Self-Assembled Copper Polypyridyl Supramolecular Metallopolymer Achieving Enhanced Anticancer Efficacy

Metallopolymers, a combination of organic polymers and metal center, contain metal atoms in repeating monomers can change its dynamic and thermodynamic properties through the directionality of coordination bonds and chemical tailoring of ligands. In the past decade, self-assembled functional supramolecular metallopolymers have aroused a surge of research interest, and have demonstrated application potential in cancer therapy. In this chapter, we have summarized the progress in the rational design of biological application of different metallopolymers. Especially, a copper polypyridyl complex was found be able to self-assemble into a supramolecular metallopolymer driven by the intermolecular interactions, which could enhance the uptake in cancer cells through endocytosis, thus effectively inhibit tumor growth in vivo without damage to the major organs. This study may provide a good example to use self-assembled metallopolymer to achieve enhanced anticancer efficacy. effectively inhibit tumor growth in vivo without damage to the major organs. This study provides a simple strategy for rational design of Cu-based metallopolymer with novel anticancer potency. For further development of self-assembled nanostructures with clinical application prospect and value in tumor diagnosis and treatment, here are some issues that should be considered. (1) Firstly, how to make the self-assembly of compounds more efficient? This may require structural opti-mization of ligands, metal centers, bonding methods, or modification of polymers or biological macromolecules to adjust the overall hydrophilicity and lipophilicity of complex molecules. (2) Secondly, how to make the assembly more selective and targeted to the tumor? It is necessary to transform the structure or add targeted groups to make the assembly more specific to tumor tissue, cells and intracellular molecules, so as to distinguish normal cells. (3) In order to use as clinic treatment, more research is needed in the aspect of medicinal properties, such as toxicology, pharmacokinetic analysis, etc., so that researchers can have a better understanding of the medicinal properties of different types of assembled structures. work


Introduction
Metallopolymers, a combination of organic polymers and metal center, contain metal atoms in repeating monomers can regulate its dynamic stability and thermodynamic properties through the variation of coordination mode of central-metal ions and chemical tailoring of ligands [1]. With purpose to improve material properties, metallopolymers have attracted increasing interest for their potential to supply advanced functional materials for a wide range of applications [2]. Supramolecular polymer, originating from the integration of polymer and supramolecule, is becoming a rapidly developing research area in recent decades [3][4][5][6][7]. Supramolecular metallopolymers received increasing attention, partly motivated by their ready-to-form self-assembly [8][9][10] and diverse applications in electrochromic materials [11], luminescent [12][13][14], accelerated guest adsorption [15], interesting magnetic properties [16], and so on [9,17]. Nitschke et al. synthesized a new type of metallopolymer, by exploring the gel self-assembly process and formation conditions, and explored the electrochemical properties, photoluminescence properties and thermochromism of the polymer [12]. Che and his co-workers synthesized cyclometalated Au III complexes, which can be self-assembled to form supramolecular polymer, through the hydrogen bonding of the guanine-like amino group of the 4-DPT ligand and the π-π stacking interaction of 2,6-diphenylpyridine. The selective growth inhibition of supramolecular polymers on tumor cells and its possible mechanism were also investigated (Figure 1) [17].
While most of supramolecular metallopolymers are "high-molecular-weight" with relatively large ligands [31], the discovery of low molecular-weight metallopolymers with tunable structures have fostered a new growth in recent years [3,4,[32][33][34][35]. Rissanen et al. reported a terpyridine-Zn (II) compound that can self-assemble into metallopolymers with fibrous structured microscopic morphology. Their studies show that this compound can realize the detection of nanomole pyrophosphate in aqueous solution and the detection of pyrophosphate in the competitive environment of cytoplasmic ions [33]. Study has showed a low-molecular-weight metallopolymer in nanofiber form demonstrating potent anticancer properties. [17,36] Nano-formulation has been showed to be able to improve the stability and selectivity of metal complexes, and hence emerges as an appealing strategy to increase anticancer activity and reduce their toxic side effects [37][38][39]. For example, Wu et al. have synthesized photosensitive triblock copolymers, which can be self-assembled to form polymer nanosystems to improve their biocompatibility and prolong their blood circulation time to achieve the purpose of regional enrichment of tumor tissue. Under the excitation of red light, the nanosystem dissociates spontaneously, releases anticancer Ru complexes and triggers the generation of a large amount of singlet oxygen to inhibit tumor growth [37]. Therefore, the formation of nanostructures of metallopolymer could have a

Synthesis and characterization
Possibly, the plane structure and NH group of the ligand could form π-π interactions and hydrogen bonds between adjacent molecules. Interestingly, in this study, we synthesized a simple Cu(II) complex, [CuCl(pip) 2 ]Cl, capable of self-assembling into a metallopolymer driven by diverse intermolecular interaction, Metallopolymers of different metal centers [24]. The solution turned to be viscous after 2 h at room temperature. Upon cooling in a refrigerator, stable green viscous solution was formed (Figure 4a). When the viscous solution stood at room temperature for 25 days, green single crystals suitable for X-ray diffraction could be obtained in a capped vial. The crystal unit of [CuCl(pip) 2 ]Cl is in a trigonal pyramidal coordination geometry (Figure 4b). Hirshfeld surface [45] clearly illustrates that rich supramolecular interactions of conjugated π-π interactions, C(N)▬H × × × Cl hydrogen bonds, and edge-to-face C▬H × × × π interactions are involved (Figure 4c and d) in the crystal packing. These results indicate that weak interactions including cooperative π-π and multiple unconventional C▬H⋯X hydrogen bonding interactions are strong enough to from the metallopolymer.
UV/Vis and FT-IR spectroscopy was employed to examine the intermolecular aggregation of [CuCl(pip) 2 ]Cl during the assembly process. As shown in Figure 5a, UV/Vis spectra of the complex in CH 3 CH 2 OH/H 2 O (5:1) exhibits a red shift with peaks from 409 to 423 nm when the concentration increased. This is ascribed to the enhancement of molecular interactions due to the increase of the concentration. The FT-IR spectra of [CuCl(pip) 2 ]Cl displayed significant difference in different solvents (Figure 5b). The red shifts in proton solvent could be attributed to the enhancement of C▬H × × × X hydrogen bond and π-π interaction.
Tyndall effect was observed in the solution of [CuCl(pip) 2 ]Cl in proton solvent CH 3 CH 2 OH/H 2 O (Figure 6), indicating the formation of self-assembled species [46], while no Tyndall phenomenon observed in aprotic solvent DMF. Therefore, the presence of proton solvent could be a determining factor for the self-assembly, as proton solvent could provide H atom feasible for the aggregation driven by hydrogen bond. In contrast, Tyndall phenomenon could not be observed in the solution of 0.8 mM (Figure 6a), possibly due to the unfavorable intermolecular distances in a dilute solution. Moreover, the mean size of the metallopolymer was found at about 95 nm (Figure 6b), indicating the presence of nanoscale aggregates in this solution.
From the TEM images, we found that metallopolymer were highly monodisperse with the size of 83 nm in diameter. And AFM measurement confirmed the spherical nanoparticle morphology of the supramolecular metallopolymer in the  proton solvent (Figure 7a and b). The metallopolymer was further confirmed by XRD (Figure 7c). The results of MALDI-TOF-MS analysis also demonstrated the presence of monomeric species, and dimer and trimer aggregation peaks of [CuCl(pip) 2 ]Cl (Figure 7d).
To further understand the phase transition, the effects of temperature, concentration and solvent on the viscosity of [CuCl(pip) 2 ]Cl were examined by Ubbelohde viscometer. Significant temperature-, solvent-and concentration-dependent changes in the viscosity were recorded (Figure 8b and c). The stacking mode of [CuCl(pip) 2 ]Cl showing hydrogen bonds (C▬H…Cl = 3.681 Å; N▬H⋯Cl = 3.279 Å) and π-π interactions (3.524-3.777 Å) (Figure 8a). High viscosity was detected when incubating in CH 3 CH 2 OH/H 2 O (5:1), which decreased obviously when the temperature increased from 5 to 75°C. However, the viscosity showed no significant increase in aprotic solvent DMF (Figure 8d). Additionally, the decreased viscosity induced by rising temperature when the temperature decreased from 75 to 5°C (Figure 8e), which demonstrated the recovering ability of the metallopolymer. The viscosity of    [44].  [CuCl(pip) 2 ]Cl increased dramatically upon increasing the concentration, which confirms the contribution of enhanced intermolecular interactions during the selfassembly of the metallopolymer (Figure 8f).

Cell proliferation inhibition of metallopolymer
Further studies were also carried out to examine the effects of the matter forms (metallopolymer and monomeric complex) on the anticancer activity of [CuCl(pip) 2 ]Cl, by in vitro and in vivo models as previously described [39,47]. As shown in Figure 9a, the metallopolymer demonstrated much higher anticancer activities against the tested cancer cells than the monomeric complex. In order to understand the reasons accounting for the different activities induced by the matter forms, we compared their cellular uptake in HepG2 hepatocellular carcinoma cells. Consistently, the metallopolymer exhibited much higher cellular uptake than monomeric complex in different time points (Figure 9b). From Figure 9c, we found that significant concentration-dependent changes in the cellular uptake were  (10 μM), and (c) cellular uptake of metallopolymer by confocal fluorescence images [44].
recorded. It is likely that metallopolymer could assembly into nanoparticles, which enter cancer cells through endocytosis in a high efficiency, thus increasing the cellular uptake and anticancer efficacy. Moreover, the results of confocal fluorescence images revealed that, the metallopolymer were internalized by cancer cells through endocytosis, and could be released into cytoplasm after 12-24 h (Figure 10).

In vivo tumor growth inhibition of metallopolymer
Furthermore, we assessed the in vivo therapeutic efficacy of the metallopolymer in HepG2 xenografts nude mice. In this study, metallopolymer was dispersed in PBS and injected into the tumor-bearing nude mice intravenously. As shown in Figure 11, the metallopolymer significantly inhibited the tumor growth, as evidenced by the decrease in tumor volume and tumor weight in a time-dependent manner. Moreover, under the effective dose, the metallopolymer showed no damage to these major organs, including heart, liver, spleen, lung and kidney (Figure 12), Figure 10. Cellular localization of metallopolymer after incubation with HepG2 cells for different periods of time [44]. demonstrating the cancer therapeutic potential and safety of this kind of selfassembled functional metallopolymer.

Conclusions and prospects
In the past decade, self-assembled functional supramolecular metallopolymers have aroused a surge of research interest, and have demonstrated application potential in cancer therapy. In this chapter, we have summarized the progress in the rational design of biological application of different metallopolymers. Especially, a simple Cu(II) complex, [CuCl(pip) 2 ]Cl, was found be able to self-assemble into surpramolecular metallopolymer driven by diverse intermolecular interactions, including π-π interactions and hydrogen bonds under a proton solvent condition. The functional metallopolymer could enter cancer cells through endocytosis, thus  H&E staining of major organs after treatments [44].

Author details
Zushuang Xiong, Lanhai Lai and Tianfeng Chen* Department of Chemistry, Jinan University, Guangzhou, China *Address all correspondence to: felixchentf@gmail.com effectively inhibit tumor growth in vivo without damage to the major organs. This study provides a simple strategy for rational design of Cu-based metallopolymer with novel anticancer potency. For further development of self-assembled nanostructures with clinical application prospect and value in tumor diagnosis and treatment, here are some issues that should be considered. (1) Firstly, how to make the self-assembly of compounds more efficient? This may require structural optimization of ligands, metal centers, bonding methods, or modification of polymers or biological macromolecules to adjust the overall hydrophilicity and lipophilicity of complex molecules. (2) Secondly, how to make the assembly more selective and targeted to the tumor? It is necessary to transform the structure or add targeted groups to make the assembly more specific to tumor tissue, cells and intracellular molecules, so as to distinguish normal cells. (3) In order to use as clinic treatment, more research is needed in the aspect of medicinal properties, such as toxicology, pharmacokinetic analysis, etc., so that researchers can have a better understanding of the medicinal properties of different types of assembled structures.