Upconversion Nanoparticles: Design, Nanochemistry, and Applications in Theranostics

Abstract

1 Introduction Theranostics is a concept of integrating imaging and therapy into a single platform for use in the next generation of personalized medicine to meet the challenges in modern health care. 1 The diagnostic role of theranostic agents reports the presence of a disease, its status, and its response to a specific treatment, while the therapeutic role of the agent can be implemented in several forms: 2 (i) The first is imaging-guided surgery for tumor resection and postsurgery evaluation. Intraoperative visualization of diseased areas is important for precision surgery, as the location of the tumor may change after presurgical imaging and during resection. 3−6 Furthermore, postsurgical assessment is valuable in ensuring complete removal of the diseased sections. (ii) The second is delivery or release of therapeutic entities to the intended site. The delivered entities can be small molecule chemotherapeutics (such as cisplatin, doxorubicin, and paclitaxel), biologics (such as protein drugs and antibodies), gene products (DNA, siRNA, and miRNA), nanotherapeutic agents, and even cells. 7−9 The release/therapy can be light-activated such as in photodynamic therapy (PDT) for destruction of the tumor or heat activated by nonradiative conversion of absorbed photon energy into heat such as in photothermal therapy (PTT), 10 which disrupts the structure of the cells and shrinks the tumor volume. 11 (iii) The third is disruption of a cellular or metabolic pathway. 2 An occupation of specific cell surface receptors by introduced theranostic agents with appropriate chemistry can disrupt cell regulation, producing a therapeutic effect. 12 Theranostics offers an opportunity to embrace multiple techniques to arrive at a comprehensive imaging/therapy regimen. Incorporation of therapeutic functions into molecular imaging contrasts plays a pivotal role in developing theranostic agents. 13 Molecular imaging using photoluminescence (PL) spectroscopy is an important technique in biochemistry and molecular biology. It has become the dominant method revolutionizing medical diagnostics, bioassays, DNA sequencing, and genomics. 14−17 It can be used to study a wide range of biological specimens, from cells to ex vivo tissue samples, and to in vivo imaging of live objects; it can also cover a broad range of length scale, from submicrometer-sized viruses and bacteria, to macroscopic-sized live biological species. 17−19 Thus, PL imaging provides a powerful noninvasive tool to visualize morphological details in tissue with subcellular resolution. However, the imperfect optical properties of conventional PL imaging agents and the challenge in incorporation of therapeutic functions onto them have severely limited their abilities for use in theranostics. PL imaging generally employs exogenous contrast agents, which encompass organic dyes, 20,21 organically modified silica, 22−24 fluorescent proteins, 25−28 metal complexes, 11,29−31 and semiconductor quantum dots (QDs). 32−35 Most of these conventional contrast agents utilize Stokes-shifted emission using excitation in the ultraviolet (UV) or blue-green visible spectral ranges. These conventional PL imaging agents when excited in such spectral range have a number of limitations: (i) low signal-to-background ratio (SBR) caused by unwanted autofluorescence as well as by strong light scattering from the biological tissues (such as fur, skin, and tissues) when excited at short wavelengths; (ii) low penetration depth of UV and visible excitation and/or emission light in biological tissues; and (iii) potential DNA damage and cell death due to long-term exposure to short wavelength, particularly UV excitation. In addition, there is also a serious concern about the toxicity of heavy metal-based QDs for bioimaging, as they contain toxic elements (e.g., cadmium, mercury, and lead). It is well-known that biological tissues have an “optical transparency window” in the near-infrared (NIR) range of 700–1100 nm. 17−19 Utilization of NIR excitation light not only allows for deeper light penetration and reduced photodamage effects, but also offers lower autofluorescence, reduced light scattering, and phototoxicity. 36−38 Two-photon-excitation or second harmonic generation contrast agents, which utilize longer wavelength light, have been recently developed to overcome these limitations for imaging of cells and small animals. 39−43 However, they require expensive ultrashort pulsed lasers (e.g., a femtosecond laser) to perform the excitation as they involve inefficient nonlinear optical processes. Lanthanide-doped upconversion nanoparticles (UCNPs) are a promising new generation of imaging agents for bioimaging. 44−57 Upconversion (UC) utilizes sequential absorption of multiple photons through the use of long lifetime and real ladder-like energy levels of trivalent lanthanide ions embedded in an appropriate inorganic host lattice to produce higher energy anti-Stokes luminescence. 51,58 It thereby converts two or more low-energy excitation photons, which are generally NIR light, into shorter wavelength emissions (e.g., NIR, visible, and UV). This process is different from nonlinear multiphoton absorption in organic dyes and QDs, which involves simultaneous absorption of two or more photons through virtual states. 59 The efficiency of a UC process is generally several orders of magnitude higher than that of nonlinear multiphoton absorption, thus enabling UC to be produced by a low-cost continuous-wave (cw) diode laser instead of the need of ultrashort pulsed lasers for nonlinear multiphoton excitation. UCNPs have multiple attributes that make them well-suited for use in theranostics comprised of imaging, drug delivery, and therapy. Their unique frequency conversion capability is usually unavailable for existing endogenous and exogenous fluorophores, thus providing UCNPs numerous distinctive characteristics for medical diagnostics and therapy. For bioimaging, some of the advantages are virtually zero autofluorescence background to improve signal-to-noise ratio, large anti-Stokes shifts allowing us to easily separate the PL from the excitation wavelength, narrow emission bandwidths allowing ease of multiplexed imaging, and high resistance to photobleaching making it suitable for long-term repetitive imaging. In addition, UCNPs are nonblinking, less light scattering, and allow for deep tissue penetration because of excitation being in the NIR region that is within the optical transparency window. Moreover, a new direction for theranostic UCNPs utilizes hierarchically built nanostructures to combine UC PL imaging with other imaging modalities such as magnetic resonance imaging (MRI), 55 computed tomography (CT), 60 single-photon emission tomography (SPECT), 61 positron emission tomography (PET), 62 as well as with therapeutics of PTT, 63 PDT, 64 gene and drug delivery, 53 for in vitro and in vivo theranostics. Indeed, significant advances in theranostic UCNPs have recently been made by the use of nanochemistry that allows for nanocontrol of their optical properties to enhance upconversion at a selected wavelength, 36 surface modification for phase transfer, 65 and surface coupling chemistry for ligands that target biomarkers. 66 Figure 1 Schematic illustration of the structure of this Review. This Review aims to summarize recent progress in design and applications of UCNPs with an emphasis on the role of nanochemistry in the advancement of this field. The organization and the scope of this Review are represented in Figure 1. In section 1, we introduce the basic concepts of UC that are utilized in sections 2 and 3. Sections 2 and 3 describe recent advances in controlling optical properties needed to achieve high UC efficiency and tunabilty of output colors, respectively. Sections 4 and 5 are dedicated to nanochemistry for controlled synthesis and surface engineering of UCNPs. Sections 6–8 summarize recent advances in theranostic applications of UCNPs in biosensing and bioassays, high contrast bioimaging, and drug delivery and therapy, respectively. Finally, section 9 concludes the overall current status, challenges, and future perspectives. 1.1 Upconversion Nanoparticles (UCNPs) Lanthanide-doped UCNPs are dilute guest–host systems where trivalent lanthanide ions are dispersed as a guest in an appropriate dielectric host lattice with a dimension of less than 100 nm. The dopants of lanthanide are optically active centers, which produce emission when excited. Through judicious selection of lanthanide dopants, UCNPs can display wavelength (color) selective upconversion, such as NIR to shorter NIR, visible (blue, green, red), or UV. Generally, the UC PL arises from the 4f–4f orbital electronic transitions with concomitant wave functions localized within a single lanthanide ion. The shielding of 4f electrons by the outer complete 5s and 5p shells results in line-like sharp emissions, which exhibit high resistance to photobleaching, and photochemical degradation. These intra-4f electronic transitions of lanthanide ions are electric dipoles forbidden by quantum mechanical selection rules, which, however, are relaxed due to local crystal field-induced intermixing of the f states with higher electronic configurations. 67,68 The primary forbidden nature of the 4f–4f transition yields very long lifetimes (up to tens of millisecond) for energy levels of lanthanide ions, thus favoring the occurrence of sequential excitations in the excited states of a single lanthanide ion as well as permitting favorable ion–ion interactions in the excited states to allow energy transfers between two or more lanthanide ions. These resulting features of lanthanide dopants determine the basic UC mechanisms (see section 1.2). The UC PL intensity generally has a nonlinear dependence on the excitation light density: 69−71 1 where I UCPL is the photoluminescence intensity, P is the power of pump laser, K is material-related coefficient, and n is the number of the excitation photons required to produce the UC PL. The log–log relation of eq 1 is very useful to determine the “n” values for the UC PL peaks at very low excitation density. However, it should be noted that “n” values can be affected by the competition process between “the decay rate” and “the upconverted rate” at the intermediate states, leading to smaller “n” values (not integral numbers) at high excitation density. 69−71 This phenomenon is termed the “saturation effect”, which needs to be carefully avoided when determining the photon processes of UC PL peaks. Because of the nonlinear nature of UC PL, the upconversion quantum yield (UCQY) or the efficiency of a UCPL peak is strongly dependent on the excitation density. UCQY is defined as the ratio of the number of the emitted upconverted photons to the number of the absorbed NIR photons, according to the following formula: 2 where α is the absorption coefficient of the host material at the excitation wavelength. By inserting eq 1 into eq 2, it can be easily obtained that: 3 According to this equation, it is clear that UC PL involving different photon processes will have distinct dependence on the excitation power density. Because the dependence of the PL intensity on the excitation density can be easily measured for a given UC PL peak, the UC QY for a given peak at any excitation density can be determined by referencing to a quantified UCQY at one particular excitation density. UCNPs have optical properties similar to those of the corresponding bulk forms. They both generally produce the same UC PL peaks due to the well-shielded 4f–4f orbital electronic transitions by the outer complete 5s and 5p shells. However, the efficiency and the relative intensity between different UC PL peaks of UCNPs are quite different from their bulk counterpart due to nanosize-induced surface effects. Because of the high surface-to-volume ratio of UCNPs, most of the lanthanide dopants are exposed to surface deactivations (due to surface defects, as well as to ligands and solvents that possess high phonon energy). Surface-related deactivations encompass two ways: (i) dopants located on or around the nanoparticle surface can be deactivated directly by neighboring surface quenching centers; and (ii) the energy contained in dopants located in the center of UCNPs can randomly migrate and travel a long distance to the dopant on/around surface or directly to the surface quenching sites. In addition to the influence of phonons from the host lattice, the optical properties of UCNPs are significantly affected by the surface properties, which are not so pronounced in bulk crystals. Indeed, UCNPs are found to exhibit much lower efficiency than their bulk form due to surface-induced deactivations. To produce high efficiency UCNPs, appropriate nanochemistry for engineering the surface of these nanoparticles plays a pivotal role (see sections 2.5–2.6). UCNPs have intrinsic attributes such as nonblinking, nonphotobleaching, high chemical stability, sharp emission bands, NIR light excitation, and large anti-Stokes emissions, making them promising for theranostic applications. The UCNPs have to meet many key technical requirements for bioapplications: (i) The first is high efficiency and multicolor emissions. High efficiency is required for all UCNPs to accomplish improved limit of detection (LOD) in sensing (see section 6), high SBR bioimaging (see section 7), and increased therapy effect (see section 8). Single-wavelength excited multicolor emissions are of critical importance for multiplexing (simultaneously detecting multiple analytes/targets in a sample) capabilities in imaging or the throughput increase of bioassays. (ii) Next is monodispersed small size, uniform shape, and stoichiometric composition (see section 4). The monodispersed small size and uniform shape are required to have identical optical properties as well as cellular uptake and biological effects for intracellular theranostics, while a precise stoichiometric composition is necessary to allow control over the concentration of lanthanide dopants to manipulate the optical attributes. (iii) The third is a nanochemically engineered surface for phase transfer and for coupling to targeting ligands. The as-synthesized UCNPs are generally hydrophobic due to their capping by long-chain hydrophobic ligands. Engineering the surface of UCNPs to allow their dispersion in aqueous phase for biological applications is required. In addition, UCNPs lack the targeting moieties to identify regions of interest in the body such as angiogenic tumor area. Appropriate coupling chemistry is required to incorporate a range of antibodies, peptides, biotin, avidin, and proteins onto the nanoparticle surface. (iv) The final requirement is biocompatibility. UCNPs should be nontoxic and biocompatible to cells and to the body. 1.2 Upconversion Mechanisms As depicted in Figure 2a–e, there are five basic UC mechanisms encompassing (a) excited-state absorption (ESA), (b) energy transfer upconversion (ETU), (c) cooperative sensitization upconversion (CSU), (d) cross relaxation (CR), and (e) photon avalanche (PA). 69,72−74 The mechanism of cooperative upconversion luminescence is not included in this review, as it is an inefficient process that is not explored in UCNPs up to now. These five basic mechanisms are discussed below. 1.2.1 Excited-State Absorption Excited-state absorption (ESA) takes the form of successive absorption of pump photons by a single ion due to the ladder-like structure of a simple multilevel system as illustrated in Figure 2a by a three-level system for two sequential photon absorption. The occurrence of this mechanism is due to the equal separation from G to E1 and from E1 to E2 as well as the reservoir capability of the intermediate level E1. When an ion is excited from the ground state to the E1 level, another pump photon has a high possibility of promoting the ion from E1 to the higher-lying state E2 due to the long lifetime of E1 state, before its decay to the ground state. Consequently, upconverted emission will occur from the E2 level. To achieve highly efficient ESA, a ladder-like arrangement of the energy states of lanthanide is required. Only a few lanthanide ions such as Er3+, Ho3+, Tm3+, and Nd3+ have such energy level structures, 58 which also find a good excitation wavelength match with the output of commercially available diode lasers (at ∼975 and/or 808 nm). Figure 2 Principal UC processes for lanthanide-doped UCNPs: (a) excited-state absorption (ESA), (b) energy transfer upconversion (ETU), (c) cooperative sensitization upconversion (CSU), (d) cross relaxation (CR), and (e) photon avalanche (PA). The red, violet, and green lines represent photon excitation, energy transfer, and emission processes, respectively. 1.2.2 Energy Transfer Upconversion Energy transfer upconversion (ETU) in Figure 2b is quite different from ESA in Figure 2a, as ESA is operated within a single lanthanide ion, while ETU involves two neighboring ions. In an ETU process, an ion 1 known as the sensitizer is first excited from the ground state to its metastable level E1 by absorbing a pump photon; it then successively transfers its harvested energy to the ground-state G and the excited-state E1 of ion 2, known as the activator, exciting ion 2 to its upper emitting state E2, while sensitizer ion 1 relaxes back to ground-state G twice. The upconversion efficiency of an ETU process is sensitive to the average distance between the neighboring sensitizer-activator, which is determined by the concentrations of dopants. In contrast to ETU, the efficiency in an ESA process is independent of the dopant concentration due to its single ion characteristic. The ETU process is of crucial importance for UCNPs, as the most efficient theranostic UCNPs to date utilize ion pairs (sensitizer/activator) of Yb3+/Tm3+, Yb3+/Er3+, and Yb3+/Ho3+ for enhanced excitation at ∼975 nm. 36,75−82 It is worth noting that biological tissues have relatively small scattering and absorption at 975 nm, which is within the “optical transparency window” of tissue. Here, Yb3+ works perfectly as a sensitizer, as it has a sufficiently large absorption cross-section in the NIR region at ∼975 nm. Moreover, its optimized concentration can be kept high (20–100% for fluoride nanoparticles) without evoking deleterious cross-relaxations (see section 1.2.4) because of the fact that Yb3+ has an only two energy level structure. To date, most efforts have been devoted to develop Yb3+-sensitized UCNPs pumped at around 975 nm. Efficient ETU can also be observed in single lanthanide doped systems utilizing lanthanide ion itself as the sensitizer, for example, in Er3+-doped LiYF4 when excited at telecom wavelength of 1490 nm, 83 or in Ho3+-doped NaGdF4 nanoparticles when excited at 1200 nm. 84 The utilization of other sensitizers can be used to quench and enhance certain emission bands. For instance, Nd3+, Ce3+, and Ho3+ have been used as sensitizers to enhance the blue emission band of Tm3+, red emission band of Ho3+, and NIR emission band of Tm3+, respectively. 85−88 1.2.3 Cooperative Sensitization Upconversion Cooperative sensitization upconversion (CSU) in Figure 2c is a process involving the interaction of three ion centers, with ion 1 and ion 3 generally being of the same type. After absorbing excitation photons, both ion 1 and ion 3 can be excited to the excited state, respectively. Both ion 1 and ion 3 then can interact with ion 2 simultaneously, cooperatively transfer the contained energy, and excite ion 2 to a higher state. The excited ion 2 can relax back to its ground state by emitting an upconveted photon. The efficiency of CSU is generally orders of magnitude lower than the ESA or ETU process, as it involves quasi-virtual pair levels during transitions, which have to be described quantum mechanically in a higher order of perturbation. Despite this, the need for confined excitation to compensate the low efficiency may provide a possibility to achieve high-resolution imaging that is unavailable from the other UC mechanisms. The CSU mechanism has been reported for Yb3+/Tb3+, 89 Yb3+/Eu3+, 90 and Yb3+/Pr3+ ion pairs. 91 1.2.4 Cross Relaxation Cross relaxation (CR) in Figure 2d is an energy transfer process, resulting from ion–ion interaction in which ion 1 transfers part of its excited energy to ion 2 through a process of E2 (ion 1) + G (ion 2) → E1 (ion 1) + E1 (ion 2). Ion 1 and ion 2 can be either the same or different, and ion 2 can also be in its excited state in some cases. The CR process is a fundamental result of ion–ion interaction; its efficiency is in close relation with the dopant concentration. CR is the main reason for the well-known “concentration quenching mechanism” of emission, but it can be intentionally used to tune the color output in UCNPs or construct efficient photon avalanche mechanism (see section 1.2.5). 1.2.5 Photon Avalanche Photon avalanche (PA) in Figure 2e is a process that produces UC above a certain threshold of excitation power. Below the threshold, very little up-converted fluorescence is produced, while the PL intensity increases by orders of magnitude above the pump threshold. Actually, the PA is a looping process that involves processes of ESA for excitation light and an efficient CR that produces feedback. The level E1 of ion 2 is initially populated by nonresonant weak ground-state absorption. The looping process starts with the ESA process to elevate ion 2 at the level E1 to the emitting level E2. An efficient CR process of E2 (ion 2) + G (ion 1) → E1 (ion 2) + E1 (ion 1) between ion 1 and ion 2 then occurs. Last, ion 1 transfers its energy to ion 2 to populate its level E1, forming a complete loop. The net effect of the looping process is that one ion 2 at metastable E1 state produces two ion 2’s at this state. When the looping process ensues, two ion 2’s at the E1 state will produce four; four will produce eight, evoking an avalanche effect for populating ion 2 in its E1 state, and thus the PA UC from the emitting level of E2. It is easy to identify PA, as it generally requires a pump threshold and a long time (seconds) to build up. Moreover, the dependence of UC PL on the pump power becomes extremely strong around the threshold pumping power. 2 Architecting Upconversion Nanoparticles with High Efficiency Photon upconversion has been known and studied over a long period of time, which was generally based on bulk materials. An extensive review of the early work on bulk materials was given by Auzel in 2004 as well as recently by other reviews on specific topics of UCNPs. 46,51,52,57,58,92−107 However, none of the previous reviews provide a comprehensive review on the design, nanochemistry, and theranostic applications of UCNPs. It is not until recently that UCNPs became available for biomedical applications due to the success achieved in producing highly efficient UCNPs. The accomplishment of high efficiency is of particular importance, as it is the basic optical function of UCNPs to perform high performance theranostic application. For this reason, we first highlight in this section recent progress made on this aspect. Figure 3 displays five general strategies that have been employed to achieve high efficiency in UCNPs: (i) selection of novel host materials; (ii) tailoring local crystal field; (iii) plasmonic enhancement; (iv) engineering energy transfers; and (v) suppression of surface-related deactivations. Figure 3 General strategies to achieve the high efficiency of UCNPs. 2.1 Selection of Novel Host Materials Selection of appropriate host materials is essential for high efficiency UC emissions. Basically, an ideal host material should be transparent in the spectral range of interest, have high optical damage threshold, and be chemically stable. Moreover, the host lattice can affect the UC efficiency in two ways: (i) by the phonon dynamics, and (ii) by the local crystal field. The phonon-induced nonradiative process is the main loss mechanism for UC emissions; the luminescence efficiency is quite sensitive to the distribution of phonon density of states in UCNPs. A nonradiative process involves multiphonon-assisted relaxations whereby the energy difference between the higher and the lower energy level is converted into many lattice phonons. 92 A good indicator of how many phonons may be needed is the cutoff phonon energy of the host lattice. The host materials investigated in nanophosphors encompass oxides (e.g., Y2O3, cutoff phonon energy ∼550 cm–1; ZrO2, cutoff phonon energy ∼500 cm–1), 108,109 fluorides (e.g., NaYF4, cutoff phonon energy ∼350 cm–1), 110 vanadates (e.g., YVO4, cutoff phonon energy ∼890 cm–1), 111 oxysulfide (e.g., Y2O2S, cutoff phonon energy ∼520 cm–1), 112 and oxy-fluorides or -chlorides (e.g., GdOCl, cutoff photon energy ∼500 cm–1). 113 Generally, the larger is the number of phonons needed to convert the excitation energy into phonon energy, the lower is the efficiency of the nonradiative process. Hence, to enhance the emission efficiency by reducing nonradiative rate, it is desirable to have the lanthanide ions incorporated into a dielectric host of very low frequency phonons. Among investigated hosts, fluoride materials have the lowest phonon cutoff energy and generally exhibit the highest UC efficiency due to the minimization of nonradiative losses in the intermediate states or the emitting states. The crystal field of a host material has a profound effect on the UC efficiency of lanthanide emitters, as the local crystal symmetry surrounding a lanthanide ion strongly influences its optical properties. A less symmetric crystal phase is generally favorable for the UC efficiency, as the intermixing of the lanthanide ion’s f states with higher electronic configurations can be more manifested. For example, hexagonal NaYF4:Yb3+/Er3+ microcrystals exhibit visible upconversion PL, which is 4.4 times higher than its cubic counterparts, 77 and monoclinic ZrO2 nanoparticles emit higher UC PL than the tetragonal phase nanoparticles. 109 Selection of an appropriate host lattice with a low phonon cutoff energy and low crystal field sym