Advances in Nanomaterial with Antimicrobial Activity

1. ADAMA SCIENCE AND TECHNOLOGY UNIVERSITY
SCHOOL OF APPLIED NATURAL SCIENCE
DEPARTMENT OF APPLIED CHEMISTRY
MATERIALS CHEMISTRY PhD PROGRAM
ADVANCES IN NANOMATERIALS WITH ANTIMICROBIAL
ACTIVITY
A Seminar Submitted for the Course: Special topics in Material
Chemistry.
Submitted by: Endale Kebede Feyie
ID NO: PGR/18356/11

2. Presentation outline
 INTRODUCTION
 MODE OF ACTION OF NANOMOLECULES
 CURRENT ANTIMICROBIAL NANOMATERIALS AND THEIR APPLICATIONS
 FACTORS AFFECTING THE EFFECTIVENESS OF NANOMATERIALS
 FUTURE PERSPECTIVES FOR ANTIMICROBIAL NANOMATERIALS
 CONCLUSION
 REFERENCES

3. 1. INTRODUCTION
 Bacterial adhesion and proliferation is a serious and increasing concern in everyday life
 Bacterial infections are a major cause of chronic infections and mortality.
 Antibiotics have been the preferred treatment method for bacterial infections because of their
cost-effectiveness and powerful outcomes.
 However, several studies have provided direct evidence that the widespread use of antibiotics
has led to the emergence of multidrug-resistant bacterial strains.
 Therefore, new strategies for controlling bacterial activity are urgently needed, and
nanomaterials constitute a very promising approach.
 Several natural and engineered nanomaterials have demonstrated strong antimicrobial
properties through diverse mechanisms
 Most of the antibiotic resistance mechanisms are irrelevant for nanoparticles (NPs)
 NPs would be less prone to promoting resistance in bacteria than antibiotics.
 Therefore, attention has been focused on new and exciting NP-based materials with
antibacterial activity.

4. 2. Mode Of Action Of Nanomolecules
 Nanoparticles are of great interest for use as potential antimicrobial agents because of their
unique optical, electronic, and magnetic properties.
 The electrostatic interaction of nanoparticles with negatively charged bacterial surfaces draws
the particles to the bacteria and promotes their penetration into the membrane.
 A strongly positive zeta potential of a nanoparticle promotes nanoparticle interactions with cell
membranes leading to membrane disruption
 nanomaterials have demonstrated strong antimicrobial properties through diverse mechanisms
including
 photocatalytic production of reactive oxygen species (ROS) that damage cell components and viruses
(e.g. TiO2, ZnO and fullerol),
 compromising the bacterial cell envelope (e.g. peptides, chitosan, carboxy fullerene, carbon
nanotubes, ZnO and silver nanoparticles (nAg)),
 interruption of energy transduction (e.g. nAg and aqueous fullerene nanoparticles (nC60)), and
 inhibition of enzyme activity and DNA synthesis (e.g. chitosan).
 The major antimicrobial mechanisms reported are summarized in Figure 1.

5. 2. Mode Of Action Of Nanomolecules
Figure 1 Various mechanisms of antimicrobial activities exerted by nanomaterials.

6. 2.1. CELL MEMBRANE DAMAGE
 Cell walls and membranes are important defensive barriers for bacterial resistance to
the external environment.
 The components of the cell membrane produce different adsorption pathways for NPs
and Gram-positive and Gram-negative bacteria.
 cell permeability is altered when in contact to nanoparticles.
 Experiments indicated the formation of a “hole” or “pore” in the living cell membranes
as a possible mechanistic hypothesis.
 a literal hole in the bilayer membrane exists which promotes the complete loss of the
plasma membrane (Leroueil, et al. 2007).

7. 2.2. RELEASE OF TOXIC IONS
 Metal and metal oxide nanoparticles can serve as a source of the metal ion (release their ions
when in contact with the cell)
 Metal ions are known to be toxic to cells as they can react with different proteins and other
cellular components interfering in metabolic pathways and causing damage
 Ag+ can precipitate the chloride in the cell and impair cell respiration(Niskanen, et al. 2010)
 Metal ions are slowly released from metal oxide and are absorbed through the cell membrane,
followed by direct interaction with the functional groups of proteins and nucleic acids, such as
mercapto (–SH), amino (–NH), and carboxyl (–COOH) groups, damaging enzyme activity,
changing the cell structure, affecting the normal physiological processes, and ultimately
inhibiting the microorganism.

8. 2.3. INTERRUPTION OF ELECTRON TRANSPORT, PROTEIN OXIDATION AND MEMBRANE
COLLAPSE
 There is strong evidence that the positive charge of nanoparticles is critical for
antimicrobial activity since bacteria cell membrane is negatively charged.
 it has been suggested that ions (e.g.: silver) can affect membrane-bound
respiratory enzymes as well as affect efflux bombs of ions that can result in cell
death (Allaker 2010).
 The contact of bacteria with CeO2 or nC60 has also given insights about key
enzymes that can be inactivated.
 In general, the cascade of events after nanoparticle contact with bacteria can
start
 With possible oxidation of respiratory enzymes,
 facilitate the production of ROS (Reactive Oxygen Species) and radical species that will
eventually affect cell physiology and promote DNA degradation (Xia, et al. 2008).

9. 2.4. GENERATION OF ROS (REACTIVE OXYGEN SPECIES)
 ROS-induced oxidative stress is an important antibacterial mechanism of NPs.
 The four ROS types are the superoxide radical (O2
−
), the hydroxyl radical (⋅ 𝑂𝐻), hydrogen peroxide (𝐻2 𝑂2), and
singlet oxygen (𝑂2), which exhibit different levels of dynamics and activity.
 For example,
 calcium oxide and magnesium oxide NPs can generate O2
−
,
 zinc oxide NPs can generate 𝐻2 𝑂2 and OH but not O2
−
.
 copper oxide NPs can produce all four types of reactive oxygen.
 Studies have indicated that O2
−
and 𝐻2 𝑂2 cause less acute stress reactions and can be neutralized by
endogenous antioxidants, such as superoxide enzymes and catalase, whereas ⋅ 𝑂𝐻 and 𝑂2 can lead to acute
microbial death.
 Oxidative stress has been confirmed as a key contributor to changing the permeability of the cell membrane,
which can result in bacterial cell membrane damage (Cheloni, Marti and Slaveykova 2016).
 ROS play a key role in the interaction between DNA and bacterial cells (Pramanik, et al. 2012).
 ROS can attack proteins and depress the activity of certain periplasmic enzymes that are essential to
maintaining normal morphology and physiological processes in bacterial cells (Padmavathy and Vijayaraghavan
2011).

10. 3. CURRENT ANTIMICROBIAL NANOMATERIALS

11. 3.1. NANO Ag
Mechanism of action
 several mechanisms have been postulated for the antimicrobial property of silver nanoparticles:
 adhesion of nanoparticles to the surface altering the membrane properties. (Sondi et al. 2004);
 nano Ag particle penetrating inside bacterial cell, resulting in DNA damage;
 dissolution of nano Ag releases antimicrobial Ag+ ions (Morones, et al. 2005).
Drawback - potential toxicity of Ag NPs in the environment and in human beings
 A simple solution is incorporating Ag+ ions in zeolite (Jedrzejczyk, et al. 2017)
 incorporation in a silica matrix to form Ag–SiO2 nanocomposites (Mosselhy, et al. 2017).
 use of nontoxic and noninflammatory capping agents like collagen, peptides and biopolymers. (Tanvir, et
al. 2017)
Improvements in efficiency
 The combination of Ag NPs with other nanomaterials with antibacterial activity such as graphene oxide
(GO) (Tuong Vi, et al. 2018)
 The proposed antimicrobial mechanism is that the GO wraps around bacteria while the Ag kills the
bacteria with its toxicity.

12. Nano Ag continued
Size effect
 In general, particles of less than 10 nm are more toxic to bacteria such as Escherichia coli and
Pseudomonas aeruginosa (Xu, et al. 2004, Gogoi, et al. 2006).
 Silver nanoparticles ranging from 1 to 10 nm inhibit certain viruses from binding to host cells by
preferentially binding to the virus’ gp120 glycoproteins (Elichiguerra, et al. 2005).
 Furthermore, triangular nano Ag nanoplates containing more reactive planes were found to be
more toxic than nano Ag rods, nano Ag spheres, or Ag+ ions (Pal, Tak and Song 2007).

13. 3.2. NANO Au
 exhibit significant antimicrobial activity. Nonetheless, the actual methods for NP production are frequently
expensive and use chemicals that are potentially harmful to the environment.
 In this context, Aljabali et al. (Aljabali, et al. 2018) reported the synthesis of Au NPs using plant extracts
 Elbagory et al. (Elbagory, et al. 2017) prepared biogenic Au NPs from the Galenia Africana and Hypoxis
hemerocallidea
 The biogenic synthesis of Au NPs is another green and nontoxic approach to produce biocompatible NPs
for biomedical applications.
 They can be functionalized with antimicrobial natural and synthetic products for enhanced microbial
activity
 Au nanoparticles functionalized with 5-fluorouracil showed more activity on Gram negative bacteria and exhibited
antifungal activities (Tiwari, et al. 2011)
 Au nanoparticles functionalized with a strongly bound capping (poly-allylamine hydrochloride) and a weakly
bound capping agent (citrate) have shown antimicrobial activities (Goodman, et al. 2004)
 are safer to the mammalian cells than the other nanometals due to the ROS-independent mechanism of
their antimicrobial activity.
 high ability of these nanoparticles for functionalization makes them ideal nanomaterials to be applied as
targeted antimicrobial agents.

14. 3.3. TiO2 NANOPARTICLES
 have a broad spectrum of activity against microorganisms, including Gram-negative and -
positive bacteria and fungi (Kubacka, Ferrer and Fernández-García 2012)
 TiO2-based nanocomposites are environmentally friendly and exert a noncontact biocidal
action.
 Milosevic et al. (Milosevic, et al. 2017) developed a simple, cost-effective and scalable wet
milling method to prepare fluorinated and N-doped TiO2 nanopowders with improved
photocatalytic properties under visible light.
 Further, the combination of N and F led to better activity against E. Coli due to synergistic
effects. These novel nanomaterials are adequate for biomedical applications, such as hospital
tools, but also food preservation or wastewater treatment.
 The antibacterial activity of TiO2 is related to ROS production, especially hydroxyl free radicals
and peroxide formed under UV-A irradiation via oxidative and reductive pathways, respectively
(Kikuchi, et al. 1997 ).

15. 3.4. Si AND SiO2 NANOPARTICLES
 Cousins et al. found that Si nanoparticles inhibited bacterial adherence to oral biofilms (Cousins, et al.
2007)
 Combination use of Si nanoparticles with the other biocidal metals such as Ag has been extensively
studied in the recent years.
 Egger et al. reported the production and investigation of antimicrobial activity of novel Ag–Si nanocomposite
revealing better antimicrobial effect of the nanocomposite against a wide range of microorganisms compared to
conventional materials, such as silver nitrate and silver zeolite (Egger, et al. 2009).
 Mukha et al. (Mukha, et al. 2010) synthesized Ag/SiO2 and Au/SiO2 nanostructures - The results showed that
Ag/SiO2 nanocomposites indicated improved antimicrobial properties against E. coli, S. aureus, and C. albicans
 Several reports showed that Si nanowires could interface with the living cells and bacteria interrupting cell
functions such as cell differentiation, adhesion and spreading. (Lee, et al. 2010, Fellahi, et al. 2013)
 Si nanowire substrates decorated with Ag or Cu nanoparticles showed strong antibacterial activity against E. coli.
And found to be biocompatible (Fellahi, et al. 2013)
 Si compounds and composites especially their nanocomposites together with metals such as Ag shows
great potential on the development of antimicrobial agents.

16. 3.5. MgO AND CaO NANOPARTICLES
 CaO and MgO indicate strong antibacterial activity related to alkalinity and active oxygen species.
 the antibacterial mechanism of CaO and MgO nanoparticles is brought about by the generation of superoxide on the
surface of these particles, and also an increase in pH value by the hydration of CaO and MgO with water (Yamamoto, et
al. 2010).
 MgO nanoparticles damage the cell membrane and then cause the leakage of intracellular contents which in turn lead to
death of the bacterial cells (Jia, Shen and Xu 2001).
 MgO nanoparticles showed the bactericidal activity against both Gram-positive and Gram-negative bacteria
(Vidic, et al. 2013)
 MgO nanoparticles alone or in combination with other antimicrobials (nisin and ZnO nanoparticles) have shown
promising activities against E. coli and Salmonella. (Jin and He 2011)
 CaO nanoparticles showed bactericidal activity against E. coli, S. typhimurium, S. aureus and B. subtilis (Jeong, et
al. 2007).
 MgO and CaO nanoparticles alone or in combination with other disinfectants show excellent antibacterial effect.
 are low cost, biocompatible and available materials. These properties make them promising antibacterial agent.
 can be utilized in environmental preservation as well as in food processing and medical treatments (Sawai and Igarashi
2002).

17. 3.6. Cu AND CuO NANOPARTICLES
 Usman et al. investigated the antimicrobial activities of Cu-chitosan nanoparticles (2–350 nm) on S.
aureus, B. subtilis, P. aeruginosa, Salmonella choleraesuis, and C. albicans. Their results indicated the high
potential of these nanoparticles as antimicrobial agents (Usman, et al. 2013)
 However, rapid oxidation of the Cu nanoparticles on exposure to the air limits their application (Usman, et
al. 2013, Mahapatra, et al. 2008)
 Mahapatra et al. tested antibacterial activity of CuO nanoparticles against Klebsiella pneumoniae, P.
aeruginosa, Salmonella paratyphi and Shigella strains.
 CuO nanoparticles (23 nm) had significant antimicrobial activity against various bacterial strains (E. coli, P.
aeruginosa, K. pneumoniae, Enterococcus faecalis, Shigella flexneri, S. typhimurium, Proteus vulgaris, and
S. aureus). (Ahamed, et al. 2014)
Mechanism
 restrict bacterial growth via passing through nanometric pores that exist on the cellular membranes of
most bacteria and then damaging the vital enzymes of bacteria triggering cell death.
 bactericidal activity of these nanoparticles depended on their size, stability, and concentration added to
the growth medium. (Azam, et al. 2012)

18. 3.7. ZnO NANOPARTICLES
 ZnO nanoparticles exhibit strong antibacterial activities on a broad spectrum of bacteria (Sawai
2003, Adams, Lyon and Alvarez 2006, Jones, et al. 2008, Huang, et al. 2008a)
 Polymeric composites comprising NPs such as ZnO have been widely investigated as food-
packaging materials.
 poly (lactic acid)-based electrospun mats incorporating ZnO-NPs and mesoporous silica doped with
ZnO have demonstrated concentration dependant viability on E. Coli. (Rokbani, Daigle and Ajji 2018)
 Another study (Mizieli´nska, et al. 2018) demonstrated that cellulose-based packaging materials
comprising polyethylene films reinforced with ZnO nanoparticles were very active against mesophilic
and psychotropic bacterial cells.
Mechanism
 The photocatalytic generation of hydrogen peroxide was suggested to be one of the primary
mechanisms (Sawai 2003).
 In addition, penetration of the cell envelope and disorganization of bacterial membrane upon
contact with ZnO nanoparticles were also indicated to inhibit bacterial growth (Brayner, et al.
2006, Huang, et al. 2008a).

19. 3.8. POLYMERIC NANOPARTICLES
 Polymeric nanoparticles kill microorganisms either by releasing antibiotics, antimicrobial peptides, and
antimicrobial agents or by contact-killing cationic surfaces such as quaternary ammonium compounds,
alkyl pyridiniums, or quaternary phosphonium
Mechanism
 cationic groups are able to disrupt the bacterial cell membrane, with some requiring hydrophobic chains
of certain lengths to penetrate and burst the bacterial membrane.
 high levels of positive charge are capable of conferring antimicrobial properties irrespective of
hydrophobic chain length, perhaps by an ion exchange mechanism between the bacterial membrane and
the charged surface (Lichter and Rubner 2009)
Examples
 Poly- 𝜀-lysine
 Quaternary Ammonium Compounds
 Cationic Quaternary Polyelectrolytes
 Peptides.
 Chitosan and chitosan derivatives

20. 3.10. CARBON NANOMATERIALS
 all carbon nanomaterials (graphene/graphene oxide, carbon nanotubes (CNTs), fullerenes, nanodiamond)
are able to show antibacterial properties. (Liu, et al. 2011)
 The antibacterial activity of CNMs is strongly dependent on surface chemistry, which determines critical
factors such as hydrophobicity or oxidation power.
Graphene and its derivatives
 Liu et al. compared the antibacterial activity of graphite, graphite oxide, graphene oxide (GO) and
reduced graphene oxide (rGO), showing that
 GO and rGO are both strongly antibacterial (Liu, et al. 2011).
 GO showed stronger antibacterial activity than rGO.
Carbon nanotubes (CNTs)
 The antibacterial activity of purified single walled CNTs was first demonstrated by Kang et al. (Kang,
Pinault, et al. 2007). They showed that:
 purified SWNTs and multi walled CNTs seriously impact bacterial membrane integrity upon direct contact.
Accordingly, metabolic activity and morphology are compromised as well (Kang, Herzberg, et al. 2008).
 single walled CNTs exhibit a stronger antibacterial activity than multi walled CNTs, probably caused by their smaller
size that facilitates membrane perturbation and provides a larger surface area.

21. continued
Fullerenes
 Fullerene water suspensions (nC60) were tested for antibacterial activity using B. subtilis by Lyon et al.
(Lyon, Adams, et al. 2016).
 fractions of nC60 containing smaller fullerene aggregates showed greater antibacterial activities.
Mechanism
 fullerenes adsorb on bacterial membranes and do not seem to cause alterations to bacterial membranes
(Lyon, Brunet, et al. 2008, Lyon, Lyon, Adams, et al. 2016)].
 Although fullerenes can act as an oxidizing agent, Lyon et al. report that the antibacterial activity is not
caused by ROS (Lyon, Adams, et al. 2016).
 Accordingly, the antibacterial action of fullerenes is most likely caused by unspecific reactions with
membrane proteins and other vital molecules (Lyon, Brunet, et al. 2008).
Nano diamond
 Nano diamonds (NDs) are assumed to have the highest biocompatibility in comparison to all other
carbon-based nanomaterials (Vaijayanthimala and Chang 2008)
 5 nm and 100 nm NDs have shows significant toxic effects on the protozoa Paramecium caudatum and
Tetrahymena thermophile. (Lyon, Adams, et al. 2016)

22. 4. FACTORS AFFECTING THE ANTIBACTERIAL MECHANISMS OF METAL NPs
 The physicochemical properties of NPs include their:
 size,
 charge,
 zeta potential,
 surface morphology, and
 crystal structure,
which are significant elements that regulate the actions of NPs on bacterial cells.
 Moreover,
 environmental conditions,
 the bacterial strain, and
 the exposure time
are other major factors that influence the antibacterial effects of NPs (Çalışkan, et al.
2014).

23. 5. FUTURE PERSPECTIVES
 Although the wet-chemical synthesis has been accepted as low cost and simple, the green method of
nanoparticle synthesis employing plant extracts has been used as a viable alternative to chemical
procedures and physical methods (Shankar, et al. 2004).
 In spite of possible limitations, nanotechnology represents an innovative strategy to develop and test new
pharmaceutical formulations based on metallic nanoparticles with efficacious antimicrobial properties.
 Characteristics of nanoparticles such as size and morphology are important not only for their
antimicrobial activity, but also for reducing tissue and eukaryotic cell toxicities.
 The possibility of developing microbial resistance cannot be ruled out. In fact, induction of horizontal
gene transfer in environmental systems has been suggested (Aruguete, Bojeong, et al. 2013).
 Therefore, pre-clinical and clinical trials are urgently needed for a better understanding of potentiality and
limitations when using nanoparticles and elucidate the mechanisms involved with the antimicrobial activity
of these particles.
 Finally, this is an important area of research that deserves our attention owing to its potential application
in the fight against multi-drug resistant microorganisms.

24. 6. CONCLUSION
 Due to ever-increasing microbial resistance to the common disinfectants and antibiotics, numerous
studies have been performed to improve antimicrobial strategies. Several valuable studies have been
documented in the field of antibacterial nanoparticles in the recent years.
 Application of nanoparticles could be considered as a suitable alternative for some antimicrobial
methods. The antimicrobial nanoparticles could benefit in the pharmaceutical and biomedical industries
for sterilization of the medical devices.
 These nanostructures could also be directed for preparation of chemical disinfectants, coating-based
applications and food preparation processes.
 Metal nanoparticles (especially metal oxide nanoparticles) show great antimicrobial effects. Improved
effectiveness of the metal oxide nanoparticles on the resistant strains of microbial pathogens as well as
their heat resistance offer them as potent antimicrobial agents.
 However, application of some of the metal oxide nanoparticles is limited because of their toxicity at higher
concentrations. It has also proposed that functionalization; ion doping and polymer conjugates of these
nanoparticles could be helpful to decrease the associated toxicity.
 Finally, it may be concluded that, the metal oxide nanoparticles with the minimized toxicity possibly will be
extensively used in the near future for eradicating several infectious conditions.
 development of the simple and low-cost inorganic/organic antimicrobial agents such as metal and metal
oxide nanoparticles as alternative of traditional antibiotics might be promising for future of pharmaceutics
and medicine.

25. THE END
Thank you!