DNA condensation induced by metal complexes

Coordination Chemistry Reviews 281 (2014) 100–113

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Coordination Chemistry Reviews journal homepage: www.elsevier.com/locate/ccr

Review

DNA condensation induced by metal complexes Guan-Ying Li, Rui-Lin Guan, Liang-Nian Ji, Hui Chao ∗ MOE Laboratory of Bioinorganic and Synthetic Chemistry, State Key Laboratory of Optoelectronic Materials and Technologies, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, PR China

Contents 1. 2. 3. 4. 5.

6.

7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Why metal complexes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metal ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polymeric metal complexes (PMCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metal complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. DNA binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. DNA coiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. DNA condensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1. Electrostatic interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2. ␲–␲ interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3. Hydrogen bonds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.4. Covalent binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Gene delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Luminescent tracking of gene vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Antitumor agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

a r t i c l e

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Article history: Received 28 April 2014 Accepted 14 September 2014 Keywords: Metal complexes DNA condensation Gene delivery Anticancer agents

a b s t r a c t DNA is stored in a highly compact, condensed phase in viruses, bacteria and eukaryotes. Co(NH3 )6 3+ is a well-known inorganic cation that can induce DNA condensation, and numerous Co(III), Co(II), Fe(II), Ca(II), Cu(II), Ni(II), Zn(II), Ru(II), Pt(II) and La(III) complexes have been evaluated as DNA condensing agents. In this review, we divided these metal complexes into four distinct classes based on the mechanism of DNA condensation: (i) complexes with high positive charges that condense DNA via electrostatic interaction, (ii) complexes with planar intercalative ligands that condense DNA via ␲–␲ interaction, (iii) complexes that bind with DNA through hydrogen bonds, and (iv) complexes that covalently bind to DNA. Some applications of these metal complex-based DNA condensing agents in gene vectors and antitumor agents are also presented. © 2014 Elsevier B.V. All rights reserved.

1. Introduction DNA is a very long and strongly charged heteropolymer. In vivo, DNA must fit into the very small space of a virus capsid, bacteria nucleoid or the nuclear region in eukaryotic cells, while the length

∗ Corresponding author. Tel.: +86 20 84110613. E-mail address: [email protected] (H. Chao). http://dx.doi.org/10.1016/j.ccr.2014.09.005 0010-8545/© 2014 Elsevier B.V. All rights reserved.

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of a stretched single DNA molecule may be up to a several dozen centimetres long, depending on the organism. Thus, DNA chains must collapse into compact and highly ordered particles containing only one or a few molecules (DNA condensation) [1]. Usually, several DNA molecules are incorporated into the condensed structure, and therefore, it is difficult to distinguish between condensation and aggregation. The term condensation is generally used when the aggregate is of finite size and has an orderly morphology. DNA condensation acts as a barrier to radiation-induced DNA damage [2]

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Fig. 1. Chemical structures of the commercial lipid agent DOTMA (1), hexaminecobalt(III) (2), speridine3+ (3) and spermine4+ (4).

and oxidative damage [3]. The radioprotective/oxiprotective effect can likely be explained by the tight DNA packing prohibiting the access of regulatory molecules to DNA; in addition, the DNA repair process is accelerated in the condensed DNA, which has a high density. The condensation of free DNA in vitro has long been of interest as a potential model for DNA packing in vivo and in gene therapies, providing a promising means of non-viral gene delivery. Many compounds have been studied for compacting DNA, such as the polycations spermidine3+ and spermine4+ [4–6], inorganic cations [5,7,8], lipids [9,10], polymers [11–14] and nanoparticles (Fig. 1) [14–18]. In the early 1980s, the classical inorganic cation Co(NH3 )6 3+ was reported to condense DNA; this cation has advantages over other DNA condensing agents [7,8]. In the first decade of the 21st century, researchers began to study metal complexes to compact DNA and for non-viral gene delivery. The morphologies and sizes of DNA condensates vary depending on the polycationinduced DNA condensate studied and the type of metal complex utilised [19–22]. The mechanistic details of DNA condensation are essential for understanding DNA packing behaviours and guiding its functions or applications. Thus, we have provided an overview of the different types of metal complexes, summarised their DNA condensing mechanisms and shown their applications as gene vectors and as new classes of antitumor agents. 2. Why metal complexes? Over the last few decades, cationic lipids and polymers have shown significant promise as DNA binding agents, DNA condensing agents and gene vectors in gene therapeutics. Much attention has been focused on the design and synthesis of cationic lipids, polymers and nanomaterials for DNA condensation studies and their applications in non-viral gene vectors [4–18]. However, metal complexes have attracted increasing interest as a novel class of DNA condensing agent [23,24]. The total amount of transition metal elements in vivo is less than 0.05%. However, there are many examples in nature that utilise transition metal ions. For example, the zinc finger protein family, consisting of nucleus-targeting molecules, have a nuclear localisation signal within the zinc finger domain, where the zinc can bind tightly [25,26]. Zinc ions have been introduced into polymer–DNA complex systems and increase the gene transfection efficiency because of their ability to associate with the phosphate diester groups that constitute the backbone of DNA [27]. In particular, metal complexes offer some advantages as DNA condensing agents. First, metal complexes exhibit a high positive charge density to neutralise the negative charge of the DNA backbone, which is a prerequisite for DNA condensation. With few exceptions, nearly all lipids or polymers achieve a positive charge through ammonium cations, which provide a +1 positive charge maximum in local sites (not total charges). However, metal ions usually have a +2 positive charge or higher. When coordinated with chelated ligands through rational design, metal complexes

can carry a high positive charge and act as high-density charged nucleation sites for DNA condensation. Second, coordination complexes offer a uniquely modular system for DNA binding. The central metal ion can interact via electrostatic attraction with the DNA phosphate backbone and (or) via base coordination with DNA base pairs. The metal centre can act as an anchor, holding the three-dimensional scaffold of ligands in place, which can also bear DNA-binding elements. Ligands that contain targeting elements can also be easily adopted through metal–ligand coordination. Third, some metal centres benefit from rich photophysical and electrochemical properties, extending their utility far beyond DNA condensing agents and gene vectors. These characteristics have allowed metal complexes to be used in a wide range of applications, such as the luminescent tracking of intracellular gene expression to fabricate smart gene vectors with photo- or electronic-trigger releases. 3. Metal ions Given the high negative surface charge, the electrostatic repulsion between DNA polyions may be a major force preventing the formation of a compact DNA structures. A sufficient protocol would be to add multivalent cations to neutralise the DNA negative charges. Bloomfield and co-workers [28] have established a simple rule for polycation-induced DNA compaction or aggregation: condensation occurs when ∼90% of the DNA charges are neutralised by counterions. Normally, multivalent cations with +3 charges or greater are required for condensing agents in aqueous solution because of charge density effects. The polyamines spermidine3+ and spermine4+ and the inorganic cation Co(NH3 )6 3+ are most commonly used to effectively condense DNA at micromolar concentrations. Metal ions can interact with the major and minor grooves of DNA through electrostatic attraction or coordination with DNA base pairs, and may play key roles in the control of the DNA conformation and topology [28–31]. Monovalent cations, such as Na+ or K+ , cannot induce DNA condensation unless an additional osmotic pressure is exerted by neutral polymers, such as polyethylene glycol (PEG), which is called a ␺-DNA or psi-DNA compact (“polymer-and salt-induced” DNA condensation) [32,33]. Thus, the forces pushing the double helices together are coming from entropic random collisions with the crowding polymers surrounding the DNA, and monovalent ions are required to neutralise the DNA charges and decrease the DNA–DNA repulsion. Divalent cations, even at molar concentrations, cannot condense double-stranded DNA (dsDNA) from dilute solutions, except under special conditions if the DNA is circular [20] or in a water–alcohol mixture [34]. However, the effects of divalent metal ions, including Cu2+ , Zn2+ , Mn2+ , Ca2+ and Mg2+ , on the DNA structural transition have been investigated, and the results indicated that DNA assumed a compact form upon interaction with divalent metal ions,

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except Mg2+ ions [35–38]. The mechanism of DNA compaction via reaction with these divalent metal ions is based on chelation and not electrostatics. The interactions between metal ions and DNA bases destabilise the DNA structure, causing bends and reducing its length, which will facilitate DNA compaction [28–31]. Metal ions, such as Cu2+ , Zn2+ , Mn2+ and Ca2+ , can interact with DNA phosphates electrostatically and form base coordination, while Mg2+ can interact only with DNA phosphates. Conversely, Mg2+ does affect the size of toroid formation when DNA is condensed by Co(NH3 )6 3+ [39]. Moreover, it has been reported that triple-stranded DNA (tsDNA), which is a more highly charged helix than dsDNA, is precipitated by alkaline-earth divalent cations (Mg2+ , Ba2+ and Ca2+ ) [40]. The DNA compacts induced by alkaline-earth divalent cations redissolve upon the addition of monovalent salts, such as NaCl. The counterintuitive observation that the more highly charged tsDNA (vs. dsDNA) is condensed by lower valence cations is likely due to the stronger attraction between the cations and the tsDNA than the dsDNA, resulting in restructuring of the hydration shell near the surface. The trivalent inorganic cation Co(NH3 )6 3+ was first reported to condense DNA by Widom and Baldwin at 1980 [7,8], and since then, its DNA condensation effect has been studied thoroughly [28,41–44]. Bloomfield and co-workers summarised it as follow [44]: (1) DNA condensation occurs only when [Co(NH3 )6 3+ ]/[DNAphosphate] is close to the average value of 0.54, which reflects the 80–90% charge neutralisation criterion for condensation. (2) The equilibrium weight average hydrodynamic radius (RH ) of the condensates first decreases, then increases with increasing Co(NH3 )6 3+ , as they undergo a transition from intramolecular (monomolecular) to intermolecular (multimolecular) condensation. (3) The uncondensed DNA fraction decays with time exponentially. The equilibrium uncondensed DNA fraction and relaxation time decrease with increasing [Co(NH3 )6 3+ ] but are insensitive to [DNA]. (4) The condensation rate in early stages is insensitive to [DNA] but proportional to Co(NH3 )6 3+ . (5) Data for low [DNA] and low [Co(NH3 )6 3+ ] at early stages of condensation are most reliable for kinetic modelling because, under these conditions, there is minimal clumping and network formation among separate condensates. The trivalent Cr3+ ion can also induce DNA condensation through binding mainly to N7(G) and to phosphate groups. Upon increasing the Cr3+ ion concentration, the shape of the condensates changes from loose flower-like structures to highly packed dense spheres [45]. 4. Polymeric metal complexes (PMCs) Cationic lipids or polymers can interact with DNA and form nanoparticles. This technique is common in gene delivery [14,46]. Lipids are one of the most efficient gene transfer vectors because they possess two elements that are crucial for gene delivery: a cationic head group to condense DNA and a lipid moiety as a fusogenic group to enhance penetration into cells. Several commercially available lipid reagents [47,10] include dioleoylphosphatidylethanolamine (DOPE), 2,3-bis(oleoyl)oxypropyltrimethyl ammonium chloride (DOTMA, Fig. 1) and dioctadecylamidoglycylspermine (DOGS). Cationic polymers are also promising DNA condensing agents in gene transfer vectors. The size of polymer–DNA aggregates has been correlated with the molecular weight of the polymer. High molecular weight polymers form DNA nanoparticles with large diameters. Poly(l-lysine) (PLL), polyethylenimine (PEI), and polymethacrylate are most commonly used as polymeric vectors [14]. Increasing interest has focused on polymeric metal complexes (PMCs) that offer some attractive features for DNA condensation. In principle, metal ions are introduced into the polymer system to promote the DNA condensing ability and transfection efficiency

or construct a responsive element under certain stimuli. Trivalent Fe(III) ions are chelated using PEI and significantly reduce the amount of PEI necessary to induce DNA condensation; however, Fe(III) ions are very inefficient at DNA condensation [48–50]. The presence of Fe(III) causes the zeta potential to change from negative to positive. Additionally, a reduction in the DNA aggregate size and a lower cytotoxicity is observed compared to native PEI–DNA complexes. In the case of Ru–PEI complexes, metal ions enhance the DNA condensing ability [51]. The introduction of Zn(II) ions can improve the transfection efficiency, especially in imidazole group containing polymers, which can chelate Zn(II) ions [52–54]. After treatment with Zn/DNA/histidylatedpolylysine complexes, the number of EGFPpositive cells significantly increases from 1% to more than 40%. Chelated Zn(II) ions promote membrane fusion, increasing endosomal zinc concentration. Furthermore, the imidazole–Zn chelation complexes exhibit pH-dependent dissociation, which is adopted to control compact DNA formation and gene expression. The lipids 11-(ferrocenylundecyl)trimethylammonium bromide (FTMA) and bis(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA) incorporate redox-active ferrocene groups and can be cycled reversibly between the oxidised state and the reduced state [55–62]. The reduced ferrocene-lipids react with DNA and form normal nanostructural vesicles similar to a classical lipid system, yielding high levels of gene expression. However, the oxidation of the reduced ferrocene-lipids leads to the formation of smaller but predominantly loose and disordered aggregates, yielding very low (background) levels of transfection efficiency. The active spatial and temporal control over gene expression can be achieved using electrochemical methods or chemical reduction of GSH. 5. Metal complexes 5.1. DNA binding Since the elucidation of the double helical structure DNA, the construction of small molecules that recognise and react with DNA has been an area of considerable interest. Due to the threedimensional structures and wide array of photophysical properties, metal complexes have become ideal templates for the design of DNA-interactive systems. The structure and chemical composition of DNA leads to several binding modes that can be reversible or irreversible: covalent binding, electrostatic interaction, intercalation and groove binding. The readers who may be interested in the metal complexes–DNA interactions are encouraged to refer to other thorough reviews [63–71]. In addition to a variety of binding modes, metal complexes also offer distinct chemical and biological activities, such as specific structural recognition of duplex DNA [72–74], defect DNA [75–78], G-quadruplex [79–85], DNA cleavage and crosslinking [86–88], DNA coiling and condensation, and in many DNA applications, photoswitching, DNA nuclease, gene vectors and anticancer agents. In addition to duplex DNA, three-way junctions are the most abundant branched structures of nucleic acid and are involved in crucial biological functions. The interaction of metallosupramolecular cylinders with three-way DNA junctions has been studied [89,90]. An X-ray crystal structure reveals that a triple-helical cylinder [Fe2 L3 ]4+ (5, Fig. 2) is similarly matched to the size and shape of the cavity in the three-way DNA junction. Both M and P enantiomers can stabilise the DNA three-way junctions, but M is more effective than P. Bulky cylinders do not provide the same stabilisation for three-way junctions. The results confirm that the precise size and shape of the cylinders plays a key role in the recognition and stabilisation of three-way junctions. These works reveal an unprecedented mode of DNA binding with metal complexes.

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Fig. 2. Chemical structures of metallo-supramolecular helical cylinders.

5.2. DNA coiling DNA binding with metal complexes through covalent metal–base pair bonds or noncovalent groove binding and intercalating binding often causes DNA conformational distortion. DNA kinking or bending, twisting, crosslink, cleavage and coiling are observed when metal complexes react with DNA. If dramatic DNA coiling occurs, condensation or aggregation of DNA is the conformational next step. Before introducing metal complexes that induce DNA condensation, we would like to provide a brief introduction for DNA coiling induced by metal complexes. The metallosupramolecular triple helical cylinder [Fe2 (C25 H20 N4 )3 ]4+ (5, Fig. 2) was developed to bind DNA to the major groove and induce DNA coiling [91]. At a low concentration of the cylinder, the average bend angle and number of bends in the DNA increases compared with free DNA. With a moderate cylinder load, the intramolecular coiling of DNA is observed, and the DNA coiling increases dramatically as the loading of the cylinder increases. In contrast, Co(NH3 )6 3+ condenses and aggregates DNA into intermolecular clusters. Supramolecular cylinders are excellent examples of small molecules in molecular structure–DNA binding effects. (1) Metal centres affect its DNA coiling effect. Fe(II) and Ru(II) cylinders (5, 6) are good DNA major groove binders and dramatically induce DNA coiling [91–93]. However, the Ni(II) cylinder (7) has a stronger preference for G-quadruplex over duplex DNA [94,95]. Additionally, no DNA coiling effects are observed in the Cu(I) cylinder (8), which can cleave DNA in the presence of peroxide and acts as an artificial nuclease [96]. (2) The shape effects of the cylinders that are the right size to fit into DNA major grooves drive the subsequent DNA conformational changes. Furthermore, the charge is not essential for the DNA coiling but does affect the integrity of the DNA duplex. New Fe(II) cylinders were prepared using substituted bridging ligands,

resulting in a wider or longer cylinder (9–14, Fig. 2) [97–101]. The overall conclusion is that widening or lengthening the Fe(II) cylinders can reduce their DNA binding strength as well as the bending and coiling effects. The “fat” cylinder with a larger cylinder radius tends to twist so that only one end of the cylinder remains within the major groove, and the “slight” cylinder mainly serves to extend the length but does not dramatically change the surface of the cylinder that interacts with the DNA bases. When introducing potential H bond acceptor units onto the surface of cylinder by replacing the central linking unit CH2 with an S or O, a similar DNA binding is observed but the stability of the triple helical cylinder is decreased by the O linked bridging ligand. However, dinuclear Ru(II) complexes (15–16) provide chemically and enantiomerically stable alternative to 5 [102]. (3) This cylinder shows enantiomeric effects in DNA binding [103–105]. The M enantiomer binds to DNA along the major groove and induces dramatic intramolecular coiling. The P enantiomer bends and coils the DNA less dramatically than the M enantiomer, but it still bends significantly compared with the free DNA. The enantiomeric difference in the structural effect may be explained by the steric effect. Only the M enantiomer fits the major groove, and the P enantiomer lies along the surface of minor groove. 5.3. DNA condensation 5.3.1. Electrostatic interaction Many metal complexes with high positive charges have been designed as DNA condensing agents (Fig. 3). Similar to Co(NH3 )6 3+ , cobalt(III) complexes also bear trivalent cationic charges, which facilitates DNA condensation. Tris(1,10-phenanthroline) cobalt(III) (17) is a typical DNA intercalating agent. One of the phenanthroline ligands partially intercalates into the adjacent base pairs and the others dispose along the major groove of DNA molecule, resulting in hydrophobic interactions that facilitate electrostatic interactions.

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Fig. 3. Chemical structures of complexes with high positive charges.

However, the DNA condensation only occurs in the presence of ethanol [106]. Atomic force microscopy (AFM) results show that, when a mixture of 2/5 molar ratio of 17/DNA nucleotide phosphate (NP) is incubated for 1 h in the absence of ethanol, both linear and circular DNA molecules are well extended, indicating a decrease in the flexibility of DNA chains. As the ethanol content is increased to 10%, the DNA chains tend to make intramolecular and intermolecular contact with many loops along the chains and the apparent persistent lengths decrease. When the ethanol content is increased to 20%, a distinct structural transition to a typical toroid is observed, measured as 124.51 nm in centre-to-centre diameter and 6.24 nm in height. Additionally, similarly sized DNA condensates are observed when the ethanol concentration is increased to 30%. At the higher ethanol concentration of 62%, a B-A transition in the secondary structure of DNA occurs. The intercalation of complex 17 can prevent B-form DNA from transitioning into the tighter Aform DNA, while Co(NH3 )6 3+ and alcohol cooperatively accelerate the conformational transition in DNA condensation [42]. Another tris-chelate Co(III) complex 18 bears the imidazole and amine group [107]. Taking advantage of electronic reorganisation and the resonance form, the release of protons from the imidazole ring can be attained in an aqueous solution, which is influenced by ionic strength effects. Upon N H bond dissociation, complex 18 is

likely to afford monovalent cationic 18 in solution, measured as a 1:1 electrolyte. Therefore, the monovalent complex does not lead to condensation of plasmid pBR322 DNA. However, changes in the DNA morphology occur 3 days post-incubation, and progressive coarsening of the non-toroid aggregates of DNA occurs 7 days post-incubation due to the eventual crystallisation induced by complex 18. In contrast to other divalent metal ions (Mg2+ , Ba2+ or Mn2+ ), mononuclear Ru(II) complexes of +2 charge can condense DNA at low concentrations [108,109]. Ru(II) complexes (19–24) containing bipyridine functionalised molecular clip ligands go through self-assembly of glycoluril receptors in water to form polycationic arrays. These arrays can rapidly condense free DNA into nanoparticles at micromolar doses. The morphologies of the DNA condensates induced by complexes 19 and 20 are different, most likely due to the different DNA binding modes, i.e., electrostatic binding mode by complex 19 to form rod-shaped DNA condensates and partial intercalating binding mode by complex 20 to form ring-shaped DNA condensates. An effective way for divalent metal complexes to increase the cationic charge density is to fabricate dinuclear or multinuclear complexes using suitable linking ligands. Using this strategy, dinuclear Ru(II) complexes (29–30, 32–33) are synthesised [110–112].

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The V-shaped complex 29 binds to the DNA groove and induces condensation of both herring sperm DNA (hsDNA) to long chain-like particle clusters and circular plasmid pBR322 DNA to particulate structures under neutral conditions. When a high concentration of NaCl (2.0 mM) is added, the relaxation of DNA condensates is observed, releasing DNA molecules as a result of the electrostatic competition between multivalent and monovalent cations with the phosphate backbone of DNA. However, the linear parallel structure of 32 does not induce DNA condensation. When replacing auxiliary bpy ligands with more hydrophobic phen ligands, both V-shaped and linear dinuclear complexes (30 and 33) can condense DNA. In addition to electrostatic interactions, the flexibility and hydrophobic effects of metal complexes are beneficial for DNA groove binding, resulting in a positive effect on the DNA condensation. More interestingly, DNA condensation by complexes 30 and 33 is enantiomeric different. The -30 condenses DNA much more efficiently than -30, while -33 is more efficient in DNA condensation than -33 [112]. This reversed chirality effect is surprising considering the similar chemical structure, but the result highlights the complexity of the DNA binding. The steric effect and the structural flexibility of the complex might be attributed to the enantiomeric effect in DNA condensation. Lanthanum metal complexes have also been investigated. Heptameric nuclear La(III) enantiomers (M-31 and P-31) selfassociate in water through specific molecular recognition process to form polycationic arrays [113]. With a width of ca. 11 A˚ and ˚ the enantiomers bind to DNA by embedding a length of ca. 21 A, perfectly in the major groove through electrostatic attraction and hydrogen bond interactions and rapidly condense free DNA into the compact state. M-31 expresses a stronger DNA affinity than P31, which may be due to the more suitable steric matching in the configuration between M-31 and DNA and M-31 fits into the major groove more deeply. More highly charged metal complexes were also synthesised to investigate their DNA condensation behaviours. A tetranuclear Ni(II) complex 25 possessing a +6 cationic charge interacts strongly with DNA through electrostatic attraction [114]. A gel electrophoresis assay shows retardation of the DNA bands (pBR322 DNA, 200 ng) in gel when the +6 charge complex concentration is increased from 5.0 to 50 ␮M as well as complete compression of the gel slot when the concentration reaches 60 ␮M. AFM images show that supercoiled pBR322 DNA is tightly packed into nearly globular nanoparticles with a mean size of approximately 500 nm when pBR322 DNA (200 ng) is incubated with 10 ␮M of complex 25 at 37 ◦ C for 4 h. The release of DNA from its globular compact state is achieved using ethylenediaminetetraacetic acid (EDTA). The release of DNA from the compact state by EDTA is attributed to two factors. On the one hand, anionic EDTA may react with the condensed DNA by neutralising the positive charge on complex 25, which would weaken the attraction between 25 and DNA and enhance the repulsion between DNA segments, resulting in the release of DNA from the vector. On the other hand, EDTA acts as a strong chelating agent to remove Ni(II) from the complex through an ion-exchange reaction, which would decompose 25 and eliminate the electrostatic attraction between 25 and the DNA, leading to the dissociation of DNA from the carrier. Noncovalent polynuclear platinum complexes (26–28) of the polyamine linker, such as spermidine or spermine, which are efficient inducers of DNA condensation, show that the increasing positive charges (+5 or +6) result in significant enhancement of the DNA condensation capability [115]. DNA condensation is studied using static light scattering, spectral analysis, gel retardation assay and AFM images, and the results indicate that the complexes are capable of inducing DNA condensation at more than 1 order of magnitude lower concentrations than conventional spermine. Considering the fact that complexes 26–28 contain no leaving

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group, they bind to DNA noncovalently with high affinity through electrostatic attractions and hydrogen-bonding effects between polyamine linkers and DNA. The DNA condensates can reversibly release upon addition of high concentrations of NaCl (0.65 M), confirming the electrostatic interaction between these noncovalent platinum complexes and DNA. Another tetranuclear complex is composed of the linear ligand in complex 32, in which case the dinuclear Ru(II) complex does not induce DNA condensation [110]. This tetranuclear Ru(II) complex 34 bears a +8 cationic charge, which is currently the highest cationic charge in DNA condensation metal complex agents [116]. A± ratio (total positive charge/total negative charge) of 3.2 is required to confine the DNA band to the gel slot in a gel retardation assay, and when the ± ratio is higher than 26.7, the average hydrodynamic diameter of the condensated DNA particles is stable at approximately 200 nm, as determined using dynamic light scattering (DLS). The AFM images also show well-distributed DNA particles with sizes of approximately 100–200 nm.

5.3.2. – interaction Electrostatic interactions between positively charged metal complexes and negatively charged DNA molecule are the major and prerequisite, but not the only, forces that drive DNA condensation. ␲–␲ interactions in the DNA bound complexes may also be a key force in DNA condensation. DNA intercalation occurs when planar aromatic compounds are inserted between adjacent base pairs in the DNA double helix as a molecular sandwich. By far, some of the most widely used intercalative agents are octahedral Ru(II) complexes containing planar or extended planar components [117,118]. Ru(II) complexes 35 and 36 containing carbonate groups (Fig. 4) can condense plasmid DNA to form DNA-complexes aggregates [117]. Note that the COOH groups not involved in the H bonds are deprotonated at physiological pH. The actual charge of 35 or 36 differs by one unit. The DNA condensation behaviour is driven by ␲–␲ stacking, most likely due to the far-reaching aromatic DIP ligands (DIP = 4,7-diphenyl-1,10phenanthroline). Although the electrostatic attractions are weaker than intercalation, it is a prerequisite to inducing DNA condensation. Two divalent Ru(II) complexes (37 and 38, Fig. 4) containing extended planar ligands [118] as classical DNA intercalators show strong intercalative binding abilities with DNA. They can condense circular pBR 322 DNA into nanoparticles at 40 ␮M. After ruling out the possibility of the photocrosslinking of Ru(II) complexes with DNA base pairs, the high DNA binding affinities through ␲–␲ stacking of planar ligands with DNA base pairs may be one of the major driving force in the condensation of DNA. Other metal complexes containing extend planar ligands have also been studied as DNA intercalators and DNA condensing agents. A benzimidazole-based mononuclear Co(III) complex [107] cannot induce DNA condensation under neutral pH condition because of the lack of a positive charge. Polybenzimidazole ligands with flexibility due to aliphatic bridging moieties are adaptable and versatile for building dinuclear or multinuclear metal complexes. Two dinuclear copper complexes, 47 and 50 (Fig. 5), linked by polybenzimidazole ligands are reported to induce aggregation of both linear ␭DNA and supercoiled plasmid DNA [119]. The forces that drive DNA condensation are comprehensive, and stating a single or simple condensing process is not allowed. These complexes bind to DNA through intercalative modes via planar benzimidazole ligands. In addition, the coordination of DNA bases to the Cu(II) ions should be considered because the complexes contain labile H2 O and Cl− ligands. The electrostatic interactions should not be ignored. In addition, the intermolecular ␲–␲ stacking of the complexes might also contribute to DNA condensation.

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Fig. 4. Chemical structures of Ru(II) complexes with planar ligands as DNA intercalators.

In further studies [22,120–123], a series of metal complexes of polybenzimidazole ligands were synthesised (39–62, Fig. 5). Strong intermolecular ␲–␲ contacts are commonly observed in these complexes. They bind to DNA via intercalative modes and are capable of condensing both linear ␭DNA and circular DNA into nanoparticles around neutral pH. The sizes of the DNA condensates are independent of the number of positive charges on the complexes, but seem dependent on the metal ion and the DNA binding affinity [122,123]. Unlike Cu2+ or Co2+ complexes, Ca2+ complexes 45 and 46 exhibit weak DNA affinities but can still induce DNA to form homogeneous spherical and compact nanoparticles at a molar ratio of 1:1. When increasing the concentration of the Ca2+ complexes, significant aggregation of the monodisperse nanoparticles is observed. The eight-coordinated structure of the Ca2+ complexes and the ␲–␲ interactions of the DNA-bound complexes are considered determinants that lead to DNA condensation. The introduction of a DNA intercalator, such as ethidium bromide (EB), is observed to competitively bind with DNA and be capable of leading to complete dissociation of the DNA condensates. This fact supports the hypothesis that intercalation is one of the key forces that drives DNA condensation via these complexes. The DNA condensates have many polymorphs, which depend on experimental conditions, such as the ratio of DNA to metal complexes and the incubation time. For complex 51 [22], at short incubation periods of <1 min, the DNA molecules are still in extended states, and no observable DNA packing occurs. Then, the extended linear or circular DNA molecules first collapse into loose assemblies and further into compact globular nanoparticles with diameters of 50–200 nm. The majority of individual loose assemblies contain one DNA molecule and are intramolecular DNA condensates, whereas the individual and globular inclusions in which the DNA is also visible mainly contain many DNA molecules and are an intermolecular DNA condensate. The difference in the DNA condensing modes may be explained by the mechanism shown in Fig. 6. The ␲–␲ interactions play an important role in the formation of the individual globular nanoparticles or net-like assemblies. First, the complex molecules bind to DNA via both electrostatic attraction and intercalation and arrange along the DNA strands, leading to increasing local concentrations of intermolecular ␲–␲ contacts of the DNA-bound complexes, which can cause the collapse of parts of the DNA strands and create nucleation sites for DNA condensation. Then, driven by the intermolecular ␲–␲ contacts, the entanglement of one DNA molecule or a few DNA molecules around a nucleation site results in the formation of individual globular DNA condensates, and the entanglement of many DNA molecules around multiple nucleation sites results in the formation of a agglomerate in which many DNA condensates associate with each other. Furthermore, the individual DNA

condensates or agglomerates can self-assemble into large, net-like species, which may be driven by intermolecular ␲–␲ contacts. As a result, the intermolecular ␲–␲ contacts in the DNA-bound complexes may be a key force driving DNA condensation. 5.3.3. Hydrogen bonds Aza heterocyclic chelated Zn(II) or Cu(II) complex (63–66, Fig. 7a) can condense both circular plasmid DNA and linear 1800 bp DNA into nanoparticles at millimole concentrations [124]. The AFM image shows the DNA nanoparticles as classical globules with an average diameter of 88.9 ± 7.9 nm and a height of 0.88 ± 0.08 nm. However, condensation in this manner occurs only at incubation temperatures up to 50 ◦ C. No detectable change in the DNA structure is observed after incubation at 37 ◦ C. After 12 h of incubation at 37 ◦ C, the condensed nanoparticles completely return to the original linear form or 99% return to the original plasmid form (with 10% supercoiled, 86.6% open circular and 2.4% linear form). This temperature-dependent reversible DNA condensation and relaxation is confirmed using AFM images (Fig. 7b), gel assays and circular dichroism (CD) spectra. The temperature effects on the DNA form may be complicated and discontinuous. One possible explanation is the competitive formation of external hydrogen bonds. As shown in Fig. 7c, the carbonyl groups of these complexes competitively interact with the H atoms in DNA base pairs to form new, weak hydrogen bonds. At high temperatures, the hydrogen bonds in DNA base pairs weaken or break. Due to the existence of electrostatic interactions and external hydrogen bonds with various orientations, DNA molecules are able to compact. When the temperature decreases, however, the broken hydrogen bonds are reformed according to the principle of complementary base pairing in DNA. The external hydrogen bonds between the carbonyls and the base pairs disappear, and the charge neutralisation could not maintain the compact conformation of condensed DNA, resulting in the release of the condensates. 5.3.4. Covalent binding Cisplatin (67, Fig. 8a) is one of the most widely used anti-cancer drugs. It is generally believed that the cytotoxicity of cisplatin derives from its covalent crosslink with DNA to form cisplatin–DNA adducts, which may interact with cellular proteins in several modes to block DNA transcription by blocking DNA polymerase or RNA polymerase [125,126]. Cisplatin-DNA adducts can also aggregate and compact. Wang et al. [127] used AgNO3 to remove the chloride in cisplatin and then studied its reaction with DNA. AFM images in Fig. 8b show that in reactions with low concentrations of cisplatin (77 ␮M, [Pt]/[NP] = 0.5), the DNA becomes more flexible and the persistence lengths decrease significantly from 52 to 15 nm. At high cisplatin concentrations (770 ␮M, [Pt]/[NP] = 5), DNA condensation

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Fig. 5. Chemical structures of benzimidazole based Co(II), Cu(II) or Ca2+ complexes as DNA intercalators.

is observed. The condensates form globules with a typical height of 6 nm and a width of 100 nm after 12 h of incubation. If cisplatin is not converted to the diaqueous derivative before reacting with DNA, a higher concentration up to 2 mM of cisplatin is needed to condense linear ␭DNA [128]. Another platinum anti-cancer drug, oxaliplatin (68a and 68b) [129], can interact with DNA in a similar way to cisplatin. Additionally, BBR 3464 (71) significantly induces DNA structural changes in terms of loops as well as overall DNA

compaction at a molar ratio that is 50 times less than that applied for cisplatin treatment [130,131]. It is reasonable to think that other similar platinum anti-cancer drugs, such as carboplatin, JM118 and ZD0473, also modify DNA structures in the same way. A group of dinuclear platinum complexes bridged with tetrazolate ligands (69 and 70) have a greater potency for DNA compaction than cisplatin when reacting with a large DNA molecule (T4 phage DNA) [132].

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Fig. 6. The hypothetic model of the DNA condensing mechanism through ␲–␲ interactions. Reproduced from Ref. [22] with permission from Elsevier.

These characteristics of platinum complexes are markedly different from those of the usual condensing agents, such as Co(NH3 )6 3+ and spermidine3+ . A reasonable model (Fig. 8c) has been proposed to explain the cisplatin-type of DNA condensation [127]: (i) first, cisplatin covalently binds with DNA and induces interstrand or intrastrand crosslinks between adjacent bases, making DNA more flexible. Thus, DNA exhibits a decreased persistence

length and few local kinks. (ii) Then, cisplatin induces the formation of loops through crosslinks of distant nucleotides brought together by DNA thermal fluctuations. At low cisplatin concentrations, DNA exhibits many micro loops, while at extremely high cisplatin concentrations, the structures with micro loops will be further crosslinked to form collections of large aggregates and, finally, condensed into a compact globule.

Fig. 7. (a) Chemical structures of Zn(II) or Cu(II) complexes with acetyl groups. (b) AFM images of DNA reacted with complex 63 at 50 ◦ C or 37 ◦ C. (c) The hypothetical model for the DNA condensing mechanism through a hydrogen bond process. Reproduced from Ref. [127] with permission from the American Chemical Society.

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Fig. 8. (a) Chemical structures of Pt(II) complexes. (b) AFM images of linear ␭DNA reacted with cisplatin: 0 ␮M (A), 77 ␮M for 6 h (B), 770 ␮M for 1 h (C) and 770 ␮M for 6 h (D). Scale bars are 500 nm. (c) The hypothetical model of the DNA condensing mechanism through a covalent bonding process. Reproduced from Ref. [130] with permission from the Oxford University Press.

6. Biological applications 6.1. Gene delivery The main potential application of DNA condensation in medicine is its use for gene delivery in gene therapy. Gene therapy may be described as the delivery of nucleic acids via a vector to patients for some therapeutic purpose [1]. Because DNA and oligonucleotides are rapidly degraded by serum nucleases in the blood when injected intravenously, the stable compact form of DNA provides protection for DNA and oligonucleotides. The basis of DNA condensation in experiments was studied a decade ago, but its application in gene delivery is just beginning. The two basic problems facing gene vectors are (1) cellular uptake of the condensed DNA particles, and (2) transfection of the

target genes. When pBR 322 DNA is condensed with Ru complexes 19 and 20 and then administered to HeLa cells for 36 h, the plasmid DNA is isolated. The restriction in digestion analysis results shows a new band at ∼550 bp, suggesting that the DNA condensates are able to target the DNA of cancer cells [108]. To directly observe the cellular uptake of DNA condensates, TEM images are helpful. Otherwise, fluorescent dyes are used to label samples, which are observed under fluorescent microscopy or confocal laser scanning microscopy (Fig. 9). As observed in the fluorescence microscopy or confocal laser scanning microscopy [22,121], the fluorescent dye labelled DNA condensates enter the cytoplasm, remain intact and mainly stay around the nucleus. In some case [109], the condensates display nuclear localisation. Flow cytometry is also a useful tool to quantify the intercellular uptake of DNA condensates. As quantified using flow cytometry, Chao et al. [116] found that over

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Fig. 9. Observation of the cellular uptake of DNA condensates using TEM images (a, complex 34, Ref. [119]), fluorescence microscopy image (b, complex 52, Ref. [22]) and confocal laser scanning microscopy images (c, complex 47, Ref. [22]). The blue arrows indicate localisation of the DNA condensates inside cells. Reproduced from Ref. [119] with permission from the Royal Society of Chemistry and Ref. [22] with permission from Elsevier.

88.5% of HeLa cells had taken up the DNA particles condensed by Ru(II) complex 34 after 4 h of incubation. The condensed DNA nanoparticles are believed to enter the cells via cellular membrane endocytosis. The positive zeta potentials surrounding the DNA nanoparticle surfaces may enhance their association with the negatively charged cellular membranes via electrostatic interactions and facilitate its endocytosis. Additionally, the sizes of the DNA condensates may have an impact on the cellular uptake. DNA nanoparticles with diameters of <30 nm or >200 nm have poor cellular uptakes [22]. Small or large DNA condensates do not facilitate cellular membrane crossing. To gain access to the transcriptional machinery of the nucleus and obtain efficient gene expression within cells, gene vectors that compact DNA into small particles should aid in the transport of DNA to the nucleus. The cytosol presents multiple barriers to DNA condensates en route to the nucleus [133]. Diffusion of free DNA in the cytoplasm is negligible. DNA condensates induced by metal complexes enter the cytoplasm around the nucleus and wait to be transported to nucleus. Only a few DNA condensates induced by metal complexes can localise to nucleus. DNA fragmentation in the cytoplasm presents another barrier [134]. Metal complexes as gene carriers may offer protection for DNA from degradation in the cytoplasm [116]. While the cytosol is the prime location for DNA condensates, gene transfer requires them to be delivered to the nucleus. To promote nuclear uptake, positively charged peptides, such as nuclear localisation sequences (NLS), have been utilised. Liu and co-workers explored the expression of genes transferred using a series of Co(II) complexes (40, 43–44, 49) in the presence of NLS (PKKKRKV) [121]. The transfection efficiency of the NLS-bound condensates is approximately 5-fold greater than the NLS-free condensates. Meanwhile, the cell viability assay of the NLS-bound condensates shows lower cytotoxicity than the NLS-free condensates. The positive-charge rich NLS peptide neutralises most of the negative charges on the DNA via electrostatic interactions, associated with the ␲–␲ interactions between the bzim groups of the complexes and the DNA base pairs, leading to a reduction in the sizes and a rise in the surface charges of the DNA condensates. The suitable sizes and large, positive surface charges facilitate the entrance of the nanoparticles into cells and promote the entrance of the DNA condensates into the nucleus through the nuclear pores. 6.2. Luminescent tracking of gene vector Although Cu(II) or Co(II) complexes can condense DNA to form nanoparticles and enter into cells to transfect target genes, they are non-emissive. Therefore, to observe their entrance and location in cells, an additional dye is needed to label free DNA or DNA particles. In addition to the side effects they may introduce into the metal complexes–DNA condensates system, organic dyes suffer

from short emissive lifetime and serious photo bleaching and, therefore, are not suitable for long-term tracking studies. Some luminescent polymers are applied for intracellular imaging, such as the linear polymers polyethylenimines (LPEI), which exhibits a 2% luminescent quantum yield and a 2.6 ns lifetime [135], and hyper branched poly(amido amine) containing ␤-cyclodextrin, which has a much weaker luminescence with a <1% quantum yield [136]. Ru(II) complexes are emissive due to their excellent photochemical properties, such as large Stokes shifts, high chemical and photo stability and relatively long lifetimes, and they have been applied as novel and promising probes in cell staining systems [63]. Ru(II) complexes (19–24) emit brightly in organic solvents with a characteristic broad emission peak between 600 and 750 nm, and emit relatively weakly in aqueous solutions [108–112]. After reacting with DNA in aqueous solutions, an increase in luminescence intensity is observed. Therefore, these Ru(II) complexes condense DNA into nanoparticles that are also luminescent. After administering to cells, these luminescent DNA condensates are observed as bright yellow (or orange or red) spots, indicating the uptake of the DNA condensates by the cells. Complex 34 [116] has been employed in long-term DNA intracellular tracking. The plasmid enhanced green fluorescent protein (pEGFP) gene is delivered by 34 to cells. As shown in Fig. 10, the DNA condensates enter the nucleus within 30 min, indicated by the red spots from the Ru(II) complex luminescence. Within 60 min, the entire nucleus is stained red, and 34 replaces the DAPI dye. The EGFP expression is detected within 12 h and increases after 24 h and 48 h. Luminescent complexes that can condense DNA could lead to a new class of non-viral gene vectors for real-time tracking. 6.3. Antitumor agents Cisplatin is an effective antineoplastic agent that is used for the treatment of cancer, including testicular, head and neck, ovarian and small cell lung neoplasms. The clinical success of cisplatin has resulted in the development of new metal complexes as potential anticancer drugs [63,66,93,137–141]. BBR 3464 (71), a 4+ cationic trinuclear platinum complex, exhibits cytotoxicity at ten to even thousands times lower dose limit than cisplatin in both cisplatin-sensitive and cisplatin-resistant cancer cells and has undergone phase II clinical trials in the treatment of ovarian and lung cancers [142,143]. Interestingly, complex 28, the noncovalent analogue of BBR 3464 has also exhibited moderate cytotoxic activity in several ovarian carcinoma cancer cell lines and induced caspase-dependent apoptosis in both primary mast cells and transformed mastocytomas by replacing the chloride leaving groups with ammonia [142]. The antitumor effects of covalent platinum complexes, such as cisplatin, oxaliplatin and BBR 3464, mainly result from their ability to damage DNA by forming various types of covalent adduct, which

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Fig. 10. Long-term tracking of complex 34-pEGFP DNA particles in cells. DAPI, complex 34 and green fluorescent protein are visualised in blue, red and green, respectively. Reproduced from Ref. [119] with permission from the Royal Society of Chemistry.

affect essential processes, such as replication and transcription, and thus, lead to cell death. Although the exact role of the DNA structural changes in terms of loop formation as well as compaction in their cytotoxic effects is yet to be revealed, a correlation between the loop structures and the cytotoxic effects could exist because the junction regions that are formed as a direct consequence of DNA crosslinking would inhibit strand separation of dsDNA, which is an essential step in DNA replication. Moreover, compaction of the DNA molecule could prevent access to the DNA sequences for enzyme molecules that are relevant to DNA transcription. Complex 28 is distinct from BBR 3464 in DNA binding. Its cytotoxic activity might be directly associated with its unique ability to condense DNA [115]. These complexes bind to and condense DNA competitively with naturally occurring DNA condensing agents, such as polyamines and putrescine, resulting in a disturbance of the correct binding of regulatory proteins, initiating the onset of apoptosis. This behaviour might lead to a new class of anticancer agents.

7. Concluding remarks High charge densities, unique three-dimensional structures and photophysical and electrochemical properties of metal complexes give them advantages in the design of DNA condensing agents and in the application of non-viral gene vectors. Metal complexes have become a new class of non-viral gene vectors in addition to cationic lipids, polymers and nanoparticles. Electrostatic attraction is a prerequisite for the approach of metal complexes to DNA, and charge neutralisation of the negatively charged DNA backbone is the major driving force in DNA condensation. Additional DNA binding effects, such as DNA intercalation and hydrogen bonds, can enhance DNA condensation. We divide metal complexes into different styles based on their DNA binding interactions and DNA condensing mechanisms. One group contains the metal complexes that bear high positive charges and can condense DNA through electrostatic interactions. Normally, a +3 or higher positive charge is needed to condense DNA. Divalent Ru(II) complexes 19–24 can condense DNA [108,109], which may be due to their self-assembly in aqueous solution, increasing the positive charges at local sites. With the exception of Co3+ , other metal ions, such as Cu2+ , Ni2+ and Ru2+ , are divalent. An efficient protocol to achieve high positive charges is to construct dinuclear or multinuclear complexes with suitable linking ligands.

A tetranuclear metal complex with a charge up to +8 (34) has been employed as a DNA condensing agent [116]. Another class of metalcomplex-based DNA condensing agents are the metal complexes that have intercalative ligands, which condense DNA through ␲–␲ interactions. Cu(II) or Co(II) complexes of polybenzimidazoles and Ru(II) DNA intercalators show strong intermolecular ␲–␲ stacking of planar ligands, which can insert between adjacent base pairs in the DNA double helix and stabilise the interactions between the complexes and DNA. Driven by ␲–␲ stacking, these metalbased DNA intercalators can condense DNA intramolecularly or intermolecularly, forming individual globular or net-like nanoparticles. Metal complexes that form hydrogen bonds with DNA can also drive DNA condensation. In this case, DNA condensation only occurs at high temperatures, and reversible DNA release is observed at 37 ◦ C. Hydrogen bonds with DNA weaken the hydrogen bonds between the DNA base pairs, and these weakened or broken bonds act as nucleation sites for DNA condensation in this exogenous DNA condensation process. Finally, metal complexes, such as cisplatin and oxaliplatin, which bind with DNA covalently, can condense DNA. The covalent binding of complexes with DNA causes DNA interstrand or intrastrand crosslinking and aggregation into compact globules. Among these interaction modes, electrostatic interaction, ␲–␲ stacking and hydrogen binding are reversible, i.e., the DNA condensates induced by these types of metal complexes can be uncompacted and released under certain conditions. However, the covalent binding with DNA of cisplatin or other platinum complexes is irreversible. Metal complexes exhibit diversity in DNA binding, resulting in DNA condensation. Their application as gene vectors has been studied, and some progress has been achieved. These particles, which have positive potentials, attract cell membranes via electrostatic interactions and enter the cytoplasm via endocytosis, forming endosomes around the nucleus. After being transported to the nucleus, the gene is transfected. The sizes and zeta potentials of the DNA condensates are efficacious for their cellular uptake and gene transfection. The types of metal ions and ligands in the complexes may also have an impact on cellular uptake and gene transfection because the structural factors are related to the sizes and zeta potentials of the DNA condensates. However, the transfection efficiency is still low, most likely due to the numerous extra- and intra-cellular obstacles. Unfortunately, none of these metal-complex-based non-viral gene vector systems have been used in vivo for DNA delivery, which offers even more barriers than DNA delivery in vitro. For example, the cationic metal complexes

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and their DNA condensates readily bind with serum proteins, such as serum albumin [144], and this protein binding hinders their cellular uptake, promotes aggregation and possibly encourages phagocytosis. Compared with traditional lipid-based gene vectors, metal complex gene vectors offer several advantages, such as diversified structures as well as photophysical, magnetic and electrochemical properties. Generally, there is a contradiction that a highly positive charge is needed to condense DNA, but this feature is also conducive to protein binding and cytotoxicity, which hinders its application in gene therapy. However, metal complexes can improve the DNA condensation capability via ␲–␲ stacking or hydrogen bonds without increasing their charges, which may reduce the side effects in gene therapy. The introduction of luminescent Ru(II) complexes combines DNA condensation, observation of cellular uptake, and long term tracking of gene vectors due to their long luminescent lifetimes and photobleaching resistance. Only luminescent Ru(II) complexes have been successfully utilised in novel gene vector systems. Other advantageous properties of metal complexes still remain under investigation. It is believed that smart DNA condensing agents based on metal complexes with optical or electronic responses will be constructed based on rational designs. Novel DNA condensing metal complexes should be used to broaden their applications. The challenges can be addressed by designing multifunctional metal-complex-based gene vector systems that respond to intracellular stimuli (e.g., changes in pH, redox state, or the presence of enzymes) or other external stimuli (e.g., heat, light, magnetic fields, etc.). The combination of metal complexes with lipids or polymers is easily adapted to the construction of multifunctional gene vector systems. Zinc ions are introduced into cationic polymers and increase the gene transfection efficiency [27,52–54]. Redox-active ferrocene has been successfully introduced into lipids and, subsequently, electrochemically transformed, changing the charge density of the lipid, thus controlling the DNA condensation and gene transfection efficiency of the lipid [55–62]. A macrocyclic Cu(II) complex-polycation system has been used as a novel theranostic vector for combining gene delivery and future positron emission tomography (PET) imaging [145]. In addition, direct construction of metal-complex-based DNA condensing agents is valuable but also challenging. Modification with cellpenetrating peptides (CPP) or NLS can promote cellular membrane penetration or nucleic orientation of metal complexes. Therefore, more studies are needed. When utilised in gene delivery systems, metal complexes must have low cytotoxicity to minimise side effects. However, some metal complexes that can condense DNA show cytotoxic activity against cancer cells, especially platinum complexes. Although the exact role of DNA condensation in the cytotoxic effect is not fully understood, it is reasonable to hypothesise that it might be related. Metal complexes compact DNA into nanoparticles, inhibiting DNA strand separation and preventing transcription of enzymes that are essential for DNA replication. These compounds might present a novel class of antitumor agents. We hope that this overview of metal-complex-based DNA condensing agents will increase understanding of DNA binding and condensation, laying the foundation for the design of new, functional DNA condensing agents that can be applied to gene delivery and novel anticancer agents. Acknowledgements This work was supported by the 973 Programme (no. 2014CB845604), NSFC (nos. 21172273, 21171177, 91122010), Programme for Changjiang Scholars and Innovative Research Team at the University of China (no. IRT1298), and the Research Fund for the Doctoral Programme of Higher Education (no. 20110171110013).

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