Metal Complexes—A Promising Approach to Target Biofilm Associated Infections

Microbial biofilms are represented by sessile microbial communities with modified gene expression and phenotype, adhered to a surface and embedded in a matrix of self-produced extracellular polymeric substances (EPS). Microbial biofilms can develop on both prosthetic devices and tissues, generating chronic and persistent infections that cannot be eradicated with classical organic-based antimicrobials, because of their increased tolerance to antimicrobials and the host immune system. Several complexes based mostly on 3D ions have shown promising potential for fighting biofilm-associated infections, due to their large spectrum antimicrobial and anti-biofilm activity. The literature usually reports species containing Mn(II), Ni(II), Co(II), Cu(II) or Zn(II) and a large variety of multidentate ligands with chelating properties such as antibiotics, Schiff bases, biguanides, N-based macrocyclic and fused rings derivatives. This review presents the progress in the development of such species and their anti-biofilm activity, as well as the contribution of biomaterials science to incorporate these complexes in composite platforms for reducing the negative impact of medical biofilms.


Introduction
Infectious diseases remain at the top of the list of mortality and morbidity causes, resulting from many converging factors such as global emergence and the spread of genetically encoded resistance to all currently used antibiotics, the delays in the discovery of novel antimicrobials, and complications associated with biofilm development on tissues and prosthetic devices [1,2]. Microbial biofilms represent the most frequent lifestyle of microbial cells in the natural environment, and in the case of pathogenic microorganisms, they protect them from adverse environmental conditions, representing both reservoirs and sources of disease outbreaks, especially in the case of medical devices [2].
The research in the field demonstrates that most bacteria, including the antibioticresistant ones, and some fungi could develop biofilms. Moreover, the formation of a mixed biofilms has been reported, such as Candida-streptococcal associations in the case of oral diseases [3]. These represent a microbial derived complex sessile community in which the microorganisms adhere irreversibly to an inert or living surface as well as an interface and to each other and are embedded in a matrix of self-generated extracellular polymeric substances (EPS) [4].
This microbial lifestyle is involved in the majority of chronic and hard to treat microbial infections, especially those associated with the healthcare system [5,6]. Microbial biofilms can form on living tissue, resulting in wound infections [7], endocarditis or lung infections survival rate under a variety of stressful conditions [13]. After developing mature biofilms, planktonic bacterial cells will disperse, attaching to new surfaces and thus starting a new life cycle [28]. This growth is the result of a complex process that involves the transport of organic and inorganic molecules together with microbial cells to a living or inert surface, a subsequent adsorption to this surface, and finally an irreversible attachment assisted by EPS production [27].
As result, biofilm development unfolds into several distinctive steps shown in Figure 1, whereas the specific mechanisms of evolution can differ based on the involved microbial populations and the local environmental conditions [29,30]. The four basic steps of biofilm development are [29][30][31][32][33][34]: (i) Adhesion in which the planktonic cells are reversibly attached to the solid biotic/abiotic surfaces; the bacterial attachment on the biotic/abiotic surfaces involves both physical and chemical interactions such as Brownian movements, van der Waals and electrostatic attraction that contribute to the initial phase of microbial adhesion, as well as interactions between these surfaces and the bacterial adhesins, represented by polymeric species exposed on the surface of cells that enable the formation of a "key-lock" bond between the cell and the surface and result in a stronger interfacial adhesion [27,[34][35][36]. (ii) Microcolony formation following initial microorganism adhesion and proliferation, with the generation of a multi-layered biofilm embedded in self-produced EPS, a complex and viscous matrix composed mainly from polysaccharides, proteins and lipids; this matrix represents the greatest barrier to diffusion for both antimicrobials and their delivery systems. In soil microbial communities, the EPS production contributes significantly to the improvement of soil quality due to its electrostatic charge that is attracting and aggregates the soil particles exerting a positive effect on the soils' mechanical conductivity [37]. Moreover, biofilm embedded microorganisms can also produce small, diffusible signalling molecules involved in the density-dependent intercellular communication mechanism called QS; this system allows microorganisms to detect a critical density and assures a coordinated behaviour within the biofilm, such as iron chelation and antibiotic defensive activities [6]. (iii) Biofilm maturation consisting of the development of a three-dimensional structure (3D) with a thickness typically less than 100 mm and a network that assures the efficient transportation of both nutrients and signaling particles in the biofilm; (iv) Detachment or dispersion corresponding to microcolonies or single cell separation that colonize other surfaces; after maturation, the migration of cells to the environment or dispersion is a result of a too dense layer formation [34].
Thus, there are several structural and physiological differences between planktonic and biofilm growth states, but two main distinctive factors are the presence of EPS and QS communication in the latter case.
Based on understanding the microbial biofilm formation mechanisms, traditional models of in vitro monospecific biofilm development have been developed. However, during infection, bacterial cells belonging to different species tend to form multicellular aggregates and biofilms can also disperse, not only as single cells, but also as aggregates [38]. Experimental and computational studies performed on Pseudomonas aeruginosa have shown that there is a competition between aggregates and single cells depending on the access to growth resources, with the balance leaning towards aggregates when competition is high. Thus, these findings show that an aggregate can outcompete the biofilm population arising from a single cell [38].
The biofilm forming abilities of microorganisms depend on several factors such as micro-environmental conditions (temperature, ionic strength and pH), the site of development, the nature of prosthetic material or tissue, nutrient type and concentration, network design and composition, strain type and heterogeneity [31].

Role of the Extracellular Polymeric Substances in Protecting Biofilm Cells
The biofilm gelatinous polymers known as EPS are 3D materials that carry intact microorganisms, attach them to a surface and protect them from environmental stress [32][33][34][35][36][37][38][39]. There are several types present in the environment, such as that bound to cell surfaces ("capsular" EPS), released into solution ("free" EPS), or associated with the hydrated matrix of biofilms [40]. Since EPS plays the role of a protective shelter or a diffusion barrier, the biofilms can resist external stressor attacks, such as by pH, ROS, antibiotics and phagocytosis [30,41].
Among these, the polysaccharides are often the most abundant species found in the biofilm matrix [45,46]. Some Pseudomonas EPS polysaccharides, such as the Pea and Peb, described in P. putida, serve structural purposes, whereas others such as alginate and cellulose play a minor role in biofilm formation and stability but are important in stress protection against ROS generated during stress [39]. The role of both alginate and cellulose in protecting against the ROS stress comes from the hydrophilic nature of polysaccharides, considering that their ability to bind water might reduce the accumulation of intracellular ROS [45].
In the EPS structure, there are several weakly acidic groups (carboxyl, phosphoryl, amide, amino, hydroxyl) that ionize in response to changes in environmental pH, ionic strength [39,40] or in interaction with a metallic ion from the environment or an antimicrobial complex. Metal-proton exchange or metal coordination is not the only mechanism involved in metal interaction with EPS, and other processes, such as cation exchange or electrostatic interactions may also occur [39,40,47]. Among the groups with coordinative abilities, hydroxyl, carboxyl, amino and phosphates represent the main sites involved in interaction with metallic ions from complexes.
Some studies indicated that both proteins and humic derivatives in EPS from activated sludge are strong ligands for Cu(II), and their carboxyl groups play an important role in Cu(II) coordination [48,49]. Furthermore, Cu(II) demonstrated a higher affinity for organic matter of biofilm in comparison with Cd(II), Zn(II) and Mn(II) [50].

Quorum Sensing Role in Biofilm Development
Biofilm development is closely interconnected with the QS mechanism, since its development comes from a cooperative behaviour of the microbial populations embedded in EPS. QS represents a cell-cell communication mechanism that coordinates gene expression as a response to the population cell density. Otherwise, QS synchronizes the switch to a biofilm lifestyle when the population density reaches a threshold level [4,28].
As result, biofilm maturation, its dispersion as well as virulence factors secretion are coordinated by density-dependent biochemical signals emitted in a synchronized way by the bacterial communities incorporated in EPS [28,31,51].
The extracellular signalling molecules are known as autoinducers (AIs), which are recognized by the receptors of producing and neighbouring cells. This signal is amplified and transmitted through appropriate regulatory systems, thus causing the expression of target genes [34,51]. Moreover, the signalling molecules allow bacteria to perceive and respond to temporal and contiguous environments [52].
The AIs are produced at basal level and gradually accumulate during microbial growth, creating a positive feedback loop that means that as the bacterial population grows, the concentration of the AIs in the surroundings increases, causing more inducer molecules to be synthesized [53]. The accumulation of critical concentrations of such species activates in response to the specific receptors. These are able to initiate in the biofilm a signaling cascade of coordinated induction/repression of multiple target genes, responsible for the optimal adaptation of biofilms to the biotic/abiotic media [52]. As result, QS enables microorganisms to respond quickly to environmental changes, such as the availability of nutrients, or the presence of other microbes or toxins in their environment [53].
The opportunistic microorganisms use the threshold AIs concentration to overcome the host defense mechanisms. To assure both infection progress and survival, microorganisms stop the synthesis of virulence factors until they reach the threshold density required for initiating the infection process [52].

Anti-Biofilm Strategies
The developed anti-biofilm strategies are acting at different levels: i.
Inhibition of the initial microbial adhesion to the substrate, by designing materials or coatings exhibiting electrochemical repulsion or by eluting substrata loaded with antimicrobial substances [54]; ii. Inhibition of adhesion by blocking the expression of adhesins or their recognition sites; iii. Inhibition of adhesion by blocking flagellar motility and impairing bacteria to reach the adhesion sites; iv. Inhibition of biofilm formation by interfering with the QS mechanisms; the quorum quenching (QQ) by using QS inhibitors can attenuate the production of bacterial toxin or biofilm formation and provide a novel therapeutic approach to control bacterial infections. The QQ can be achieved either by blocking the QS molecules biosynthesis, by destruction of QS molecules, or by inhibition of the binding of AIs to QS receptors [55]. QSIs often exhibit a synergic anti-biofilm activity with antibiotics [56]. v.
Inhibition of biofilm maturation by blocking the production of extracellular polymeric substances (EPS); vi. Inhibition of maturation by inhibiting the growth and multiplication of cells in biofilm by using different approaches such as application of infrared and light pulsing, direct-current electrical stimulation, ultrasound and alternating electric fields [57]; use of drug delivery systems [58]; local delivery of catheter locks, intratracheal locks etc. [59]; vii. Targeting non-growing dormant and persister biofilm cells (by metabolic interference or lytic substances) or interfering with the formation of persister cells (by inhibiting the bacterial regulatory tetra and penta-guanosine phosphate nucleotides, which activate the inhibitors of cell growth) [60]. viii. Elimination of the biofilm by disorganization of the protective extracellular matrix based on EPS-degrading enzymes, anti-EPS antibodies, and nucleic acid binding proteins, matrix destabilizing agents such as ethylenediaminetetraacetic acid (H 4 EDTA), which are targeting the biofilm extracellular polymeric substance, leading to biofilm dispersion or by the mechanical debridement of biofilms by using ultrasound and surgical procedures [61][62][63].
Although many strategies have been proposed to control biofilm development, however, very few became available to the clinicians [2].

Complexes with Antibiofilm Activity
One of the most promising approaches for the treatment of biofilm-associated infection is based on designing agents that exhibit multiple mechanisms of action. The special characteristic of biofilms presented above together with their resistance to classical antibiotics requires such strategies like complexes that so far demonstrated their utility, at least in vitro.
So far, for antibiotics, the achievement of an anti-biofilm activity was reached either by modification of the conjugated moieties to the basic antimicrobial backbone or by a combinatory therapy [54,55,64].

Complexes with Antibiotics
When the activity of antibiotics and antifungals was outclassed both by the emergence of resistance and by the reduced efficiency against biofilms, solutions were sought to overcome these problems. One of these solutions was provided by antibiotic complexation to biocations and especially to transition metal ions, which easily change their oxidation state and as a result can interact with target biomolecules involved in the destruction of the biofilm by redox processes.
Among bacteria, Pseudomonas aeruginosa represents an important nosocomial pathogen that is responsible for a large spectrum of infections, such as endocarditis, cystic fibrosis, burn, wound and urinary tract infections. Its pathogenicity is related to virulence factors such as biofilm formation as well as exotoxins, elastase, alginate and siderophores production [145]. The major limitation of therapy in chronic pulmonary infection is the P. aeruginosa biofilm formation in the lung, this being over 1000-fold more resistant to antimicrobials compared to planktonic bacteria [146]. As result, complex [Cu(Hcip)(H 2 O) 2 ]SO 4 ·2H 2 O (1) (Hcip: ciprofloxacin-a fluoroquinolone antibiotic) was studied as anti-biofilm species able to provide a high concentration of Hcip in the lungs. [65]. Besides structure determination for (1·EtOH), another study demonstrated that at sub-minimum inhibitory concentration (MIC), this complex exhibits a significant reduction in motility, biofilm formation, alginate, violacein and pyocyanin production and sensitivity to H 2 O 2 in a concentration dependent manner [66]. Considering the biological effects of complex (1) and its inhibitory activity on QS at low concentrations, quantified through the expression of QS genes lasI and lasR, this may be used as an effective approach in the management of infections caused by this microorganism.
Complex {[ZnCl 2 (fcz) 2 ]·2C 2 H 5 OH}n (2) (fcz: fluconazole-a triazole antifungal) showed both strong inhibition of C. albicans clinical isolates biofilm formation at subinhibitory concentration and the ability to reduce its adherence to human non-small cell lung cancer A549 cells in vitro. Moreover, this compound inhibits pyocyanin production and biofilm formation in P. aeruginosa, results that recommend its further examination in the mixed Candida-P. aeruginosa infections [67].
Compound [Mn(H 2 O) 6 ] 0.5 [Mn(smx) 3 ] (3) (smx: sulfamethoxazole-an antibiotic from the second generation of sulfonamides) was fully characterized by single crystal X-ray diffraction and proved to be an inhibitor of both the planktonic and biofilm embedded Staphylococcus aureus strain [68]. Complexes of Hg(II), Cu(II), Cd(II) and Ag(I) with this ligand were reported as anti-biofilm inhibitors for E. coli [69], while its species with Au(I), Cu(II), Ag(I), Hg(II) and Cd(II) were found to be active against biofilm produced by Mycobacterium abscessus, M. fortuitum, and M. massiliense strains, the most active being [Au(smx)(PPh 3 )] (4) (Ph: phenyl) species for all tested strains [70]. Similar activity was evidenced for Au(I) sulfadiazine complexes, an activity associated with the inhibition of cyclic-di-guanosine monophosphate (c-di-GMP) synthesis, which is an important signaling molecule for the rapidly growing mycobacteria (RGM) biofilm formation. These RGM are found in non-sterile water and are often associated with severe post-surgical infections and affect immunocompromised patients [70].
Amoxicillin (amx) is a bacteriolytic β-lactam antibiotic that inhibits the carboxypeptidase and transpeptidase required for peptidoglycan biosynthesis. Several studies revealed that its complexation is important to enhance antibacterial activity [71,72], and as a result, its species with Cu(II), Zn(II) and Fe(III) in 1:1 molar ratio were synthesized and studied against E. coli. The Cu(II) and Fe(III) complexes were more potent compared with Zn(II) complex with amx both on planktonic and biofilm embedded strains, the involved mechanism being oxidation by the redox active cations [73].
Hence, the coordination of fluoroquinolone, sulfonamide and β-lactam antibiotics as well as triazole antifungals to both essential and non-essential metal ions lead to an improved anti-biofilm activity of these antibiotic classes. The activity improvement could be related to both the coordinative and the redox ability of metallic ions that can interfere with either EPS or QS. The species with redox active metallic ions are by far more active as a result of ROS generation.
Several complexes of type [Cu(cbl)(PPh 3 ) 2 X] (17) (X: Cl, Br, I) with β-carboline (cbl) at sub-MIC concentration interfered significantly with the QS regulated functions in Chromobacterium violaceum (violacein), P. aeruginosa (elastase, pyocyanin and alginate production) and S. marcescens (prodigiosin). Aside from the inhibitory effect on the EPS production and swarming motility, these complexes also demonstrated potent broad-spectrum inhibition of biofilm formed by P. aeruginosa, E. coli, C. violaceum, S. marcescens, K. pneumoniae and L. monocytogenes [84]. Also, various N-heterocyclic carbene (nhc) complexes with Ag(I) and Cu(I) of type [M(nhc)Cl] (18) can inhibit biofilm formation of L. monocytogenes, S. aureus, S. epidermidis, P. aeruginosa and E. coli at low concentrations. The Ag(I) complexes of this series bearing aromatic groups on lipophilic nhc ligands exhibits the broadest anti-biofilm activity [85]. Moreover, a collection of Cu(I) complexes bearing nhc derivatives with different substituents was developed to prevent Streptococcus mutans biofilm formation, the most active being the less lipophilic and less sterically hindered compound [86]. A similar activity was achieved for Ag(I) species with nhc ligands in the case of E. coli and C. albicans biofilms [87].
Complexes of type [M(fphen)(dach)]X 2 (23) (M: Cu, Pt, Pd, fphen: functionalized 1,10-phenanthrolines, dach: 1S,2S-or 1R,2R-diaminocyclohexane, X: Cl, ClO 4 ) showed significant activity against biofilms associated with a MRSA clinical isolate and were more active in the biofilm removal than vancomycin, an antibiotic currently used in the treatment of MRSA infections. The dach have no influence on activity and Cu(II) complexes, which were more active comparing with Pt(II) and Pd(II) ones as a result of nuclease activity characteristic for Cu(II) complexes bearing phen derivatives [91].
Good activity was demonstrated for a series of complexes of type [Mn(snh) 2 X 2 ] (25) (snh: substituted nitrogen heterocycle like pyridine or imidazole substituted with HO, CHO, CO or COOH groups, X: Cl, NO 3 , 1 2 SO 4 ) in P. aeruginosa biofilm eradication. The structure-activity relationship analysis evidenced an enhanced activity for pyridine derived ligands for hydroxyl as a substituent and nitrate as a counterion. In addition, complexes are non-toxic on primary human fibroblasts, exhibit catalase like activity and the ability to easily reach at Mn(III), associated with the compound's ability to interact with biological target involved in biofilm destruction through redox processes [93].
By combining the relative low toxicity of essential ions (Co(II), Ni(II), Cu(II), Zn(II)) with the biological activity of N-heterocycles (pyridine, pyrimidine, imidazole and pyrazole derivatives), several valuable anti-biofilm species were developed. One of the potential mechanisms of action revealed by different studies is ROS generation.
The antimicrobial activity is enhanced when two or more isolated or fused N-heterocycle rings are present in different ligand molecules. Moreover, complexes bearing mixed ligands, both from this class of derivatives, proved to be more active compared with those having only one type of N-heterocycle in their structure.
It is worthy of mentioning the wide spectrum of most of Cu(II) complexes as well as the good activity against resistant strains such as MRSA, ESBL E. coli and multi-drug-resistant P. aeruginosa.

Complexes with Schiff Bases
In recent years, researchers have drawn significant attention toward Schiff bases and their metal complexes considering their numerous applications in the biological field, such as their antiviral, antimicrobial, antimalarial, and antitumor properties. Furthermore, some of complexes exhibit a good anti-biofilm activity besides the antimicrobial one against planktonic bacteria.
A The best activity was achieved for Cu(II) and Zn(II) species and, moreover, Cu(II) complexes of the series also exhibited a promising antiproliferative activity on human laryngeal carcinoma (HEp 2) and HT 29 cell lines.
Compounds with the mixed ligands [Cu(BS7)(phen)] (40) (H 2 BS7: 3-methoxy-2oxidobenzylidenebenzohydrazide) were characterised by single crystal X-ray diffraction, and it was found that it repressed both P. aeruginosa and S. aureus biofilm formation. Moreover, the compound exhibited the ability to intercalate into the DNA strands [108].
Overall, the available data indicate that several Cu(II) complexes with multidentate Schiff bases having N,O or N,O,O donor sets and bulky substituents like phenyl, pyridine, triazole or pyrazole exhibit a very good anti-biofilm activity on a wide range of Grampositive and Gram-negative bacteria. The activity is enhanced for the species bearing besides Schiff base another chelate ligand such as 1,10-phenantroline. However, the specific mechanisms of action for this type of complex remains to be elucidated in future research.

Complexes with Biguanide Derivatives
From the perspective of anti-biofilm activity, the complexes with biguanide derivatives have also shown promising potential. The biguanides are valuable ligands that can coordinate in neutral, anionic or cationic form. Due to their chelate coordination through the imide groups they form stable complexes with transition metal ions as neutral or anionic species [109].
The metformin (N,N -dimethylbiguanide, Hdmbg) compound used for type II diabetes treatment by decreasing the glucose release from the hepatic tissue is also the best known of these derivatives as complex formatters [110]. Furthermore, the dmbg moiety incorporated into a polymeric material was used as an efficient catheter coating that prevented the development of S. aureus and E. coli biofilms [111], while a novel nano-system based on a polybiguanide moiety was recently developed as a biocompatible and effective inhibitor of MRSA biofilms both in vitro and in vivo [112].
This anti-biofilm potential of biguanides motivated the research for the design of complexes with such ligands. Among these, complexes [M(Hdmbg) 2 ]X 2 (41) (M: Mn(II), Ni(II), Cu(II), Zn(II); X: CH 3 COO [113], and ClO 4 [114]) with this ligand demonstrated the ability to inhibit S. aureus and P aeruginosa biofilm development on inert substratum, the most active being Cu(II) and Zn(II) complexes. Moreover, all complexes exhibited very low cytotoxicity levels on human cervical cancer (HeLa) cells.
The compounds [M(Htbg) 2 ]Cl 2 (46) (M: Ni, Pd, Pt) proved to have good efficiency against the biofilm embedded S. aureus, B. subtilis and E. coli cells at sub-MIC values. The most efficient compounds showing the largest spectrum of anti-biofilm activity were Pd(II) and Pt(II) complexes. Moreover, the Pt(II) compound exhibited the most significant antiproliferative activity on the human cervical cancer (SiHa) cell line, inducing a cell cycle arrest in the G2/M phase [119].
Recent studies indicated that the biguanide incorporation into a macrocycle ligand generated promising complexes [M(dmbgMc)] (49) (M: Ni, Cu; H 2 dmbgMc: ligand resulted from Hdmbg condensation with ammonia/hydrazine and formaldehyde) for applications in the treatment of infections produced by pathogenic microorganisms, including those complicated by biofilm development. A broad anti-biofilm spectrum on P. aeruginosa, E. faecium, E. faecalis, C. albicans and C. parapsilosis was registered for Ni(II) complex with macrocycle resulted in ammonia system [123].
A good anti-biofilm activity was obtained by combining the biguanides ability to disrupt biofilm with their chelate properties. These generate several valuable species with both essential (Cu(II), Zn(II)) and non-essential (Ni(II), Pd(II), Pt(II), Ir(III)) metal ions. The anti-biofilm activity is enhanced for perchlorate species and biguanides bearing bulky substituents as a result of the favourable balance between their water solubility and lipophilicity. The ROS generation was reported as one of the potential mechanisms of action for Cu(II) species.
The complex [Cu(mc3)]Cl 2 (52) (mc3: macrocycle synthesised by the condensation reaction between substituted carbohydrazone and thiosemicarbazide) was found to be able to disrupt the biofilm produced by MRSA [126] while the antibiofilm activity of EDTAbased phenylene macrocycle (edtaod) on L. monocytogenes, P. aeruginosa, S. typhimurium and S. aureus was enhanced by its complexation with Cu(II) and Fe(III), activity that is similar and closely related with the molecular volume of EDTA complexes [127].
These reports evidenced a good anti-biofilm activity for both mono-and bisazamacrocycle complexes of Ni(II), Cu(II) and Zn(II) against a wide range of Gram-negative and Gram-positive bacterial strains. However, future studies are needed to explore their specific mechanisms of action.
The complex [Cu(cur) 2 ] (60) (cur: curcumin) inhibited biofilm formation in the case of S. aureus and significantly repressed the expression of lasI and lasR genes, demonstrating its QS inhibitory effect [136].
Moreover, the complex with mixed ligands [Zn(tsa)(tmeda)] 2 (65) (Htsa: thiosalicylic acid; tmeda: N,N,N ,N -tetramethylethylenediamine) is very active on the old biofilms of S. aureus, as indicated in the studies performed by confocal laser scanning microscopy which revealed its bactericidal activities, possibly by membrane alterations, as demonstrated by the propidium iodide (PI) uptake [142].
There are few reports concerning the anti-biofilm activity of some multidentate ligands bearing carboxylate/thiocarboxylate, hydroxy or amino groups. The good activity reported for species bearing sulfur as donor atoms opens promising leads that will likely boost future research in the field.
The anti-biofilm activity of complexes together with identified mechanisms of action is presented in Figure 2.

Materials as Carriers for Metal Ions or Complexes with Anti-Biofilm Activity
In order to overcome the problems associated with the use of antibiotics, some polymer species complexed with proper metal ions or loaded with biological active complexes as well as nanomaterials with anti-biofilm properties and biocompatibility/environmental safety have also been developed. Several dendrimers and polymers appropriately modified with coordinative groups able to chelate metal ions were designed for this purpose. Moreover, several attempts were made for the complexes' incorporation into organic or inorganic matrices.
Dendrimers are branched three-dimensional macromolecules based on a nitrogen, phosphorus and silicone backbone, and carry groups able to coordinate metallic ions, properties that afford applications as metal ion carriers for anti-biofilm purposes. A proper selection of dendritic scaffold, generation type and nature of donor atom can provide a potent system that can overcome the limitations of traditional therapies with antibiotics [147]. As result, a second-generation poly(propylene imine) dendrimer modified with acridine and loaded with Cu(II) was developed first as an antimicrobial with low cytotoxicity against the human epithelial type 2 (HEp-2) cell line. Afterwards, a cotton fabric modified with this dendrimer was proved to exhibit anti-biofilm activity against B. cereus and P. aeruginosa strains, and no cytotoxicity on the HEp-2 cell line [148].
Another dendrimer from first generation of polyamidoamine (PAMAM) functionalised with 1,8-naphthalimide moiety was loaded with Cu(II) and attached to the cotton surface. The study showed that this material prevented the biofilm formation in the case of B. subtilis, B. cereus and A. johnsonii, the best effect being observed for the last strain [149].
Furthermore, a material based on a second generation PAMAM dendrimer modified with 4-(N,N-dimethylaminoethyloxy)-1,8-naphthalimide and conjugated with cis-Cu(NO 3 ) 2 moiety was developed and deposited on cotton fabric. The obtained composite exhibits inhibitory activity against B. cereus, P. aeruginosa and C. lipolytica biofilms [150]. On the other hand, a water-soluble carbosilane dendrimer, decorated with iminopyridine groups and conjugated with Cu(OH 2 )(ONO 2 ) 2 moiety (69), was developed as a potent species against both planktonic and biofilm embedded S. aureus and E. coli cells [151].
A series of 2,6-pyridinedicarboxylate-based polyesters employing several diols with different aliphatic chains were synthesised and complexed with Cu(II) and Ag(I). The composites were tested for their antibacterial potential and were found to effectively resist P. aeruginosa attachment and colonization, the silver-based polymers being superior in comparison with their copper analogues [154].
Schiff-base ligands were grafted on a natural biopolymer of ε-poly-L-lysine functionalized mesoporous silica SBA-15 for the selective coordination of Ag(I). This nano-species (72) exhibited inhibitory effect on E. coli, S. aureus, M. tuberculosis and C. albicans. Besides killing the C. albicans cells, this system inhibited biofilm formation and eliminated preformed biofilms, with no development of resistance during continuous serial passaging. The antifungal activity is related to the disruption of bacterial cell membranes and increased levels of intracellular ROS. In mouse models of multidrug-resistant C. albicans infection, nano-species exhibited an efficient in vivo fungicidal efficacy superior to the antifungal drugs, amphotericin B and fluconazole. Moreover, treatment induced negligible toxicity against normal tissues [155].
Anti-biofilm agents based on Ga(III) or zinc Zn(II) complexed with protoporphyrin IX or mesoprotoporphyrin IX were found to be highly effective in the inhibition of both planktonic bacterial growth and biofilm formation. These complexes were incorporated in poly(ether urethane) (PEU) polymer films in order to obtain a system for their controlled sustained release by using poly(ethylene glycol) (PEG) as a pore-forming agent. All complex-loaded PEU films exhibited in vitro a ≥ 90% reduction of S. epidermidis and P. aeruginosa in both suspended and biofilm culture. Moreover, the cytotoxicity and endotoxin evaluation demonstrated no adverse responses, while in vivo studies further substantiated the anti-biofilm efficacy of these composites [156].
The development of an effective treatment for MRSA infections is complicated by the fact that antibiotics can be degraded by β-lactamases, and the antibiotics cannot penetrate the full depth of biofilms. Considering the nanoparticle-based carriers' ability to deliver antibiotics with better biofilm penetration, a platform for β-lactam antibiotics and β-lactamase inhibitors co-delivery based on metalcarbenicillin framework-coated mesoporous silica nanoparticles (MSN) was developed. Carbenicillin, a β-lactam antibiotic, was used as a ligand for Fe(III) in order to generate a metalcarbenicillin framework able to block the pores of the MSN. The study evidenced that this system achieved a better penetration in the depth of biofilms and exhibited an inhibitory effect on the MRSA biofilm both in vitro and in vivo [159].
Despite the current tendency to use drug delivery systems (DDSs) based on biocompatible and biodegradable matrices, the studies concerning the use of DDS for anti-biofilm species are rather few. The available studies are reporting Cu(II) or Ag(I) coordination to dendrimers or natural or synthetic polymers providing N as donor atoms or the incorporation of some complexes into polymeric (linear or branched) or silica matrices, or even in organic-inorganic composites.

Conclusions
Biofilm development on viable tissues and prosthetic devices represents an important challenge for the medical field, due to their involvement in biofilm-associated infections, which are persistent and hard or often impossible to treat. Therefore, finding efficient agents capable of surpassing the numerous mechanisms of biofilms tolerance to high doses of antimicrobials represents one of the hot fields of research for both microbiologists and chemists. Metal complexes offer promising leads for the development of biofilm disrupting agents due to their multi-target, complex mechanism of action. The current data reveal the efficiency of metal complexes against biofilms formed by epidemiologically important resistant strains, such as MRSA. From the point of view of ligands, it was observed that nitrogen-based ligands mostly involved in chelation lead to an enhanced anti-biofilm activity, and Cu(II) complexes with these species exhibit the most promising activity associated with biofilm disruption. However, most studies in the field are focused only on assessing the in vitro anti-biofilm activity, and very few address the elucidation of the intimate mechanisms of action; the current studies identified the QS inhibition or ROS/NOS generation as some of the main mechanisms involved in biofilm disruption by metal complexes. From these data it is obvious that this represents an open field, and there are many aspects that must be elucidated by further studies.
One of the most promising leads for the design of new complexes with anti-biofilm activity are the redox active metal ions such Cu(II), Fe(III) and Mn(II) but the less-studied ones such VO(IV) and Ru(II) should be also considered. All of these ions have ROS or NOS generation as a common mechanism of action. The best anti-biofilm activity is achieved then these ions are combined with multidentate ligands, especially bearing N as donor atoms, assuring enhanced stability. Furthermore, the perchlorate anion that easily generates single crystals seems to enhance the anti-biofilm activity in complexes bearing neutral organic ligands. The most active compounds show an improved activity after incorporation in organic, inorganic or composite matrices. The majority of the current literature refers to the in vitro study of the anti-biofilm activity of complexes, this explaining the paucity of novel anti-biofilm agents in medical practice. Thus, there is an urgent need for additional in vivo studies in this field in order to elucidate the safety, efficacy and toxicity of these species in order to develop new valuable drugs for the treatment of biofilm-associated infections.