Biomedical Applications of Gold Nanoparticles

VADIM V. SUMBAYEV, INNA M. YASINSKA, BERNHARD F. GIBBS

Medway School of Pharmacy, University of Kent, UNITED KINGDOM

E-mail: V.Sumbayev@kent.ac.uk

Abstract: - Gold nanoparticles display unique properties which allow them to influence a number of different

biochemical processes. Interestingly, these effects are dependent on the particle surface chemistry which varies

considerably based on the particle size. In addition, unlike most of other nanomaterials, gold nanoparticles do

not trigger the formation of pro-inflammatory multiprotein complexes, known as inflammasomes. Furthermore,

they display clear anti-inflammatory activity. Here we discuss the biological effects of intact and functionalised

gold nanoparticles and analyse their possible biochemical mechanisms.

Key Words: - Gold Nanoparticles, Surface Chemistry, Resonance, Nanoparticle Size, Inflammation, Immune

Responses, Cancer.

1 Introduction

In recent years there has been increasing evidence

showing the successful biomedical applications of

gold nanoparticles (AuNPs). Although colloidal

gold has been known for at least 25 centuries (1)

and was first applied by Paracelsus to treat mental

disorders and syphilis (1) in 16th century, the use of

gold particles of extremely small size was only first

published in 1971 (2). In this case, W.P. Faulk and

G.M. Taylor conjugated antibodies with colloidal

gold which was used for direct microscopic

visualisation of Salmonella surface antigens. This

was the starting point of biomedical applications of

colloidal gold conjugates displaying biological

activity (1) and the concept has now progressed to

the point where AuNPs in native and functionalized

(conjugated) form are widely used in both

biomedical research and therapy. Currently, AuNPs

are used in diagnostics, bioimaging, biosensors,

photothermal and photodynamic therapy as well as

in drug/genetic material delivery.

All these applications have become possible due to

the unique physical and chemical properties of

AuNPs.

2 Aim In this report we aim to analyse the properties of

AuNPs which account for their interactions with

biological systems. We will analyse the properties

and reactivity of AuNPs which determine their

biocompatibility and potential toxicological effects.

3.1. Physical and chemical properties

of AuNPs.

AuNPs, as for other nanomaterials, can be subjected

to a detailed analysis of their optical and surface

chemical properties (1). Optical properties of

AuNPs are normally used to determine the strategies

of their applications. The interaction of AuNPs with

light is determined by their size, physical

dimensions as well as environment. Oscillating

electrical fields of light beams circulating near

AuNPs interact with free electrons causing a joint

oscillation of electron charge which is in resonance

with the respective frequency of visible light. These

resonant oscillations are recognised as surface

plasmons. A plasmon can be defined as a quantum

of plasma oscillation. Normally, plasmons are

collective oscillations of the free electron gas

density at certain (for example optical) frequencies.

Plasmons can also couple with a photon, thus

creating another quasi-particle known as a plasma

polariton (3-5).

Most of the plasmon properties can be derived

directly from Maxwell's equations since they stand a

quantization of classical plasma oscillations (3-5).

The resonance and the optical properties of AuNPs

depend on their size. Both extinction (Aext) and

scattering efficiency (at 90° angle (I90(λ)) – these

are the main optical characteristics of NPs) depend

on their size. If we assume that the AuNP has d=2a

in an aqueous environment (their concentration is a

constant value) where the refractive index equals

nm(λ), and calculate its Aext as well as I90(λ), the

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dependence of these parameters on particle size

becomes obvious (see equations (1) and (2)).

In this case ρ – is the density of the metal,

Qext = Cext/πa2 – is the scattering efficiency

factor, S11(ka, θ) – normalised scattering

efficiency at the angle of 90°, k = 2πnm/λ – the

wavenumber in the water.

As such, smaller AuNPs mostly absorb light

while bigger particles tend to scatter it (3-5).

For example, in monodisperse AuNPs with

d<30 nm the surface plasmon resonance

phenomenon causes absorption of light in the

blue-green region of the spectrum (~450 nm)

while red light (>680 nm) is reflected, resulting

in a red colour. Particles with greater size (>50

nm) tend to absorb red while blue light is

reflected, giving rise to a pale blue or purple

colour. A simplified diagram representing

dependence of the AuNP colour on their size is

presented in the Figure 1.

Figure 1. Dependence of the AuNP colour on

particle size.

A recently discovered a phenomenon reported

by Huang et al showed that AuNPs smaller than

10 nm display unique advantages over NPs

larger than 10 nm in terms of their ability to

interact with living cells. It was found that the

properties of the surface of smaller AuNPs

differ from those of larger nanomaterials and

this is generally reflected in changes in the

frequency and intensity of the corresponding

surface plasmon resonance peaks (see above)

(6).

Chemical and biological effects of AuNPs are

not only determined by their size and

optical/resonance properties but also by their

surface chemistry. Gold surfaces demonstrate

high affinity to thiol groups and even to

disulfides. The adsorption of thiols on Au(111)

at low coverage could be considered as the

simplest case where the influence of the van der

Waals interactions are minimised. Au(111)

undergoes a 23×√3 reconstruction where face-

centered cubic (fcc) domains alternate with

hexagonal close-packed (hcp) domains,

separated by smaller regions with Au atoms on

bridge positions (Figure 2) (8).

Figure 2. Au(111) with examples of

determining indices for a plane using

intercepts at each axis.

The sulphur of thiol groups and disulfides form

semi-covalent interactions with gold with the

strength of approximately 45kcal/mol (7, 8).

This property of AuNPs is used to coat them

with different compounds. For example,

PEGylation (coupling of the AuNPs with poly-

ethylene glycol (PEG)) of AuNPs is normally

performed through SH groups or dihydrolipoic

acid (DHLA, Figure 3) (9).

Figure 3. Structure of PEG used to couple

with AuNPs.

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ISBN: 978-1-61804-164-7 343

Sulphur, contained in the two amino acids

cysteine and methionine, was found to interact

with gold surfaces (7).

Additionally, AuNP surfaces can form weaker

hydrophobic interactions with compounds such

as lipopolysaccharide (LPS) – the main

component of the cell wall of Gram-negative

bacteria (10).

Biological effects of AuNPs.

Due to their ability to bind to sulphur-

containing proteins and as well as forming

hydrophobic interactions, AuNPs interact with

many biologically active compounds. This

applies not only to purified proteins, which

could be easily immobilised on the gold surface

through Au-S interactions, but also to purified

non-protein compounds displaying hydrophobic

motifs (for example LPS). AuNPs quickly

interact with plasma/serum proteins forming a

so-called “protein corona”. In this case some of

the signalling proteins which interact with

receptors associated with plasma cell

membranes can be bound to AuNPs. This

applies mostly to blood cells which participate

in host immune defence and hypersensitivity

reactions. Most of these cells display the ability

to endocytose particles and

microorganisms/viral capsides and thus could

potentially internalise NPs (an exception in this

case would possibly be erythrocytes) (11).

As a result, the ligand could still bind the

receptor but it binds as a ligand-AuNP complex.

This kind of interaction produces a localised

decrease in the Gibbs free energy, which

induces the membrane to wrap around the

AuNP forming a closed vesicle structure. This

may lead to an internalisation of the AuNPs,

together with the protein corona, via

endocytosis (where phagocytosis takes place in

the majority of cases). Internalised AuNPs,

depending on the corona, were found to

influence major intracellular signalling events

and biochemical reactions by direct actions on

the nucleus and mitochondria as well as by

interacting with actin filaments. However, the

behaviour of AuNPs in the endolysosomal

vesicles remains unknown. In some cases

Cathepsin L (a proteolytic enzyme) could

cleave the nanoparticle ligands. In macrophages

degradative enzymes slowly decompose the

core structure. However, in most cases when

released into the cytosol, AuNPs affect cell

behaviour (reviewed in (11)). These

internalisation mechanisms are schematically

presented in the Figure 4.

Figure 4. Scheme demonstrating

internalisation of AuNP-ligand complexes

into cells. Arrows show the intracellular

elements affected by the internalised

nanomaterials.

Therefore, the ability of AuNPs to interact with

proteins and other biologically active

compounds depends on the ligand structure as

well as the positioning and reactivity of the

gold-interacting elements.

For example, we recently demonstrated that

AuNPs, depending on their size, could affect

the interleukin-1β (IL-1β)-dependent pro-

inflammatory responses of human myeloid cells

(12). IL-1β is a highly inflammatory cytokine

which mediates the crosstalk between innate

and adaptive immunity. IL-1β acts through the

plasma membrane-associated IL-1 receptor type

1 (IL-1R1) and induces the activation of

antigen-presenting cells leading to the

generation of host adaptive immune defences.

However, IL-1β production can also occur

during erroneous inflammatory reactions

against host-derived endogenous ligands. Such

a process often leads to the development of

different types of inflammatory autoimmune

disorders, such as rheumatoid arthritis, psoriasis

and many others. These disorders pose a serious

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ISBN: 978-1-61804-164-7 344

medical burden worldwide, where patients face

a lifetime of illness and treatment (13-14). Our

results indicated that this type of anti-

inflammatory activity of AuNPs is associated

with an extracellular interaction with IL-1β.

Importantly, this effect does not downregulate

the ability of the IL-1β to interact with its

plasma membrane-associated receptor but

attenuates IL-1β-receptor complex-dependent

intracellular signalling events and subsequently

abrogates the pro-inflammatory response (12).

In contrast, stem cell factor (SCF)-mediated

triggering THP-1 human myeloid leukaemia

cells were not affected by AuNPs. SCF is a

hematopoietic growth factor which acts by

signalling through the tyrosine kinase receptor

Kit (or CD117). SCF-Kit signalling regulates

survival, proliferation and differentiation

myeloid lineage hematopoietic cells.

Furthermore, since myeloid leukaemia cells

express the Kit receptor, SCF may play an

important role in myeloid leukaemia

progression too (12).

Structural analysis of human IL-1β and SCF

indicates that IL-1β has both cysteine and

methionine-derived sulphur atoms which are

able to interact with 5 nm AuNPs, some of

which are likely to interact with the surface of

the AuNPs. However, in the case of SCF these

sulphur atoms are not easily accessible to the

nanomaterial surface (Figure 5).

Figure 5. Positioning of sulphur atoms in

folded human IL-1β (A) and SCF (B). Using

PDB files from the RCSB protein data bank

(4G6M – IL-1β and 2E9W – SCF) space

structures of IL-1β and SCF were analysed.

Sulphur atoms are depicted in blue.

Unlike other nanomaterials, AuNPs do not

provoke inflammatory responses. It has recently

been found that poorly soluble uric acid, the

final product of human purine catabolism forms

monosodium urate (MSU) crystals, which

induce formation of a multiprotein complex

known as the inflammasome. This contains

Nalp3 protein binding to the apoptosis spec-like

protein 2 (ASC2) forming the complex which

activates caspase 1, the cysteine protease

catalysing maturation of pro-IL-1β into the

biologically active IL-1β (15, 16).

Recently, Si and Ni nanoparticles were

demonstrated to induce formation of the Nalp3

inflammasome and IL-1β maturation (15). Our

results confirmed this observation with SiO2

nanoparticles (12). All these observations allow

to hypothesise that such an effect could be a

result of the presence of chemically active

surface in the cell. Schematically this

hypothesis is presented in the Figure 6.

Figure 6. Possible mechanism of surface-

dependent activation of the Nalp3

inflammasome. Xanthine oxidase (XOD)

converts hypoxanthine into xanthine and further

into uric acid. Sodium is transported into the

cell through the sodium-glucose symport

performed by the respective protein transporter

(SGT). The distribution of sodium and

potassium on both sides of the plasma

membrane is regulated by the sodium-

potassium ATPase (antiport – NKA). Uric acid,

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ISBN: 978-1-61804-164-7 345

formed by XOD, could therefore easily interact

with sodium forming non-soluble monosodium

urate (MSU). Possible similarities of the Nalp3

activating action of MSU and SiNPs are

presented.

Our results demonstrated that AuNPs,

regardless the size, do not activate the Nalp3

inflammasome while SiO2 NPs do. The reason

for this could be related to the different type of

reconstruction undergone by the Au and Si/Ni

surfaces. In fact, Si/Ni surfaces are likely to

share a number of chemical properties with the

MSU surface (15-17).

Biomedical applications of AuNPs based on

their physical/chemical properties and

biological effects

Considering the information described above

one could clearly estimate possible applications

of these nanomaterials. First of all, given the

fact that AuNPs are solid nanomaterials which

do not undergo destruction and also can’t

replicate themselves it is obvious that the exact

number of AuNPs in the media could be

calculated using simple equations.

(3)

1

1 1 3

1 31

;

( ) ( ) ( ); 196.97

; 19.3 ;

4 ( ); ;

3 2

For example, 100 l of 35 nm 5 mM AuNP contain:

total AuAuNP

AuNP

total Au Au Au AuNP Au

AuNP Au AuNP Au

AuNPAuNP

total Au

mN

m

mol g gm C MW V l MW

l mol mol

gm V

cm

d cmV R R

m

ρ ρ

π

µ

=

= × × =

= × =

= =

3 4 5

7 3 3 161

3

5

1

( ) ( ) ( ) 5 10 9.8485 10 ;

4 351 19.3 ( 10 ) 4.33051780840371875 ;

3 2

9.8485 10

4.3

Au Au AuNP

Au AuNP

total AuAuNP

AuNP

mol g mol gC MW V l l g

l mol l mol

gm AuNP V cm g

cm

m gN

m

ρ

− − −

− −

−

= × × = × × 196.97 × 10 = ×

= × = × × 3.14 × × = × 10

× = =

16227420840549 2.27420840549

3051780840371875particles particles

g−= = × 10

× 10

Here: CAu is the gold concentration, MWAu is

the molecular weight of gold, VAuNP is the

volume of the nanoparticles, ρAu is the density

of gold and R is the radius of each particle,

expressed in nm.

Given the physical/chemical properties of the

AuNPs, discussed in the above sections, they

are currently widely studied and applied to

biomedical research and practice. The

biomedical applications of AuNPs are

summarised in the Figure 7.

Figure 7. Summary of the biomedical

applications of AuNPs.

Given the fact that AuNPs display anti-

inflammatory activity and do not induce IL-1β

maturation they could be considered as one of

the most biocompatible nanomaterials.

Therefore, they are now widely considered for

use in therapy such as plasmonic photothermal

therapy. The principle of this method is the use

of ligand-conjugated AuNPs to label target

cells. AuNPs are then irradiated with laser

pulses which creates local heating and destroys

the labelled cells (reviewed in (1)).

Another application is photodynamic therapy

using photosensitizers together with light of a

specific wavelength. Given the ability of the

AuNPs to become conjugated with such

photodynamic dyes it is possible for them to

beinternalised into the target cells (involving

the same principles described above).

Photodynamic therapy is mostly applicable for

treatment of oncological and infectious diseases

(reviewed in (1)).

AuNPs are also increasingly being employed

for use in biomedical research/analysis,

especially in bioimaging and visualisation. The

ability of Au nanoconjugates to specifically

label target cells and microorganisms can be

widely applied in such as transmission electron

microscopy and in biophotonic methods

including X-ray and magnetic resonance

tomography as well as fluorescence correlation

microscopy (1).

Au surfaces could be used for immobilisation of

different analytes, for example protein

conformation-specific peptides. Such

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ISBN: 978-1-61804-164-7 346

conjugates would allow recognition of specific

conformations of proteins and receptors thus

allowing for rapid screening of receptor ligand

properties of different compounds, for example

environmental endocrine disruptors interfering

with the actions of male and female sex

hormones. This approach is therefore an

excellent strategy for the generation of

plasmonic biosensors (1, 18, 19).

Importantly, given the properties of AuNPs,

their biocompatibility and absence of severe

pro-inflammatory effects they could be

considered as potential drug delivery platforms.

In the same way they may be used for the

delivery of specific genetic materials in both

research and therapy. In the same way, they are

considered as possible delivery platforms for

delivery of antigens through adjuvant-free and

adjuvant containing nanovaccines. This is a

developing field and has been supported by the

discovery of the ability of AuNPs to bind to

various ligands (for example LPS via

hydrophobic interactions).

4 Conclusion

Our analytical work describes the unique

physical and chemical properties of AuNP

surfaces. Both their plasmonic properties and

surface chemistry allow them to form stable

interactions with biologically active

compounds. They can also bind to ligands with

lower strengths through hydrophobic

interactions. Despite these interactions, AuNPs

are relatively inert and do not display noticeable

pro-inflammatory activities, in stark contrast to

many other nanomaterials. All these properties

support the use of AuNPs in biomedical

applications such as plasmonic photothermal

and photodynamic therapy, bioimaging,

generation of plasmonic biosensors as well as

drug/gene delivery.

Despite these many potential applications, there

is still a pressing need to determine any long-

term toxicological effects of AuNPs in order to

determine their biocompatibility.

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