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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5368_Библиотеки_им_академика_М_И_Перельмана
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192 R. D. Mellor
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Fig. 9.3 LaMer model of metal nanoparticle formation. This model is fundamental to understanding the formation of metal nanoparticles, including AuNPs, in a solution. It elucidates the key stages
of nucleation and growth that occur during chemical synthesis
(Fumitaka et al. 2001; Lévy et al. 2021; Amendola et al. 2006), thermal decomposition (Cho et al. 2016; Bakrania et al. 2009), and mechanical milling (Rak et al.
2014). However, chemical synthesis methods stand out as the most widely employed
due to their ability to yield a vast array of physicochemical properties with a high
degree of specificity.
Most chemical synthesis methods for AuNPs generally follow a common
sequence of steps, as illustrated in Fig. 9.3, which depicts the LaMer model of
metal nanoparticle formation:
3+
1. Reduction of Au
: This step involves the reduction of Au
sourced from a gold salt like HAuCl
, to elemental Au0 . This reduction is rapid
4
3+
ions, typically
and continues until the concentration of gold atoms in solution reaches a state of
supersaturation.
2. Nucleation: Gold atoms, now in their elemental form, nucleate into small clusters.
The number of nucleation sites plays a critical role in determining the number
concentration of AuNPs. More nucleation events lead to smaller particles, while
fewer events result in larger particles.
3. Growth: Subsequent growth occurs through the coalescence of gold clusters and
the diffusion of remaining soluble gold atoms onto the surface of existing gold
agglomerates.
In the following sections, we will delve into four of the most commonly
employed methods for synthesizing ultrasmall AuNPs: the Turkevich/Frens method,
reduction by sodium borohydride, the Brust–Schiffrin approach, and seeded growth.
To aid comprehension, Table
names.
9.1 provides explanations for abbreviated reagent
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Table 9.1 Abbreviations for reagents used in AuNP synthesis
Abbreviation Meaning
BDAC Benzyldimethylhexadecylammonium chloride
CTAB Cetyltrimethylammonium bromide
CTAC Cetyltrimethylammonium chloride
GSH Glutathione
HAuCl
4
HQL 8-Hydroxyquinoline
MPA Mercaptopropionic acid
NaBH
4
NaI Sodium iodide
ODA Octadecylamine
PVP Polyvinylpyrrolidone
TOAB Tetraoctylammonium bromide
Chloroauric acid
Sodium borohydride
Fig. 9.4 Methods of AuNP synthesis
Turkevich/Frens Synthesis (Fig. 9.4a), represents a classical method for produc-
ing AuNPs. It was initially developed by Turkevich et al. in 1951 (Turkevich et al.
1951), producing AuNPs with sizes ranging from 15 to 24 nm. The method was later
refined by Frens in 1973 (Frens 1973
Citrate serves as both a reducing and capping agent in this synthesis, although due to
citrate’s limited reducing ability at room temperature, the reaction is typically carried
out at elevated temperatures, usually at or near boiling point. The size of the resulting
AuNPs is predominantly controlled by the ratio of citrate to gold, with more citrate
leading to faster nucleation and smaller particles. Other factors influencing par ticle
size and distribution include pH (Tyagi et al. 2016), temperature (Tran et al.
and the order of reagent addition (Ojea-Jiménez et al. 2011). While it is possible to
synthesize AuNPs with an average diameter of 4 nm by modifying the Turkevich
method (Yang et al. 2003), it is more common for particles to exceed 10 nm in
diameter.
), extending the size range to 16–147 nm.
2016),
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Sodium Borohydride (Fig. 9.4b) serves as a strong reducing agent in AuNP
synthesis enabling reactions to be conducted at room temperature and facilitating
the formation of smaller AuNPs, often below 5 nm. Similar to the Turkevich method,
citrate can be included; however, when NaBH4 is the reducing agent, citrate
functions solely as a capping agent (Al-Johani et al.
2017). Alternatively, other
hydrophilic capping agents such as alginate (Beishenaliev et al. 2021) or chitosan
(Abrica-González et al.
2019) can replace citrate. This synthesis can also take place
in non-polar solvents like chloroform, utilizing hydrophobic capping agents like
CTAB (Fan and Jiang
1997) and ODA (Mellor et al. 2021; Chen et al. 2000). In
some cases, capping agents may be entirely omitted to produce ‘bare’ AuNPs
(Shellaiah et al.
2016).
Brust–Schiffrin Synthesis (Fig. 9.4c) represents a two-phase approach for pro-
ducing alkanethiol-capped AuNPs that are soluble in hydrophobic solvents. Typically, TOAB is employed to transfer AuCl
phase, often toluene. NaBH
is then used to reduce the gold salt in the presence of a
4
-
from the aqueous phase to an organic
4
capping agent, traditionally dodecanethiol (Brust et al. 1994). However, alternative
alkanethiols such as pentanethiol (Briñas et al. 2013) or hexanethiol (Dichello et al.
2017), surfactants like CTAB or CTAC (Mapanao et al. 2021), or even ionizable
molecules such as MPA (Kuroda et al. 2012) can replace dodecanethiol.
Seeded Growth Synthesis (Fig. 9.4d) involves the initial production of Au
clusters, either using NaBH4 or Turkevich/Frens, to be used as seeds. These seeds
are then introduced into a growth solution, where the particle number concentration
can be precisely controlled by varying the number of nuclei introduced. The final
particle size is regulated by the gold concentration in the growth solution. Seeded
growth is not particularly well suited to the formation of ultrasmall AuNPs, it is
commonly used for producing particles over a wide size range (Bastús et al. 2011;
Ziegler and Eychmüller 2011) and can also yield a variety of shapes by employing
different shape-directing agents, such as CTAC for spheres (Zheng et al. 2014) and
cubes (Oh et al.
CTAC/HQL for bipyramids/javelins (Chateau et al.
and Vo-Dinh
2012), CTAC/NaI for triangles (Bhattarai et al. 2017), CTAB/
2015), PVP for stars (Khoury
2008), and BDAC/CTAB for rods (Nikoobakht and El-Sayed 2003).
The methods outlined in the previous sectio ns are summarized in Table 9.2.
0
9.5 Approaches to Clustering
Many approaches have been reported for forming ultrasmall-in-nano constructs;
while they all follow what is essentially the same schematic, depicted in Fig. 9.1,
and have some common characteristics, they differ in the approach used to cluster
the ultrasmalls and in the application of the nano construct. Some of these
approaches are outlined in the following sections and summarized in Table 9.3.
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)
Fan and Jiang (1997)
Mellor et al. (2021)
Chen et al. (2000)
3
3
3
O Turkevich et al. (1951)
O Frens (1973)
O Yang et al. (2003)
O Al-Johani et al. (2017)
O Beishenaliev et al. (2021)
2
2
2
O Abrica-González et al. (2019)
2
2
2
O Shellaiah et al. (2016)
O Piella et al. (2016)
O Cortez-Lemus et al. (2015)
2
2
O Xia et al. (2016)
2
2
Kuroda et al. (2012)
O
O Bastús et al. (2011)
O Ziegler and Eychmüller (2011)
2
H
O Zheng et al. (2014)
2
2
2
(continued)
Method of synthesis Size range (nm) Shape Surface chemistry Polarity Solvent Ref.
Turkevich 15–24 Sphere Citrate Hydrophilic H
Table 9.2 Methods of AuNP synthesis. Focusing mainly on ultrasmall spheres, with several prominent examples of methods for synthesizing larger or
non-spherical particles
Frens 16–147 Sphere Citrate Hydrophilic H
3.6–13 Sphere Citrate/tannic acid Hydrophilic H
3.5–15 Sphere PDEAEM Hydrophilic H
2–330 Sphere Citrate Hydrophilic H
Sodium borohydride 3–5 Sphere Citrate Hydrophilic H
Turkevich/Frens 4 Sphere Citrate Hydrophilic H
Sodium borohydride 3.5–14 Sphere Chitosan Hydrophilic H
Sodium borohydride 3.3–12 Sphere Alginate Hydrophilic H
Sodium borohydride 3–14 Sphere CTAB Hydrophobic CHCl
Sodium borohydride 4.7 Sphere ODA Hydrophobic CHCl
Sodium borohydride 3 Sphere ODA Hydrophobic CHCl
Sodium borohydride 3–5 Sphere Bare Hydrophilic H
Turkevich/Frens—
modified
Turkevich/Frens—
modified
Turkevich/Frens—
modified
Brust–Schiffrin 1–3 Sphere Dodecanethiol Hydrophobic Toluene Brust et al. (1994
Brust–Schiffrin 5 Sphere Pentanethiol Hydrophobic Toluene Briñas et al. (2013)
Brust–Schiffrin 3 Sphere MPA Variable Toluene/
Brust–Schiffrin 10 Sphere CTAB/CTAC Hydrophobic Toluene Praharaj et al. (2007)
Brust–Schiffrin 2 Sphere Hexanethiol Hydrophobic Toluene Dichello et al. (2017)
Seeded growth 8.4–180.5 Sphere Citrate Hydrophilic H
Seeded growth 15–300 Sphere Citrate Hydrophilic H
Seeded growth 5–150 Sphere CTAC Hydrophilic H
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2014)
al. (
O Bhattarai et al. (2017)
O Oh et al. (2012)
O Chateau et al. (2015)
2
2
2
O Nikoobakht and El-Sayed (2003)
O Liu et al. (2013c)
O Yu et al. (2017)
2
O Xi and Haes (2019)
2
2
2
O Liu et al. (2013d)
2
Hydrophilic H
Method of synthesis Size range (nm) Shape Surface chemistry Polarity Solvent Ref.
Seeded growth 60 Triangle CTAC/NaI Hydrophilic H
Table 9.2 (continued)
CTAB/CTAC/
HQL
javelin
Seeded growth 76 Cube CTAC Hydrophilic H
Seeded growth 40–300 Bipyramid/
Seeded growth 10–100 Rod BDAC/CTAB Hydrophilic H
Other—GSH reduction 2.5 Sphere GSH Hydrophilic H
Seeded growth 45–116 Star PVP Hydrophilic DMF Khoury and Vo-Dinh (2008)
Other—GSH reduction 2.3 Sphere GSH/cysteamine Hydrophilic H
Other—HEPES reduction 23 Star HEPES Hydrophilic H
Other—TBAB reduction 2–7 Sphere Oleylamine Hydrophobic DCM Yang et al. (2018)
Other—TBAB reduction 3–10 Sphere Oleylamine Hydrophobic Hexane Wu et al. (2013)
Other—mechanochemical 1–4 Sphere Various Various None Rak et
Other—thermal reduction 2 Sphere PEG Hydrophilic H
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Table 9.3 Methods of generating ultrasmall-in-nano constructs
Ultrasmall
(surface
chemistry, and
size)
ODA
4.67 ± 1.74 nm
NA
2–8nm
Tannic acid
and/or citrate
3, 5, and 13 nm
PSS
~3 nm (varies
with article)
AcetalDextranpMBA-AuNPs
2.1 ± 0.5 nm
11-MUA or
GSH
2–5nm
GSH
~2 nm
Citrate/lysine
4.1 ± 0.8 nm
NA
6.1 ± 1.8 nm
Nr not reported
Nano (clustering
principle, and size)
Crosslinking with
EGBMA
254–278 nm
Coating of DSPC:
cholesterol liposomes
100–120 nm
Single-stranded
DNA-coated
AuNPs + complementary linker
50–150 nm
Ionic interactions
with PL
~100 nm (varies
with article)
Encapsulation in
PEG-PCL
111.1 ± 38 nm
Encapsulation in
PCPP
40–500 nm
Encapsulation in
PAA HCl
120 nm
Interaction with
PLA(2K)-PEG
(10K)-PLA(2K)
83.0 ± 4.6 nm
Self-assembly with
PCL-PHEMA and
PMEO
MA
2
300 nm
SPR Reversible Ref.
710 nm Yes Mellor et al. (2021)
760 nm Yes Rengan et al. (2015)
Nr Yes Chou et al. (2014)
530 nm Yes Mapanao et
Nr Nr Higbee-Dempsey et al.
>650 nm Yes Cheheltani et al. (
Nr Nr Yahia-Ammar et al. (2016)
Broad,
NIR
absorbance
800 nm Nr Deng et al. (2015)
Yes Tam et al. (2010)
2021), Cassano et al.
, b), and Santi et al.
(
2019a
(
2020)
(
2020)
2018, 2020,
al. (
2016)
9.5.1 Small Molecule Crosslinking
Mellor et al. (2021) utilized a labile dithiol compound known as ethylene glycol
bis-mercaptoacetate (EGBMA) to induce crosslinking among ultrasmall AuNPs
with octadecylamine (ODA) surface coatings, all measuring less than 5 nm in
diameter. Importantly, these clustered nanoparticles were observed to revert to
their ultrasmall state under physiological conditions (Fig. 9.5). This unique behaviour made the nanostructures highly promising for biomedical applications.
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Fig. 9.5 Absorbance spectra (a) and TEM micrographs (b) of ultrasmall AuNPs and ultrasmall-innano constructs. TEM micrographs depict constructs at various stages: initial ultrasmall AuNPs (i),
ultrasmall-in-nano constructs (ii), and liberated AuNPs (iii). Scalebar = 20 nm
Notably, these nanostructures exhibited strong absorbance within the
phototherapeutic window, necessary for non-invasive detection and heating of the
particles. To enhance their utility, the nanoparticles were additionally labelled with a
Raman reporter molecule biphenyl-4-thiol (BPT), enabling precise detection within
complex biological matrices.
9.5.2 Coating of Liposomes
Rengan et al. (2015) developed a unique formulation, characterized as ultrasmall-onnano. This formulation involved an initial creation of nano-sized liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol.
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Subsequently, these liposomes were coated with ultrasmall AuNPs measuring
2–8 nm. The research showcased the remarkable potential of these particles, administered via intratumoral injection, to effectivel y eradicate cancer cells using PTT,
resulting in complete ablation of the tumour mass upon exposure to a 750 nm laser.
Furthermore, the study explored the fate of these coated liposomes within the
body. It was observed that these particles underwent degradation within hepatocytes
and were subsequently cleared from the body through the hepatobiliary and renal
pathways. Notably, on days 1, 7, and 14 post-inj ection, the %ID detected in the liver
decreased significantly, measuring 52%, 9.8%, and 3%, respectively. Similarly, in
the kidneys, %ID decreased to 2.7%, 0.25%, and 0.22% on the same days. This
reduction in gold levels within just 14 days was a substantial finding, leading the
authors to postulate that renal excretion would be further enhanced when the
constructs underwent both photothermal and enzymatic degradation.
9.5.3 DNA Assembly
Chou et al. (2014) devised a core-satellite architecture consisting of 13 nm cores,
accompanied by 3 nm or 5 nm satellites, all coated with thiolated, single-stranded
DNA. The assembly was achieved through the use of complementary sequence
linker DNA and capping ligand.
By selecting specific DNA sequences and capping ligands, the researchers were
able to finely tune several crucial characteristics of these AuNPs. These included the
ability of the constructs to encapsulate small molecules, their propensity for cellular
uptake, their elimination from the body, and their potential for tumour targeting.
In their investigation, the study quantified the gold content in urine 48 h after the
systemic injection of the AuNP cores, satellites, and core-satellite const ructs. Notably, they observed that the smallest particles, specifically the 3 nm satellites with a
1 kDa PEG coating, exhibited the highest renal excretion levels, accounting for 15%
ID. Conversely, as particle size or PEG molecular weight increased, renal excretion
levels decreased.
Additionally, when assessing the core-satellite constructs, the researchers found
that urine levels were directly proportional to the size of the satellite tested. Importantly, these levels were lower than those observed for the corres ponding satellites
when administered alone. This finding strongly suggests that only the satellites are
being excreted, while the 13 nm cores remain in the body.
9.5.4 Encapsulation/Ionic Interaction
Voliani and their research team, in a series of publications (Mapanao et al. 2018,
2020, 2021; Cassano et al. 2019a, b; Santi et al. 2020), introduced what they call a
‘passion fruit’-like nanostructure formed through ionic interactions between poly
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(l-lysine) (PL) and poly(sodiu m 4-styrene sulfonate) (PSS)-coated ultrasmall AuNPs
with an average size of approximately 3 nm. Their investigations covered various
aspects of this nano construct:
1. Tumour Target ing (Mapanao et al. 2018): After modifying the nano construct
with a specific peptide, Voliani’s team assessed its ability to target tumours.
2. Biocompatibility and Excretion (Cassano et al. 2019a): In murine models, the
researchers monitored the biodistribution and excretion over a 10-day period
following intravenous injection via the tail vein. Their observations indicated a
decline in gold concentration in the liver over time. Furthermore, gold was
consistently detected in both the urine and faeces throughout the experiment,
resulting in a cumulative excretion of approximately 16% of the initial dose over
the 10-day period.
3. Photothermal Therapy Suitability (Cassano et al. 2019b): The team explored the
potential of their ultrasmall-in-nano construct for photothermal therapy
applications.
4. Pharmacokinetics Following Inhalation (Mapanao et al. 2020): Their research
included a study of pharmacokinetics after inhalation, revealing accumulation in
lung tissue, translocation to secondary organs, and nearly complete excretion
within 10 days.
Higbee-Dempsey et al. (2020) synthesized ultrasmall p-MBA-AuNPs coated
with thiolated dextran, measuring 2.1 nm in diameter. To render the dextran hydrophobic, they introduced acetyl groups through covalent bonding. Subsequently,
these hydrophobic AcetalDextran-pMBA-AuNPs were combined with poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-PCL), leading to the formation of
micelles and the dense packing of the ultrasmall cores.
Under acidic conditions, the acetyl groups underwent cleavage, transforming the
polymer into a hydrophilic form. This process resulted in the demicellization of the
nanoparticles and the dispe rsion of the gold cores. The research group observed the
gradual clearance of gold from the organs of mice over a period of 3 months,
following a single bolus injection via the tail vein. Specifically, they reported an
86% reduction in gold levels in the liver and a 72% reduction in the spleen over a
span of 12 weeks. Moreover, the presence of gold was detected in urine and faeces,
with levels decreasing over time. It is worth noting that these reported levels
represent samples collected on the day of sacrifice and do not provide a cumulative
assessment of excretion between time points. Nonetheless, it is reasonable to infer
that gold excretion occurred continuously throughout the study period.
Cheheltani et al. (2016) successfully encapsulated ultrasmall AuNPs coated with
glutathione (GSH) (2–5 nm in size) within a biodegradable poly di
(carboxylatophenoxy)phosphazene (PCPP ) polymer matrix. The size of these particles and, consequently, the position of their surface plasmon resonance (SPR) peak
in the near-infrared (NIR) region were controlled by varying the amount of polyethylene glycol-polylysine block copolymer in the formulation.
Their research demonstrates the potential utility of these constructs as contrast
agents for both computed tomography (CT) and photoacoustic (PA) imaging, as
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demonstrated through in vitro and in vitro experiments. Furthermore, Cheheltani and
colleagues showcased the degradability of these nanoparticle clusters when exposed
to serum.
Yahia-Ammar et al. (2016) first synthesized ultrasmall AuNPs with a size of
approximately 2 nm and coated with GSH. Subsequently, these particles underwent
encapsulation through the addition of poly(allyl amine hydrochloride) (PAA HCl).
The results revealed that the clustered nanoparticles exhibited significantly enhanced
fluorescence compared to their ultrasmall counterparts, showcasing a remarkable
increase in quantum yield from 7% to 25%. Furthermore, the study demonstrated a
notable improvement in the cellular uptake of these encapsulated particles. This
enhanced cellular delivery capability was successfully applied to facilitate the
transport of both peptides and antibodies.
Tam et al. (2010) conducted a study in which ultrasmall AuNPs with a diameter
of 4.1 nm, capped with lysine and citrate, were clustered through ionic interactions.
These clusters were formed in the presence of a biodegradable triblock copolymer
consisting of polylactic acid and polyethylene glycol, denoted as PLA(2 K)-PEG
(10 K)-PLA(2 K).
Upon clustering, there was a significant shift in the absorbance spectrum of the
AuNPs. The absorbance maximum, originally at 520 nm, shifted into the nearinfrared (NIR) region, with consistent absorbance observed within the
700 to 900 nm range. Furthermore, the resear chers investigated the stability of
these nanoclusters under different pH conditions. Limited degradation occurred
after 4 weeks when exposed to a neutral pH environment. In contrast, nearly
complete degradation was observed within just 1 week at a pH of 5. This discrepancy
in degradation rates was attributed to the inherent stability of PLA under neutral pH
conditions. Nanocluster degradation was confirmed in vitro by TEM and scattering
spectra from hyperspectral images of treated and untreated murine macrophage cells
over 168 h.
Deng et al. (2015) investigated the encapsulation and ionic interactions involving
6.1 nm AuNPs, which approach the upper limit of what can be considered ultrasmall
particles, as acknowledged by the researchers. However, their approach shows
potential applicability to even smaller particles to ensure efficient excretion.
Their syst em is based on the self-assembly of AuNPs within a novel amphipathic
polymer, a comb-like structure composed of hydrophobic poly(ε -caprolactone)/poly
(2-hydroxyethyl methacrylate) (PCL-PHEMA) and hydrophilic poly
(2-(2-methoxyethoxy) ethyl methacrylate) (PMEO2MA).
The research team assessed the PTT potential of these particles by subjecting
solutions with varying concentrations to an 808 nm near-infrared (NIR) laser at a
power density of 1.5 W cm
(0.4 mg mL
-1
), the particles demonstrated efficient heating, reaching temperatures
-2
. Remarkably, at the highest concentration
as high as 71 °C after 5 min of laser irradiation.
Additionally, the researchers loaded the particles with DOX (doxorubicin), which
was released upon laser irradiation, showcasing the dual capabilities of this construct
in both chemotherapy and phototherapy.
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