Nanomaterial-Based Therapeutics For Antibiotic-Resistant Bacterial Infections

Reviews
Nanomaterial-based therapeutics for

antibiotic-resistant bacterial infections
Jessa Marie V. Makabenta 1, Ahmed Nabawy 1, Cheng-Hsuan Li 1,
Suzannah Schmidt-Malan2, Robin Patel2 and Vincent M. Rotello 1 ✉
Abstract | Antibiotic-resistant bacterial infections arising from acquired resistance and/or
through biofilm formation necessitate the development of innovative ‘outside of the box’
therapeutics. Nanomaterial-based therapies are promising tools to combat bacterial infections
that are difficult to treat, featuring the capacity to evade existing mechanisms associated with
acquired drug resistance. In addition, the unique size and physical properties of nanomaterials
give them the capability to target biofilms, overcoming recalcitrant infections. In this Review,
we highlight the general mechanisms by which nanomaterials can be used to target bacterial
infections associated with acquired antibiotic resistance and biofilms. We emphasize design
elements and properties of nanomaterials that can be engineered to enhance potency. Lastly,
we present recent progress and remaining challenges for widespread clinical implementation
of nanomaterials as antimicrobial therapeutics.
Osteomyelitis The emergence of antibiotic resistance in bacteria has as a barrier against host immune responses and some
Bone infection. resulted in the challenge of recalcitrant infections1,2. conventional antimicrobial agents7,9. More importantly,
Multidrug-resistant (MDR) bacteria are a global crisis, biofilms exhibit a diversity of altered phenotypes, includ-
Infective endocarditis increasing morbidity and mortality of infected individu ing slow growth rates, the presence of persister cells and
Infection of endocardium,
typically of heart valves.
als and negatively impacting the clinical outcome of a spatial and chemical heterogeneities that contribute to
wide range of groups, including those in intensive care resistance to many available antibiotics10,11.
Persister cells units or undergoing surgery, transplantation or can- Antibiotics are currently the main therapeutic strat-
Subpopulation of dormant, cer treatment2,3. A 2017 report from the WHO Global egy for treating both planktonic and biofilm infections12.
antibiotic-tolerant bacterial
Antimicrobial Surveillance System highlighted antibiotic They target processes necessary for growth and/or sur-
cells that are able to resume
growth after antimicrobial
resistance as a worldwide challenge4. The estimated cost vival of bacteria, including cell wall and cell membrane
stress is relieved. of treating antibiotic-resistant infections is substantial synthesis or maintenance, or the production of DNA,
(~US$50,000 per individual), with an estimated US$20 RNA or essential proteins. Many antibiotics are derived
Debridement billion societal cost annually5. The use, and in some from products that have been deployed by microorgan-
Surgical removal of damaged
or dead tissue from an infected
situations misuse, of antibiotics, combined with the scar- isms to combat one another for billions of years. The
wound. city of new therapeutics entering the antibiotic pipeline, offensive molecules that have evolved throughout this
further exacerbates this public health threat6. warfare have generated defence responses; bacteria have
Planktonic (free-floating) bacteria are central players developed the intrinsic ability to escape the activity of
in multiple health threats, including sepsis3. Infections many traditional antibiotics13. Eradicating MDR bacteria
associated with planktonic bacteria present acute threats may require multiple or high dosages of antibiotic agents
and are rapidly becoming more challenging to treat or the use of ‘last-resort’ antibiotics12. Adding to the ther-
1
Department of Chemistry,
owing to rising rates of acquired antibiotic resistance. apeutic challenge, when bacteria are present in biofilms,
University of Massachusetts This challenge is amplified when bacteria form bio- biofilm-associated resistance becomes a compounding
Amherst, Amherst, MA, USA. films, which are associated with recurring and chronic factor, often requiring physical removal of the biofilm
2
Division of Clinical bacterial infections7. The ability of bacteria to protect through aggressive debridement, for example, accom-
Microbiology, Department themselves within biofilms complicates treatment of panied by high doses of antibiotics14,15. These strategies
of Laboratory Medicine and numerous infection types, including chronic wounds, can result in long and expensive treatments, with the
Pathology, Mayo Clinic,
osteomyelitis and infective endocarditis8. Antibiotic resist- possibility of adverse effects and uncertain outcomes.
Rochester, MN, USA.
✉e-mail: ance associated with the biofilm state is distinct from Nanomaterials access antimicrobial modalities
[email protected] acquired resistance, but can compound and exacerbate that are novel to bacteria and thus are not in their
https://doi.org/10.1038/ therapeutic challenges9. Bacterial cells produce extra- natural defensive arsenal (Box 1). Recent advances in
s41579-020-0420-1 cellular polymeric substances (EPS), which may serve nanomaterial-based systems provide new opportunities
naTure RevIewS | MICRObIOLOgy volume 19 | January 2021 | 23

Reviews
Box 1 | What are nanomaterials?

Nanomaterials Polymers
Nanomaterials are organic, inorganic or hybrid particles that are generally Polymeric nanomaterials can be either natural or synthetic. Natural
100 nm or smaller; however, some particles 500 nm or smaller are also polymers are used to fabricate cationic and pH-switchable
considered as nanomaterials139. They can be in different forms, including antimicrobials55,99. Synthetic polymers can mimic the activity of
nanoparticles, nanowires and nanorods. Nanomaterials have an almost antimicrobial peptides23,149. Moreover, polymeric micelles are used as
unlimited range of structures and morphologies, from rods to pyramids to nanocarriers to increase the solubility, stability and efficacy and improve
fibrous networks to spheres, with hollow or solid interiors bearing rough the pharmacokinetic profiles of drugs150. Dendrimers are regular polymeric
or smooth surfaces20. Materials in the nanoscale realm possess distinctive molecules comprising a central core, branch-like structures radiating from
physico-chemical characteristics, including size, shape and surface, the core and an outer surface bearing functional groups. Glycopeptide
compared with their bulk counterparts140. The unique properties of dendrimers have been shown to inhibit P. aeruginosa biofilms151.
nanomaterials have revolutionized many technologies and industries,
Nanocomposites
including medicine. Being comparable in size to biomolecules and
Nanocomposites are hybrids of inorganic and organic nanoparticles.
bacterial intracellular structures, nanomaterials can be engineered as
For example, incorporation of silver nanoparticles into the cationic
new therapeutic modalities19. Representative classes of nanomaterials
polymer poly(2-dimethylamino)ethyl methacrylate resulted in synergistic
for antimicrobial application include metal-based nanoparticles,
antimicrobial activity against P. aeruginosa and Staphylococcus aureus152.
carbon-based nanomaterials, polymers, nanocomposites, nanoemulsions,
liposomes and smart nanomaterials. Nanoemulsions
Nanoemulsions are nano-sized emulsions composed of interdispersed
Metal-based nanoparticles
hydrophobic and aqueous layers, commonly used to encapsulate
Metal-based nanoparticles are composed of either pure metals (for
bioactive compounds, such as antibiotics and essential oils, to increase
example, gold, silver and iron) or their compounds (for example, oxides).
solubility and stability153,154.
Their primary mechanisms of toxicity involve reactive oxygen species
production and impairment of membrane function31,33. Metal-based Liposomes
nanoparticles have been demonstrated to be active in treating several Liposomes are vesicles composed of one or more phospholipid bilayers
multidrug-resistant bacterial infections141–144. Silver-based nanomaterials with an aqueous inner core. Being membrane-based structures, they have
are the most established metal antimicrobials; although the exact good biocompatibility and are useful antimicrobial delivery vehicles155.
mechanism of action of silver nanoparticles is unknown, two widely They can encapsulate hydrophilic drugs in their aqueous interior and/or
proposed modes of action are disruption of membranes by leached silver entrap hydrophobic drugs in their phospholipid membrane98,156.
ions and ion-mediated killing145,146.
Smart nanomaterials
Carbon-based nanomaterials Smart materials can respond to endogenous stimuli, such as pH and
Carbon-based nanomaterials include carbon quantum dots35, bacterial toxins, or external stimuli, such as light, temperature or
nanotubes147 and 2D materials, including graphene148. Their bactericidal ultrasound, to produce changes in their characteristics that allow them to
action involves physical and chemical damage; however, specific exert antimicrobial action52,157,158. For instance, hybrid micelles composed
mechanisms are yet to be understood. In one study, multiwalled carbon of polyethylene glycol, poly(aspartamide), 2-(diisopropylamonio)
nanotubes prevented formation of Klebsiella oxytoca, Pseudomonas ethylamine, azithromycin and cis-aconityl-d-tyrosine can shrink in size,
aeruginosa and Staphylococcus epidermidis biofilms by blocking bacterial reverse surface charge and release drug cargo in response to the acidic
adhesion26. environment of P. aeruginosa biofilms157.
to address MDR planktonic as well as biofilm infections, Mechanisms against planktonic bacteria
acting either as inherent therapeutics or as nanocarriers The array of sizes and shapes adopted by nanomaterials
for antimicrobial agents16. The unique physico-chemical offers unique capabilities for targeting bacteria19 (Fig. 1).
properties of nanomaterials, such as size, shape and sur- Nanomaterials can use multiple bactericidal mechanisms,
face chemistry, influence their therapeutic activity17. The including direct cell wall and/or membrane damage, gen-
sizes and shapes of different nanomaterials are similar eration of reactive oxygen species (ROS) and binding to
to those of bacterial biomolecules, affording a variety intracellular components. Most antibiotics target cell
of interactions that can be regulated through surface walls or membranes, or disrupt intracellular processes.
functionalization. High surface-to-volume ratios and Nanomaterials can target these cellular features, albeit
multivalent interactions are important for creating anti- in different ways, and offer advantages in combating
bacterial nanomaterials16,17. Nanomaterials can evade antibiotic-resistant pathogens relative to small-molecule
existing resistance mechanisms and may be less prone drugs (Table 1). Furthermore, nanomaterials can be used
to select for resistance than conventional antibiotics18 as nanocarriers for delivery of therapeutic agents19,20.
(Box 2). Moreover, nanomaterials have the ability to erad- The mechanisms used by nanomaterials arise from their
icate bacteria in biofilms17. From the aforementioned unique physico-chemical properties, in particular mul-
points taken together, nanotechnology provides a new tivalent interactions with bacterial cells. Van der Waals
toolkit for the creation of treatment strategies for MDR forces, receptor–ligand interactions, hydrophobic inter-
infections. actions and electrostatic attractions have roles in the
In this Review, we illustrate how nanomaterials could interfaces between nanomaterials and bacteria21.
be used to combat MDR bacterial infections. We discuss
Nanocarriers properties and design elements that result in therapeutic Cell wall and membrane disruption. The bacterial cell
A drug delivery platform in the efficacy, providing insight into how nanomaterials might envelope has evolved to serve as a physical barrier to
nanoscale range (2–500 nm).
Common nanocarriers include
be tailored to optimize activity against planktonic and antimicrobials. Teichoic acids, present in the cell wall of
liposomes, polymers and biofilm bacteria. Lastly, we highlight the status of clinical Gram-positive bacteria, and lipopolysaccharide, found
micelles. development of antibacterial nanomaterials. in the outer membrane of Gram-negative bacteria, have
24 | January 2021 | volume 19 www.nature.com/nrmicro

Reviews
phosphate groups that render bacterial surfaces negatively self-assemble into cationic micellar nanoparticles, kill-
charged. This highly polar environment limits penetra- ing methicillin-resistant Staphylococcus aureus (MRSA).
tion of hydrophobic antimicrobials across membranes, These polymeric nanoparticles interact with bacteria
compromising their activity against bacteria21. through electrostatic interactions, resulting in disruption
Bacterial cell surfaces are more negatively charged of the membranes and cell lysis28. ‘Nanoknives’, materi-
than are those of mammalian cells, facilitating prefer- als with sharp-pointed edges, are particularly effective
ential electrostatic interactions with positively charged in compromising bacterial membrane integrity. In one
materials22. The charge density and hydrophobicity study, single-walled carbon nanotubes and graphene
of the nanomaterial surface are important factors in oxide ruptured the cell surface of Ralstonia solanacearum,
designing nanomaterials to selectively disrupt bacterial leading to cytoplasmic leakage and bacterial death29. The
membranes23–25. Highly cationic nanomaterials can bind ability of bacteria to develop resistance against therapeu-
to the surface of mammalian cells, as can nanomaterials tics that damage the cell envelope is likely to be limited,
with overly hydrophobic surfaces, reducing selectivity. making these strategies promising for long-term use with
Cationic nanomaterials with good amphiphilic balance minimal risk of emergence of bacterial resistance27,30.
(that is, the optimized balance between hydrophobicity
and cationic charge) can provide potent antimicrobial Generation of ROS. ROS are by-products of cellular
effects with low levels of haemolysis and cytotoxicity23. oxidative metabolic processes that affect cell differenti-
A range of nanomaterial-b ased strategies focus ation, signalling, survival and cell death31. Accumulation
on targeting the negatively charged surface of plank- of excessive ROS results in lethal oxidative stress. ROS
tonic bacteria23,24,26,27. One study synthesized biode- can damage cells through multiple mechanisms, in par-
gradable cationic and amphiphilic polycarbonates that ticular through reaction of superoxide and hydroxyl
Box 2 | Antibiotic resistance mechanisms and nanomaterials

Bacteria have acquired multiple survival mechanisms that
enable them to evade killing by antibiotics (see the figure).
The biofilm state itself confers resistance against antibiotics, Antimicrobials
which is separate from genetically acquired antibiotic Reduced uptake
Efflux pump
resistance96,159. Nanomaterials are novel to bacteria and can
circumvent resistance mechanisms that affect traditional Deactivating enzymes
antibiotics, and they can target bacterial biofilms.
Resistance gene transfer and target modification
Bacteria communicate and share genetic information with Target modification Inactive metabolic state
one another, resulting in spread of resistance genes across
bacterial populations10,160. Expression of resistance genes
can lead to the chemical modification of either antibiotics
or antibiotic targets, inhibiting their mechanism of action
and conferring protection. Unlike traditional antibiotics Adsorbed
Deactivating or trapped
that have specific targets, nanomaterials can have multiple enzymes
killing mechanisms because they access multiple targets,
rendering emergence of resistance less likely than with
traditional antibiotics18.
Deactivating enzymes Resistance
gene transfer
Resistant bacteria may harbour extracellular and/or
intracellular enzymes that degrade or limit the binding
of antibiotics. The multiple mechanisms of action of
nanoparticles and their abiological structure allow them
Susceptible cell Resistant cell Persister cell
to escape deactivation by these enzymes18.
Reduced antibiotic uptake
Some Gram-negative bacteria have evolved to limit entry of antibiotics
nanomaterials, particularly membrane damage, do not require bacteria to be
through porin gene mutations31. Nanomaterials, however, enter bacterial cells
in a state of active growth, rendering these agents active against persisters27.
through non-porin mechanisms, such as endocytosis and membrane fusion18.
As an example, liposomal nanoparticles loaded with antimicrobial agents Extracellular polymeric substances limit antibiotic penetration
enter through membrane fusion and rapidly release high concentrations of The protective nature of the extracellular polymeric substances of
antimicrobial agents to membranes or into the cytoplasm58. bacterial biofilms restricts penetration of some antibiotics, such as
aminoglycosides, owing to electrostatic repulsion160,162. Although other
Efflux pumps
antibiotic groups can diffuse into the inner layers of biofilms, complex
Efflux pumps, which are often upregulated in antibiotic-resistant bacterial
gradients of nutrients and waste can diminish their antimicrobial effects163.
cells, actively transport antimicrobial agents outside bacterial cells.
The unique surface chemistry of nanomaterials allows facile penetration
Nanomaterials can block efflux pumps, increasing accumulation of
into biofilms and interaction with deeply embedded bacterial cells.
antibiotics inside bacterial cells132,133.
The amphiphilic balance of many nanomaterials helps them exert
Inactive metabolic state multiple interactions with extracellular polymeric substances, including
Persister cells are subpopulations of metabolically inactive bacteria with hydrophobic and electrostatic interactions, maximizing adsorption by
reduced susceptibility to antimicrobials161. The killing mechanisms of many and diffusion across biofilms17,18.

Reviews
Fenton-inactive metals
radicals with thiols in proteins, deactivating membrane Silver and other Fenton-inactive metals increase the for-
Also called ‘redox-inactive receptors32. There are several mechanisms by which mation of ROS in bacteria through their ability to dis-
metals’, these are a class of nanoparticles generate ROS: direct ROS production rupt cellular donor ligands coordinating with iron, such
transition metals (such as Ag from the nanoparticle surface or from leached ions; as cysteine, and to induce release of iron from [4Fe–4S]
and Hg) that cannot undergo
redox reaction and hence
interaction with intracellular organelles; and oxidation clusters; this iron release then increases ROS formation31.
cannot inherently produce through interaction with redox-active biomolecules, Gold nanoparticles (AuNPs) have also shown
toxic reactive oxygen species. including NADPH oxidase33. Some metal-based nano- enzyme-like activities39. Mesoporous silica can provide
particles use ROS generation as their major antibacte- support and enhance the stability and catalytic activity
rial mechanism (reviewed in refs31,33,34) owing to their of AuNPs40. AuNPs bound on the surface of bifunction-
inherent photocatalytic activity (that is, photodynamic alized mesoporous silica nanoparticles (MSNs) have
therapy)31,35,36. been shown to display peroxidase-like and oxidase-like
An example of ROS-based antibacterial activity is activities, killing both Gram-positive and Gram-negative
the release of free copper (Cu+) ions from copper iodide bacteria, dual enzyme-like activity that increases effi-
(CuI) nanoparticles, generating ROS and damaging bac- ciency of ROS production, imparting oxidative stress
terial DNA and intracellular proteins of Escherichia coli to bacteria41.
and Bacillus subtilis37. Silver–zinc oxide nanocomposites
likewise exhibited antibacterial activity against S. aureus Damage to intracellular components. Cellular homoe-
and antibiotic-resistant E. coli, which was ascribed to ostasis and intracellular signalling pathways are central
potent ROS generation and release of silver (Ag+) and to the function and survival of bacteria. Nanomaterials
zinc (Zn2+) ions. These combined processes then gen- can be engineered to interfere with these processes, ulti-
erated a cascade of bactericidal effects, including dam- mately leading to cell death. These disruptions include
aged cell membranes, protein dysfunction, inhibition of alteration in gene expression, protein synthesis and DNA
DNA replication and leakage of intracellular contents38. damage42,43. As an example, AuNPs were functionalized
Size comparison of bacteria and nanomaterials 100 nm
x20
magnification
Hydrophobic
therapeutics
Polymer
2,000 nm
Nanoemulsion Bacteria
250 nm Liposome
750 nm × 2,500 nm
Metal NP
2 nm Ribosome
Therapeutics
Lipid
Gram-negative envelope: Gram-positive envelope:

Liposome
200 nm Cytoplasmic membrane Cytoplasmic membrane
Peptidoglycan Peptidoglycan
Outer membrane
Antimicrobial mechanisms
of nanomaterials
5 nm 50S H2O O2
2 nm
NP
NP
NP
30S •OH O2–
10 nm 10 nm Ribosome 20 nm
Membrane disruption Binding to intracellular components Generation of ROS Delivery of therapeutics through
(for example, DNA and ribosomes) membrane fusion
Fig. 1 | Nano versus micro — antimicrobial mechanisms of nanomaterials. cytoplasmic leakage. Nanomaterials can bind various intracellular
Bacteria typically have diameters ranging from 0.2 to 10 µm. Different components, such as ribosomes, proteins and/or DNA, disrupting their
nanomaterials and preparation methods provide a wide range of particle sizes functions. Nanomaterials with catalytic activities increase the production of
(2–500 nm) that facilitate maximal contact and strong interactions with reactive oxygen species (ROS), such as hydroxyl radicals and superoxides,
bacterial membranes. Nanomaterials display a variety of bactericidal causing oxidative cellular stress. Nanomaterials can also be used for delivery
mechanisms. Electrostatic interactions of nanomaterials with the negatively of therapeutic agents; some nanomaterials readily enter bacterial cells
charged groups present on bacterial surfaces result in membrane damage and through membrane fusion, facilitating delivery of their cargo. NP, nanoparticle.

Reviews
Table 1 | Overcoming resistance mechanisms that elicit multiple mechanisms of action, and through
targeted release of cargo, preventing exposure of bacte-
Mechanism Antibiotics Potential ways ria to subinhibitory doses of the drug51,52. For instance,
of action nanomaterials evade
Class Select resistance
resistance the antibiotic gentamicin loaded into poly(lactide-co-
mechanisms glycolide) (PLGA) nanoparticles exhibited increased
Cell wall or β-Lactams Drug-modifying Physical damage antimicrobial activity against P. aeruginosa in vitro and
membrane enzymes126, binding to the cell envelope in vivo48. Subsequently, levofloxacin loaded into silver
disruption site modifications127, limits development of core-embedded MSNs (Ag@MSNs@LEVO) afforded an
porin changes128 resistance29,63,84, flexible
design space and unique effective treatment of MDR isolates of E. coli; the combi-
Glycopeptides Binding site physico-chemical nation of antibiotic levofloxacin with Ag@MSNs yielded
modifications129 properties can be used a synergistic antimicrobial effect. The silver component
Peptide Outer membrane to maximize disruptive of the system not only functioned as a carrier but also
antibiotics modifications130 interactions16
imparted antimicrobial effects through silver ion gen-
Damage to Aminoglycosides Drug-modifying Entry through membrane eration. In an in vivo mouse peritonitis model, treatment
intracellular enzymes130 fusion overcomes resistance with Ag@MSNs@LEVO reduced bacterial burden by
components from limited antimicrobial
Macrolides Efflux pumps131, entry58, ability to block three orders of magnitude, with concomitant reduction
binding site efflux pumps132,133, multiple of damage to the spleen and peritoneum, with no tox-
modifications128 active groups available that icity observed53. In a related approach, ampicillin was
Quinolones Binding site target general rather than attached to the surface of AuNPs and AgNPs, yielding
modifications128, specific pathways18 broad-spectrum bactericidal agents that evade resist-
porin changes128
ance mechanisms of MDR strains of P. aeruginosa and
Enterobacter aerogenes, and of MRSA49.
with 4,6-diamino-2-pyrimidinethiol (DAPT), an ana- Therapeutic selectivity and enhancement of delivery
logue of 2-pyrimidinethiol (found in E. coli), to generate efficiency can be achieved through release of a drug in
pyrimidine-capped AuNPs (Au–DAPT)44. These nano- response to specific stimuli52,54. Bacterial infection sites
particles inhibited proliferation of MDR strains of E. coli are weakly acidic and can be targeted52,55,56. For example,
and Pseudomonas aeruginosa. The mechanisms of action vancomycin was encapsulated in a pH-responsive, sur-
of Au–DAPT were elucidated through the following: gel face charge-switching triblock copolymer, poly(d,l-
electrophoresis showing the ability of nanoparticles to lactic-co-glycolic acid)-b-poly(l-histidine)-b-polyethylene
bind bacterial DNA; electron microscopy images dis- glycol (PLGA–PLH–PEG). Therapeutic cargo was
playing leakage of nucleic acids and Au–DAPT binding released only on interaction with the acidic infec-
to ribosomes and chromosomes; an E. coli-free tran- tion site, providing a target for vancomycin delivery56.
scription/translation system demonstrating protein PLGA was chosen owing to its low toxicity and ease
synthesis inhibition; and colorimetric assays showing of surface fine-tuning; PEG reduced off-target inter-
selective chelation of Mg2+, destabilizing the cell mem- actions, prolonging the circulation time; and PLH
brane. Similarly, polymer-coated silver nanoparticles provided the charge-switchable characteristic of the
(AgNPs) killed E. coli cells by inhibiting both the Krebs polymer. The selective protonation of the imidazole
cycle and amino acid metabolism45. Polymers were used groups of PLH in weakly acidic conditions allowed a
to modify the surface of AgNPs to increase interactions stimuli-responsive effect. Biomaterials can also provide
with bacterial cells. The mechanism of action was con- charge-switching behaviour, with pH-triggered release
firmed by observation of downregulation of the expres- of vancomycin achieved with chitosan nanoparticles55.
sion of genes associated with tricarboxylic acid cycle Furthermore, bacterial toxins can be used as a trigger for
(aceF and frdB) and amino acid metabolism (gadB, metL release of antimicrobials. Lecithin, a phospholipid, and
and argC), ultimately leading to cell death. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG
(DSPE-PEG3400) were used to coat a mixture of fatty
Delivery of therapeutic agents. Several therapeutics acids, forming liposome-based nanoreactors that release
incorporating nanotechnology called ‘nanodrugs’ — calcium peroxide and rifampin in the presence of
liposomal nanoformulations in particular — have been α-toxin, a pore-forming toxin produced by S. aureus57.
approved by the FDA and made available for clinical use This strategy selectively targeted pathogenic bacteria as
to treat different diseases, including cancer46. Similarly, demonstrated by its antimicrobial activity against MRSA
Peritonitis
Inflammation of the
nanomaterials may be used as carriers for delivery of and minimal effect on non-pathogenic B. subtilis.
peritoneum, the tissue layer antimicrobial agents47. Therapeutics can be encapsu- Overall, nanomaterials provide multiple bacteri-
lining the inner wall of the lated inside nanomaterials or bound to their surfaces48,49. cidal pathways to combat bacteria and evade antibiotic
abdomen, often as a result Nanomaterials protect these agents from enzymes and resistance mechanisms. Appropriate engineering of size,
of bacterial infection.
molecules that might otherwise degrade them. This pro- shape and surface properties provides a broad design
Chitosan tection can increase the therapeutic efficiency of a drug, space for novel antimicrobial agents.
A linear polysaccharide, resulting in lower dosage requirements to augment thera-
obtained from the outer peutic effects and reduce host toxicity50. The use of deliv- Combating planktonic bacteria
skeleton of insects and ery systems can also enhance the stability, solubility and Drug-resistant hospital-acquired (nosocomial) infec-
shellfish, composed of
randomly distributed
biocompatibility of otherwise pharmacologically chal- tions are challenging to treat. A group of pathogens
d-glucosamine and lenging antibiotics. Use of nanocarriers can minimize comprising Enterococcus faecium, S. aureus, Klebsiella
N-acetyl-d-glucosamine units. selection of resistance through delivery of therapeutics pneumoniae, Acinetobacter baumannii, P. aeruginosa

Reviews
a SNAPP S16 b
Areas of PLGA
LPS SNAPP-induced PVA
OM and LPS
destabilization Esculentin(1-21) or
OM Esculentin(1021)-1c
PG
Optimal
SNAPP membrane aerosolization
CM properties
channels in CM
SNAPP influx into

Ion efflux through the cytoplasm
SNAPP membrane
channel
c d
Gen
MSN Gen@MSN
Aptamer Tagged AMP
Gen@MSN-LU
AuNP AuNP–Apt AuNP–Apt–AMP
Cholesterol
Bacterial killing
DOPC
Cellular uptake DOPE
Salmonella 18:0 PEG-2000PE

AuNP–Apt–AMP S. aureus
UBI29–41
HO O O OH H
N
e f OH O O N
Br
Low activity HO
OH 35 OH H O O
O
O
•OH OH HO OH HO OH HN
Neutral pH
DA95B5
Carboxy- H2O2 N+ Cl–
methyl-
dextran
Micelle
Bacterium
Iron oxide core Biofilm
•OH
Acidic pH Dispersal
H2O2 •OH
High activity Substrate Substrate
and Enterobacter species — collectively termed ‘ESKAPE against last-resort antibiotics6. In this regard, nanoma-
pathogens’ — is responsible for most nosocomial infec- terials can provide a lifeline for therapeutic design, as
tions, complicating the conditions of patients who are limited to no resistance development is observed with
often immunocompromised2,3,6. The approaches for nanomaterial-based strategies18,28,30,58.
treating infections caused by these pathogens are limited Numerous studies have explored the utility of nano-
owing to the rapid rate of resistance development, even materials against the ESKAPE pathogens59–62 (Fig. 2).

Reviews
▶ Fig. 2 | Examples of nanomaterial-based strategies used to combat bacterial Combating intracellular bacteria
infections. Nanomaterial-based strategies can be used to combat planktonic bacterial Bacteria can reside within mammalian cells, giving
infections (parts a,b), intracellular infections (parts c,d) and biofilm infections (parts e,f). rise to recurring systemic infections68. For example,
a | Structurally nanoengineered antimicrobial peptide polymers (SNAPPs) exhibited Salmonella enterica subsp. enterica serovar Typhimurium
promising antimicrobial activity in vitro and in vivo. SNAPPs interacted with the outer
is a common facultative intracellular pathogen that
membrane (OM), peptidoglycan (PG) and cytoplasmic membrane (CM) layers of bacteria
through electrostatic interactions, ultimately leading to cell lysis. b | Intratracheal
causes life-threatening food-borne infections in mil-
administration of the antimicrobial esculentin 1a formulated to be delivered to the lions of people worldwide each year69. Salmonella spe-
lungs using poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles reduced Pseudomonas cies can survive and replicate inside host cells, including
aeruginosa lung infection in a mouse model. c | Histidine–aptamer-conjugated gold macrophages. Intracellular localization of bacteria
nanoparticles (AuNPs) loaded with His-tagged antimicrobial peptides (AuNP–Apt–AMPs) adds a level of complexity to treatment, because many
were active for treatment of mammalian cells infected with Salmonella enterica subsp. antibiotics have limited ability to cross mammalian
enterica serovar Typhimurium. d | Gentamicin-loaded mesoporous silica nanoparticles cell membranes and can also be actively exported out
(Gen@MSNs) with a bacterial toxin-responsive lipid bilayer surface shell and bacterium- by the host cell70,71. Nanomaterials can mitigate this
targeting peptide fragment ubiquicidin 29–41 (UBI29–41) (Gen@MSN-LU) allowed targeted challenge through their ability to penetrate eukary-
release of antibiotic for killing of intracellular Staphylococcus aureus. e | A carboxymethyl
otic cells, as well as through their high drug loading
dextran-coated iron oxide nanoparticle, ferumoxytol, catalysed reactive oxygen species
production of H2O2 in a pH-dependent manner as a treatment for oral biofilms. f | Dextran
capacity (Fig. 2).
(green) and poly[(3-acrylamidopropyl)trimethylammonium chloride]-co-(butyl methacrylate) In one example of nanomaterial-based treatment
(light blue) forms DA95B5, a micelle with a bactericidal core and non-fouling dextran of intracellular infections, enrofloxacin-loaded docos-
shell used to treat wound biofilms. Electrostatic interaction of the nanoparticles with anoic acid solid lipid nanoparticles increased intracel-
biofilms weakens bacterial attachment while gradually dispersing the extracellular lular accumulation of enrofloxacin up to ~40-fold and
polymeric substance matrix. DOPC, dioleoyl-sn-glycero-3-phosphocholine; DOPE, enhanced Salmonella killing inside macrophages72.
dioleoylphosphatidylethanolamine. LPS, lipopolysaccharide; PEG, polyethylene glycol; In another approach, colistin, a poorly permeable antibi-
PVA, poly(vinyl) alcohol. Part a reprinted from ref.63, Springer Nature Limited. Part b otic, was formulated into liposomes functionalized with
reprinted with permission from ref.67, American Chemical Society. Part c reprinted with a bacterially derived protein to promote internalization
permission from ref.78, Elsevier. Part d reprinted with permission from ref.74, American
into eukaryotic cells, providing therapeutics with high
Chemical Society. Part e reprinted from ref.112, CC BY 4.0 (https://creativecommons.org/
licenses/by/4.0/). Part f reprinted with permission from ref.120, American Chemical
oral bioavailability73. In another strategy, gentamicin was
Society. loaded into MSN with bacterial toxin-responsive lipid
bilayer surface shells. Functionalization of the MSN
surface shell with bacterium-targeting peptide ubiqui-
One study reported the activity of structurally nano- cidin 29–41 allowed targeted treatment of intracellular
engineered antimicrobial peptide polymers (SNAPPs) S. aureus infection74.
against MDR Gram-n egative ESKAPE pathogens Mycobacterium tuberculosis, the causative agent of
in vitro and in an in vivo mouse peritonitis model63. tuberculosis, is another example of an intracellular path-
Researchers designed artificial antimicrobial peptide- ogen that survives within host macrophages75. Several
inspired peptide polymer nanoparticles consisting studies have demonstrated the activity of nanomaterials
of lysine and valine residues that self-assemble into against intracellular Mycobacterium species. One study
star-shaped unimolecular structures, mimicking anti- reported a library of cationic star-shaped polycarbonate
microbial peptides. SNAPPs elicit multiple proposed nanostructures with wide-spectrum antimicrobial
bactericidal mechanisms, including damage to outer activity and low rates of haemolysis76. Mannose func-
and inner cell membranes, disruption of ion efflux or tionalization of the polycarbonates allowed mannose
influx regulation and induction of an apoptotic-like cell receptor-mediated entry of the nanostructures into the
death pathway. The proposed multimodal antimicro- macrophages, consequently restricting the growth of
bial activity of SNAPPs renders the barrier to resistance intracellular Mycobacterium bovis BCG. In another study,
high. From comparison of the concentration that results biodegradable multimetallic microparticles, consisting
in death of 50% of the mammalian cell population (that of AgNPs and zinc oxide nanoparticles encapsulated
is, the half-maximal inhibitory concentration) and within PLGA, were used to deliver the antituberculo-
the concentration that kills half of bacterial isolates sis drug rifampin into M. tuberculosis-infected alveolar
(minimum bactericidal concentration), SNAPPs had macrophages, demonstrated through an in vitro intra-
a therapeutic index higher than that of colistin, a drug cellular infection model77. The ability of AgNPs and zinc
of last resort for MDR Gram-negative bacterial infec- oxide nanoparticles to interact with and compromise
tions. Furthermore, MDR A. baumannii did not acquire bacterial membrane stability furthered the antimicrobial
resistance to SNAPPs after multiple passages in subin- effects of the system.
hibitory concentrations. Liposome-based nanoparticles Nanomaterial-b ased strategies to combat other
are another promising system, restoring potency of intracellular pathogens have also been developed.
the antibiotics cefepime, imipenem and ceftazidime For example, AuNP–DNA aptamer conjugates
Therapeutic index against MDR P. aeruginosa64, chloramphenicol against loaded with antimicrobial peptides showed activity
A quantitative measure of MRSA65 and amikacin against K. pneumoniae66 through against intracellular Salmonella species78 and Vibrio
the relative safety of a drug efficient drug delivery. Similarly, delivery of antimi- vulnificus 79 in in vivo mouse infection models. In
determined by the dosage that crobial peptides was achieved with the use of PLGA another example, gentamicin-loaded AuNPs deco-
produces a therapeutic effect
without host toxicity and the
nanoparticles, providing a successful treatment strat- rated with phosphatidylcholine eradicated intracellular
concentration that results in egy for P. aeruginosa lung infection in an in vivo mouse Listeria monocytogenes and P. aeruginosa in infected
dangerous side effects. model67. macrophages47.

Reviews
Box 3 | Nanomaterial properties and design elements

Thoughtful engineering of nanomaterial surfaces modulates interactions Furthermore, biofilm penetration can be heightened by surface
between nanoparticles (NPs) and bacteria. The interface between NPs and modification83,167,168. As shown in the figure, anionic and zwitterionic NPs
bacteria is characterized by hydrophobic and electrostatic interactions have poor matrix penetration, whereas cationic NPs with an appropriate
and van der Waals forces that can be modulated by tuning nanomaterial hydrophobic balance can penetrate the extracellular polymeric substance.
properties95. Tuning the size, surface and shape of a nanomaterial can Several strategies have taken advantage of the acidic pH of biofilms to
maximize antibacterial activity, biofilm penetration, biocompatibility, switch from anionic or zwitterionic NPs to cationic NPs87,157. The insets
biodistribution and the therapeutic index164. in the figure are confocal images showing the biofilm penetration profile
Size of quantum dots (QDs) with different surface charges (scale bar 20 μm).
The size of nanomaterials regulates bactericidal activity. Small NPs Shape
(2–10 nm) cause more membrane damage than larger ones because Contact killing can be influenced by NP shape; sharp and pointed NPs can
of high surface area contact with bacterial cells coupled with greater puncture bacterial cell membranes, leading to cytoplasmic leakage29,148,164,169.
curvatures165,166. NPs smaller than 350 nm can diffuse through pores within Comparisons of the activities of spherical, rod-shaped and truncated
biofilms85,86 (see the figure). triangular silver nanoplates against those of planktonic cells of Escherichia
Surface coli reveal that truncated triangular silver NPs exhibit enhanced
Nanomaterial surfaces can be functionalized with chemical groups that bactericidal effects. This is due to the number of NP facets directly
enable multivalent interactions with bacterial cells and the extracellular interacting with the bacterial surface. Triangular NPs have more facets
polymeric substances matrix of biofilms17. NPs have surface charge- than the two other shapes, causing more membrane damage to bacteria169.
dependent bacterial toxicity (that is, the more positively charged the Rod-shaped nitric oxide-releasing silica NPs result in better biofilm
surface, the more toxic the NP becomes)23. Careful placement of eradication than their spherical counterparts, an outcome attributed
the positive charge and hydrophobic moieties can enhance antibacterial to the higher particle aspect ratio of rod-shaped NPs in comparison with
activity of polymeric NPs, while maintaining minimal cytotoxicity. spherical NPs170.
S O O S O S O S O
CdSe CdSe CdSe + CdSe +
S O OH S O S O S O
S
S
S
O O O OH O N O N
Zn
Zn
3 n 3 3
Zn
Zn
COOH-QD PEG-QD Hexyl-QD TTMA-QD
Anionic NPs
Neutral NPs Cationic NPs
<350 nm pH-responsive
NPs >350 nm NPs <350 nm
Biofilm
Surface
COOH-QD, carboxyl-functionalized quantum dot; hexyl-QD, dimethylhexyl ammonium-functionalized quantum dot; PEG-QD, polyethylene glycol-functionalized
quantum dot; TTMA-QD, trimethyl ammonium-functionalized quantum dot. Figure adapted with permission from ref.138, The Royal Society of Chemistry.
Therapeutic strategies against biofilms biopolymers including nucleic acids, proteins and pol-
MDR biofilm infections present a particularly difficult ysaccharides that provide a 3D protective scaffold for
therapeutic challenge80. The matrix composed of EPS may bacteria. The matrix is rich in negatively charged com-
provide a barrier to some cellular and small-molecule ponents and hydrophobic groups, with pores filled with
(for example, antibiotic) assaults. Bacteria embedded water facilitating the transport of nutrients10. Tuning
within the matrix are capable of synergistic interactions, the surface functionality and design of nanomaterials
cell-to-cell communications and the transfer of resist- can facilitate biofilm penetration82,83 (Box 3). Size and
ance genes10,11. Furthermore, the deeper layers of the electrostatic interactions are important factors influ-
matrix have low oxygen and nutrient supply, inducing encing the biofilm penetration profile of nanomaterials.
formation of dormant persister cells, which promote Generally, uncharged nanoparticles smaller than 350 nm
antimicrobial tolerance and resistance80,81. have greater mobility across pores within biofilms,
Overcoming the physical barrier presented by bio- whereas cationic nanoparticles have a good distribution
films is needed to combat biofilms. The EPS comprise throughout the matrix52,84–86.

Reviews
Targeting resident pathogens. On biofilm penetration, Nanomaterials can also deliver therapeutics to bac-
nanomaterials can interact with bacteria and exert the terial cells embedded within the EPS matrix. For exam-
therapeutic mechanisms discussed earlier for plank- ple, although the potent antimicrobial carvacrol oil, an
tonic bacteria (Fig. 3a). For instance, the efficient biofilm essential oil found in oregano and thyme, poorly pen-
penetration profile and bacterial membrane-damaging etrates biofilms, one study used carvacrol to eradicate
activity of poly(oxanorborneneimide)-based cationic biofilms using biodegradable oil-in-water crosslinked
polymeric nanoparticles eradicated MDR biofilms of polymeric nanocomposites, which eliminated MDR
P. aeruginosa, Enterobacter cloacae complex and MRSA84. biofilms of both Gram-negative and Gram-p ositive
In another approach, the use of stimuli-responsive nan- bacteria while maintaining minimal cytotoxicity
oparticles led to the activation of bactericidal effects in towards mammalian cells 30. The polymer scaffold
a spatiotemporally controlled manner. pH-responsive contained guanidinium, maleimide and tetraethylene
silver nanoantibiotics were developed using self- glycol monomethyl ether groups. The cationic property of
assembled silver nanoclusters and the charge-switchable the nanocomposite was attributed to guanidinium. The
ligand PEG-poly(aminopropyl imidazole-aspartate)- presence of maleimide groups provided crosslinking
polyalanine87. Protonation of the imidazole groups in the sites and imparted biodegradation points onto the nano-
low-pH microenvironment of biofilms induced disas- composite, while the tetraethylene glycol monomethyl
sembly of pH-responsive silver nanoantibiotics owing to ether conferred hydrophilicity on the assembly. Careful
electrostatic repulsion with silver ions. Disassembly into design of the polymer increased the solubility, stability,
smaller silver nanoclusters enabled biofilm penetration, biodegradability and antimicrobial potency of carvacrol
killing deeply embedded MRSA cells. Similarly, appli- oil, while assisting its penetration of the biofilm matrix.
cation of an external magnetic field facilitated biofilm Similarly, nanoscale liposomes delivered the antibiotic
penetration by AgNPs88; superparamagnetic iron oxide amikacin through size-dependent biofilm penetration
nanoparticles were coated with silver rings and the mag- as a strategy to treat chronic P. aeruginosa biofilm lung
netic field generated allowed biofilm penetration, with infections89, an approach that is currently in phase III
silver conferring antibacterial activity. clinical trials.
a b
Disruption of pre-existing
Antibacterial NPs physico-chemical
interactions in EPS
Delivery of EPS-degrading enzymes
Nanocarriers
Dispersed EPS matrix
Dead bacteria
Fig. 3 | Eradicating biofilms using nanoparticles. Biofilms comprise cells with phenotypic heterogeneity embedded
across the 3D matrix of their self-secreted extracellular polymeric substances (EPS). The ability of nanoparticles (NPs) to
penetrate throughout the matrix allows them to interact with cells, both susceptible (blue) and resistant (red), entrenched
within the EPS (part a) and/or initiate disruptive interactions with the matrix that weaken physico-chemical interactions
responsible for keeping the 3D structure of biofilms intact (part b). NPs can then either exert their inherent antimicrobial
action or deliver therapeutic agents, such as antibiotics or essential oils, to kill the bacteria within the biofilms. NPs can
alternatively deliver EPS-degrading enzymes that promote dispersion of biofilms, facilitating their disruption.

Reviews
Disrupting the EPS matrix. Beyond killing bacteria, the Combating biofilm infections
EPS matrix can be disrupted for the purpose of treat- The number of biofilm-related infections continues to
ing biofilms90. EPS scaffold remaining after treatment grow year by year103,104. Bacteria can form biofilms in
can be inhabited and populated by other microorgan- and on tissues and organs, including on skin, in the oral
isms. Different nanomaterial-based approaches can cavity and on linings of the gastrointestinal and respira-
be used to disperse EPS matrix, including mechanical tory tracts8,80. Biofilms largely contribute to chronic and
disruption and delivery of matrix-degrading enzymes persistent infections. With advances in our understand-
(for example, DNase, hydrolase and protease) (Fig. 3b). ing of medical biofilms, nanotherapeutic strategies have
For instance, PLGA nanoparticles loaded with cip- emerged to potentially address biofilm infections.
rofloxacin were functionalized with DNase I to target
P. aeruginosa biofilms91. DNase I degraded extracellular Oral biofilms. The oral cavity is a major site for biofilms;
DNA, rendering the 3D network fragile and suscepti- Streptococcus mutans is a common oral biofilm patho-
ble to ciprofloxacin. Similarly, AuNPs functionalized gen. The acidic microenvironment of dental biofilms
with proteinase K dispersed Pseudomonas fluorescens (that is, plaque) results in destruction of tooth enamel,
biofilms92. Alternatively, magnetic iron oxide nanopar- causing dental caries105,106. Nanoparticle-based strategies
ticles disrupted MRSA biofilms with the application of have been used to address oral biofilm-associated infec-
direct current and alternating current magnetic fields93. tions, taking advantage of the highly acidic oral biofilm
Application of a rotating direct current magnetic field microenvironment. Liposomes coated with quaternary
mechanically damaged the biofilm matrix. Magnetic ammonium-modified chitosan have been used to deliver
iron oxide nanoparticles traversing across the 3D net- the antibiotic doxycycline to Porphyromonas gingivalis
work acted as ‘shield breakers’, destroying biofilms oral biofilms107. The residual amines of chitosan pro-
through static friction. Exposure of magnetic iron vide pH-responsive groups that are protonated under
oxide nanoparticles to an alternating current magnetic acidic conditions, providing pH-dependent activity.
field resulted in a localized increase in temperature Similarly, nanocarriers fabricated with pH-responsive
that dispersed embedded cells. As the mechanisms of block copolymers that can bind to negatively charged
action of these magnetic iron oxide nanoparticles do hydroxyapatite have been used to deliver farnesol108
not include killing of bacteria, this system offers a long- and chlorhexidine109 for treatment of dental caries.
term antibiofilm strategy that may escape resistance Nanoparticles that induce ROS production and EPS
development. matrix degradation are also being investigated for oral
A promising strategy for targeting biofilm growth biofilm treatment. For instance, catalytic nanoparticles
is interruption of bacterial communication systems consisting of biocompatible Fe3O4 were used to catalyse
essential for coordinated activities, including colo in situ generation of free radicals from H2O2, resulting in
nization and biofilm development. Bacteria com- a reduction of S. mutans biofilms110. Coating iron oxide
municate through quorum sensing, a process that can nanoparticles with FDA-approved polymers, such as
be sabotaged to prevent formation of biofilms or dextran, increased their stability in an aqueous formu-
induce their dispersion94–96. One study demonstrated lation and enhanced biocompatibility with oral soft
that hampering quorum sensing can silence bacte- tissues111. The iron-supplying nanotherapeutic ferumox-
rial communication97. Silicon dioxide nanoparticles ytol was ‘reinvented’ from an iron-deficiency drug into a
decorated with β-c yclodextrin blocked communica- topical oral biofilm therapeutic112. This FDA-approved
tion between Vibrio fischeri cells. V. fischeri exhibits iron-based nanoparticle possesses a pH-dependent
bioluminescence controlled by population density, peroxidase-like property that provides localized catalytic
which can be monitored through the quorum sens- activity (Fig. 2e). This work demonstrated that ferumox-
ing signalling molecule acylhomoserine lactone. The ytol diffuses within biofilm matrices and generates free
β-cyclodextrin group attached to silicon dioxide nano radicals from H2O2, resulting in in situ bacterial death
particles binds to acylhomoserine lactone, quench- and EPS degradation. Both a human-derived ex vivo
ing its activity. As a result, the luminous output of model and an in vivo rat dental caries model revealed effi-
V. fischeri was reduced. Furthermore, downregulation cacy in preventing acid damage of enamel and suppres-
of luminescence genes, luxA and luxR, was observed. sion of dental caries without altering the oral microbiota,
Other studies have demonstrated inhibition of bio- and safety towards gingival and mucosal tissues.
film formation and virulence factors by deactivating
quorum sensing molecules using liposome-b ased Wound biofilms. Wound infections affect ∼300 million
nanop articles 98 , chitosan nanoparticles 99,100 and people worldwide, with treatment costs estimated as
metal-based nanoparticles101,102. high as $US25 billion in the USA alone113,114. In these
Nanomaterial penetration profiles predict suc- infections, necrotic tissue fosters attachment of bacteria
cess of biofilm elimination. Size and amphiphilicity and provides nutrients that enhance bacterial prolif-
Quorum sensing mainly influence nanoparticle distribution across the eration and biofilm formation, which impedes wound
A process whereby bacteria biofilm. The exact interactions of nanoparticles with healing by inhibiting re-epithelialization and prolonging
communicate and perform the EPS depend on the type of biofilm, which differs inflammation15,103,115. AgNPs incorporated in hydrogels
coordinated activities in by species and in some cases by strain of bacteria. or in wound wraps are used to treat wound infections116.
response to a particular cell
population density determined
Controllable parameters of nanomaterials provide Other types of nanoparticles have also been increas-
by specific signalling a flexible toolkit to address the diversity of biofilm ingly studied for the treatment of biofilm-infected
molecules. infections. wounds117,118. For example, copper particles incorporated

Reviews
Table 2 | Nanomaterial-based therapeutics in clinical trials

Trade name Nanoparticle Active agent Target Clinical Clinical trial Ref.
type pathogens or trial number
infection phase
Arikace Liposomal Amikacin Gram negative III NCT01315691 124
Pulmaquin Liposomal Ciprofloxacin Gram negative III NCT02104245 125
Silvasorb AgNP Silver Topical infection III NCT00659204 134
AgTive (central venous AgNP Silver Central venous IV NCT00337714 135
catheters impregnated catheter-related

with AgNPs) infection
NA Polymeric Doxycycline Chronic II NCT02726646 136
nanoparticle periodontitis
NA Polymeric Ammonium Oral infection II NCT01167985 137
nanoparticle polyethyleneimine
AgNP, silver nanoparticle; NA, not applicable.
into biodegradable nanofibres prevented the formation Successful clinical translation will require stand-
of and eradicated preformed biofilms of P. aeruginosa ardized guidelines for evaluating biocompatibility and
and S. aureus. Further in vitro and in vivo studies are nanotoxicology. Most formulations undergoing clinical
under way to demonstrate the applicability of this strat- testing are nanocarriers for antibiotic delivery or anti-
egy for wound dressings119. Another strategy uses the microbial AgNPs (Table 2). Two liposomal nanoformu-
amphiphilic core–shell polymeric nanoparticle DA95B5, lations for controlled delivery of antibiotics are currently
which removes preformed biofilms of MRSA through in phase III clinical trials. Arikace, a liposomal formula-
nanoscale bacterial ‘debridement’120 (Fig. 2f). DA95B5 can tion of the antibiotic amikacin, was designed to increase
diffuse through the EPS, disrupting biofilms by weak- the therapeutic efficacy of amikacin as well as to allevi-
ening attachment of bacteria to the matrix. An in vivo ate its renal and neurological toxicity124. Pulmaquin is
mouse excisional wound biofilm model demonstrated a nanoliposome-based formulation designed for rapid
effective dispersal of MRSA biofilms. DA95B5-soaked and delayed release of ciprofloxacin125. Many challenges
hydrogel pad dressings reduced bacterial counts in mice still hamper nanodrug translation into clinical settings,
by up to ~4 log. Notably, the nanoparticle exhibited min- including safety concerns; however, it is likely only a
imal in vitro eukaryotic cell lysis and low in vivo toxic- matter of time until these novel therapeutics provide
ity. Combination of these nanoparticles with molecules solutions for currently unmet clinical demands46,122.
that accelerate wound healing, including growth factors,
anti-inflammatory molecules and extracellular matrix Conclusions and perspectives
mimics, may further nanoparticle-based strategies for Nanomaterials present an emerging ‘outside of the box’
the treatment of wound infections. As an example, a pH- approach for treatment of recalcitrant MDR planktonic
responsive antimicrobial nanofibre network, formed by bacterial and biofilm infections. The tunable properties of
the self-assembly of the octapeptide IKFQFHFD, was nanomaterials, particularly their surface functionalities,
incorporated into a hydrogel and loaded with cypate provide design spaces that can be fine-tuned to maximize
and proline121. The octapeptide possesses an inherent the therapeutic effect while minimizing host toxicity.
antimicrobial property through cell wall and membrane In this Review, we have provided examples of how
disruption; cypate is a photothermal drug that is thought nanoparticles can combat bacteria in both planktonic
to disrupt EPS matrix; and the procollagen component and biofilm forms using a wide range of mechanisms.
proline is added to aid in collagen and extracellular Nanomaterials can access multimodal antibacterial
matrix re-formation. The hydrogel eradicated MRSA mechanisms that are novel, slowing or stopping the gen-
biofilms and facilitated healing in chronic wounds as eration of drug resistance. Nanoparticles have potential as
demonstrated in an in vivo diabetic mouse model. topical treatments for oral and wound biofilm-associated
infections. Strategies combining bactericidal effects and
Towards clinical translation biofilm dispersion, however, are ideal to ensure complete
There has been a rapid increase in the exploration of eradication of biofilms. Stimuli-responsive nanoparti-
antimicrobial nanomaterials for treatment of MDR cles that take advantage of unique microenvironments
Cypate planktonic bacterial and biofilm infections. Most stud- at infection sites, such as pH and pathogen-derived
A near-infrared fluorescent dye
ies have been conducted in vitro, with fewer proceeding metabolites, provide one of the many avenues to tar-
belonging to the carbocyanine
dye family, widely used for to animal models and fewer still proceeding to human get MDR bacteria using nanomaterials. Systemic safety
metabolite labelling and in vivo trials46,122. Developing appropriate in vitro and in vivo and long-term effects of nanoparticles on the body are
imaging. models that demonstrate efficacy and safety of nan- still among the major barriers to clinical use. Current
oparticles will provide clinical feasibility for their use. studies are determining the pharmacokinetic profile of
Proline
A proteinogenic amino acid
Several reviews have summarized appropriate in vitro nanoparticles to better understand their fate in the body.
vital for the biosynthesis of and in vivo models to explore, depending on the type of The generation of effective antimicrobial nanoma-
collagen. infection being targeted7,103,123. terials requires interdisciplinary collaborations among

Reviews
chemists, biomedical researchers (including microbi- strategies offer a promising alternative to antibiotics for
ologists) and engineers. Likewise, partnership between difficult-to-treat infections, alleviating challenges faced
fundamental, translational and industrial agencies will in the post-antibiotic era.
be instrumental in moving antimicrobial nanomateri-
als to the clinic. Overall, nanomaterial-based treatment Published online 19 August 2020
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Nanomaterial-based therapeutics for

naTure RevIewS | MICRObIOLOgy volume 19 | January 2021 | 23

Box 1 | What are nanomaterials?

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Box 2 | Antibiotic resistance mechanisms and nanomaterials

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Size comparison of bacteria and nanomaterials 100 nm

Gram-negative envelope: Gram-positive envelope:

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SNAPP inﬂux into

Cellular uptake DOPE

Salmonella 18:0 PEG-2000PE

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Box 3 | Nanomaterial properties and design elements

COOH-QD PEG-QD Hexyl-QD TTMA-QD

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Delivery of EPS-degrading enzymes

Dispersed EPS matrix

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Table 2 | Nanomaterial-​based therapeutics in clinical trials

Pulmaquin Liposomal Ciprofloxacin Gram negative III NCT02104245 125

Silvasorb AgNP Silver Topical infection III NCT00659204 134

AgTive (central venous AgNP Silver Central venous IV NCT00337714 135

catheters impregnated catheter-​related

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Table 2 | Nanomaterial-based therapeutics in clinical trials

catheters impregnated catheter-related