Tải bản đầy đủ - 0 (trang)
Chapter 6. Chemical Preparation of Gold Nanoparticles on Surfaces Catherine Louis

Chapter 6. Chemical Preparation of Gold Nanoparticles on Surfaces Catherine Louis

Tải bản đầy đủ - 0trang

November 15, 2012


9in x 6in

Gold Nanoparticles for Physics, Biology and Chemistry

C. Villiers

Gold can also be used as an electron carrier: the idea being to transport the

electrons generated from Redox enzymatic reactions to electrodes via gold

nanoparticles; measurement of the resulting current gives information on the

presence and amount of the enzyme substrate. In these devices, the enzyme

is directly conjugated to the surface of the particles which are fixed to the

electrode of the chip. The enzyme binds preferentially to the particle and

not to the electrode surface because the available surface is larger allowing

a greater amount of enzyme to be accommodated; furthermore, it seems

that the shape of the small particles facilitates a close contact between the

enzyme and the conductive surface and thus the electronic exchanges.

11.3.2 Gold nanoparticles as a cellular tracker

The identification of specific cells in a complex mixture or the localization of

intracellular compartments such as endosomes, lysosomes or mitochondria

in cells is a permanent challenge in many biological fields. The use of electron microscopy is probably one of the best-known illustrations of intracellular protein localization based on AuNPs. AuNPs are providing a powerful

contrast enhancement for transmission electron microscopy (TEM). Antibodies are covalently attached to AuNPs so that they can be easily traced.

The interaction of these antibodies with cell proteins is investigated: the

antibody can target a protein localized in a cellular or intracellular structure

and due to the presence of the AuNP these events can be monitored by

TEM. In the absence of such NP labelling, it is very difficult to characterize

cellular compartments such as endosomes, lysosomes, Golgi apparatus, etc.

By electron microscopy due either to very low contrast or to the absence

of specific morphological characteristics. Cell or tissue slices are fixed on a

support (cover slide or culture dish) and then permeabilized allowing particles to pass through the cellular membrane in order to interact specifically

with the antigen against which the antibody is directed.

Gold nanoparticles provide an excellent contrast for observation using

a transmission electron microscope, whereas it is much more difficult to

observe the same nanoparticles with an optical microscopy due to the resolution limit which is theoretically a few hundredths of a nm. Recently,

however, Klein et al. showed that the detection of particles of 60 nm is reliable, succeeding in counting such nanoparticles with a confocal microscope.



November 15, 2012


9in x 6in

Gold Nanoparticles for Physics, Biology and Chemistry

Gold Nanoparticles for Sensors and Drug Delivery

Furthermore, AuNPs observed in back-scattering mode induce a phase shift

in the light signal, due to the cellular environment which thereby facilitates

their observation.64 Of course, cell labelling is also possible without permeabilization; in these conditions, nanoparticles do not enter the cells and

can only interact with components localized on the outer face of the cellular

membrane. The optical limitations mentioned above remain after permeabilization but this method has the advantage of monitoring proteins or structures in real-time since the cellular integrity is not altered, allowing dynamic

analysis. For example, if an AuNP is attached to an antibody that targets a

membrane protein, it will be fixed with high affinity to this membrane and

will follow its movements in the cell. Therefore, if the nanoparticle is found

inside the cell after incubation, it means that the protein has been internalized by the cell and in these conditions; a precise localization may be

performed using TEM.65 Dynamic analysis of the intracellular movements

of the protein can be simply carried out by modifying the incubation time.

In theory, these techniques combined with the small size of AuNPs, make

it possible to monitor all the endocytic processes: phagocytosis, pinocytosis, receptor mediated endocytosis etc. But attention must be paid to the

necessary absence of impact of this tracker on endocytosis: nanoparticles

should neither perturb the process nor bind to proteins which could influence their internalization. Indeed, it is worth noting that despite their small

size, nanoparticles could interfere with the internalization of receptors.66

AuNPs may be a good alternative to fluorochromes for cellular imaging

by confocal microscopy: fluorescent probes are often used because of their

ease of use, but are very unstable due to photobleaching, whereas AuNPs

are very stable.

Nanoparticle visualization is also possible by photo-acoustic imaging:

when the distance between particles decreases, the wavelength of the localized plasmon resonance shifts to a higher value. When the beads are equally

distributed at the cell surface, the distance between the particles is too great

to induce interaction between the surface plasmons: in these conditions,

when they are illuminated at a wavelength above the plasmon resonance

frequency, no signal is recorded. Whereas, when antibody binding occurs

on proteins which are aggregated at the cell surface, the gold nanoparticles

are close to each other and their resulting interaction leads to light absorption

and a photo-acoustic signal can be measured. Consequently, the gathering



November 15, 2012


9in x 6in

Gold Nanoparticles for Physics, Biology and Chemistry

C. Villiers

of receptors (capping) at the cell surface, which may be the result of the

binding of their ligands, can be monitored by the variation of photo-acoustic


11.4 AuNPs Used for Treatment

As indicated in the first part of this chapter, many molecules can be bound

to AuNPs, and this feature is widely exploited for medical treatment. In this

case,AuNPs can be considered as a vector for a precise delivery of molecules

at the required place. If immunization is the goal, antigens must be targeted

to areas of high immunological activity; if tumour treatment is sought, active

molecules must exclusively concentrate at the level of malignant cells in

order first to facilitate their eradication and second to reduce adverse effects

which may result from the presence of these very harmful molecules near

non malignant cells. The delivery of such molecules can be realised using

gold nanoparticles as carriers. For this purpose, they are fixed with a stable

link to the particles that drive them into the area of interest and, at this stage,

the molecule can be released by breaking the bond that links them to the

particle either by proteolysis or by hydrolysis. These different possibilities

are discussed in the following paragraphs.

11.4.1 Gene gun

The principle of the gene gun is to send gold nanoparticles with sufficient

kinetic energy so that they penetrate inside the cell membrane, and carry

into the cells the molecules fixed on their surface or on the shell. The main

interest of this technique is to deliver molecules into the cells without receptor limitation and whatever the nature of the cells. Particle acceleration is

obtained using various devices such as bullets, gas cartridges or electric

discharges.67 The gene gun is used for the introduction of coding DNA

into the cellular cytoplasm. One of the most important applications of the

gene gun is the introduction of plasmid in plants, the use of such material being particularly suitable for crossing highly resistant cellulosic walls

found around plant cells.68

The gene gun is also used to inject DNA into animal cells.69 Indeed, skin

is a highly immune-reactive tissue containing abundant antigen-presenting



November 15, 2012


9in x 6in

Gold Nanoparticles for Physics, Biology and Chemistry

Gold Nanoparticles for Sensors and Drug Delivery

cells70 and, consequently, it provides a favourable site for DNA immunization. Such immunization was obtained after injection of plasmide coding

for the antigen alone or together with immune activators.71

11.4.2 AuNPs for targeting cells

The purpose of cell targeting is to precisely deliver a molecule to an organ

or a group of cells and simultaneously avoid all aspecific interactions with

non-targeted cells or tissues. Indeed, all cells can ingest nanoparticles but

the amount of material found inside the cells may vary dramatically according to the cell type and the internalization pathway involved: pinocytosis,

receptor mediated endocytosis, phagocytosis, etc. In the case of particle

targeting, the interaction with the cells must be as specific as possible.

The amount of material internalized by pinocytosis and phagocytosis is

directly related to two factors: the concentration of the particles and their

stealth, i.e. their capacity to be invisible in particular for immune cells. The

biocompatibility of the particles has been previously documented in this

chapter: when AuNPs are invisible to the immune system, the phagocytosis

by macrophages or dendritic cells is reduced, and the particles remain in the

circulating fluids allowing their routing to and interaction with the targeted

cells. Specific interaction with targeted cells is much more important than

aspecific endocytosis. Specific binding of AuNPs to membrane proteins

expressed by the targeted cells ensures a great specificity but such interaction does not systematically lead to the internalisation of the particles.

For example, some membrane proteins induce an intracellular signalization after binding of their ligand but no internalization as is the case for

toll-like receptors (the proteins in charge of the detection of foreign materials; viral ARN non-human glycoproteins, etc). Whereas other proteins

are internalized upon binding of their ligand, as is the case for transferring

receptors for example. There is no possibility of inducing the internalization

of a protein that normally does not have this capacity. However, according

to the nature of tumour treatment performed using AuNPs, internalization

is not always required: drug delivery may require internalization whereas

hyperthermia is effective without internalization. The challenge is to target

AuNPs to receptors present only on tumour cells, but the main problem is

that only a few proteins are expressed exclusively by these cells and most of



November 15, 2012


9in x 6in

Gold Nanoparticles for Physics, Biology and Chemistry

C. Villiers

Table 11.1. Cell surface proteins which may be used as docking receptors for

particle targeting.∗

Tumour or tissue

Pancreatic adenocarcinoma


lymphatic vessels



Breast or head and neck


Endothelium (liver cancer)

Endithelium (colon


Endothelium (lung


Endotheliums (solid

tumour) (pancreatic


Endothelium (solid tumour)

Endothelium (solid tumour)

Endothelium (solid tumour)

Endothelium (solid tumour)

Endothelium (solid tumour)

Targeted protein/receptor

Cell surface plectin-1

P32 or gC1q receptor


Kelly et al.72

Fogal et al.73

Aminopeptidase N (CD13) Pastorino et al.74


Lewis et al.75


Platt et al.76



Cheng et al.77

Kondo et al.78


Gosk et al. 79

αvβ3 and αVβ5

Benezra et al.80

Liu et al.81




Annexin I


Nilsson et al.82

Christian et al.83

Carson-Walter et al.84

Oh et al.85

Christian et al.86

∗ The list of cell proteins which may be used as potential docking for AuNPs is not exhaustive and is

adapted from the reviews of Ruoslahti et al.87

these are not accessible from the outside of the cells without permeabilization. Another possibility is to target proteins present on the endothelial cells

forming the neovascularization of the tumour as many of them have already

been characterized, some of the different possibilities are summarized in

Table 11.1.

Some proteins, such as aminopetidase N88 or endosialin83 for example,

were found to be over-expressed by tumours. In these cases, antibodies

specific to the over-expressed proteins are attached to the nanoparticles,

allowing them to target tumours.89 Another possibility is to drive the AuNPs



Tài liệu bạn tìm kiếm đã sẵn sàng tải về

Chapter 6. Chemical Preparation of Gold Nanoparticles on Surfaces Catherine Louis

Tải bản đầy đủ ngay(0 tr)