyridine complex with a 50:50 mixture of poly(D,L-lactide-co-glycolide) and Poloxamer 188 into 2.The counterions were omitted for clarity.