Soft Matter Composites Interfacing with Biomolecules, Cells, and Tissues - Bioinspired Approaches for Human-Centric Technologies - page 62

lead to indirect damages by means of reactive oxygen species (ROS) production. In
this respect, singlet oxygen, hydrogen peroxide, superoxide radical anion, and
hydroxyl radical are known to target lipids, DNA, RNA, and proteins, causing
severe effects, including malfunction of membranes, proteins, and DNA replication
machinery (Cabiscol et al. 2000 ). The issue of Ag + -related ROS production
remains, however, quite controversial, as some research works addressed strong
correlation (Choi and Hu 2008 ; Inoue et al. 2002 ; Hwang et al. 2008 ), while other
experimental data displayed no significant trends (Sintubin et al. 2011 ; Xiu
et al. 2011 ). This is likely due to the ability of microorganisms to resist oxidative
stress by adopting several molecular strategies, which include direct immediate
detoxification carried out by enzymes (i.e., catalase, superoxide dismutase, and
peroxidase) (Fang 2004 ) and a long-term detoxification controlled by a transcrip-
tional expression of several proteins (including OxyR, SoxRS, and PerR). These
strategies enable bacteria a high survival probability against ROS-related stress.
However, it should be considered that a detailed and universal description of the
antibacterial mechanisms of AgNPs is still not available, also due to general lack of
standardized materials and protocols to be employed for the assays. In particular,
the synthesis of high-quality AgNPs, in terms of narrow size and shape distribution,
remained a challenge for several years, and only in the recent years some good
results were achieved (Burda et al. 2005 ; Wennemers 2012 ; Liang et al. 2010 ;
Belser et al. 2009 ; Upert et al. 2012 ). A typical reaction is governed, in fact, by
different thermodynamic factors. Capturing the distinct stages of a controlled
atomic nucleation around few atoms represented a serious challenge, which has
been only solved recently. However, most of the data available to date about the
bactericidal properties of AgNPs have been obtained with particles having almost
uncontrolled physicochemical properties or particles that were not characterized.
Together with the absence of an analytical approach for particles characterization
and testing, this hindered the possibility to have a confident explanation of the
various phenomena. In this respect, an important point is that AgNPs should be
characterized by means of different techniques (e.g., dynamic light scattering,
UV-visible spectroscopy, transmission electron, and/or scanning electron micro-
scopy), both in aqueous medium and after incubation in the bacterial culture
medium. The specific components of the media may, in fact, interact with the
particle surface (e.g., forming a protein corona), significantly changing their orig-
inal physicochemical properties and, consequently, also the observed biological
outcomes (Walczyk et al. 2010 ; Monopoli et al. 2011a , b ). In particular, the
colloidal stability of AgNPs in biological growth media is an important parameter
to keep under control: NPs may form aggregates/agglomerates and precipitates,
consequently compromising the effective dose of silver and, also, the NPs efficacy.
Furthermore, also the medium proteins and salts may bind free Ag + (released from
the AgNPs surface), reducing the overall final dose available.
Another point is the kinetic of silver oxidation that may be strongly affected by
the specific medium used. In this latter case, a correct procedure includes the use of
different methods for quantifying, in situ, the Ag + release from the NPs surface. For
instance,

the inductively coupled plasma spectrometry-based techniques (i.e.,