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Construction and optimization of biofunctional upconversion nanoplatforms

Ding, Y.

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Citation for published version (APA):Ding, Y. (2018). Construction and optimization of biofunctional upconversion nanoplatforms.

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Download date: 28 Sep 2020

Introduction

1

CHAPTER 1

Introduction

Chapter 1

2

1.1 Luminescent nanomaterials

With the rapid development of nanotechnology, various luminescent nanomaterials, such

as quantum dots (QDs), fluorescent silica nanoparticles, and upconversion nanoparticles

(UCNPs), have been developed and investigated intensively. Compared with conventional

organic dyes, the luminescent nanomaterials are less susceptible to photobleaching, and

have higher chemical stability. They also have unique properties distinct from their bulk

counterparts owing to the size effects, surface effects, etc. The optical properties of these

nanomaterials can be modulated by rational structural design and surface engineering.

These materials are particularly attractive in biological and biomedical applications; the

large surface to volume ratio, for example, allows for the loading of a great amount of

drugs on the surface of the nanomaterials. In this thesis, we will focus on lanthanide ions

doped UCNPs.

1.2 Lanthanide doped upconversion nanoparticles

1.2.1 Upconversion mechanism

As a typical anti-Stokes process, upconversion involves the absorption of two or more

photons and the emission of one individual photon with a higher energy. Lanthanide ions

have attracted particular attention in this regard, partly due to their rich long-lived

metastable energy levels. When doped into glasses or crystals, lanthanide ions are capable

of converting near-infrared (NIR) light into ultraviolet (UV) and/or visible light. It can be

realized with an economical continuous-wave (CW) laser diode, in sharp contrast with

coherent upconversion processes (e.g., two-photon absorption) for which an expensive

high-power pulsed laser is needed.

Figure 1. Schematic illustration of upconversion mechanisms. (A) Excited-state absorption.

(B) Energy transfer upconversion. (C) Photo-avalanche.

Introduction

3

The upconversion discussed here is a nonlinear optical process, which may involve

different mechanisms. The most well-known ones are excited-state absorption (ESA),

energy transfer upconversion (ETU), and photon-avalanche (PA), as shown in Figure 1. In

the ESA process, the active ion absorbs a first photon to populate the intermediate energy

level (E1), and then another photon to go to a higher excited state (E2). In order to suppress

nonradiative cross-relaxation (CR) processes between the active ions, a low doping

concentration of the active ions is usually necessary for this mechanism to be dominant.

ETU is the most efficient upconversion mechanism. A sensitizer ion absorbs one photon

and then transfers the energy nonradiatively to the activator in the close vicinity. The

activator in the long-lived E1 state is further excited to E2 when it receives the

corresponding energy transferred from another excited neighboring sensitizer. The PA

process involves ESA and CR processes, usually occurring in highly-doped systems with

the excitation power above a certain threshold. This mechanism is regarded as much less

efficient than the other two, and is seldom found in nanomaterials.

1.2.2 Composition of lanthanide doped upconversion nanoparticles

Lanthanide doped UCNPs are composed of optically inert host nanocrystals and optically

active lanthanide dopants. The crystal field of the host allows for 4f-4f transitions of the

dopant ions to produce upconversion luminescence, which is otherwise forbidden. To

minimize the phonon assisted nonradiative relaxation of the lanthanide ions and thus

improve the upconversion efficiency, a low phonon energy is preferred. Till now, fluorides

(phonon energy of ~350 cm-1

), such as NaYF4 and NaLuF4, are favored over oxides

(phonon energy larger than 500 cm-1

). The lanthanide ions can act as a sensitizer or

activator. The former absorbs the excitation energy and transfers it to the latter which emits.

Due to the relatively large absorption cross section among the lanthanides1 (~10

-20 cm

-2)

and its energy level structure which is well-matched with various activators, Yb3+

has been

the most popular sensitizer. Its simple energy level structure with only one long-lived

excited state (2F5/2) renders efficient energy transfer (ET) from Yb

3+ to the ideal ETU

activators with ladder-like energy levels, including Er3+

, Ho3+

and Tm3+

. Yb3+

, Er3+

co-doped hexagonal phase NaYF4 (also known as β-NaYF4) is generally considered as the

most efficient upconversion material.2,3

1.3 Upconversion luminescence enhancement in nanoparticles

Compared with conventional fluorescent tags such as organic dyes and QDs, UCNPs are

particularly attractive in various biological and biomedical fields owing to prominent

Chapter 1

4

advantages such as narrow emission bands, a large anti-Stokes shift, absence of

photobleaching and photoblinking, deep tissue penetration, and minimal background

fluorescence. However, the low upconversion quantum efficiency significantly impedes

broad applications of UCNPs. Therefore, large efforts have been devoted to enhancing

upconversion luminescence.

1.3.1 Lattice and dopant modulation

As the local crystal field provided by the host lattice is of vital importance for generating

upconversion luminescence, lattice structure adjustment has naturally become one of the

possibilities to enhance the upconversion luminescence. Various cations, including rare

earth ions and alkali, alkaline earth and transition metal ions (e.g., Sc3+

, Li+, Ca

2+ and Fe

3+),

have been used to replace the cations in the host material to tailor the local crystal field, and

indeed enhanced upconversion luminescence was observed.4-7

The improvement of

crystallinity might also contribute to the luminescence enhancement. In addition to the type

of the dopants, the concentration of the dopants has also been investigated extensively to

enhance upconversion luminescence efficiency. In general, a relatively high concentration

of the sensitizer (e.g., 20% for Yb3+

) is applied, but a low doping concentration of the

activator (e.g., 2% for Er3+

) is chosen to minimize deleterious cross relaxation energy loss.

Recently, the concentration quenching effect of Er3+

was eliminated by making an inert

NaYF4 shell, and the optimal doping concentration of Er3+

was increased to 100%, i.e.,

NaErF4@NaYF4.8

1.3.2 Surface passivation

Surface passivation is an effective way to improve upconversion luminescence properties,

as upconversion luminescence can be quenched by the surface related entities, like surface

defects, contaminants, ligands and solvents.9 It can be realized by an epitaxial shell on the

surface of the UCNPs to isolate the upconversion area from these quenchers, which has

become a typical approach to alleviate the excited energy dissipation to the surface

quenchers and thus enhance upconversion luminescence efficiency by up to two orders of

magnitude.9-12

Zhang and Zhao et al. realized direct imaging of NaYF4: Yb3+

,

Er3+

@NaGdF4 nanocrystal core-shell structure at the subnanometer level, and observed a

linear increase in the overall upconversion emission intensity upon the growth of the inert

shell layer by layer.13

Capobianco et al. reported an active core/active shell strategy, where

sensitizer Yb3+

was doped into the NaGdF4 shell which absorbs extra energy other than the

Yb3+

in the core and transfers the energy to the NaGdF4: Yb3+

, Er3+

core.14

The active

core/active shell structured UCNPs exhibited higher upconversion luminescence intensity

Introduction

5

than the corresponding bare core and active core/inert shell structured UCNPs.

1.3.3 Surface plasmon enhancement

Surface plasmon resonance is another effective approach to increase upconversion

luminescence by a factor up to several hundreds. Gold or silver of various forms, such as

nanoparticles, nanorods and nanowires, has been explored in this regard.15-18

The plasmon

resonance wavelength can match either the excitation or the emission wavelength of the

UCNPs. In the former case, the upconversion luminescence might be enhanced by the

increase of the absorption cross sections and ET rates, while in the latter case the

enhancement is mainly realized by the Purcell effect.19

The enhancement is influenced not

only by the plasmon resonance wavelength, but also the structure and the excitation power

density. Schietinger et al. coupled a single NaYF4: Yb3+

, Er3+

nanocrystal and gold

nanosphere with atomic force microscope, and obtained 3.8 times enhancement of the

overall upconversion luminescence by adjusting the position of the gold nanosphere

relative to the NaYF4: Yb3+

, Er3+

nanocrystal and the polarization axis of the excitation

light.20

It should be noted that the upconversion luminescence can also be quenched by the

surface plasmon at very short distances, and thus a spacer layer (e.g., silica, Al2O3, and

polymers) is commonly used to enhance the luminescence.17,21,22

1.3.4 NIR dye sensitization

The pioneering work of dye-sensitized upconversion luminescence enhancement was

reported by Hummelen et al. in 2012.23

An organic NIR dye, IR 806, was anchored onto 16

nm NaYF4: Yb3+

, Er3+

UCNPs to serve as an antenna. The overlap between the emission

spectrum of IR 806 and the absorption spectrum of the UCNPs allows for Förster-type ET

from excited IR 806 to the Yb3+

sensitizers in the UCNPs, which further sensitize Er3+

ions

to generate upconversion luminescence. The wide absorption band and large extinction

coefficient of the NIR dye (390 l g-1

cm-1

of IR 806 at 806 nm vs. 7×10-5

l g-1

cm-1

of the

UCNPs at 975 nm) contribute to the extension of the excitation range (from ~980 nm to

720-1000 nm) and significant enhancement of the luminescence intensity (by three orders

of magnitude) of the UCNPs. Inspired by the above work, some other organic dyes and

upconversion nanosystems were designed and utilized in the dye-sensitization

upconversion scheme.24-27

By utilizing cascaded ET between multiple dyes anchored on the

surface of NaYF4: Yb3+

, Tm3+

UCNPs, Hyeon et al. broadened the excitation range of the

UCNPs significantly from the narrow NIR region to the entire visible range (including red,

green and blue).28

The synergistic effect of dye-sensitization and core-shell enhancement

can further improve upconversion luminescence properties.25,27

Chapter 1

6

1.4 Synthesis of upconversion nanoparticles

The synthesis of high-quality lanthanide doped UCNPs with controllable size, shape and

crystalline phase is of great significance for modulating their luminescence properties and

exploring their potential applications in various fields. Up to now, numerous methods have

been developed for the synthesis of UCNPs, such as the widely-used hydro/solvo-thermal

method, thermal decomposition method, and coprecipitation method.

Figure 2. Scanning electron microscope (SEM) images of lanthanide doped (A) β-NaYF4

nanotube arrays, (B) flower-patterned β-NaYF4 hexagonal disk arrays and (C) β-NaYF4

nanorod arrays synthesized by the hydrothermal method.30

Transmission electron

microscope (TEM) images of (D) LaF3 triangular nanoplates, (E) NaYF4: Yb3+

, Er3+

nanocrystals synthesized by thermal decomposition,33,34

and (F) LaF3: Yb3+

, Er3+

nanocrystals synthesized by coprecipitation.43

1.4.1 Hydro/solvo-thermal method

The hydro/solvo-thermal method is one of the most popular strategies for preparing

lanthanide doped UCNPs. The hydro/solvo-thermal reactions typically occur in water or

other solvents under high pressures and temperatures above the critical point of the solvent

to increase the solubility and reactivity of the inorganic chemicals, such as lanthanide

chloride/nitrate/oxide, NH4F, and NaF. A typical example was provided by Li et al. who

proposed a general synthesis strategy based on phase transfer and separation at the

interfaces of the liquid, solid and solution phases, and various nanocrystals with different

Introduction

7

chemistries and properties were obtained, including noble metals, semiconductors,

magnetic/dielectric oxides, and lanthanide doped luminescent nanocrystals.29

By using

oleic acid (OA) as a stabilizing agent, Zhao et al. synthesized a series of uniform

nanostructured arrays of NaYF4 nanotubes, flower-patterned disks and nanorods (Figure

2A-C).30

Water-dispersible UCNPs were also prepared with this method using appropriate

capping ligands, such as 6-aminocaproic acid and polyethylenimine (PEI).31,32

Compared to

other synthetic methods, highly crystalline nanocrystals could be obtained at a lower

temperature (generally, lower than 250 C) without the need of post-treatment. The

disadvantages of this method lie in the necessity of using specialized reaction vessels

(typically, Teflon-lined autoclaves) and that it is not possible to observe the growth process

of the nanocrystals.

1.4.2 Thermal decomposition method

The thermal decomposition method is generally based on the thermolysis of

organometallic precursors (typically, CF3COONa and Ln(CF3COO)3) at elevated

temperatures in high boiling point organic solvents. The commonly used noncoordinating

solvent is 1-octadecene (ODE), and the surfactant is normally OA, oleylamine (OM), or

trioctylphosphine oxide (TOPO).33-37

This method was first reported by Yan et al. to

synthesize LaF3 triangular nanoplates via the thermal decomposition of La(CF3COO)3 at

280 C in OA/ODE (Figure 2D).33

It was then employed by Chow et al. to synthesize

hexagonal-phase NaYF4: Yb3+

, Er3+

/Tm3+

UCNPs (with an average size of 10.5 or 11.3 nm)

via the decomposition of CF3COONa and Ln(CF3COO)3 (Ln=Y, Yb, Er and Tm) at 330 C

in OM (Figure 2E).34

Up to now, this method has been extended as a common route for the

synthesis of various lanthanide doped oxides, fluorides, oxyfluorides, etc.38-42

The rapid

nucleation induced by thermal decomposition of the metallic precursors facilitates the

formation of monodisperse nanocrystals, and the high temperature reaction in the presence

of the organic ligands results in nanocrystals with high crystallinity and relatively small

sizes. The drawback of this method is that the thermal decomposition of the metallic

trifluoacetate salts may produce very toxic fluorinated and oxyfluorinated carbon species

that are unfriendly to both the laboratory personnel and the environment. Besides, further

surface modification of the UCNPs is required to render them water-dispersible for

biomedical applications.

1.4.3 Coprecipitation method

Coprecipitation is a convenient and cost-effective technique for synthesizing relatively

small UCNPs with a narrow size distribution. For example, with ammonium

Chapter 1

8

di-n-octadecyldithiophosphate as capping ligand to control the nanocrystal growth and to

prevent the nanoparticles from aggregation, Yi et al. synthesized Yb3+

, Er3+

/Ho3+

/Tm3+

co-doped LaF3 upconversion nanocrystals with an average diameter of 5.4 nm and a

standard deviation of 0.9 nm (Figure 2F).43

This method has also been used to synthesize

other UCNPs, such as NaYF4: Yb3+

, Er3+

/Tm3+

, CaMoO4: Ho3+

, Yb3+

, Mg2+

, LuPO4: Yb3+

,

Tm3+

and YbPO4: Er3+

.44-47

Heat treatment is typically needed to obtain highly luminescent

UCNPs.

1.4.4 Other methods

Some other methods, such as the sol-gel method,48,49

combustion synthesis,50,51

ionic-liquid based synthesis52,53

and the microwave assisted method,54

have also been

developed for the synthesis of lanthanide doped UCNPs. Each method has its merits and

drawbacks. For example, the sol-gel method does not need expensive reaction precursors

and apparatuses, but calcination is often required to improve upconversion luminescence,

and the poor size control and aggregation of the particles might also be problematic.

Combustion synthesis is a time and energy saving method, but the obtained nanoparticles

might aggregate severely.

1.5 Surface modification of upconversion nanoparticles

For biomedical applications, the UCNPs should be dispersible in aqueous solutions.

However, monodispersed UCNPs with a relatively small size and efficient upconversion

luminescence are usually prepared in the presence of hydrophobic surfactants, and do not

possess intrinsic aqueous disposability and biofunctional moieties. Therefore, surface

modification is always required to render the UCNPs water dispersible and provide them

with reactive groups for bioconjugation. In the past decades, various surface modification

strategies have been developed, such as ligand exchange, polymer encapsulation, silica

coating, ligand-free method and ligand oxidation.

1.5.1 Ligand exchange

Ligand exchange replaces the original hydrophobic ligands with hydrophilic ones. The

hydrophilic ligands can be multi-chelating or single-chelating with stronger coordination

interactions with the lanthanide ions. For example, the OA-capped UCNPs can be

transferred to the water phase by replacing the OA ligand with poly(acrylic acid) (PAA),55

mercaptopropionic acid (MPA),56

poly(ethyleneglycol) (PEG)-phosphate57

and citrate58

. Yin

et al. developed a general and robust ligand exchange approach to transfer hydrophobic

Introduction

9

nanocrystals from nonpolar organic solvents to an aqueous solution.59

Polyelectrolytes,

such as PAA, poly(allylamine) (PAAM) and poly(sodium styrene sulfonate) (PSS), were

used to replace the hydrophobic ligands (OA or TOPO) at an elevated temperature. Murray

et al. reported a versatile strategy for transferring hydrophobic nanocrystals to various polar,

hydrophilic solvents by replacing the original OA and/or OM ligands with NOBF4.60

The

ligand exchange reaction can be completed at room temperature within 5 minutes. The

BF4−-capped colloidal nanocrystals can be stable for years without observable aggregation

or precipitation, and they can be readily functionalized with various capping molecules.

The advantage of the ligand exchange method is that it normally has little effect on the size,

shape, crystalline phase and luminescent properties of the UCNPs.

1.5.2 Amphiphilic polymer encapsulation

Amphiphilic polymer encapsulation is also an effective strategy to transfer the

hydrophobic UCNPs to an aqueous phase. The hydrophobic part of the amphiphilic

polymers can intercalate the hydrophobic alkyl chains of the surface ligands via van der

Waals interactions, and the outer hydrophilic part of the amphiphilic polymer molecules

provides the UCNPs with water dispersibility and functional groups for further

bioconjugation. A variety of amphiphilic polymers have been used for this purpose,

examples include octylamine-poly(acrylic acid)-poly(ethylene glycol) (OA-PAA-PEG),61

poly((ethylene glycol)-block-lactic acid) (PEG-b-PLA),62

poly (L-lysine) (PLL)63

and

poly(maleic anhydride-alt-1-octadecene) (PMAO)64

. For example, Prud’homme et al.

stabilized OA and phosphine (TOP) capped NaYF4: Yb3+

, Er3+

UCNPs in water, buffer or

culture media with proteins by encapsulating them with three amphiphilic polymers:

PEG-b-PLA, poly((ethylene glycol)-block-ploy(caprolactone) (PEG-b-PCL), and

poly((ethylene glycol)-block-poly(lactic-coglycolic acid) (PEG-b-PLGA).62

van Veggel et

al. transferred oleate-capped NaYF4: Yb3+

, Er3+

/Tm3+

@NaYF4 core/shell UCNPs from a

hydrophobic to an aqueous phase by coating them with PEG-modified PMAO and then

cross linking the PMAO units with bis(hexamethylene)triamine (BHMT).64

The

cross-linked PMAO-BHMT coated UCNPs exhibited very high stability at different pH

values, physiological buffers and biological growth media. By using the amphiphilic

polymer encapsulation method, the original hydrophobic ligands on the surface of the

UCNPs are retained, and thus the luminescent properties of the UCNPs are less affected.

However, the amphiphilic polymer layer increases the hydrodynamic diameters of the

UCNPs, which are crucial for their biomedical applications. In addition, the amphiphilic

Chapter 1

10

polymers are often expensive or not commercially available, leading to high costs or

complex experimental procedures.

1.5.3 Silica coating

Silica coating (or surface silanization) is a popular inorganic surface modification

strategy due to the unique properties of silica such as good water dispersibility, high

chemical stability, excellent biocompatibility, optical transparency and easy surface

functionalization. Reverse microemulsion is typically used to coat hydrophobic ligands

capped UCNPs with silica. The silica shell is produced by hydrolysis and condensation of

tetraethoxysilane (TEOS) in a nano-reactor generated by a mixture of ammonia,

cyclohexane, surfactant (Igepal CO-520, TritonX-100) and TEOS. Reverse microemulsion

to transfer OA capped NaYF4: Yb3+

, Er3+

UCNPs to aqueous media was reported by

coating the UCNPs with 8 and 5.3 nm thick silica shells, respectively.65,66

Mesoporous

silica was also used to coat lanthanide doped UCNPs to further increase the specific surface

areas of the UCNPs and thus more functional molecules or drugs can be loaded.67,68

It

should be noted that hydrophilic UCNPs can also be encapsulated with silica by the Stöber

method.69

In addition to the size increase of the UCNPs, special attention should be paid to

obtain a uniform silica coating without inducing aggregation of the nanoparticles during

silanization process of the UCNPs.

1.5.4 Other methods

In addition to the above popularly used methods, some other surface modification

methods have been developed, such as ligand removal and ligand oxidation. Capobianco et

al. reported a facile approach to obtain water dispersible NaYF4: Yb3+

, Er3+

UCNPs by

removing the original oleate ligand via acid treatment.70

The detachment of the protonated

oleate ligand leaves abundant metallic ions on the surface of the UCNPs, and thus various

biocompatible molecules with functional groups such as -COOH and -NH2 can readily be

anchored to the UCNPs via coordination interaction for further bioapplications. Li and Yan

et al. obtained water dispersible UCNPs by oxidizing the OA ligand into azelaic acid or

azelaic aldehyde with Lemieus-von Rudloff reagent and ozone, respectively.71,72

The ligand

oxidation method is simple and straightforward, and it also provides the UCNPs with

carboxylic groups for further bioconjugation. However, it suffers from a long reaction time

and low yield.

Introduction

11

1.6 Biomedical applications of upconversion nanoparticles

Traditional fluorescent materials, such as organic dyes and QDs, have been explored

extensively in various biological and biomedical fields. Despite great efforts, practical

applications are still impeded by some intrinsic drawbacks, such as photobleaching,

background emission interference and photodamage to biological tissues. In these aspects,

lanthanide doped UCNPs are particularly attractive. They are characterized by narrow

emission bands, absence of photobleaching and photoblinking, deep tissue penetration, and

minimized background fluorescence. Up to now, UCNPs have shown great potential in

various biomedical fields, including bioimaging, bioassays, biosensing, photodynamic

therapy (PDT) and photothermal therapy (PTT).

1.6.1 Bioimaging

High contrast cellular imaging has been reported extensively in the past decade using

UCNPs modified with various capping ligands (such as silica, small molecules and

polymers) or biomolecular recognition moieties (such as folic acid, peptides and antibodies).

For example, Prasad et al. used 3-mercaptoproionic acid modified NaYF4: Yb3+

, Tm3+

UCNPs (20-30 nm) to image human pancreatic cancer cells.56

Complete absence of

background autofluorescence demonstrated the applicability of UCNPs for high contrast

luminescence imaging. Lee and Suh et al. performed long-term real-time imaging and

tracking of PEG-phospholipids capped NaYF4: Yb3+

, Er3+

UCNPs (~40 nm) in living HeLa

cells at the single vesicle level for 6 h continuously.73

The transport dynamics of the

UCNPs was found to consist of multiple phases within a single trajectory, and the active

transport by motor proteins was clearly visualized. Following the spatiotemporal

distribution of endocytosed UCNPs on a longer time scale, the full intracellular pathway of

the UCNPs was demonstrated to consist of endocytosis, active transport, and exocytosis.74

Mao and Xu et al. realized targeted immunolabeling and upconversion luminescence

imaging of HeLa cells using rabbit anti-CEA8 antibodies functionalized NaYF4: Yb3+

,

Er3+

@SiO2 UCNPs (~45 nm).75

The application potential of UCNPs for in vivo imaging was first explored in nematode

worms by Lim et al.76

They inoculated 150 nm sized Y2O3: Yb3+

, Er3+

UCNPs into live

Caenorhabditis elegans, and tracked the movement of the particles through their digestive

system by imaging under 980 nm excitation. After that, mice (and rats) models have

become the most popular small animal models used for in vivo upconversion luminescence

imaging. Zhang et al. injected PEI capped NaYF4: Yb3+

, Er3+

UCNPs (~50 nm)

Chapter 1

12

subcutaneously into the groin and upper leg of anaesthetized Wistar rats, and observed

visible emission from a depth of up to 10 mm under NIR excitation.77

Compared with QDs,

the UCNPs exhibited minimized autofluorescence, high detection sensitivity and deep light

penetration in biological tissues. Li et al. demonstrated tumor targeting function of

chlorotoxin peptide functionalized NaYF4: Yb3+

, Er3+

/Ce3+

UCNPs (25×55 nm) by injecting

intravenously UCNPs to Balb-c nude mice bearing xenograft glioma tumors (tumor size

about 0.5-1.0 cm).78

The tumor was visualized by red upconversion luminescence at about

24 h post-injection. Multicolor in vivo upconversion luminescence imaging has also been

realized by imaging subcutaneously injected OA-PAA-PEG modified NaYF4: Yb3+

,

Er3+

/Tm3+

UCNPs (~30 nm).61

It was further applied for multiplexed in vivo lymph node

mapping and cancer cell tracking in mice.

Multimodal imaging has also attracted considerable attention as it can combine the

advantages of individual imaging modalities so as to provide more accurate and

comprehensive information. To this end, various multimodal imaging contrast agents have

been developed. For example, Gd3+

containing UCNPs and nanocomposites composed of

UCNPs and iron oxide have been frequently used for dual-modal optical/magnetic

resonance imaging (MRI).79,80

By introducing 18

F radionuclides into NaYF4: Gd3+

, Yb3+

,

Er3+

UCNPs, a tri-modal upconversion/MRI/positron emission tomography (PET) imaging

system was constructed.58

Li et al. realized four-modal imaging in mice with core-shell

structured NaLuF4: Yb3+

, Tm3+

@NaGdF4(153

Sm) UCNPs, where Yb3+

/Tm3+

enables

upconversion luminescence imaging, Gd3+

MRI imaging, 153

Sm single-photon emission

computed tomography (SPECT), and all the lanthanide ions contribute to X-ray computed

tomography (CT).81

1.6.2 Bioassay and biosensing

UCNPs have also aroused great interest in bioassays and biosensing. Temperature

sensing, detection of various biomolecules and metal ions, sensing of small gas molecules,

and some other biosensing systems have all been reported. Except for some heterogeneous

bioassays and temperature sensing, most of the other bioassay and biosensing systems are

developed on the basis of nonradiative Förster resonance energy transfer (FRET). FRET is

illustrated schematically in Figure 3A. It can be seen that FRET affects the temporal

behavior of the donor emission by providing an additional nonradiative relaxation channel.

The FRET efficiency depends not only on the spectral overlap between the emission of the

energy donor and the absorption of the energy acceptor, but also on the distance between

the energy donor and acceptor. Considering a simplified dipole-dipole interaction, the

Introduction

13

FRET efficiency is inversely proportional to the sixth power of the donor-acceptor distance.

The distance sensitivity of the FRET efficiency is thus widely taken as the basis for FRET

based bioassays and biosensors. Inner filter effects have also been reported to sense pH,

gases, metal ions, and so on. It mainly involves the absorption of the upconversion

luminescence by some specific molecules, and it may therefore be considered as a

reabsorption process or radiative ET process, as depicted schematically in Figure 3B.

Reabsorption also depends on the spectral overlap between the emission of the energy

donor and the absorption of the energy acceptor. The main difference between the two ET

mechanisms is that the emission dynamics of the energy donor is not affected in case of

reabsorption, and that the reabsorption rate is less sensitive to the donor-acceptor distance

(the radiation density of the donor on the acceptor is inversely proportional to the square of

the distance). Therefore, the variation in the spectral overlap is generally used as the basis

for constructing inner filter effect-based biosensors.

Figure 3. Schematic illustration of (A) nonradiative FRET and (B) radiative reabsorption

processes.

In 2006, Li et al. developed a heterogeneous sandwich-hybridization assay for DNA.82

NaYF4: Yb3+

, Er3+

UCNPs and Fe3O4 magnetic nanoparticles were modified with

3’-propylthiol-terminated probe DNA and 5’-propylthiol-terminated capture DNA,

respectively. The presence of target DNA led to the formation of magnetic-upconversion

nanocomposites through the base-match interaction of the oligonucleotides. Sensitive

detection of the nucleic acids was thus achieved by monitoring the upconversion

luminescence intensity of the nanocomposites in combination with magnetic separation and

concentration technology.

In 2010, Capobianco et al. reported the first upconversion nanothermometer in liquids

and live cells.83

It was based on the temperature dependence of the upconversion

luminescence from water-dispersible PEI-capped NaYF4: Yb3+

, Er3+

UCNPs (~18 nm). The

Chapter 1

14

intensity ratio of the green emission originating from the 2H1/2-

4I15/2 and

4S3/2-

4I15/2

electronic transitions of Er3+

ions centered around 525 and 545 nm, respectively, was used

to provide a thermometric scale in solution. The thermometer was then used to measure the

temperature gradient in water as well as the temperature changes of an individual cancer

cell from 25 C to 45 C.

In 2005, Li et al. reported a FRET-based biosensor for the detection of trace amounts of

avidin.84

Biotin conjugated Na(Y1.5Na0.5)F6: Yb3+

, Er3+

UCNPs (~50 nm) and Au

nanoparticles (with the absorption peak at ~520 nm) were used as the energy donor and

energy acceptor, respectively. In the presence of avidin, the biotin-UCNPs and biotin-Au

nanoparticles were brought into close proximity through sensitive and selective interaction

between avidin and biotin, and the green upconversion luminescence was quenched via

FRET. The results indicate the large potential of UCNPs-based FRET system for biological

analyses.

In 2010, Wolfbeis et al. developed an ammonia probe based on the inner filter effect.85

The sensor film was composed of NaYF4: Yb3+

, Er3+

UCNPs (60-90 nm), pH indicator

phenol red, and a polystyrene matrix that was impermeable to protons. Ammonia can

penetrate the polystyrene film and increase the local pH, thus increasing the absorption of

phenol red at 560 nm substantially. It caused the green upconversion luminescence to be

screened off, but had no effect on the red emission. The ratio of the green to red emission

was used to determine the concentration of ammonia in aqueous solutions.

It should be pointed out that amongst the UCNPs-based bioassays and biosensing,

FRET-based bioassays and biosensing are the most popular, evidenced by numerous

publications in this regard. However, the underlying ET mechanism in related systems still

remains vague.

1.6.3 Photo-dynamic/thermal therapy

Unlike traditional chemotherapy, radiotherapy or surgery, PDT and PTT are two newly

emerging cancer treatment techniques that destroy cancer cells with light. In PDT, cancer

cells are killed by reactive oxygen species (ROS) generated by photosensitizers, while in

PTT, they are destroyed via thermal ablation induced by photothermal agents. As the

photosensitizers and photothermal agents are generally excited by UV or visible light which

have a small tissue penetration depth, UCNPs are attractive as NIR light transducers for

treatment of deep tumors. The UCNPs can convert NIR irradiation into UV or visible light,

which in turn can excite the photosensitizers or photothermal agents in the close vicinity to

produce ROS or heat via ET (FRET and/or reabsorption).

Introduction

15

The application potential of UCNPs in PDT was first demonstrated in vitro by Zhang et

al. in 2007.86

They doped merocyanine 540 (the photosensitizer) into the porous silica shell

coated on NaYF4: Yb3+

, Er3+

UCNPs, and then modified the nanoparticles with antibodies

to realize targeted cancer cell killing under 974 nm laser excitation. In 2012, Zhang et al.

showed enhanced PDT efficacy by simultaneous activation of two photosensitizers

(merocyanine 540 and zinc (II) phthalocyanine) with NaYF4: Yb3+

, Er3+

UCNPs as the light

transducer.87

By targeting the folic acid modified nanocomplex to melanoma tumors in

mice, PDT induced tumor growth inhibition was observed in vivo. Recently, TiO2 was

incorporated with NaYF4: Yb3+

, Tm3+

UCNPs to serve as a photosensitizer instead of

molecular photosensitizers.88

The controllable and highly reproducible photosensitizer

loading prevented any leakage of the photosensitizer, and ensured significant ROS

generation upon NIR excitation for effective and repeatable PDT results both in vitro and in

vivo.

In 2011, Shao and Liu et al. fabricated multifunctional magnetic-upconversion

nanocomposites by a layer-by-layer self-assembly method.89

Superparamagnetic Fe3O4

nanoparticles (~5 nm) were adsorbed on the surface of NaYF4: Yb3+

, Er3+

UCNPs (~160

nm) by electrostatic attraction, and then a thin gold shell (photothermal agent) was formed

by seed-induced growth. After further modification with folic acid, the nanocomposites

showed molecular and magnetic targeted PTT efficacy of cancer cells under NIR light

exposure. In 2013, Bu and Shi et al. constructed a multifunctional upconversion

nanotheranostic platform by adsorbing citrate-stabilized CuS nanoparticles (photothermal

agent) to NH2-modified NaYbF4: Er3+

, Gd3+

@SiO2 UCNPs.90

This nanotheranostic system

not only enabled effective thermal ablation, but also boosted localized radiation dose to

enhance radiation damage in vitro and in vivo. Liu et al. simultaneously loaded two types of

dye molecules, Rose Bengal (photosensitizer) and NIR-absorbing IR 825 dye

(photothermal agent), into the bovine serum albumin (BSA) layer of NaGdF4: Yb3+

,

Er3+

@BSA nanoparticles, and killed both in vitro and in vivo cancer cells via combined

PDT and PTT under NIR light irradiation.91

1.7 Outline of the thesis

The thesis comprises five chapters. In this first chapter, we have introduced the unique

optical properties of UCNPs as well as their synthesis and modification methods towards

their biological and biomedical applications. Despite the significant advancements achieved

over the years, great challenges remain in relation to expanding their practical applications.

Chapter 1

16

These involve questions like how to improve the sensitivity of the sensing with UCNPs and

how to use UCNPs to treat effectively cancers at an early stage. Aiming at the construction

and optimization of biofunctional upconversion nanoplatforms, the present thesis will study

the cancer targeting behavior of functional UCNPs and investigate the ET mechanism

involved in the theranostic nanoplatform construction and upconversion luminescence

modulation.

Chapter 2 deals with the surface biofunctionalization of NaYF4: Yb3+

, Er3+

UCNPs and

relevant cancer targeting processes. Early stage tumor models, i.e., three-dimensional

multicellular tumor spheroid (MCTS, ~ 0.5 mm) of human breast cancer MCF-7 cell

grafted on chick embryo chorioallantoic membrane (CAM), are employed to evaluate the

surface effects on the targeting process and efficiency. In Chapter 3 we construct

multimagnetic-beads-embedded Fe3O4/NaYF4: Yb3+

, Er3+

magnetic upconversion

luminescent nanocomposites, and demonstrate their application potential in magnetic

targeted upconversion luminescence bioimaging in both human breast cancer MCF-7 cells

and mouse fibroblast 3T3 cells. Chapter 4 reports a quantitative study on the

shell-thickness-dependent interplay between dynamic (nonradiative) and static (radiative)

ET in nanosystems. The proposed model is validated in a typical biofunctional

upconversion nanoplatform composed of NaYF4: Yb3+

, Er3+

/NaYF4 UCNPs and

energy-acceptor photosensitizing Rose Bengal (RB) molecules. Different from the ET from

UCNPs to surface molecules, ET from a surface anchored NIR dye (indocyanine green) to

UCNPs is also very interesting from a fundamental science and application point of view.

The core/shell structure dependence of the relevant ET efficiency is addressed in Chapter 5.

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