Gold Nano Recent Advances

2256 Chem. Soc. Rev., 2012, 41, 2256–2282 This journal is c The Royal Society of Chemistry 2012

Cite this: Chem. Soc. Rev., 2012, 41, 2256–2282

Gold nanoparticles in biomedical applications: recent advances

and perspectives

Lev Dykmanaand Nikolai Khlebtsov*

ab

Received 19th June 2011

DOI: 10.1039/c1cs15166e

Gold nanoparticles (GNPs) with controlled geometrical, optical, and surface chemical properties

are the subject of intensive studies and applications in biology and medicine. To date, the ever

increasing diversity of published examples has included genomics and biosensorics, immunoassays

and clinical chemistry, photothermolysis of cancer cells and tumors, targeted delivery of drugs

and antigens, and optical bioimaging of cells and tissues with state-of-the-art nanophotonic

detection systems. This critical review is focused on the application of GNP conjugates to

biomedical diagnostics and analytics, photothermal and photodynamic therapies, and delivery

of target molecules. Distinct from other published reviews, we present a summary of the

immunological properties of GNPs. For each of the above topics, the basic principles, recent

advances, and current challenges are discussed (508 references).

1. Introduction

Gold was one of the first metals discovered by humans, and

the history of its study and application is estimated to be a

minimum of several thousand years old. The first information

on colloidal gold can be found in tracts by Chinese, Arabic,

and Indian scientists, who obtained colloidal gold as early as

in the fifth and fourth centuries B.C. and used it, in particular,

for medical purposes (the Chinese ‘‘gold solution’’ and the

Indian ‘‘liquid gold’’).

In the Middle Ages in Europe, colloidal gold was studied

and employed in alchemists’ laboratories. Specifically, Paracelsus

wrote about the therapeutic properties of quinta essentia auri,

which he prepared by reducing auric chloride with alcohol or

oil plant extracts. He used ‘‘potable gold’’ to treat some mental

a Institute of Biochemistry and Physiology of Plants andMicroorganisms, RAS, 13 Pr. Entuziastov, Saratov 410049,Russian Federation. E-mail: [email protected]

b Saratov State University, 83 Ul. Astrakhanskaya, Saratov 410012,Russian Federation

Lev Dykman

Dr Lev A. Dykman, leadingresearcher of the Immuno-chemistry Lab at the Instituteof Biochemistry and Physiol-ogy of Plants and Micro-organisms Russian Academyof Sciences. He has publishedmore than 200 scientific worksincluding one monograph oncolloidal gold nanoparticles.His current scientific interestsinclude immunochemistry,fabrication of gold nano-particles and their applicationsto biological and medicalstudies. In particular, his

research is aimed at interaction of nanoparticles and conjugateswith immune-responsible cells and at the delivery of engineeredparticles to target organs, tissues, and cells.

Nikolai Khlebtsov

Professor Nikolai G. Khlebtsov,head of the NanobiotechnologyLab at the Institute of Bio-chemistry and Physiology ofPlants and MicroorganismsRussian Academy of Sciences.He also holds a BiophysicsChair at the Saratov StateUniversity. He has publishedmore than 300 scientificworks, including two mono-graphs. His current scientificinterests include biophotonicsand nanobiotechnology ofplasmon-resonant particles,biomedical applications of

metal nanoparticles, static and dynamic light scattering by smallparticles and clusters, programming and computer simulation oflight scattering and absorption by various metal and dielectricnanostructures. Prof. Khlebtsov also serves as an AssociateEditor of the Journal of Quantitative Spectroscopy and Radia-tive Transfer.

Chem Soc Rev Dynamic Article Links

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This journal is c The Royal Society of Chemistry 2012 Chem. Soc. Rev., 2012, 41, 2256–2282 2257

disorders and syphilis. Paracelsus once proclaimed that chemistry

is for making medicines, not for making gold out of metals.

His contemporary Giovanni Andrea applied aurum potabile to

the treatment of lepra, ulcer, epilepsy, and diarrhea. In 1583,

alchemist David de Planis-Campy, surgeon to the King of

France Louis XIII, recommended his ‘‘elixir of longevity’’—an

aqueous colloidal gold solution—as a means of life prolongation.

The first book on colloidal gold preserved to our days was

published by philosopher and doctor of medicine Francisco

Antonii in 1618.1 It contains information on the preparation

of colloidal gold and on its medical applications, including

practical suggestions.

In 1880, a method was put forward to treat alcoholism by

intravenous injection of a colloidal gold solution (‘‘gold

cure’’).2 In 1927, the use of colloidal gold was proposed to

ease the suffering of inoperable cancer patients.3 Colloidal

gold in color reactions toward spinal-fluid and blood-serum

proteins has been taken advantage of since the first half of the

twentieth century.4 Colloidal solutions of the 198Au gold

isotope (half-life time, 65 h) were therapeutically successful

at cancer care facilities.5 More recent examples of colloidal

gold applications include catalytic processes and electron

transport in biomacromolecules,6 transport of substances into

cells by endocytosis,7 investigation of cell motility,8 and improve-

ment of PCR efficiency.9

Despite the centuries-old history, a ‘‘revolution in immuno-

chemistry,’’10 associated with the use of gold particles in

biological research, took place in 1971, when British researchers

W. P. Faulk and G. M. Taylor published an article titled ‘‘An

immunocolloid method for the electron microscope.’’11 In that

article, a technique was described to conjugate antibodies with

colloidal gold for direct electron microscopic visualization of

Salmonella surface antigens, representing the first time that a

colloidal gold conjugate served as an immunochemical marker.

From this point on, the use of colloidal-gold biospecific

conjugates in various fields of biology and medicine became

very active. There has been a wealth of reports dealing with the

application of functionalized gold nanoparticles (GNPs; conju-

gates with recognizing biomacromolecules, e.g., antibodies, lectins,

enzymes, or aptamers)12–14 to the studies of biochemists, micro-

biologists, immunologists, cytologists, physiologists, morphologists,

and many other specialist researchers.

The range of uses of GNPs in current medical and biological

research is extremely broad. In particular, it includes genomics;

biosensorics; immunoassay; clinical chemistry; detection and

photothermolysis of microorganisms and cancer cells; targeted

delivery of drugs, peptides, DNA, and antigens; and optical

bioimaging and monitoring of cells and tissues with the use of

state-of-the-art nanophotonic recording systems. GNPs have

been proposed for use in practically all medical applications,

including diagnostics, therapy, prophylaxis, and hygiene

(e.g., in water purification15). Extensive information on the most

important aspects of preparation and use of colloidal gold in

biology and medicine can be found elsewhere.16–30 Such a

broad range of application is based on the unique physical and

chemical properties of GNPs. Specifically, the optical properties

of GNPs are determined by their plasmon resonance, which is

associated with the collective excitation of conduction electrons

and is localized in a wide region (from visible to infrared,

depending on particle size, shape, and structure).31 Scheme 1

shows a simplified scheme for the current biomedical applications

of GNPs, which reflects the structure of this review. However,

since biodistribution and toxicity have been extensively reviewed

and discussed in a number of recent publications (see, e.g., ref. 32

and references therein), we restrict ourselves to a short comment

in the Conclusions section.

Considering the great body of existing information and the

high speed of its renewal, we chose in this review to generalize

the data that have accumulated during the past few years for

the most promising directions in the use of GNPs in current

medical and biological research.

2. GNPs in diagnostics

2.1 Visualization and bioimaging methods

GNPs have been actively used in various visualization and

bioimaging methods to identify chemical and biological

agents.33,34 Historically, electron microscopy (mainly its trans-

mission variant, TEM) has for a long time (starting in 197111)

been the principal method to detect biospecific interactions

with the help of colloidal gold particles (owing to their high

electron density). Although GNPs can intensely scatter and

emit secondary electrons, they have not received equally wide

acceptance in scanning electron microscopy.35 It is no mere

chance that the first three-volume book on the use of colloidal

gold36 was devoted mostly to the application of GNPs in

TEM. A peculiarity of current use of the electron microscopic

technique is the application of high-resolution transmission

electron microscopes and systems for the digital recording and

processing of images.37 The major application of immunoelectron

microscopy in present-day medical and biological research is

the identification of infectious agents and their surface antigens38–40

(Fig. 1a). The techniques often employed for the same purposes

include scanning atomic-force41 (Fig. 1b), scanning electron,42

and fluorescence43 microscopies.

Alongside the use of ‘‘classic’’ colloidal gold with quasi-

spherical particles (nanospheres) as labels for microscopic

studies, the past few years have seen the application of

nonspherical cylindrical particles (nanorods), nanoshells, nano-

cages, nanostars, and other types of particles, referred to by

Scheme 1 Generalized scheme for the biomedical application of

GNPs. Along with basic applications in diagnostics and therapy, this

review briefly discusses the immunological properties of GNPs.


the broad term ‘‘noble-metal plasmon-resonant particles’’25

(Fig. 2). Table 1 illustrates some plasmonic properties and

possible biomedical applications of the gold nanomaterials

shown in Fig. 2. This is by no means a detailed description of

geometrical or optical parameters (size, shape, structure,

absorption and scattering cross sections, their spectra, etc.).

In fact, we only provide typical ranges for the major plasmon

wavelength and its sensitivity to the dielectric environment in

terms of plasmonic shift per refractive index unit (RIU).

Accordingly, Fig. 2 and Table 1 should be considered as an

attempt to show a cross section of the work in this area, rather

than a comprehensive list.

Recently, the popularity of visualization methods employing

GNPs and optical microscopy,55 in particular confocal laser

microscopy, has also been on the rise. Confocal microscopy is

a technique for detecting microobjects with the aid of an

optical system ensuring that light emission is recorded only

when it comes from objects located in the system’s focal point.

This allows one to scan samples according to height and,

ultimately, to create their 3D images by superposition of

scanograms. In this method, the use of GNPs and their

antibody conjugates permits real-time detection of gold penetration

into living (e.g., cancerous) cells at the level of a single particle

and even estimation of their number.56–59

Confocal images can be obtained with, e.g., detection of

fluorescence emission (confocal fluorescence microscopy) or

resonance elastic or two-photon (multiphoton) light scattering

by plasmonic nanoparticles (confocal resonance-scattering or

two-photon luminescence microscopy). These techniques are

based on detecting microobjects with the optical microscope, in

which the luminescence of an object is excited owing to simulta-

neous absorption of two (or more) photons, the energy of each of

which is lower than that needed for fluorescence excitation. The

basic advantage of this method is the increase in contrast through

a strong reduction in the background signal. Specifically, two-

photon luminescence of GNPs enables molecular markers of

cancer to be visualized on or inside cells,60–63 Bacillus spores,64

and the like. Fig. 3a shows an example of combined bioimaging

of cancer cells with the help of adsorption, fluorescence, and

luminescence plasmon resonance labels.

Dark-field microscopy remains one of the most popular GNP-

aided bioimaging methods. It is based on light scattering by

microscopic objects, including those whose sizes are smaller than

the resolution limit for the light microscope (Fig. 3b and c). In

dark-field microscopy, the light entering the objective lens is solely

that scattered by the object at side lighting (similarly to the Tyndall

effect); therefore, the scattering object looks bright against a dark

background.

Compared with fluorescent labels, GNPs have greater potential

to reveal biospecific interactions with the help of dark-field

microscopy,65 because the particle scattering cross section is three

to five orders of magnitude greater than the fluorescence cross

section for a single molecule. This principle was for the first time

employed byMostafa El-Sayed’s group66 for their new method of

simple and reliable diagnosis of cancer with the use of GNPs. The

method is founded on the preferential binding of GNPs con-

jugated with tumor-antigen-specific antibodies to the surface

of cancerous cells, as compared with binding to healthy cells.

With dark-field microscopy, therefore, it is possible to ‘‘map

out’’ a tumor with an accuracy of several cells (Fig. 3b and c).

Subsequently, gold nanorods,67 nanoshells,68 nanostars,69 and

nanocages70 were used for these purposes.

The use of self-assembled monolayers or island films, as well

as nonspherical and/or composite particles, opens up fresh

opportunities for increased sensitivity of detection of bio-

molecular binding on or near the nanostructure surface. The

principle of amplification of a biomolecular binding signal

depends on the strong local electromagnetic fields arising near

nanoparticles with spiky surface sites or in narrow (on the

order of 1 nm or smaller) gaps between two nanoparticles.

This determines the increased plasmon-resonance sensitivity to

the local dielectric environment71 and the high scattering

intensity as compared to the sensitivity and intensity of

equivolume spheres. Therefore, such nanostructures show

considerable promise for use in biomedical diagnostics assisted

by dark-field microscopy.72–74

In dark-field microscopy, GNPs are employed for detection

of microbial cells and their metabolites,75 bioimaging of

cancerous cells76–78 and revelation of receptors on their surface,79,80

study of endocytosis,81 and other purposes. In most biomedical

applications, the effectiveness of conjugate labeling of cells is

assessed qualitatively. One of the few exceptions is the work of

Khanadeev et al.,82 in which a method was suggested for the

quantitative estimation of the effectiveness of GNP labeling of

cells and its use was illustrated with the specific example of

labeling of pig embryo kidney cells with gold nanoshell con-

jugates. Another approach was developed by Fu et al.83

Apart from the just mentioned ways to record biospecific

interactions with the help of diverse versions of optical micro-

scopy and GNPs, the development of other state-of-the-art

detection and bioimaging methods is currently active. These are

referred to collectively as biophotonic methods.31,84 Biophotonics

combines all studies related to the interaction of light with

biological cells and tissues. Existing biophotonic methods include

optical coherence tomography,85–87 X-ray and magnetic

resonance tomography,88,89 photoacoustic microscopy90 and

tomography,91 fluorescence correlation microscopy,92 and other

techniques. These methods also successfully use variously sized

and shaped GNPs. In our opinion, biophotonic methods

employing nonspherical gold particles may hold particular

promise for bioimaging in vivo.34,93,94

Fig. 1 TEM image of a Listeria monocytogenes cell labeled with an

antibody–colloidal gold conjugate (a) and a scanning atomic-force

microscopy image of tobacco mosaic virus labeled with an antibody–

colloidal gold conjugate (b). Adapted from Bunin et al.39 (a) and from

Drygin et al.41 (b) by permission from Springer.


Fig. 2 Various types of plasmon-resonant nanoparticles: 16 nm nanospheres (a);25 gold nanorods (b);44 gold bipyramids (c);45 gold nanorods

surrounded by silver nanoshells (d);25 ‘‘nanorice’’ (gold-coated Fe2O3 nanorods) (e);46 SiO2/Au nanoshells (f)25 (the inset shows a hollow

nanoshell47); nanobowls with bottom cores (g);48 spiky SiO2/Au nanoshells (h)49 (the inset shows a gold nanostar50); gold tetrahedra, octahedra,

and cubooctahedra (i);51,52 gold nanocubes (j);51 silver nanocubes and gold–silver nanocages obtained from them (in the insets) (k);53 and gold

nanocrescents (l).54 Adapted from the data of the cited papers by permission from The Royal Society of Chemistry, Elsevier, IOP Publishing,

Springer, Wiley Interscience, and The American Chemical Society.

Table 1 Properties and possible biomedical applications of plasmonic nanoparticles

Particle Major resonances/nm Plasmonic shift/RIU/nm Possible biomedical applications

Colloidal Au nanospheres (3–100 nm) 510–570 45–90 EM, OI, HA, SA, PPT,a DCAu nanorods (thickness, 10–20 nm; aspect ratio, 2–10) 650–1200 150–290 OI, OA, PPT, HIA,Au bipyramids 650–1100 150–540 OI, BSAu(core)/Ag(shell) NRs 550–1000 — OI, SERSFe2O3 (core)/Au (shell) nanorice 1100–1300 790–810 SERS, BSSiO2(core)/Au(shell) 600–1100 160b

315c OI, SA, PPTHollow Au shell 125 OI, PPTNanobowls 560–1000 — SERSSpiky SiO2 nanoshells and Au nanostars 600–850, —

675–770 240–665 OI, SERSAu polyhedralsd 550–750 — EM, OIAu cubes 550–700 83 EM, OIAu–Ag nanocages 450–1000 410–620 OIA, BS, SERS, PPTAu nanocrescentse 980–2600 240–880 BS

Designations: RIU—refractive index unit, EM—electron microscopy, OI—optical imaging, HA—homogeneous assays, SA—solid phase assays,

PPT—plasmonic photothermal therapy, DC—drug carriers, OA—optoacoustical applications, BS—biosensing, SERS—surface enhanced Raman

scattering. a PPT applications of clusterized Au nanospheres. b Monolayer data. c Suspension data. d Tetrahedra, octahedra, and cubooctahedra.e Nanolithography array.


2.2 Analytical diagnostic methods

2.2.1 Homophase techniques. Beginning in 1980s, colloidal

gold conjugated with recognizing biomacromolecules was com-

ing into use in various analytical methods in clinical diagnostics.

In 1980, Leuvering et al.95 put forward a new immunoanalysis

method that they called sol particle immunoassay (SPIA). A new

SPIA version was advanced by Mirkin et al.96 for the colori-

metric detection of DNA. Both versions (protein and DNA

SPIA) use two principles: (1) The sol color and absorption

spectrum change little when biopolymers are adsorbed on

individual particles.25 (2) When particles move closer to each

other to distances smaller than 0.1 of their diameter, the red color

of the sol changes to purple or gray and the absorption spectrum

broadens and red-shifts.97 This change in the absorption spectrum

can easily be detected spectrophotometrically or visually

(Fig. 4a and b98,99).

The authors employed an optimized variation of SPIA (by

using larger gold particles and monoclonal antibodies to various

antigenic sites) to detect human chorionic gonadotropin.100

Subsequently, the assay was used for the immunoanalysis of

Shistosoma101 and Rubella102 antigens; estimation of immuno-

globulins,103,104 thrombin (by using aptamers),105 and glucose;106

direct detection of cancerous cells;107 detection of Leptospira cells

in urine;108 detection of Alzheimer’s disease markers;109 determi-

nation of protease activity;110 and other purposes. The simulta-

neous use of antibody conjugates of gold nanorods and

nanospheres for the detection of tumor antigens was described

by Liu et al.111 Wang et al.112 provided data on the detection of

hepatitis B virus in blood with gold nanorods conjugated to

specific antibodies.

All SPIA versions proved easy to implement and were highly

sensitive and specific. However, investigators came up against the

fact that antigen–antibody reactions on sol particles do not

necessarily lead to system destabilization (aggregation). Some-

times, despite the obvious complementarity of the pair, changes

in solution color (and, correspondingly, in absorption spectra)

were either absent or slight. Dykman et al.113 presented a model

for the formation of a second protein layer on gold particles

without loss of sol aggregative stability. The spectral changes

arising from biopolymer adsorption on the surface of metallic

particles are comparatively small114 (Fig. 4c and d). However, as

shown by Englebienne,115 even such minor changes in absorption

spectra, resulting from a change in the structure of the biopolymer

layer (specifically, its average refractive index) near the GNP

surface, could be recorded and used for assay in biological

applications.

For increasing the sensitivity of the analytical reaction, new

interaction-recording techniques have been proposed, including

photothermal spectroscopy,116 laser-based double beam absorption

spectroscopy,117 hyper-Rayleigh scattering,118 differential light-

scattering spectroscopy,119 and dynamic light scattering.120 In

addition, two vibrational spectroscopymethods—surface enhanced

infrared absorption spectroscopy121 and surface enhanced Raman

spectroscopy122—have been suggested for use in SPIA.

The ability of gold particles interacting with proteins to

aggregate with a solution color change served as a basis for the

development of a colorimetric method for protein determination.123

A new SPIA version using microplates and an ELISA reader,

with colloidal-gold-conjugated trypsin as a specific agent for

proteins, was devised by Dykman et al.124

Fig. 3 Confocal image of HeLa cells in the presence of GNPs (a). Blue, the nuclei stained with Hoechst 33258; red, the actin cytoskeleton labeled

with Alexa Fluor 488 phalloidin; green, unlabeled GNPs. The image was taken by two-photon microscopy.63 Dark-field microscopy of cancerous

(b) and healthy (c) cells by using GNPs conjugated with antibodies to epidermal growth factor.66 Adapted from the data of the cited papers by

permission from The American Chemical Society.

Fig. 4 Sol particle immunoassay. (a) Scheme for the aggregation of

conjugates as a result of binding by target molecules and (b) the

corresponding changes in the spectra and in sol color. (c) Scheme for

the formation of a secondary layer without conjugate aggregation and

(d) the corresponding differential extinction spectra at 600 nm.

Adapted from the data of Khlebtsov et al.,97 Wu et al.,98 and

Englebienne115 by permission from The American Chemical Society

and The Royal Society of Chemistry.


At present, colorimetric DNA detection includes two strategies:

(1) The use of GNPs conjugated with thiol-modified

ssDNA96,125–130 or aptamers.131 (2) The use of unmodified

GNPs132–135 (Table 2).

The first strategy is based on the aggregation of conjugates

of 10–30 nm GNPs with thiol-modified ssDNA probes after

the addition of target polynucleotides to the system. A cross-

linking variant of the first strategy uses two types of probes

complementary to both terminal target sites. Hybridization of

the targets and probes leads to the formation of GNP aggre-

gates, which is accompanied by a change in the absorption

spectrum of the solution and can readily be detected visually,

photometrically,137 or by dynamic light scattering.130,138

In contrast to the cross-linking aggregation, Maeda and

coworkers139,140 developed a non-cross-linking diagnostic system

involving GNP conjugates of only one type, with 30 or 50 thiol-

modified probes. The aggregation of GNPs occurred at high

ionic strength (1 M NaCl) and only with complementary

probes and targets, whereas noncomplementary targets prevented

aggregation. Contrary to the observations of the Maeda group,139

Baptista et al.129,141 reported enhanced colloidal stability after the

addition of complementary targets to a DNA conjugate solution

even at high ionic strength (2 M NaCl), whereas noncomple-

mentary targets did not prevent aggregation at 2 M NaCl. The

apparent contradictions between these data were explained by

Song et al.142 through the difference in surface functionalization

density.

Consider now the second DNA-sensing strategy, which

utilizes unmodified GNPs. This approach is based on the

observation by Li and Rothberg133 that at high ionic strength,

ssDNA protects unmodified gold nanoparticles from aggregation,

whereas dsDNA does not. This method was employed by

Shawky et al.143 to detect hepatitis C virus. Recently, Xia et al.144

described another variant of the second strategy, which uses

ssDNA, unmodified GNPs, and a cationic polyelectrolyte.

All the above-cited reports on DNA detection used citrate-

stabilized anionic GNPs. He et al.135 described a new version

of unmodified-particle assay employing as-prepared positively

charged CTAB-coated gold nanorods. After mixing nanorods

with the probe and target ssDNA under appropriate hybridi-

zation conditions, the authors observed particle aggregation,

as determined by absorption and scattering spectra. In contrast,

the addition of noncomplementary targets did not cause any spectral

changes. According to He et al.,135 the detection limit (DL) of

a 21-mer ssDNA was about 0.1 nM, whereas Ma et al.145

reported a 0.1 pM DL for an optimized version of He et al.’s

assay135 and 30-mer ssDNA. Quite recently, Pylaev et al.44

applied CTAB-coated positively charged GNPs in combination

with spectroscopic and dynamic scattering methods for the

detection of DNA targets. Thus, there exists a more than

Table 2 Detection of DNA with the use of gold nanoparticles

Particles Probe Target Detection method Detection limit Ref.

ModifiedGNSs(GNSs withchemicallyattachedprobes)

50-HS–(CH2)6–N13–N15-30 ssDNA Cross-linking aggregation,

UV–AS, spot test10 fmol 125, 1997

50-SH–(dT)10-30 Biotinylated PCR

productHybridization of targets with poly-Aand streptavidin followed by a striptest with GNSs

2 fmol (500 copiesof PSA cDNA)

126, 2003

50-N15–(CH2)10–SH-30a;50-SH–(CH2)10–N15-3

0bssDNA Bio-bar-code assay, scanometric

detection of silver-enhanced spots500 zmol (10 copiesin 30 mL)

127, 2004

50-N15–C-30–(CH2)3SH;

HS(CH2)6–50-N18-3

0PCR product Non-cross-linking aggregation in

1 M NaCl, VE, spot test100–250 nM;1–2 nM

128, 2006;140, 2005

50-HS–N16-30 1-round PCR

productStabilization of nanoprobes bytargets in 2M NaCl, VE, UV–AS

300 nM 129, 2006

50-N12–C3–SS-30;

50-SS–C6–N15-30

ssDNA Aggregation, DLS 5 pM 130, 2008

50-SH–(CH2)6–N15-30;

50-Rox–N15–(CH2)3–SH-30ssDNA SERS 10 zM 136, 2010

UnmodifiedGNSs

Rhodamine red–50-N15-30 ssDNA Fluorescence quenching/retaining

with nontarget/target ssDNA0.5 pM 133, 2004

50-N15-30 ssDNA, PCR

productStabilization/aggregation withnontarget/target ssDNA, UV–AS

2 pM 134, 2004

ssDNA, aptamers +conjugated polyelectrolytec

ssDNA, thrombin,cocaine, Hg

Aggregation in the case of nontargetmolecules, VE, UV–AS

1 pM (DNA);10 nM (thrombin);10 mM (cocaine);50 mM (Hg)

144, 2010

50-N21-30, 50-N23-3

0 ssDNA Aggregation of positively chargedGNSs, UV–AS, DLS

10 pM 44, 2011

UnmodifiedGNRs

50-N21-30 ssDNA Aggregation, UV–LS 1.7 nM 135, 2008

50-N30-30 ssDNA Aggregation (optimized), UV–AS 0.1 pM 145, 2010

Designations: Nm—m-bases oligonucleotide; GNSs—gold nanospheres (colloidal gold nanoparticles with a roughly spherical shape); UV–AS—UV–vis

absorption spectroscopy; VE—visual evaluation; UV–LS—UV–vis light scattering spectroscopy; PSA—prostate-specific antigen; DLS—dynamic light

scattering; GNRs—gold nanorods. a Particle probe. b Substrate probe. c Poly [(9,9-bis (60-N,N,N-trimethylammonium)hexyl)fluorene-alt-1,4-phenylene]

bromide.


three-order difference in the reported estimations of the detection

sensitivity of colorimetric methods (0.1–100 pM). Further work

is called for, as the existing aggregation models are inconsistent

with the detection limits of about 0.1–1 pM DNA.44

The above-mentioned SPIA formats have served for the

detection of the DNA of mycobacteria,129,146,147 staphylococci,148

streptococci,149 and chlamydiae150 in clinical samples.

The sensitivity of DNA detection can be increased with

optical methods that utilize plasmonic enhancement of the

local electromagnetic field near particle-cluster hotspots. For

example, Hu et al.136 developed a sensitive DNA biosensor

based on multilayer metal–molecule–metal nanojunctions and

the SERS technique. With regard to an HIV-1 DNA sequence,

the sensor could detect a concentration as low as 10�19 M

(10�23 mol) with the ability of single base mismatch discrimination.

Another way to reach PCR-like sensitivity involves a bio-bar-code

approach combined with a silver-enhanced spot test.127

2.2.2 Dot immunoassay. At the early stages of immuno-

assay development, preference was given to liquid-phase techni-

ques, in which bound antibodies were precipitated or unbound

antigen was removed by adsorption with dextran-coated activated

charcoal. Currently, the most popular techniques are solid-

phase ones (first used for protein radioimmunoassay), because

they permit the analysis to be considerably simplified and the

background signal to be reduced. Themost widespread solid-phase

carriers are polystyrene plates and nitrocellulose membranes.

Membrane immunoassays (dot and blot assays) commonly

employ radioactive isotopes (125I, 14C, 3H) and enzymes

(peroxidase, alkaline phosphatase, etc.) as labels. In 1984, four

independent reports were published151–154 in which colloidal

gold was proposed as a label in a solid-phase immunoassay.

The use of GNP conjugates in solid-phase assays is based on

the fact that the intense red coloration of a gold-containing

marker allows the results of a reaction run on a solid carrier to

be determined visually. Immunogold methods in a dot–blot

assay outperform other techniques (e.g., enzyme immunoassay)

in terms of sensitivity (Table 3), rapidity, and cost.155,156 After

an appropriate immunochemical reaction is run, the sizes of

GNPs can be increased by enhancement with salts of silver157

or gold (autometallography),158 considerably expanding the

application limits of the method. An optimized solid-phase

assay using a densitometry system afforded a dynamic detection

range of 1 mM to 1 pM159 with a detection limit of 100 aM,

which was lowered to 100 zM by silver enhancement. The use

of state-of-the-art instrumental detection methods, such as

photothermal deflection of a probing laser beam, caused by

heating of the local environment near absorbing particles

subjected to light pulses from a heating laser (LISNA assay),160

also ensures a very broad detection range (within three orders of

magnitude, to the extent of several individual particles on a spot).

In specific staining, a membrane with applied material under

study is incubated in a solution of antibodies (or other

biospecific probes) labeled with colloidal gold.18 As probes,

‘‘gold’’ dot or blot assays use immunoglobulins, Fab and scFv

antibody fragments, staphylococcal protein A, lectins, enzymes,

avidin, aptamers, and other probes. Sometimes, several labels

are used simultaneously (e.g., colloidal gold and peroxidase or

alkaline phosphatase) for the detection of multiple antigens on a

membrane.161

Colloidal gold in membrane assays has served for the diagnosis

of parasitic,162–166 viral,167–170 and fungal171,172 diseases, tuber-

culosis,173 melioidosis,174 syphilis,175 brucellosis,176 shigellosis,177

E. coli infections,178 salmonellosis,179 and early pregnancy;180

blood group determination;181 dot–blot hybridization;182 detection

of antibiotics;183 diagnosis of myocardial infarction184 and

hepatitis B;185 and other purposes.

The immunodot assay is one of the simplest methods developed

to analyze membrane-immobilized antigens. In some cases, it

permits quantitative determination of antigens. Most commonly,

the immunodot assay is employed to study soluble antigens.186

However, there have been several reports in which corpuscular

antigens (whole bacterial cells) served as a research object in dot

assays with enzyme labels.187 Bogatyrev et al.188,189 were the first

to perform a dot assay of whole bacterial cells, with the reaction

products being visualized with immunogold markers (‘‘cell–gold

immunoblotting’’). This technique was applied to the serotyping

of nitrogen-fixing soil microorganisms of the genus Azospirillum

and subsequently to the rapid diagnosis of enteric infections.190

Gas et al.191 used a dot assay with GNPs to detect whole cells of

the toxic phytoplankton Alexandrium minutum.

Khlebtsov et al.192 first presented experimental results for the

use of gold nanoshells as biospecific labels in dot assays. Three

types of gold nanoshells were examined that had silica core

diameters of 100, 140, and 180 nm and a gold shell thickness of

about 15 nm (Fig. 5, data for 140/15 nm nanoshells not shown).

The biospecific pair was normal rabbit serum (target molecules)

and sheep antirabbit immunoglobulins (target-recognizing

molecules). When the authors performed a standard protocol

for a nitrocellulose-membrane dot assay, with 15 nm gold

nanospheres as labels, the minimum detectable quantity of

rabbit IgG was 15 ng. Replacing colloidal gold conjugates with

nanoshells increased the assay sensitivity to 0.2 ng for 180/15 nm

gold nanoshells and to 0.4 ng for 100/15 and 140/15 nm

nanoshells. Such noticeable increases in sensitivity with nano-

shells, as against nanospheres, can be explained by the different

optical properties of the particles.193

A very promising analytical approach is the use of colloidal gold

to analyze large arrays of antigens in micromatrices (immuno-

chips).194,195 These enable an analyte to be detected in 384

samples simultaneously at a concentration of 60–70 ng L�1 or,

with account taken of the microlitre amounts of sample and

detecting immunogold marker, with a detection limit of lower

than 1 pg.

The sensitivity of protein dot-analysis can be greatly improved

with a combination of activated glass slides and a CCD

camera196,197 or a flatbed scanner.198 In the case of very dilute

Table 3 Sensitivity limits for immunodot/blot methods implementedon nitrocellulose filters by using various labels (according to ref. 155)

LabelSensitivity limit(pg of protein per fraction)

125I 5Horseradish peroxidase 10Alkaline phosphatase 1Colloidal gold 1Colloidal gold + silver 0.1Fluorescein isothiocyanate 1000


samples, a dot-immunogold filtration assay183 has been suggested.

Finally, the sensitivity of a standard ELISA can be enhanced

up to the single-molecule detection limit199 by using GNPs in

colorimetric analysis of ELISA samples.200,201

2.2.3 Immunochromatographic assays. In 1990, several

companies began to manufacture immunochromatographic

test systems for hand-held diagnostics. Owing to their high

specificity and sensitivity, these strip tests have found a wide

utility in the detection of narcotics, toxins, highly dangerous

infections, and urogenital diseases.202–208 Methods have been

developed for the diagnosis of tuberculosis,209 helicobacteriosis,210

staphylococcal infection,211 hepatitis B,212 prostatitis,213 and early

pregnancy,214 for DNA hybridization;215 for the detection of

pesticides,216 aflatoxin,217 hexoestrol,218 and cephalexin219 in

environmental constituents; and for other purposes.

The immunochromatographic assay is based on eluent

movement along the membrane (lateral diffusion), giving rise

to specific immune complexes at different membrane sites; the

complexes are visualized as colored bands.220 As labels, these

systems use enzymes, colored lattices, but mostly GNPs.221,222

The sample being examined migrates along the test strip at

the cost of capillary forces. If the sample contains the sought-

for substance or immunochemically related compounds, there

occurs a reaction with colloidal-gold-labeled specific antibodies

as the sample passes through the absorber. The reaction is

accompanied by the formation of an antigen–antibody

complex. The colloidal preparation enters into a competitive

binding reaction with the antigen immobilized in the test zone

(as a rule, the detection of low-molecular-weight compounds

employs conjugates of haptens with protein carriers for

immobilization). If the antigen concentration in the sample

exceeds the threshold level, the conjugate does not possess free

valences for interaction in the test zone and the colored band

corresponding to the formation of the complex is not revealed.

When the sample does not contain the sought-for substance or

when the concentration of that substance is lower than the

threshold level, the antigen immobilized in the test zone of the

strip reacts with the antibodies on the surface of colloidal gold,

causing a colored band to develop.

When the liquid front moves on, the gold particles with

immobilized antibodies that have not reacted with the antigen

in the strip test zone bind to antispecies antibodies in the

control zone of the test strip. The appearance of a colored

band in the control zone confirms that the test was done

correctly and that the system’s components are diagnostically

active. Otherwise, the test is invalid. A negative test result—the

appearance of two colored bands (in the test zone and in

the control zone)—indicates that the antigen is absent from

the sample or that its concentration is lower than the threshold

level. A positive test result—the appearance of one colored

band in the control zone—shows that the antigen concen-

tration exceeds the threshold level (Fig. 6).220

Studies have shown that such assay systems are highly

stable, their results are reproducible, and they correlate with

alternative methods. Densitometric characterization of the

dissimilarity degree for detected bands yields values ranging

from 5 to 8%, allowing reliable visual determination of the

analysis results. These assays are very simple and convenient

to use.

Nowadays, GNP-assisted immunochromatographic analysis is

used actively in such fields as the rapid diagnostics of pseu-

dorabies,223 tuberculosis,224 and botulism225 and the detection

of pesticides,226 antibiotics,227 and toxins,228 in biological

liquids and the environment.

In summary, being effective diagnostic tools, rapid tests

allow qualitative and quantitative determination, in a matter

of minutes, of antigens, antibodies, hormones, and other

Fig. 5 Dot assay of normal rabbit serum (1) by using suspensions of

conjugates of 15 nm GNPs and SiO2/Au nanoshells (100 and 180 nm

silica core diameters and 15 nm Au shell) with sheep antirabbit

antibodies. The amount of IgG in the first square of the top row is

1 mg and decreases from left to right in accordance with twofold

dilutions. The lower rows (2) correspond to the application of 10 mg ofbovine serum albumin to each square as a negative control. The

detected analyte quantity is 15 ng for 15 nm GNPs and 0.4 and 0.2 ng

for 100/15 and 180/15 nm nanoshells, respectively. Adapted from

ref. 193 by permission from IOP Publishing.

Fig. 6 Results of an immunochromatographic assay: positive, negative,

and invalid determination because of the absent coloration in the

control zone. Adapted from ref. 219 by permission from The American

Chemical Society.


diagnostically important substances in humans and animals.

Rapid tests are highly sensitive and accurate, as they can

detect more than 100 diseases (including tuberculosis, syphilis,

gonorrhea, chlamydiosis, various types of viral hepatitis, etc.)

and the whole gamut of narcotic substances used, with the

reliability of detection being high. An important advantage of

these tests is their use in diagnostics in vitro, which does not

require a patient’s presence.

However, immunochromatographic test strips are not devoid

of weak points, related to reliability, sensitivity, and cost-effectiveness.

Reliability and sensitivity depend, first, on the quality of monoclonal

antibodies used in a test and, second, on the antigen concen-

tration in a biomaterial. The quality of monoclonal antibodies

depends on the methods of their preparation, purification, and

fixation on a carrier. The antigen concentration depends on

the disease state and the biomaterial quantity. For increasing

the analysis sensitivity, it has been proposed to employ the

silver enhancement procedure229 or gold nanorods as labels.230

In addition, semiquantitative and quantitative instrumental

formats of immunochromatographic analysis have been developed

that use special readers for recording the intensity of a label’s

signal in the test zone of a test strip.220

2.2.4 Plasmonic biosensors. Collective oscillations of

conductive electron plasma in metals are called ‘‘plasmons.’’231

Depending on the boundary conditions, these oscillations can be

categorized into three types:232 bulk plasmons (3D plasma);

propagating surface plasmons (PSP), or surface plasmon polaritons

(2D dielectric/metal interfaces); and localized surface plasmons

(LSP), excited in nanoparticles (Fig. 7).25 Bulk plasmons cannot

be excited by visible light, as their energy is about 10 eV for noble

metals.231

The term ‘‘surface plasmon polaritons’’ designates collective

charge density oscillations at the metal/dielectric interface,

propagating in a waveguide-like fashion along the surface

(Fig. 7b). In the normal direction, the exciting electromagnetic

field is rapidly falling off with distance. At a given photon

energy �ho, the wave vector �ho/c should be increased to ensure

effective coupling for the transformation of incident photons

into propagating surface plasmons.232 Therefore, direct excitation

of surface plasmon polaritons with freely propagating light is not

possible. However, this problem can be solved by two approaches:

the grating couplingmethod and the attenuated reflectionmethod,

which use a lattice waveguide structure or total reflection inside a

prism, respectively.232

In metal nanoparticles, the electron plasma is restricted in

all three dimensions. Accordingly, localized surface plasmons

differ from propagating surface plasmons because of the

different boundary conditions to the Maxwell electromagnetic

equations. In a small metal nanoparticle, the incident optical

field exerts a force on conductive electrons and displaces them

from their equilibrium positions to create uncompensated

charges at the nanoparticle surface (Fig. 7c). As the main

effect producing the restoring force is the polarization of the

particle surface, the excited nanoparticle behaves like a resonance

oscillator possessing a localized plasmon resonance (LSPR).

The principal difference between the propagating and localized

plasmons is that the former can be directly excited by light

waves without any additional coupling set up. On the other

hand, in both LSP and PSP cases, the excited plasmons ‘‘feel’’

the dielectric environment. It is the property that is the basis

for the LSP and the PSP.

The optical response of nanoparticles or their aggregates

(especially ordered ones) depends on particle size, shape, and

composition71,233 interparticle distance,234,235 and the properties

of the particles’ local dielectric environment,236,237 which

enables sensor ‘‘tuning’’ to be controlled. The theory behind

the creation of LSP biosensors and their use in practice have

been considered in ref. 238–254. In general, all sensing strate-

gies are based on the change in the intensity of the LSPR and

its spectral shift caused by a change in the local dielectric

environment owing to biospecific interactions near the particle

surface. A unique local-sensing property of the LSPR is

related to the rapid decay of its local field, which only probes

a nanoscale volume around the particle. In particular, the local

nature of the LSPR allows one to detect single-molecular

interactions near the particle surface by using various single-

particle detecting schemes.254 Fig. 8a illustrates a sensitive

detection of about 18 streptavidin molecules after their binding

to a single biotin-functionalized gold nanorod (74� 33 nm).255

Nusz et al.255 also reported a 1 nM low detection limit (Fig. 8b)

and discussed several ways to approach single-molecular detection.

In recent years, gold and silver nanoparticles and their

composites have served widely as effective optical transducers

of biospecific interactions.256 In particular, the resonant

optical properties of nanometre-sized metallic particles have been

successfully used to develop plasmonic biochips and bio-

sensors belonging to a broad family of sensors, viz. colorimetric,

refractometric, electrochemical, and piezoelectric.220,257,258

Such devices are of much interest in biology (determination

of nucleic acids, proteins, and metabolites), medicine (screening

of drugs, analysis of antibodies and antigens, diagnosis of

Fig. 7 Schematic representation of the bulk (a), propagating surface

(b), and localized surface (c) plasmons.

Fig. 8 (a) Single-gold-nanorod light scattering spectra in water (1),

with biotin, and after binding of about 18 streptavidin molecules.

(b) LSPR peak shift as a function of the streptavidin concentration.

The low detection limit is below 1 nM. Adapted from ref. 255 by

permission from The American Chemical Society.


infections), and chemistry (rapid environmental monitoring,

assays of solutions and disperse systems). Of particular signifi-

cance is the detection of specified nucleic acid (gene) sequences

and the construction of new materials that relies on the formation

of 3D ordered structures during hybridization in solutions of

complementary oligonucleotides attached covalently to metallic

nanoparticles.259

For more than 10 years, biospecific interactions have been

studied in systems in which GNPs are represented as ordered

structures: self-assembled (thin films)260 or as part of polymer

matrices.261 Such structures have been actively employed for

detection of biomolecules and infectious agents, development

of DNA chips, and other purposes. In this case, investigators

directly implement the possibility, in principle, of using the

strong enhancement of the optical signal from the probe

(GNPs conjugated to biospecific macromolecules) resulting

from the strengthening of the exciting local field in the

aggregate formed from gold nanoclusters. At present, biosensors

are built with novel, unique technologies, including monolayer

self-assembly of metallic particles,262 fiber-based LSPR sensing,263

and nanolithography.264 The reported types of LPR studies

of biospecific interactions include biotin–streptavidin, antigen–

antibody, lectin–carbohydrate, toxin–receptor, aptamer–protein,

and DNA hybridization.254 For further information about LPR

sensing, the readers are referred to a recent excellent review by

Mayer and Hafner.254

In experimental work with SPR biosensors, three stages can

be singled out:239 (1) one of the reagents (target-recognizing

molecules) is covalently attached to the sensor surface. (2) The

other reagent (target molecules) is added at a definite concen-

tration to the sensor surface along with the flow of the buffer.

The process of complex formation is then recorded. (3) The

sensor is regenerated (dissociation of the formed complexes).

As this takes place, the following conditions should be met:

� Reagent immobilization on the substrate should not lead

to a critical change in the conformation of native molecules.

� The relatively small difference between the refractive

indices of most biological macromolecules forces one to use

a high local concentration of binding sites on the sensor

surface (10–100 mM).

� The reagent being added should be vigorously agitated to

achieve effective binding to the immobilized molecules. Unbound

reagent should be promptly removed from the sensor surface to

avoid nonspecific sorption.

Apart from that, the sensitivity, stability, and resolution of a

sensor depend directly on the characteristics of the optical

system being used for recording. The most popular sensor

system of this type is BIAcoret.265,266 The measurement

principle in planar, prismatic, or mirror biosensors is similar

to the principle of the method of frustrated total internal

reflection, traditionally employed to measure the thickness and

refractive index of ultrathin organic films on metallic (reflecting)

surfaces.257 The excitation of the plasmon resonance in a

planar gold layer occurs when polarized light is incident on

the surface at a certain angle. The excited electromagnetic

waves and charged density waves propagate along the metal/

dielectric interface. These propagating electromagnetic fields

are localized near the interface because of the exponential

decrease in amplitude perpendicularly to the dielectric with a

typical attenuation length of up to 200 nm (the effect of total

internal reflection, Fig. 9). The reflection coefficient at a

certain angle and light wavelength depends on the dielectric

properties of the thin layer at the interface, which are

ultimately determined by the mass of the captured target

molecules at the sensing surface.

Various types of GNP-aided biosensors have been developed

for the immunodiagnostics of tick-borne encephalitis,268 the

papilloma269 and HIV270 viruses, and Alzheimer’s disease;271,272

the detection of organophosphorus substances and pesticides,273

antibiotics,274 allergens,275 cytokines,276 carbohydrates,277 and

immunoglobulins;278 the detection of tumor279 and bacterial280

cells; the detection of brain cell activity;281 and other purposes.

GNP-based biosensors are used not only in immuno-

assay281–284 but also for the supersensitive detection of nucleotide

sequences.96,259,285 In their pioneering works, Raschke et al.286

and McFarland et al.287 obtained record-high sensitivity of

such sensors in the zeptomolar range, and they showed the

possibility of detecting spectra of resonance scattering from

individual particles as an analytical tool. This opens up the

way to the recording of intermolecular interactions at the level

of individual molecules.288,299 To make the response stronger,

investigators often use avidin–biotin, barnase–barstar, and

other systems.290 In addition, GNPs are applied in other

analytical methods (diverse versions of chromatography, electro-

phoresis, and mass spectrometry).291

SPR and LSPR biosensors were compared in side-by-side

experiments by Yonzon et al.292 for concanavilin A binding to

monosaccharides and by Svedendahl et al.293 for biotin–strep-

tavidin binding. It was found that both techniques demon-

strate similar performance. As the bulk refractive index

sensitivity is known to be higher for SPR, the above similarity

was attributed to the long decay length of propagating plasmons,

as compared to localized ones. The overall comparison of SPR

and LSPR sensors can be found in ref. 251 and 254.

Future development of low-cost SPR and LSPR biomedical

sensors needs increasing the detection sensitivity and creating

substrates that can operate in biological fluids and can be easy

to functionalize with probing molecules, to clean, and to reuse.

Fig. 9 Typical setup for analyte detection in a BIAcoret-type SPR

biosensor. The instrument detects changes in the local refractive index

near a thin gold layer coated by a sensor surface with probing

molecules. SPR is observed as a minimum in the reflected light

intensity at an angle dependent on the mass of captured analyte.

The minimum SPR angle shifts from A to B when the analyte binds to

the sensor surface. The sensogram is a plot of resonance angle versus

time that allows for real-time monitoring of an association/dissociation

cycle. Adapted from ref. 267 by permission from Springer.


3. GNPs in therapy

3.1 Plasmonic photothermal therapy

Photothermal damage to cells is currently one of the most

promising research avenues in the treatment of cancer and

infectious diseases.294 The essence of this phenomenon is as

follows: GNPs have an absorption maximum in the visible or

near-IR region and get very hot when irradiated with corres-

ponding light. If, in this case, they are located inside or around

the target cells (which can be achieved by conjugating gold

particles to antibodies or other molecules), these cells die.

The thermal treatment of cancerous cells has been known in

tumor therapy since the 18th century, employing both local

heating (with microwave, ultrasonic, and radio radiation) and

general hyperthermia (heating to 41–47 1C for 1 h).295 For

local heating to 70 1C, the heating time may be reduced to

3–4 min. Local and general hyperthermia leads to irreversible

damage to the cells, caused by disruption of cell membrane

permeability and protein denaturation. Naturally, the process

also injures healthy tissues, which imposes serious limitations

on the use of this method.

The revolution in thermal cancer therapy is associated with

laser radiation, which enabled controlled and limited injury to

tumor tissues to be achieved.296 Combining laser radiation

with fiber-optic waveguides produced excellent results and was

named interstitial laser hyperthermia.297 The weak point of

laser therapy is its low selectivity, related to the need for high-

powered lasers for effective stimulation of tumor cell death.298

A variant of photothermal therapy was also proposed in which

photothermal agents help to achieve selective heating of the

local environment.299 Selective photothermal therapy relies on

the principle of selective photothermolysis of a biological tissue

containing a chromophore—a natural or artificial substance with

a high coefficient of light absorption.

GNPs were first used in photothermal therapy in 2003.300,301

Subsequently, it was suggested to call this method plasmonic

photothermal therapy (PPTT).295 Pitsillides et al.302 first described

a new technique for selective damage to target cells that is founded

on the use of 20 and 30 nm gold nanospheres irradiated with 20 ns

laser pulses (532 nm) to create local heating. For pulse photo-

thermia in a model experiment, those authors were the first to

employ a sandwich technology for labeling T-lymphocytes with

GNP conjugates.

In a particularly promising method, GNPs have found

application in the photothermal therapy of chemotherapy-resistant

cancers.303 Unlike photosensitizers (see below), GNPs are unique in

being able to keep their optical properties in cells for a long time

under certain conditions. Successive irradiation with several laser

pulses allows control of cell inactivation by a nontraumatic means.

The simultaneous use of the scattering and absorbing properties of

GNPs permits PPTT to be controlled by optical tomography.68

Fig. 10 shows an example of successful therapy of an implanted

tumor in a model experiment with mice.304

The further development of PPTT and its acceptance in

actual clinical practice305 depends on success in solving many

problems, the most important ones being (1) the choice of

nanoparticles with optimal optical properties, (2) the enhancement

of nanoparticle accumulation in tumors and the lowering of

total potential toxicity, and (3) the development of methods

for the delivery of optical radiation to the targets and the

search for alternative radiation sources combining high permeability

with a possibility of heating GNPs.

The first requirement is determined by the concordance of the

spectral position of the absorption plasmon resonance peak with

the spectral window for biological tissues in the 700–900 nm near-

IR region.306 Khlebtsov et al.234 made a resumptive theoretical

analysis of the photothermal effectiveness of GNPs, depending

on their size, shape, structure, and aggregation extent. They

showed that although gold nanospheres themselves are

Fig. 10 Scheme and the results of an experiment on the photothermal destruction of an implanted tumor in a mouse (2–3 weeks after injection of

MDA-MB-435 human cancer cells). Laser irradiation (a, b; 810 nm, 2 W cm�2, 5 min) was performed at 72 h after injection of gold nanorods

functionalized with poly(ethylene glycol) (PEG) (a, c; 20 mg Au per kg) or of buffer (b, d). It can be seen that the tumor continued developing after

particle-free irradiation (control b), as it did after particle or buffer administration without irradiation (controls a and d), and that complete

destruction was obtained only in the experiment (a). Designations: NIR, near-IR region; NRs, nanorods. Adapted in part from ref. 304 by

permission from The American Association for Cancer Research.


ineffective in the near-IR region, aggregates formed from them

can be very effective at sufficiently short interparticle distances

(shorter than 0.1 of the particle diameter). Such clusters can be

formed both on the surface of and inside cells.307 Experimental

data indicating an enhancement of PPTT through clusterization

have been presented in ref. 308–310. Specifically, Huang et al.308

demonstrated that small aggregates composed of 30 nm particles

enable cancerous cells to be destroyed at a radiation power

20-fold lower than that in the particle-free control.

The gold nanoshell and nanorod parameters optimal for PPTT

have also been defined.234,311 By now, there have been quite a few

publications dealing with the application in PPTT of gold nano-

rods;67,312–314 nanoshells;301,315–320 and a comparatively new particle

class, gold–silver nanocages.93,321,322 Experimental data com-

paring the heating efficiencies of nanorods, nanoshells, and

nanocages have been reported in ref. 53, 323 and 324.

Regarding the optimization of particle parameters, one should

be aware of three matters of principle. First, the absorption cross

section is not the sole parameter determining the effectiveness of

PPTT.325 Rapid heating of nanoparticles or aggregates gives rise

to vapor bubbles,326 which can cause cavitation damage to cells

irradiated with visible309 or near-IR327 light. The effectiveness of

vapor bubble formation increases substantially when nanoparticle

aggregates are formed.301,307 Possibly, it is this effect, and not

enhanced absorption, that bears responsibility for greater damage

to cells, with all other factors being the same.325 Finally, particle

irradiation with high-power resonant nanosecond IR pulses can

lead to particle destruction as early as after the first pulse (see, e.g.,

ref. 328 and 329 and references therein to earlier publications). In

a series of recent investigations, Lapotko et al.325,330 (see also

references therein) paid their attention to the fact that the heating

of GNPs and their destruction can sharply reduce the photo-

thermal effectiveness of ‘‘cold’’ particles, which have been tuned to

the laser wavelength. The use of femtosecond pulses offers no

solution to this problem because of the low energy supplied, and

for this reason, it is necessary to exert close control over the

preservation of nanoparticles’ properties for the chosen irradiation

mode. Furthermore, bubble formation strongly depends on the

media as well as on laser intensity, thus making cellular damage

poorly controlled.

We now shift to consider the second question, associated

with targeted nanoparticle delivery to a tumor. This question

has two important aspects: increasing the particle concen-

tration in the target and lowering the side effects caused by

GNP accumulation in other organs, primarily in the liver and

spleen (see below). Usually, there are two delivery strategies.

One is based on the conjugation of GNPs to PEG; the other,

on the conjugation with antibodies developed to specific

marker proteins of tumor cells. PEG acts to enhance the

bioavailability and stability of nanoparticles, ultimately

prolonging the time of their circulation in the blood stream.

Citrate-coated gold nanospheres, CTAB-coated nanorods,

and nanoshells are less stable in saline solutions. When

nanoparticles are conjugated to PEG, their stability is improved

considerably and salt aggregation is prevented.

In vivo PEGylated nanoparticles preferentially accumulate

in tumor tissue owing to the increased permeability of the

tumor vessels331 and are retained in it owing to the decreased

lymph outflow. In addition, PEGylated nanoparticles are less

accessible to the immune system (stealth technologies). This

delivery method is called passive, as distinct from the active

version, which uses antibodies332,333 (Fig. 11). The active

method of delivery is more reliable and effective, employing

antibodies to specific tumor markers, most often to epidermal

growth factor receptor (EGFR) and its varieties (e.g.,

Her2),315,334,335 as well as to tumor necrosis factor (TNF).336

Particular promise is offered by the simultaneous use of

GNP–antibody conjugates for both diagnosis and PPTT

(methods of what is known as theranostics).337–339 In addition

to antibodies, active delivery may also use folic acid, which

serves as a ligand for the numerous folate receptors of tumor

cells,313,340,341 and hormones.342

In the very recent past, the effectiveness of targeted nano-

particle delivery to tumors has again become a subject for

detailed study and discussion.343 In experiments with liposomes

labeled with anti-Her2344 and GNPs labeled with transferrin,345

it was shown that functionalization improves nanoparticle pene-

tration into cells but produces no appreciable increase in

particle accumulation in tumors. Huang et al.343 examined

the biodistribution and localization of gold nanorods labeled

with three types of probing molecules, including (1) an scFv

peptide that recognizes EGFR; (2) an amino terminal frag-

ment peptide that recognizes the urokinase plasminogen acti-

vator receptor; and (3) a cyclic RGD peptide that recognizes

the avb3 integrin receptor. The authors showed that when

injected intravenously, all three ligands induce insignificant

increases in nanoparticle accumulation in cell models and in

tumors but greatly affect extracellular distribution and intra-

cellular localization. They concluded that for PPTT, direct

administration of particles to the tumor can be more effective

than intravenous injection.

The final important question in current PPTT concerns

effective delivery of radiation to a biological target. Because

the absorption of biological tissue chromophores in the visible

region is lower than that in the near-IR region by two orders

of magnitude,294 the use of IR radiation radically decreases the

nontarget heat load and enhances the penetration of radiation

into the tissue interior. Nevertheless, the depth of penetration

usually does not exceed 5–10 mm,84,311 so it is necessary to

look for alternative solutions. One approach consists in using pulse

(nanoseconds) irradiation modes in preference to continuous

ones, which enables irradiation power to be enhanced without

Fig. 11 Scheme for a PPTT employing active delivery of GNPs to

cancer cells.


increasing side effects. Another approach involves fiber-optic

devices for endoscopic or intratissue delivery of radiation.

The strong and weak points of such an approach are evident.

Finally, for hyperthermia, it is possible to use radiations with

greater depths of penetration, e.g., radiofrequency346–349 or

nonthermal air plasma.350

GNPs conjugated to antibiotics and antibodies have also

served as photothermal agents for selective damage to protozoa

and bacteria.351–354 Information on certain issues in the use of

PPTT can be found in several books and reviews.295,355–359 Of

particular note is the most recent comprehensive review by

Kennedy et al.294

In summary, gold nanostructures with a plasmon resonance

offer considerable promise for selective PPTT of cancer and

other diseases. Without doubt, several questions await further

study, including the stability and biocompatibility of nano-

particle bioconjugates, their chemical interaction in physiological

environments, the period of circulation in blood, penetration

into the tumor, interaction with the immune system, and

nanoparticle excretion. We expect that the success of the initial

stages of nanoparticle use for selective PPTT can be effectively

enhanced at the clinical stage, provided that further studies are

made on the optimal procedural parameters. In particular, one

can mention the efforts of J. Feldmann’s group360 related to

thermoplasmonics—a field that is not well understood yet and

that is nowadays is a trend for hyperthermia and delivery upon

light-to-heat conversion.

3.2 Photodynamic therapy with GNPs

The photodynamic method of treating oncological diseases

and certain skin or infectious diseases is based on the application

of light-sensitizing agents called photosensitizers (including

dyes) and, as a rule, of visible light at a specific wavelength.361

Most often, sensitizers are introduced intravenously, but contact

and oral administration is also possible. The substances used

in photodynamic therapy (PDT) can selectively accumulate in

tumors or other target tissues (cells). The affected tissues are

irradiated with laser light at a wavelength corresponding to the

peak of dye absorption. In this case, apart from the usual heat

emission through absorption,21 an essential role is played by

another mechanism, related to the photochemical generation

of singlet oxygen and the formation of highly active radicals,

which induce necrosis and apoptosis in tumor cells. PDT also

disrupts the nutrition of the tumor and leads to its death by

damaging its microvessels. The major shortcoming of PDT is

that the photosensitizer remains in the organism for a long

time, leaving patient tissues highly sensitive to light. On the

other hand, the effectiveness of dye use for selective tissue

heating21 is low because of the small cross section of chromo-

phore absorption.

It is well known362 that metallic nanoparticles are effective

fluorescence quenchers. However, it has been shown recently363,364

that fluorescence intensity can be enhanced by a plasmonic

particle if the molecules are placed at an optimal distance from

the metal. In principle, this idea can improve the effectiveness

of PDT.

Several investigators have proposed methods for the delivery

of drugs as part of polyelectrolyte capsules on GNPs

(which decompose when acted upon by laser radiation and

deliver the drug to the targets365,366) or by using nanoparticles

surrounded by a layer of polymeric nanogel.367,368 Apart from

that, the composition of nanoconjugates includes photoactive

substances,369peptides (e.g., CALLNN), and proteins (e.g.,

transferrin), which facilitate intracellular penetration.345,370,371

Recently, Bardhan et al.372 suggested the use of composite

nanoparticles, including, in addition to gold nanoshells, magnetic

particles, a photodynamic dye, PEG, and antibodies. Finally,

according to the data of Kuo et al.,373,374 nanoparticles

conjugated with photodynamic dyes can demonstrate a synergetic

antimicrobial effect, though the absence of such an effect has

also been reported.375

3.3 GNPs as a therapeutic agent

In addition to being used in diagnostics and cell photothermolysis,

GNPs have been increasingly applied directly for therapeutic

purposes.29 In 1997, Abraham and Himmel376 reported success

in colloidal gold treatment of rheumatoid arthritis in humans.

In 2008, Abraham published a great body of data from a

decade of clinical trials of Aurasols—an oral preparation for

the treatment of severe rheumatoid arthritis.377 Tsai et al.378

described positive results obtained when rats with collagen-

induced arthritis were intraarticularly injected with colloidal

gold. The authors explain the positive effect by an enhance-

ment of antiangiogenic activity resulting from the binding of

GNPs to vascular endothelium growth factor, which brought

about a decrease in macrophage infiltration and in inflammation.

Similar results were obtained by Brown et al.,379,380 who

subcutaneously injected GNPs into rats with collagen- and

pristane-induced arthritis.

A series of papers by a research team from Maryland

University have described the application of a colloidal gold

vector to the delivery of TNF to solid tumors in rats.381–384

When injected intravenously, GNPs conjugated to TNF accu-

mulated rapidly in tumor cells and could not be detected in

cells of the liver, spleen, and other healthy animal organs. The

accumulation of GNPs in the tumor was proven by a record-

able change in its color, because it became bright red-purple

(characteristic of colloidal gold and its aggregates), which

was coincident with the peak of the tumor-specific activity

of TNF (Fig. 12). The colloidal gold–TNF vector was less

toxic and more effective in tumor reduction than was native

TNF, because the maximal antitumor reaction was attained

at lower drug doses. A medicinal preparation based on the

GNP–TNF conjugate, which is called AurImmunet and

intended for intravenous injection, is already in the third stage

of clinical trials.

In experiments in vitro and in vivo, Bhattacharya et al.385

and Mukherjee et al.386 demonstrated the antiangiogenic

properties of GNPs. The particles were found to interact with

heparin-binding glycoproteins, including vascular permeability

factor/vascular endothelial growth factor and basic fibroblast

growth factor. These substances mediate angiogenesis, including

that in tumor tissues, and inhibit tumor activity by changing

the conformation of the molecules.387 Because intense angio-

genesis (the formation of new vessels in organs or tissues) is a

major factor in the pathogenesis of tumor growth, the presence


of antiangiogenic properties in GNPs makes them potentially

promising in oncotherapy.388,389 The same research team has

shown that GNPs enhance the apoptosis of chronic lympho-

cytic leukemia cells resistant to programmed death390 and

inhibit the proliferation of multiple myeloma cells.391

Quite recently, Wang et al.392 revealed that PEG-coated

gold nanorods have an unusual property: they can induce

tumor cell death by accumulating in mitochondria and sub-

sequently damaging them. Unexpectedly, for normal and stem

cells, such an effect is either absent or less pronounced.

4. GNPs as drug carriers

4.1 Targeted delivery of anticancer drugs

One of the most promising aspects of GNP use in medicine,

currently under intense investigation, is targeted drug

delivery.393–395 The most popular objects for targeted delivery

are antitumor preparations396 and antibiotics.

GNPs have been conjugated to a variety of antitumor

substances382–411 listed in Table 4.

Conjugation is done both by simple physical adsorption of

preparations on GNPs and by using alkanethiol linkers. The

action of the conjugates is evaluated both in vitro (primarily),

with tumor cell cultures, and in vivo, with mice bearing implanted

tumors of various nature and localizations (Lewis lung carcinoma,

pancreatic adenocarcinoma, etc.). For creation of a delivery

system, target molecules (e.g., cetuximab) are applied along

with the active substance so as to ensure better anchoring and

penetration of the complex into the target cells.399 It was also

suggested that multimodal delivery systems be used,412 in

which GNPs are loaded with several drugs (both hydrophilic

and hydrophobic) and with auxiliary substances (target mole-

cules, PDT dyes, etc.;413,414 Fig. 13). Most researchers have

noted the high effectiveness of GNP-conjugated antitumor

preparations.415

4.2 Delivery of other substances and genes

Besides antitumor substances, other objects employed to

deliver GNPs are antibiotics and other antibacterial agents.

Gu et al.416 prepared a stable vancomycin–colloidal gold

Fig. 12 Accumulation of the GNP–TNF conjugate in the tumor after

1–5 h. Diseased mice were intravenously injected with 15 mg of the

GNP–TNF vector. The belly images were obtained at the indicated

times and show changes in tumor color within 5 h. The red arrow

shows vector accumulation in the tumor, and the blue arrows mark the

accumulation in the tissues around the tumor. Adapted from ref. 382

by permisson from Wiley Interscience.

Table 4 Antitumor substances conjugated with GNPs

Drugs ParticlesMethods offunctionalization Auxiliary substances Cell lines or animals Ref.

Paclitaxel GNSs, 26 nm Paclitaxel–SH PEG–SH, TNF MC-38; C57/BL6 mice implantedwith B16/F10; melanoma cells

382, 2006

Methotrexate GNSs, 13 nm Physical adsorption — LL2, ML-1, MBT-2, TSGH 8301,TCC-SUP, J82, PC-3, HeLa

397, 2007

Daunorubicin GNSs, 5 nm,16 nm

3-Mercaptopropionicacid as a linker

— K562/ADM 398, 2007

Gemcitabine GNSs, 5 nm Physical adsorption Cetuximab(monoclonalantibodies)

PANC-1, AsPC-1, MIA Paca2 399, 2008

6-Mercaptopurine GNSs, 5 nm Physical adsorption — K-562 400, 2008Dodecylcysteine GNSs,

3–6 nmPhysical adsorption — EAC 401, 2008

5-Fluorouracil GNSs, 2 nm Thiol ligand — MCF-7 402, 2009c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CO2H)]

GNSs, 13 nm Amide linkages DNA HeLa, U2OS, PC3 403, 2009

Cisplatin GNSs, 5 nm PEG–SH as a linker Folic acid, PEG–SH OV-167, OVCAR-5,HUVEC, OSE

404, 2010

Oxaliplatin GNSs, 30 nm PEG–SH as a linker PEG–SH A549, HCT116, HCT15,HT29, RKO

405, 2010

Kahalalide F GNSs, 20,40 nm

Physical adsorption — HeLa 406, 2009

Tamoxifen GNSs, 25 nm PEG–SH as a linker PEG–SH MDA-MB-231, MCF-7, HSC-3 407, 2009Herceptin GNRs, lmax=760 nm 11-Mercaptoundecanoic

acid as a linker— BT474, SKBR3, MCF-7 408, 2009

b-Lapachon GNSs, 25 nm Physical adsorption Cyclodextrin as a drugpocket, anti-EGFR,PEG–SH

MCF-7 409, 2009

Doxorubicin GNSs, 12 nm Physical adsorption Folate-modified PEG KB 410, 2010Prospidin GNSs, 50 nm Physical adsorption — HeLa 411, 2010


complex and showed its effectiveness toward various (including

vancomycin-resistant) enteropathogenic strains of Escherichia

coli, Enterococcus faecium, and Enterococcus faecalis. Similar

results were presented by Rosemary et al.:417 a complex

formed between ciprofloxacin and gold nanoshells had high

antibacterial activity against E. coli. Selvaraj and Alagar418

reported that a colloidal gold conjugate of the antileukemic

drug 5-fluorouracil exhibited noticeable antibacterial and

antifungal activities against Micrococcus luteus, Staphylococcus

aureus, Pseudomonas aeruginosa, E. coli, Aspergillus fumigatus,

and A. niger. Noteworthy is the fact that in all those cases, the

drug–GNP complexes were stable, which could be judged by the

optical spectra of the conjugates.

By contrast, Saha et al.419 (antibiotics: ampicillin, streptomycin,

and kanamycin; bacteria: E. coli,M. luteus, and S. aureus) and

Grace and Pandian420,421 (aminoglycoside antibiotics: genta-

micin, neomycin, and streptomycin; quinolone antibiotics:

ciprofloxacin, gatifloxacin, and norfloxacin; bacteria: E. coli,

M. luteus, S. aureus, and P. aeruginosa) failed to make stable

complexes with GNPs. Nevertheless, those authors showed

that depending on the antibiotic used, the increase in the

activity of an antibiotic–colloidal gold mixture, as compared

to that of the native drug, ranged from 12 to 40%. From these

data, it was concluded that the antibacterial activity of the

antibiotics is enhanced at the cost of GNPs. However, the

question as to the mechanisms involved in such possible

enhancement remained unclarified, which was noted by the

authors themselves. Burygin et al.422 experimentally proved

that free gentamicin and its mixture with GNPs do not

significantly differ in antimicrobial activity in assays on solid

and in liquid nutrient media. They proposed that a necessary

condition for enhancement of antibacterial activity is the

preparation of stable conjugates of nanoparticles coated with

antibiotic molecules. Specifically, Rai et al.423 suggested the

use of the antibiotic cefaclor directly in the synthesis of GNPs.

As a result, they obtained a stable conjugate that had high

antibacterial activity against E. coli and S. aureus.

Other drugs conjugated to GNPs are referred to much more

rarely in the literature. However, some of those works deserve

mention. Nie et al.424 demonstrated high antioxidant activity

of GNPs complexed with tocoferol and suggested potential

applications of the complex. Bowman et al.425 provided data

to show that a conjugate of GNPs with the preparation TAK-

779 exhibits more pronounced activity against HIV than the

native preparation at the cost of the high local concentration.

Joshi et al.426 described a procedure for oral and intranasal

administration of colloidal-gold-conjugated insulin to diabetic

rats, and they reported a significant decrease in blood sugar,

which was comparable with that obtained by subcutaneous

insulin injection, Finally, Chamberland et al.427 reported a

therapeutic effect of the antirheumatism drug etanercept con-

jugated to gold nanorods.

In conclusion, it is necessary to mention gene therapy, which

can be seen as an ideal strategy for the treatment of genetic

and acquired diseases.428 The term ‘‘gene therapy’’ is used in

reference to a medical approach based on the administration,

for therapeutic purposes, of gene constructs to cells and the

organism.429 The desired effect is achieved either as a result of

expression of the introduced gene or through partial or

complete suppression of the function of a damaged or over-

expressing gene. There have also been recent attempts at

correcting the structure and function of an improperly func-

tioning (‘‘sick’’) gene. In such a case, too, GNPs can serve as

an effective means of delivery of genetic material to the

cytoplasm and the cell nucleus.430–433 Furthermore, gold

glyconanoparticles have been proposed for use as a carrier

of tumor-associated antigens in the development of anticancer

vaccines.434

5. Immunological properties of GNPs

5.1 Production of antibodies by using GNPs

Since the 1920s, the immunological properties of colloidal

metals (in particular, gold) have been attracting much research

interest. This interest was mainly due to the physicochemical

(nonspecific) theory of immunity proposed by J. Bordet, who

had postulated that immunogenicity, along with antigenic

specificity, depends predominantly on the physicochemical

properties of antigens, first of all on their colloidal state. In

1929, Zilber and Friese successfully obtained agglutinating

sera to colloidal gold.435 (Curiously, another attempt to prepare

antisera to colloidal gold was made almost 80 years later, in

2006.436) Yet, several authors have shown that the introduction

of a complete antigen together with colloidal metals promotes

the production of antibodies.437 Moreover, some haptens may

cause antibody production when adsorbed to colloidal particles.438

Numerous data pertaining to the influence of colloidal gold on

nonspecific immune responses are given in one of the best early

reviews.439 Specifically, it was noted (with reference to the

1911 work of Gross and O’Connor) that at 2 h after an

Fig. 13 Schemes for different versions of drug delivery systems.

Adapted from ref. 412.


intravenous injection of 5 mL of colloidal gold into rabbits,

there occurs a sizable increase in total leukocytes in 1 mL of

blood (from 10 000 to 19 800) against a slight decline in

mononuclear cells (from 5200 to 4900) and a considerable

increase in polynuclear cells (from 4700 to 14900). On injection

of other colloidal metals, no such phenomena are observed.

Unfortunately, with advances in immunology and denial of

many postulates of Bordet’s theory, the interest in the immuno-

logical properties of colloids decreased. There is no doubt,

though, that the data obtained on the enhancement of immune

response to antigens adsorbed on colloidal particles were utilized

to develop various adjuvants.440

It is known that antibody biosynthesis is induced by substances

possessing sufficiently developed structures (immunogenicity).

These include proteins, polysaccharides, and some synthetic

polymers. However, many biologically active substances (vita-

mins, hormones, antibiotics, narcotics, etc.) have relatively small

molecular masses and, as a rule, do not elicit a pronounced

immune response. In standard methods of antibody preparation

in vivo, this limitation is overcome by chemically attaching such

substances (haptens) to high-molecular-weight carriers (most

often proteins), which makes it possible to obtain specific antisera.

However, such antisera usually contain attendant antibodies

to the carrier’s antigenic structures.441

In 1986, Japanese researchers442 first reported success in

generating antibodies against glutamate by using colloidal gold

particles as a carrier. Subsequently, a number of papers were

published whose authors applied and further developed this

technique to obtain antibodies to the following haptens and

complete antigens: amino acids;443,444 platelet-activating

factor;445,446 quinolinic acid;447 biotin;448 recombinant

peptides;449,450 lysophosphatide acid;451 endostatin;452 the capsid

peptides of the hepatitis C,453 influenza,454 and foot-and-mouth

disease455 viruses; a-amidated peptides;456 actin;457 antibiotics;458

azobenzene;459 Ab-peptide;460 clenbuterol;461 Yersinia surface

antigens;462 transmissible gastroenteritis virus;463 tuberculin;464

and the peptides of the malaria (Plasmodium) surface protein.465

In all these studies, the haptens were directly conjugated to

colloidal gold particles, mixed with complete Freund’s adjuvant

or alum, and used for animal immunization. As a result, high-

titer antisera were obtained that did not need further purification

from contaminant antibodies (Fig. 14).

In 1993, Pow and Crook466 suggested attaching a hapten

(g-aminobutyric acid) to a carrier protein before conjugating

this complex to colloidal gold. This suggestion was supported

in articles devoted to the raising of antibodies to some

peptides,467–471 amino acids,472–475 phenyl-b-D-thioglucoronide,476

and diminazene.477 The antibodies obtained in this way possessed

high specificities to the antigens under study and higher (as Pow

and Crook466 put it, ‘‘extremely high’’) titers—from 1 : 250000

to 1 : 1000000, as compared with the antibodies produced

routinely.

In 1996, Demenev et al.478 showed for the first time the

possibility of using colloidal gold particles included in the

composition of an antiviral vaccine as carriers for the protein

antigen of the tick-borne encephalitis virus capsid. According

to the authors’ data, the offered experimental vaccine had

higher protective properties than its commercial analogs,

despite the fact that the vaccine did not contain adjuvants.

In 2011, Wang et al.479 suggested a new therapeutic vaccine

based on the combination of myelin-associated inhibitors and

GNPs for the treatment of rat medullispinal traumas. Also, for

GNP-assisted antigens, two groups reported new administration

ways: through the skin and mucous coats.480,481

Scheme 2 summarizes the literature data on antigens and

haptens that have been conjugated with GNP carriers and

then used for immunization of animals. The titers of the

antibodies have been increased owing to GNPs.

A considerable number of papers devoted to the use of

GNPs for creating DNA vaccines have emerged as well. The

principle of DNA immunization is as follows: gene constructs

coding for the proteins to which one needs to obtain anti-

bodies are introduced into the organism being treated. If the

gene expression is effective, these proteins serve as antigens for

the development of an immune response. Among the nano-

particles used as DNA carriers, colloidal gold particles are

especially popular with researchers.482,483

5.2 Adjuvant properties of GNPs

Dykman et al.458,464 proposed a technology for the preparation

of antibodies to various antigens, which uses colloidal gold as

a carrier and as an adjuvant. In their method, antigens are

adsorbed directly on the GNP surface, with no cross-linking

reagents added. It was found that animal immunization with

colloidal gold–antigen conjugates (with or without Freund’s

complete adjuvant) yielded specific, high-titer antibodies to a

variety of antigens, with no concomitant antibodies. GNPs

can stimulate antibody synthesis in rabbits, rats, and mice, and

the required amount of antigen is reduced, as compared to

that needed with some conventional adjuvants (Table 5).

It was demonstrated that GNPs used as an antigen carrier

activate the phagocytic activity of macrophages and influence

the functioning of lymphocytes, which apparently may be

responsible for their immunomodulating effect. It was also

revealed that GNPs and their conjugates with low- and high-

molecular weight antigens stimulate the respiratory activity of

cells of the reticuloendothelial system and the activity of

macrophage mitochondrial enzymes.484 This stimulation is

possibly one of the causative factors determining the adjuvant

properties of colloidal gold. That GNPs act as both an

adjuvant and a carrier (i.e., they present haptens to T cells)

Fig. 14 Schematic representation of immunogen localization on the

surface of keyhole limpet hemocyanin (KLH) and GNPs, used as

antigen carriers. (A) Antibodies toward the peptide–KLH conjugate

are produced to the epitopes of both peptide and KLH. (B) Antibodies

toward the peptide–GNP conjugate are produced only to the epitopes

of the peptide. Adapted from ref. 455 by permission from IOP

Publishing.


seems the most interesting aspect of immunogenic properties

exhibited by colloidal gold. In particular, GNPs conjugated to

antigens were found to influence the activation of T cells,

inducing a tenfold increase in proliferation, as compared with

that observed on the addition of the native antigen. This fact

indicates that there is a fundamental possibility of targeted

activation of T cells followed by macrophage activation and

pathogen killing.

However, not a single paper available to us has reported

data on the mechanism of such properties of gold particles. In

our opinion, the reasoning given by Pow and Crook466 on the

preferable macrophage response to corpuscular antigens, as

opposed to soluble ones, is certainly valid. This fact has also

been confirmed by researchers studying the mechanism of

action of DNA vaccines and using gold particles to deliver

genetic material to cells.485,486 The role of Kupffer and Langer-

hans cells in the development of immune response was shown

in those investigations. The influence of dendritic cells on the

development of immune response upon injection of a GNP-

conjugated antigen was discussed by Vallhov et al.487 In

addition, those authors noted that when using nanoparticles

in medical practice, one has to ensure that there are no

lipopolysaccharides on their surface. The interaction of cells

of the immune system with GNPs was examined recently by

Villiers et al.488 and by Zolnik et al.489

The penetration of GNPs complexed with peptides into the

cytoplasm of macrophages, causing their activation, was

shown electron microscopically by Bastus et al.490,491 They

established that after the conjugates have interacted with the

TLR-4 receptors of macrophages, the nanoparticles penetrate

into the cells, this being accompanied by secretion of the

proinflammatory cytokines TNF-a, IL-1b, and IL-6 and by

suppression of macrophage proliferation. Dobrovolskaia and

McNeil492 do not exclude the possibility of another (nonin-

flammatory) way of GNP penetration into macrophages,

namely by means of interaction with scavenger receptors.

Franca et al.493 showed that irrespective of size, GNPs are

internalized by macrophages via multiple routes, including

both phagocytosis and pinocytosis. If either route is blocked,

the particles enter the cells via the other route. GNPs with

hydrodynamic sizes below 100 nm can be phagocytozed.

Phagocytosis of anionic gold colloids by RAW264.7 cells is

mediated by macrophage scavenger receptor A. Ma et al.494

investigated the inhibitory effect of PEG-coated GNPs on NO

production and its molecular mechanism in lipopolysaccharide-

stimulated macrophages. It was found that GNPs inhibited

lipopolysaccharide-induced NO production and inducible nitric

synthase expression in macrophages. Yen et al.495 noted that

on the administration of GNPs, the number of macrophages

decreases and their size increases, this being accompanied by

Scheme 2 Conjugates of antigens and haptens used for immunization of animals.

Table 5 The antibody titers obtained during immunization of rabbits with Yersinia antigen

Preparation 1st immunization 2nd immunization Boosting

Colloidal gold + antigen (1 mg) 1 : 32 1 : 256 1 : 10 240Complete Freund’s adjuvant + antigen (100 mg) 1 : 32 1 : 256 1 : 10 240Physiological saline + antigen (100 mg) 1 : 2 1 : 16 1 : 512


elevated production of IL-1, IL-6, and TNF. The influence of

colloidal gold on immunocompetent cells was examined in vivo

also by Tian et al.496 and by Lou et al.497 They showed that

injection of nonconjugated GNPs into mice enhances the

proliferation of lymphocytes and normal killers, as well as

an increase in IL-2 production.

Recently, many articles have appeared (see above) discussing

problems in GNP use for targeted drug delivery. In our

opinion, this question should be dealt with very carefully,

with account taken of the possibility of production in animals

or humans of antibodies specific to the administered drug

adsorbed on gold particles. We believe that the discovery of

adjuvant properties in GNPs has created favorable conditions

for designing next-generation vaccines.

Alongside GNPs, other nonmetallic nanoparticles also can

serve as antigen carriers. The published examples include

liposomes, proteosomes, microcapsules, fullerenes, carbon

nanotubes, dendrimers, and paramagnetic particles.464 In

our view, especially promising carriers are synthetic and

natural polymeric biodegradable nanomaterials [polymethyl

methacrylate, poly(lactid-co-glycolid acid), chitosan, gelatin].

With the use of such nanoparticles, the immunogenicity of a

loaded substance and its representation in a host immune

system will be changed. A nanoparticle conjugate with an

absorbed or a capsulated antigen can serve as an adjuvant for

the optimization of immune response after vaccination. The

evident advantages of biodegradable nanoparticles are their

utilization in the vaccinated organism, high loading efficiency

for the target substance, enhanced ability to cross various

physiological barriers, and low systemic side effects. In all

likelihood, the immune actions of biodegradable nanoparticles

and GNPs as corpuscular carriers are similar. Keeping in mind

the recent data for the low toxicity of GNPs and their efficient

excretion by the hepatobiliary system, we expect that both

nanoparticle classes—GNPs and biodegradable nanoparticles—will

compete on equal footing for being used in the development of

next–generation vaccines.

6. Conclusions

Owing to the success of the rapid development of technologies

for the chemical synthesis of GNPs during the past decade,

investigators currently have at their disposal an enormous

diversity of available particles with required parameters in

respect of size, shape, structure, and optical properties. More-

over, the question that is now on the agenda is the primary

modeling of a nanoparticle with desired properties and the

subsequent development of a procedure for the synthesis of a

theoretically predicted nanostructure.

From the standpoint of medical applications, much signifi-

cance was held by the development of effective technologies

for the functionalization of GNPs with molecules belonging to

various classes, which ensure nanoparticle stabilization in vivo

and targeted interaction with biological targets. At this juncture,

the best stabilizers are thiolated derivatives of PEG and other

molecules. Specifically, PEG-coated particles can circulate in

the blood stream for longer times and are less susceptible to

the attack of the cellular components of the immune system.

However, the creation of ‘‘stealth’’ conjugates, able to bind to

biological targets in an effective and target-oriented way,

remains an unresolved problem.

It is now generally recognized that GNP conjugates are

excellent labels to use in bioimaging, which can be realized by

various biophotonic technologies, including dark-field resonance

scattering microscopy, confocal laser microscopy, diverse versions

of two-photon scattering and own luminescence of GNPs, optical

coherence and acoustic tomography, and so on.

GNP conjugates have found numerous applications in

analytical research based on both state-of-the-art instrumental

methods (SERS, SEIRA, LISNA, etc.) and simple solid-phase

or homophase techniques (dot assay, immunochromato-

graphy, etc.). The following two examples are illustrative: (1)

by using GNP–antibody conjugates, it is possible to detect a

prostate-specific antigen with a sensitivity that is millionfold

greater than that in the ELISA.498 (2) The sharp dependence

of the system’s color on interparticle distances enables mutant

DNAs to be detected visually in a test known as the North-

western spot test.125 Along with the literature examples of clinical

diagnostics of cancer, Alzheimer’s disease, AIDS, hepatitis,

tuberculosis, diabetes, and other diseases, new diagnostic appli-

cations of GNPs should be expected. Progress in this direction

will be determined by success achieved in improving the sensi-

tivity of analytical tests with retention of the simplicity of

detection. The limitations of homophasic methods with visual

detection are due to the need to use a large number [approxi-

mately 1010 (ref. 21)] of nanoparticles. Even at the minimal ratio

between target molecules and particles (1 : 1), the detection limit

will be ca. 0.01 pM—considerably (millionfold) higher than the

quantity of target molecules to be detected, e.g., in typical biopsy

samples.21 Thus, sensitivity can be improved either by enhancing

the signal (PCR, autometallography, etc.) or by using sensitive

instrumental methods. For instance, single-particle instrumental

methods289 have a single-molecule detection limit that is attain-

able in principle. Specifically, the SERS is a trend technique to

detect very low concentrations of solutes (see, e.g., the recent

review and reports by Liz-Marzan and coworkers499–501 regarding

SERS detection of biological molecules). However, the topical

problem is to create multiplex sensitive tests that do not

require equipment and can be performed by the end user

under nonlaboratory conditions. A prototype of such tests is

Pro Strips,TM which can simultaneously detect five threads:

antrax, ricin toxin, botulinum toxin, Y. pestis (plague), and

staphylococcal enterotoxin B. The physical basis of the new

tests may be associated with the dependence of the plasmon

resonance wavelength on the local dielectric environment or

on the interparticle distance.

GNP-assisted PPTT of cancer, first described in 2003, is

now in the stage of clinical trials.305 The actual clinical success

of this technology will depend on how quickly it will be

possible to solve several topical problems: (1) development

of effective methods for the delivery of radiation to tumors

inside the organism by using fiber-optic technologies or non-

optical heating methods, (2) improvement of the methods of

conjugate delivery to tumors and enhancement of the contrast

and accumulation uniformity, and (3) development of methods

for controlling the process of photothermolysis in situ.

GNP-aided targeted delivery of DNA, antigens, and drugs

is one of the most promising directions in biomedicine.


Specifically, research conducted by Warren Chan’s teams at

Toronto University502 has shown the size-dependent possibility

of delivery of GNPs complexed with herceptin to cancer cells

with much greater effectiveness than that obtained with a pure

preparation. The recent critical reexamination of the PPTT

concept, based on the intravenous targeted delivery of GNPs

conjugated with molecular probes to the molecular receptors

of cancer,343 indicates that there is a pressing need to continue

research in this direction. The discovery of adjuvant properties

in GNPs has created favorable conditions for the development

of next-generation vaccines.

Finally, there is need to continue and expand work in the

area of GNP biodistribution and toxicity. Although the past

five years have seen significant research activity22,32,503–505

several important aspects remain to be studied. It is commonly

recognized now that the final conclusions concerning biodistri-

bution and toxicity can be affected by many factors, including

particle size and shape, functionalization methods, animal

types, particle administration doses and methods, and so on.

For instance, Alkilany and Murphy504 addressed the important

question of the origin of toxicity of GNP suspensions. The

well-known example of CTAB-coated gold nanorods clearly

shows that one should discriminate between the toxicity of

GNPs themselves and that of a dispersion medium in which

the GNPs are dispersed (‘‘supernatant control’’).504

Although bulk gold is known to be chemically inert, the

nanotoxicity of GNPs with sizes smaller than 3–5 nm may be

different from that of both organogold complexes and larger

GNPs. In particular, GNPs with diameters of 1–2 nm have

potentially higher toxicity because of the possibility of irreversible

binding to cell biopolymers. For example, Tsoli et al.506 and

Pan et al.507 reported that 1.4 nmGNPs were toxic to various cell

lines, whereas no toxicity was found for 15 nm GNPs. Yet,

numerous experiments with cell cultures did not reveal noticeable

toxicity of 3 to 100 nm colloidal particles, provided that the

limiting dose did not exceed a value of approximately 1012

particles per mL.

The available data on the toxicity of GNPs in vivo are rare

and somewhat controversial. One can only speculate that

noticeable toxicity does not occur during short-period

(approximately weeklong) administration of GNPs at a daily dose

not greater than 0.5 mg kg�1. Perhaps this estimate may sound

too strong if we compare it with the data reported by von

Maltzahn et al.304 Those authors used gold nanorods at a total

dose of 20 mg kg�1 and did not observe any toxicity in either

tumor-bearing or tumor-free mice.

Whereas there are more than 40 reports on GNP toxicity

in vitro, those studies cannot be used as good predictors of

possible toxicity in vivo. For example, Chen et al.454 observed

that the toxicity of the same GNPs was contradictory for

models in vivo and in vitro.

As pointed out by Alkilany and Murphy,504 one should

introduce standards for GNP characterization not only prior

to administration to a biological model but also after mixing

with biological media. Otherwise, there may be a change in

GNP charge, desorption of stabilizing molecules, and, eventually,

GNP aggregation.

It should be emphasized that GNPs are not biodegradable.

Therefore, the biodistribution and excretion kinetics have to

be studied comprehensively for different animal models. The

organs of the reticuloendothelial system are the basic primary

target for the accumulation of GNPs within the size range

10–100 nm, and the uniformity of biodistribution increases

with a decrease in particle size. As the excretion of accumu-

lated particles from the liver and spleen can take up to

3–4 months, the question as to the injected doses and possible

inflammation processes is still of great importance. Bioaccu-

mulated GNPs can interfere with different diagnostic techniques,

or accumulated GNPs can exhibit catalytic properties. All these

concerns, together with potential toxicity, are big limitations of

GNPs on a successfully clinical translation. Nowadays, despite

the huge numbers of studies regarding the synthesis and functio-

nalization of GNPs (different shapes, coatings, sizes, charges,

etc.), there are very few nanomaterial-based pharmaceuticals on

the market (see Rivera Gil et al.508).

In summary, there should be a coordinated research program

to establish correlations between the particle parameters (size,

shape, and functionalization with various molecular probes),

the experimental parameters (model; doses; method and time

schedule of administration; observation time; organs, cells and

subcellular structures examined; etc.), and the observed biological

effects.

Acronyms

CTAB cetyltrimethylammonium bromide

EGFR epidermal growth factor receptor

GNP(s) gold nanoparticle(s)

PEG poly(ethylene glycol)

PDT photodynamic therapy

PPTT plasmonic photothermal therapy

SPIA sol particle immunoassay

SPR surface plasmon resonance

TEM transmission electron microscopy

TNF tumor necrosis factor

Acknowledgements

We acknowledge support from the Russian Foundation for Basic

Research and from the Presidium of the Russian Academy of

Sciences within the program ‘‘Basic Sciences forMedicine.’’We also

acknowledge support by grants from theMinistry of Education and

Science of the Russian Federation (no. MK-1057.2011.2, 2.1.1/

2950, 14.740.11.260, and 02.740.11.0484) and by a grant from the

Government of the Russian Federation designed to support

research projects supervised by leading scientists at Russian institu-

tions of higher education. We thank D. N. Tychinin (IBPPM

RAS) for his help in preparation of the manuscript.

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