## 5 Coating Materials with Embedded Antibiotics and Other Antibofilm Agents
### Biofilm Control by Inhibitors or Modulators of Bacterial Signaling Processes
#### 5.5.2 Antibiofilm Coatings Are Interfering with c-di-GMP Signaling
However, the utilization of NO-donor compounds that spontaneously release NO in solution to treat biofilm infections is hampered for two reasons. First, NO is a lipophilic gas that can freely penetrate across cell membranes, and due to the high reactivity imparted by its free radical character, it damages non-selectively both bacterial cells and host cells. Second, the high reactivity and the abundance of available molecular targets cause the half-life time of NO in biological systems to be truly short. Therefore, the successful application of the NO-induced biofilm dispersal approach critically depends on the development of delivery systems capable of targeted on-demand NO release. An important and highly versatile class of NO donors are the diazeniumdiolates (NONOates). These compounds containing the [N(O)NO] functional group are readily available by reacting secondary amines and high pressure of NO. Dialkylamino diazeniumdiolates undergo proton-initiated decomposition leading to secondary amines and NO according to the mechanism depicted in Scheme 12 [66]. Although the oxygen atoms are the preferential sites of protonation, only protonation at the nitrogen atom bearing the two alkyl radicals results in heterolytic N-N bond cleavage leading to eventual NO release. [PERSON] et al. [67] developed a new class of targeted NO-donor prodrugs based on cephalosporiorn-3'-diazeniumdiolates. Due to the clever design of these prodrugs, NO release from a NONOate donor is prevented in the absence of some bacteria-specific enzymes. Thus, bacteria-triggered enzymatic NO release and targeted NO delivery were achieved. 3'-Diazeniumdiolates-substituted cephalosporiorines spontaneously release the NONOate leaving group following nucleophilic attack from specific bacterial \(\beta\)-lactamases and cleavage of the \(\beta\)-lactamase ring. Moreover, it seems that not only secreted \(\beta\)-lactam antibiotics inactivating \(\beta\)-lactamases can activate the release of NO-donor NONOates since NO release from these prodrugs was also triggered by non-\(\beta\)-lactamase-producing _E. coli_ extracts. The authors suggested that other bacterial enzymes, most probably transpeptidases or the transpeptidase domain of penicillin-binding proteins, which are key enzymes involved in bacterial cell wall synthesis at the same time being the target enzymes of \(\beta\)-lactam antibiotics [68], can also activate the release of the NO-donor leaving group from these prodrugs. The innovative 3'-diazeniumdiolates-substituted cephalosporiorines were tested on several pathogens, including mixed species biofilms from cystic fibrosis (CF) sputum, and they proved to be highly effective in biofilm dispersal. Moreover, they can potentiate the antimicrobial activity of therapeutic agents currently used in the treatment of chronic lung diseases, including chronic obstructive pulmonary disease (COPD) and reduce biofilm tolerance to antibiotics. For instance, when co-administered with tobramycin or ciprofloxacin, they significantly decrease the number of colony-forming units (CFU) in _P. aeruginosa_ biofilms and also enhance the susceptibility to azithromycin of _Haemophilus influenzae_ biofilms [69]. Figure 12: Mechanism of proton-driven NO release from diazeniumdiolates [66]. Adapted with permission from Decomposition of amino diazeniumdiolates (NONOates): Molecular mechanisms. Copyright (2014) Elsevier. Besides acting as a signal that triggers biofilm dispersal, NO can be used to actively inhibit bacterial proliferation or kill bacteria when it is administered in a controlled manner and in the appropriate concentration to avoid cytotoxic effects on host cells. So, there are some challenges to be overcome for the safe and effective application of NO donors in clinical practice. First, it is a problem of NO donor stability under physiological conditions and second the ability to constantly deliver NO amounts ranging in the therapeutic window for a prolonged period of time. Keeping this in mind, [PERSON] et al.