Recent advances of upconversion nanoparticles in theranostics and bioimaging applications

Journal Pre-proof Recent advances of upconversion nanoparticles in theranostics and bioimaging applications Rafia Rafique, Suresh Kumar Kailasa, Tae Jung Park PII:

S0165-9936(19)30316-4

DOI:

https://doi.org/10.1016/j.trac.2019.115646

Reference:

TRAC 115646

To appear in:

Trends in Analytical Chemistry

Received Date: 21 May 2019 Revised Date:

24 July 2019

Accepted Date: 24 August 2019

Please cite this article as: R. Rafique, S.K. Kailasa, T.J. Park, Recent advances of upconversion nanoparticles in theranostics and bioimaging applications, Trends in Analytical Chemistry, https:// doi.org/10.1016/j.trac.2019.115646. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. All rights reserved.

Graphical Abstract

Schematic diagram of theranostics and bioimaging using the upconversion nanoparticles modified with various polymer/ligands.

Recent advances of upconversion nanoparticles in theranostics and bioimaging applications

Rafia Rafiquea, Suresh Kumar Kailasab and Tae Jung Parka*

a

Department of Chemistry, Institute of Interdisciplinary Convergence Research, Chung-Ang

University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea b

Applied Chemistry Department, Sardar Vallabhbhai National Institute of Technology, Surat-395

007, India

*Corresponding author, Tel. +82-2-820-5220; Fax. +82-2-825-4736 E-mail address: [email protected] (Tae Jung Park)

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ABSTRACT In recent years, upconversion nanoparticles (UCNPs) have attracted considerable research interest because of their unique optical merits (unique optical merits: sharp emissions, large anti-Stokes shifts, long lifetimes, and negligible photobleaching) that make them as ideal candidates for unified applications in biomedical fields including drug delivery, bioimaging, and cancer therapy. In this review, we illustrate the recent efforts on fabrication of UCNPs with organic, inorganic and biomolecules that create UCNPs as the next generation biomedical agents for drug delivery and cancer therapy. The introduction summarizes the recent developments and uses of UCNPs in analytical and biomedical research areas. We discuss the optical properties and the surface modifications of UCNPs and their synergistic theranostic applications in cancer therapies (chemo and photothermal) in vivo and in vitro studies. Further, we also discuss the cytotoxicity of UCNPs. Finally, the prospects and future challenges of UCNPs in drug delivery, bioimaging and cancer therapy are described.

Keywords: Surface chemistry; Upconversion nanoparticles; Optical properties; Drug delivery; Cancer therapy; In vitro and in vivo studies.

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Table of contents 1. Introduction 2. Optical properties of UCNPs 3. Cellular internalization mechanism of UCNPs by various cell lines 4. Tuning of upconversion photoluminescence for effective bioimaging 4.1. Influence of UCNPs lifetime for constructing imaging nanoprobes 5. Effect of ligand chemistry on UCNPs for in vitro drug delivery and biomedical applications 5.1. Polymer-coated UCNPs 5.2. Silica-coated UCNPs 5.3. Metal oxide-coated UCNPs 5.4. Functionalization of UCNPs with different molecules 5.5. Surface modification of UCNPs with various coatings 5.6. Modification of UCNPs with different dopant ions 6. In vivo biomedical applications of UCNPs 6.1. UCNPs-based NIR-responsive phototherapies 6.2. UCNPs-based pH-responsive chemo- and phototherapies 7. Toxicity assessment 8. Conclusions and future challenges

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Abbreviations ATP AuNPs AuNRs AlPC BPS BSA C18PMH Ca2+ Ce6 cF CNQDs Co2+ CSLM CT CTAC CuS CuxS Cy7 DA.HCl Dextran-g-DOPE DMMA DNA DOPE DOX DSPE-PEG-2000 DOTAP DTPA EBNA1 EBV EDTMP EIPA EPR FA Fe3O4 FRET GND GQD HA HMSC HPG ICG ICP-MS LRET MB MC540 Mn2+

Adenosine triphosphate Gold nanoparticles Gold nanorods Aluminum phthalocyanine chloride Black phosphorus sheets Bovine serum albumin PEG-grafted poly(maleic anhydride-alt-1- octadecene) polymer Calcium ion Chlorin e6 Caged fluorescein Carbon nitride quantum dots Cobalt ion Confocal scanning laser microscopy Computed tomography Cetyltrimethylammonium chloride Copper sulfide Copper chalcogenides Cyanine 7 4-(2-aminoethyl)-1,2- benzenediol hydrochloride Dextran-grafted L-α-phosphatidylethanolamine, dioleoyl Dimethylmaleic anhydride Deoxyribonucleic acid L-α-phosphatidylethanolamine, dioleoyl Doxorubicin (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethylene glycol)-2000]) 1,2-dioleoyl-3- trimethylammonium propane Diethylenetriaminepentaacetic acid dianhydride Epstein–Barr nuclear antigen 1 Epstein–Barr virus Ethylenediamine tetra(methylenephosphonic acid) Ethylisopropylamiloride Enhanced permeability and retention Folic acid Iron oxide Forster resonance energy transfer Gold nanodots Graphene quantum dot Hyaluronic acid Hollow mesoporous silica capsule Hyperbranched polyglycerol Indocyanine green Inductively coupled plasma mass spectrometry luminescence resonance energy transfer Methylene blue Merocyanine 540 Manganese ion 4

MRI mSiO2 MPPA MTT Nd NIR NOBF4 OM P4 PAA PAH PAH PDT PEG PEI PET P-gp pHLIP PL PLGA PLL PN PNP POM Ppa PSs PS-b-PAA Pt(IV) PTT PVP RB RGD ROS SA SDT SPR TAT TDPA-Zn2+ TiO2 TPGS TL-CPT TMB TPP TRITC UC UCNPs ZnPc ZwitLipo β-CD

Magnetic resonance imaging Mesoporous silica Pyropheophorbide-a methyl ester 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Neodymium Near infrared Nitrosonium tetrafluoroborate Oleylamine EBNA1-targeting peptide Polyacrylic acid Polycyclic aromatic hydrocarbon Poly-(allylamine hydrochloride) Photodynamic therapy Polyethylene glycol Polyethylenimine Positron emission tomography P-glycoprotein pH low insertion peptide Photoluminescence Poly(lactic-co-glycolic acid) Poly-L-lysine Perinaphthenone PEG-NMAB(S-(o-Nitro-m-methoxy-pazide Alkoxy Benzyl Ester)- PLA(polylactic acid) Polyoxometalate Pyropheophorbide Photosensitizers polystyrene-block-poly(acrylic acid) Platinum(IV) Photothermal therapy Polyvinylpyrrolidone Rose Bengal Arg-Gly-Asp Reactive oxygen species Succinic anhydride Sonodynamic therapy Surface plasmon resonance Transactivator of transcription Zinc-dipicolylamine Titanium dioxide Tocopheryl PEG 100 succinate Thioketal linker camptothecin Mesitylene Triphenylphosphine 5(6)-tetramethyl-rhodamine isothiocyanate Upconversion Upconversion nanoparticles Zinc phthalocyanine Zwitterionic phospholipids β-Cyclodextrin 5

A549 CAL-27 CT-26wt H22 HeLa HepG2 HGC KB L929 MCF-7 MRC-5 NCI-H460 NPC43 OECM-1 OSCC THP-1 U87MG

Human lung adenocarcinoma epithelial Centre Antoine Lacassagne-27 Colon carcinoma cell lines Murine hepatocarcinoma cell line Human cervical carcinoma Hepatocellular carcinoma cells Human gingival cells (normal) KERATIN-forming tumor cells National Collection of Type Cultures clone 929, clone of strain L (fibroblast cells) Michigan Cancer Foundation-7 Human normal lung fibroblast Human lung cancer cell lines Nasopharyngeal carcinoma Human oral epidermoid carcinoma Meng-1 Oral squamous cell carcinoma Human myelomonocytic cell line Uppsala 87 malignant glioma

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1. Introduction Usually, theranostics is a single platform concept that involves imaging and therapy using nanomaterials as theranostic agents, which plays an essential role in generating personalized medicine to various diseases [1]. The main function of theranostic agents is to examine and report the status of disease during the specific treatment [2]. Recently, theranostics has proven to be a versatile platform in cancer research [3]. Nanomaterials in theranostics provide the following advantages (i) the growth of tumor that can be hampered during diagnostic treatment, and (ii) the subsequent tumor treatment would be simplified due to the reduction of tumor size and growth. Importantly, theranostic agents exhibited several features including (i) evaluation of tumor resection and post-surgery by imaging, (ii) visualization of tumor infected areas, which is more important for surgery with high precision, (iii) delivery of therapeutic agents (drugs - doxorubicin, cisplatin, and paclitaxel; biomolecules – antibodies and protein drugs; gene-based agents - siRNA, DNA, and miRNA), respectively [4,5]. Further, tumor cells were greatly destructed using nanomaterials as theranostic agents via either light-induced release/therapy (photodynamic therapy) or heat activated process (photothermal therapy), resulting to disrupt the structure of tumor cells and to shrink the volume of tumor, which ultimately inhibits the metabolic pathways in cancer cells [6-9]. The theranostics applications of nanomaterials were greatly improved by modifying their morphology, surface chemistry and optical properties [1,2,10,11]. In view of this, nanomaterials, such as semiconductor nanocrystals [12], metal complexes [13-15], surface modified silica [16,17] and organic dyes [18,19] have been used as probes for diagnosis of cancer. These materials have also been used as imaging probes for visualization of cancer cells, offering an accurate cancer diagnosis via real-time, non-invasive and highresolution images, which allows to capture specific information that relates to the tumor metabolism and structure [20]. Unfortunately, these materials have limitations toxic nature 7

due to the presence of heavy metals, poor signal-to-background ratio by either autofluorescence or strong light scattering from the tissues at short wavelengths; inability to penetrate into the tissues due to their optical properties in UV and visible range (excitation and emission) and significant damage to DNA and normal cell death due to the exposure of UV light with shorter wavelength for long time, which restrict their promising applications to in vitro or in vivo studies [21,22]. To overcome these issues, nanomaterials with near-infrared (700–1100 nm) excitation properties have proven to be promising theranostic agents in cancer therapy due to their negligible autofluorescence, phototoxicity, and deep penetration into the tissues without any tissues damage [20,23]. In the past few years, UCNPs have received considerable attention in various research fields because of their remarkable properties (short emission wavelengths, high ability to absorb NIR light, narrow emission bands, negligible auto-fluorescence, and low toxicity), which renders them more appealing optical nanomaterials than conventional fluorescence nanomaterials for various bioapplications [23]. Lanthanide-based UCNPs exhibit a distinct process in which low-energy light (excitation) is converted to higher-energy light to generate anti-Stokes emission via sequential absorption of photons [24-26]. Recently, UCNPs have been extensively employed as promising theranostic agents for bioimaging, cancer diagnostics, and therapy [27-29]. More importantly, UCNPs have shown high ability to absorb NIR laser that allow efficient visualization of cancer tissues with negligible photodamage to cells as compared to UV light absorbing nanomaterials. To date, UCNP-coated nanocomposites have been extensively used as theranostic agents for cytotoxicity, in vitro tumor-targeted imaging, and cellular internalization or tracking studies [1]. In view of the rapid advancements in this field, it is important to summarize recent studies pertaining to the effect of surface chemistry, doped elements in

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UCNPs that play key role for bioimaging, drug delivery in vitro and in vivo cellular studies and cancer therapy (PTT and PDT) [1,17,28]. To support this, recently several reviews have been summarized the potential application of UCNPs in sensing, bioimaging, drug delivery, in vivo and in vitro studies, PDT and PPT [30-34]. These reviews have well summarized about the biosensing, bioimaging and drug delivery applications of UCNPs. UCNPs have been fabricated with various coating groups and structures such as biomolecules, PSs, polymers, inorganic nanoparticles, and anticancer drugs for use in cancer therapy [25,35]. The concept of UCNPs-based biomedical-oriented research has emerged as an promising materials for various applications because of their advantages: (i) they show high stability and dispersibility in solvents over a large pH range, (ii) they can be easily functionalized with desired biomolecules, (iii) they exhibit minimal nonspecific adsorption and show anticancer activity, (iv) perturbation of the native molecules in the biological environment by the UCNPs and coated groups is negligible, and (v) they can be used for efficient UC luminescence imaging. Thus, there is a necessity to summarize the recent reports on the modification of UCNPs with wide variety ligands and their applications in bioimaging, cancer therapy (PDT and PTT), in vivo and in vitro studies, which significantly promote the use of UCNPs in theranostic applications. In this review, we summarize the recent reports on the modifications of UCNPs using organic, inorganic and biomolecules in bioimaging, and theranostic applications. Section 2 illustrates the optical properties of UCNPs. Section 3 summarizes the internalization of UCNPs by various cell lines. Tuning of PL of UCNPs for bioimaging applications is discussed in Section 4. The role of surface chemistry on UCNPs for in vitro drug delivery and biomedical applications is overviewed in Section 5. Section 6 summarizes the in vivo studies, NIR- and pH-responsive cancer therapies using UCNPs as theranostics agents. Section 7

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explores the cytotoxicity of UCNPs. Finally, we discuss the prospects and future challenges of UCNPs in biomedical fields.

2. Optical properties of UCNPs UCNPs are generally fabricated by using three precursors such as inorganic host matrix, sensitizer ions, and activator ions that exhibit distinctive emission and UC PL. Rare earth elements with inorganic composition (e.g., NaYF4, NaErF4, NaGdF4, NaLuGdF4, Y2O3, Y2O2S, and GdOCl) act as promising host nanocomposites because of their outstanding properties such as low phonon energy, high stability (chemical and thermal), and high transparency [1]. Moreover, Yb3+ ions, which show high two-photon absorption (~980 nm, 2

F7/2 → 2F5/2), and other rare earth elements (Er3+, Tm3+, Ho3+, Tb3+, Eu3+, Dy3+, Sm3+, and

Gd3+) have been effectively used as an efficient sensitizer and activators for the synthesis of UCNPs, respectively [1,36,37]. In particular, energy is effectively transferred from Yb3+ ions to activator ions upon excitation with a laser of 980 nm [38]. UCNPs have also been fabricated using Nd3+ ion as a sensitizer; these ions facilitate the energy transfer process [4F3/2 (Nd3+) → 2F5/2 (Yb3+) → activator] when excited with wavelength of 808 nm light [39-41]. The degree of energy transfer can be increased by controlling the inorganic host matrix, sensitizer ions, and activator ions in UCNPs at excitation wavelengths of 808 and 980 nm [26,32]. Notably, the emission colors of UCNPs can be significantly controlled by optimizing the concentration of sensitizers and activators [33] and by incorporating other metal ions [3436]. Importantly, UCNPs exhibited colorful emissions, which make them as promising materials for bioapplications [36]. Several research groups have explored the use of UCNPs containing other lanthanide ions (Gd3+, Yb3+, Ho3+, and Sm3+) as contrast probes for specific features in MRI, CT, and PET for the evaluation of biological tissues [42-45]. These great

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optical features render the UCNPs well-suited candidates for the diagnosis of various diseases and in tracing drug molecules in tissues. Bioimaging is an integral part for monitoring of tumor progression and assessment in cancer diagnosis. For example, MRI, CT, and PET techniques have been recognized as promising diagnostics tools for cancer patient management [46]. Over the last few decades, lanthanide-doped UCNPs have also been extensively utilized as potential bioimaging agents in various imaging techniques such as fluorescence [47], ultrasound [48], Raman [49], and multimodality imaging [50,51] for imaging of various biological tissues. This review is intended to provide details on the use of UCNPs as promising candidates for PL imaging of cancer cells. PL imaging techniques have good spatial resolution but low tissue penetration depth because of signal attenuation. The use of UCNPs involves NIR light, which causes less photodamage and penetrates deep. Moreover, NIR light exhibits low phototoxicity, autofluorescence, and light scattering [52-54]. Recently, several reports have illustrated that the use of UCNPs in imaging of biological tissues can facilitate multimodal imaging for cancer detection, unlike other PL imaging agents such as quantum dots [55,56]. For the enhancement of their imaging capability, UCNPs have been functionalized with different polymers and molecules and doped with different ions [1,23]. Table 1 presents the optical properties and in vitro applications of UCNPs with different compositions. The studies revealed that the optical properties and biocompatibility of UCNPs were greatly tuned by either modifying their surfaces with organic/inorganic materials or doping with different metal ions. For example, Gd3+ ions were incorporated into UCNPs for serving as T1-based MRI contrast agents in the imaging of cancer tissues [1]. To enhance the PL and MRI capabilities of the Gd3+-incorporated nanostructures, various rare earth elements such as Er3+, Yb3+, and Tm3+ were co-doped into the Gd3+-based host lattice, offering efficient UC PL and MRI.

Furthermore,

cisplatin

was

introduced 11

into

the

silica

shell

of

UCNPs

(NaYF4:Yb3+/Er3+@NaGdF4) for the implementation of radiosensitization-based synergistic chemo-/radiotherapy for tumors [57]. Superparamagnetic Fe3O4 NPs were coated on the surface of lanthanide-dopedUCNPs for effective UC PL and T2-enhanced MRI imaging [58]. Cheng’s group fabricated a NaYF4:Yb3+/Er3+@Fe3O4@Au nanostructured platform with several optical and magnetic properties,

for

multimodality

imaging

[58].

Xia

and

co-workers

prepared

NaYF4:Yb3+/Tm3+@FexOy core-shell nanostructured materials for T2 MRI and UC PL bimodal lymphatic imaging [59]. The imaging data showed that UCNPs act as potential imaging contrast agents for guiding clinical lymph nodal studies and diagnosis in the absence of skin surgery. Zhu’s group synthesized multifunctional Fe3O4@NaLuF4:Yb3+, Er3+/Tm3+ core-shell NPs for in vivo and in vitro multimodal imaging studies [60]. In vitro studies confirmed that the as-prepared UCNPs were noncytotoxic in nature, suggesting that the UCNPs can be used as a multifunctional probe for trimodality imaging (MRI, CT, and UC PL). These studies revealed that the modification of UCNPs surfaces with organic ligands or inorganic compounds could enhance the optical and magnetic properties, which make them as potential candidates for cellular internalization with enhanced UC PL intensity. Similarly,

ICG

was

incorporated

into

core-shell

structure

of

UCNPs

(NaYF4:Yb3+,X3+@NaYbF4@NaYF4:Nd3+ (X = null, Er3+, Ho3+, Tm3+, or Pr3+) for enhanced PL properties [61]. The authors used ICG as an antenna to capture the energy and transfer to Nd3+ and Yb3+ initially and then to Yb3+ and X3+ for enhanced UC luminescence. They also modified the UCNPs surfaces with an amphiphilic polymer DSPE-PEG-2000 for high dispersion in water, which allow them to use as probes for effective internalization of cells and for strong and clear UC PL emission when excited at 800 nm laser. Another study explored that the use of Cy7 as an antenna for producing dye-sensitized UC PL via sequential

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energy transfer from Cy7 to Nd3+-Nd3+-Yb3+-Er3+ [62]. It was observed that UC PL increased 17 times when excited at 800 nm laser and used as probes for imaging living HeLa cells and the lymph of mice. Wu et al. [63] described that the use of IR-806 as an antenna for broadening the absorption range and boosting the UC PL efficiency of UCNPs. They doped Yb3+ into the core-shell structure of UCNPs, facilitating energy transfer from the dye to UCNP core. The authors then modified the surface of core-shell UCNPs with Pluronic F-127, which increased the dispersion capability of UCNPs in water, rendering them as promising candidates for in vivo studies.

3. Cellular internalization mechanism of UCNPs by various cell lines Previous reports [64,65] have described that cellular internalization of UCNPs by cells are based on the nanoparticle-cell interactions via different receptor-mediated endocytosis mechanisms, namely, clathrin, caveolae, and micropinocytosis. Conventional (neutral, anionic), cationic, and pH-sensitive UCNPs usually follow the clathrin-receptordependent endocytic pathway for the efficient internalization [66] and release of embedded drugs in the acidic environment of early endosomes (pH ≈ 6.5), late endosomes (pH ≈ 5.5), and lysosomes (pH ≈ 4.5), as shown in Fig. 1. The endocytic pathway allows those UCNPs that display optimum size (nm) and a rational surface design to internalize with receptordirected ligands (i.e., FA). The internalization enhances the cell imaging capability, drug delivery capability, and therapeutic efficiency of the UCNPs [67,68]. For example, Lv et al. [69] tuned the size of Y2O3:Yb3+, Er3+-CuxS-DOX NPs to 90, 150, and 200 nm and determined their cellular uptake and intranuclear drug accumulation properties. The CSLM results of HeLa cells showed stronger UC signals when the cells were incubated with 90 nm nanoparticles in comparison with incubation with large-sized particles, suggesting the size-

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dependence of cellular internalization. Further, the concentration of UCNPs and incubation time also play an important role in determining the effective tissue penetration by the nanoparticles through the endocytosis process in both the nucleus and cytoplasm of cells [69,70]. The internalization pathways of as-prepared UCNPs in MCF-7 cell lines were investigated [71] by using different endocytosis inhibitors such as EIPA, micropinocytosismediated [72], chlorpromazine (clathrin-mediated), and filipin (caveolae-mediated) [73]. The UC intensity was not changed by the EIPA-treated cell lines, indicating the endocytosis process was not micropinocytosis-mediated. However, the cellular fluorescence intensity was significantly decreased when treated with other inhibitors, suggesting nanoparticle internalization in the clathrin- and caveolae-mediated pathways [71]. The surface charges of as-prepared UCNPs affect their ionic interaction with cells [74]. One study revealed that positively charged UCNP-PEI has higher affinity to be taken up by cancer cells as compared to UCNP-PVP (neutral charge) and UCNP-PAA (negative charge). By employing clathrin- or caveolae-expressed plasmids and their chemical inhibitors, the authors further confirmed that the cellular internalization route of UCNP-PEI was either clathrin- or caveolae-mediated endocytosis, which was confirmed by time lapse or ICP-MS studies, respectively. These observations suggested that PEI coated UCNPs were entered in the HeLa cell mainly through the clathrin-mediated endocytosis process [74]. UCNPs following this pathway are destined for targeting cancerous tissues through lysosomal accumulation due to tumor acidic environment, which is followed by degradation of the particles (HMSCs-C@Gd2O3:Yb3+/Tm3+-DOX) [75]. During these processes, first, the endocytic vesicle forms through membrane invagination and transforms into the early endosome to late endosome for the entire nanoparticle digestion, while the endosome transforms into the lysosome for the lysosomal path followers, where they disassemble under enzymatic degradation [76-78]. Polymer-coated UCNPs can respond to the tumor 14

extracellular and intracellular pH gradients through chemically defined mechanisms because of different surface charges, which can efficiently promote cell internalization [79] and drug accumulation at the tumor site via either enhanced permeability and retention effect [80] or the endocytosis process [81], as apparent in Fig. 1. The cell membranes are usually negatively charged, and therefore, the positively charged UCNPs can quickly internalize through the counter charge effect [82], as shown in Fig. 1. The negatively charged UCNPs are commonly internalized through the endocytosis-mediated receptors, and inside the highly protonated tumor extracellular microenvironment, showing charge conversion behavior that leads to change their surface charge to positive for efficient internalization and accumulation in cancerous cells [48,66]. Tumors possess a very complex and distinct physiological environment that resists therapeutic approaches. To overcome this barrier, UCNPs have been grafted with specific ligands such as antibodies/peptides, namely, anti-MUC1, bispecific (BsAb), and anti-EpCAM antibody for tyrosine kinase, EphA2, and EpCAM receptors overexpressed in different cancer cell lines, TAT and TCP1 peptides for binding the α- and β-receptors present on the cell nucleus [83-86], folate [87], and glucose [88,89], respectively. This facilitates the quick internalization the anticancer carrier through a receptor-dependent or receptor-independent endocytic pathway. These targeted molecules can reside inside cells because of the high affinity and specific recognition between ligands and receptors (as mentioned above) on cancer cell lines through the endosomal targeted pathway for the disassembly of the ligand layer and the release of internal content. Ligand-targeted internalization occurs more rapidly as compared to untargeted internalization because these receptors are upregulated in cancer cells [90]. Suitable targeting agents (ligands, polymers, or biomolecules) could then enter the lysosomes or endosomes through controlled delivery to improve conditions related to these discrete organelles, including cancer cells [90]. A literature review revealed that the surface 15

coating, composition, and size of UCNPs play a significant role in their cellular entry through the plasmon membrane and the corresponding endocytosis pathways, not only for the purpose of targeted cell labeling but also for enhancing therapeutic effectiveness through controlled drug delivery.

4. Tuning of upconversion photoluminescence for effective bioimaging Considerable effort has been devoted to the enhancement of PL properties of UCNPs [91-93]. Toward this end, in one study, CaF2 was doped into UCNPs for enhancing PL intensities by reducing multi-phonon de-excitation, which improves energy transfer rates [54, 94-96]. It was noticed that the tissue depth penetration was greatly improved by doping of Tm3+ ion into UCNPs, exhibiting 4 times higher tissue depth penetration and emission peak as compared to CaF2:Er3+/Yb3+ [96]. Similarly, the high PL quantum yield was achieved by suppressing surface defects and the cross relaxation between UCNPs and doped ions [97,98]. Briefly, core/shell UCNPs (NaGdF4:Nd3+/NaGdF4) were synthesized with high PL quantum yields [97]. The NIR-to-NIR downconversion PL peaks were observed at 900, 1050, and 1300 nm when excited at 740 nm. The as-synthesized UCNPs act as promising bioimaging agents for in vitro and in vivo investigations at NIR region, showing higher biological optical transparency window, spectral sharpness, and high photostability, which renders them as extremely promising optical probes for bioimaging applications. Silica-coated UCNPs doped with Gd3+ ions have shown good performance in cell labeling, optical bioimaging, and MRI [99]. NaGdF4-doped UCNPs are one of the best host lattices because of high positive contrast and UC PL capability, which makes them potentially suitable for multimodal imaging. Sometimes, uncoated UCNPs, irrespective of their composition, size, and shape, can be degraded in an acidic cellular environment [54]. This can not only reduce their luminescence

16

capabilities for imaging but also generate proinflammatory effects. To overcome this issue, EDTMP was introduced as promising ligand for the strong complexation with lanthanide ions, thereby preserving the fluorescence capabilities of UCNPs and preventing inflammatory effects in the tumor environment [100]. Ultra-small UCNPs show poor luminescence because of fewer emitters, more lattice defects, and vibration energy diffusion [1]. To overcome these drawbacks, Mn2+ ions were doped into a UCNP system that exhibited strong UC luminescence [S1]. The UC luminescence spectra of doped UCNPs were ≈59.1 times and ≈39.3 times greater than those of undoped UCNPs (NaLuF4 and NaYbF4) [S1]. The high UC luminescence was found to be highly beneficial for imaging of HeLa cells (100 µg/mL), and exhibited deep tissue penetration capability, high sensitivity, and negligible autofluorescence. In one study, NaGdF4 was used as a core for UC luminescence and AuNPs were used as strong contrast agents [S2]. The authors evaluated the bioimaging capability of NaGdF4:Er,Yb/silica/AuNPs on HeLa cells. The presence of AuNPs endowed the nanoparticles with plasmonic properties, which facilitated their uptake by cells, and a strong contrast signal was obtained in the confocal reflection imaging mode upon excitation with a 514-nm laser. Furthermore, strong luminescence cellular imaging was also observed upon excitation with a 975-nm laser because of the presence of UCNP core in NaGdF4:Er,Yb/silica/AuNPs. HeLa cells displayed red and green emission colors after incubation with NaGdF4:Er3+/Yb3+/silica/AuNPs and UCNPs [S2]. It was revealed that Nd3+ ions exhibited a stronger PL intensity than Yb3+ ions because of their higher absorption cross section, and therefore, they captured a larger number of excitation photons [S3]. Similarly, doping of Mn2+ ions into UCNPs was remarkably enhanced the UC luminescence intensity, especially in red emission bands [S4]. These enhanced optical properties are due to the crystal size control that facilitate changes in the phase structure of UCNPs. These results confirmed that the UC PL of UCNPs could be 17

considerably enhanced either by doping various metal ions or through surface modifications, leading to a larger number of UCNPs being internalized by cells, which allows to generate high-resolution cell images. The modified UCNPs exhibited higher NIR laser absorption capability, which exhibits several advantages including high resolution images, multicolor and multimodal imaging, negligible autofluorescence, deeper penetration ability, and reduces cell damage, respectively.

4.1. Influence of UCNPs lifetime for constructing imaging nanoprobes FRET has become one of the important techniques to study various biomolecular interactions in biochemical, and physical laboratories. These assays abled to generate farfield signals through light emissions using probe, which further captured by a sensor to observe biological phenomenon [S5]. The development of UCNPs as optical probes have greatly improved the FRET technique capability due to larger Stokes shift, photo-stability, tunable and narrow emission bands. In this system, UCNPs served as an energy donor, and different kinds of fluorescent or non-fluorescent materials were functionalized on the surface of UCNPs to quench/convert the upconverting emissions [S6,S7]. The critical distance for the successful energy transfer should be within 10 nm [S6,S8]. UCNPs contain several Ln3+ ions as emitting centers in each particle, and an optimized distance lead to high energy transfer and signal-to-background noise ratio. Up to now, several studies have been studied the effect of size, doping of metal ions and surface chemistry for generation of UCNPs with high radiative quantum efficiency, which can enhance the lifetimes of UCNPs by reducing surface defects [S9-S13]. Lanthanide-doped UCNPs enjoyed long lifetime arising from 4f−4f electronic transition states due to occurrence of sequential excitation within single lanthanide ion as 18

well as favoring ion−ion interaction in the excited state to permit successful energy transfer between lanthanide ions [S14]. The decay time kinetics of UCNPs is one of the important features of its temporal characteristics, not only for their characterization and optimization, but also for their applications [S15]. The lanthanides decay time is about 103-fold and 106fold longer than autofluorescent biological samples and other fluorescent molecules (organic dyes and fluorescent proteins) [S16]. This spectral feature of UCNPs were significantly improved by modifying their surfaces and changing the doping elements, which enhances signal-to-noise ratio and detection/imaging sensitivity with low background fluorescence [S5]. In one study, efficient energy transfer has been noticed in the core-shell UCNPs (NaYbF4:Gd3+, Tm3+@NaGdF4@CaF2:Ce3+) when excited at 980 nm laser [S17]. The CaF2 shell can be exploited to fine-tune of 4f-5d transitions in the Ce3+ ion as a dopant. The energy transfer was occurred from Gd3+ to Ce3+ at the core-shell interface, which lead to generate UCL emission with long lifetime in the UV region. In another study, benzene-bridge rattle organosilica coated (ROS)-UCNPs (NaLuF4: Gd3+, Yb3+, Er3+) exhibited dominant red (654 nm) and green (541 nm) emissions under 980 nm laser excitation [S18]. The UCNPs with aromatic bridging group and rattle structure shell have shown several features such as efficient energy transfer to enhance ZnPC (PS) storage, avoid aggregation, and controlled energy transfer. This results to decrease distance between PS and UCNPs, which not only improve the UC/CT imaging but also promote the PDT efficacy. In this system, UCL lifetime at 654 nm was significantly decreased (98%) due to the energy transfer from UCNP to ZnPC whereas UCL lifetime at 541 nm was decreased to some extent from 0.14 to 0.13 ms, which could be utilized for imaging. These excellent results were achieved by optimizing the distance between UCNP@ROS and ZnPC, thereby allowing efficient radiative or/and nonradiative energy transfer processes for PDT and bioimaging [S18]. The life-time decay (radiative and non-radiative) was enhanced by optimizing various parameters (dopant levels, 19

magnetic or electronic field, temperature, pressure, and solvent or by intrinsic material) [S19S24]. Based on the above observations, optical properties and lifetime of UCNPs were greatly improved by optimizing various parameters, surface chemistry and doping elements.

5. Effect of ligand chemistry on UCNPs for in vitro drug delivery and biomedical applications Recently, the use of UCNPs in biomedical research has increased considerably because of their remarkable properties including biocompatibility, long lifetime, multicolor emissions and high water dispersibility, respectively. Several research groups have been investigated their potential long-term risks at the cellular and animal level after interaction with the cell membrane, nucleus, or mitochondria [S25,S26]. Adverse effects including the death of healthy cells, perturbation in physiological conditions, and protein regulation or mutagenesis have been investigated on various cells [S27,S28]. Therefore, it is imperative to perform toxicological studies in vitro before the in vivo or clinical applications of UCNPs. Numerous studies have been evaluated the biocompatibility of UCNPs in in vitro cytotoxicity by MTT assay. The MTT assay was evaluated by measuring the enzymatic activity of mitochondria in cells using spectrophotometry [S29]. Although these assays may not predict the in vivo cytotoxicity accurately, they can provide general information on the cellular internalization and cytotoxicity mechanisms of nanoparticles. Herein, we briefly discuss the surface modified UCNPs with different molecules (PSs, polymers, inorganic nanoparticles, and anticancer drugs) for assessing their biocompatibility, imaging capability, and anticancer effects, as shown in Scheme 1.

5.1. Polymer-coated UCNPs 20

UCNPs are widely considered to be the most promising nanoplatform for various biomedical applications due to their outstanding properties [S30]. However, the short-term colloidal stability and limited water dispersibility of as-prepared nanoparticles considerably limit their use in the real world [S31]. In recent years, UCNPs have been coated with different polymers such as PAA, PEI, PVP, and PEG to overcome the above-mentioned drawbacks and to endow them with attractive properties, including biocompatibility, different surface charges, hydrophilicity, and long-term colloidal stability [74,S31]. For example, Jin’s group coated three polymers (PAA, PEI, and PVP) on the surfaces of UCNPs and studied their cytotoxicity on various cell lines (U87MG and HeLa cells) [74]. They observed that the PAA-coated UCNPs had no major impact on the cell toxicity even at high concentration (500 µg/mL), whereas PEI- and PVP-coated UCNPs were significantly reduced cell viability to 70%–80% at the same concentration (500 µg/mL). Interestingly, the cytotoxicities of PEIand PVP- coated UCNPs were drastically reduced upon decreasing their concentration to 250 µg/mL. In another study, hydrophilic PEG-functionalized UCNPs (size: 30 nm) were synthesized and loaded with DOX for multifunctional applications, namely, for delivering chemotherapy and performing bioimaging for cancer diagnostics [S32]. Moreover, FA, which is useful for detecting cancer cells via the folate receptors, was also attached along with DOX for targeted release [S33]. These results revealed that FA/PEGylated-UCNPs/DOX exhibit higher cytotoxicity toward KERATIN-forming tumor cells (KB) and HeLa cells as compared to PEGylated-UCNPs/DOX, signifying the potential use of FA onto the surface of UCNPs. In another study, PEI and PEG were coated on the surface of UCNPs enhanced the stability, water dispersibility, and biocompatibility [S34]. Interestingly, compared with mSiO2-UCNPs (≈75%), PEG-coated mSiO2-coated UCNPs (80 nm size) exhibited better cell viability (≈100%) to L929 fibroblast cells at a dose of 500 µg/mL at incubation time of 24 h [S35]. In another study, mSiO2 was coated onto the 21

surfaces of UCNPs (β‐NaYF4:Yb3+, Er3+@β‐NaGdF4:Yb3+) for forming core-shell nanospheres [81], and the surfaces of the nanospheres were modified with hydrophilic PEG for their use in drug delivery and in vitro cytotoxicity studies. The MRI studies of the functionalized UCNPs revealed that the contrast brightening was increased remarkably with Gd3+ ion concentration. The uptake of mSiO2‐UCNPs-PEG nanocomposites by cells was observed to be significant, and the cells exhibited green emission upon excitation with NIR laser at 980-nm. However, the cell viability was decreased to 30%–40% after loading the anticancer drug DOX on the surface of PEG-coated mSiO2-UCNPs (size: ≈80 nm) [81,S35], suggesting the modifcation of UCNPs surface with drug molecule was drastically decreased cell viability. The emergence of smart stimuli-responsive drug delivery systems has enhanced the anticancer effects and clinical utility of UCNPs in various cancer therapies without any side effects [S36,S37]. Cao et al. [44] fabricated PEG-coated UCNPs (10-nm) for in vitro and cytotoxicity studies in Kunming mice. The cell viability exceeded 90% event at 500 µg/mL concentration after incubation for 24 h. A comprehensive understanding of the surface chemistry of UCNPs is necessary for fabricating of UCNPs with outstanding bioapplications.

5.2. Silica-coated UCNPs Recently, rapid advances have been made in the development of silica-coated UCNPs as promising agents in cancer theranostics [S38]. These UCNPs offer several advantages such as excellent stability, technological versatility, low cost, biocompatibility, easy surface functionalization, and high payload capability [S39,S40]. On the basis of cytotoxicity data, SiO2-coated UCNPs (size: 24.6 nm, 50 µg/mL) showed nearly 100% viability to HeLa cells after 24 and 48 h of incubation, whereas bare UCNPs (size: 22.3 nm) showed a cell viability of around 80% after 48 h of incubation, confirming the cytotoxic nature of UCNPS was 22

greatly minimized by modifying their surfaces with silica [S41]. In another study, mSiO2coated UCNPs (size: 46.5 nm) not only showed high loading capacity for PSs but also prevented the direct contact of PSs with the bioenvironment and protected them from the degradation at harsh tumor environment [S3]. These modified UCNPs showed a cell viability of 99.4% (L929 cells) after treatment with 500 µg/mL of UCNPs without NIR laser treatment, indicating high in vitro biocompatibility. These results were consistent with those of another study conducted by Zhang’s group [S42]. In this study, the UC luminescence of UCNPs was increased by two orders of magnitude when Nd3+ ions were introduced as a sensitizer. It was also noticed that the introduction of Ca2+ ions into UCNPs leads to positively reinforced energy migration and to reduce lattice defects in the crystals. The surface of UCNPs was further coated with mSiO2 (Fig. 2b) for loading of DOX for chemotherapy. The molecular valves of UCNP@mSiO2 were grafted with blue-light-cleavable ruthenium (Ru) for preventing DOX from leaking before reaching the target. Under 808-nm laser excitation, the blue upconverted light triggered the cleavage of Ru and uncapped the pores to release DOX to kill cancer cells (Fig. 2a). The UC luminescence of UCNPs was decreased after the Ru complex grafted onto the surface of UCNPs (Fig. 2c), indicating the successful quenching of the blue upconverted light. As a result, effective drug release was observed upon exciting the UCNPs with 808-nm light, resulting in the cancer cells being killed with increasing irradiation time (Fig. 2d). This observation confirmed that the antitumor activities of UCNPs were greatly improved for effective chemotherapy, which was confirmed by capturing the florescence images (Fig. 2e–i). Recently, the biocompatibility of mSiO2-coated UCNPs to MCF-7 cell lines did not reduce cellular viability even at 200 µg/mL after exposure for 24 h [S43]. In another study, UCNPs were fabricated on the surface of hollow mSiO2 capsules and these capsules were generated by etching Fe3O4 nanocomposites [75]. These UCNP-mSiO2 capsules exhibited good dispersity and had a large surface area, which made them as 23

promising candidates for the sustained loading and release of DOX in a pH-sensitive environment. Cytotoxicity studies revealed that the surface modified UCNPs exhibited high biocompatibility to L929 fibroblast cells without any DOX release even at 250 µg/mL (viability: 105%) after 24 h of incubation. However, DOX-loaded capsules showed significant toxicity to HeLa cells (≈50%) at 7.9 µg/mL [75]. Recently, mSiO2 was decorated on the surface of AuNRs by using the seed crystal growth approach, and the formed mSiO2AuNRs were assembled with lanthanide-doped UCNPs via electrostatic adsorption [82]. The AuNRs exhibited SPR peaks at 520 and 660 nm, which were overlapped with the luminescence peaks of UCNPs and generated heat via energy transfer. Subsequently, MC540, a PS for inducing ROS production, was loaded onto a silica shell coated UCNPs and used as potential candidates for efficient cancer phototherapies. Similarly, silica was coated onto the surfaces of UCNPs (size: 47 nm) and the biocompatibility of silica-coated UCNPs was investigated on A549 [99]. The A549 cell viability exceeded 99% even at 200 µg/mL of SiO2-UCNPs, suggesting the potential use of SiO2-UCNPs for bioassays.

5.3. Metal oxide-coated UCNPs Metal oxide-coated UCNPs possess multifunctional properties including high tumor targeting efficiency, drug loading capability, and anticancer effects [35,48,S44]. These properties will be discussed precisely in this section. Dual-core-shell (Fe3O4-NaYF4@TiO2) nanostructured materials were synthesized and loaded with DOX for synergetic SDT [48]. Each nanocomposite had a unique role: TiO2 acted as a sonosensitizer for SDT and NaYF4 NPs served as a UC luminescence agent. The cellular uptake capability of UCNPs was increased considerably upon modification of UCNPs with HA. The cytotoxicity of asprepared HA-Fe3O4-UCNP@TiO2 and HA-Fe3O4-UCNP@TiO2-DOX NPs were investigated 24

on MCF-7 cells, and showed the cell viabilities of 75% and 35% after just 5 min of treatment (100 µg/mL) under ultrasonic radiation. In this nanocomposite, HA exhibited excellent tumor-nucleus-targeting capability for killing of cancer cells. In another study, plasmonic AuNRs dimers were assembled onto the surfaces of UCNPs for multimodal imaging-guided combination phototherapy [S45]. In this study, two typical building blocks—AuNRs and UCNPs—were hierarchically combined via DNA hybridization. Subsequently, Ce6 was fabricated on the surface of UCNPs-AuNRs for effective PDT. Under 980-nm laser irradiation, Ce6 was excited by the energy transfer from UCNPs, since the emission wavelength of UCNPs was exactly matched with the absorption peak of Ce6. Thus, the nanorod dimers acted as a strong PTT agent, and the Ce6-triggered UCNPs considerably enhanced the PDT efficacy for synergistic therapeutic capability. Upon incubation with HeLa cells after combined therapies, this nanoassembly showed a cell viability of only 4%, which indicated its significant anticancer effects [S45]. Similarly, in a different study, AuNRs were assembled on the surfaces of UCNPs (NaYF4:Yb3+/Er3+) for effective PDT [S46]. In this work, MB was incorporated into a SiO2 shell for the use in plasmon-enhanced PDT. The AuNRs were greatly enhanced the UC luminescence and increased the generation of ROS through the SPR effect. The results revealed that the multiple benefits (MB loading content, and optimized distance SPR-enhanced ROS generation capability) offered by coating silica shell on UCNPs, confirming the surface chemistry modification on UCNPs play a key role in theranostic applications. Further, the cytotoxicity of MB- and FA-functionalized UCNPs was evaluated on OECM-1 cells, and observed that UCNPs (400 µg/mL) exhibited >90% cell viability without NIR laser irradiation, however the cell viability was deceased to 30% after NIR laser irradiation [S46]. These results demonstrated that the modification of UCNPs with AuNRs plays key role in enhancing their cancer theranostics applications via ROS generation [S46]. In another study, silica-coated UCNPs acted as a loading agent for AuNPs, which were 25

readily amenable to biomonitoring [S2]. These reports illustrated that the surface modified UCNPs with metal oxides, silica and AuNPs exhibited outstanding properties (effective drug release, decrease in cancer cell viability and ROS generation) in cancer therapy.

5.4. Functionalization of UCNPs with different molecules To enhance drug-loading capacity of UCNPs, UCNPs have been functionalized with different molecules/ligands for specific bioconjugation with cancer biomarkers or drugs. An example of such a molecule is β-CD, which acts as a host molecule that shows high potentiality for internalizing guest molecules. It is having binding constant in the range of 100.5 - 105 M-1 in water [S47]. It is highly soluble in water solubility and negligible toxicity [S48]. To enhance water dispersibility and biocompatibility of UCNPs, a novel β-CD derivative i.e., 6-phosphate-6-deoxy-β-cyclodextrin was synthesized and then functionalized on the surface of UCNPs [S49]. It was observed that the β-PCD-UCNPs showed less cytotoxicity (>80%) to HeLa cells at a dose of 500 µg/mL after 48 h of incubation. On the other hand, ZwitLipo were used as potential ligands for the surface modification of UCNPs (size: 39 nm) [S50]. It was observed that 500 µg/mL of ZwitLipo-UCNPs showed negligible toxicity to HeLa cells even after 24 h of incubation [S50]. Mesoporous γ-AlO(OH) nanocomposite was coated on the surfaces of UCNPs and used as carriers for DOX delivery and cell imaging studies [S51]. The DOX drug was successfully loaded onto the surfaces of UCNP-Al, and it was released from the surfaces under mild acidic conditions. In vitro cell cytotoxicity studies revealed that the DOX-loaded UCNP-Al nanocomposites exhibited a higher degree of cytotoxicity compared to free DOX, suggesting an increase in the cell uptake of DOX from the modified UCNPs. Chen et al. [S52] fabricated a multifunctional nanoplatform by modifying the surfaces of UCNPs with PAA and BSA and then loading two 26

dyes, RB (PS) and IR825 (NIR-absorbing dye), as shown in Fig. 3a. The developed nanoplatform did not show any toxicity to 4T1 cancer cells even at a high concentration (400 µg/mL, Fig. 3b) in the absence of laser irradiation. The uptake of UCNP@BSA-RB&IR825 was studied by using CSLM, which confirms that the strong UC PL signals in the cytoplasm of 4T1 cells (Fig. 3c). This multifunctional nanoplatform has shown high ability to absorb green light at 980 nm, resulting in efficient photodynamic therapy, which results to kill cancer cells. Noticeably, the use of IR825 on UCNPs improves the resolution between the UC excitation and emission wavelengths, which leads to a strong photothermal effect (Fig. 3d–f). Chen et al. confirmed that the modified UCNPs (500 µg/mL) exhibited high toxicity to 4TI murine breast cancer cells (≈10%, 24 h of incubation) when exposure to NIR lasers at 980- and 808-nm. The surface chemistry of UCNPs plays vital role in developing the drug carriers with multidrug resistance for effective chemotherapy. Toward this end, TPGS was coated on the surface of UCNPs and used as an inhibition agent of P-gp, which is overexpressed on the cell membrane [S53]. Since P-gp has strong resisted the accumulation of anticancer drugs in cells [S53]. The synthesized TPGS-UCNPs showed excellent water dispersibility, P-gp inhibition, and cell viability even at 500 µg/mL concentration (≈100%, 24 h incubation). By contrast, while facilitating drug accumulation, DOX-resistant MCF-7 cells exhibit low cell viability (<20%, 200 µg/mL concentration) because of a reduction in P-gp expression. The EBNA1 is a well-known latent protein of EBV-infected tumor cells [S54]. Recently, P4 was decorated on the surfaces of UCNPs to overcome the diagnostic limitations, including poor sensitivity and short fluorescence lifetime. The as-prepared UCNPs were applied to EBV negative (MRC-5 and HeLa, without P4) and EBV positive (NPC43 and C666-1) cancer cells and evaluated their potential applications in cancer therapy. Interestingly, it was observed that EBNA1-P4 uncoated UCNPs showed cell viabilities >90%, ≈80%, >90%, and >80% to 27

MRC-5, HeLa, NPC43, and C666-1 cells, respectively, whereas EBNA1-P4 coated UCNPs exhibited viabilities of >90%, ≈100%, ≈30%, and ≈20% to the cells, respectively [S54]. These cytotoxicity results revealed that the modification of UCNPs with specific biomolecule was drastically changed their biocompatible nature, showing high toxicity nature towards specific cells. Further, EDTMP was coated on the surface of UCNPs for various applications such as high stability, imaging with good resolution and minimization of in vitro and in vivo proinflammatory effects [100]. Remarkably, it was noticed that EDTMP-coated UCNPs had no impact on the cell viability and cell morphology but they enhanced the cellular uptake of UCNPs in THP-1, indicating the UCNPs are the safe and effective platform for cancer therapy. A variety of materials have been coated on the surface of UCNPs to realize desirable and effective structural features. Briefly, polypeptide (poly(Asp-Lys)-b-Asp)-wrapped mSiO2 decorated on the surface of UCNPs for the long-term tracking and real-time monitoring of ATP-responsive drug release [S55]. Furthermore, UCNPs were functionalized with a TDPAZn2+ on their exterior surface and then loaded with chemotherapeutic drug in the interior mesopores. In this system, LRET was observed due to multivalent interactions between the polypeptide and TDPA-Zn2+ complex, which facilitates to monitor drug release capability in real time. From these results, it can be noticed that the proposed UCNP@mSiO2-polypeptide hybrid nanoparticles have shown high potentiality for stimuli-responsive drug delivery as well as for monitoring biochemical changes in live cancer and stem cells. The addition of ATP was quickly displaced the surface-bound peptide because of its high binding affinity with TDPA-Zn2+, resulting in drug release and elimination of LRET. The as-prepared nanoparticles had low toxic effect on HeLa cells (≈80% viability) in the absence of ATP, indicating the effectiveness of ATP-responsive cancer therapy [S55]. These studies revealed that the ligand chemistry on UCNPs was remarkably improved the UCNPs properties such as 28

easy functionalization, good stability with respect to pH, time, and salinity with low nonspecific proteins interactions, and adhesion in the bioimaging process [S56].

5.5. Surface modification of UCNPs with various coatings Recently, UCNPs were decorated with various components to enhance their biocompatibility and anticancer effects for a short duration. In particular, components such as CuS NPs were decorated on the surface of UCNP@mSiO2 for employing them as a novel photothermal agent [S57]. The fabricated core-shell-satellite UCNPs (NaGdF4:Yb, Er, Mn, Co@mSiO2-CuS) exhibited biocompatible nature due to the surface modification with silicaCuS nanocomposites. To improve their potential uses in PDT and chemotherapies, the nanostructures were further modified with ZnPc and DOX for cancer therapy. The MRI properties of the modified UCNPs were improved by doping with Co2+ ions, and their red emission was also enhanced by co-doping with Mn2+ ions. Additionally, ZnPc was also loaded on the doped UCNPs for generating ROS through the absorption of red UC luminescence from the UCNPs, which can be used as promising agents for effective PDT treatment. It was noticed that the heat was produced by NIR light irradiation, resulting effective PTT as well as release of DOX from UCNPs surfaces. When L929 fibroblast cells (normal cell lines) were incubated with modified UCNPs for 24 h, the cell viability exceeded 90%. By contrast, HeLa cells showed a viability of 10% after 24 h of incubation under NIR light irradiation because of their synergistic PDT, PTT, and chemotherapy effects on cancer cells. In another study, novel multifunctional GdOF:Ln@SiO2 (Ln = 10%Yb/1%Er/4%Mn) mesoporous nanocomposites were synthesized and used as therapeutic agents for photothermal chemotherapy [50]. The red emission was successfully transferred to the 29

conjugated PDT agent (ZnPc) because of the co-doping of Yb/Er/Mn ions into the GdOF host, allowing to produce highly active singlet oxygen, which facilitates a significant thermal effect and a remarkable DOX release from the surfaces of UCNPs. The developed nanoplatform exhibited unique functions in anticancer therapies multiple imaging ability (UC luminescence, MRI, computed tomography imaging), high loading drug capacity (mSiO2), PDT agent (ZnPc), PTT agent (carbon dots), DOX (anticancer drug) and FA (tumor targeting), respectively. Xu’s group modified UCNPs with a Pt(IV) prodrug and PEG-PAHDMMA polymer and then used as an anticancer agent for chemotherapy [66]. This anionic polymer transforms into a cationic polymer in a mild acidic tumor environment, yielding to generate charge repulsion between UCNPs and polymer, which results to release positive UCNP-Pt(IV). In addition, UV-light-emitting UCNPs and reductive glutathione together in tumor cells could generate highly toxic Pt(II) from Pt(IV), which results to a drastic decrease in HeLa cell viability (≈10% viability, 500 µg/mL of UCNPs). CNQDs are found to be a highly biocompatible and photoreactive polymeric nature with high chemical stability in different solvents [S58]. The emission peak of CNQDs was observed at 536 nm and emitted bright green light due to the absorption of light from UCNPs, leading to generate high amount of ROS, which yields effective PDT [S59]. In this study, UCNPs were modified with PLL and then functionalized with CNQDs. The modified UCNPs-PLL@CNQDs (size: 45 nm; concentration: 500 µg/mL) showed cell viabilities of >90% and ≈30% to CAL-27 oral cancer cells without and with 808-nm laser irradiation [S59]. These results revealed that the modified UCNPs showed high ability to absorb NIR light that enhances their cytotoxic nature via PTT. In view of their anticancer effects, the NIRmediated UCNPs were synthesized by modifying their surfaces with mSiO2 [35]. The modified UCNPs@mSiO2 nanocomposites were used as a novel nanocarrier for anticancer therapy. The TiO2 (3.0–3.2 eV) was introduced as a PS because of its UV properties. 30

Consequently, a large amount of ROS was generated when TiO2 absorbed UV light under irradiation with a 980-nm laser. Further, in this system, a UV light cleavable linker (TC) was fabricated as a “gate” to encapsulate DOX on the surface of mSiO2 for controlled release. Under NIR excitation at 980 nm, TiO2 could effectively absorb UV light and generate singlet oxygen species, thereby increasing the drug release via degradation of TC linker. As a result, the modified UCNPs exhibited superior cytotoxic nature towards HeLa cells (cell viability ~20%), which confirms that the surface modified UCNPs acted as promising agents for PPT and chemotherapy.

5.6. Surface modification of UCNPs with different dopant ions In the past several years, researchers have been tremendously improved the optical properties of UCNPs by doping different metal ions into UCNPs [S60-S62]. For example, recently, Co2+ and Mn2+ ions were doped into the core of UCNPs and used as promising probes for MRI applications [S57]. To endow UCNPs with T2 MRI properties, Co2+ ions were doped into UCNPs. Similarly, Mn2+ ions were also doped into UCNPs to enhance the red emission for bright fluorescence imaging of cancer cells. Although ultra-small UCNPs are biocompatible and safe for investigation of biodistribution profiles [S63], unfortunately they are poor luminescence nature because of the presence of fewer emitters, more lattice defects, and vibration energy diffusion. To overcome such drawbacks, a combinatorial strategy was adopted to modify the crystal lattice and migration system of UCNPs by doping with Ca2+ ions [S42]. Calcium ions were incorporated into each layer of UCNPs (core-shellshell) for effective crystal growth with uniform size, which results to generate strong emission peaks of UCNPs with reduced the quenching effect. In another approach, Ding and

31

co-workers doped Yb3+/Er3+ ions into K(Mn, Zn)F3 mesoporous microspheres to obtain single-band red emission UCNPs for potential biomedical applications [S64]. In another study, a new approach was adopted to fabricate Nd3+ free-photo-switchable UCNPs (PUCNPs) for realizing improved photo-switching properties [S65]. It was observed that the strong blue emission was generated by Tm3+ ion under the excitation of 980 nm wavelength. However, this emission was completely switched off when irradiated at 808-nm light. Further, the red emission was observed upon the irradiation with 808-nm laser, allowing them to use as promising materials for biomedical applications via photoinduced “off-on” therapy. Once a tumor site was imaged, the 808-nm light was switched to 980-nm light to induce UV-blue emissions. This UV emission was successfully absorbed by TiO2 NPs to activate PDT (Fig. 4a). The A549 cell imaging profiles revealed that the generation of blue light emission upon excitation with 980-nm light, and excitation with both 980- and 808nm lasers induced red emissions (Fig. 4b). The cell viability of A549 decreased to 15% after treatment with 400 µg/mL of PUCNP@TiO2 (24 h incubation) under excitation with only 980-nm light. The 808-nm laser was used only for the diagnosis of the tumor site (Fig. 4c). In another study, a decoupling theranostic approach was developed using a Yb3+/ Er3+-doped core, an undoped first shell, and an Nd3+-doped second shell; in the approach, the 806- and 980-nm NIR light functionally decoupled the diagnostics and therapy [S66]. Heating-free 806-nm laser irradiation extended the tissue depth action, and 980-nm NIR irradiation triggered ROS generation by Ce6 (PS); these irradiations helped visualize the area of interest and damage cancer cells, respectively.

6. In vivo biomedical applications of UCNPs

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Recently, UCNPs have shown to be ideal probes for real-time monitoring and distribution of multiple targets in tumor cells [S67]. Further, in vivo fluorescence imaging of tumor cells plays a vital role in estimating the tumor locations and tumor size in the living organisms. In this view, several reports have been explored the use of UCNPs as ideal candidates for in vivo imaging of various cells including tumor cells [S68]. The designed UCNPs have been used as multiple probes (drug carrier, PDT and PTT) for diagnosis of various cancer diseases.

6.1. UCNPs-based NIR-responsive phototherapies PDT is an effective approach for various drug delivery applications, including the PSs transportation to tumor site for photodynamic cancer treatment [S69]. In this system, cytotoxic ROS species are generated by exposing PSs to the specific wavelength light, resulting drastic damage to tissue or cell damage. Generally, organic molecules have been used as PSs for various applications however they have some demerits such as visible light sensitization and poor solubility. To overcome the limitations, scientists have been introduced UCNPs-based approaches for PDT due to their high efficiency at NIR irradiation through FRET process. The loading of PS on UCNPs surface is not only increased the water dispersibility of functionalized UCNPs but also enhanced blood circulation and tumor accumulation time of PSs due to their conjugation with peptide, antibodies, polymers or hydrophilic materials (MnO2) [S70,S71]. The PSs can be sensitized by NIR light which further improve the deep tissue therapeutic efficacy. For example, a single 808 nm NIR laser was triggered a dual-PSs nanocomposite by loading HA (blue- light) and TiO2 (UV-light) on the surface of UCNPs (NaYF4: 40% Yb3+, 0.5% Tm3+@NaYF4: 10% Yb3+ @NaNdF4: 10% Yb3+@NaYF4) [S72]. This system was based on FRET process from absorbing the UCNPs

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emission light to PS molecule. In this strategy, HA was introduced to enhance inhibited ion efficiency and in vivo targeted therapy of HeLa tumor-bearing balb/c nude mice. The asprepared UCNPs@TiO2@HA showed efficient tumor ablation due to the presence of HA as a targeting ligand in comparison with single PS system (UCNPs@TiO2). Lv et al. designed a UCNPs (NaGdF4: 19% Yb3+, 1% Er3+@ NaGdF4: 5% Yb3+@ NaNdF4: 5% Yb3+@ NaGdF4: 5% Yb3+) based multifunctional nanoplatform (UCNPs-BPS) to achieve PDT through FRET process [S73]. In this approach, authors used 808 nm laser light for PDT and noticed that the drastic reduction of tumor volume just after 14 days of treatment [S73]. It is worthy to note that UCNPs (Ce3+-Yb3+, Tm3+)-DOX has shown to be as an active and stable system for PDT and chemotherapy of cancer under NIR irradiation, exhibiting higher performance as compared to the commonly used PSs [S74]. The CeO2 was used as pH-/H2O2-triggered dissociable agent to regulate tumor hypoxia for enhancing antitumor effect. PTT is another kind of noninvasive and novel in vitro and in vivo cancer therapeutic techniques, and attracted considerable attention in biomedical research due to less toxicity, controllable operation, and highly selective tumor elimination efficiency based on the photothermal conversion effect of PTT agent [S75]. Recently, several studies have been illustrated the potential use of UCNPs with combined PTT and PDT agents for synergistic cancer therapy. Sun and co-workers explored the potentiality of combined PDT and PTT for synergistic cancer therapy using multifunctional nanoparticle (NR dimer-UCNPs-Ce6) [S45]. In this approach, AuNRs dimer acted as a PTT agent which were synthesized from complementary thiolated-oligonucleotides to increase photothermal efficiency under 808 nm laser irradiation in comparison with building block NRs. Whereas UCNPs-Ce6-DNA1 served as both imaging and PDT agents under 980 nm light exposure. Importantly, NR dimerUCNPs-Ce6 exhibited multimodal imaging ability even after 24 h injection time of HeLa tumor-bearing mice, indicating the remarkable tumor-targeting ability of functionalized 34

UCNPs. In addition, tumor inhibition results revealed that PTT and PDT were achieved at a safe NIR power dosage for in vivo tumor therapy. Some other studies also demonstrated that the PTT effect could be generated by UCNPs via either non-radiative or radiative relaxation process which can convert NIR photon energy to thermal energy [S76-S78]. Therefore, it is postulated that nanomaterials with weak and strong emissions would be accompanied by strong and weak photothermal effect, respectively. Interestingly, bright emissions and strong photothermal effect have also achieved by doping rare earth ions with different concentrations in UCNPs [S79]. This PTT effect enabled only at tumor site in the presence of smart nanoprobe and NIR irradiation. Meanwhile, UCNPs could be served as excellent multifunctional bioimaging and therapeutic agents for synergistic cancer therapy.

6.2. UCNPs-based pH-responsive chemo- and phototherapies Recently, pH-responsive cancer therapies have been extensively explored due to the specific cancer cell microenvironment Fig. 1 as compared to other organelles (Fig. 1). Wang’s group fabricated a pH-responsive drug delivery nanoplatform, abbreviated as UCNP@mSiO2@DOX-ZnO, where mSiO2 housed DOX and ZnO QDs acted as gatekeeper on the surface of UCNP@mSiO2 to control the drug release [S80]. The ZnO QDs dissolved in low pH environment, which results to release DOX in the mesopores. The in vitro and in vivo studies revealed that the diagnostic and therapeutic effectiveness of the as-prepared modified UCNPs have found to be high for HeLa cell lines and H22 tumor-bearing balb/c mice. Superparamagnetic particles can also impart UCNPs with MRI capability along with anticancer effect, such as manganese oxide (MnO2) or iron oxide [S81-S83]. MnO2 can degrade to Mn2+ ions within the tumor microenvironment, endowing UCNPs with MRI capacity. Recently, MnO2 sheets were grown on the surface of UCNPs (NaGdF4: 20% Yb3+, 35

2% Er3+@ NaGdF4: 20% Yb3+) and loaded with Ce6 and DOX to achieve MRI signals, PDT and chemotherapy [S83]. In this case, MnO2 was degraded to relieve tumor hypoxia and to improve PDT by decomposing endogenous O2 or H2O2, indicating self-enhanced therapy. Liu’s group fabricated Nd3+-sensitized UCNPs and then functionalized with negatively charged PAA shell which was further linked with DOX via electrostatic interaction [S84]. The UCNPs@PAA-DOX has shown remarkable property to hold high drug for pH-triggered chemotherapy of H22 tumor-bearing mice [S84]. The bond between DOX and PAA was broken due to protonation of PAA in tumor microenvironment, resulting to release DOX for efficient anticancer treatment. In one study, pH-responsive drug molecules were functionalized on the surface of UCNPs and used as a drug delivery agent for pH-driven PDT [S85]. The Nd3+-sensitized UCNPs were functionalized with pHLIP for efficient tumor targeting PDT under 808 nm excitation [S85]. pHLIP can bring as-prepared nanocomposite into cancer cells under low pH environment, realizing PDT both in vitro and in vivo. In another strategy, a switchable UCNPs/DNA nanocomposite was constructed (NaYF4: 20% Yb3+, 0.2% Er3+@PAA-DNA) by modifying two types of FA-DNA sequences [S86]. The shorter FA-DNA sequence was protected from longer DNA sequence in healthy tissues, which avoids unexpected cellular internalization. As a result, UCNPs@PAA-DNA was effectively internalized into tumor environment followed by folding of longer DNA under acidic condition. Moreover, FA was used as guider to reach the target. Simultaneously, Ce6 molecules on the longer DNAUCNPs surface were effectively improved PDT efficacy. These studies revealed that the modification of UCNPs with unique chemical species provides larger scope for precise tumor-targeting and highly efficient synergistic therapies in clinical biology.

36

7. Toxicity assessment Even though UCNPs have shown remarkable applications in biomedical research area, it is also very important to study their possible toxicity with the cell biology or biology system. In this connection, several researchers have been investigated the long-term in vivo toxicity of bare- and coated-UCNPs that can help to use UCNPs as ideal candidates for theranostic applications. The in vivo toxicity of UCNPs was investigated in Caenorhabditis elegans worms, mice and zebra embryos [S25,S87,S88]. These results indicated that there was no obvious toxicity of UCNPs, and the cell viability was not decreased in the presence of UCNPs, which confirms that the biocompatible nature of UCNPs. In particular, Xiong and co-workers demonstrated that the long-term behavior of UCNPs@PAA in mice through serum biochemistry, hematology and histology analysis and body weight measurement with an administration of 15 mg/kg UCNPs@PAA dosage for about 3 months [S26]. The structure and function of the organs were found to be normal and comparable with the control groups. In another study, Cheng and co-workers fabricated ultra-small PAA- and PEG-coated UCNPs and determined their in vitro and in vivo cytotoxicities, biodistribution, and long-term stability in different physiological solutions [S25]. No toxic and side effects were noticed, implying that the polymer-coated UCNPs exhibited high degree of biocompatibility and stability. Recently, sun et al. investigated that the in vitro and in vivo applications of ultrasmall PEG-coated UCNPs (NaGdF4:Dy3+) [S89]. The novel probe possessed diagnostic ability but also excretion property via renal pathway. The biodistribution of these UCNPs was tracked by MR and X-ray CT imaging, indicating the accumulation of particles in the spleen and liver after 24 h post-injection time. Most of the particles (PEG-NaGdF4:Dy3+) were excreted out of mouse after 30 days of post-injection time to avoid potential toxicity [S89]. The UCNP-based in vitro and in vivo therapeutic applications revealed that UCNPs have shown outstanding properties in NIR-triggered drug release, drug delivery, PDT, and 37

PTT. Additionally, drug, PTT and PDT agents on the surface of UCNPs provide impressive therapeutic improvement. Besides this, UCNPs and organic/inorganic ligands offer a new inspiration for simultaneous medical therapy and diagnosis to treat malignant diseases or cancers due to their biocompatibility, stability, and targeting drug delivery. However, more in vivo therapeutic applications are required in the future with a high drug payload with controlled release in the target area.

8. Conclusions and future challenges In summary, UCNPs have exhibited outstanding potential applications in biomedical sciences because of their unique properties (nontoxicity, low background signals, extraordinary photostability, NIR-to-visible and NIR-to-NIR UC luminescence, and bioimaging/multimodal imaging capability). These properties make them as potential candidates for bioimaging of cancer cells and theranostics applications. A literature survey reveals that the emission peak and color of UCNPs can be effectively tuned by doping with either rare earth elements or transition metal ions. Importantly, other inorganic nanocomposites (mSiO2, TiO2, Al(OH)3, CuxS, and Au nanoparticles) have been successfully coated on the surfaces of UCNPs to form core-shell structures, and these structures have then been functionalized with a wide variety of polymers to enhance their biocompatibility and MRI capability. Furthermore, the drug loading capacity of UCNPs has been considerably improved by modifying their surfaces with specific ligands (β-CDs, biomolecules and polymers), rendering them as promising drug vehicles for the controlled release of DOX and considerably enhancing their uptake by cells. Studies have shown that UCNPs exhibit tremendous optical properties for the imaging of cells, which is useful for the elucidation of structural and functional information of cells. Moreover, surface-modified UCNPs exhibit 38

nontoxicity and are effectively internalized by cells because their bio-mimicking properties with cell walls. Owing to their surface functionalization and core-shell structures, UCNPs can serve as promising PDT agents for the PDT treatment of various cancer tumors. Multimodality theranostics probes can be designed readily by using a combination of UCNPbased imaging and a therapeutic agent. The UCNPs UC efficiency, surface functionalization, and wavelength matching between UC luminescence and PS absorption should be considered when UCNP-PS systems are constructed for efficient UC-PDT treatment. On the basis of the mode of PS attachment or loading, the acquired UC-PDT may exhibit the following advantages: (1) UCNPs-PS can be engineered with functional compounds or targeting agents to prevent the premature release of the PS and increase its tumor-targeting and accumulating capability, thereby reducing the overall phototoxicity. (2) The large surface-to-volume ratio of UCNPs can effectively increase the amount of PS that can be loaded. (3) The UCNP-PS system may make the PS amphiphilic, allowing the UCNP-PS to travel unimpeded through the blood stream into the tumor tissue. (4) The UCNP-PS may exploit the enhanced EPR effect, which is induced by abnormal and leaky tumor neovasculature and poor lymphatic drainage of the tumor tissue, to diffuse into and remain in the tumor tissue. Despite the remarkable applications of UCNPs in bioimaging and drug delivery, research must focus on the development of facile and one- or two-step reactions for the fabrication of ultra-small UCNPs with high UC luminescence efficiency. Furthermore, UCNPs with a high degree of biocompatibility, high UC luminescence, and superior paramagnetism should be fabricated for use in MRI studies. Additionally, the following key challenges should be addressed in the near future for the wide application of UCNPs in various fields of science: (i) synthesis of water-soluble UCNPs with high crystallinity, (ii) development of techniques for using UCNPs for the imaging of single molecule, (iii) modification of UCNP surfaces with specific ligands for the parallel detection of multiple 39

molecular species with minimal volumes, and (iv) evaluation of the molar mass, extinction coefficient and UC efficiency of the as-prepared UCNPs for the designing and development of biomedical materials. Thus, considerable research efforts should be devoted to the design and fabrication of UCNPs with superior optical and magnetic properties for their wide range of applications in biomedical sciences.

Acknowledgments This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSI) (NRF-2017R1A2B4009581).

Appendix A. Supplementary material The references above 100 are provided in supplementary data and cited in the main text as [S1, S2, … S119], which can be found in the online version at https://doi.org/10.1016/j.trac.2019.XX.XXX

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53

Figure legends Scheme 1. Modification of UCNPs with different chemical compositions and their applications to in vitro cytotoxicity studies and cellular uptake.

Fig. 1. Cellular internalization of UCNPs by the endocytic pathway and direct diffusion against a negatively charged cell membrane. The complete degradation of nanoparticles is initialized by their potential interaction with the highly protonated and enzymatic environment of cellular organelles (endosomes and lysosomes).

Fig. 2. (a) Fabrication of UCNPs (NaGdF4:Yb3+, Tm3+, Ca2+@NaYbF4:Ca2+@NaNdF4:Gd3+, Ca2+) and coating of mSiO2 and DOX for chemotherapy applications. (b) TEM image of UCNP@mSiO2 NPs. (c) UC spectra of UCNPs, UCNP@mSiO2, UCNP@mSiO2-Ru nanoparticles (1 mg/mL) under 808-nm laser irradiation. (d) In vitro 4T1 cell viabilities after treatment with as-prepared particles at different time points (in minutes) and under identical conditions. Fluorescence microscopy images of photodamage detected using fluorescent probes at 50 µg/mL of DOX-UCNP@mSiO2-Ru at (e) 0, (f) 10, (g) 30, (h) 60, and (i) 90 min (reproduced with permission from the American Chemical Society [142]).

Fig. 3. (a) Synthesis of UCNPs with PAA, BSA, RB, and IR825 for both PDT and PTT based on the UCNP@BSA-RB&IR825 nanocomposite. (b) Relative in vitro 4T1 cell viabilities after incubation with different concentrations of UCNPs for 24 h. (c) CSLM images of 4T1 cells treated with various concentrations of UCNP@BSA-RB&IR825 for 4 h. (d) Relative 4T1 cell viabilities after incubation with different concentrations of UCNP@BSA-RB and after 980-nm laser excitation for 10 min. (e) Relative 4T1 cell viabilities treated with 54

different concentrations of UCNP@BSA-IR825 after 808-nm laser excitation for 5 min. (f) In vitro cell viabilities induced by combined PDT-PTT treatment; the cell viabilities clearly show that a significant number of cancer cells are killed (reproduced with permission from Elsevier [152]).

Fig. 4. (a) Preparation of photoswitchable UCNP@TiO2 for PDT, biomonitoring, and imageguided therapy. (b) A549 cells incubated with PUCNP@TiO2 for imaging under 800- and 980-nm laser irradiation. Scale bar shows 20 µm. (c) In vitro A549 cell viabilities after treatment with PUCNP@TiO2 under 800- and 980-nm laser excitation (reproduced with permission from the American Chemical Society [165]).

55

Table 2. Summary of the composition, optical properties, drug delivery and in vivo biomedical applications of UCNPs Optical properties

Organic or inorganic ligand/ polymer

Composition of UCNPs

Size (nm)

In vivo

Therapeutic cargo

Therapy

References

Excitation (nm)

Emission (nm)

PLL

NaYF4: 18% Yb3+, 2% Er3+@NaYF4: 25% Yb3+, 25% Nd3+@NaYF4

808

522, 540, 662

148.9

CAL 27 tumorbearing mice

AuNR@mSiO2-MC

PTT, PDT

[82]

DNA1, DNA2, PS-b-PAA

NaGdF4: 18% Yb3+, 2% Er3+

980, 808

500-700 (980),

≈100

HeLa tumor-bearing Balb/c nude mice

Ce6, AuNRs

PDT, PTT

[S45]

PAA, BSA, RGD

NaGdF4: 20% Yb3+, 2% Er3+

980, 808

≈525, ≈545, 660

≈62

4T1 tumor-bearing nude mice

RB&IR825

PTT, PDT

[S52]

PVP

NaYF4: 40% Yb3+, 0.5% Tm3+@NaYF4: 10% Yb3+ @NaNdF4: 10% Yb3+@NaYF4

808

290, 360, 450, 480

≈135

HeLa tumor-bearing mice

TiO2- Hyaluronic acid, Hypocrellin A

PDT

[S72]

PAA

NaGdF4: 19% Yb3+, 1% Er3+@ NaGdF4: 5% Yb3+@ NaNdF4: 5% Yb3+@ NaGdF4: 5% Yb3+

808

543, 654

≈50

Female balb/c mice (U14 tumor)

BPS-NH2

PDT

[S73]

mSiO2, PEGRGD

Ce3+-Yb3+, Tm3+

980

445, 660, 800

≈160

U87MG tumorbearing mice

DOX, Cerium oxide UCNP

Chemotherapy, PDT

[S74]

PEI

NaYF4: 20% Yb3+, 2% Er3+@ NaYF4@NaLnF4 (Ln= Yb3+ or Er3+)

975

500-600, 600700, 14501650

≈26

4T1 tumor-bearing mice

NaLnF4

PTT

[S79]

mSiO2, MnO2PEG

NaGdF4: 20% Yb3+, 2% Er3+@ NaGdF4: 20% Yb3+

980

510-530, 530570, 630-680

53.2

U14 tumor-bearing mice

Ce6, DOX

Chemotherapy, PDT

[S83]

C18PMH-PEGNH2, pHLIP

NaYbF4: Nd3+@NaGdF4: Yb3+, Er3+@NaGdF4

808

500-600

≈40-50

4T1 tumor-bearing balb/c mice

Ppa

PDT

[S85]

1

PVP

NaYF4: 40% Yb3+, 0.5% Tm3+@NaGdF4: 2% Yb3+

980

348, 365, 453, 480

≈100

HeLa tumor-bearing Balb/c nude mice

TiO2

PDT

[S99]

PEG

NaYF4: Yb3+, % Er3+ @NaGdF4

980

520-540, 540560, 640-680

≈70

U87MG tumorbearing nude mice

Ce6

PDT

[S100]

PEG, APTES, FA

NaYF4: 20% Yb3+, 0.5% Tm3+

980

362, 450, 485, 650

20-40

Female balb/c nude mice

DOX, TiO2

Chemotherapy, PDT

[S101]

APTES, FA

NaGdF4: 20% Yb3+, 0.5% Tm3+@SiO2

980

362, 452, 478,

≈100

MCF-7 tumorbearing nude mice

TiO2

PDT

[S102]

mSiO2

NaGdF4: 19.5% Yb3+, 0.5% Tm3+ @NaGdF4: Yb3+ @NaNdF4: 10% Yb3+@NaGdF4

808

348, 365, 453, 480

≈56

Female balb/c

TiO2

PDT

[S103]

TPGS

NaYbF4: 2% Yb3+

980

521, 541, 654

≈21.7

MCF-7 tumorbearing nude mice

DOX

Chemotherapy

[S104]

PEI, TRITC

NaYF4: 40% Yb3+, 0.5% Tm3+

980

336, 363, 445, 470

≈86

4T1 tumor-bearing mice

GQD

PDT

[S105]

Caspase-3, cRGD-PEGDSPE

NaYF4: 18% Yb3+, 2% Er3+

980

510-570, 640680

≈120

4T1 tumor-bearing mice

DEVD peptideDOX, MPPA

Chemotherapy, PDT

[S106]

mSiO2, CTAC

NaYF4: 20% Yb3+, 2% Er3+

980

523, 541, 655

≈28

CT26 tumor-bearing balb/c mice

MC540

PDT

[S107]

mSiO2-NH2, PAA

NaYF4: 20% Yb3+, 2% Er3+@NaYF4: 10% Yb3+ @NaNdF4: 10% Yb3+@NaYF4

808

510-530

≈100

H22 tumor-bearing mice

CuS, DOX

PTT, chemotherapy

[S108]

DSPE-PEG

NaYF4: 20% Er3+

808

540, 650, 1520

≈17

Pancreatic tumorbearing mice

ICG

PTT

[S109]

PEI

NaGdF4: Yb3+, Er3+@NaGdF4: Nd3+, Yb3+

980, 808

500-700

≈106

4T1 breast cancer bearing mice

RB-HA

PDT

[S110]

2

mSiO2, HA, CD44

NaYF4: 18% Yb3+, 2% Er3+@ NaGdF4

980

540

≈90

MCF-7 tumorbearing mice

DOX, CuS

Chemotherapy, PTT

[S111]

PDA

NaYF4: 20% Yb3+, 2% Er3+@NaYF4: Yb3+ @ NaNdF4: %Yb3+@NaYF4

808

521,542

≈50

H22 tumor-bearing mice

ICG

PTT, PDT

[S112]

OM, PEG

NaYF4: 20% Yb3+, 1.6% Er3+, 0.4% Tm3+

980

530-560, 645675, 770-830

68

NCI-H460 tumorbearing mice

TL-CPT, Ce6

Chemotherapy, PDT

[S113]

DA, PEG, FA

NaGdF4: x% Dy3+

808

-

≈10.59

4T1 tumor-bearing balb/c mice

PDA

PTT

[S114]

APTES, BSA, FA

GdOF: 20% Yb3+, 2% Er3+

980

520, 540, 661

≈47

U14 tumor-bearing mice

GND, DOX

PTT, chemotherapy

[S115]

mSiO2, Lascorbic acid

NaGdF4: 18% Yb3+, 2% Er3+@NaGdF4: 30% Nd3+, 10% Yb3+

808

510-530, 530570, 630-680

68.3

U14 tumor-bearing mice

POM

Chemotherapy, PTT

[S116]

Phospholipids (EggPC), OA, cholesterol, FA

NaYF4: 20% Yb3+, 2% Er3+

980

500-700

195

CT-26wt tumorbearing balb/c nude mice

DOTAP, PN, MB, RB, Re(CO)3(bpy)Br, TPP, ZnPC, AIPC

PDT

[S117]

PEG, PNP

NaYF4: 0.3% Tm3+@CaF2

980

325-800

≈350

MCF-7 tumor bearing nude mice

DOX

Chemotherapy

[S118]

PEI, DMMA, oxidized starch

NaYF4: Yb3+, Tm3+

980

350, 475

125.8

4T1 tumor-bearing female balb/c mice

DOX, Fe2+

Chemotherapy

[S119]

3

Table 1. Summary of the composition, optical properties, and in vitro applications of UCNPs

Optical properties Organic or inorganic ligand/ polymer

SiO2, FA Ligand-free HA

Composition of UCNPs

NaYF4: 18% Yb3+, 2% Tm @NaYF4@mSiO2-TiO2linker NaYF4: 20% Yb3+, 3% Er3+ NaYF4: 24% Yb3+, 6% Er3+Fe3O4@TiO2 3+

975

PEI, PEG, PAH, DMMA, SA Linker-COOH, β-CD Alginate, PEG, FA

NaYF4: 20% Yb3+, 1% Tm3+Pt(IV)

980

PAA TPP

980 980 980 808 980

PAA, PVP, PEI

NaYF4: 18% Yb3+, 2% Er3+

980

HPG

NaYF4: 20% Yb3+, 2% Er3+

980

PEG

3+

3+

NaYF4: 20% Yb , 2% Er @

Viability (%)

≈65

HeLa

HeLa

250, 24

≈100

[35]

548, 662 520, 540, 650 476, 650, 700, 802 289, 349, 361, 450, 475

≈80

HeLa KB, MCF7

HeLa

1000, 24

>80

[37]

MCF-7

100, 24

>85

[48]

540, 660

343, 360, 449, 476

NaYbF4: 0.5% Tm3+@CaF2

FA

C (µg/mL), Time (h)

980

PAA-PEI-HA

NaYF4: 20% Yb3+, 2% Er3+@mSiO2 NaLuGdF4: 18% Yb3+, 2% Er3+, x% Cr3+ La(OH)3: 2% Yb3+, 1% Er3+CuxS NaYF4: 18% Yb3+, 2% Er3+@ NaYF4: 10% Yb3+@NaNdF4: 10% Yb3+@NaYF4: 10% Yb3+ NaYF4: 20% Yb3+, 0.2% Tm3+@TiO2

Cell lines

Emission (nm)

980

980

Cell viability studies

In vitro cell imaging

Excitation (nm)

980

Bioapplications Size (nm)

423

522–565, 655–670 520–560, 650–700 540 347, 362, 452, 476 531–563, 633–685 407, 524, 542, 656 521, 542, 1

Reference

27

HeLa

-

-

-

[54]

56

HeLa

HeLa, L929

500, -

>80, >97

[66]

75

A549

A549

512, 24

≈60

[67]

40

KB, HeLa

KB, HeLa

300, 24

>90, >95

[68]

90

HeLa

HeLa

500, 24

94.3%

[69]

52

MCF-7

MCF-7

200, 48

≈100

[70]

25

MCF-7

MCF-7

100, -

>90

[71]

150

HeLa

HeLa, U87MG

500, 24

≈100, ≈80, <80

[74]

≈34

MCF-7

MCF-7

800, 24

>90

[79]

≈78

HeLa

L929

200, 24

≈100

[81]

PLL, SiO2 PEGphospholipids, TAT peptide PEG, bispecific antibody

NaGdF4: 2% Yb3+@mSiO2 NaYF4: 17% Yb3+, 3% Er3+mSiO2-MC@Au

808

NaYF4: 20% Yb3+, 2% Er3+

980

NaYF4: 20% Yb3+, 4% Tm3+

980

3+

Tween20 PEI-cF Citrate Citrate

NaGdF4: 20% Yb , 2% Er3+@NaGdF4 NaYbF4: 0.5%Tm3+@CaF2 NaYbF4: 0.1% Tm3+@CaF2@ NaDyF4 CaF2: 20% Yb3+, 2% Er3+/ Tm3+

980 975 980

654 410, 522, 540, 662 525, 540, 655 400-500, 800 525, 541, 654 362 475, 650, 700, 800

149

CAL-27

CAL-27

250, 48

>80

[82]

118

HeLa

-

-

-

[83]

≈43

PC3, LNCaP

-

-

-

[86]

42.1

HeLa

HeLa

500, 48

>90

[87]

52

HeLa

HeLa

500, 24

80

[94]

16

HeLa

RAW 264.7

1000, 24

≈100

[95]

920

655, 790

≈11

HeLa

HeLa

50, 18

>100

[96]

Ligand-free

NaGdF4: 3%Nd3+@NaGdF4

740

≈860–900, ≈1050, ≈1330

15

HeLa

HeLa

300, 48

≈100

[97]

PAA, silica sol, pluronic PE6800 copolymer

YVO4: 2% Er3+, 20% Yb3+

980

520, 550, 650

40

-

-

-

-

[98]

Silica

GdF3: 20% Yb3+, 2% Er3+, x% Li+@silica

980

≈47

HeLa

A549

200, 24

>99

[99]

19.9

HeLa

-

-

-

[S1]

≈22

HeLa

-

-

-

[S2]

46

HeLa

L929

500, 24

99

[S3]

55

HeLa

L929, HeLa

800, 24

≈100

[S4]

30

HeLa

KB cells

800, 24

≈100

[S32]

≈80

L929

L929

500, 24

≈100

[S35]

Ligand-free Silica NOBF4, Ce6, MC540 Oleic acid, gelatin C18PMH-PEG PEG-FA

NaLnF4: x% Mn2+, 18% Yb3+, 2% Er3+ NaGdF4: 20% Yb3+, 2% Er3+@silica@Au NaGdF4: 20% Yb3+, 2% Er3+@NaGdF4:x% Nd3+, x% Yb3+@mSiO2 NaGdF4: 18% Yb3+, 2% Er3+, x% Mn2+-ZnPC NaYF4: 20% Yb3+, 2% Er3+ NaYF4: 18% Yb3+, 2% Er3+@mSiO2

980 975 808 980 980 980

518–536, 544–556, 649–671 525, 543, 656 520, 540, 650 510–570, 630–680 510–560, 640–680 500–700 521, 542, 654 2

SiO2

NaYF4: 20% Yb3+, 2% Er3+ and NaYF4: 20% Yb3+, 0.2% Tm3+@SiO2

980

SiO2

NaGdF4:Yb3+, Tm3+, Ca @NaYbF4:Ca2+@mSiO2 Ru

808

MB, FA

NaYF4:Yb3+, Er3+

980

β-PCD-AdRGD peptide Zwitterionicphospholipid micelle

NaYF4: 20% Yb3+, 2% Er3+

980

NaGdF4: 20% Yb3+, 2% Er3+@NaGdF4

980

AlO(OH) PAA, BSA, RGD PEI, P4 peptide Polypeptide G2d2-TDPAZn2+ SiO2, ZnPC

PLL

2+

NaYF4: 20% Yb3+, 2% Er3+AlO(OH) NaGdF4: 20% Yb3+, 2% Er3+@BSA-RB&IR825 NaGdF4: 20% Yb3+, 2% Er3+@NaGdF4 NaYF4: 30% Yb3+, 0.5% Tm3+@ NaYF4: 18% Yb3+, 2% Er3+@mSiO2 NaGdF4: 20% Yb3+, 2% Er3+, x% Mn2+, 2% Co2+ @mSiO2CuS NaYF4: 18% Yb3+, 2% Tm3+@NaYF4: 21% Yb3+, 21% Nd3+@CNQDs

(Er) 540, 550, 654, (Tm) 474, 645, 697, 800 368, 450, 475, 649, 696 408, 520, 540, 654 520, 540, 650

≈24

HeLa

HeLa

50, 48

≈100

[S41]

32

4T1

4T1

100, 24

>90

[S42]

22–35

OECM-1

OECM-1

250, 24

>90

[S46]

118

MCF-7, HeLa

HeLa

500, 48

>80

[S49]

≈39

HeLa

HeLa

500, 24

>90

[S50]

45

HeLa

HeLa

100, 48

≈100

[S51]

≈62

4T1

4T1

200, 24

>90

[S52]

980

520, 540, 654

≈27

HeLa, C666-1

HeLa, MRC-5, NPC43, C666-1

-, 24

≈100, >90, ≈30, ≈20

[S54]

980

365, 475, 656

87

HeLa, MCF-7

HeLa

20, -

>80

[S55]

980

521, 540, 655

54

HeLa

HeLa, L929

500, 24

>90

[S57]

808

348, 365, 453, 480

≈45

CAL-27

CAL-27

250, 24

>90

[S59]

980 980, 808

514, 560, 635-680 520, 540, 655 ≈525, ≈545, 660

PVP, TiF4

NaErF4@NaYF4@NaYbF4: 0.5% Tm3+@NaYF4@TiO2

980, 800

(980) 380, 410 (800) 660

≈40

A549

A549

800, 24

>85

[S65]

Dextran-gDOPE

NaYF4: 20% Yb3+, 2% Er3+

980

350–700

150

MCF-7

MCF-7

250, 24

>90

[S90]

3

BSA.DTPAGd

NaGdF4: 20% Yb3+, 2% Er3+

980

PEG

Ba2GdF7: 18% Yb3+, 2% Er3+

980

PLGA

NaYF4: 17% Yb3+, 3% Er3+

980

Malonic acid

NaLuGdF4: 18% Yb3+, 2% Er3+ and NaLuGdF4: 19.5% Yb3+, 0.5% Tm3+

980

Chitosan

NaYF4: 17% Yb3+, 3% Er3+

980

PEG, ZnPC

NaErF4@NaLuF4

808

PAA

NaYF4: 20% Yb3+, 3% Er3+

980

PVP, TiF4

Nd3+: NaYF4: 20% Yb3+, 2% Er3+@ NaYF4: 10% Yb3+@HTiO2

808

520, 540, 656 523, 540, 651 520, 539, 653 (Er) 523, 542, 656 (Tm) 477, 805 520–550, 630–690 540, 654

43

-

HeLa

200, 24

>90

[S91]

24

HepG2

HepG2

5000, 24

≈90

[S92]

100

HGC, OSCC

HGC, OSCC

50, 24

>88, 60

[S93]

≈80

HeLa

HeLa

600, 12

≈75

[S94]

>90, 66

[S95]

500, 24

≈85

[S96]

527, 540, 655

≈62

HeLa

1000, 12

>80

[S97]

409, 439, 522, 542, 655, 663

≈100

HeLa

HGC, OSCC HeLa HeK293, HeLa, A549, SCC7 SKOV-3, GES-1, HeLa

50, 24

≈32

HGC, OSCC HeLa

800, 24

≈100

[S98]

120

4

Research Highlights Doping and fabrication of shell structures on UCNPs play a key for bioapplications. UCNPs surfaces were successfully modified with various polymer/ligands. Modified UCNPs acted as good candidates for photodynamic and photothermal therapy. Functionalized UCNPs exhibited non-toxicity and improved cell uptake ability. UCNPs acted as efficient probes for MRI and in vivo and in vitro bioimaging studies.